Ion-Pair-Enhanced Double-Click Driven Cell Adhesion and Altered Expression of Related Genes.

Bio-orthogonal ligations that crosslink living cells with a substrate or other cells require high stability and rapid kinetics to maintain the nature of target cells. In this study, we report water-soluble cyclooctadiyne (WS-CODY) derivatives that undergo an ion-pair enhanced double-click reaction. The cationic side chain of WS-CODY accelerated the kinetics on the azide-modified cell surface due to proximity effect. Cationic WS-CODY was able to crosslink azide-modified, poorly adherent human lung cancer PC-9 cells not only to azide-grafted glass substrates but also to other cells within 5-30 min. We discovered that cell-substrate crosslinking induced the ITGA5 gene expression, whereas cell-cell crosslinking induced the CTNNA1 gene, according to the adhesion partner. Ion-pair-enhanced WS-CODY can be applied to a wide range of cells with established azide modifications and is expected to provide a powerful tool to regulate cell-substrate and cell-cell interactions.

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.

Reverse engineering liver buds through self-driven condensation and organization towards medical application.

The self-organizing tissue-based approach coupled with induced pluripotent stem (iPS) cell technology is evolving as a promising field for designing organoids in culture and is expected to achieve valuable practical outcomes in regenerative medicine and drug development. Organoids show properties of functional organs and represent an alternative to cell models in conventional two-dimensional differentiation platforms; moreover, organoids can be used to investigate mechanisms of development and disease, drug discovery and toxicity assessment. Towards a more complex and advanced organoid model, it is essential to incorporate multiple cell lineages including developing vessels. Using a self-condensation method, we recently demonstrated self-organizing "organ buds" of diverse systems together with human mesenchymal and endothelial progenitors, proposing a new reverse engineering method to generate a more complex organoid structure. In this section, we review characters of organ bud technology based on two important principles: self-condensation and self-organization focusing on liver bud as an example, and discuss their practicality in regenerative medicine and potential as research tools for developmental biology and drug discovery.

Cytoadhesion of Plasmodium falciparum-infected erythrocytes to chondroitin-4-sulfate is cooperative and shear enhanced.

Infections with the human malaria parasite Plasmodium falciparum during pregnancy can lead to severe complications for both mother and child, resulting from the cytoadhesion of parasitized erythrocytes in the intervillous space of the placenta. Cytoadherence is conferred by the specific interaction of the parasite-encoded adhesin VAR2CSA with chondroitin-4-sulfate (CSA) present on placental proteoglycans. CSA presented elsewhere in the microvasculature does not afford VAR2CSA-mediated cytoadhesion of parasitized erythrocytes. To address the placenta-specific binding tropism, we investigated the effect of the receptor/ligand arrangement on cytoadhesion, using artificial membranes with different CSA spacing intervals. We found that cytoadhesion is strongly dependent on the CSA distance, with half-maximal adhesion occurring at a CSA distance of 9 ± 1 nm at all hydrodynamic conditions. Moreover, binding to CSA was cooperative and shear stress induced. These findings suggest that the CSA density, together with allosteric effects in VAR2CSA, aid in discriminating between different CSA milieus.

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.

Biophysical Approach to Mechanisms of Cancer Prevention and Treatment with Green Tea Catechins.

Green tea catechin and green tea extract are now recognized as non-toxic cancer preventives for humans. We first review our brief historical development of green tea cancer prevention. Based on exciting evidence that green tea catechin, (-)-epigallocatechin gallate (EGCG) in drinking water inhibited lung metastasis of B16 melanoma cells, we and other researchers have studied the inhibitory mechanisms of metastasis with green tea catechins using biomechanical tools, atomic force microscopy (AFM) and microfluidic optical stretcher. Specifically, determination of biophysical properties of cancer cells, low cell stiffness, and high deformability in relation to migration, along with biophysical effects, were studied by treatment with green tea catechins. The study with AFM revealed that low average values of Young's moduli, indicating low cell stiffness, are closely associated with strong potential of cell migration and metastasis for various cancer cells. It is important to note that treatments with EGCG and green tea extract elevated the average values of Young's moduli resulting in increased stiffness (large elasticity) of melanomas and various cancer cells. We discuss here the biophysical basis of multifunctions of green tea catechins and green tea extract leading to beneficial effects for cancer prevention and treatment.

Brief report: reconstruction of joint hyaline cartilage by autologous progenitor cells derived from ear elastic cartilage.

In healthy joints, hyaline cartilage covering the joint surfaces of bones provides cushioning due to its unique mechanical properties. However, because of its limited regenerative capacity, age- and sports-related injuries to this tissue may lead to degenerative arthropathies, prompting researchers to investigate a variety of cell sources. We recently succeeded in isolating human cartilage progenitor cells from ear elastic cartilage. Human cartilage progenitor cells have high chondrogenic and proliferative potential to form elastic cartilage with long-term tissue maintenance. However, it is unknown whether ear-derived cartilage progenitor cells can be used to reconstruct hyaline cartilage, which has different mechanical and histological properties from elastic cartilage. In our efforts to develop foundational technologies for joint hyaline cartilage repair and reconstruction, we conducted this study to obtain an answer to this question. We created an experimental canine model of knee joint cartilage damage, transplanted ear-derived autologous cartilage progenitor cells. The reconstructed cartilage was rich in proteoglycans and showed unique histological characteristics similar to joint hyaline cartilage. In addition, mechanical properties of the reconstructed tissues were higher than those of ear cartilage and equal to those of joint hyaline cartilage. This study suggested that joint hyaline cartilage was reconstructed from ear-derived cartilage progenitor cells. It also demonstrated that ear-derived cartilage progenitor cells, which can be harvested by a minimally invasive method, would be useful for reconstructing joint hyaline cartilage in patients with degenerative arthropathies.

Laser ablation for protein crystal nucleation and seeding.

With the recent development in pulsed lasers with ultrashort pulse widths or wavelengths, spatially precise, low-damage processing by femtosecond or deep-UV laser ablation has shown promise for the production of protein single crystals suitable for X-ray crystallography. Femtosecond laser processing of supersaturated solutions can shorten the protein nucleation period or can induce nucleation at low supersaturation, which improves the crystal quality of various proteins including membrane proteins and supra-complexes. In addition to nucleation, processing of protein crystals by femtosecond or deep-UV laser ablation can produce single crystalline micro- or macro-seeds without deterioration of crystal quality. This tutorial review gives an overview of the successful application of laser ablation techniques to nucleation and seeding for the production of protein single crystals, and also describes the advantages from a physico-chemical perspective.

Mechanical Characterization of Mucus on Intestinal Tissues by Atomic Force Microscopy.

Mucus is part of the innate immune system that defends the mucosa against microbiota and other infectious threats. The mechanical characteristics of mucus, such as viscosity, elasticity, and lubricity, are critically involved in its barrier function. However, assessing the mechanical properties of mucus remains challenging because of technical limitations. Thus, a new approach that characterizes the mechanical properties of mucus on colonic tissues needs to be developed. Here, we describe a novel strategy to characterize the ex vivo mechanical properties of mucus on colonic tissues using atomic force microscopy. This description includes the preparation of the mouse colon sample, AFM calibration, and determining the elasticity (Young's modulus, E [kPa]) of the mucus layer in the colon.

Noninvasive total counting of cultured cells using a home-use scanner with a pattern sheet.

The inherent variability in cell culture techniques hinders their reproducibility. To address this issue, we introduce a comprehensive cell observation device. This new approach enhances the features of existing home-use scanners by implementing a pattern sheet. Compared with fluorescent staining, our method over- or underestimated the cell count by a mere 5%. The proposed technique showcased a strong correlation with conventional methodologies, displaying R2 values of 0.91 and 0.99 compared with the standard chamber and fluorescence methods, respectively. Simulations of microscopic observations indicated the potential to estimate accurately the total cell count using just 20 fields of view. Our proposed cell-counting device offers a straightforward, noninvasive means of measuring the number of cultured cells. By harnessing the power of deep learning, this device ensures data integrity, thereby making it an attractive option for future cell culture research.

Spatiotemporal Control of Polymorphic Phase Transition of Glycine Crystals by Three-Dimensional Femtosecond Laser Ablation Processing.

Spatiotemporal control of the polymorphic phase transition of glycine crystals was demonstrated by three-dimensional (3D) processing with a focused femtosecond laser pulse as an external stimulus. We found that the transition from a metastable form (ß-form) to more stable ones (α- or γ-form) could be triggered from the irradiated area of not only the surface but also inside of glycine crystals. This 3D processing with a focused femtosecond laser pulse enabled us to precisely monitor the transition dynamics from a targeted position to the entire part of crystals. The systematic study with the space-selective phase transition method revealed that the phase transition inside of glycine crystals was significantly slower (e.g., ∼50 times) than that at the crystal surface, which indicates the crucial role of water molecules in air on the phase transition dynamics. We foresee that this laser method can be a practical tool for monitoring spatiotemporal dynamics of phase transition.

Mie-Resonant Nanophotonic-Enhancement of Asymmetry in Sodium Chlorate Chiral Crystallization.

Studies on chiral spectroscopy have recently demonstrated strong enhancement of chiral light-matter interaction in the chiral near-field of Mie resonance in high-refractive-index dielectric nanostructures by studies on chiral spectroscopy. This situation has motivated researchers to demonstrate effective chiral photosynthesis under a chiral near-field beyond circularly polarized light (CPL) as a chiral source. However, the effectivity of the chiral near-field of Mie resonance for chiral photosynthesis has not been clearly demonstrated. One major challenge is the experimental difficulty in evaluating enantiomeric excess of a trace amount of chiral products synthesized in the near-field. Here, by adopting sodium chlorate chiral crystallization as a phenomenon that includes both synthesis and the amplification of chiral products, we show that crystallization on a Mie-resonant silicon metasurface excited by CPL yields a statistically significant large crystal enantiomeric excess of ∼18%, which cannot be achieved merely by CPL. This result provides implications for efficient chiral photosynthesis in a chiral near-field.

The impact of crystal phase transition on the hardness and structure of kidney stones.

Calcium oxalate kidney stones, the most prevalent type of kidney stones, undergo a multi-step process of crystal nucleation, growth, aggregation, and secondary transition. The secondary transition has been rather overlooked, and thus, the effects on the disease and the underlying mechanism remain unclear. Here, we show, by periodic micro-CT images of human kidney stones in an ex vivo incubation experiment, that the growth of porous aggregates of calcium oxalate dihydrate (COD) crystals triggers the hardening of the kidney stones that causes difficulty in lithotripsy of kidney stone disease in the secondary transition. This hardening was caused by the internal nucleation and growth of precise calcium oxalate monohydrate (COM) crystals from isolated urine in which the calcium oxalate concentrations decreased by the growth of COD in closed grain boundaries of COD aggregate kidney stones. Reducing the calcium oxalate concentrations in urine is regarded as a typical approach for avoiding the recurrence. However, our results revealed that the decrease of the concentrations in closed microenvironments conversely promotes the transition of the COD aggregates into hard COM aggregates. We anticipate that the suppression of the secondary transition has the potential to manage the deterioration of kidney stone disease.

Advanced Interferometry with 3-D Structured Illumination Reveals the Surface Fine Structure of Complex Biospecimens.

Interference reflection microscopy (IRM) is a powerful, label-free technique to visualize the surface structure of biospecimens. However, stray light outside a focal plane obscures the surface fine structures beyond the diffraction limit (dxy ≈ 200 nm). Here, we developed an advanced interferometry approach to visualize the surface fine structure of complex biospecimens, ranging from protein assemblies to single cells. Compared to 2-D, our unique 3-D structure illumination introduced to IRM enabled successful visualization of fine structures and the dynamics of protein crystal growth under lateral (dx-y ≈ 110 nm) and axial (dx-z ≤ 5 nm) resolutions and dynamical adhesion of microtubule fiber networks with lateral resolution (dx-y ≈ 120 nm), 10 times greater than unstructured IRM (dx-y ≈ 1000 nm). Simultaneous reflection/fluorescence imaging provides new physical fingerprints for studying complex biospecimens and biological processes such as myogenic differentiation and highlights the potential use of advanced interferometry to study key nanostructures of complex biospecimens.

Cell polarity linked to gravity sensing is generated by LZY translocation from statoliths to the plasma membrane.

Organisms have evolved under gravitational force, and many sense the direction of gravity by means of statoliths in specialized cells. In flowering plants, starch-accumulating plastids, known as amyloplasts, act as statoliths to facilitate downstream gravitropism. The gravity-sensing mechanism has long been considered a mechanosensing process by which amyloplasts transmit forces to intracellular structures, but the molecular mechanism underlying this has not been elucidated. We show here that LAZY1-LIKE (LZY) family proteins involved in statocyte gravity signaling associate with amyloplasts and the proximal plasma membrane. This results in polar localization according to the direction of gravity. We propose a gravity-sensing mechanism by which LZY translocation to the plasma membrane signals the direction of gravity by transmitting information on the position of amyloplasts.

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.

Spatiotemporal Control of Ice Crystallization in Supercooled Water via an Ultrashort Laser Impulse.

Focused irradiation with ultrashort laser pulses realized the fine spatiotemporal control of ice crystallization in supercooled water. An effective multiphoton excitation at the laser focus generated shockwaves and bubbles, which acted as an impulse for inducing ice crystal nucleation. The impulse that was localized close to the laser focus and accompanied by a small temperature elevation allowed the precise position control of ice crystallization and its observation with spatiotemporal resolution of micrometers and microseconds using a microscope. To verify the versatility of this laser method, we also applied it using various aqueous systems (e.g., plant extracts). The systematic study of crystallization probability revealed that laser-induced cavitation bubbles play a crucial role in inducing ice crystal nucleation. This method can be used as a tool for studying ice crystallization dynamics in various natural and biological phenomena.

Preparation of mechanically patterned hydrogels for controlling the self-condensation of cells.

Synthetic protocols providing mechanical patterns to culture substrate are essential to control the self-condensation of cells for organoid engineering. Here, we present a protocol for preparing hydrogels with mechanical patterns. We describe steps for hydrogel synthesis, mechanical evaluation of the substrate, and time-lapse imaging of cell self-organization. This protocol will facilitate the rational design of culture substrates with mechanical patterns for the engineering of various functional organoids. For complete details on the use and execution of this protocol, please refer to Takebe et al. (2015) and Matsuzaki et al. (2014, 2022).1,2,3.

Cell membrane fluidity and ROS resistance define DMSO tolerance of cryopreserved synovial MSCs and HUVECs.

OBJECTIVES: Synovial mesenchymal stem cells (MSCs) have high freeze-thaw tolerance, whereas human umbilical vein endothelial cells (HUVECs) have low freezing tolerance. The differences in cell type-specific freeze-thaw tolerance and the mechanisms involved are unclear. This study thus aimed to identify the biological and physical factors involved in the differences in freeze-thaw tolerance between MSCs and HUVECs. MATERIALS AND METHODS: For biological analysis, MSC and HUVEC viability after freeze-thawing and alteration of gene expression in response to dimethyl sulfoxide (DMSO, a cryoprotectant) were quantitatively evaluated. For physical analysis, the cell membrane fluidity of MSCs and HUVECs before and after DMSO addition was assessed using a histogram for generalized polarization frequency. RESULTS: HUVECs showed lower live cell rates and higher gene expression alteration related to extracellular vesicles in response to DMSO than MSCs. Fluidity measurements revealed that the HUVEC membrane was highly fluidic and sensitive to DMSO compared to that of MSCs. Addition of CAY10566, an inhibitor of stearoyl-coA desaturase (SCD1) that produces highly fluidic desaturated fatty acids, decreased the fluidity of HUVECs and increased their tolerance to DMSO. The combination of CAY10566 and antioxidant glutathione (GSH) treatment improved HUVEC viability from 57 to 69%. Membrane fluidity alteration may thus contribute to pore-induced DMSO influx into the cytoplasm and reactive oxygen species production, leading to greater cytotoxicity in HUVECs, which have low antioxidant capacity. CONCLUSIONS: Differences in freeze-thaw tolerance originate from differences in the cell membranes with respect to fluidity and antioxidant capacity. These findings provide a basis for analyzing cell biology and membrane-physics to establish appropriate long-term preservation methods aimed at promoting transplantation therapies.

Low Surface Potential with Glycoconjugates Determines Insect Cell Adhesion at Room Temperature.

Cell-coupled field-effect transistor (FET) biosensors have attracted considerable attention because of their high sensitivity to biomolecules. The use of insect cells (Sf21) as a core sensor element is advantageous due to their stable adhesion to sensors at room temperature. Although visualization of the insect cell-substrate interface leads to logical amplification of signals, the spatiotemporal processes at the interfaces have not yet been elucidated. We quantitatively monitored the adhesion dynamics of Sf21 using interference reflection microscopy (IRM). Specific adhesion signatures with ring-like patches along the cellular periphery were detected. A combination of zeta potential measurements and lectin staining identified specific glycoconjugates with low electrostatic potentials. The ring-like structures were disrupted after cholesterol depletion, suggesting a raft domain along the cell periphery. Our results indicate dynamic and asymmetric cell adhesion is due to low electrostatic repulsion with fluidic sugar rafts. We envision the logical design of cell-sensor interfaces with an electrical model that accounts for actual adhesion interfaces.