Dimeric peptoids as antibacterial agents.

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.

HtrA, fatty acids, and membrane protein interplay in Chlamydia trachomatis to impact stress response and trigger early cellular exit.

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.

A hybridized mechano-electroporation technique for efficient immune cell engineering.

Immune cell engineering, which involves genetic modification of T cells, natural killer cells, and macrophages, is shifting the paradigm in immunotherapy for treating hematologic malignancies. These modified cells can be viewed as living drugs and offer advantages, including dynamic functionality, active local trafficking, and boosting the immune system while recognizing and eliminating malignant cells. Among the current technologies employed for the modification of immune cell functions, electroporation stands as a predominant approach, but it suffers from heterogeneity arising from the treatment of a bulk population of immune cells during the manufacturing procedures. To address this challenge of the field, here we present a hybrid approach to induce consecutive gentle mechanical and electric shocks. This approach enhances the treatment homogeneity and improves outcomes in difficult-to-load immune cells. The hybrid approach aims to enhance the treatment homogeneity by passing individual immune cells through a microengineered filter membrane with micropores smaller than the cell diameter. This facilitates the creation of transient pores in the cell membrane, followed by efficient delivery of biomolecules through the complementary use of a gentle electric shock. Using this hybrid mechano-electroporation (HMEP) system, we could successfully deliver fluorescein isothiocyanate (FITC) dextran molecules from the smallest (4 kDa) to the largest (2000 kDa) size and EGFP expressing plasmid DNA into different immune cell types. We also provide insight into the delivery performance of the HMEP system in comparison with the benchtop electroporation since both methods hinge on membrane disruption as their permeabilization mechanism. Immune cells treated with the HMEP protocol demonstrated higher delivery efficiencies while maintaining cell viability compared to those experiencing conventional electroporation. Therefore, membrane-based mechanoporation can be a cost-effective and efficient approach to pre-treat the hard-to-deliver immune cells before electroporation, elevating the treatment homogeneity and delivery of exogenous cargoes to a higher level.

Triglyceride-Tethered Membrane Lipase Sensor.

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.

E-Cigarette Aerosol Condensate Leads to Impaired Coronary Endothelial Cell Health and Restricted Angiogenesis.

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.

Protonophoric and mitochondrial uncoupling activity of aryl-carbamate substituted fatty acids.

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.

Structure-Activity Relationship and Mechanistic Studies of Bisaryl Urea Anticancer Agents Indicate Mitochondrial Uncoupling by a Fatty Acid-Activated Mechanism.

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.

Cholic Acid-Based Antimicrobial Peptide Mimics as Antibacterial Agents.

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.

A rationally designed synthetic antimicrobial peptide against Pseudomonas-associated corneal keratitis: Structure-function correlation.

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.

Subtle changes in pH affect the packing and robustness of fatty acid bilayers.

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.

A special issue of the Australian society for Biophysics.

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.

Preparing Ion Channel Switch Membrane-Based Biosensors.

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.

Langmuir-Schaefer Deposition to Create an Asymmetrical Lipopolysaccharide Sparsely Tethered Lipid Bilayer.

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.

Determining the Pore Size of Multimeric Peptide Ion Channels Using Cation Conductance Measures of Tethered Bilayer Lipid Membranes.

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.

Bioinformatic Analysis of Na+, K+-ATPase Regulation through Phosphorylation of the Alpha-Subunit N-Terminus.

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.

Calcium Ion Binding at the Lipid-Water Interface Alters the Ion Permeability of Phospholipid Bilayers.

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.

Announcing the call for the Special Issue on "The Australian Society for Biophysics (ASB) - 2021 Meeting".

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.

Mitochondrial uncoupler SHC517 reverses obesity in mice without affecting food intake.

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.

Headspace analysis of E-cigarette fluids using comprehensive two dimensional GC×GC-TOF-MS reveals the presence of volatile and toxic compounds.

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.

Tethered Bilayer Lipid Membranes to Monitor Heat Transfer between Gold Nanoparticles and Lipid Membranes.

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.