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Surface modification using zwitterionic 2-methacryloyloxyethylphosphorylcholine (MPC) polymers is one of the most reasonable ways to prepare medical devices that can suppress undesired biological reactions such as blood coagulation. Usable MPC polymers are hydrophilic and water soluble, and their surface modification strategy involves exploiting the copolymer structures by adding physical or chemical bonding moieties. In this study, we developed copolymers composed of MPC, hydrophobic anchoring moiety, and chemical cross-linking unit to clarify the role of hydrophobic interactions in achieving biocompatible and long-term stable coatings. The four kinds of MPC copolymers with cross-linking units, such as 3-methacryloxypropyl trimethoxysilane (MPTMSi), and four different hydrophobic anchoring moieties, such as 3-(methacryloyloxy)propyltris(trimethylsiloxy)silane (MPTSSi) named as PMMMSi, n-butyl methacrylate (BMA) as PMBSi, 2-ethylhexyl methacrylate (EHMA) as PMESi, and lauryl methacrylate as PMLSi, were synthesized and coated on polydimethylsiloxane, polypropylene (PP), and polymethyl pentene. These copolymers were uniformly coated on the substrate materials PP and poly(methyl pentene) (PMP), to achieve hydrophilic and electrically neutral coatings. The results of the antibiofouling test showed that PMBSi repelled the adsorption of fluorescence-labeled bovine serum albumin the most, whereas PMLSi repelled it the least. Notably, all four copolymers suppressed platelet adhesion similarly. The variations in protein adsorption quantities among the four copolymer coatings were attributed to their distinct swelling behaviors in aqueous environments. Further investigations, including 3D scanning force microscopy and neutron reflectivity measurements, revealed that the PMLSi coating exhibited a higher water intake under aqueous conditions in comparison to the other coatings. Consequently, all copolymer coatings effectively prevented the invasion of platelets but the proteins penetrated the PMLSi network. Subsequently, the dynamic stability required to induce shear stress was evaluated using a circulation system. The results demonstrated that the PMMMSi and PMLSi coatings on PMP and PP exhibited exceptional platelet repellency and maintained high stability during circulation. This study highlights the potential of hydrophobic moieties to improve hemocompatibility and stability, offering potential applications in medical devices.
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Interações Hidrofóbicas e Hidrofílicas , Fosforilcolina/química , Fosforilcolina/análogos & derivados , Polímeros/química , Animais , Reagentes de Ligações Cruzadas/química , Materiais Revestidos Biocompatíveis/química , Adesividade Plaquetária/efeitos dos fármacos , Propriedades de Superfície , Soroalbumina Bovina/química , Humanos , Metacrilatos/química , Fosfolipídeos/química , BovinosRESUMO
The invention of 3D atomic force microscopy (3D-AFM) has enabled visualizing subnanoscale 3D hydration structures. Meanwhile, its applications to imaging flexible molecular chains have started to be experimentally explored. However, the validity and principle of such imaging have yet to be clarified by comparing experiments and simulations or cross-observations with an alternative technique. Such studies are impeded by the lack of an appropriate model. Here, this difficulty is overcome by fabricating 3D carbon nanotube (CNT) structures flexible enough for 3D-AFM, large enough for scanning electron microscopy (SEM), and simple enough for simulations. SEM and 3D-AFM observations of the same model provide unambiguous evidence to support the possibility of imaging overlapped nanostructures, such as suspended CNT and underlying platinum (Pt) nanodots. Langevin dynamics simulations of such 3D-AFM imaging clarify the imaging mechanism, where the flexible CNT is laterally displaced to allow the AFM probe access to the underlying structures. These results consistently show that 3D-AFM images are affected by the friction between the CNT and AFM nanoprobe, yet it can be significantly suppressed by oscillating the cantilever. This study reinforces the theoretical basis of 3D-AFM for imaging various 3D self-organizing systems in diverse fields, from life sciences to interface sciences.
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Self-organizing solid-binding peptides on atomically flat solid surfaces offer a unique bio/nano hybrid platform, useful for understanding the basic nature of biology/solid coupling and their practical applications. The surface behavior of peptides is determined by their molecular folding, which is influenced by various factors and is challenging to study. Here, the effect of charged amino acids is studied on the self-assembly behavior of a directed evolution selected graphite-binding dodecapeptide on graphite surface. Two mutations, M6 and M8, are designed to introduce negatively and positively charged moieties, respectively, at the anchoring domain of the wild-type (WT) peptide, affecting both binding and assembly. The questions addressed here are whether mutant peptides exhibit molecular crystal formation and demonstrate molecular recognition on the solid surface based on the specific mutations. Frequency-modulated atomic force microscopy is used for observations of the surface processes dynamically in water at molecular resolution over several hours at the ambient. The results indicate that while the mutants display distinct folding and surface behavior, each homogeneously nucleates and forms 2D self-organized patterns, akin to the WT peptide. However, their growth dynamics, domain formation, and crystalline lattice structures differ significantly. The results represent a significant step toward the rational design of bio/solid interfaces, potent facilitators of a variety of future implementations.
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Aminoácidos , Microscopia de Força Atômica , Peptídeos , Mutação Puntual , Peptídeos/química , Aminoácidos/química , Propriedades de Superfície , Grafite/químicaRESUMO
Star polymers consisting of three helical poly(phenylacetylene) chains with a precisely controlled molecular weight (molar mass dispersity < 1.03) were successfully synthesized by the living polymerization of phenylacetylene derivatives with a Rh-based multicomponent catalyst system comprising trifunctional initiators, which have three phenylboronates centered on a benzene ring, the Rh complex [Rh(nbd)Cl]2, diphenylacetylene, triphenylphosphine, and a base. The analysis of chiroptical properties of the optically active star polymers obtained by the living polymerization of optically active phenylacetylene derivatives revealed that the star polymers exhibited chiral amplification properties owing to their unique topology compared with the corresponding linear polymers.
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Water molecules on oxide surfaces influence the chemical reactivity and molecular adsorption behavior of oxides. Herein, three-dimensional atomic force microscopy (3D-AFM) and molecular dynamics simulations are used to visualize the surface hydroxyl (OH) groups and their hydration structures on sapphire (001) and α-quartz (100) surfaces at the atomic-scale. The obtained results revealed that the spatial density distributions and hydrogen-bonding strengths of surface OH groups affect their local hydration structures. In particular, the force curves obtained by 3D-AFM suggest that the hydration forces of water molecules intensify at sites where water molecules strongly interact with the surface OH groups. The insights obtained in this study deepen our understanding of the affinities of Al2O3 and SiO2 for water molecules and contribute to the use of 3D-AFM in the investigation of atomic-scale hydration structures on various surfaces, thereby benefiting a wide range of research fields dealing with solid-liquid interfaces.
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Scanning ion conductance microscopy (SICM) is a promising tool for visualizing the dynamics of nanoscale cell surface topography. However, there are still no guidelines for fabricating nanopipettes with ideal shape consisting of small apertures and thin glass walls. Therefore, most of the SICM imaging has been at a standstill at the submicron scale. In this study, we established a simple and highly reproducible method for the fabrication of nanopipettes with sub-20 nm apertures. To validate the improvement in the spatial resolution, we performed time-lapse imaging of the formation and disappearance of endocytic pits as a model of nanoscale time-lapse topographic imaging. We have also successfully imaged the localization of the hot spot and the released extracellular vesicles. The nanopipette fabrication guidelines for the SICM nanoscale topographic imaging can be an essential tool for understanding cell-cell communication.
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Vesículas Extracelulares , Microscopia , Cintilografia , Comunicação Celular , Membrana Celular , ÍonsRESUMO
Atomic force microscopy (AFM) is capable of nanoscale imaging but has so far only been used on cell surfaces when applied to a living cell. Here, we describe a step-by-step protocol for nanoendoscopy-AFM, which enables the imaging of nanoscale structures inside living cells. The protocol consists of cell staining, fabrication of the nanoneedle probes, observation inside living cells using 2D and 3D nanoendoscopy-AFM, and visualization of the 3D data. For complete details on the use and execution of this protocol, please refer to Penedo et al. (2021)1 and Penedo et al. (2021).2.
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Nanotecnologia , Microscopia de Força Atômica/métodos , Nanotecnologia/métodos , Membrana Celular/químicaRESUMO
Nanocellulose is emerging as a sustainable building block in materials science. Surface modification via polymer grafting has proven to be effective in tuning diverse material properties of nanocellulose, including wettability of films and the reinforcement effect in polymer matrices. Despite its widespread use in various environments, the structure of a single polymer-grafted nanocellulose remains poorly understood. Here, we investigate the morphologies of polymer-grafted CNFs at water-mica and air-mica interfaces by using all-atom molecular dynamics simulation and atomic force microscopy. We show that the morphologies of the polymer-grafted CNFs undergo a marked change in response to the surrounding environment due to variations in the conformation of the surface polymer chains. Our results provide novel insights into the molecular structure of polymer-grafted CNFs and can facilitate the design and development of innovative biomass-based nanomaterials.
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Nanoestruturas , Polímeros , Polímeros/química , Silicatos de Alumínio , Estrutura MolecularRESUMO
Corrosion of Al alloy often starts from the nanoscale corrosion around the surface-exposed Al-Fe intermetallic particles (IMPs) and leads to a serious damage limiting its application range in the automobile industry. To solve this issue, understanding of the nanoscale corrosion mechanism around the IMP is essential, yet it is impeded by the difficulties in directly visualizing nanoscale distribution of reaction activity. Here, this difficulty is overcomed by open-loop electric potential microscopy (OL-EPM) and investigate nanoscale corrosion behavior around the IMPs in H2 SO4 solution. The OL-EPM results reveal that the corrosion around a small IMP settles down in a short time (<30 min) after transient dissolution of the IMP surface while that around a large IMP lasts for a long time especially at its edges and results in a severe damage of the IMP and matrix. This result suggests that an Al alloy with many small IMPs gives a better corrosion resistance than that with few large IMPs if the total Fe content is the same. This difference is confirmed by corrosion weight loss test using Al alloys with different IMP sizes. This finding should give an important guideline to improve the corrosion resistance of Al alloy.
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Alzheimer's disease is associated with the aggregation of the misfolded neuronal peptide, amyloid-ß42 (Aß42). Evidence has suggested that several reasons are responsible for the toxicity caused by the aggregation of Aß42, including the conformational restriction of Aß42. In this study, one of the toxic conformers of Aß42, which contains a Glu-to-Pro substitution (E22P-Aß42), was explored using atomic force microscopy and molecular docking to study the aggregation dynamics. We proposed a systematic model of fibril formation to better understand the molecular basis of conformational transitions in the Aß42 species. Our results demonstrated the formation of amorphous aggregates in E22P-Aß42 that are stem-based, network-like structures, while the formation of mature fibrils occurred in the less toxic conformer of Aß42, E22-Aß42, that are sphere-like flexible structures. A comparison was made between the biophysical properties of E22P-Aß42 and E22-Aß42 that revealed that E22P-Aß42 had greater stiffness, dihedral angle, number of ß sheets involved, and elasticity, compared with E22-Aß42. These findings will have considerable implications toward our understanding of the structural basis of the toxicity caused by conformational diversity in Aß42 species.
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Doença de Alzheimer , Peptídeos beta-Amiloides , Humanos , Peptídeos beta-Amiloides/toxicidade , Peptídeos beta-Amiloides/química , Simulação de Acoplamento Molecular , Microscopia de Força Atômica , Amiloide , Proteínas Amiloidogênicas , Fragmentos de Peptídeos/químicaRESUMO
Although various spectroscopic methods have been developed to capture ion-concentration profile changes, it is still difficult to visualize the ion-concentration profile and surface topographical changes simultaneously during the charging/discharging of lithium-ion batteries (LIBs). To tackle this issue, we have developed an operando scanning ion conductance microscopy (SICM) method that can directly visualize an ion-concentration profile and surface topography using a SICM nanopipette while controlling the sample potential or current with a potentiostat for characterizing the polarization state during charging/discharging. Using operando SICM on the negative electrode (anode) of LIBs, we have characterized ion-concentration profile changes and the reversible volume changes related to the phase transition during cyclic voltammetry (CV) and charge/discharge of the graphite anode. Operando SICM is a versatile technique that is likely to be of major value for evaluating the correlation between the electrolyte concentration profile and nanoscale surface topography changes.
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Nanocellulose is attracting attention in the field of materials science as a sustainable building block. Nanocellulose-based materials, such as films, membranes, and foams, are fabricated by drying colloidal dispersions. However, little is known about how the structure of a single nanocellulose changes during the complex drying process. Here, all-atom molecular dynamics simulations and atomic force microscopy is used to investigate the structural dynamics of single nanocellulose during drying. It is found that the twist morphology of the nanocellulose became localized along the fibril axis during the final stage of the drying process. Moreover, it is shown that conformational changes at C6 hydroxymethyl groups and glycoside bond is accompanied by the twist localization, indicating that the increase in the crystallinity occurred in the process. It is expected that the results will provide molecular insights into nanocellulose structures in material processing, which is helpful for the design of materials with advanced functionalities.
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Numerous studies have investigated the various cellular responses against genotoxic stress, including those mediated by focal adhesions. We here identified a novel type of focal adhesion remodelling that occurs under genotoxic stress conditions, which involves the replacement of active focal adhesion kinase (FAK) with FAK-related non-kinase (FRNK). FRNK stabilized focal adhesions, leading to strong cell-matrix adhesion, and FRNK-depleted cells were easily detached from extracellular matrix upon genotoxic stress. This remodelling occurred in a wide variety of cells. In vivo, the stomachs of Frnk-knockout mice were severely damaged by genotoxic stress, highlighting the protective role of FRNK against genotoxic stress. FRNK was also found to play a vital role in cancer progression, because FRNK depletion significantly inhibited cancer dissemination and progression in a mouse cancer model. Furthermore, in human cancers, FRNK was predominantly expressed in metastatic tissues and not in primary tissues. We hence conclude that this novel type of focal adhesion remodelling reinforces cell adhesion and acts against genotoxic stress, which results in the protection of normal tissues, but in turn facilitates cancer progression.
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Adesões Focais , Neoplasias , Camundongos , Animais , Humanos , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Adesões Focais/metabolismo , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Adesão Celular , Neoplasias/genética , Neoplasias/metabolismo , Movimento Celular/fisiologia , Fosforilação , Células CultivadasRESUMO
In this study, a low-molecular-weight organogelator derived from (l)-amino acids was designed and synthesized. Gelation assays using (l)-amino acid derivatives were performed to confirm the gelation ability, which was found to be high in several compounds. The (l)-alanine derivatives were determined to be excellent gelators, forming good gels even when smaller amounts were added. These results led to a library of amino acid-derived organogelators. In addition, the thermal properties of the (l)-alanine derivatives with high gelation performance were measured. Differential scanning calorimetry measurements revealed that the thermal stability of the gels could be controlled by changing the gelator concentration. The surface states of the obtained gels were observed by field-emission scanning electron microscopy and atomic force microscopy measurements, which confirmed the structure of the self-molecular aggregates. Self-molecular aggregates were observed to be helical or sheet-like, and the gels were constructed by forming aggregates by self-molecular recognition.
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Understanding the mechanisms of self-organization of short peptides into two- and three-dimensional architectures are of great interest in the formation of crystalline biomolecular systems and their practical applications. Since the assembly is a dynamic process, the study of structural development is challenging at the submolecular dimensions continuously across an adequate time scale in the natural biological environment, in addition to the complexities stemming from the labile molecular structures of short peptides. Self-organization of solid binding peptides on surfaces offers prospects to overcome these challenges. Here we use a graphite binding dodecapeptide, GrBP5, and record its self-organization process of the first two layers on highly oriented pyrolytic graphite surface in an aqueous solution by using frequency modulation atomic force microscopy in situ. The observations suggest that the first layer forms homogeneously, generating self-organized crystals with a lattice structure in contact with the underlying graphite. The second layer formation is mostly heterogeneous, triggered by the crystalline defects on the first layer, growing row-by-row establishing nominally diverse biomolecular self-organized structures with transient crystalline phases. The assembly is highly dependent on the peptide concentration, with the nucleation being high in high molecular concentrations, e.g., >100 µM, while the domain size is small, with an increase in the growth rate that gradually slows down. Self-assembled peptide crystals are composed of either singlets or doublets establishing P1 and P2 oblique lattices, respectively, each commensurate with the underlying graphite lattice with chiral crystal relations. This work provides insights into the surface behavior of short peptides on solids and offers quantitative guidance toward elucidating molecular mechanisms of self-assembly helping in the scientific understanding and construction of coherent bio/nano hybrid interfaces.
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Synthesis of highly stable two-dimensional single-layer assemblies (SLAs) is a key challenge in supramolecular science, especially those with long-range molecular order and well-defined morphology. Here, thin (thickness <2â nm) triangular AuI -thiolate SLAs with high thermo-, solvato- and mechano- stability have been synthesized via a double-ligand co-assembly strategy. Furthermore, the SLAs show assembly-level elastic and anisotropic deformation responses to external stimuli as a result of the long-range anisotropic molecular packing, which provides SLAs with new application potentials in bio-mimic nanomechanics.
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We report a case in which sub-stoichiometric binding of an actin-binding protein has profound structural and functional consequences, providing an insight into the fundamental properties of actin regulation. Rng2 is an IQGAP contained in contractile rings in the fission yeast Schizosaccharomyces pombe Here, we used high-speed atomic force microscopy and electron microscopy and found that sub-stoichiometric binding of the calponin-homology actin-binding domain of Rng2 (Rng2CHD) induces global structural changes in skeletal muscle actin filaments, including shortening of the filament helical pitch. Sub-stoichiometric binding of Rng2CHD also reduced the affinity between actin filaments and muscle myosin II carrying ADP and strongly inhibited the motility of actin filaments on myosin II in vitro. On skeletal muscle myosin II-coated surfaces, Rng2CHD stopped the actin movements at a binding ratio of 11%. Rng2CHD also inhibited actin movements on myosin II of the amoeba Dictyostelium, but in this case, by detaching actin filaments from myosin II-coated surfaces. Thus, sparsely bound Rng2CHD induces apparently cooperative structural changes in actin filaments and inhibits force generation by actomyosin II.
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Dictyostelium , Schizosaccharomyces , Actinas/metabolismo , Actomiosina/metabolismo , Dictyostelium/metabolismo , Miosinas de Músculo Esquelético/metabolismo , Miosina Tipo II/metabolismo , Citoesqueleto de Actina/metabolismo , Schizosaccharomyces/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas do Citoesqueleto/metabolismo , Difosfato de Adenosina/metabolismoRESUMO
Stimuli-responsive polyelectrolyte brushes adapt their physico-chemical properties according to pH and ion concentrations of the solution in contact. We synthesized a poly(acrylic acid) bearing cysteine residues at side chains and a lipid head group at the terminal, and incorporated them into a phospholipid monolayer deposited on a hydrophobic silane monolayer. The ion-specific, nanoscale response of polyelectrolyte brushes was detected by using three-dimensional scanning force microscopy (3D-SFM) combined with frequency modulation detection. The obtained topographic and mechanical landscapes indicated that the brushes were uniformly stretched, undergoing a gradual transition from the brush to the bulk electrolyte in the absence of divalent cations. When 1 mM calcium ions were added, the brushes were uniformly compacted, exhibiting a sharper brush-to-bulk transition. Remarkably, the addition of 1 mM cadmium ions made the brush surface significantly rough and the mechanical landscape highly heterogeneous. Currently, cadmium-specific nanoscale compaction of the brushes is attributed to the coordination of thiol and carboxyl side chains with cadmium ions, as suggested for naturally occurring, heavy metal binding proteins.
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Cellulose, a renewable structural biopolymer, is ubiquitous in nature and is the basic reinforcement component of the natural hierarchical structures of living plants, bacteria, and tunicates. However, a detailed picture of the crystalline cellulose surface at the molecular level is still unavailable. Here, using atomic force microscopy (AFM) and molecular dynamics (MD) simulations, we revealed the molecular details of the cellulose chain arrangements on the surfaces of individual cellulose nanocrystals (CNCs) in water. Furthermore, we visualized the three-dimensional (3D) local arrangement of water molecules near the CNC surface using 3D AFM. AFM experiments and MD simulations showed anisotropic water structuring, as determined by the surface topologies and exposed chemical moieties. These findings provide important insights into our understanding of the interfacial interactions between CNCs and water at the molecular level. This may allow the establishment of the structure-property relationship of CNCs extracted from various biomass sources.