Understanding membrane transport processes such as ion occlusion reactions of ion pumps and transporters and the ion gating of channels requires knowledge of lipid bilayer electrostatics. A simple example of the effect of membrane electrostatics on ion transport is the much higher permeability of the membrane to hydrophobic anions, such as tetraphenylborate (TPB-), compared to hydrophobic cations, such as tetraphenylphosphonium (TPP+) or tetraphenylarsonium (TPA+). This has been attributed to the membrane dipole potential, of which a major contributor has been determined to be oriented water dipoles in the lipid headgroup region of the membrane. From the ratio of the TPB- to TPP+ or TPA+ conductances, the magnitude and polarity of the dipole potential can be estimated. Using the voltage-sensitive dye RH421 in conjunction with the stopped-flow technique and solid-supported membrane electrophysiology here we show that the transport of these ions is not simply a diffusion through the membrane but rather occurs in jumps between discrete binding sites within the membrane. The hydrophobic anion TPB- causes much greater RH421 spectral changes than TPA+. This could be explained by a combination of a stronger interaction of TPB- with RH421 and a deeper binding of TPB- within the membrane compared to TPA+. The experimental methods, used here for the first time to study the kinetics of ion transport across membranes, are potentially applicable to investigations of the membrane permeability of charged drug molecules, in particular anticancer agents.

There is no safe level of exposure to air pollution, including particulate matter smaller than 2.5 µm (PM2.5), to human health. Whilst it is well known that exposure to heavily polluted air is associated with several liver disorders, it is unclear how long-term exposure to low-level traffic-derived PM2.5 affects liver health. BALB/c mice (5 weeks, male) were exposed to traffic-derived PM2.5 (10 µg/mouse/day, intranasally) daily for 4, 8 and 12 weeks. Markers of inflammation and fibrosis were measured at each time point. Changes in liver proteome and lipid profiles were measured using proteomics and lipidomics at 12 weeks. Low-dose PM2.5 exposure increased macrophage infiltration, pro-inflammatory cytokine production, and increased collagen deposition at 12 weeks. Despite liver lipid metabolism being increased, the abundance of triglycerides, precursor diacylglycerols, and ceramide was also significantly increased by PM2.5 exposure, whereas glycogen content was reduced. Proteomics analysis revealed 64 proteins to be significantly changed in PM2.5-exposed mice, and KEGG pathway enrichment analysis indicated their involvement in lipid metabolism, alcohol-related liver disease, neutrophil extracellular trap formation, and transcriptional dysregulation related to cancer. In conclusion, prolonged exposure to low-dose traffic-derived PM2.5 promotes pathological changes in the liver, suggestive of an increased risk of metabolic dysfunction-associated fatty liver disease. Future studies can enable the identification of the signalling pathways underlying low-dose PM2.5-induced lipid accumulation in the liver.

Tertiary amides such as peptoids are a novel class of peptidomimetics that offer enhanced structure, activity, and stability compared to many naturally occurring antimicrobial peptides. Guanidino compounds have gained interest in medicinal chemistry as cell-penetrating molecules. This work investigates the changes in the antibacterial activity of modified guanidino groups on the structure of active guanidino tertiary amides by incorporating lipophilic, hydrophobic, and extra cationic groups, thereby combining the properties of the tertiary amide in the peptoid backbone with the important role of addition of extra cationic and lipophilic residues, such as those in AMPs, but supported by guanidine backbones. A library of active antibacterial bromo-phenyl and dichloro-phenyl-based guanidinium tertiary amides, including three series, was designed. These compounds exhibited MICs of 1-2 μg mL-1, 4-8 μg mL-1, and 16.5-35.6 μg mL-1 against S. aureus, E. coli, and P. aeruginosa, respectively. Tertiary amides with their guanidine bearing an alkylated cationic group of 3C (19a and 20a) and 6C (19b and 20b) length resulted in the most active molecules against all tested strains. Additionally, at 8× MIC, compound 19b was the most effective S. aureus biofilm disruptor, disrupting 75% of the biofilm, while compound 19g was the most active molecule against E. coli biofilm, with 50% disruption. The membrane permeability and QCM-D studies suggested that the designed cationic tertiary amides could depolarize and disrupt the bacterial cell membrane. The most potent peptoids were non-toxic, with HC50 of more than 50 μg mL-1.

The global health impact of viruses highlights the urgent need for innovative antiviral strategies. This study investigated the synergistic potential of two anthranilamide-based peptide mimics (RK610 and RK758) in combination, and one peptide mimic (RK610) in combination with the cationic peptides Mel4 and melimine, against murine hepatitis virus (a coronavirus; MHV-1), influenza virus (H1N1), and Herpes simplex virus (HSV-1). Checkerboard assays demonstrated RK610+RK758 had synergy against MHV-1 and H1N1 (∑FICI values of 0.14 and 0.5, respectively), while RK610+Mel4 showed potent synergy against HSV-1 (∑FICI = 0.18). Co-treatment outperformed sequential application. Transmission electron microscopy confirmed structural damage to virions, while cytotoxicity assays indicated that all tested combinations were non-toxic in MDCK, A9, and Vero cells, except for melimine and RK610+melimine with A9 cells. Biophysical analyses using DOPC (100 %) and DOPC: POPS (70:30) lipids provided mechanistic ideas into peptide-mimic interactions with lipid envelopes. Tethered bilayer lipid membranes (tBLMs) in conjunction with electrical impedance spectroscopy revealed that both peptide mimics and their combinations reduced membrane conductance, regardless of lipid composition. Quartz crystal microbalance with dissipation monitoring (QCM-D) revealed that RK610 and RK758 induced mass addition at the outer layer, significantly increasing with POPS. In DOPC, RK610 increased the surface pressure in a Langmuir-Blodgett trough while RK758 reduced it. The 610 + 758 and 610+Mel4 combinations raised maximum pressures. In DOPC + POPS, RK758 destabilized the monolayer (35 mN/m, no plateau), whereas combinations restored stability. These findings highlight peptide mimics as broad-spectrum antiviral agents that show synergy to target viral envelopes, paving the way for safer and low-resistance therapeutics.

ABSTRACT Chlamydia trachomatis is an intracellular bacterial pathogen that undergoes a biphasic developmental cycle, consisting of intracellular reticulate bodies and extracellular infectious elementary bodies. A conserved bacterial protease, HtrA, was shown previously to be essential for Chlamydia during the reticulate body phase, using a novel inhibitor (JO146). In this study, isolates selected for the survival of JO146 treatment were found to have polymorphisms in the acyl-acyl carrier protein synthetase gene ( aasC ) . AasC encodes the enzyme responsible for activating fatty acids from the host cell or synthesis to be incorporated into lipid bilayers. The isolates had distinct lipidomes with varied fatty acid compositions. A reduction in the lipid compositions that HtrA prefers to bind to was detected, yet HtrA and MOMP (a key outer membrane protein) were present at higher levels in the variants. Reduced progeny production and an earlier cellular exit were observed. Transcriptome analysis identified that multiple genes were downregulated in the variants especially stress and DNA processing factors. Here, we have shown that the fatty acid composition of chlamydial lipids, HtrA, and membrane proteins interplay and, when disrupted, impact chlamydial stress response that could trigger early cellular exit. IMPORTANCE Chlamydia trachomatis is an important obligate intracellular pathogen that has a unique biphasic developmental cycle. HtrA is an essential stress or virulence protease in many bacteria, with many different functions. Previously, we demonstrated that HtrA is critical for Chlamydia using a novel inhibitor. In the present study, we characterized genetic variants of Chlamydia trachomatis with reduced susceptibility to the HtrA inhibitor. The variants were changed in membrane fatty acid composition, outer membrane proteins, and transcription of stress genes. Earlier and more synchronous cellular exit was observed. Combined, this links stress response to fatty acids, membrane proteins, and HtrA interplay with the outcome of disrupted timing of chlamydial cellular exit.

Building upon our previous study on peptoid-based antibacterials which showed good activity against Gram-positive bacteria only, herein we report the synthesis of 34 dimeric peptoid compounds and the investigation of their activity against Gram-positive and Gram-negative pathogens. The newly designed peptoids feature a di-hydrophobic moiety incorporating phenyl, bromo-phenyl, and naphthyl groups, combined with variable lengths of cationic units such as amino and guanidine groups. The study also underscores the pivotal interplay between hydrophobicity and cationicity in optimizing efficacy against specific bacteria. The bromophenyl dimeric guanidinium peptoid compound 10j showed excellent activity against S. aureus 38 and E. coli K12 with MIC of 0.8 μg mL-1 and 6.2 μg mL-1, respectively. Further investigation into the mechanism of action revealed that the antibacterial effect might be attributed to the disruption of bacterial cell membranes, as suggested by tethered bilayer lipid membranes (tBLMs) and cytoplasmic membrane permeability studies. Notably, these promising antibacterial agents exhibited negligible toxicity against mammalian red blood cells. Additionally, the study explored the potential of 12 active compounds to disrupt established biofilms of S. aureus 38. The most effective biofilm disruptors were ethyl and octyl-naphthyl guanidinium peptoids (10c and 10 k). These compounds 10c and 10 k disrupted the established biofilms of S. aureus 38 with 51 % at 4x MIC (MIC = 17.6 μg mL-1 and 11.2 μg mL-1) and 56 %-58 % at 8x MIC (MIC = 35.2 μg mL-1 and 22.4 μg mL-1) respectively. Overall, this research contributes insights into the design principles of cationic dimeric peptoids and their antibacterial activity, with implications for the development of new antibacterial compounds.

The cytoplasmic lysine-rich N-termini of the Na+,K+- and H+,K+-ATPases have been designated as R domains because of their possible role in regulation of ion pumping activity. Prior to the publication of any crystal structures of either protein, it was suggested, based on mutagenesis studies, that regulation of the Na+,K+-ATPase could involve the formation and breakage of a salt bridge between Lys30 of the N-terminus and Glu233 in the first M2-M3 cytoplasmic loop of the alpha subunit. In structural studies of the Na+,K+-ATPase, the N-terminus has never been able to be resolved, probably because it is intrinsically disordered. Nevertheless, after theoretically predicting the N-terminus conformation, and adding it to the structure, the distance between residues Lys30 and Glu233 makes it doubtful that a direct long-lived interaction between them is involved in pump regulation. As an alternative hypothesis, we propose that regulation could occur via an interaction of the positively charged lysine residues of the N-terminus with negatively charged lipid headgroups (notably phosphatidylserine) on the cytoplasmic surface of the surrounding membrane, and that such an interaction could potentially be regulated by an electrostatic switch mechanism, whereby serine and tyrosine residues of the N-terminus are phosphorylated by protein kinases. Phosphorylation of these residues would decrease the positive charge of the N-terminus, allowing its release from the membrane. Experimental and theoretical data will be presented which support this hypothesis.

The Na+, K+-ATPase is an integral membrane protein which uses the energy of ATP hydrolysis to pump Na+ and K+ ions across the plasma membrane of all animal cells. It plays crucial roles in numerous physiological processes, such as cell volume regulation, nutrient reabsorption in the kidneys, nerve impulse transmission, and muscle contraction. Recent data suggest that it is regulated via an electrostatic switch mechanism involving the interaction of its lysine-rich N-terminus with the cytoplasmic surface of its surrounding lipid membrane, which can be modulated through the regulatory phosphorylation of the conserved serine and tyrosine residues on the protein's N-terminal tail. Prior data indicate that the kinases responsible for phosphorylation belong to the protein kinase C (PKC) and Src kinase families. To provide indications of which particular enzyme of these families might be responsible, we analysed them for evidence of coevolution via the mirror tree method, utilising coevolution as a marker for a functional interaction. The results obtained showed that the most likely kinase isoforms to interact with the Na+, K+-ATPase were the θ and η isoforms of PKC and the Src kinase itself. These theoretical results will guide the direction of future experimental studies.

Sensors that can quickly measure the lipase activity from biological samples are useful in enzyme production and medical diagnostics. However, current lipase sensors have limitations such as requiring fluorescent labels, pH control of buffer vehicles, or lengthy assay preparation. We introduce a sparsely tethered triglyceride substrate anchored off of a gold electrode for the impedance sensing of real-time lipase activity. The tethered substrate is self-assembled using a rapid solvent exchange technique and can form an anchored bilayer 1 nm off the gold electrode. This allows for an aqueous reservoir region, providing access to ions transported through membrane defects caused by triglyceride enzymatic hydrolysis. Electrical impedance spectroscopy techniques can readily detect the decrease in resistance caused by enzymatically induced defects. This rapid and reliable lipase detection method can have potential applications in disease studies, monitoring of lipase production, and as point-of-care diagnostic devices.

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Cardiovascular disease (CVD) is a leading cause of mortality worldwide, with cigarette smoking being a major preventable risk factor. Smoking cessation can be difficult due to the addictive nature of nicotine and the withdrawal symptoms following cessation. Electronic cigarettes (e-Cigs) have emerged as an alternative smoking cessation device, which has been increasingly used by non-smokers; however, the cardiovascular effects surrounding the use of e-Cigs remains unclear. This study aimed to investigate the effects of e-Cig aerosol condensate (EAC) (0 mg and 18 mg nicotine) in vitro on human coronary artery endothelial cells (HCAEC) and in vivo on the cardiovascular system using a mouse model of ‘e-vaping’. In vitro results show a decrease in cell viability of HCAEC when exposed to EAC either directly or after exposure to conditioned lung cell media (p < 0.05 vs. control). Reactive oxygen species were increased in HCAEC when exposed to EAC directly or after exposure to conditioned lung cell media (p < 0.0001 vs. control). ICAM-1 protein expression levels were increased after exposure to conditioned lung cell media (18 mg vs. control, p < 0.01). Ex vivo results show an increase in the mRNA levels of anti-angiogenic marker, FKBPL (p < 0.05 vs. sham), and endothelial cell adhesion molecule involved in barrier function, ICAM-1 (p < 0.05 vs. sham) in murine hearts following exposure to electronic cigarette aerosol treatment containing a higher amount of nicotine. Immunohistochemistry also revealed an upregulation of FKBPL and ICAM-1 protein expression levels. This study showed that despite e-Cigs being widely used for tobacco smoking cessation, these can negatively impact endothelial cell health with a potential to lead to the development of cardiovascular disease.

Contact lens wearers are at an increased risk of developing Pseudomonas-associated corneal keratitis, which can lead to a host of serious ocular complications. Despite the use of topical antibiotics, ocular infections remain a major clinical problem, and a strategy to avoid Pseudomonas-associated microbial keratitis is urgently required. The hybrid peptide VR18 (VARGWGRKCPLFGKNKSR) was designed to have enhanced antimicrobial properties in the fight against Pseudomonas-induced microbial keratitis, including contact lens-related keratitis. In this paper, VR18's modes of action against Pseudomonas membranes were shown by live cell Raman spectroscopy, live cell NMR, live-cell fluorescence microscopy and measures taken using sparsely tethered bilayer lipid membrane bacterial models to be via a bacterial-specific membrane disruption mechanism. The high affinity and selectivity of the peptide were then demonstrated using in vivo, in vitro and ex vivo models of Pseudomonas infection. The extensive data presented in this work suggests that topical employment of the VR18 peptide would be a potent therapeutic agent for the prevention or remedy of Pseudomonas-associated microbial keratitis.

Connecting molecular interactions to emergent properties is a goal of physical chemistry, self-assembly, and soft matter science. We show that for fatty acid bilayers, vesicle rupture tension, and permeability to water and ions are coupled to pH via alterations to lipid packing. A change in pH of one, for example, can halve the rupture tension of oleic acid membranes, an effect that is comparable to increasing lipid unsaturation in phospholipid systems. We use both experiments and molecular dynamics simulations to reveal that a subtle increase in pH can lead to increased water penetration, ion permeability, pore formation rates, and membrane disorder. For changes in membrane water content, oleic acid membranes appear to be more than a million times more sensitive to protons than to sodium ions. The work has implications for systems in which fatty acids are likely to be found, for example in the primitive cells on early Earth, biological membranes especially during digestion, and other biomaterials.

Swept frequency electrical impedance spectroscopy (EIS) can be used in conjunction with tethered bilayer lipid membranes to monitor the membrane permeability of ions in real-time (Deplazes et al. J Phys Chem Lett 11:6353-6358, 2020). Conductance readings, as determined by EIS, are a measure of the ability of ions to be transported across membranes. Recording the change in conductance as a function of cation concentration and a comparison between a range of cations permits conclusions to be made about the specificity of cation transport through pores. An estimate for upper pore size and cation selectivity of ion channels can be established using this method.

Because they are firmly anchored to a noble metal substrate, tethered bilayer lipid membranes (tBLMs) are considerably more robust than supported lipid bilayers such as black lipid membranes (BLMs) (Cranfield et al. Biophys J 106:182-189, 2014). The challenge to rapidly create asymmetrical tBLMs that include a lipopolysaccharide outer leaflet for bacterial model membrane research can be overcome by the use of a Langmuir-Schaefer deposition protocol. Here, we describe the procedures required to assemble and test asymmetric lipopolysaccharide (LPS) tethered lipid bilayers.

On behalf of the Australian Society for Biophysics (ASB) and the Editors of this Special Issue, I would like to express our appreciation to Editor-in-Chief, Damien Hall, for arranging the publication of this Special Issue. The ASB is about five times smaller than our sister the Biophysical Society for Japan (BSJ) and tenfold smaller than the US Biophysical Society (USBS), but our meetings are notable because of the encouragement the Society gives to emerging biophysicists. It can be a terrifying experience for a PhD student to have to face a roomful of professors and senior academics, but invariably they appreciate the experience. Another feature of the ASB meetings is the inclusion of contributions from the Asian Pacific region. We now have formal ties with our New Zealand colleagues and our meetings with the BSJ contain joint sessions (see below). In 2020, despite the impact of COVID-19 (see Adam Hill's Commentary), there is a joint session with the University of California Davis. This Special Issue comprises 2 Editorials, 3 Commentaries, and 25 reviews.

There is a significant and urgent need for the development of novel antibacterial agents to tackle the increasing incidence of antibiotic resistance. Cholic acid-based small molecular antimicrobial peptide mimics are reported as potential new leads to treat bacterial infection. Here, we describe the design, synthesis and biological evaluation of cholic acid-based small molecular antimicrobial peptide mimics. The synthesis of cholic acid analogues involves the attachment of a hydrophobic moiety at the carboxyl terminal of the cholic acid scaffold, followed by the installation of one to three amino acid residues on the hydroxyl groups present on the cholic acid scaffold. Structure–activity relationship studies suggest that the tryptophan moiety is important for high antibacterial activity. Moreover, a minimum of +2 charge is also important for antimicrobial activity. In particular, analogues containing lysine-like residues showed the highest antibacterial potency against Gram-positive S. aureus. All di-substituted analogues possess high antimicrobial activity against both Gram-positive S. aureus as well as Gram-negative E. coli and P. aeruginosa. Analogues 17c and 17d with a combination of these features were found to be the most potent in this study. These compounds were able to depolarise the bacterial membrane, suggesting that they are potential antimicrobial pore forming agents.

Aryl-urea substituted fatty acids are protonophores and mitochondrial uncouplers that utilise a urea-based synthetic anion transport moiety to carry out the protonophoric cycle. Herein we show that replacement of the urea group with carbamate, a functional group not previously reported to possess anion transport activity, produces analogues that retain the activity of their urea counterparts. Thus, the aryl-carbamate substituted fatty acids uncouple oxidative phosphorylation and inhibit ATP production by collapsing the mitochondrial proton gradient. Proton transport proceeds via self-assembly of the deprotonated aryl-carbamates into membrane permeable dimeric species, formed by intermolecular binding of the carboxylate group to the carbamate moiety. These results highlight the anion transport capacity of the carbamate functional group.

Monitoring the changes in membrane conductance using electrical impedance spectroscopy is the platform of membrane-based biosensors in order to detect a specific target molecule. These biosensors represent the amalgamation of an electrical conductor such as gold and a chemically tethered bilayer lipid membrane with specific incorporated ion channels such as gramicidin-A that is further functionalized with detector molecules of interest.