The tether that anchors the lipid bilayer to the solid surface is a key component of tethered lipid membranes, which mimic biological membranes. The obtained lipid bilayers with high electrical sealing properties facilitate the application of electrochemical impedance spectroscopy (EIS) to assess conductivity and defectiveness resulting, for example, from pore-forming toxins. However, it remains uncertain whether the tether compound in the lipid membrane impacts only EIS sensitivity or also influences analyte (protein) function. Thus, we investigated how the lipid membrane properties change by varying the amount and the length of the tether compound. Following this, the membranes were exposed to the pore-forming toxin pneumolysin produced by Streptococcus pneumoniae. Several methods were used, including surface plasmon resonance for binding analysis, surface-enhanced infrared absorption spectroscopy for structural insights, EIS for quantifying the pores, and atomic force microscopy for morphological characterization. The study highlights two key observations. First, the composition of mixed self-assembled monolayers deviates from that of the preparative solution. Second, the quantity of tether compounds present in the lipid membrane influences protein oligomerization due to the minor steric hindrance introduced by these compounds. These insights should be helpful in utilizing tethered lipid membranes as a tool to mimic cell membrane viscosity.

Cholesterol-dependent cytolysins (CDCs) are a family of pore-forming toxins produced by numerous pathogenic bacteria and represent important virulence factors. CDCs share a conserved structural organization and require membrane cholesterol for pore formation. Although the molecular mechanism of pore formation has been extensively characterized, the cholesterol requirement in modulating CDCs activity remains incompletely understood. Using tethered bilayer lipid membranes (tBLMs) and electrochemical impedance spectroscopy, we found that lower cholesterol concentrations were sufficient to support PLY-mediated membrane disruption in membranes containing lipids with less hydrated headgroups. In contrast to the reduced activity of PLY on human erythrocytes at acidic pH, substantial PLY-mediated damage was observed in tBLMs under acidic conditions, accompanied by a reduced cholesterol requirement. Whether this effect results from pH-dependent changes in PLY itself or in the physicochemical properties of membrane lipids remains to be determined. Finally, temperature did not alter the cholesterol threshold for PLY activity but enhanced membrane disruption, likely by reducing membrane viscosity. Although tBLMs do not fully recapitulate the complexity of biological membranes, their well-defined composition and structural properties make them a valuable platform for dissecting protein-membrane interactions that are difficult to resolve in cellular systems. These findings identify membrane composition and environmental conditions as key determinants of the cholesterol requirement for PLY activity.

Solid and liquid stilbene forms were characterized using a range of techniques, including atomic force microscopy, coherent anti-Stokes Raman scattering microspectroscopy and optical spectroscopy. The obtained experimental results were analyzed by means of quantum chemical calculations and using a non-negative matrix factorization algorithm. It was confirmed that pure cis-stilbene formed a homogeneous fluid film on a glass substrate, whereas pure trans-stilbene formed crystals. Mixtures of trans-stilbene and polystyrene were shown to form stable solid films, which were non-homogeneous on the microscopic scale: stilbene molecules self-organized into microcrystals, which floated on the surface of polystyrene glass.

Argonaute (Ago) proteins are present in all three domains of life (bacteria, archaea and eukaryotes). They use small (15-30 nucleotides) oligonucleotide guides to bind complementary nucleic acid targets and are responsible for gene expression regulation, mobile genome element silencing, and defence against viruses or plasmids. According to their domain organization, Agos are divided into long and short Agos. Long Agos found in prokaryotes (long-A and long-B pAgos) and eukaryotes (eAgos) comprise four major functional domains (N, PAZ, MID and PIWI) and two structural linker domains L1 and L2. The majority (∼60%) of pAgos are short pAgos, containing only the MID and inactive PIWI domains. Here we focus on the prokaryotic Argonaute AfAgo from Archaeoglobus fulgidus DSM4304. Although phylogenetically classified as a long-B pAgo, AfAgo contains only MID and catalytically inactive PIWI domains, akin to short pAgos. We show that AfAgo forms a heterodimeric complex with a protein encoded upstream in the same operon, which is a structural equivalent of the N-L1-L2 domains of long pAgos. This complex, structurally equivalent to a long PAZ-less pAgo, outperforms standalone AfAgo in guide RNA-mediated target DNA binding. Our findings provide a missing piece to one of the first and the most studied pAgos.

Neuronal cell death induced by cell membrane damage is one of the major hallmarks of neurodegenerative diseases. Neuroinflammation precedes the loss of neurons; however, whether and how inflammation-related proteins contribute to the loss of membrane integrity remains unknown. We employed a range of biophysical tools, including high-speed atomic force microscopy, fluorescence spectroscopy, and electrochemical impedance spectroscopy, to ascertain whether the pro-inflammatory protein S100A8 induces alterations in biomimetic lipid membranes upon interaction. Our findings underscore the crucial roles played by divalent cations and membrane charge. We found that apo-S100A8 selectively interacts with anionic lipid membranes composed of phosphatidylserine (PS), causing membrane disruption through a detergent-like mechanism, primarily affecting regions where phospholipids are less tightly packed. Interestingly, the introduction of Ca2+ ions inhibited S100A8-induced membrane disruption, suggesting that the disruptive effects of S100A8 are most pronounced under conditions mimicking intracellular compartments, where calcium levels are low, and PS concentrations in the inner leaflet of the membrane are high. Overall, our results present a mechanistic basis for understanding the molecular interactions between S100A8 and the plasma membrane, emphasizing S100A8 as a potential contributor to the onset of neurodegenerative diseases.

Pro-inflammatory, calcium-binding protein S100A9 is localized in the cytoplasm of many cells and regulates several intracellular and extracellular processes. S100A9 is involved in neuroinflammation associated with the pathogenesis of Alzheimer's disease (AD). The number of studies on the impact of S100A9 in co-aggregation processes with amyloid-like proteins is increasing. However, there is still a lack of data on how this protein interacts with lipid membranes. We employed atomic force microscopy (AFM), dynamic light scattering (DLS), and fluorescence measurements (Laurdan and Thioflavin-T) to study the interaction between protein and the membrane surface. We used lipid vesicles in bulk and planar tethered lipid bilayers as biomimetic membrane models. We demonstrated that the protein accumulates on negatively charged lipid bilayers but with no further loss of the bilayer's integrity. The most important result is that the initial adsorption and accumulation of apo-form of S100A9 on the lipid membrane surface is lipid phase-sensitive. The breaking down of raft-like and disappearance of gel-like domains indicate that protein incorporates into the hydrophobic part of the lipid bilayer. We observed the most noticeable loss of integrity in lipid bilayers constructed from a lipid mixture (brain total lipid extract). Understanding the function and interactions of these proteins in cellular environments might expand the development of new diagnostic and therapeutic approaches for AD or other related diseases.

The main function of a membrane is to control the exchange of matter between the surrounding regions. As such, accurate modeling of membranes is important to properly describe their properties. In many cases in both biological systems and technical applications, the membranes are composite structures where transport properties may vary between the different sub-regions of the membrane. In this work we develop a method based on Mesh analysis that is asymptotically exact and can describe diffusion in composite membrane structures. We do this by first reformulating a generalized Fick's law to include the effects from activity coefficient, diffusion coefficient, and solubility using a single condensed parameter. We then use the derived theory and Mesh analysis to, in essence, retrieve a finite element method approach. The calculated examples are based on a membrane structure that reassembles that of the brick and mortar structure of stratum corneum, the upper layer of our skin. Resulting concentration profiles from this procedure are then compared to experimental results for the distribution of different probes within intact stratum corneum, showing good agreement. Based on the derived approach we further investigate the impact from a gradient in the fluidity of the stratum corneum mortar lipids across the membrane, and find that it is substantial. We also show that anisotropic organisation of the lipid mortar can have large impact on the effective permeability compared to isotropic mortar lipids. Finally, we examine the effects of corneocyte swelling, and their lateral arrangement in the membrane on the overall membrane permeability.

A systematic spectroscopic characterization of highly homogeneous water suspensions of 'buckydiamonds' comprising sp3cubic nanodiamond (ND) core covered with disordered sp2shell densely decorated with oxygen-containing groups demonstrates the excitation-wavelength-dependent photoluminescence (PL) given by at least four types of specific structures on the ND surface (hydroxyl, C=O containing ketones, carboxylic anhydrides, and carboxyl groups). PL properties of NDs suspensions possess concentration-dependent behavior revealing tendency of NDs to agglomerate. PL of NDs has been found to be strongly sensitive to pH of the environment in wide range of pH values, i.e. 2-11. We disclosed the mechanisms of pH sensitivity of the 'buckydiamond' and proved that it can serve as all-optical sensor of tiny pH variations suitable for further exploitation for pH sensing locally in the area where NDs have been delivered for any purpose, e.g. bioimaging or therapeutic needs.

Microbially induced calcite precipitation (MICP) is an efficient and eco-friendly technique that has attracted significant interest for resolving various problems in the soil (erosion, improving structural integrity and water retention, etc.), remediation of heavy metals, production of self-healing concrete or restoration of different concrete structures. The success of most common MICP methods depends on microorganisms degrading urea which leads to the formation of CaCO3 crystals. While Sporosarcina pasteurii is a well-known microorganism for MICP, other soil abundant microorganisms, such as Staphylococcus bacteria have not been thoroughly studied for its efficiency in bioconsolidation though MICP is a very important proccess which can ensure soil quality and health. This study aimed to analyze MICP process at the surface level in Sporosarcina pasteurii and a newly screened Staphylococcus sp. H6 bacterium as well as show the possibility of this new microorganism to perform MICP. It was observed that Staphylococcus sp. H6 culture precipitated 157.35 & PLUSMN; 3.3 mM of Ca2+ ions from 200 mM, compared to 176 & PLUSMN; 4.8 mM precipitated by S. pasteurii. The bioconsolidation of sand particles was confirmed by Raman spectroscopy and XRD analysis, which indicated the formation of CaCO3 crystals for both Staphylococcus sp. H6 and S. pasteurii cells. The water-flow test suggested a significant reduction in water permeability in bioconsolidated sand samples for both Staphylococcus sp. H6 and S. pasteurii. Notably, this study provides the first evidence that CaCO3 precipitation occurs on the surface of Staphylococcus and S. pasteurii cells within the initial 15-30 min after exposure to the biocementation solution. Furthermore, Atomic force microscopy (AFM) indicated rapid changes in cell roughness, with bacterial cells becoming completely coated with CaCO3 crystals after 90 min incubation with a biocementation solution. To our knowledge, this is the first time where atomic force microscopy was used to visualize the dynamic of MICP on cell surface.

In this work, we aimed at fast and scalable manufacturing of low-cost SERS substrates using ultrashort-pulse laser-induced plasma-assisted ablation (LIPAA) of soda-lime glass. A two-step approach of amorphous nanostructure formation on the glass surface and subsequent deposition of 170 nm silver layer resulted in homogenous SERS-active substrates covered with nanostructures of around 100 nm in diameter, forming 1-3 mu m size dendrimers. The average enhancement factor (EF) evaluated using thiophenol was 3.0 x 105, while the surface activity was found to be consistent at any given point of substrates. Relatively modest EF was explained by amorphous, rounded-shaped features on soda-lime glass formed during the LIPAA-induced melting and subsequent solidification. That resulted in a lesser amount of hot spots but increased surface homogeneity. The use of soda-lime glass to fabricate SERS substrates promises a cheaper and scalable alternative to more widely used sapphire and other material substrates opening prospects for low-cost routine SERS testing with high reproducibility in chemistry, medical, forensic, and environmental sciences.

CRISPR-Cas systems are prokaryotic adaptive immune systems that protect against phages and other invading nucleic acids. The evolutionary arms race between prokaryotes and phages gave rise to phage anti-CRISPR (Acr) proteins that act as a counter defence against CRISPR-Cas systems by inhibiting the effector complex. Here, we used a combination of bulk biochemical experiments, X-ray crystallography and single-molecule techniques to explore the inhibitory activity of AcrIF6 and AcrIF9 proteins against the type I-F CRISPR-Cas system from Aggregatibacter actinomycetemcomitans (Aa). We showed that AcrIF6 and AcrIF9 proteins hinder Aa-Cascade complex binding to target DNA. We solved a crystal structure of Aa1-AcrIF9 protein, which differ from other known AcrIF9 proteins by an additional structurally important loop presumably involved in the interaction with Cascade. We revealed that AcrIF9 association with Aa-Cascade promotes its binding to off-target DNA sites, which facilitates inhibition of CRISPR-Cas protection.

Abstract Atomic force microscopy (AFM) image analysis of supported bilayers, such as tethered bilayer membranes (tBLMs) can reveal the nature of the membrane damage by pore-forming proteins and predict the electrochemical impedance spectroscopy (EIS) response of such objects. However, automated analysis involving pore detection in such images is often non-trivial and can require AI-based object detection techniques. The specific object-detection algorithm we used to determine the defect coordinates in real AFM images was a convolutional neural network (CNN). Defect coordinates allow to predict the EIS response of tBLMs populated by the pore-forming toxins using finite element analysis (FEA) modeling. We tested if the accuracy of the CNN algorithm affected the EIS spectral features sensitive to defect densities and other physical parameters of tBLMs. We found that the EIS spectra can be predicted sufficiently well, however, systematic errors of characteristic spectral points were observed and need to be taken into account. Importantly, the comparison of predicted EIS curves with experimental ones allowed to estimate important physical parameters of tBLMs such as the specific resistance of submembrane reservoir. This reservoir separates phospholipid bilayer from the solid support. We found that the specific resistance of the reservoir amounts to $$10^{4.25 \pm 0.10}$$ 10 4.25 ± 0.10 $$\Omega \cdot cm$$ Ω · c m which is approximately two orders of a magnitude higher compared to the specific resistance of the buffer bathing tBLMs studied in this work. We hypothesize that such effect may be related in part due to decreased concentration of ionic carriers in the submembrane due to decreased relative dielectric permittivity in this region.

Fragmented multi-layered graphene films were directly synthesized via chemical vapor deposition (CVD) on dielectric substrates with a pre-deposited copper catalyst. We demonstrate that the thickness of the sacrificial copper film, process temperature, and growth time essentially influence the integrity, quality, and disorder of the synthesized graphene. Atomic force microscopy and Kelvin probe force microscopy measurements revealed the presence of nano-agglomerates and charge puddles. The potential gradients measured over the sample surface confirmed that the deposited graphene film possessed a multilayered structure, which was modelled as an ensemble of randomly oriented conductive prolate ellipsoids. THz time domain spectroscopy measurements gave theacconductivity of the graphene flakes and homogenized graphitic films as being around 1200 S cm-1and 1000 S cm-1, respectively. Our approach offers a scalable fabrication of graphene structures composed of graphene flakes, which have effective conductivity sufficient for a wide variety of THz applications.

“Blinking” behavior of fluorophores, being harmful for the majority of super-resolved techniques, turns into a key property for stochastic optical fluctuation imaging and its modifications, allowing one to look at the fluorophores already used in conventional microscopy, such as graphene quantum dots, from a completely new perspective. Here we discuss fluorescence of aggregated ensembles of graphene quantum dots structured at submicron scale. We study temperature dependence and stochastic character of emission. We show that considered quantum dots ensembles demonstrate rather complicated temperature-dependent intermittent emission, that is, “blinking” with a tendency to shorten “blinking” times with the increase of temperature. We verify “blinking” mechanism demonstrating hysteresis of the optical response under pulsed excitation timed to expected rates of dots transition to “dark” nonemitting states. Experimental results are well fitted by a simple qualitative model of transitions to the “dark” states. The obtained results suggest that this type of standardized quantum dots and even their submicron-size agglomerations can be useful as controlled fluorophores for super-resolution microscopy and, particularly, for SOFI-like microscopy.

Argonaute (Ago) proteins are found in all three domains of life. The best-characterized group is eukaryotic Argonautes (eAgos), which are the core of RNA interference. The best understood prokaryotic Ago (pAgo) proteins are full-length pAgos. They are composed of four major structural/functional domains (N, PAZ, MID, and PIWI) and thereby closely resemble eAgos. It was demonstrated that full-length pAgos function as prokaryotic antiviral systems, with the PIWI domain performing cleavage of invading nucleic acids. However, the majority of identified pAgos are shorter and catalytically inactive (encode just MID and inactive PIWI domains), thus their action mechanism and function remain unknown. In this work we focus on AfAgo, a short pAgo protein encoded by an archaeon Archaeoglobus fulgidus . We find that in all previously solved AfAgo structures, its two monomers form substantial dimerization interfaces involving the C-terminal β-sheets. Led by this finding, we have employed various biochemical and biophysical assays, including SEC-MALS, SAXS, single-molecule FRET, and AFM, to show that AfAgo is indeed a homodimer in solution, which is capable of simultaneous interaction with two DNA molecules. This finding underscores the diversity of prokaryotic Agos and broadens the range of currently known Argonaute-nucleic acid interaction mechanisms.

In this work, the assembly of functional tethered bilayer membranes (tBLMs) on silver surface was demonstrated for the first time. To avoid degradation of thin silver films the non-corrosive mixture of thiols composed of WC14 molecular anchor and 3-mercapto-1-propanol (3M1P) serving as a backfiller was used to self-assemble monolayers which allowed fusion of multilamellar vesicles and tBLM formation. tBLMs on silver films exhibited high stability and low residual defect densities as measured by the electrochemical impedance spectroscopy (EIS). Silver supported tBLMs demonstrated all features typical for interactions of pore-forming proteins and bilayer membranes. Specifically, the EIS spectral response was typical for the formation of water filled defects when tBLMs were exposed to the solution of vaginolysin (VLY), the cholesterol dependent cytolysin. Increase of defect density was observed only in case tBLMs contained significant amount, e.g., 50 %, of cholesterol, thus confirming functional reconstitution of vaginolysin into tBLMs. This observation is consistent with the biological nature of interaction between the bilayer and the cholesterol dependent cytolysins. The reconstitution of VLY was confirmed by the direct imaging of the protein entities in tBLMs by the atomic force microscopy (AFM). Notably, the quantitative estimate of the functional protein-pores in tBLMs measured by EIS, was found to be close to the number of VLY entities in tBLMs measured by the AFM. Our work demonstrates that silver can serve as less expensive alternative to gold in designing tBLM based devices, for example phospholipid biosensors, as well as the experimental platform for studying membrane protein interactions by electrochemical, and microscopy techniques.

Integration of living cells with nonbiological surfaces (substrates) of sensors, scaffolds, and implants implies severe restrictions on the interface quality and properties, which broadly cover all elements of the interaction between the living and artificial systems (materials, surface modifications, drug-eluting coatings, etc.). Substrate materials must support cellular viability, preserve sterility, and at the same time allow real-time analysis and control of cellular activity. We have compared new substrates based on graphene and pyrolytic carbon (PyC) for the cultivation of living cells. These are PyC films of nanometer thickness deposited on SiO2 and black silicon and graphene nanowall films composed of graphene flakes oriented perpendicular to the Si substrate. The structure, morphology, and interface properties of these substrates are analyzed in terms of their biocompatibility. The PyC demonstrates interface biocompatibility, promising for controlling cell proliferation and directional intercellular contact formation while as-grown graphene walls possess high hydrophobicity and poor biocompatibility. By performing experiments with C6 glioma cells we discovered that PyC is a cell-friendly coating that can be used without poly-l-lysine or other biopolymers for controlling cell adhesion. Thus, the opportunity to easily control the physical/chemical properties and nanotopography makes the PyC films a perfect candidate for the development of biosensors and 3D bioscaffolds.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder causing memory loss, language problems and behavioural disturbances. AD is associated with the accumulation of fibrillar amyloid‐β (Aβ) and the formation of neurofibrillary tau tangles. Fibrillar Aβ itself represents a danger‐associated molecular pattern, which is recognized by specific microglial receptors. One of the key players is formation of the NOD‐, LRR‐ and pyrin domain‐containing 3 (NLRP3) inflammasome, whose activation has been demonstrated in AD patient brains and transgenic animal models of AD. Here, we investigated whether Aβ oligomers or protofibrils that represent lower molecular aggregates prior to Aβ deposition are able to activate the NLRP3 inflammasome and subsequent interleukin‐1 beta (IL‐1β) release by microglia. In our study, we used Aβ preparations of different sizes: small oligomers and protofibrils of which the structure was confirmed by atomic force microscopy. Primary microglial cells from C57BL/6 mice were treated with the respective Aβ preparations and NLRP3 inflammasome activation, represented by caspase‐1 cleavage, IL‐1β production, and apoptosis‐associated speck‐like protein containing a CARD speck formation was analysed. Both protofibrils and low molecular weight Aβ aggregates induced a significant increase in IL‐1β release. Inflammasome activation was confirmed by apoptosis‐associated speck‐like protein containing a CARD speck formation and detection of active caspase‐1. The NLRP3 inflammasome inhibitor MCC950 completely inhibited the Aβ‐induced immune response. Our results show that the NLRP3 inflammasome is activated not only by fibrillar Aβ aggregates as reported before, but also by lower molecular weight Aβ oligomers and protofibrils, highlighting the possibility that microglial activation by these Aβ species may initiate innate immune responses in the central nervous system prior to the onset of Aβ deposition.