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Targeting the cancer cell mitochondrion is a promising approach for developing novel anticancer agents. The experimental anticancer agent N,N'-bis(3,5-dichlorophenyl)urea (SR4) induces apoptotic cell death in several cancer cell lines by uncoupling mitochondrial oxidative phosphorylation (OxPhos) using a protein-free mechanism. However, the precise mechanism by which SR4 depolarizes mitochondria is unclear because SR4 lacks an acidic functional group typically found in protein-independent uncouplers. Recently, it was shown that structurally related thioureas can facilitate proton transport across lipid bilayers by a fatty acid-activated mechanism, in which the fatty acid acts as the site of protonation/deprotonation and the thiourea acts as an anion transporter that shuttles deprotonated fatty acids across the phospholipid bilayer to enable proton leak. In this paper, we show that SR4-mediated proton transport is enhanced by the presence of free fatty acids in the lipid bilayer, indicating that SR4 uncouples mitochondria through the fatty acid-activated mechanism. This mechanistic insight was used to develop a library of substituted bisaryl ureas for structure-activity relationship studies and subsequent cell testing. It was found that lipophilic electron-withdrawing groups on bisaryl ureas enhanced electrogenic proton transport via the fatty acid-activated mechanism and had the capacity to depolarize mitochondria and reduce the viability of MDA-MB-231 breast cancer cells. The most active compound in the series reduced cell viability with greater potency than SR4 and was more effective at inhibiting adenosine triphosphate production.

Calcium ions (Ca2+) play a fundamental role in membrane-associated physiological processes. Ca2+ can also significantly modulate the physicochemical properties of phospholipid bilayers, but whether this occurs at physiologically relevant concentrations is difficult to determine because of the uncertainty in the reported affinity of Ca2+ for phospholipid bilayers. In this article, we determine the apparent affinity of Ca2+ for zwitterionic phospholipid bilayers using tethered bilayer lipid membranes (tBLMs) used in conjunction with swept-frequency electrical impedance spectroscopy (EIS). We report that Ca2+ binds to phospholipid bilayers at physiologically relevant concentrations and modulates membrane permeability. We present direct experimental evidence that this effect is governed by specific interactions with select lipid headgroup moieties, which is supported by data from molecular dynamics (MD) simulations. This is the first reported use of tBLM/EIS to estimate cation-membrane affinity. Combined with MD simulations, this technique provides a novel methodology to elucidate the molecular details of cation-membrane interactions at the water-phospholipid interface.

GALA is a 30-residue amphipathic peptide that self-assembles into multimeric transmembrane pores in a pH-dependent fashion. In this study, we characterize the size, multimeric structure, and cation selectivity of GALA pores in planar phospholipid bilayers using electrical impedance spectroscopy and molecular dynamics simulations. We demonstrate that in planar bilayers GALA pores are likely formed by six peptide monomers rather than eight to 12 monomers as previously reported for lipid vesicles. We further show that in planar bilayers, GALA pores exhibit previously unreported cation selectivity. We propose that the difference between the predicted pore structures in planar bilayers and lipid vesicles exemplifies the importance of phospholipid bilayer structural properties on the aggregation of transmembrane helical structures.

Despite the central role of Na+ and K+ in physiological processes, it is still unclear whether they interact or alter the physical properties of simple zwitterionic phospholipid bilayers at physiol...

International audience; Phospholipase-A (PLA) enzymes catalyze the hydrolysis of ester bonds in select glycerophospholipids. Sensors for rapidly measuring the PLA activity in biological samples have relevance in the study of venom compositions and in medical diagnostics for the diagnosis of diseases such as acute pancreatitis. Current PLA sensor technologies are often restricted by the time it takes to prepare an assay, the necessity of using fluorescent labels, or the fact they might require strict pH control of the buffer vehicles used. Here we present a tethered bilayer lipid membrane (tBLM) impedance sensor array for the rapid and real-time detection of PLA, which includes the ability to selectively detect phospholipase-A(2)(PLA(2)) from phospholipase-A(1) (PLA(1)) isoforms. Comparing the activity of PLA(1) and PLA(2) in an array of tBLMs composed of ether phospholipids, ester phospholipids or ether-ester phospholipids allows for the rapid and reliable distinction between the isoforms, as measured using swept-frequency electrical impedance spectroscopy. After testing the assay using pure enzymes, we demonstrate the capacity of the sensor to identify specific PLA(2)-type, calcium-dependent activity from the venom of the South American bullet ant, Paraponera clavata, at a concentration of 1 mu g/mL. The specificity of the phospholipase activity was corroborated using matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry. As further validation, we tested the activities of a PLA(1) isoform in the presence of different buffers commonly used in biology and biochemistry experiments. Sensitivity testing shows that PLA(1) can be detected at an activity as low as 0.06 U/mL. The rapid and reliable detection of phospholipases presented in this study has potential applications in the study of animal venoms as well as in lipase bioreactors and point-of-care devices.

Peptide-based nanofibres are a versatile class of tunable materials with applications in optoelectronics, sensing and tissue engineering. However, the understanding of the nanofibre surface at the molecular level is limited. Here, a series of homologous dilysine–diphenylalnine tetrapeptides were synthesised and shown to self-assemble into water-soluble nanofibres. Despite the peptide nanofibres displaying similar morphologies, as evaluated through atomic force microscopy and neutron scattering, significant differences were observed in their ability to support sensitive primary neurons. Contact angle and labelling experiments revealed that differential presentation of lysine moieties at the fibre surface did not affect neuronal viability; however the mobility of phenylalanine residues at the nanofibre surface, elucidated through solid- and gel-state NMR studies and confirmed through tethered bilayer lipid membrane experiments, was found to be the determining factor in governing the suitability of a given peptide as a scaffold for primary neurons. This work offers new insights into characterising and controlling the nanofibre surface at the molecular level., The mobility of hydrophobic moieties at a peptide nanofibre surface determines its suitability as a scaffold for sensitive primary cells.

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.

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.

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.

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.

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.

This Commentary describes a call for submissions for the upcoming Special Issue focused on the research topics presented at the Australian Society of Biophysics (ASB) in 2020 and 2021. Submissions from past and present ASB members who could not attend these meetings are also welcome as contributions to this special issue.

Respiring mitochondria establish a proton gradient across the mitochondrial inner membrane (MIM) that is used to generate ATP. Protein-independent mitochondrial uncouplers collapse the proton gradient and disrupt ATP production by shuttling protons back across the MIM in a protonophoric cycle. Continued cycling relies on the formation of MIM-permeable anionic species that can return to the intermembrane space after deprotonation in the mitochondrial matrix. Previously described protonophores contain acidic groups that are part of delocalised π-systems that provide large surfaces for charge delocalisation and facilitate anion permeation across the MIM. Here we present a new class of protonophoric uncoupler based on aryl-urea substituted fatty acids in which an acidic group and a π-system are separated by a long alkyl chain. The aryl-urea group in these molecules acts as a synthetic anion receptor that forms intermolecular hydrogen bonds with the fatty acid carboxylate after deprotonation. Dispersal of the negative charge across the aryl-urea system produces lipophilic dimeric complexes that can permeate the MIM and facilitate repeated cycling. Substitution of the aryl-urea group with lipophilic electron withdrawing groups is critical to complex lipophilicity and uncoupling activity. The aryl-urea substituted fatty acids represent the first biological example of mitochondrial uncoupling mediated by the interaction of a fatty acid and an anion receptor moiety, via self-assembly., A new mitochondrial uncoupler that forms membrane permeable dimers through interactions of remote acidic and anion receptor groups.

Maternal smoking is currently a public health concern and has been associated with a number of complications in the offspring. E-cigarettes are gaining popularity as a “safer” alternative to tobacco cigarettes during pregnancy, however, there are a limited number of studies to suggest that it is actually “safe.” Balb/C female mice were exposed to ambient air (n = 8; Sham), or tobacco cigarette smoke (n = 8; SE) before gestation, during gestation and lactation. A third group was exposed to cigarette smoke before gestation followed by e-cigarette aerosols during gestation and lactation (n = 8; Switch). Male offspring (12-week old, n = 10–14/group) underwent behavioral assessments to investigate short-term memory, anxiety, and activity using the novel object recognition and elevated plus maze tests. Brains were collected at postnatal day (P)1, P20, and Week 13 for global DNA methylation, epigenetic gene expression, and neuronal cell counts. The offspring from mothers switching to e-cigarettes exhibited no change in exploration/activity but showed a decrease in global DNA methylation, Aurora Kinase (Aurk) A and AurkB gene expression and a reduction in neuronal cell numbers in the cornu ammonis 1 region of the dorsal hippocampus compared with the SE group. Continuous tobacco cigarette smoke exposure during pregnancy resulted in marked neurological deficits in the offspring. Switching to e-cigarettes during pregnancy reduced these neurological deficits compared with cigarette smoke exposure. However, neurological changes were still observed, so we therefore conclude that e-cigarette use during pregnancy is not advised.

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.

Here we report a protocol to investigate the heat transfer between irradiated gold nanoparticles (GNPs) and bilayer lipid membranes by electrochemistry using tethered bilayer lipid membranes (tBLMs) assembled on gold electrodes. Irradiated modified GNPs, such as streptavidin-conjugated GNPs, are embedded in tBLMs containing target molecules, such as biotin. By using this approach, the heat transfer processes between irradiated GNPs and model bilayer lipid membrane with entities of interest are mediated by a horizontally focused laser beam. The thermal predictive computational model is used to confirm the electrochemically induced conductance changes in the tBLMs. Under the specific conditions used, detecting heat pulses required specific attachment of the gold nanoparticles to the membrane surface, while unbound gold nanoparticles failed to elicit a measurable response. This technique serves as a powerful detection biosensor which can be directly utilized for the design and development of strategies for thermal therapies that permits optimization of the laser parameters, particle size, particle coatings and composition.

Aims Mitochondrial uncouplers decrease caloric efficiency and have potential therapeutic benefits for the treatment of obesity and related metabolic disorders. Herein we investigate the metabolic and physiologic effects of a recently identified small molecule mitochondrial uncoupler named SHC517 in a mouse model of diet-induced obesity. Methods SHC517 was administered as an admixture in food. The effect of SHC517 on in vivo energy expenditure and respiratory quotient was determined by indirect calorimetry. A dose-finding obesity prevention study was performed by starting SHC517 treatment concomitant with high fat diet for a period of 12 days. An obesity reversal study was performed by feeding mice western diet for 4 weeks prior to SHC517 treatment for 7 weeks. Biochemical assays were used to determine changes in glucose, insulin, triglycerides, and cholesterol. SHC517 concentrations were determined by mass spectrometry. Results SHC517 increased lipid oxidation without affecting body temperature. SHC517 prevented diet-induced obesity when administered at 0.05% and 0.1% w/w in high fat diet and reversed established obesity when tested at the 0.05% dose. In the obesity reversal model, SHC517 restored adiposity to levels similar to chow-fed control mice without affecting food intake or lean body mass. SHC517 improved glucose tolerance and fasting glucose levels when administered in both the obesity prevention and obesity reversal modes. Conclusions SHC517 is a mitochondrial uncoupler with potent anti-obesity and insulin sensitizing effects in mice. SHC517 reversed obesity without altering food intake or compromising lean mass, effects that are highly sought-after in anti-obesity therapeutics.

There has been an increasing interest in the development of antimicrobial peptides (AMPs) and their synthetic mimics as a novel class of antibiotics to overcome the rapid emergence of antibiotic resistance. Recently, phenylglyoxamide-based small molecular AMP mimics have been identified as potential leads to treat bacterial infections. In this study, a new series of biphenylglyoxamide-based small molecular AMP mimics were synthesised from the ring-opening reaction of N-sulfonylisatin bearing a biphenyl backbone with a diamine, followed by the conversion into tertiary ammonium chloride, quaternary ammonium iodide and guanidinium hydrochloride salts. Structure&ndash, activity relationship studies of the analogues identified the octanesulfonyl group as being essential for both Gram-positive and Gram-negative antibacterial activity, while the biphenyl backbone was important for Gram-negative antibacterial activity. The most potent analogue was identified to be chloro-substituted quaternary ammonium iodide salt 15c, which possesses antibacterial activity against both Gram-positive (MIC against Staphylococcus aureus = 8 &mu, M) and Gram-negative bacteria (MIC against Escherichia coli = 16 &mu, M, Pseudomonas aeruginosa = 63 &mu, M) and disrupted 35% of pre-established S. aureus biofilms at 32 &mu, M. Cytoplasmic membrane permeability and tethered bilayer lipid membranes (tBLMs) studies suggested that 15c acts as a bacterial membrane disruptor. In addition, in vitro toxicity studies showed that the potent compounds are non-toxic against human cells at therapeutic dosages.

Peptide hydrogels show great promise as extracellular matrix mimics due to their tuneable, fibrous nature. Through incorporation of polar cationic, polar anionic or polar neutral amino acids into the Fmoc-diphenylalanine motif, we show that electrostatic charge plays a key role in the properties of the subsequent gelators. Specifically, we show that an inverse relationship exists for biocompatibility in the solution state versus the gel state for cationic and anionic peptides. Finally, we use tethered bilayer lipid membrane (tBLM) experiments to suggest a likely mode of cytotoxicity for tetrapeptides which exhibit cytotoxicity in the solution state.

Plasmon resonance frequency irradiated gold nanoparticles (GNPs) have gained interest as a laser-targeted treatment for infections, tumors and for the controlled release of drugs in situ. Questions still remain, however, as to the efficiency of heat delivery within biological tissues and how this can be reliably determined. Here, we demonstrate how a nanomaterial-electrode interface that mimics cell membranes can detect the localized heat transfer characteristics arising from plasmon resonance frequency-matched laser excitation of GNPs. We demonstrate that the lipid bilayer membrane can be affected by conjugated GNP induced hyperthermia when irradiated with a laser power output as low as 135 nW/μm2. This is four orders of magnitude lower power than previously reported. By restricting the lateral movement of the lipids in the bilayer membrane, it was shown that the change in membrane conductance as a result of the heat transfer was due to the creation of transient lipidic toroidal pores within the membrane. We further demonstrate that the heat transfer from the GNPs alters diffusion rates of monomers of the gramicidin-A peptide within the lipid leaflets. This work highlights how targeted low laser power GNP hyperthermia treatments, in vivo, could play a dual role of interfering with both cell membrane morphology and dynamics, along with membrane protein function.

© 2017 American Chemical Society. Cyclotides are cyclic disulfide-rich peptides that are chemically and thermally stable and possess pharmaceutical and insecticidal properties. The activities reported for cyclotides correlate with their ability to target phosphatidylethanolamine (PE)-phospholipids and disrupt cell membranes. However, the mechanism by which this disruption occurs remains unclear. In the current study we examine the effect of the prototypic cyclotides, kalata B1 (kB1) and kalata B2 (kB2), on tethered lipid bilayer membranes (tBLMs) using swept frequency electrical impedance spectroscopy. We confirmed that kB1 and kB2 bind to bilayers only if they contain PE-phospholipids. We hypothesize that the increase in membrane conduction and capacitance observed upon addition of kB1 or kB2 is unlikely to result from ion channel like pores but is consistent with the formation of lipidic toroidal pores. This hypothesis is supported by the concentration dependence of effects of kB1 and kB2 being suggestive of a critical micelle concentration event rather than a progressive increase in conduction arising from increased channel insertion. Additionally, conduction behavior is readily reversible when the peptide is rinsed from the bilayer. Our results support a mechanism by which kB1 and kB2 bind to and disrupt PE-containing membranes by decreasing the overall membrane critical packing parameter, as would a surfactant, which then opens or increases the size of existing membrane defects. The cyclotides need not participate directly in the conductive pore but might exert their effect indirectly through altering membrane packing constraints and inducing purely lipidic conductive pores.

Antibiotic resistance is a major global health concern. There is an urgent need for the development of novel antimicrobials. Recently, phenylglyoxamide-based small molecular antimicrobial peptide mimics have been identified as potential new leads to treat bacterial infections. Here, we describe the synthesis of novel phenylglyoxamide derivatives via the ring-opening reaction of N-sulfonylisatins with primary amines, followed by conversion into hydrochloride, quaternary ammonium iodide or gunidinium salts. The antibacterial activity of the compounds against Staphylococcus aureus was evaluated by in vitro assays. Structure-activity relationship studies revealed that 5-bromo-substituent at the phenyl ring, octyl group appended to the ortho sulfonamide group or guanidine hydrochloride salt as the terminal group significantly contributed to potency. The most potent compound, the gunidinium salt 35 d, exhibited a minimum inhibitory concentration value of 12 μM and a therapeutic index of 15. It also demonstrated its potential to act as antimicrobial pore-forming agent. Overall, the results identified 35 d as a new lead antimicrobial compound.

© 2018 Elsevier Ltd Microcystin-LR (MC-LR) is a potent cyanobacterial toxin responsible for animal and human poisonings worldwide. MC-LR is found in organisms throughout the foodweb, however there is conjecture regarding whether it biomagnifies. Few studies have investigated how MC-LR interacts with lipid membranes, a determinant of biomagnification potential. We tested whether 1 μM MC-LR irreversibly associates with lipid bilayers or causes the creation of pore defects upon short and long-term exposure. Using tethered bilayer lipid membranes (tBLMs), we observed an increase in membrane conduction in tBLMs, representing an interaction of microcystin-LR with the lipid bilayer and a change in membrane packing properties. However, there were minimal changes in membrane capacitance upon short and long-term exposure, and MC-LR exhibited a rapid off-rate. Upon 24 h exposure to the toxin, no lipophilic multimeric complexes were detected capable of altering the toxin's off-rate. There was no evidence of the creation of new pores. This study demonstrates that MC-LR does not irreversibly imbed itself into lipids membranes after short or long-term exposure and suggests MC-LR does not biomagnify through the food web via lipid storage.

© 2019, International Union for Pure and Applied Biophysics (IUPAB) and Springer-Verlag GmbH Germany, part of Springer Nature. Maintaining gradients of solvated protons and alkali metal ions such as Na+ and K+ across membranes is critical for cellular function. Over the last few decades, both the interactions of protons and alkali metal ions with phospholipid membranes have been studied extensively and the reported interactions of these ions with phospholipid headgroups are very similar, yet few studies have investigated the potential interdependence between proton and alkali metal ion binding at the water–lipid interface. In this short review, we discuss the similarities between the proton–membrane and alkali ion–membrane interactions. Such interactions include cation attraction to the phosphate and carbonyl oxygens of the phospholipid headgroups that form strong lipid–ion and lipid–ion–water complexes. We also propose potential mechanisms that may modulate the affinities of these cationic species to the water–phospholipid interfacial oxygen moieties. This review aims to highlight the potential interdependence between protons and alkali metal ions at the membrane surface and encourage a more nuanced understanding of the complex nature of these biologically relevant processes.

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This review identifies the ways in which tethered bilayer lipid membranes (tBLMs) can be used for the identification of the actions of antimicrobials against lipid bilayers. Much of the new research in this area has originated, or included researchers from, the southern hemisphere, Australia and New Zealand in particular. More and more, tBLMs are replacing liposome release assays, black lipid membranes and patch-clamp electrophysiological techniques because they use fewer reagents, are able to obtain results far more quickly and can provide a uniformity of responses with fewer artefacts. In this work, we describe how tBLM technology can and has been used to identify the actions of numerous antimicrobial agents.

The analysis of electronic cigarrete (E-cigarette) fluids by high performance liquid chromatography or gas chromatography (GC) coupled to mass spectrometry (MS), GC hyphenated to flame-ionisation detection, or nuclear magnetic resonance spectroscopy poses many challenges due to the complex matrix and extremely high number of compounds present. In order to overcome these challenges, this study focused on the detection of the multiple complex compounds classes produced by the pyrolysis of E-cigarette liquids using comprehensive two dimensional gas chromatography (GCxGC) coupled to time of flight (TOF)-MS. Gas samples were prepared by heating E-liquids inside aluminium tins for 5 min. The tins were placed in a sand bath, which was temperature controlled at 200 °C. The samples were collected using thermal desorption tubes connected to volatile organic compound (VOC) sampling pump attached and subsequently analysed using GCxGC-TOF-MS. The greater peak resolution obtained when using GCxGC-TOF-MS allowed to distinguish many toxic compounds and VOCs that could not be detected by the other methods mentioned above. As a result, a comprehensive list of volatile compounds emitted from E-cigarette fluids when heated was established, which might allow a better understanding of potential health effects of vaping. Heating E-liquids to moderate temperature results in the emission of over 1000 volatile compounds of which over 150 are toxic. These compounds are either present in the liquid or can be formed during storage or heating leading to a more complex volatile profile of E-cigarette liquids than previously assumed. The application of GCxGC-TOF-MS allows the elucidation of this profile and therefore a better understanding of possible health implications.

This study explains the importance of the phosphate moiety and H3O+ in controlling the ionic flux through phospholipid membranes. We show that despite an increase in the H3O+ concentration when the pH is decreased, the level of ionic conduction through phospholipid bilayers is reduced. By modifying the lipid structure, we show the dominant determinant of membrane conduction is the hydrogen bonding between the phosphate oxygens on adjacent phospholipids. The modulation of conduction with pH is proposed to arise from the varying H3O+ concentrations altering the molecular area per lipid and modifying the geometry of conductive defects already present in the membrane. Given the geometrical constraints that control the lipid phase structure of membranes, these area changes predict that organisms evolving in environments with different pHs will select for different phospholipid chain lengths, as is found for organisms near highly acidic volcanic vents (short chains) or in highly alkaline salt lakes (long chains...

© The Royal Society of Chemistry 2016. Antimicrobial peptides (AMPs) are a key component of the human immune system. Synthetic AMP mimics represent a novel strategy to counteract the increasing incidence of antimicrobial resistance. Here, we describe the synthesis of novel glyoxamide derivatives via ring-opening reactions of N-hexanoyl, N-benzoyl and N-naphthoylisatins with N,N-dimethylethane-1,2-diamine and N,N-dimethylpropane-1,3-diamine. These were converted to both the hydrochloric acid (HCl) or quaternary ammonium iodide (MeI) salts and their antibacterial activity against Staphylococcus aureus was investigated by their zone-of-inhibition and minimum inhibitory concentration (MIC). The HCl salt 22b exhibited the lowest MIC of 16 μg mL-1, whereas the corresponding MeI salt 22c had a MIC of 39 μg mL-1. We also investigated the in vitro toxicity of active compounds against the MRC-5 normal human lung fibroblasts and their activity against established biofilm in S. aureus.

Copyright © 2018 American Chemical Society. Melimine and its derivatives are synthetic chimeric antimicrobial agents based on protamine and melittin. The binding of solubilized melimine and its derivative, with a cysteine on N-terminus, (cys-melimine) on tethered bilayer lipid membranes (tBLMs) was examined using ac electrical impedance spectroscopy. The addition of melimine and cys-melimine initially increased membrane conduction, which subsequently falls over time. The results were obtained for tBLMs comprising zwitterionic phosphatidylcholine, anionic phosphatidylglycerol, or tBLMs made using purified lipids from Escherichia coli. The effect on conduction is more marked with the cysteine variant than the noncysteine variant. The variation in membrane conduction most probably arises from individual melimines inducing increased ionic permeability, which is then reduced as the melimines aggregate and phase-separate within the membrane. The actions of these antimicrobials are modeled in terms of altering the critical packing parameter (CPP) of the membranes. The variations in the peptide length of cys-melimine were compared with a truncated version of the peptide, cys-mel4. The results suggest that the smaller molecule impacts the membrane by a mechanism that increases the average CPP, reducing membrane conduction. Alternatively, an uncharged alanine-replacement version of melimine still produced an increase in membrane conduction, further supporting the CPP model of geometry-induced toroidal pore alterations. All the data were then compared to their antimicrobial effectiveness for the Gram-positive and Gram-negative strains of bacteria, and their fusogenic properties were examined using dynamic light scattering in 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine lipid spheroids. We conclude that a degree of correlation exists between the antimicrobial effectiveness of the peptides studied here and their modulation of membrane conductivity.

At the 2017 meeting of the Australian Society for Biophysics, we presented the combined results from two recent studies showing how hydronium ions (H3O+) modulate the structure and ion permeability of phospholipid bilayers. In the first study, the impact of H3O+ on lipid packing had been identified using tethered bilayer lipid membranes in conjunction with electrical impedance spectroscopy and neutron reflectometry. The increased presence of H3O+ (i.e. lower pH) led to a significant reduction in membrane conductivity and increased membrane thickness. A first-order explanation for the effect was assigned to alterations in the steric packing of the membrane lipids. Changes in packing were described by a critical packing parameter (CPP) related to the interfacial area and volume and shape of the membrane lipids. We proposed that increasing the concentraton of H3O+ resulted in stronger hydrogen bonding between the phosphate oxygens at the water–lipid interface leading to a reduced area per lipid and slightly increased membrane thickness. At the meeting, a molecular model for these pH effects based on the result of our second study was presented. Multiple μs-long, unrestrained molecular dynamic (MD) simulations of a phosphatidylcholine lipid bilayer were carried out and showed a concentration dependent reduction in the area per lipid and an increase in bilayer thickness, in agreement with experimental data. Further, H3O+ preferentially accumulated at the water–lipid interface, suggesting the localised pH at the membrane surface is much lower than the bulk bathing solution. Another significant finding was that the hydrogen bonds formed by H3O+ ions with lipid headgroup oxygens are, on average, shorter in length and longer-lived than the ones formed in bulk water. In addition, the H3O+ ions resided for longer periods in association with the carbonyl oxygens than with either phosphate oxygen in lipids. In summary, the MD simulations support a model where the hydrogen bonding capacity of H3O+ for carbonyl and phosphate oxygens is the origin of the pH-induced changes in lipid packing in phospholipid membranes. These molecular-level studies are an important step towards a better understanding of the effect of pH on biological membranes.

© 2018 American Chemical Society. Electronic cigarette (e-cigarette) use is on the rise worldwide and is particularly attractive to young people and as a smoking substitute by pregnant woman. There is a perception in pregnant women and women of child-bearing age that the use of e-cigarettes (vaping) is safer than smoking tobacco cigarettes during pregnancy. However, there is little evidence to support this perception. Here, we examined the offspring from mouse dams that had been exposed during and after pregnancy to ambient air (sham) (n = 8), e-cigarette aerosols with nicotine (n = 8), or e-cigarette aerosols without nicotine (n = 8). Offspring underwent cognitive testing at 12 weeks of age and epigenetic testing of brain tissues at 1 day, 20 days, and 13 weeks after birth. The findings showed deficits in short-term memory, reduced anxiety, and hyperactivity in offspring following maternal e-cigarette exposure using the novel object recognition and elevated plus maze tests. In addition, global DNA methylation was increased in the brains of offspring soon after birth. Using a quantitative-PCR array specific to chromatin modification enzymes on genomic DNA and histones,13 key genes were identified to be significantly altered in the offspring brains from the e-cigarette groups compared to the nonexposed groups. The changes to genes Aurka, Aurkb, Aurkc, Kdm5c, Kdm6b, Dnmt3a, Dnmt3b, and Atf2, all associated with modulating neurological activity, were validated using RT-qPCR. In conclusion, in a mouse model, maternal exposure to e-cigarette aerosols resulted in both cognitive and epigenetic changes in offspring. This suggests that the use of e-cigarettes during pregnancy may have hitherto undetected neurological consequences on newborns.

© 2017 the Owner Societies. This work seeks to identify the mechanisms by which hydronium ions (H3O+) modulate the structure of phospholipid bilayers by studying the interactions of H3O+ with phospholipids at the molecular level. For this, we carried out multiple microsecond-long unrestrained molecular dynamics (MD) simulations of a POPC bilayer at different H3O+ concentrations. The results show that H3O+ accumulates at the membrane surface where it displaces water and forms strong and long-lived hydrogen bonds with the phosphate and carbonyl oxygens in phospholipids. This results in a concentration-dependent reduction of the area per lipid and an increase in bilayer thickness. This study provides an important molecular-level insight into the mechanism of how H3O+ modulates the structure of biological membranes and is a critical step towards a better understanding of the effect of low pH on mammalian and bacterial membranes.

© 2017 Elsevier Masson SAS Antimicrobial peptides (AMPs) and their synthetic mimics have received recent interest as new alternatives to traditional antibiotics in attempts to overcome the rise of antibiotic resistance in many microbes. AMPs are part of the natural defenses of most living organisms and they also have a unique mechanism of action against bacteria. Herein, a new series of short amphiphilic cationic peptidomimetics were synthesized by incorporating the 3′-amino-[1,1′-biphenyl]-3-carboxylic acid backbone to mimic the essential properties of natural AMPs. By altering hydrophobicity and charge, we identified the most potent analogue 25g that was active against both Gram-positive Staphylococcus aureus (MIC = 15.6 μM) and Gram-negative Escherichia coli (MIC = 7.8 μM) bacteria. Cytoplasmic permeability assay results revealed that 25g acts primarily by depolarization of lipids in cytoplasmic membranes. The active compounds were also investigated for their cytotoxicity to human cells, lysis of lipid bilayers using tethered bilayer lipid membranes (tBLMs) and their activity against established biofilms of S. aureus and E. coli.

© The Royal Society of Chemistry. Antimicrobial resistance in bacteria is becoming increasingly prevalent, posing a critical challenge to global health. Bacterial biofilm formation is a common resistance mechanism that reduces the effectiveness of antibiotics. Thus, the development of compounds that can disrupt bacterial biofilms is a potential strategy to combat antimicrobial resistance. We report herein the synthesis of amphipathic guanidine-embedded glyoxamide-based peptidomimetics via ring-opening reactions of N-naphthoylisatins with amines and amino acids. These compounds were investigated for their antibacterial activity by the determination of minimum inhibitory concentration (MIC) against S. aureus and E. coli. Compounds 35, 36, and 66 exhibited MIC values of 6, 8 and 10 μg mL−1 against S. aureus, respectively, while compounds 55 and 56 showed MIC values of 17 and 19 μg mL−1 against E. coli, respectively. Biofilm disruption and inhibition activities were also evaluated against various Gram-positive and Gram-negative bacteria. The most active compound 65 exhibited the greatest disruption of established biofilms by 65% in S. aureus, 61% in P. aeruginosa, and 60% in S. marcescens respectively, at 250 μM concentration, while compound 52 inhibited the formation of biofilms by 72% in S. marcescens at 250 μM. We also report here the in vitro toxicity against MRC-5 human lung fibroblast cells. Finally, the pore forming capability of the three most potent compounds were tested using tethered bilayer lipid membrane (tBLM) technology.

© 2014 by the Biophysical Society. This article reports on the construction and predictive models for a platform comprised of an engineered tethered membrane. The platform provides a controllable and physiologically relevant environment for the study of the electroporation process. The mixed self-assembled membrane is formed via a rapid solvent exchange technique. The membrane is tethered to the gold electrode and includes an ionic reservoir separating the membrane and gold surface. Above the membrane, there is an electrolyte solution, and a gold counterelectrode. A voltage is applied between the gold electrodes and the current measured. The current is dependent on the energy required to form aqueous pores and the conductance of each pore. A two-level predictive model, consisting of a macroscopic and a continuum model, is developed to relate the pore dynamics to the measured current. The macroscopic model consists of an equivalent circuit model of the tethered membrane, and asymptotic approximations to the Smoluchowski-Einstein equation of electroporation that is dependent on the pore conductance and the energy required to form aqueous pores. The continuum model is a generalized Poisson-Nernst-Planck (GPNP) system where an activity coefficient to account for steric effects of ions is added to the standard PNP system. The GPNP is used to evaluate the conductance of aqueous pores, and the electrical energy required to form the pores. As an outcome of the setup of the device and the two-level model, biologically important variables can be estimated from experimental measurements. To validate the accuracy of the two-level model, the predicted current is compared with experimentally measured current for different tethering densities.

In this work, we present experimental data, supported by a quantitative model, on the generation and effect of potential gradients across a tethered bilayer lipid membrane (tBLM) with, to the best of our knowledge, novel architecture. A challenge to generating potential gradients across tBLMs arises from the tethering coordination chemistry requiring an inert metal such as gold, resulting in any externally applied voltage source being capacitively coupled to the tBLM. This in turn causes any potential across the tBLM assembly to decay to zero in milliseconds to seconds, depending on the level of membrane conductance. Transient voltages applied to tBLMs by pulsed or ramped direct-current amperometry can, however, provide current-voltage (I/V) data that may be used to measure the voltage dependency of the membrane conductance. We show that potential gradients >∼150 mV induce membrane defects that permit the insertion of pore-forming peptides. Further, we report here the novel (to our knowledge) use of real-time modeling of conventional low-voltage alternating-current impedance spectroscopy to identify whether the conduction arising from the insertion of a polypeptide is uniform or heterogeneous on scales of nanometers to micrometers across the membrane. The utility of this tBLM architecture and these techniques is demonstrated by characterizing the resulting conduction properties of the antimicrobial peptide PGLa.