Arginine catabolism is essential to polymyxin dependence in Acinetobacter baumannii.

Polymyxins are often the only effective antibiotics against the "Critical" pathogen Acinetobacter baumannii. Worryingly, highly polymyxin-resistant A. baumannii displaying dependence on polymyxins has emerged in the clinic, leading to diagnosis and treatment failures. Here, we report that arginine metabolism is essential for polymyxin-dependent A. baumannii. Specifically, the arginine degradation pathway was significantly altered in polymyxin-dependent strains compared to wild-type strains, with critical metabolites (e.g., L-arginine and L-glutamate) severely depleted and expression of the astABCDE operon significantly increased. Supplementation of arginine increased bacterial metabolic activity and suppressed polymyxin dependence. Deletion of astA, the first gene in the arginine degradation pathway, decreased phosphatidylglycerol and increased phosphatidylethanolamine levels in the outer membrane, thereby reducing the interaction with polymyxins. This study elucidates the molecular mechanism by which arginine metabolism impacts polymyxin dependence in A. baumannii, underscoring its critical role in improving diagnosis and treatment of life-threatening infections caused by "undetectable" polymyxin-dependent A. baumannii.

HIV and influenza fusion peptide interactions with (dis)ordered lipid bilayers: Understanding mechanisms and implications for antimicrobial and antiviral approaches.

The interactions of viral fusion peptides from influenza (E4K and Ac-E4K) and human immunodeficiency virus (gp41 and Ac-gp41) with planar lipid bilayers and monolayers was investigated herein. A combination of surface-sensitive techniques, including quartz crystal microbalance with dissipation (QCM-D), Langmuir-Blodgett area-pressure isotherms with Micro-Brewster angle microscopy, and neutron reflectometry, was employed. Differences in the interactions of the viral fusion peptides with lipid bilayers featuring ordered and disordered phases, as well as lipid rafts, were revealed. The HIV fusion peptide (gp41) exhibited strong binding to the DOPC/DOPS bilayer, comprising a liquid disordered phase, with neutron reflectometry (NR) showing interaction with the bilayer's headgroup area. Conversely, negligible binding was observed with lipid bilayers in a liquid ordered phase. Notably, the influenza peptide (E4K) demonstrated slower binding kinetics with DOPC/DOPS bilayers and distinct interactions compared to gp41, as observed through QCM-D. This suggests different mechanisms of interaction with the lipid bilayers: one peptide interacts more within the headgroup region, while the other is more involved in transmembrane interactions. These findings hold implications for understanding viral fusion mechanisms and developing antimicrobials and antivirals targeting membrane interactions. The differential binding behaviours of the viral fusion peptides underscore the importance of considering membrane composition and properties in therapeutic strategy design.

Advances in sample environments for neutron scattering for colloid and interface science.

This review describes recent advances in sample environments across the full complement of applicable neutron scattering techniques to colloid and interface science. Temperature, pressure, flow, tensile testing, ultrasound, chemical reactions, IR/visible/UV light, confinement, humidity and electric and magnetic field application, as well as tandem X-ray methods, are all addressed. Consideration for material choices in sample environments and data acquisition methods are also covered as well as discussion of current and potential future use of machine learning and artificial intelligence.

Epitaxial graphene growth on cubic silicon carbide on silicon with high temperature neutron reflectometry: an operando study.

The growth of graphene on silicon carbide on silicon offers a very attractive route towards novel wafer-scale photonic and electronic devices that are easy to fabricate and can be integrated in silicon manufacturing. Using a Ni/Cu catalyst for the epitaxial growth of graphene has been successful in the mitigation of the very defective nature of the underlying silicon carbide on silicon, leading to a consistent graphene coverage over large scales. A more detailed understanding of this growth mechanism is warranted in order to further optimise the catalyst composition, preferably via the use of operando characterization measurements. Here, we report in situ neutron reflectometry measurements of (Ni, Cu)/SiC films on silicon wafers, annealed from room temperature to 1100 °C, which initiates graphene formation at the buried (Ni, Cu)/SiC interface. Detailed modelling of the high temperature neutron reflectometry and corresponding scattering length density profiles yield insights into the distinct physical mechanisms within the different temperature regimes. The initially smooth solid metallic layers undergo intermixing and roughening transitions at relatively low temperatures below 500 °C, and then metal silicides begin to form above 600 °C from interfacial reactions with the SiC, releasing atomic carbon. At the highest temperature range of 600-1100 °C, the low neutron scattering length density at high temperature is consistent with a silicon-rich, liquid surface phase corresponding to molten nickel silicides and copper. This liquid catalyst layer promotes the liquid-phase epitaxial growth of a graphene layer by precipitating the excess carbon available at the SiC/metal interface.

Polysaccharide-Targeting Lipid Nanoparticles to Kill Gram-Negative Bacteria.

The rapid increase and spread of Gram-negative bacteria resistant to many or all existing treatments threaten a return to the preantibiotic era. The presence of bacterial polysaccharides that impede the penetration of many antimicrobials and protect them from the innate immune system contributes to resistance and pathogenicity. No currently approved antibiotics target the polysaccharide regions of microbes. Here, describe monolaurin-based niosomes, the first lipid nanoparticles that can eliminate bacterial polysaccharides from hypervirulent Klebsiella pneumoniae, are described. Their combination with polymyxin B shows no cytotoxicity in vitro and is highly effective in combating K. pneumoniae infection in vivo. Comprehensive mechanistic studies have revealed that antimicrobial activity proceeds via a multimodal mechanism. Initially, lipid nanoparticles disrupt polysaccharides, then outer and inner membranes are destabilized and destroyed by polymyxin B, resulting in synergistic cell lysis. This novel lipidic nanoparticle system shows tremendous promise as a highly effective antimicrobial treatment targeting multidrug-resistant Gram-negative pathogens.

Galinstan Liquid Metal Electrical Contacts for Monolayer-Modified Silicon Surfaces.

Galinstan is the brand name for a low-melting gallium-based alloy, which is a promising nontoxic alternative to mercury, the only elemental metal found in the liquid state at room temperature. Liquid alloys such as Galinstan have found applications as electromechanical actuators, sensors, and soft contacts for molecular electronics. In this work, we validate the scope of Galinstan top contacts to probe the electrical characteristics of Schottky junctions made on Si(111) and Si(211) crystals modified with Si-C-bound organic monolayers. We show that the surface-to-volume ratio of the Galinstan drop used as a macroscopic contact defines the junction stability. Further, we explore chemical strategies to increase Galinstan surface tension to obtain control over the junction area, hence improving the repeatability and reproducibility of current-voltage (I-V) measurements. We explore Galinstan top contacts as a means to monitor changes in rectification ratios caused by surface reactions and use these data, most notably the static junction leakage, toward making qualitative predictions on the DC outputs recorded when these semiconductor systems are incorporated in Schottky-based triboelectric nanogenerators. We found that the introduction of iron particles leads to poor data repeatability for capacitance-voltage (C-V) measurements but has only a small negative impact in a dynamic current measurement (I-V).

Underscreening in concentrated electrolytes: re-entrant swelling in polyelectrolyte brushes.

Hypersaline environments are ubiquitous in nature and are found in myriad technological processes. Recent empirical studies have revealed a significant discrepancy between predicted and observed screening lengths at high salt concentrations, a phenomenon referred to as underscreening. Herein we investigate underscreening using a cationic polyelectrolyte brush as an exemplar. Poly(2-(methacryloyloxy)ethyl)trimethylammonium (PMETAC) brushes were synthesised and their internal structural changes and swelling response was monitored with neutron reflectometry and spectroscopic ellipsometry. Both techniques revealed a monotonic brush collapse as the concentration of symmetric monovalent electrolyte increased. However, a non-monotonic change in brush thickness was observed in all multivalent electrolytes at higher concentrations, known as re-entrant swelling; indicative of underscreening. For all electrolytes, numerical self-consistent field theory predictions align with experimental studies in the low-to-moderate salt concentration regions. Analysis suggests that the classical theory of electrolytes is insufficient to describe the screening lengths observed at high salt concentrations and that the re-entrant polyelectrolyte brush swelling seen herein is consistent with the so-called regular underscreening phenomenon.

Spatz: the time-of-flight neutron reflectometer with vertical sample geometry at the OPAL research reactor.

The Spatz neutron beam instrument is the second time-of-flight neutron reflectometer to be installed at the OPAL research reactor. The instrument was formerly the V18 BioRef reflectometer at the BER-II reactor in Berlin and was transferred to Australia in 2016. Subsequently the instrument was re-installed in the neutron guide hall of the OPAL reactor at the end position of the CG2B cold-neutron guide and recommissioned. The instrument performance has not been compromised by the move, with reflectivity achieved down to 10-7 and good counting statistics within a reasonable time frame using a wavelength range of 2-20 Å. Several different samples at the solid-air interface and the solid-liquid interface have been measured to demonstrate the instrument's capabilities.

Sterol Structural Features' Impact on the Spontaneous Membrane Insertion of CLIC1 into Artificial Lipid Membranes.

Background: A membrane protein interaction with lipids shows distinct specificity in terms of the sterol structure. The structure of the sterol's polar headgroup, steroidal rings, and aliphatic side chains have all been shown to influence protein membrane interactions, including the initial binding and subsequent oligomerization to form functional channels. Previous studies have provided some insights into the regulatory role that cholesterol plays in the spontaneous membrane insertion of the chloride intracellular ion channel protein, CLIC1. However, the manner in which cholesterol interacts with CLIC1 is yet largely unknown. Method: In this study, the CLIC1 interaction with different lipid:sterol monolayers was studied using the Langmuir trough and neutron reflectometry in order to investigate the structural features of cholesterol essential for the spontaneous membrane insertion of the CLIC1 protein. Molecular docking simulations were also performed to study the binding affinities between CLIC1 and the different sterol molecules. Results: This study, for the first time, highlights the vital role of the free sterol 3ß-OH group as an essential structural requirement for the interaction of CLIC1 with cholesterol. Furthermore, the presence of additional hydroxyl groups, methylation of the sterol skeleton, and the structure of the sterol alkyl side chain have also been shown to modulate the magnitude of CLIC1 interaction with sterols and hence their spontaneous membrane insertion. This study also reports the ability of CLIC1 to interact with other naturally existing sterol molecules. General Significance: Like the sterol molecules, CLIC proteins are evolutionarily conserved with almost all vertebrates expressing six CLIC proteins (CLIC1-6), and CLIC-like proteins are also present in invertebrates and have also been reported in plants. This discovery of CLIC1 protein interaction with other natural sterols and the sterol structural requirements for CLIC membrane insertion provide key information to explore the feasibility of exploiting these properties for therapeutic and prophylactic purposes.

The membrane activity of the antimicrobial peptide caerin 1.1 is pH dependent.

Antimicrobial peptides are an important class of membrane-active peptides that can provide alternatives or complements to classic antibiotics. Among the many classes of AMPs, the histidine-rich family is of particular interest since they may induce pH-sensitive interactions with cell membranes. The AMP caerin 1.1 (Cae-1), from Australian tree frogs, has three histidine residues, and thus we studied the pH dependence of its interactions with model cell membranes. Using NMR spectroscopy and molecular dynamics simulations, we showed that Cae-1 induced greater perturbation of the lipid dynamics and water penetrations within the membrane interior in an acidic environment compared with physiological conditions. Using 31P solid-state NMR, the packing, chemical environment, and dynamics of the lipid headgroup were monitored. 2H solid-state NMR showed that Cae-1 ordered the acyl chains of the hydrophobic core of the bilayer. These results supported the molecular dynamics data, which showed that Cae-1 was mainly inserted within the lipid bilayer for both neutral and negatively charged membranes, with the charged residues pulling the water and phosphate groups inward. This could be an early step in the mechanism of membrane disruption by histidine-rich antimicrobial peptides and indicated that Cae-1 acts via a transmembrane mechanism in bilayers of neutral and anionic phospholipid membranes, especially in acidic conditions.

The Assembly Mechanism and Mesoscale Architecture of Protein-Polysaccharide Complexes Formed at the Solid-liquid Interface.

Protein-polysaccharide composite materials have generated much interest due to their potential use in medical science and biotechnology. A comprehensive understanding of the assembly mechanism and the mesoscale architecture is needed for fabricating protein-polysaccharide composite materials with desired properties. In this study, complex assemblies were built on silica surfaces through a layer-by-layer (LbL) approach using bovine beta-lactoglobulin variant A (ßLgA) and pectin as model protein and polysaccharide, respectively. We demonstrated the combined use of quartz crystal microbalance with dissipation monitoring (QCM-D) and neutron reflectometry (NR) for elucidating the assembly mechanism as well as the internal architecture of the protein-polysaccharide complexes formed at the solid-liquid interface. Our results show that ßLgA and pectin interacted with each other and formed a cohesive matrix structure at the interface consisting of intertwined pectin chains that were cross-linked by ßLgA-rich domains. Although the complexes were fabricated in an LbL fashion, the complexes appeared to be relatively homogeneous with ßLgA and pectin molecules spatially distributed within the matrix structure. Our results also demonstrate that the density of ßLgA-pectin complex assemblies increased with both the overall and local charge density of pectin molecules. Therefore, the physical properties of the protein-polysaccharide matrix structure, including density and level of hydration, can be tuned by using polysaccharides with varying charge patterns, thus promoting the development of composite materials with desired properties.

A polytherapy based approach to combat antimicrobial resistance using cubosomes.

A depleted antimicrobial drug pipeline combined with an increasing prevalence of Gram-negative 'superbugs' has increased interest in nano therapies to treat antibiotic resistance. As cubosomes and polymyxins disrupt the outer membrane of Gram-negative bacteria via different mechanisms, we herein examine the antimicrobial activity of polymyxin-loaded cubosomes and explore an alternative strategy via the polytherapy treatment of pathogens with cubosomes in combination with polymyxin. The polytherapy treatment substantially increases antimicrobial activity compared to polymyxin B-loaded cubosomes or polymyxin and cubosomes alone. Confocal microscopy and neutron reflectometry suggest the superior polytherapy activity is achieved via a two-step process. Firstly, electrostatic interactions between polymyxin and lipid A initially destabilize the outer membrane. Subsequently, an influx of cubosomes results in further membrane disruption via a lipid exchange process. These findings demonstrate that nanoparticle-based polytherapy treatments may potentially serve as improved alternatives to the conventional use of drug-loaded lipid nanoparticles for the treatment of "superbugs".

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.

Substrate-dependent arrangements of the subunits of the BAM complex determined by neutron reflectometry.

In Gram-negative bacteria, the ß-barrel assembly machinery (BAM) complex catalyses the assembly of ß-barrel proteins into the outer membrane, and is composed of five subunits: BamA, BamB, BamC, BamD and BamE. Once assembled, - ß-barrel proteins can be involved in various functions including uptake of nutrients, export of toxins and mediating host-pathogen interactions, but the precise mechanism by which these ubiquitous and often essential ß-barrel proteins are assembled is yet to be established. In order to determine the relative positions of BAM subunits in the membrane environment we reconstituted each subunit into a biomimetic membrane, characterizing their interaction and structural changes by Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) and neutron reflectometry. Our results suggested that the binding of BamE, or a BamDE dimer, to BamA induced conformational changes in the polypeptide transported-associated (POTRA) domains of BamA, but that BamB or BamD alone did not promote any such changes. As monitored by neutron reflectometry, addition of an unfolded substrate protein extended the length of POTRA domains further away from the membrane interface as part of the mechanism whereby the substrate protein was folded into the membrane.

Covalent Linkages of Molecules and Proteins to Si-H Surfaces Formed by Disulfide Reduction.

Thiols and disulfide contacts have been, for decades, key for connecting organic molecules to surfaces and nanoclusters as they form self-assembled monolayers (SAMs) on metals such as gold (Au) under mild conditions. In contrast, they have not been similarly deployed on Si owing to the harsh conditions required for monolayer formation. Here, we show that SAMs can be simply formed by dipping Si-H surfaces into dilute solutions of organic molecules or proteins comprising disulfide bonds. We demonstrate that S-S bonds can be spontaneously reduced on Si-H, forming covalent Si-S bonds in the presence of traces of water, and that this grafting can be catalyzed by electrochemical potential. Cyclic disulfide can be spontaneously reduced to form complete monolayers in 1 h, and the reduction can be catalyzed electrochemically to form full surface coverages within 15 min. In contrast, the kinetics of SAM formation of the cyclic disulfide molecule on Au was found to be three-fold slower than that on Si. It is also demonstrated that dilute thiol solutions can form monolayers on Si-H following oxidation to disulfides under ambient conditions; the supply of too much oxygen, however, inhibits SAM formation. The electron transfer kinetics of the Si-S-enabled SAMs on Si-H is comparable to that on Au, suggesting that Si-S contacts are electrically transmissive. We further demonstrate the prospect of this spontaneous disulfide reduction by forming a monolayer of protein azurin on a Si-H surface within 1 h. The direct reduction of disulfides on Si electrodes presents new capabilities for a range of fields, including molecular electronics, for which highly conducting SAM-electrode contacts are necessary and for emerging fields such as biomolecular electronics as disulfide linkages could be exploited to wire proteins between Si electrodes, within the context of the current Si-based technologies.

Phytantriol-Based Cubosome Formulation as an Antimicrobial against Lipopolysaccharide-Deficient Gram-Negative Bacteria.

Treatment of multidrug-resistant (MDR) bacterial infections increasingly relies on last-line antibiotics, such as polymyxins, with the urgent need for discovery of new antimicrobials. Nanotechnology-based antimicrobials have gained significant importance to prevent the catastrophic emergence of MDR over the past decade. In this study, phytantriol-based nanoparticles, named cubosomes, were prepared and examined in vitro by minimum inhibitory concentration (MIC) and time-kill assays against Gram-negative bacteria: Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Phytantriol-based cubosomes were highly bactericidal against polymyxin-resistant, lipopolysaccharide (LPS)-deficient A. baumannii strains. Small-angle neutron scattering (SANS) was employed to understand the structural changes in biomimetic membranes that replicate the composition of these LPS-deficient strains upon treatment with cubosomes. Additionally, to further understand the membrane-cubosome interface, neutron reflectivity (NR) was used to investigate the interaction of cubosomes with model bacterial membranes on a solid support. These results reveal that cubosomes might be a new strategy for combating LPS-deficient Gram-negative pathogens.

The Location of the Antimicrobial Peptide Maculatin 1.1 in Model Bacterial Membranes.

Maculatin 1.1 (Mac1) is an antimicrobial peptide (AMP) from the skin secretions of Australian tree frogs. In this work, the interaction of Mac1 with anionic phospholipid bilayers was investigated by NMR, circular dichroism (CD) spectroscopy, neutron reflectometry (NR) and molecular dynamics (MD). In buffer, the peptide is unstructured but in the presence of anionic (DPC/LMPG) micelles or (DMPC/DMPG/DHPC) bicelles adopts a helical structure. Addition of the soluble paramagnetic agent gadolinium (Gd-DTPA) into the Mac1-DPC/LMPG micelle solution showed that the N-terminus is more exposed to the hydrophilic Gd-DTPA than the C-terminus in micelles. 2H and 31P solid-state NMR showed that Mac1 had a greater effect on the anionic lipid (DMPG). A deuterium labeled Mac1 used in NR experiments indicated that the AMP spanned across anionic (PC/PG) bilayers, which was compatible with MD simulations. Simulations also showed that Mac1 orientation remained transmembrane in bilayers and wrapped on the surface of the micelles regardless of the lipid or detergent charge. Thus, the peptide orientation appears to be more susceptible to curvature than charged surface. These results support the formation of transmembrane pores by Mac1 in model bacterial membranes.

Characterization of BamA reconstituted into a solid-supported lipid bilayer as a platform for measuring dynamics during substrate protein assembly into the membrane.

In Gram-negative bacteria, the multi-protein ß-barrel assembly machine (BAM) complex is a nanomachine playing a vital role in the process of assembling ß-barrel proteins into the outer membrane (OM). The core component of this multiprotein complex, BamA, is an evolutionarily conserved protein that carries five polypeptide-transport-associated (POTRA) domains that project from the outer membrane. BamA is essential for chaperoning the insertion of proteins into the OM surface of bacterial cells. In this work, we have reconstituted a membrane containing BamA on a gold substrate and characterized structure of each component and movement in different situation at the nanoscale level using quartz-crystal microbalance with dissipation and neutron reflectometry (NR). The purified BamA in n-dodecyl ß-D-maltoside (DDM) was first engineered onto a nickel-NTA (Nα, Nα-bis-(carboxymethyl)-l-lysine) modified gold surface followed by DDM removal and bilayer assembly. The system was then used to monitor the binding and insertion of a substrate membrane protein. The data shows the total reach of BamA was 120 Å and the embedding of membrane had no effect on the BamA morphology. However, the addition of the substrate enabled the periplasmic POTRA domain of BamA to extend further away from the membrane surface. This dynamic behaviour of BamA POTRA domains is consistent with models invoking the gathering of transported substrates from the periplasmic space between the inner and outer membranes in bacterial cells. This study provides evidence that NR is a reliable tool for diverse investigations in the future, especially for applications in the field of membrane protein biogenesis.

The antimicrobial peptide maculatin self assembles in parallel to form a pore in phospholipid bilayers.

Little is known experimentally about the detailed orientation of membrane-bound maculatin 1.1 (Mac1), an antimicrobial peptide from the skin secretions of Australian tree frogs. In this work multiple 15N-labelled or 2H-labelled Mac1 with dodecylphosphocholine (DPC) micelles and isotropic DMPC/DHPC (q = 0.5) bicelles were investigated by solution NMR, circular dichroism (CD) spectroscopy, neutron reflectometry and molecular dynamics (MD) simulations in explicit solvent. In buffer, the 15N-1H HSQC and CD spectra were indicative of the peptide being random coiled. In the presence of micelles or isotropic bicelles, a unique and helical peptide structure that was confirmed by CD was found. The titration of the soluble paramagnetic agent gadolinium (Gd-DTPA) into the Mac1-DPC solution led to enhanced relaxation of all 15N labelled residues. The peptide N-terminus was more exposed to Gd-DTPA than the C-terminus in micelles, while only the Gly-4 and Ala-18 resonances were significantly reduced in the presence of isotropic bicelles. MD simulations of Mac1 fully inserted into a DPC micelle converged towards a solvent exposed orientation and a topology where Mac1 was wrapped around the DPC micelle with the more hydrophobic side facing inward. MD simulations of Mac1 fully inserted into a phosphatidylcholine (PC) bilayer converged towards a kinked transmembrane orientation with water molecules penetrating around Lys-8. A deuterium labelled Mac1 used in neutron reflectometry experiments suggested a preferred orientation in zwitterionic PC bilayers. These results give insight into the membrane disrupting activity of Mac1 against cell membranes.

Spontaneous S-Si bonding of alkanethiols to Si(111)-H: towards Si-molecule-Si circuits.

We report the synthesis of covalently linked self-assembled monolayers (SAMs) on silicon surfaces, using mild conditions, in a way that is compatible with silicon-electronics fabrication technologies. In molecular electronics, SAMs of functional molecules tethered to gold via sulfur linkages dominate, but these devices are not robust in design and not amenable to scalable manufacture. Whereas covalent bonding to silicon has long been recognized as an attractive alternative, only formation processes involving high temperature and/or pressure, strong chemicals, or irradiation are known. To make molecular devices on silicon under mild conditions with properties reminiscent of Au-S ones, we exploit the susceptibility of thiols to oxidation by dissolved O2, initiating free-radical polymerization mechanisms without causing oxidative damage to the surface. Without thiols present, dissolved O2 would normally oxidize the silicon and hence reaction conditions such as these have been strenuously avoided in the past. The surface coverage on Si(111)-H is measured to be very high, 75% of a full monolayer, with density-functional theory calculations used to profile spontaneous reaction mechanisms. The impact of the Si-S chemistry in single-molecule electronics is demonstrated using STM-junction approaches by forming Si-hexanedithiol-Si junctions. Si-S contacts result in single-molecule wires that are mechanically stable, with an average lifetime at room temperature of 2.7 s, which is five folds higher than that reported for conventional molecular junctions formed between gold electrodes. The enhanced "ON" lifetime of this single-molecule circuit enables previously inaccessible electrical measurements on single molecules.