Simultaneous detection of force and tunneling current in electrolyte solution by using qPlus sensor.

We have developed a sensor for simultaneous measurement of atomic force microscopy (AFM) and scanning tunneling microscopy (STM) under liquid environments. The sensor, which is based on the qPlus sensor, is equipped with an insulated conductive tip. Owing to its electrical insulation except for the tip apex, the developed sensor enabled simultaneous detection of tip-sample interaction force and tunneling current, suppressing the Faradaic leakage current. As a fundamental demonstration, we performed simultaneous AFM/STM imaging in an electrolyte solution by using the developed sensor.

Sialylation shapes mucus architecture inhibiting bacterial invasion in the colon.

In the intestine, mucin 2 (Muc2) forms a network structure and prevents bacterial invasion. Glycans are indispensable for Muc2 barrier function. Among various glycosylation patterns of Muc2, sialylation inhibits bacteria-dependent Muc2 degradation. However, the mechanisms by which Muc2 creates the network structure and sialylation prevents mucin degradation remain unknown. Here, by focusing on two glycosyltransferases, St6 N-acetylgalactosaminide α-2,6-sialyltransferase 6 (St6galnac6) and ß-1,3-galactosyltransferase 5 (B3galt5), mediating the generation of desialylated glycans, we show that sialylation forms the network structure of Muc2 by providing negative charge and hydrophilicity. The colonic mucus of mice lacking St6galnac6 and B3galt5 was less sialylated, thinner, and more permeable to microbiota, resulting in high susceptibility to intestinal inflammation. Mice with a B3galt5 mutation associated with inflammatory bowel disease (IBD) also showed the loss of desialylated glycans of mucus and the high susceptibility to intestinal inflammation, suggesting that the reduced sialylation of Muc2 is associated with the pathogenesis of IBD. In mucins of mice with reduced sialylation, negative charge was reduced, the network structure was disturbed, and many bacteria invaded. Thus, sialylation mediates the negative charging of Muc2 and facilitates the formation of the mucin network structure, thereby inhibiting bacterial invasion in the colon to maintain gut homeostasis.

Polar zinc oxide surface in electrolyte solutions: an atomic view of reconstruction, hydration and surface states.

The stabilization mechanism of the Zn-terminated (Zn-) ZnO(0001) surface in electrolyte solutions has been investigated by using atomic-resolution liquid-environment atomic force microscopy (AFM) and an electrochemical method. The electrochemically measured pH dependence of the flat band potential of the Zn-ZnO(0001) surface indicated the adsorption of OH groups onto the (0001) surface in the wide pH range of 1-13. Atomic-scale AFM images of the Zn-ZnO(0001) surface showed a well-ordered hydroxide superstructure in an alkaline solution but a disordered structure in an acidic solution, which is probably attributed to the rapid diffusion of the adsorbed OH groups. Furthermore, the density of the O-terminated step edge on the Zn-ZnO(0001) surface in an acidic solution was higher than that in an alkaline solution. From these findings, we concluded that the excess positive charges of the Zn-ZnO(0001) surface are compensated by the adsorbed OH groups and the O-terminated step edges. In acidic solutions, a higher density of the O-terminated step edge is required for charge compensation. In addition, it was found that potential-dependent reversible surface reconstruction occurs in the local transition area with disordered step orientation by electrochemical AFM. We concluded that the reconstruction compensates the excess surface charges of the local transition area which are induced and varied by potential-dependent local surface states.

Stiffening of Cancer Cell Membranes Is a Key Biophysical Mechanism of Primary and Tertiary Cancer Prevention with Green Tea Polyphenols.

Over the past 30 years, research of green tea polyphenols, especially (-)-epigallocatechin gallate (EGCG), has revealed that consumption of green tea is a practical and effective primary cancer prevention method for the general population. More recently, we believe that green tea polyphenols are beneficial for tertiary cancer prevention using green tea alone or combined with anticancer drugs because EGCG has the potential to inhibit metastatic progression and stemness, and enhance antitumor immunity. In an effort to identify a common underlying mechanism responsible for EGCG's multifunctional effects on various molecular targets, we studied the biophysical effects of EGCG on cell stiffness using atomic force microscopy. We found that EGCG acts to stiffen the membranes of cancer cells, leading to inhibition of signaling pathways of various receptors. Stiffening of membranes with EGCG inhibited AXL receptor tyrosine kinase, a stimulator of cell softening, motility and stemness, and expression of programmed cell death-ligand 1. This review covers the following: i) primary cancer prevention using EGCG or green tea, ii) tertiary cancer prevention by combining EGCG and anticancer drugs, iii) inhibition of metastasis with EGCG by stiffening the cell membrane, iv) inhibition of AXL receptor tyrosine kinase, a stimulator of cell softening and motility, with EGCG, v) inhibition of stemness properties with EGCG, and vi) EGCG as an alternative chemical immune checkpoint inhibitor. Development of new drugs that enhance stiffening of cancer cell membranes may be an effective strategy for tertiary cancer prevention and treatment.

Quantitative evaluation of the impact of artificial cell adhesion via DNA hybridization on E-cadherin-mediated cell adhesion.

Programmable cell adhesion with DNA hybridization is a promising approach for fabricating various tissue architectures without sophisticated instrumentation. However, little is known about how this artificial interaction influences the binding of cell adhesion proteins, E-cadherin. In this work, we designed a planar and fluid lipid membrane displaying E-cadherin and/or single-strand DNA with well-defined densities. Visualization of cells on membranes by fluorescence and interference microscopy revealed cell adhesion to be a two-step process: artificial adhesion by DNA hybridization within a few minutes followed by biological adhesion via cadherin-cadherin binding within hours. Furthermore, we discovered that DNA hybridization can substantially facilitate E-cadherin-mediated cell adhesion. The promotive effect is probably due to the enforced binding between E-cadherin molecules in geometrical confinement between two membranes. Our in vitro model of cell adhesion can potentially be used to design functional synthetic molecules that can regulate cell adhesion via cell adhesion proteins for tissue engineering.

A molecular dynamics study proposing the existence of statistical structural heterogeneity due to chain orientation in the POPC-cholesterol bilayer.

We performed molecular dynamics simulations of a lipid bilayer consisting of POPC and cholesterol at temperatures from 283 to 308K and cholesterol concentrations from 0 to 50% mol/mol. The purpose of this study was to look for the existence of structural differences in the region delimited by these parameters and, in particular, in a region where coexistence of liquid disordered and liquid ordered phases has been proposed. Our interest in this range of concentration and temperature responds to the fact that polyene ionophore activity varies considerably along it. Two force fields, CHARMM36 and Slipids, were compared in order to determine the most suitable. Both force fields predict non-monotonic behaviors consistent with the existence of phase transitions. We found the presence of lateral structural heterogeneity, statistical in nature, in some of the bilayers occurring in this range of temperatures and sterol concentrations. This heterogeneity was produced by correlated ordering of the POPC tails and not due to cholesterol enrichment, and lasts for tens of nanoseconds. We relate these observations to the action of polyenes in these membranes.

Morphology and dynamics of domains in ergosterol or cholesterol containing membranes.

The effect of cholesterol and ergosterol on supported lipid bilayers composed of 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and egg sphingomyelin (eSM) in a 1/1 M ratio was studied using atomic force microscopy. The addition of ergosterol or cholesterol to these membranes considerably modifies both the structure and the dynamics of the domains present in them. The height of the eSM enriched domains increases with concentration of both sterols, but more markedly with ergosterol. The height of the POPC enriched domains increases with concentration in a similar manner for both sterols. This effect is larger for eSM than for POPC when ergosterol, not cholesterol, is present. Domain coverage increases with both sterols at 5 mol% but decreases at 20 mol% and almost disappears at 40 mol%. The size of the eSM enriched domains decreases with sterol concentration, more markedly with cholesterol. Bilayer rupture forces show that overall stiffness increases with the addition of 5 mol% cholesterol, but only for the eSM enriched domains with ergosterol at the same concentration. At larger sterol concentrations the stiffness of both regions becomes reduced. At 40 mol% sterol concentration, both membranes present the same rupture force value. To gain mechanistic insight into these observations we performed Quantum Mechanical calculations and Molecular Dynamics simulations of the sterol molecules. We found that conformational freedom for the sterol molecules is quite different. This difference might be behind the observed phenomena. Finally, the different action of sterols on membrane properties is related to the sterol-dependent ionophoretic activity of polyene antibiotics.

Cell softening in malignant progression of human lung cancer cells by activation of receptor tyrosine kinase AXL.

To study the role of cell softening in malignant progression, Transwell assay and atomic force microscope were used to classify six human non-small cell lung cancer cell lines into two groups: a high motility-low stiffness (HMLS) group and a low motility-high stiffness (LMHS) group. We found a significant role of activity of the AXL receptor tyrosine kinase, which belongs to the TAM (Tyro3, AXL, Mer) family, in the stimulation of motility and cell softening. HMLS cells expressed higher AXL levels than LMHS cells and contained phosphorylated AXL. H1703 LMHS cells transfected with exogenous AXL exhibited increased motility and decreased stiffness, with low levels of actin stress fibre formation. Conversely, the AXL-specific inhibitor R428 and AXL-targeting siRNA reduced motility and increased stiffness in H1299 HMLS cells. Knockdown of AXL stimulated actin stress fibre formation, which inhibited tumour formation in a mouse xenograft model. The Ras/Rac inhibitor SCH 51344, which blocks disruption of actin stress fibres, exerted similar effects to AXL inactivation. We therefore propose that the Ras/Rac pathway operates downstream of AXL. Thus, AXL activation-induced cell softening promotes malignant progression in non-small cell lung cancer and represents a key biophysical property of cancer cells.

Adsorption of galloyl catechin aggregates significantly modulates membrane mechanics in the absence of biochemical cues.

Physical interactions of four major green tea catechin derivatives with cell membrane models were systemically investigated. Catechins with the galloyl moiety caused the aggregation of small unilamellar vesicles and an increase in the surface pressure of lipid monolayers, while those without did not. Differential scanning calorimetry revealed that, in a low concentration regime (≤10 µM), catechin molecules are not significantly incorporated into the hydrophobic core of lipid membranes as substitutional impurities. Partition coefficient measurements revealed that the galloyl moiety of catechin and the cationic quaternary amine of lipids dominate the catechin-membrane interaction, which can be attributed to the combination of electrostatic and cation-π interactions. Finally, we shed light on the mechanical consequence of catechin-membrane interactions using the Fourier-transformation of the membrane fluctuation. Surprisingly, the incubation of cell-sized vesicles with 1 µM galloyl catechins, which is comparable to the level in human blood plasma after green tea consumption, significantly increased the bending stiffness of the membranes by a factor of more than 60, while those without the galloyl moiety had no detectable influence. Atomic force microscopy and circular dichroism spectroscopy suggest that the membrane stiffening is mainly attributed to the adsorption of galloyl catechin aggregates to the membrane surfaces. These results contribute to our understanding of the physical and thus the generic functions of green tea catechins in therapeutics, such as cancer prevention.

A relationship between three-dimensional surface hydration structures and force distribution measured by atomic force microscopy.

Hydration plays important roles in various solid-liquid interfacial phenomena. Very recently, three-dimensional scanning force microscopy (3D-SFM) has been proposed as a tool to visualise solvated surfaces and their hydration structures with lateral and vertical (sub) molecular resolution. However, the relationship between the 3D force map obtained and the equilibrium water density, ρ(r), distribution above the surface remains an open question. Here, we investigate this relationship at an interface of an inorganic mineral, fluorite, and water. The force maps measured in pure water are directly compared to force maps generated using the solvent tip approximation (STA) model and from explicit molecular dynamics simulations. The results show that the simulated STA force map describes the major features of the experimentally obtained force image. The agreement between the STA data and the experiment establishes the correspondence between the water density used as an input to the STA model and the experimental hydration structure and thus provides a tool to bridge the experimental force data and atomistic solvation structures. Further applications of this method should improve the accuracy and reliability of both interpretation of 3D-SFM force maps and atomistic simulations in a wide range of solid-liquid interfacial phenomena.

Quantitative Evaluation of Cancer Cell Adhesion to Self-Assembled Monolayer-Patterned Substrates by Reflection Interference Contrast Microscopy.

Adhesion of cancer cells with different metastatic potential and anticancer drug resistance has been quantitatively evaluated by using self-assembled monolayer (SAM)-patterned substrates and reflection interference contrast microscopy (RICM). Cell-adhesive SAM spots with optimized diameter could prevent cell-cell adhesion and thus allowed the systematic evaluation of statistically reliable numbers of contact area between single cancer cells and substrates by RICM. The statistical image analysis revealed that highly metastatic mouse melanoma cells showed larger contact area than lowly metastatic cells. We also found that both cancer cell types exhibited distinct transition from the "strong" to "weak" adhesion states with increase in the concentration of (-)-epigallocatechin gallate (EGCG), which is known to exhibit cancer preventive activity. Mathematical analysis of the adhesion transition revealed that adhesion of the highly metastatic mouse melanoma cells showed more EGCG tolerance than that of lowly metastatic cells. Moreover, time-lapse RICM observation revealed that EGCG weakened cancer cell adhesion in a stepwise manner, probably via focal adhesion complex. These results clearly indicate that contact area can be used as a quantitative measure for the determination of cancer phenotypes and their drug resistance, which will provide physical insights into the mechanism of cancer metastasis and cancer prevention.

Visualizing Nanoscale Distribution of Corrosion Cells by Open-Loop Electric Potential Microscopy.

Corrosion is a traditional problem but still one of the most serious problems in industry. To reduce the huge economic loss caused by corrosion, tremendous effort has been made to understand, predict and prevent it. Corrosion phenomena are generally explained by the formation of corrosion cells at a metal-electrolyte interface. However, experimental verification of their nanoscale distribution has been a major challenge owing to the lack of a method able to visualize the local potential distribution in an electrolytic solution. In this study, we have investigated the nanoscale corrosion behavior of Cu fine wires and a duplex stainless steel by in situ imaging of local corrosion cells by open-loop electric potential microscopy (OL-EPM). For both materials, potential images obtained by OL-EPM show nanoscale contrasts, where areas of higher and lower potential correspond to anodic areas (i.e., corrosion sites) and cathodic areas, respectively. This imaging capability allows us to investigate the real-time transition of local corrosion sites even when surface structures show little change. This is particularly useful for investigating reactions under surface oxide layers or highly corrosion-resistant materials as demonstrated here. The proposed technique should be applicable to the study of other redox reactions on a battery electrode or a catalytic material. The results presented here open up such future applications of OL-EPM in nanoscale electrochemistry.

Kinesin-Driven Active Substrate Giving Stochastic Mechanical Stimuli to Cells for Characterization.

We present a new platform to give stochastic mechanical stimuli to cells for their characterization. There nano- and micrometer scaled fluctuations are generated by an engineered motor protein system of kinesin-microtubules (MTs) on a solid surface. Cells have abilities to deform in many ways during homeostatic metabolism, tissue forming processes, cancer developments, and so on. Namely, cells in biological tissues are exposed to noise-like stochastic movements at nano- and micrometer-scales, which mainly come from the mechanical environment surrounding the cells. Although cells seem to have the potential to respond to such types of mechanical stimuli, the influences on cellular behaviors are poorly understood. As a first attempt to verify an effect of noise-like mechanical stimuli in vitro, we prepared a system to give stochastic mechanical stimuli to cells using a technique of in vitro motility assay for a kinesin-MT system. An active substrate was obtained by integrating movements of MTs on a kinesin-coated glass surface via cross-linkage, and stochastic mechanical stimuli at the cell-scale were successfully applied to the seeded cells. There, traveling distances of the cells over one cell length were observed until they started to adhere. When metastatic melanoma cells were exposed to the stochastic mechanical stimuli, unusually long protrusions or extensions of cell bodies were observed. Cellular aggregations were also promoted through the movements on this active substrate which could disturb the landing and enhance the collisions of the cells. This approach giving mechanical stimuli to cells in a stochastic manner at nano- and micrometer-scales might allow us to uncover unknown behaviors of cells, which might contribute to research fields requiring our understanding on the mechanical nature of cells, such as cancer diagnosis and regenerative medicine.

Dual frequency open-loop electric potential microscopy for local potential measurements in electrolyte solution with high ionic strength.

Recent development of open-loop electric potential microscopy (OL-EPM) has enabled to measure local potential distribution at a solid/liquid interface. However, the operating environment of OL-EPM has been limited to a weak electrolyte solution (<1 mM). This has significantly limited its application range in biology and chemistry. To overcome this limitation, we have developed dual frequency (DF) mode OL-EPM. In the method, an ac bias voltage consisting of two frequency components at f(1) and f(2) is applied between a tip and sample. The local potential is calculated from the amplitudes of the f(1) and |f(1) - f(2)| components of the electrostatic force. In contrast to the conventional single frequency (SF) mode OL-EPM, the detection of the 2f(1) component is not required in DF mode. Thus, the maximum bias modulation frequency in DF mode is twice as high as that in SF mode. The high bias modulation frequency used in DF mode prevents the generation of electrochemical reactions and redistribution of ions and water, which enables to operate OL-EPM even in a strong electrolyte solution. In this study, we have performed potential measurements of nanoparticles on a graphite surface in 1 and 10 mM NaCl solution. The results demonstrate that DF mode OL-EPM allows measurements of local potential distribution in 10 mM electrolyte solution.

Multifrequency high-speed phase-modulation atomic force microscopy in liquids.

We have developed a new technique, called multifrequency high-speed phase-modulation atomic force microscopy (PM-AFM) in constant-amplitude (CA) mode based on the simultaneous excitation of the first two flexural modes of a cantilever. By performing a theoretical investigation, we have found that this technique enables the simultaneous imaging of the surface topography, energy dissipation and elasticity (nonlinear mapping) of materials. We experimentally demonstrated high-speed imaging at a scan speed of 5 frames/s for a polystyrene (PS) and polyisobutylene (PIB) polymer-blend thin-film surface in water.

Nanoscale potential measurements in liquid by frequency modulation atomic force microscopy.

We have developed a method for local potential measurements in liquid using frequency modulation atomic force microscopy. In this method, local potential is calculated from the first and second harmonic vibrations of a cantilever induced by applying an ac bias voltage between a tip and a sample. The use of an ac bias voltage with a relatively high frequency prevents uncontrolled electrochemical reactions and redistribution of ions and water. The nanoscale resolution of the method is demonstrated by imaging potential distribution of a dodecylamine thin film deposited on a graphite surface in 1 mM NaCl solution.