Increasing Antibiotic Susceptibility: The Use of Cationic Gold Nanoparticles in Gram-Negative Bacterial Membrane Models.

Antibiotic resistance will be one of the most prominent challenges to health-care systems in the coming decades, with the OECD predicting that up to 2.4 million deaths will be caused between 2015 and 2050 by drug-resistant bacterial infections in first-world countries alone, with infections costing health-care systems billions of dollars each year. Developing new methods to increase bacterial susceptibility toward drugs is an important step in treating resistant infections. Here, the synergistic effects of gold nanoparticles and the antibiotic drug colistin sulfate have been examined. A tethered lipid bilayer membrane was used to mimic a Gram-negative bacterial cell membrane. Exposing the membrane to gold nanoparticles prior to adding the antibiotic significantly increased the effect of the antibiotic on the membrane. Cationic gold nanoparticles could thus be used to enhance bacterial susceptibility to antibiotics, leading to a more potent treatment.

Solid-supported lipid bilayers - A versatile tool for the structural and functional characterization of membrane proteins.

The cellular membrane is central to the development of single-and multicellular life, as it separates the delicate cellular interior from the hostile environment. It exerts tight control over entry and exit of substances, is responsible for signaling with other cells in multicellular organisms and prevents pathogens from entering the cell. In the case of bacteria and viruses, the cellular membrane also hosts the proteins enabling invasion of the host organism. In a very real sense therefore, the cellular membrane is central to all life. The study of the cell membrane and membrane proteins in particular has therefore attracted significant attention. Due to the enormous variety of tasks performed by the membrane, it is a highly complex and challenging structure to study. Ideally, membrane components would be studied in isolation from this environment, but unlike water soluble proteins, the amphiphilic environment provided by the cellular membrane is key to the structure and function of the cell membrane. Therefore, model membranes have been developed to provide an environment in which a membrane protein can be studied. This review presents a set of tools that enable the comprehensive characterization of membrane proteins: electrochemical tools, surface plasmon resonance, neutron scattering, the surface forces apparatus and atomic force microscopy are discussed, with a particular focus on experimental technique and data evaluation.

Soft and Hard Interactions between Polystyrene Nanoplastics and Human Serum Albumin Protein Corona.

Upon contact with biological fluids, the surface of nanoparticles is surrounded by many types of proteins, forming a so-called "protein corona". The physicochemical properties of the nanoparticle/corona complex depend predominantly on the nature of the protein corona. An understanding of the structure of the corona and the resulting complex provides insight into the structure-activity relationship. Here, we structurally evaluate the soft and hard components of the protein corona, formed from polystyrene (PS) nanoplastics and human serum albumin (HSA). Using circular dichroism spectroscopy to elucidate the structure of HSA within the complex, we establish the effect of nanoparticle size and pH on the nature of the protein corona formed- whether hard or soft. Despite the weak interaction between PS and the HSA corona, small angle neutron scattering revealed the formation of a complex structure that enhanced the intermolecular interactions between HSA proteins, PS particles, and the HS/PSA complexes. Fractal formation occurred under conditions where the interaction between PS and HSA was strong, and increasing HSA concentrations suppressed the degree of aggregation. The size of the nanoparticles directly influenced the nature of the protein corona, with larger particles favoring the formation of a soft corona, due to the decreased PS-HSA attraction.

Investigating the Structure of Self-Assembled Monolayers Related to Biological Cell Membranes.

Tethered bilayer lipid membranes are solid supported lipid membranes, where the inner leaflet is covalently linked to the solid supported substrate through anchorlipids. These anchorlipids form a self-assembled monolayer, which serves as the basis of the membrane and also provides submembrane space. The molecular structure and composition of this monolayer has thus significant influence on the membrane structural and functional properties. The density of the self-assembled monolayer can be tailored by adding small molecules to the monolayer. Here, the structure of fully tethered and sparsely tethered monolayers, where the anchorlipid has been diluted with a small surface-active thiol, has been analyzed using neutral impact collision ion scattering spectroscopy, X-ray photoelectron spectroscopy, and metastable induced electron spectroscopy. Combination of these three techniques allowed description of the self-assembly process in detail. The monolayers have been characterized in terms of layer thickness and orientation of the lipids.

A tethered bilayer lipid membrane that mimics microbial membranes.

A model membrane system has been developed, which mimics the outer membrane of Gram negative bacteria. The structure is based on a tethered monolayer which has been fused with vesicles containing lipopolysaccharide molecules. The effect of the composition of the monolayer and the lipids in the outer layer on the structural and electrical properties of the membrane has been investigated. By using electrochemical impedance spectroscopy as well as neutron scattering techniques, it could be shown that a relatively high tethering density and a small amount of diluting lipids in the outer membrane leaflet leads to the formation of a stable solid supported membrane. The influence of divalent ions on the membrane stability has been probed as well as the interaction of the bilayer with the antibiotic colistin. A number of different architectures were developed, suited to both the study of bacterial membrane proteins and the screening of antimicrobial activity of potential drug candidates.

Synthesis and Characterization of Novel Anchorlipids for Tethered Bilayer Lipid Membranes.

Tethered bilayer lipid membranes are versatile solid-supported model membrane systems. Core to these systems is an anchorlipid that covalently links a lipid bilayer to a support. The molecular structure of these lipids can have a significant impact on the properties of the resulting bilayer. Here, the synthesis of anchorlipids containing ester groups in the tethering part is described. The lipids are used to form bilayer membranes, and the resulting structures are compared with membranes formed using conventional anchorlipids or sparsely tethered membranes. All membranes showed good electrical sealing properties; the disulphide-terminated anchorlipids could be used in a sparsely tethered system without significantly reducing the sealing properties of the lipid bilayers. The sparsely tethered systems also allowed for higher ion transport across the membrane, which is in good correlation with higher hydration of the spacer region as seen by neutron scattering.

Cell-Free Synthesis of a Functional Membrane Transporter into a Tethered Bilayer Lipid Membrane.

Eukaryotic cell-free synthesis was used to incorporate the large and complex multispan plant membrane transporter Bot1 in a functional form into a tethered bilayer lipid membrane. The electrical properties of the protein-functionalized tethered bilayer were measured using electrochemical impedance spectroscopy and revealed a pH-dependent transport of borate ions through the protein. The efficacy of the protein synthesis has been evaluated using immunoblot analysis.

Oxidative Damage to Biomimetic Membrane Systems: In Situ Fe(II)/Ascorbate Initiated Oxidation and Incorporation of Synthetic Oxidized Phospholipids.

Damage to cellular membranes from oxidative stress has been implicated in aging related diseases. We report the effects of oxidative damage on the structure and properties of biomimetic phospholipid membrane systems. Two oxidation methods were used, in situ oxidation initiated using Fe(II) and ascorbate, and the incorporation of a synthetic "oxidized" phospholipid, PoxnoPC, into biomimetic membranes. The biomimetic systems employed included multibilayer stacks, tethered bilayers, and phospholipid monolayers studied using a combination of reflectometry, attenuated total reflection infrared spectroscopy, electrochemical impedance spectroscopy, and neutron diffraction. We show that oxidation with Fe(II) and ascorbate caused an increase in the order of the membrane, attributed to cross-linking of the phospholipids, and a change in the electrical permeability of the membrane, but no significant impact on the thickness or completeness of the membrane. Incorporation of PoxnoPC, on the other hand, had a larger impact on the structure of the membrane. Inversion of the aldehyde-terminated truncated sn-2 chain of PoxnoPC into the head group region was observed, along with a slight decrease in the thickness and order of the membrane.

Interaction of silver nanoparticles with tethered bilayer lipid membranes.

Silver nanoparticles are well-known for their antibacterial properties. However, the detailed mechanism describing the interaction between the nanoparticles and a cell membrane is not fully understood, which can impede the use of the particles in biomedical applications. Here, a tethered bilayer lipid membrane has been used as a model system to mimic a natural membrane and to study the effect of exposure to small silver nanoparticles with diameters of about 2 nm. The solid supported membrane architecture allowed for the application of surface analytical techniques such as electrochemical impedance spectroscopy and atomic force microscopy. Exposure of the membrane to solutions of the silver nanoparticles led to a small but completely reversible perturbation of the lipid bilayer.

Microplastics in biosolids: A review of ecological implications and methods for identification, enumeration, and characterization.

Biosolids, or treated sludge, are by-products of the wastewater treatment processes and are commonly used in agricultural applications to enrich soil nutrients. However, it contains microplastics, plastic particles with a diameter below 1 mm. Microplastics exist and accumulate in the environment, which can have major impacts on the ecosystem. Despite their abundance in the environment, there are to date no standardized methods for their enumeration and characterization. A literature review was conducted focusing on the occurrence of microplastics at wastewater treatment plants, particularly in the solid waste stream, and their influence on the soil ecosystem where biosolids is applied. We found a conflicting evidence to which extent microplastics negatively impact the ecosystem. Some reported either a direct negative impact of microplastics or because of microplastic interaction with other soil contaminants. Meanwhile, other studies showed no effect or at certain amount of microplastics on the ecosystem. We also found that microplastics size, shape, type, concentration, and exposure time play a critical role in their ecological impacts. However, currently, there is no unified approach for microplastics identification and characterization in solid waste resulting in a various and incomparable data. Therefore, utilizing standardized methods for microplastics analysis must be considered as the initial step to better understand the impact of microplastics onto the environment. We suggest a method's scaling comparison as a practical approach to select and develop techniques based on cost, time, data obtained, accuracy, and sensitivity criteria. Further research into the ecotoxicity of microplastics and continuous monitoring of biosolid applications are also necessary.

Promotion of osteogenic cell response using quasicovalent immobilized fibronectin on titanium surfaces: introduction of a novel biomimetic layer system.

PURPOSE: Despite the undeniable potential of cell adhesion molecules such as fibronectin to support osteogenic cell responses and consecutive dental implant healing, the most beneficial mode of application onto titanium implant surfaces still requires investigation. Unspecific fibronectin adsorption on titanium dioxide (TiO(2)) surfaces can result in low-loading, high-desorption rates and protein-metal interactions with impaired biologic activity. The aim of the present study was to monitor the osteogenic cell responses (cell adhesion, proliferation, and differentiation) specifically to fibronectin biofunctionalized TiO(2). MATERIALS AND METHODS: An innovative biomimetic streptavidin-biotin layer system allows flexible, but stable, specific binding of biotinylated biomolecules such as fibronectin on TiO(2) surfaces. Transparent glass disks were sputtered with TiO(2). The biomimetic layer system was immobilized by self-assembly and consisted of silane, biotin-derivate, streptavidin, and biotinylated fibronectin (bFN). For the control group, unbiotinylated fibronectin was directly coated onto TiO(2). Early cell adhesion dynamics were quantified using automated processing of light microscopy images within the first 24 hours. Relative mRNA expression of integrin-ß1, cyclin D1, runt-related gene 2, alkaline phosphatase, and osteocalcin was obtained using quantitative real-time polymerase chain reactions 3 and 7 days after incubation. RESULTS: Although untreated TiO(2) preserved a rather immature osteogenic phenotype, both unbiotinylated fibronectin and bFN promoted osteogenic cell adhesion and cell differentiation. In particular, runt-related gene 2 expression was significantly promoted by bFN after 3 days. In contrast, cyclin D1 expression was decreased for unbiotinylated fibronectin and bFN after 7 days. CONCLUSIONS: The introduced biomimetic layer system contributes a coherent immobilization approach of adhesion molecules with promotion of osteogenic cell response in vitro.

Model architectures for bacterial membranes.

The complex composition of bacterial membranes has a significant impact on the understanding of pathogen function and their development towards antibiotic resistance. In addition to the inherent complexity and biosafety risks of studying biological pathogen membranes, the continual rise of antibiotic resistance and its significant economical and clinical consequences has motivated the development of numerous in vitro model membrane systems with tuneable compositions, geometries, and sizes. Approaches discussed in this review include liposomes, solid-supported bilayers, and computational simulations which have been used to explore various processes including drug-membrane interactions, lipid-protein interactions, host-pathogen interactions, and structure-induced bacterial pathogenesis. The advantages, limitations, and applicable analytical tools of all architectures are summarised with a perspective for future research efforts in architectural improvement and elucidation of resistance development strategies and membrane-targeting antibiotic mechanisms. Supplementary Information: The online version contains supplementary material available at 10.1007/s12551-021-00913-7.

Nanoscale patterning of solid-supported membranes by integrated diffusion barriers.

Ultraflat nanostructured substrates have been used as a template to create patterned solid-supported bilayer membranes with polymerizable tethered lipids acting as diffusion barriers. Patterns in the size range of 100 nm were successfully produced and characterized. The diffusion barriers were embedded directly into the phospholipid bilayer and could be used to control the fluidity of the membrane as well as to construct isolated membrane corrals. By using nanosphere lithography to structure the templates it was possible to systematically adjust the lipid diffusion coefficients in a range comparable to those observed in cellular membranes. Single colloids applied as mask in the patterning process yielded substrates for creation of isolated fluid membrane patches corralled by diffusion barriers. Numerous potential applications for this new model system can be envisioned, ranging from the study of cellular interactions or of molecular diffusion in confined geometries to biosensor arrays.

Probing protein-membrane interactions using solid supported membranes.

Tethered bilayer lipid membranes have been used as a model system to mimic the interactions between the whey protein ß-lactoglobulin and a lipid interface. The approach allowed for a detailed study of the lipid-protein interactions, the results being of possible importance in food and cosmetic applications. For such applications, lipid-protein interactions and the interfacial behavior are vital factors in controlling and manipulating process conditions such as emulsion stabilization and gelification. Lipid composition as well as the structural properties of the protein governed their interactions, which were probed by a combination of surface plasmon spectroscopy, neutron reflectivity, and electrochemical impedance spectroscopy. Comparison of results obtained using native and a partially unfolded protein indicated that the protein preferentially forms loosely packed layers at the lipid interface.

Adsorption and conformation behavior of biotinylated fibronectin on streptavidin-modified TiO(X) surfaces studied by SPR and AFM.

It is well-known that protein-modified implant surfaces such as TiO(2) show a higher bioconductivity. Fibronectin is a glycoprotein from the extracellular matrix (ECM) with a major role in cell adhesion. It can be applied on titanium oxide surfaces to accelerate implant integration. Not only the surface concentration but also the presentation of the protein plays an important role for the cellular response. We were able to show that TiO(X) surfaces modified with biotinylated fibronectin adsorbed on a streptavidin-silane self-assembly multilayer system are more effective regarding osteoblast adhesion than surfaces modified with nonspecifically bound fibronectin. The adsorption and conformation behavior of biotinylated and nonbiotinylated (native) fibronectin was studied by surface plasmon resonance (SPR) spectroscopy and atomic force microscopy (AFM). Imaging of the protein modification revealed that fibronectin adopts different conformations on nonmodified compared to streptavidin-modified TiO(X) surfaces. This conformational change of biotinylated fibronectin on the streptavidin monolayer delivers a fibronectin structure similar to the conformation inside the ECM and therefore explains the higher cell affinity for these surfaces.

Dendron growth from vertically aligned single-walled carbon nanotube thin layer arrays for photovoltaic devices.

Single-walled carbon nanotube arrays attached to conductive transparent electrodes have previously shown promise for use in photovoltaic devices, whilst still retaining light transmission. Here, chemical modification of these thin (<200 nm) arrays with PAMAM-type dendrons has been undertaken to enhance the photoresponse of these devices. The effect of modification on the electrode was measured by differential pulse voltammetry to detect the dendrons, and the effect on the nanotubes was measured by Raman spectroscopy. Solar simulator illumination of the cells was performed to measure the effect of the nanotube modification on the cell power, and determine the optimal modification. Electrochemical impedance spectroscopy was also used to investigate the equivalent electronic circuit elements of the cells. The optimal dendron modification occurred with the second generation (G-2.0), which gave a 70% increase in power over the unmodified nanotube array.

Reviewing nanoplastic toxicology: It's an interface problem.

Multiple international agencies have recently raised environmental and health concerns regarding plastics in nanoforms (nanoplastics), but there is insufficient knowledge of their properties to allow for an accurate risk assessment to be conducted and any risks managed. For this reason, research into the toxicity of nanoplastics has focused strongly on documenting their impacts on biological organisms. One scope of this review is to summarise the recent findings on the adverse effects on biological organisms and strategies which can be adopted to advance our understanding of nanoplastic properties and their toxicity. Specifically, a mechanistic approach has already been employed in nanotoxicology, which focuses on the cause-and-effect relationships to establish a tool that predicts the biological impacts based on nanoparticle characteristics. Identifying the chemical and biological bases behind the observed biological effects (such as in vitro cellular response) is a major challenge, due to the intricate nature of nanoparticle-biological molecule complexes and an unawareness of their interaction with other biological targets, particularly at interfacial level. An exemplary case includes protein corona formation and ecological molecule corona (eco-corona) for nanoplastics. Therefore, the second scope of this review is to discuss recent findings and importance of (for both non-plastic and plastic nanoparticles) coronae formation and structure. Finally, we discuss the opportunities provided by model system approaches (model protein corona and lipid bilayer) to deepen the understanding of the above-mentioned perspectives, and corroborate the findings from in vitro experiments.

Cellular interactions with polystyrene nanoplastics-The role of particle size and protein corona.

Plastic waste is ubiquitously spread across the world and its smaller analogs-microplastics and nanoplastics-raise particular health concerns. While biological impacts of microplastics and nanoplastics have been actively studied, the chemical and biological bases for the adverse effects are sought after. This work explores contributory factors by combining results from in vitro and model mammalian membrane experimentation to assess the outcome of cell/nanoplastic interactions in molecular detail, inspecting the individual contribution of nanoplastics and different types of protein coronae. The in vitro study showed mild cytotoxicity and cellular uptake of polystyrene (PS) nanoplastics, with no clear trend based on nanoplastic size (20 and 200 nm) or surface charge. In contrast, a nanoplastic size-dependency on bilayer disruption was observed in the model system. This suggests that membrane disruption resulting from direct interaction with PS nanoplastics has little correlation with cytotoxicity. Furthermore, the level of bilayer disruption was found to be limited to the hydrophilic headgroup, indicating that transmembrane diffusion was an unlikely pathway for cellular uptake-endocytosis is the viable mechanism. In rare cases, small PS nanoplastics (20 nm) were found in the vicinity of chromosomes without a nuclear membrane surrounding them; however, this was not observed for larger PS nanoplastics (200 nm). We hypothesize that the nanoplastics can interact with chromosomes prior to nuclear membrane formation. Overall, precoating PS particles with protein coronae reduced the cytotoxicity, irrespective of the corona type. When comparing the two types, the extent of reduction was more apparent with soft than hard corona.

The Membrane Composition Defines the Spatial Organization and Function of a Major Acinetobacter baumannii Drug Efflux System.

Acinetobacter baumannii is one of the world's most problematic nosocomial pathogens. The combination of its intrinsic resistance and ability to acquire resistance markers allow this organism to adjust to antibiotic treatment. Despite being the primary barrier against antibiotic stress, our understanding of the A. baumannii membrane composition and its impact on resistance remains limited. In this study, we explored how the incorporation of host-derived polyunsaturated fatty acids (PUFAs) is associated with increased antibiotic susceptibility. Functional analyses of primary A. baumannii efflux systems indicated that AdeB-mediated antibiotic resistance was impacted by PUFA treatment. Molecular dynamics simulations of AdeB identified a specific morphological disruption of AdeB when positioned in the PUFA-enriched membrane. Collectively, we have shown that PUFAs can impact antibiotic efficacy via a vital relationship with antibiotic efflux pumps. Furthermore, this work has revealed that A. baumannii's unconditional desire for fatty acids may present a possible weakness in its multidrug resistance capacity. IMPORTANCE Antimicrobial resistance is an emerging global health crisis. Consequently, we have a critical need to prolong our current arsenal of antibiotics, in addition to the development of novel treatment options. Due to their relatively high abundance at the host-pathogen interface, PUFAs and other fatty acid species not commonly synthesized by A. baumannii may be actively acquired by A. baumannii during infection and change the biophysical properties of the membrane beyond that studied in standard laboratory culturing media. Our work illustrates how the membrane phospholipid composition impacts membrane protein function, which includes an important multidrug efflux system in extensively-drug-resistant A. baumannii. This work emphasizes the need to consider including host-derived fatty acids in in vitro analyses of A. baumannii. On a broader scope, this study presents new findings on the potential health benefits of PUFA in individuals at risk of contracting A. baumannii infections or those undergoing antibiotic treatment.

Assembly of the m2 tetramer is strongly modulated by lipid chain length.

The influenza virus matrix protein 2 (M2) assembles into a tetramer in the host membrane during viral uncoating and maturation. It has been used as a model system to understand the relative contributions of protein-lipid and protein-protein interactions to membrane protein structure and association. Here we investigate the effect of lipid chain length on the association of the M2 transmembrane domain into tetramers using Förster resonance energy transfer. We observe that the interactions between the M2 helices are much stronger in 1,2-dilauroyl-sn-glycero-3-phosphocholine than in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers. Thus, lipid chain length and bilayer thickness not only modulate peptide interactions, but could also be a major determinant of the association of transmembrane helices into functional membrane protein oligomers.