Publications

Takashi Nishio, Yuko Yoshikawa, Wakao Fukuda, Naoki Umezawa, Tsunehiko Higuchi, Shinsuke Fujiwara, Tadayuki Imanaka and *Kenichi Yoshikawa,
Branched-Chain Polyamine Found in Hyperthermophiles Induces Unique Temperature-Dependent Structural Changes in Genome-Size DNA,
ChemPhysChem 19, 2299-2304 (2018).

[Summary] A pentavalent branched‐chain polyamine, N4‐bis(aminopropyl)spermidine 3(3)(3)4, is a unique polycation found in the hyperthermophilic archaeon Thermococcus kodakarensis, which grows at temperatures between 60 and 100 °C. We studied the effects of this branched‐chain polyamine on DNA structure at different temperatures up to 80 °C. Atomic force microscopic observation revealed that 3(3)(3)4 induces a mesh‐like structure on a large DNA (166 kbp) at 24 °C. With an increase in temperature, DNA molecules tend to unwind, and multiple nano‐loops with a diameter of 10–50 nm are generated along the DNA strand at 80 °C. These results were compared to those obtained with linear‐chain polyamines, homocaldopentamine 3334 and spermidine, the former of which is a structural isomer of 3(3)(3)4. These specific effects are expected to neatly concern with its role on high‐temperature preference in hyperthermophiles.

Hiroki Sakuta, Shunsuke Seo, Shuto Kimura, Marcel Hörning, Koichiro Sadakane, Takahiro Kenmotsu, Motomu Tanaka and *Kenichi Yoshikawa,
Optical Fluid Pump: Generation of Directional Flow via Microphase Segregation/Homogenization,
The Journal of Physical Chemistry Letters 9, 5792-5796 (2018).

[Summary] We report the successful generation of directional liquid-flow under stationary laser irradiation at a fixed position in a chamber. We adopt a homogeneous solution consisting of a mixture of water and triethylamine (TEA), with a composition near the critical point for phase segregation. When geometrical asymmetry is introduced around the laser focus in the chamber, continuous directional flow is generated, accompanied by the emergence of water-rich microdroplets at the laser focus. The emerging microdroplets tend to escape toward the surrounding bulk solution and then merge/annihilate into the homogeneous solution. The essential features of the directional flow are reproduced through a simple numerical simulation using fluid dynamic equations.

Keisuke Danno, Takuto Nakamura, Natsumi Okoso, Naohiko Nakamura, Kohta Iguchi, Yoshiaki Iwadate, Takahiro Kenmotsu, Masaya Ikegawa, Shinji Uemoto and *Kenichi Yoshikawa,
Cracking pattern of tissue slices induced by external extension provides useful diagnostic information,
Scientific Reports 8, 12167/1-6 (2018).

[Summary] Although biopsy is one of the most important methods for diagnosis in diseases, there is ambiguity based on the information obtained from the visual inspection of tissue slices. Here, we studied the effect of external extension on tissue slices from mouse liver with different stages of disease: Healthy normal state, Simple steatosis, Non-alcoholic steatohepatitis and Hepatocellular carcinoma. We found that the cracking pattern of a tissue slice caused by extension can provide useful information for distinguishing among the disease states. Interestingly, slices with Hepatocellular carcinoma showed a fine roughening on the cracking pattern with a characteristic length of the size of cells, which is much different than the cracking pattern for slices with non-cancerous steatosis, for which the cracks were relatively straight. The significant difference in the cracking pattern depending on the disease state is attributable to a difference in the strength of cell-cell adhesion, which would be very weak under carcinosis. As it is well known that the manner of cell-cell adhesion neatly concerns with the symptoms in many diseases, it may be promising to apply the proposed methodology to the diagnosis of other diseases.

Yukinori Nishigami, Takuya Ohmura, Atsushi Taniguchi, Shigenori Nonaka, Junichi Manabe, Takuji Ishikawa, Masatoshi Ichikawa,,
Influence of cellular shape on sliding behavior of ciliates,
Communicative & Integrative Biology 11, e1506666 (2018).

[Summary] Some types of ciliates accumulate on solid/fluid interfaces. This behavior is advantageous to survival in nature due to the presence of sufficient nutrition and stable environments. Recently, the accumulating mechanisms of Tetrahymena pyriformis at the interface were investigated. The synergy of the ellipsoidal shape of the cell body and the mechanosensing feature of the cilia allow for cells to slide on interfaces, and the sliding behavior leads to cell accumulation on the interfaces. Here, to examine the generality of the sliding behavior of ciliates, we characterized the behavior of Paramecium caudatum, which is a commonly studied ciliate. Our experimental and numerical results confirmed that P. caudatum also slid on the solid/fluid interface by using the same mechanism as T. pyriformis. In addition, we evaluated the effects of cellular ellipticity on their behaviors near the wall with a phase diagram produced via numerical simulation.

Jose M. Carnerero, Shinsuke Masuoka, Hikari Baba, Yuko Yoshikawa, Rafael Prado-Gotor and *Kenichi Yoshikawa,
Decorating a Single Giant DNA with Gold Nanoparticles,
RSC Advances 8, 26571-26579 (2018).

[Summary] We decorated a single giant DNA (1.66 × 105 base pairs) with gold nanoparticles through the simple procedure of mild warming, without denaturation of the DNA molecule. Single-molecule observation with fluorescence microscopy revealed that individual decorated DNA molecules stay in the bulk solution by avoiding aggregation and precipitation, and exhibit translational and conformational fluctuation, i.e., Brownian motion. An analysis of the intra-chain fluctuation of single DNA molecules revealed that the apparent spring constant and damping coefficient of a DNA chain increased by ca. 13- and 5-fold, respectively, upon decoration with gold nanoparticles. Observation by transmission electron microscopy revealed that gold nanoparticles were stably attached to the DNA skeleton. UV-visible measurements revealed the absence of any detectable change in surface plasmon resonance, suggesting that the gold nanoparticles assemble without the formation of a densely packed aggregate. CD measurements showed that the secondary structure of decorated DNA is still essentially the B-form.

Naoki Nakatani, Hiroki Sakuta, Masahito Hayashi, Shunsuke Tanaka, Kingo Takiguchi, Kanta Tsumoto and *Kenichi Yoshikawa,
Specific Spatial Localization of Actin and DNA in a Water/Water Microdroplet: Self‐Emergence of a Cell‐Like Structure,
ChemBioChem 19, 1370-1374 (2018).

[Summary] The effect of binary hydrophilic polymers on a pair of representative bio‐macromolecules in a living cell has been examined. The results showed that these bio‐macromolecules exhibited specific localization in cell‐sized droplets that were spontaneously formed through water/water microphase segregation under crowding conditions with coexisting polymers. In these experiments, a simple binary polymer system with poly(ethylene glycol) (PEG) and dextran (DEX) was used. Under the conditions of microphase segregation, DNA was entrapped within cell‐sized droplets rich in DEX. Similarly, F‐actin, linearly polymerized actin, was entrapped specifically within microdroplets rich in DEX, whereas G‐actin, a monomeric actin, was distributed evenly inside and outside these droplets. This study has been extended to a system with both F‐actin and DNA, and it was found that DNA molecules were localized separately from aligned F‐actin proteins to create microdomains inside microdroplets, reflecting the self‐emergence of a cellular morphology similar to a stage of cell division.

Hayato Kikuchi, Keiji Nose, Yuko Yoshikawa and *Kenichi Yoshikawa,
Double-strand breaks in genome-sized DNA caused by mechanical stress under mixing: Quantitative evaluation through single-molecule observation,
Chemical Physics Letters 701, 81-85 (2018).

[Summary] It is becoming increasingly apparent that changes in the higher-order structure of genome-sized DNA molecules of more than several tens kbp play important roles in the self-control of genome activity in living cells. Unfortunately, it has been rather difficult to prepare genome-sized DNA molecules without damage or fragmentation. Here, we evaluated the degree of double-strand breaks (DSBs) caused by mechanical mixing by single-molecule observation with fluorescence microscopy. The results show that DNA breaks are most significant for the first second after the initiation of mechanical agitation. Based on such observation, we propose a novel mixing procedure to significantly decrease DSBs.

Takuya Ohmura, Yukinori Nishigami, Atsushi Taniguchi, Shigenori Nonaka, Junichi Manabe, Takuji Ishikawa, and Masatoshi Ichikawa,
Simple mechanosense and response of cilia motion reveal the intrinsic habits of ciliates,
Proceedings of the National Academy of Sciences 115, 3231-3236 (2018).

[Summary] An important habit of ciliates, namely, their behavioral preference for walls, is revealed through experiments and hydrodynamic simulations. A simple mechanical response of individual ciliary beating (i.e., the beating is stalled by the cilium contacting a wall) can solely determine the slidingmotion of the ciliate along the wall and result in a wall-preferring behavior. Considering ciliate ethology, this mechanosensing system is likely an advantage in the single cell’s ability to locate nutrition. In other words, ciliates can skillfully use both the sliding motion to feed on a surface and the traveling motion in bulk water to locate new surfaces according to the single “swimming” mission.

Tatsuaki Tsuruyama,
Information Thermodynamics of the Cell Signal Transduction as a Szilard Engine,
Entropy 20, 224 (2018).

[Summary] A cell signaling system is in a non-equilibrium state, and it includes multistep biochemical signaling cascades (BSCs), which involve phosphorylation of signaling molecules, such as mitogen-activated protein kinase (MAPK) pathways. In this study, the author considered signal transduction description using information thermodynamic theory. The ideal BSCs can be considered one type of the Szilard engine, and the presumed feedback controller, Maxwell’s demon, can extract the work during signal transduction. In this model, the mutual entropy and chemical potential of the signal molecules can be redefined by the extracted chemical work in a mechanicochemical model, Szilard engine, of BSC. In conclusion, signal transduction is computable using the information thermodynamic method.

Ai Kanemura, Yuko Yoshikawa, Wakao Fukuda, Kanta Tsumoto, Takahiro Kenmotsu and *Kenichi Yoshikawa,
Opposite effect of polyamines on In vitro gene expression: Enhancement at low concentrations but inhibition at high concentrations,
PLOS ONE 13, 1-11 (2018).

[Summary] ［Background］Polyamines have various biological functions including marked effects on the structure and function of genomic DNA molecules. Changes in the higher-order structure of DNA caused by polyamines are expected to be closely related to genetic activity. To clarify this issue, we examined the relationship between gene expression and the higher-order structure of DNA under different polyamine concentrations.［Principal findings］We studied the effects of polyamines, spermidine SPD(3+) and spermine SP(4+), on gene expression by a luciferase assay. The results showed that gene expression is increased by ca. 5-fold by the addition of SPD(3+) at 0.3 mM, whereas it is completely inhibited above 2 mM. Similarly, with SP(4+), gene expression is maximized at 0.08 mM and completely inhibited above 0.6 mM. We also performed atomic force microscopy (AFM) observations on DNA under different polyamine concentrations. AFM revealed that a flower-like conformation is generated at polyamine concentrations associated with maximum expression as measured by the luciferase assay. On the other hand, DNA molecules exhibit a folded compact conformation at polyamine concentrations associated with the complete inhibition of expression. Based on these results, we discuss the plausible mechanism of the opposite effect, i.e., enhancement and inhibition, of polyamines on gene expression.［Conclusion and significance］It was found that polyamines exert opposite effect, enhancement and inhibition, on gene expression depending on their concentrations. Such an opposite effect is argued in relation to the conformational change of DNA: enhancement is due to the parallel ordering of DNA segments that is accompanied by a decrease in the negative charge of double-stranded DNA, and inhibition is caused by the compaction of DNA into a tightly packed state with almost perfect charge-neutralization.

*Tatsuaki Tsuruyama,
Information Thermodynamics Derives the EntropyCurrent of Cell Signal Transduction as a Model of aBinary Coding System,
Entropy 20, 145 (2018).

[Summary] The analysis of cellular signaling cascades based on information thermodynamics has recently developed considerably. A signaling cascade may be considered a binary code system consisting of two types of signaling molecules that carry biological information, phosphorylated active, and non-phosphorylated inactive forms. This study aims to evaluate the signal transduction step in cascades from the viewpoint of changes in mixing entropy. An increase in active forms may induce biological signal transduction through a mixing entropy change, which induces a chemical potential current in the signaling cascade. We applied the fluctuation theorem to calculate the chemical potential current and found that the average entropy production current is independent of the step in the whole cascade. As a result, the entropy current carrying signal transduction is defined by the entropy current mobility.

Yugo Harada, Keisuke Koyoshi, Hiroki Sakuta, Koichiro Sadakane,Takahiro Kenmotsu, and *Kenichi Yoshikawa,
Emergence of Pendular and Rotary Motions of a Centimeter-SizedMetallic Sheet under Stationary Photoirradiation,
The Journal of Physical Chemisty C 122, 2747-2752 (2018).

[Summary] We report that both rhythmic pendular motion and rotary motion are generated under stationary irradiation by a green laser for a centimeter-sized metallic sheet floating on an aqueous solution. For a hammer-shaped aluminum sheet, regular pendular motion is caused by CW laser irradiation when the “handle” of the pendulum is in contact with the wall of the glass containing vessel. This rhythmic pendular motion occurs as on/off switching from a stationary state with an increase in laser power. We discuss the mechanism of such stable pendular motion in terms of limit-cycle oscillation with the aid of phenomenological coupled differential equations, by incorporating the effects of a decrease in interfacial tension with an increase in temperature under laser absorption and of the dissipation of heat into the environment. Stable rotary motion of the metallic sheet was also generated, driven by stationary laser irradiation. The chirality of the rotary motion, either clockwise or anticlockwise, for the metallic object could be selected through the introduction of chiral symmetry breaking in its morphology.

Aya Miyagawa-Hayashino,corresponding, Hajime Yoshifuji, Koji Kitagori, Shinji Ito, Takuma Oku, Yoshitaka Hirayama, Adeeb Salah,Toshiki Nakajima, Kaori Kiso, Norishige Yamada, Hironori Haga, and *Tatsuaki Tsuruyama,
Increase of MZB1 in B cells in systemic lupus erythematosus: proteomic analysis of biopsied lymph nodes.,
Arthritis Research & Therapy 20, 13 (2018).

[Summary] Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease in which dysregulation of B cells has been recognized. B-cell subsets from NZB/W F1 mice, which exhibit autoimmune disease resembling human SLE, were analyzed by flow cytometry. Endoplasmic reticulum (ER) stress was induced by tunicamycin and the serum concentration of anti-dsDNA antibodies was determined by ELISA. MZB1, which comprises part of a B-cell-specific ER chaperone complex and is a key player in antibody secretion, was one of the differentially expressed proteins identified by LC-MS and confirmed by immunoblotting. Immunohistochemically, larger numbers of MZB1+ cells were located mainly in interfollicular areas and scattered in germinal centers in specimens from SLE patients compared with those from controls. MZB1 colocalized with CD138+ plasma cells and IRTA1+ marginal zone B cells. MZB1mRNA was increased by 2.1-fold in B cells of SLE patients with active disease (SLE Disease Activity Index 2000 ≥ 6) compared with controls. In aged NZB/W F1 mice, splenic marginal zone B cells and plasma cells showed elevated MZB1 levels. Tunicamycin induced apoptosis of MZB1+ cells in target organs, resulting in decreased serum anti-dsDNA antibody levels. Additionally, MZB1+ cells were increased in synovial tissue specimens from patients with rheumatoid arthritis.

Salomé Mielke, Taichi Habe, Mariam Veschgini, Xianhe Liu, Kenichi Yoshikawa, Marie Pierre Krafft and Motomu Tanaka,
Emergence of Strong Nonlinear Viscoelastic Response of Semifluorinated Alkane Monolayers,
Langmuir 34, 2489−2496 (2018).

[Summary] Viscoelasticity of monolayers of fluorocarbon/hydrocarbon tetrablock amphiphiles di(FnHm) ((CnF2n+1CH2)(Cm–2H2m–3)CH–CH(CnF2n+1CH2)(Cm–2H2m–3)) was characterized by interfacial dilational rheology under periodic oscillation of the moving barriers at the air/water interface. Because the frequency dispersion of the response function indicated that di(FnHm) form two-dimensional gels at the interface, the viscosity and elasticity of di(FnHm) were first analyzed with the classical Kelvin–Voigt model. However, the global shape of stress response functions clearly indicated the emergence of a nonlinearity even at very low surface pressures (π ≈ 5 mN/m) and small strain amplitudes (u0 = 1%). The Fourier-transformed response function of higher harmonics exhibited a clear increase in the intensity only from odd modes, corresponding to the nonlinear elastic component under reflection because of mirror symmetry. The emergence of strong nonlinear viscoelasticity of di(FnHm) at low surface pressures and strain amplitudes is highly unique compared to the nonlinear viscoelasticity of other surfactant systems reported previously, suggesting a large potential of such fluorocarbon/hydrocarbon molecules to modulate the mechanics of interfaces using the self-assembled domains of small molecules.

Satoshi Takatori, Hikari Baba, Takatoshi Ichino, Chwen-Yang Shew and Kenichi Yoshikawa,
Cooperative standing-horizontal-standing reentrant transition for numerous solid particles under external vibration,
Scientific Reports 8(437), 1-11 (2018).

[Summary] We report the collective behavior of numerous plastic bolt-like particles exhibiting one of two distinctstates, either standing stationary or horizontal accompanied by tumbling motion, when placed on ahorizontal plate undergoing sinusoidal vertical vibration. Experimentally, we prepared an initial state inwhich all of the particles were standing except for a single particle that was placed at the center of theplate. Under continuous vertical vibration, the initially horizontal particle triggers neighboring particlesto fall over into a horizontal state through tumbling-induced collision, and this effect gradually spreadsto all of the particles, i.e., the number of horizontal particles is increased. Interestingly, within a certainrange of vibration intensity, almost all of the horizontal particles revert back to standing in associationwith the formation of apparent 2D hexagonal dense-packing. Thus, phase segregation between highand low densities, or crystalline and disperse domains, of standing particles is generated as a result ofthe reentrant transition. The essential features of such cooperative dynamics through the reentranttransition are elucidated with a simple kinetic model. We also demonstrate that an excitable wave withthe reentrant transition is observed when particles are situated in a quasi-one-dimensional confinementon a vibrating plate.

*Tatsuaki Tsuruyama,
Channel capacity of coding system on Tsallis entropy and q-Statistics,
Entropy 19, 682 (2017).

[Summary] The field of information science has greatly developed, and applications in various fields　have emerged. In this paper, we evaluated the coding system in the theory of Tsallis entropy for transmission of messages and aimed to formulate the channel capacity by maximization of the Tsallis entropy within a given condition of code length. As a result, we obtained a simple relational expression between code length and code appearance probability and, additionally, a generalized formula of the channel capacity on the basis of Tsallis entropy statistics. This theoretical frameworkmay contribute to data processing techniques and other applications.

Chwen-Yang Shew, Soutaro Oda and *Kenichi Yoshikawa,
Localization switching of a large object in a crowded cavity: A rigid/soft object prefers surface/inner positioning,
The Journal of Chemical Physics 149, 204901/1-11 (2017).

[Summary] For living cells in the real world, a large organelle is commonly positioned in the inner region away from membranes, such as the nucleus of eukaryotic cells, the nucleolus of nuclei, mitochondria, chloroplast, Golgi body, etc. It contradicts the expectation by the current depletion-force theory in that the larger particle should be excluded from the inner cell space onto cell boundaries in a crowding media. Here we simply model a sizable organelle as a soft-boundary large particle allowing crowders, which are smaller hard spheres in the model, to intrude across its boundary. The results of Monte Carlo simulation indicate that the preferential location of the larger particle switches from the periphery into the inner region of the cavity by increasing its softness. An integral equation theory is further developed to account for the structural features of the model, and the theoretical predictions are found consistent with our simulation results.

Yongjun Chen, Koichiro Sadakane, Hiroki Sakuta, Chenggui Yao and *Kenichi Yoshikawa,
Spontaneous Oscillations and Synchronization of Active Droplets on a Water Surface via Marangoni Convection,
Langmuir 33, 12362-12368 (2017).

[Summary] Shape-oscillations and synchronization are intriguing phenomena in many biological and physical systems. Here, we report the rhythmic mechanical oscillations and synchronization of aniline oil droplets on a water phase, which is induced by Marangoni convection during transfer of the solute. The repetitive increase and decrease in the surface concentration in the vicinity of the contact line leads to the oscillations of droplets through an imbalance in surface tensions. The nature of the oscillations depends on the diameter of the droplet, the depth of the bulk aqueous phase, and the concentration of the aqueous phase. A numerical simulation reproduces the essential behaviors of active oscillations of a droplet. Droplets sense each other through a surface tension gradient and advection, and hydrodynamic coupling in the bulk solution induces the synchronization of droplet oscillations.

Kaori Kiso, Hajime Yoshifuji, Takuma Oku, Masaki Hikida, Koji Kitagori, Yoshitaka Hirayama, Toshiki Nakajima, Hironori Haga, Tatsuaki Tsuruyama, *Aya Miyagawa-Hayashino,
Transgelin-2 is upregulated on activated B-cells and expressed in hyperplastic follicles in lupus erythematosus patients.,
PLoS One 12, 9 (2017).

[Summary] Transgelin-2 (TAGLN2) is an actin-binding protein that controls actin stability and promotes T cell activation. We found that TAGLN2-expressing B-cells were localized in the germinal center (GC) and TAGLN2 mRNA was significantly upregulated after IgM+IgG stimulation in human B-cells, suggesting that TAGLN2 was upregulated upon B-cell activation. Lymph nodes (LNs) from patients with systemic lupuserythematosus (SLE), in which the intense GC activity have been recognized, showed increased TAGLN2 expression in B-cells compared to control LNs. Moreover, TAGLN2+B-cells were distributed widely not only in the GC but also in the perifollicular areas in SLE LNs. CD19+ B-cells and CD19+CD27+ memory-B cells in peripheral blood of SLE patients showed no increase in TAGLN2 mRNA. TAGLN2 colocalized with F-actin and moved together to the periphery upon stimulation. TAGLN2-knockdown in Raji cells resulted in impaired phosphorylation of PLCγ2 leading to inhibition of cell migration. Microarray analysis of TAGLN2-knockdown Raji cells showed decreased expression of the genes associated with immune function including CCR6 and as well as of those associated with regulation of the actin cytoskeleton including ABI2. These results suggest that TAGLN2 might regulate activation and migration of B-cells, in particular, the entry of activated B-cells into the follicle.

Kazusa Beppu, Ziane Izri, Jun Gohya, Kanta Eto, Masatoshi Ichikawa, *Yusuke T. Maeda,
Geometry-driven collective ordering of bacterial vortices,
Soft Matter 13, 5038-5043 (2017).

[Summary] Controlling the phases of matter is a challenge that spans from condensed materials to biological systems. Here, by imposing a geometric boundary condition, we study the controlled collective motion of Escherichia coli bacteria. A circular microwell isolates a rectified vortex from disordered vortices masked in the bulk. For a doublet of microwells, two vortices emerge but their spinning directions show transition from parallel to anti-parallel. A Vicsek-like model for confined self-propelled particles gives the point where the two spinning patterns occur in equal probability and one geometric quantity governs the transition as seen in experiments. This mechanism shapes rich patterns including chiral configurations in a quadruplet of microwells, thus revealing a design principle of active vortices.

Hiroaki Ito, Masahiro Makuta, Yukinori Nishigami, and *Masatoshi Ichikawa,
Active materials integrated with actomyosin,
Journal of the Physical Society of Japan 86, 101001/1-6 (2017).

[Summary] Muscles are the engine of our body, and actomyosin is the engine of a cell. Both muscle and the actomyosin use thesame proteins, namely, actin, and myosin, which are the pair of cytoskeleton and motor proteins generating a force torealize deformation. The properties of force generation by actomyosin at a single-molecule level have been studied formany years. Moreover, the active properties of higher-order structures integrated by actomyosin are attracting theattention of researchers. Here, we review the recent progress in the study of reconstituted actomyosin systems in vitrotoward real-space models of nonequilibrium systems, collective motion, biological phenomena, and active materials.

Masami Noda, Yue Ma, Yuko Yoshikawa, Tadayuki Imanaka, Toshiaki Mori, Masakazu Furuta, Tatsuaki Tsuruyama and Kenichi Yoshikawa,
A single-molecule assessment of the protective effect of DMSO against DNA double-strand breaks induced by photo-and g-ray-irradiation, and freezing,
Scientific Reports 7, 8557/ 1-8 (2017).

[Summary] Dimethyl sulfoxide (DMSO) is widely used as a cryoprotectant for organs, tissues, and cell suspension in storage. In addition, DMSO is known to be a useful free radical scavenger and a radio-protectant. To date, many in vitro assays using cultured cells have been performed for analysing the protective effect of DMSO against genomic DNA damage; however, currently it has been rather difficult to detect DNA double strand breaks (DSBs) in a quantitative manner. In the present study, we aimed to observe the extent of DNA damage by use of single molecular observation with a fluorescence microscope to evaluate DSBs induced by photo- and γ-ray-irradiation, or freeze/thawing in variable concentrations of DMSO. As a result, we found that 2% DMSO conferred the maximum protective effect against all of the injury sources tested, and these effects were maintained at higher concentrations. Further, DMSO showed a significantly higher protective effect against freezing-induced damage than against photo- and γ-ray-irradiation-induced damage. Our study provides significant data for the optimization of DNA cryopreservation with DMSO, as well as for the usage of DNA as the protective agent against the injuries caused by active oxygen and radiations.

Marcel Hörning, François Blanchard, Akihiro Isomura, andKenichi Yoshikawa,
Dynamics of spatiotemporal line defects and chaos control in complex excitable systems,
Scientific Reports 7, 7757/1-9 (2017).

[Summary] Spatiotemporal pattern formation governs dynamics and functions in various biological systems. In the heart, excitable waves can form complex oscillatory and chaotic patterns even at an abnormally higher frequency than normal heart beats, which increase the risk of fatal heart conditions by inhibiting normal blood circulation. Previous studies suggested that line defects (nodal lines) play a critical role in stabilizing those undesirable patterns. However, it remains unknown if the line defects are static or dynamically changing structures in heart tissue. Through in vitro experiments of heart tissue observation, we reveal the spatiotemporal dynamics of line defects in rotating spiral waves. We combined a novel signaling over-sampling technique with a multi-dimensional Fourier analysis, showing that line defects can translate, merge, collapse and form stable singularities with even and odd parity while maintaining a stable oscillation of the spiral wave in the tissue. These findings provide insights into a broad class of complex periodic systems, with particular impact to the control and understanding of heart diseases.

Aoi Yoshida, Shoto Tsuji, Hiroaki Taniguchi, Takahiro Kenmotsu, Koichiro Sadakane, *Kenichi Yoshikawa,
Manipulating Living Cells to Construct a 3D Single-Cell Assembly without an Artificial Scaffold,
Polymers 9, 319/2-10 (2017).

[Summary] Artificial scaffolds such as synthetic gels or chemically-modified glass surfaces that have often been used to achieve cell adhesion are xenobiotic and may harm cells. To enhance the value of cell studies in the fields of regenerative medicine and tissue engineering, it is becoming increasingly important to create a cell-friendly technique to promote cell-cell contact. In the present study, we developed a novel method for constructing stable cellular assemblies by using optical tweezers in a solution of a natural hydrophilic polymer, dextran. In this method, a target cell is transferred to another target cell to make cell-cell contact by optical tweezers in a culture medium containing dextran. When originally non-cohesive cells are held in contact with each other for a few minutes under laser trapping, stable cell-cell adhesion is accomplished. This method for creating cellular assemblies in the presence of a natural hydrophilic polymer may serve as a novel next-generation 3D single-cell assembly system with future applications in the growing field of regenerative medicine.

Daigo Yamamoto, Ryota Yamamoto, Takahiro Kozaki, Akihisa Shioi, Syuji Fujii and *Kenichi Yoshikawa,
Periodic Motions of Solid particles with Various Morphology under a DC Electrostatic Field,
Chemistry Letters 46, 1470-1472 (2017).

[Summary] This paper describes the generation of periodic motions of solid particle in an oil phase under a direct current (DC) voltage. We found that a dimer and trimer composed of spherical polystyrene (PS) particles exhibit a novel periodic motion, spin. These particles maintain their stable motion without any support from mechanical devices such as rotational axes or electronic switching devices. We expect that the simple DC micromotion is applicable for mechanical and fluidic devices employing microrobots and microfluidics.

Keisuke Mae, Hidetoshi Toyama, Erika Okita Nawa, Daigo Yamamoto, Akihisa Shioi, Yongjun Chen, *Kenichi Yoshikawa, Fumiyuki Toshimitsu, Naotoshi Nakashima and Kazunari Matsuda,
Self-Organized Micro-Spiral of Single-Walled Carbon Nanotubes,
Scientific Reports 7, 5267/1-12 (2017).

[Summary] Single-walled carbon nanotubes (SWCNTs) are reported to spontaneously align in a rotational pattern by drying a liquid droplet of toluene containing polyfluorene as a dispersant. By situating a droplet of an SWCNT solution around a glass bead, spiral patterns are generated. The parallel alignment of SWCNTs along one stripe of such a pattern is confirmed using scanning electron microscopy and polarized optical microscopy. The orientation order increases toward the outer edge of a stripe. The stripe width in the pattern is proportional to the solute concentration, and the width and position of the stripes follow geometric sequences. The growth of the rotational pattern is also observed in real time. The process of spiral pattern formation is visualized, indicating the role of the annihilation of counter-traveling accompanied by continuous depinning. The geometric sequences for the stripe width and position are explained by the near-constant traveling speed and solute enrichment at the droplet periphery.

Yuta Shimizu, Yuko Yoshikawa, Takahiro Kenmotsu, Seiji Komeda and *Kenichi Yoshikawa,
Conformational transition of DNA by dinuclear Pt(II) complexes causes cooperative inhibition of gene expression,
Chemical Physics Letters 678, 123-129 (2017).

[Summary] Recently, it was reported that a cationic tetrazolato-bridged dinuclear Pt(II) complex, 5-H-Y, is a promising anticancer drug candidate. Here, we investigated the effects of a series of tetrazolato-bridged dinuclear Pt(II) complexes on the higher-order structure of DNA by using fluorescence and atomic force microscopies. The results showed that these dinuclear Pt(II) complexes cause marked shrinkage on the conformation of genomic DNA. We also found highly cooperative inhibitory effects of these drugs on in vitro gene expression. The unique mechanism of action of these dinuclear Pt(II) complexes is discussed in terms of their bridging effect on DNA segments.

Adeeb Salah, Hajime Yoshifuji, Shinji Ito, Koji Kitagori, Kaori Kiso, Norishige Yamada, Toshiki Nakajima, Hironori Haga, Tatsuaki Tsuruyama, and *Aya Miyagawa-Hayashino,
High Expression of Galectin-3 in Patients with IgG4-Related Disease: A Proteomic Approach.,
Pathology Research International 2017, 9312142 (2017).

[Summary] Immunoglobulin G4-related disease (IgG4-RD) is a multiorgan condition manifesting itself in different forms. This study aimed to investigate protein expression profiles and to find the possible biomarker for IgG4-RD by liquid chromatography mass spectrometry (LC-MS) using tissue sections in IgG4-RD patients.Protein expression profiles in five IgG4-related pancreatitis and three normal pancreatic samples were compared using LC-MS and were validated by quantitative real-time PCR (qRT-PCR), immunoblotting, and immunohistochemistry. ELISA was employed in the serum of 20 patients with systemic IgG4-RD before and during steroid treatment.LC-MS indicated that the levels of 17 proteins were significantly higher and 12 others were significantly lower in IgG4-related pancreatitis patients compared to controls. Among these proteins, galectin-3 levels were 13-fold higher in IgG4-related pancreatitis (P < 0.01). These results were confirmed by immunoblotting and qRT-PCR. The average number of galectin-3 + cells in various organs of IgG4-RD patients, including salivary glands, lungs, and lymph nodes, was higher than in controls. Serum galectin-3 levels were higher in patients with IgG4-RD compared with healthy donors and remained high during steroid therapy.Galectin-3 was overexpressed in IgG4-RD and the levels were indirectly related to clinical activity.

Kanta Tsumoto and *Kenichi Yoshikawa,
The Aqueous Two Phase System (ATPS) Deserves Plausible Real-World Modeling for the Structure and Function of Living Cells,
MRS Advances 2, 2407-2413 (2017).

[Summary] An aqueous two phase system (ATPS) is composed of binary hydrophilic polymers, for example, polyethylene glycol (PEG) and dextran, under an immiscible condition, and can also exhibit micro-segregation to produce cell-sized microcompartments like water-in-water microdroplets. Without membranes, interestingly, the microdroplet can serve as a micro-vessel (reactor) that contains various biochemical macromolecules like DNAs and proteins. We here present that PEG/dextran ATPS micro-segregation can provide an effective soft boundary to separate these biochemical macromolecules from the external environment. Trapped DNAs and proteins were concentrated inside such small spaces, and therefore, their interaction could be highly promoted to cause passive aggregation and controlled cross-linking if a certain cross-linker was added. We believe that the ATPS microdroplets might be associated with complicated structures and functions of living cells.

*Tomo Kurimura, Yoshiko Takenaka, Satoru Kidoaki, and *Masatoshi Ichikawa,
Fabrication of gold microwires by drying goldnanorods suspensions,
Advanced Materials Interfaces 1601125, 1-5 (2017).

[Summary] The ramification pattern of gold nanorods is fabricated by drying its suspensionbetween two glass slides. The aspect ratio of the nanorods and the pinningon the contact line among air, water, and substrate are important. Afterbeing baked, this pattern also conducts electricity. The method of patterningis useful for microwiring without the additional need to pattern the wires intospecific shapes.

Rinko Kubota, Yusuke Yamashita, Takahiro Kenmotsu, Yuko Yoshikawa, Kenji Yoshida, Yoshiaki Watanabe, Tadayuki Imanaka and *Kenichi Yoshikawa,
Double-Strand Breaks in Genome-Sized DNA Caused by Ultrasound,
ChemPhysChem 18, 959-964 (2017).

[Summary] DNA double-strand breaks (DSBs) caused by ultrasound were evaluated in a quantitative manner by single-molecule fluorescence microscopy. We compared the effect of time-interval (or pulse) sonication to that of continuous wave (CW) sonication at a fixed frequency of 30 kHz. Pulses caused fewer DSBs than CW sonication under the same total input ultrasound energy when the pulse repetition period was above the order of a second. In contrast, pulses caused more DSBs than CW sonication for pulse widths shorter than a second. These effect of ultrasound on DNA were interpreted in terms of the time-dependent decay in the probability of breakage during the duration of a pulse. We propose a simple phenomenological model by considering a characteristic decay in the probability of DSBs during single-pulse sonication, which reproduces the essence of the experimental trend. In addition, a data analysis revealed a characteristic scaling behavior between the number of pulses and the number of DSBs.

Tatsuaki Tsuruyama,
Kinetic Stability Analysis of Protein Assembly on the Center Manifold around the Critical Point.,
BMC Systems Biology 11 (2017).

[Summary] Background: Non-linear kinetic analysis is a useful method for illustration of the dynamic behavior of cellular biological systems. To date, center manifold theory (CMT) has not been sufficiently applied for stability analysis of biological systems. The aim of this study is to demonstrate the application of CMT to kinetic analysis of protein assembly and disassembly, and to propose a novel framework for nonlinear multi-parametric analysis. We propose a protein assembly model with nonlinear kinetics provided by the fluctuation in monomer concentrations during their diffusion. Results: When the diffusion process of a monomer is self-limited to give kinetics non-linearity, numerical simulations suggest the probability that the assembly and disassembly oscillate near the critical point. We applied CMT to kinetic analysis of the center manifold around the critical point in detail, and successfully demonstrated bifurcation around the critical point, which explained the observed oscillation.Conclusions: The stability kinetics of the present model based on CMT illustrates a unique feature of protein assembly, namely non-linear behavior. Our findings are expected to provide methodology for analysis of biological systems.

Seiji Komeda, Hiroki Yoneyama, Masako Uemura, Akira Muramatsu, Wakao Fukuda, Tadayuki Imanaka, Toshio Kanbe, Yuko Yoshikawa and *Kenichi Yoshikawa,
Specific Conformational Change in Giant DNA Caused by Anticancer Tetrazolato-Bridged Dinuclear Platinum(II) Complexes: Middle-Length Alkyl Substituents Exhibit Minimum Effect,
Inorganic Chemistry 56, 802–811 (2017).

[Summary] Derivatives of the highly antitumor-active compound [{cis-Pt(NH3)2}2(μ-OH)(μ-tetrazolato-N2,N3)]2+ (5-H-Y), which is a tetrazolato-bridged dinuclear platinum(II) complex, were prepared by substituting a linear alkyl chain moiety at C5 of the tetrazolate ring. The general formula for the derivatives is [{cis-Pt(NH3)2}2(μ-OH)(μ-5-R-tetrazolato-N2,N3)]2+, where R is (CH2)nCH3 and n = 0 to 8 (complexes 1–9). The cytotoxicity of complexes 1–4 in NCI-H460 human non-small-cell lung cancer cells decreased with increasing alkyl chain length, and those of complexes 5–9 increased with increasing alkyl chain length. That is, the in vitro cytotoxicity of complexes 1–9 was found to have a U-shaped association with alkyl chain length. This U-shaped association is attributable to the degree of intracellular accumulation. Although circular dichroism spectroscopic measurement indicated that complexes 1–9 induced comparable conformational changes in the secondary structure of DNA, the tetrazolato-bridged complexes induced different degrees of DNA compaction as revealed by a single DNA measurement with fluorescence microsopy, which also had a U-shaped association with alkyl chain length that matched the association observed for cytotoxicity. Complexes 7–9, which had alkyl chains long enough to confer surfactant-like properties to the complex, induced DNA compaction 20 or 1000 times more efficiently than 5-H-Y or spermidine. A single DNA measurement with transmission electron microscopy revealed that complex 8 formed large spherical self-assembled structures that induced DNA compaction with extremely high efficiency. This result suggests that these structures may play a role in the DNA compaction that was induced by the complexes with the longer alkyl chains. The derivatization with a linear alkyl chain produced a series of complexes with unique cellular accumulation and DNA conformational change profiles and a potentially useful means of developing next-generation platinum-based anticancer drugs. In addition, the markedly high ability of these complexes to induce DNA compaction and their high intracellular accumulation emphasized the difference in mechanism of action from platinum-based anticancer drugs

Masa Tsuchiya, Alessandro Giuliani, Midori Hashimoto, Jekaterina Erenpreisa and *Kenichi Yoshikawa,
Self-Organizing Global Gene Expression Regulated through Criticality: Mechanism of the Cell-Fate Change,
Plos One, 1-47 (2016).

Akira Muramatsu, Yuta Shimizu, Yuko Yoshikawa, Wakao Fukuda, Naoki Umezawa, Yuhei Horai, Tsunehiko Higuchi, Shinsuke Fujiwara, Tadayuki Imanaka and *Kenichi Yoshikawa,
Naturally occurring branched-chain polyamines induce a crosslinked meshwork structure in a giant DNA,
The Journal of Chemical Physics 145, 235103/1-7 (2016).

[Summary] We studied the effect of branched-chain polyamines on the folding transition of genome-sized DNA molecules in aqueous solution by the use of single-molecule observation with fluorescence microcopy. Detailed morphological features of polyamine/DNA complexes were characterized by atomic force microscopy (AFM). The AFM observations indicated that branched-chain polyamines tend to induce a characteristic change in the higher-order structure of DNA by forming bridges or crosslinks between the segments of a DNA molecule. In contrast, natural linear-chain polyamines cause a parallel alignment between DNA segments. Circular dichroism measurements revealed that branched-chain polyamines induce the A-form in the secondary structure of DNA, while linear-chain polyamines have only a minimum effect. This large difference in the effects of branched- and linear-chain polyamines is discussed in relation to the difference in the manner of binding of these polyamines to negatively charged double-stranded DNA.

*Tatsuaki Tsuruyama, Takuya Hiratsuka, Wulamujiang Aini, Takuro Nakamura,
STAT5A modulates chemokine receptor CCR6 expression and enhances pre-B cell growth in a CCL20-dependent manner. J cellular biochemistry.,
J Cell Biochem 117, 2630-2642 (2016).

[Summary] Signal transducer and activator of transcription 5A (STAT5A) contributes to B-cell responses to cytokines through suppressor of cytokine signaling (Socs) genes in innate immunity. However, its direct roles in B-cell responses to chemokines are poorly understood. In this study, we examined the role of STAT5A in the innate immune response. We found that STAT5A upregulated the transcription of C-C motif receptor 6 (Ccr6) to induce responses to its ligand, CCL20. STAT5A transcriptional activity proceeded through binding to the interferon-g activation site (GAS) element in the CCR6 promoter in the genome of pre-B cells. High levels of STAT5A and CCR6 increased CCL20-dependent colony growth of pre-B cells. In human B-lymphoblastic lymphoma with inflammation, STAT5A phosphorylation was correlated with CCR6 expression STAT5A enhanced the response of pre-B cells to CCL20 to promote their growth.

*Shunsuke F. Shimobayashi, Mafumi Hishida, Tomo Kurimura and Masatoshi Ichikawa,
Nanoscale hydration dynamics of DNA-lipid blend dry films: DNA-size dependency,
Physical Chemistry Chemical Physics 2016, 18, 31664-31669 (2016).

[Summary] In this study, nanoscale hydration dynamics of DNA–lipid blend dry films are investigated *via* small angle X-ray diffraction. Compared to the hydration of lipid films, fragmented short DNA strands and counterions in stacked lipid layers dramatically accelerate both the relaxation of the lamellar distance to a metastable interval and the subsequent peeling-off process of lipid bilayers. Moreover, genome-sized long DNA and counterions accelerate the relaxation process, but suppress the peeling-off process and simultaneously induce a damped-oscillation of the lamellar interval; this is probably due to the viscoelastic properties of the entangled long DNA dissolved in hydrated water between the stacked lipid bilayers. This study's findings can pave the way for producing cell-sized liposomes, which efficiently encapsulate any arbitrary sized DNA through natural swelling.

Jerzy Górecki, Jonna N Gorecka, Bogdan Nowakowski, Hiroshi Ueno, Tatsuaki Tsuruyama and *Kenichi Yoshikawa,
Sensing Parameter of a Time Dependent Inflow with an Enzymatic Reaction,
Advances in Unconventional Computing 2, 85-104 (2016).

[Summary] Functionality of living organisms is based on decision making. Chemical reactions stand behind information processing in biological systems. Therefore, it is interesting to consider reaction models that show ability to make decisions by evolving towards significantly different states, depending on conditions at which those reactions proceed. It has been recently demonstrated that a system exhibiting cooperative or sigmoidal response with respect to the input exhibits the potential to function as a discriminator of the amplitude or the frequency of its external periodic perturbation. Here we consider a few models of allosteric enzymatic reactions and discuss their applicability for sensing the frequency or the amplitude of the time dependent input in a form of reagent inflow. The output is coded in a product oscillation type. On the basis of numerical simulations we compare results for a full reaction model with its reduced, easier to analyze version

Tomo Kurimura, Seori Mori, Masako Miki and *Kenichi Yoshikawa,
Rotary motion of a micro-solid particle under a stationary difference of electric potential,
The Journal of Chemical Physics 145, 034902/1-4 (2016).

[Summary] The periodic rotary motion of spherical sub-millimeter-sized plastic objects is generated under a direct-current electric field in an oil phase containing a small amount of anionic or cationic surfactant. Twin-rotary motion is observed between a pair of counter-electrodes; i.e., two vortices are generated simultaneously, where the line between the centers of rotation lies perpendicular to the line between the tips of the electrodes. Interestingly, this twin rotational motion switches to the reverse direction when an anionic surfactant is replaced by a cationic surfactant. We discuss the mechanism of this self-rotary motion in terms of convective motion in the oil phase where nanometer-sized inverted micelles exist. The reversal of the direction of rotation between anionic and cationic surfactants is attributable to the difference in the charge sign of inverted micelles with surfactants. We show that the essential features in the experimental trends can be reproduced through a simple theoretical model, which supports the validity of the above mechanism.

Soutaro Oda, Yoshitsugu Kubo, Chwen-Yang Shew and *Kenichi Yoshikawa,
Fluctuations induced transition of localization of granular objects caused by degrees of crowding,
Physica D 336, 39-46 (2016).

[Summary] Fluctuations are ubiquitous in both microscopic and macroscopic systems, and an investigation of confined particles under fluctuations is relevant to how living cells on the earth maintain their lives. Inspired by biological cells, we conduct the experiment through a very simple fluctuating system containing one or several large spherical granular particles and multiple smaller ones confined on a cylindrical dish under vertical vibration. We find a universal behavior that large particles preferentially locate in cavity interior due to the fact that large particles are depleted from the cavity wall by small spheres under vertical vibration in the actual experiment. This universal behavior can be understood from the standpoint of entropy

Jerzy Górecki, Jonna N. Gorecka, Bogdan Nowakowski, Hiroshi Ueno and *Kenichi Yoshikawa,
How many enzyme molecules are needed for discrimination oriented applications?,
Physical Chemistry Chemical Physics 18, 20518-20527 (2016).

[Summary] Chemical reactions establish a molecular mechanism for information processing in living organisms. Here we consider a simple enzymatic reaction model that can be used to discriminate parameters characterizing periodic reagent inflow. Numerical simulations based on the kinetic equations show that there exist a range of inflow frequencies and amplitudes in which the time evolution of the system is very sensitive to small changes in the values of these parameters. However, the kinetic equations are derived for the thermodynamic limit, whereas in a real biological medium, like a cell, the number of enzyme molecules is an integer and finite. We use stochastic simulations to estimate discriminator reliability as a function of the number of enzyme molecules involved. For systems with 10000 molecules the functionality predicted by kinetic equations is confirmed. If the number of molecules is decreased to 100, discrimination becomes unreliable

Takuya Hiratsuka, Yuji Takei, Rei Ohmori, Yuuki Imai, Makoto Ozeki, Keiji Tamaki, Hironori Haga, Takashi Nakamura, *Tatsuaki Tsuruyama,
ZFP521 contributes to pre-B-cell lymphomagenesis through modulation of the pre-B-cell receptor signaling pathway,
Oncogene 35, 3227-3238 (2016).

[Summary] ZFP521 was previously identified as a putative gene involved in induction of B-cell lymphomagenesis. However, the contribution of ZFP521 to lymphomagenesis has not been confirmed. In this study, we sought to elucidate the role of ZFP521 in B-cell lymphomagenesis. To this end, we used a retroviral insertion method to show that ZFP521 was a target of mutagenesis in pre-B-lymphoblastic lymphoma cells. The pre-B-cell receptor (pre-BCR) signaling molecules BLNK, BTK and BANK1 were positively regulated by the ZFP521 gene, leading to enhancement of the pre-BCR signaling pathway. In addition, c-myc and c-jun were upregulated following activation of ZFP521. Stimulation of pre-BCR signaling using anti-Vpreb antibodies caused aberrant upregulation of c-myc and c-jun and of Ccnd3, which encodes cyclin D3, thereby inducing the growth of pre-B cells. Stimulation with Vpreb affected the growth of pre-B cells, and addition of interleukin (IL)-7 receptor exerted competitive effects on pre-B-cell growth. Knockdown of BTK and BANK1, targets of ZFP521, suppressed the effects of Vpreb stimulation on cell growth. Furthermore, in human lymphoblastic lymphoma, analogous to pre-B-cell lymphoma in mice, the expression of ZNF521, the homolog of ZFP521 in humans, was upregulated. In conclusion, our data showed that the ZFP521 gene comprehensively induced pre-B-cell lymphomagenesis by modulating the pre-B-cell receptor signaling pathway.

Chika Tongu, Takahiro Kenmotsu, Yuko Yoshikawa, Anatoly A. Zinchenko, Ning Chen and *Kenichi Yoshikawa,
Divalent Cation Shrinks DNA but Inhibits its Compaction with Trivalent Cation,
The Journal of Chemical Physics 144, 205101/1-7 (2016).

[Summary] Our observation reveals the effects of divalent and trivalent cations on the higher-order structure of giant DNA (T4 DNA 166 kbp) by fluorescence microscopy. It was found that divalent cations, Mg(2+) and Ca(2+), inhibit DNA compaction induced by a trivalent cation, spermidine (SPD(3+)). On the other hand, in the absence of SPD(3+), divalent cations cause the shrinkage of DNA. As the control experiment, we have confirmed the minimum effect of monovalent cation, Na(+) on the DNA higher-order structure. We interpret the competition between 2+ and 3+ cations in terms of the change in the translational entropy of the counterions. For the compaction with SPD(3+), we consider the increase in translational entropy due to the ion-exchange of the intrinsic monovalent cations condensing on a highly charged polyelectrolyte, double-stranded DNA, by the 3+ cations. In contrast, the presence of 2+ cation decreases the gain of entropy contribution by the ion-exchange between monovalent and 3+ ions.

Hiroki Sakuta, Nobuyuki Magome, Yoshihito Mori and *Kenichi Yoshikawa,
Negative/Positive Chemotaxis of a Droplet: Dynamic Response to a Stimulant Gas,
Applied Physics Letters 108, 203703/1-4 (2016).

[Summary] We report here the repulsive/attractive motion of an oil droplet floating on an aqueous phase caused by the application of a stimulant gas. A cm-sized droplet of oleic acid is repelled by ammonia vapor. In contrast, a droplet of aniline on an aqueous phase moves toward hydrochloric acid as a stimulant. The mechanisms of these characteristic behaviors of oil droplets are discussed in terms of the spatial gradient of the interfacial tension caused by the stimulant gas.

Shu Hashimoto, Aoi Yoshida, Taeko Ohta, Hiroaki Taniguchi, Koichiro Sadakane and *Kenichi Yoshikawa,
Formation of Stable Cell-Cell Contact without a Solid/Gel Scaffold: Non-invasive Manipulation by Laser under Depletion Interaction with a Polymer,
Chemical Physics Letters 655-656, 11-16 (2016).

[Summary] We report a novel method for constructing a stable three-dimensional cellular assembly in the absence of a solid or gel scaffold. A targeted cell was transferred to another cell, and the two were kept in contact for a few minutes by optical manipulation in an aqueous medium containing a hydrophilic polymer. Interestingly, this cell–cell adhesion was maintained even after elimination of the polymer. We discuss the mechanism of the formation of stable multi-cellular adhesion in terms of spontaneous rearrangement of the components embedded in the pair of facing membranes.

Tomo Kurimura and *Masatoshi Ichikawa,
Noise-supported actuator: Coherent resonance in the oscillations of a micrometersized object under a direct current-voltage,
Applied Physics Letters 108, 144101/1-4 (2016).

[Summary] Noise supported regular motion in a micro-fluidic environment is studied. Recently, it was reported that an aqueous droplet in an oil phase exhibited rhythmic back-and-forth motion under stationary direct current voltage between the cone-shaped electrodes, where the oscillating water droplet moves on a limit cycle orbital. We now confirm that a combination of the limit cycle nature and white noise supports and enhances the regular motion of the object through coherent resonance. The present result will open a way to design an efficient machinery in microfluidic and micromechanical devices.

Shunsuke F. Shimobayashi, Masatoshi Ichikawa and *Takashi Taniguchi,
Direct observations of transition dynamics from macro- to micro-phase separation in asymmetric lipid bilayers induced by externally added glycolipids,
Europhysics Letters 113, Number 5, 56005-p1-p6 (2016).

[Summary] We present the first direct observations of morphological transitions from macro- to micro-phase separation using micrometer-sized asymmetric lipid vesicles exposed to externally added glycolipids (GM1:monosialotetrahexosylganglioside). The transition occurs via an intermediate stripe morphology state. During the transition, monodisperse micro-domains emerge through repeated scission events of the stripe domains. Moreover, we numerically confirmed such transitions using a time-dependent Ginzburg-Landau model, which describes both the intramembrane phase separation and the bending elastic membrane. The experimental and simulation results are in quantitative agreement.

Yuki Oda, Koichiro Sadakane, Yuko Yoshikawa, Tadayuki Imanaka, Kingo Takiguchi, Masahito Hayashi, Takahiro Kenmotsu and *Kenichi Yoshikawa,
Highly Concentrated Ethanol Solution Behaves as a Good Solvent for DNA as Revealed by Single-Molecule Observation,
ChemPhysChem 17(4), 471-473 (2016).

Array

Yukinori Nishigami, *Hiroaki Ito, Seiji Sonobe, and *Masatoshi Ichikawa,
Non-periodic oscillatory deformation of an actomyosin microdroplet encapsulated within a lipid interface,
Scientific Reports 6, 18964/1-11 (2016).

[Summary] Active force generation in living organisms, which is mainly involved in actin cytoskeleton and myosin molecular motors, plays a crucial role in various biological processes. Although the contractile properties of actomyosin have been extensively investigated, their dynamic contribution to a deformable membrane remains unclear because of the cellular complexities and the difficulties associated with in vitro reconstitution. Here, by overcoming these experimental difficulties, we demonstrate the dynamic deformation of a reconstituted lipid interface coupled with self-organized structure of contractile actomyosin. Therein, the lipid interface repeatedly oscillates without any remarkable periods. The oscillatory deformation of the interface is caused by the aster-like three-dimensional hierarchical structure of actomyosin inside the droplet, which is revealed that the oscillation occurs stochastically as a Poisson process.

*Hiroaki Ito, Yukinori Nishigami, Seiji Sonobe, and *Masatoshi Ichikawa,
Wrinkling of a spherical lipid interface induced by actomyosin cortex,
Physical Review E 92, 062711/1-8 (2015).

[Summary] Actomyosin actively generates contractile forces that provide the plasma membrane with the deformation stresses essential to carry out biological processes. Although the contractile property of purified actomyosin has been extensively studied, to understand the physical contribution of the actomyosin contractile force on a deformable membrane is still a challenging problem and of great interest in the field of biophysics. Here, we reconstitute a model system with a cell-sized deformable interface that exhibits anomalous curvature-dependent wrinkling caused by the actomyosin cortex underneath the spherical closed interface. Through a shape analysis of the wrinkling deformation, we find that the dominant contributor to the wrinkled shape changes from bending elasticity to stretching elasticity of the reconstituted cortex upon increasing the droplet curvature radius of the order of the cell size, i.e., tens of micrometers. The observed curvature dependence is explained by the theoretical description of the cortex elasticity and contractility. Our present results provide a fundamental insight into the deformation of a curved membrane induced by the actomyosin cortex.

Tomohiro Yanao, Sosuke Sano and *Kenichi Yoshikawa,
Chiral selection in wrapping, crossover, and braiding of DNA mediated by asymmetric bend-writhe elasticity,
AIMS Biophysics 2(4), 666–694 (2015).

[Summary] Wrapping, crossover, and braiding of DNA are the motifs of fundamental interest ingenome packaging, gene regulation, and enzyme recognition. This study explores elasticmechanisms for the selection of chirality in wrapping, crossover, and braiding of DNA based on acoarse-grained model. The DNA model consists of two elastic chains that mutually intertwine in aright-handed manner forming a double-stranded helix with the distinction between major and minorgrooves. Although individual potential energy functions of the DNA model have no asymmetry interms of left and right twist, the model as a whole exhibits an asymmetric propensity to writhe in theleft direction upon bending due to the right-handed helical geometry. Monte Carlo simulations of thismodel suggest that DNA has a propensity to prefer left-handed wrapping around a spherical coreparticle and also around a uniform rod due to the asymmetric elastic coupling between bending andwrithing. This result indicates an elastic origin of the uniform left-handed wrapping of DNA innucleosomes and also has implications on the wrapping of double-stranded DNA around rod-likemolecules. Monte Carlo simulations of the DNA model also suggest that two juxtaposed DNAmolecules can braid each other spontaneously under moderate attractive interactions with thepreference for left-handed braiding due to the asymmetric coupling between bending and writhing.This result suggests the importance of asymmetric elasticity in the selection of chirality in braidingof a pair of DNA molecules.

Hiroshi Ueno, Tatsuaki Tsuruyama, Bogdan Nowakowski, Jerzy Górecki, and *Kenichi Yoshikawa,
Discrimination of time-dependent inflow properties with a cooperative dynamical system.,
Chaos 25, 103115 (2015).

[Summary] Many physical, chemical, and biological systems exhibit a cooperative or sigmoidal response with respect to the input. In biochemistry, such behavior is called an allosteric effect. Here, we demonstrate that a system with such properties can be used to discriminate the amplitude or frequency of an external periodic perturbation. Numerical simulations performed for a model sigmoidal kinetics illustrate that there exists a narrow range of frequencies and amplitudes within which the system evolves toward significantly different states. Therefore, observation of system evolution should provide information about the characteristics of the perturbation. The discrimination properties for periodic perturbation are generic. They can be observed in various dynamical systems and for different types of periodic perturbation.

Tatsuaki Tsuruyama, Wulamujiang Aini, Takuya Hiratsuka,
Reassessment of H&E-stained clot specimens and immunohistochemistry of phosphorylated Stat5 for histologic diagnosis of MDS/MPN.,
Pathology 47, 673-677 (2015).

[Summary] Few studies have comprehensively analysed histopathological findings of bone marrow clots for diagnosis of haematopoietic cell dysplasia. In particular, a limited number of studies have assessed the use of haematoxylin and eosin (H&E) staining, which is generally considered less informative than May-Giemsa staining. In the current study, the utility of bone marrow clot specimens for diagnosis was examined using H&E staining and immunohistochemistry. Patients with myelodysplastic syndromes (MDS) and myelodysplastic/myeloproliferative neoplasm (MDS/MPN), including chronic myelomonocytic leukaemia (CMML), atypical chronic myeloid leukaemia (aCML) lacking Philadelphia chromosome, and juvenile myelomonocytic leukaemia (JMML), were selected for histological evaluation. H&E stained specimens were advantageous for observation of atypical basophilic staining of the cytoplasm and nucleus related to dysplasia. This finding was significantly supported for both MDS and MDS/MPN (p < 0.05 versus May-Giemsa staining); therefore, we concluded that H&E staining could be used for identification of dysplastic cells. In addition, despite the loss of tissue structure, phosphorylated Stat5 immunostaining was sufficiently useful for the observation of myelodysplastic blasts. Thus, clot specimens are useful for diagnosis of haematopoietic dysplasia by pathologists.

Masanobu Tanaka, Marcel Hörning, Hiroyuki Kitahata and *Kenichi Yoshikawa,
Elimination of a spiral wave pinned at an obstacle by a train of plane waves: Effect of diffusion between obstacles and surrounding media,
Chaos 25, 103127 (2015).

[Summary] In excitable media such as cardiac tissue and Belousov-Zhabotinsky reaction medium, spiral waves tend to anchor (pin) to local heterogeneities. In general, such pinned waves are difficult to eliminate and may progress to spatio-temporal chaos. Heterogeneities can be classified as either the absence or presence of diffusive interaction with the surrounding medium. In this study, we investigated the difference in the unpinning of spiral waves from obstacles with and without diffusive interaction, and found a profound difference. The pacing period required for unpinning at fixed obstacle size is larger in case of diffusive obstacles. Further, we deduced a generic theoretical framework that can predict the minimal unpinning period. Our results explain the difference in pacing periods between for the obstacles with and without diffusive interaction, and the difference is interpreted in terms of the local decrease of spiral wave velocity close to the obstacle boundary caused in the case of diffusive interaction.

Shogo Okubo, Shuhei Shibata, Yuriko Sasa Kawamura, Masatoshi Ichikawa and *Yasuyuki Kimura,
Dynamic clustering of driven colloidal particles on a circular path,
Physical Review E 92, 032303 (2015).

[Summary] We studied the collective motion of particles forced to move along a circular path in water by utilizing anoptical vortex. Their collective motion, including the spontaneous formation of clusters and their dissociation,was observed. The observed temporal patterns depend on the number of particles on the path and the variationof their sizes. The addition of particles with different sizes suppresses the dynamic formation and dissociation ofclusters and promotes the formation of specific stationary clusters. These experimental findings are reproducedby numerical simulations that take into account the hydrodynamic interaction between the particles and the radialtrapping force confining the particles to the circular path. A transition between stationary and nonstationaryclustering of the particles was observed by varying their size ratio in the binary-size systems. Our simulationreveals that the transition can be either continuous or discontinuous depending on the number of different-sizeparticles. This result suggests that the size distribution of particles has a significant effect on the collectivebehavior of self-propelled particles in viscous fluids.

Yue Ma, Naoki Ogawa, Yuko Yoshikawa, Toshiaki Mori, Tadayuki Imanaka, Yoshiaki Watanabe and *Kenichi Yoshikawa,
Protective Effect of Ascorbic Acid against Double-strand Breaks in Giant DNA: Marked Differences among the Damage Induced by Photo-irradiation, Gamma-rays and Ultrasound,
Chemical Physics Letters 638, 205-209 (2015).

[Summary] tThe protective effect of ascorbic acid against double-strand breaks in DNA was evaluated by single-molecule observation of giant DNA (T4 DNA; 166 kbp) through fluorescence microscopy. Samples wereexposed to three different forms of radiation: visible light, -ray and ultrasound. With regard to irradiationwith visible light, 1 mM AA reduced the damage down to ca. 30%. Same concentration of AA decreased thedamage done by -ray to ca. 70%. However, AA had almost no protective effect against the damage causedby ultrasound. This significant difference is discussed in relation to the physico-chemical mechanism ofdouble-strand breaks depending on the radiation source.

Yongjun Chen, Kosuke Suzuki and *Kenichi Yoshikawa,
Self-organized Target and Spiral Patterns through the “Coffee Ring” Effect,
Journal of Chemical Physics 143, 084702 (2015).

[Summary] We studied the precipitation pattern of fullerene C60 nanocrystals generated through the evaporation of a confined liquid bridge. In contrast to the usual “coffee ring” pattern, both target and spiral patterns were observed. The characteristics of the pattern critically depended on the concentration of the solution, the temperature, and the level of vacuum. In addition, the morphology of the microscopic precipitates varied greatly as a function of these experimental parameters. This pattern formation can be interpreted as a two-step rhythmic nucleation/precipitation of fullerene crystals during receding motion of the contact line. Symmetric motion of the contact line produces a target pattern, and the propagation of distortion of the liquid interface caused by a disturbance generates a spiral pattern.

Naoki Umezawa, Yuhei Horai, Yuki Imamura, Makoto Kawakubo, Mariko Nakahira, Nobuki Kato, Akira Muramatsu, Yuko Yoshikawa, *Kenichi Yoshikawa and Tsunehiko Higuchi,
Structurally Diverse Polyamines: Solid-Phase Synthesis and Interaction with DNA,
ChemBioChem 16, 1811-1819 (2015).

[Summary] A versatile solid-phase approach based on peptide chemistry was used to construct four classes of structurally diverse polyamines with modified backbones: linear, partially constrained, branched, and cyclic. Their effects on DNA duplex stability and structure were examined. The polyamines showed distinct activities, thus highlighting the importance of polyamine backbone structure. Interestingly, the rank order of polyamine ability for DNA compaction was different to that for their effects on circular dichroism and melting temperature, thus indicating that these polyamines have distinct effects on secondary and higher-order structures of DNA.

Xiaojun Ma, Tomohiro Aoki, Tatsuaki Tsuruyama, and Shuh Narumiya,
Definition of prostaglandin E2-EP2 signals in the colon tumor microenvironment which amplify inflammation and tumor growth.,
Cancer Reserach 75, 2822-32 (2015).

[Summary] Inflammation in the colon contributes significantly to colorectal cancer development. While aspirin reduces the colorectal cancer risk, its action mechanism, especially in inflammation in tumor microenvironment, still remains obscure. Here, we examined this issue by subjecting mice deficient in each prostaglandin (PG) receptor to colitis-associated cancer model. Deficiency of PGE receptor subtype EP2 selectively reduced, and deficiency of EP1 and EP3 enhanced, the tumor formation. EP2 is expressed in infiltrating neutrophils and tumor-associated fibroblasts in stroma, where it regulates expression of inflammation- and growth-related genes in a self-amplification manner. Notably, expression of cytokines such as TNFα and IL6, a chemokine, CXCL1, a PG-producing enzyme, COX-2, and Wnt5A was significantly elevated in tumor lesions of wild-type mice but this elevation was significantly suppressed in EP2-deficient mice. Intriguingly, EP2 stimulation in cultured neutrophils amplified expression of TNFα, IL6, CXCL1, COX-2, and other proinflammatory genes synergistically with TNFα, and EP2 stimulation in cultured fibroblasts induced expression of EP2 itself, COX-2, IL6, and Wnt genes. EP2 expression in infiltrating neutrophils and tumor-associated fibroblasts was also found in clinical specimen of ulcerative colitis-associated colorectal cancer. Bone marrow transfer experiments suggest that EP2 in both cell populations is critical for tumorigenesis. Finally, administration of a selective EP2 antagonist potently suppressed tumorigenesis in this model. Our study has thus revealed that EP2 in neutrophils and tumor-associated fibroblasts promotes colon tumorigenesis by amplifying inflammation and shaping tumor microenvironment, and suggests that EP2 antagonists are promising candidates of aspirin-alternative for chemoprevention of colorectal cancer.

Hisako Takigawa-Imamura, Ritsuko Morita, Takafumi Iwaki, Takashi Tsuji and *Kenichi Yoshikawa,
Tooth germ invagination from cell–cell interaction: Working hypothesis on mechanical instability,
Journal of Theoretical Biology 382, 284 (2015).

[Summary] In the early stage of tooth germ development, the bud of the dental epithelium is invaginated by the underlying mesenchyme, resulting in the formation of a cap-like folded shape. This bud-to-cap transition plays a critical role in determining the steric design of the tooth. The epithelial-mesenchymal interaction within a tooth germ is essential for mediating the bud-to-cap transition. Here, we present a theoretical model to describe the autonomous process of the morphological transition, in which we introduce mechanical interactions among cells. Based on our observations, we assumed that peripheral cells of the dental epithelium bound tightly to each other to form an elastic sheet, and mesenchymal cells that covered the tooth germ would restrict its growth. By considering the time-dependent growth of cells, we were able to numerically show that the epithelium within the tooth germ buckled spontaneously, which is reminiscent of the cap-stage form. The difference in growth rates between the peripheral and interior parts of the dental epithelium, together with the steric size of the tooth germ, were determining factors for the number of invaginations. Our theoretical results provide a new hypothesis to explain the histological features of the tooth germ.

Daigo Yamamoto, Tsuyoshi Takada, Masashi Tachibana, Yuta Iijima, Akihisa Shioi and Kenichi Yoshikawa,
Micromotors working in water through artificialaerobic metabolism,
Nanoscale 7, 13186–13190 (2015).

[Summary] Most catalytic micro/nanomotors that have been developed so far use hydrogen peroxide as fuel, while some use hydrazine. These fuels are difficult to apply because they can cause skin irritation, and often form and store disruptive bubbles. In this paper, we demonstrate a novel catalytic Pt micromotor that does not produce bubbles, and is driven by the oxidation of stable, non-toxic primary alcohols and aldehydes with dissolved oxygen. This use of organic oxidation mirrors living systems, and lends this new motor essentially the same characteristics, including decreased motility in low oxygen environments and the direct isothermal conversion of chemical energy into mechanical energy. Interestingly, the motility direction is reversed by replacing the reducing fuels with hydrogen peroxide. Therefore, these micromotors not only provide a novel system in nanotechnology, but also help in further revealing the underlining mechanisms of motility of living organisms.

*Tsutomu Hamada, Rie Fujimoto, Shunsuke F. Shimobayashi, Masatoshi Ichikawa, *Masahiro Takagi,
Molecular behavior of DNA in a cell-sized compartment coated by lipids,
Physical Review E 91, 62717 (2015).

[Summary] The behavior of long DNA molecules in a cell-sized confined space was investigated. Microscopic observation revealed that the adsorption of coiled DNA onto the membrane surface depended on the size of the vesicular space.

Masa Tsuchiya, Alessandro Giuliani, Midori Hashimoto, Jekaterina Erenpreisa and Kenichi Yoshikawa,
Emergent Self-Organized Criticality in Gene Expression Dynamics: Temporal Development of Global Phase Transition Revealed in a Cancer Cell Line,
PLOS ONE 10(6), e0128565 (2015).

[Summary] BackgroundThe underlying mechanism of dynamic control of the genome-wide expression is a fundamental issue in bioscience. We addressed it in terms of phase transition by a systemic approach based on both density analysis and characteristics of temporal fluctuation for the time-course mRNA expression in differentiating MCF-7 breast cancer cells.MethodologyIn a recent work, we suggested criticality as an essential aspect of dynamic control of genome-wide gene expression. Criticality was evident by a unimodal-bimodal transition through flattened unimodal expression profile. The flatness on the transition suggests the existence of a critical transition at which up- and down-regulated expression is balanced. Mean field (averaging) behavior of mRNAs based on the temporal expression changes reveals a sandpile type of transition in the flattened profile. Furthermore, around the transition, a self-similar unimodal-bimodal transition of the whole expression occurs in the density profile of an ensemble of mRNA expression. These singular and scaling behaviors identify the transition as the expression phase transition driven by self-organized criticality (SOC).Principal FindingsEmergent properties of SOC through a mean field approach are revealed: i) SOC, as a form of genomic phase transition, consolidates distinct critical states of expression, ii) Coupling of coherent stochastic oscillations between critical states on different time-scales gives rise to SOC, and iii) Specific gene clusters (barcode genes) ranging in size from kbp to Mbp reveal similar SOC to genome-wide mRNA expression and ON-OFF synchronization to critical states. This suggests that the cooperative gene regulation of topological genome sub-units is mediated by the coherent phase transitions of megadomain-scaled conformations between compact and swollen chromatin states.Conclusion and SignificanceIn summary, our study provides not only a systemic method to demonstrate SOC in whole-genome expression, but also introduces novel, physically grounded concepts for a breakthrough in the study of biological regulation.

Hiroaki Ito, Navina Kuss, Bastian E. Rapp, Masatoshi Ichikawa, Thomas Gutsmann, Klaus Brandenburg, Johannes M. B. Pöschl, and *Motomu Tanaka,
Quantification of the Influence of Endotoxins on the Mechanics of Adult and Neonatal Red Blood Cells,
Journal of Physical Chemistry B 119, 7837−7845 (2015).

[Summary] In this study, we physically modeled the influence of endotoxin-induced sepsis symptoms on human red blood cells (RBCs) by quantifying the impact of endotoxins on the cell mechanics by the analysis of Fourier-transformed mean square amplitude of shape fluctuation, called flicker spectroscopy. With the aid of a microfluidic diffusion chamber, we noninvasively determined principal mechanical parameters of human RBCs in the absence and presence of endotoxins for individual RBCs for the first time. Because of the elongation of saccharide chain length of endotoxins, we found an increase in the morphological transition from discocytes to echinocytes, and monotonic changes in the mechanical parameters. Since septic shocks often cause lethal risks of neonates, we measured the mechanical parameters of neonatal RBCs, and compared them to those of adult RBCs. The quantitative comparison reveals that neonatal RBCs are more susceptible to the effect of endotoxins than adult RBCs. Furthermore, coincubation with the antiseptic peptide P19-2.5 (Aspidasept) with endotoxin results in a slight suppression of the impact of the endotoxin. The strategy proposed in our study can potentially be applied for the quantitative diagnosis of RBCs based on mechanical readouts.

Daigo Yamamoto, Chika Nakajima, Akihisa Shioi, Marie Pierre Krafft, Kenichi Yoshikawa ,
The evolution of spatial ordering of oil drops fast spreading on a water surface,
Nature Communications 6, 7189/1-6 (2015).

[Summary] The design of dynamically self-assembled systems is of high interest in science and technology. Here, we report a unique cascade in the self-ordering of droplets accompanied by a dewetting transition. The dynamic self-emergent droplets are observed when a thin liquid layer of an immiscible fluorocarbon oil (perfluorooctyl bromide, PFOB) is placed on a water surface. Due to the gradual evaporation of PFOB, a circular PFOB-free domain appears as a result of a local dewetting transition. A circular pearling structure is generated at the rim with the growth of the dewetting hole. As the next stage, linear arrays of droplets are generated in a radial manner from the centre of the hole. These one-dimensional arrangements then evolve into two-dimensional hexagonal arrays of microdroplets through collective rhythmical shrinking/expanding motions. The emergence of such dynamic patterns is discussed in terms of the nonlinear kinetics of the dewetting transition under thermodynamically dissipative conditions.

Yongjun Chen, Shun N. Watanabe and *Kenichi Yoshikawa,
Roughening Dynamics of Radial Imbibition in a Porous Medium,
Journal of Physical Chemistry C 199(22), 12508–12513 (2015).

[Summary] We report radial imbibition of water in a porous medium in a Hele–Shaw cell, including forced imbibition and spontaneous imbibition. Washburn’s law is confirmed in our experiment. Radial imbibition follows scaling dynamics. For forced radial imbibition, anomalous roughening dynamics is found when the front invades the porous medium, and the roughening dynamics depend on the flow rate of the injected fluid. The growth exponents increase linearly with an increase in the flow rate while the roughness exponents decrease with an increase in the flow rate. For spontaneous imbibition, we found a growth exponent (β = 0.6) that was independent of the pressure applied at the liquid inlet, and the roughness exponent decreased with an increase in pressure. Thus, it has become evident that the roughening dynamics of radial imbibition is markedly different from one-dimensional imbibition with a planar interface window.

Yoshitsugu Kubo, *Shio Inagaki, *Masatoshi Ichikawa, and Kenichi Yoshikawa,
Mode bifurcation of a bouncing dumbbell with chirality,
Physical Review E 91, 052905/1-9 (2015).

[Summary] We studied the behavior of a dumbbell bouncing upon a sinusoidally vibrating plate. By introducing chiral asymmetry to the geometry of the dumbbell, we observed a cascade of bifurcations with an increase in the vibration amplitude: spinning, orbital, and rolling. In contrast, for an achiral dumbbell, bifurcation is generated by a change from random motion to vectorial inchworm motion. A simple model particle was considered in a numerical simulation that reproduced the essential aspects of the experimental observation. The mode bifurcation from directional motion to random motion is interpreted analytically by a simple mechanical discussion.

*Takafumi Iwaki, Tomomi Ishido, Ken Hirano, Alexei Lazutin, Valentina V. Vasilevskaya, Takahiro Kenmotsu and Kenichi Yoshikawa,
Marked difference in conformational fluctuation between giant DNA molecules in circular and linear forms,
Journal of Chemical Physics 142, 145101 (2015).

[Summary] We performed monomolecular observations on linear and circular giant DNAs (208 kbp) in an aqueous solution by the use of fluorescence microscopy. The results showed that the degree of conformational fluctuation in circular DNA was ca. 40% less than that in linear DNA, although the long-axis length of circular DNA was only 10% smaller than that of linear DNA. Additionally, the relaxation time of a circular chain was shorter than that of a linear chain by at least one order of magnitude. The essential features of this marked difference between linear and circular DNAs were reproduced by numerical simulations on a ribbon-like macromolecule as a coarse-grained model of a long semiflexible, double-helical DNA molecule. In addition, we calculated the radius of gyration of an interacting chain in a circular form on the basis of the mean field model, which provides a better understanding of the present experimental trend than a traditional theoretical equation.

Kanta Tsumoto, Masafumi Arai, Naoki Nakatani, Shun N. Watanabe and *Kenichi Yoshikawa,
Does DNA Exert an Active Role in Generating Cell-Sized Spheres in an Aqueous Solution with a Crowding Binary Polymer?,
Life 5(1), 459-466 (2015).

[Summary] We report the spontaneous generation of a cell-like morphology in an environment crowded with the polymers dextran and polyethylene glycol (PEG) in the presence of DNA. DNA molecules were selectively located in the interior of dextran-rich micro-droplets, when the composition of an aqueous two-phase system (ATPS) was near the critical condition of phase-segregation. The resulting micro-droplets could be controlled by the use of optical tweezers. As an example of laser manipulation, the dynamic fusion of two droplets is reported, which resembles the process of cell division in time-reverse. A hypothetical scenario for the emergence of a primitive cell with DNA is briefly discussed.

Takahiro Umeki, Masahiko Ohata, *Hiizu Nakanishi, Masatoshi Ichikawa,
Dynamics of microdroplets over the surface of hot water,
Scientific Reports 5, 8046/1-6 (2015).

[Summary] When drinking a cup of coffee under the morning sunshine, you may notice white membranes of steam floating on the surface of the hot water. They stay notably close to the surface and appear to almost stick to it. Although the membranes whiffle because of the air flow of rising steam, peculiarly fast splitting events occasionally occur. They resemble cracking to open slits approximately 1 mm wide in the membranes, and leave curious patterns. We studied this phenomenon using a microscope with a high-speed video camera and found intriguing details: i) the white membranes consist of fairly monodispersed small droplets of the order of 10 μm; ii) they levitate above the water surface by 10 ~ 100 μm; iii) the splitting events are a collective disappearance of the droplets, which propagates as a wave front of the surface wave with a speed of 1 ~ 2 m/s; and iv) these events are triggered by a surface disturbance, which results from the disappearance of a single droplet.

Kingo Takiguchi, Makiko Egishi, Yohko Tanaka-Takiguchi, Masahito Hayashi and Kenichi Yoshikawa,
Specific Transformation of Assembly with Actin Filaments and Molecular Motors in a Cell-Sized Self-Emerged Liposome,
Origins of Life and Evolution of Biospheres 44(4), 325-329 (2015).

[Summary] Eukaryotes, by the same combination of cytoskeleton and molecular motor, for example actin filament and myosin, can generate a variety of movements. For this diversity, the organization of biological machineries caused by the confinement and/or crowding effects of internal living cells, may play very important roles.

Anatoly A. Zinchenko and *Kenichi Yoshikawa,
Compaction of Double-Stranded DNA by Negatively Charged Proteins and Colloids,
Current Opinion in Colloid & Interface Science 20, 60-65 (2015).

[Summary] A great number of past in vivo and in vitro studies on DNA condensation/compaction made clear DNA conformational behavior and its influence on DNA bioactivity in systems containing either polymers or colloids which are positively charged or electrically neutral. However, there still remains a lack of understanding about interaction between DNA and like-charged species. Such knowledge is important for deeper insight into DNA behavior in cellular environment, where DNA encounters negatively charged biopolymers such as RNA and RNase at relatively high concentrations, and should contribute to an overall understanding of the intrinsic mechanism that underlies the spatiotemporal dynamics of biomacromolecules in various cellular processes. In this review, we focus on DNA condensation/compaction by negatively-charged species, compare it with established earlier model systems of DNA condensation/compaction, and discuss recent advances in the field. We show that DNA compaction by negatively charged polymers and colloids exhibits some similarities with that induced by neutral polymers, but stress that several new features imposed by the electrostatics of polyanions should be considered to grasp the whole picture.

*Chwen-Yang Shew and Kenichi Yoshikawa,
A toy model for nucleus-sized crowding confinement,
Journal of Physics: Condensed Matter 27(6), 064118/1-7 (2015).

[Summary] We conduct Monte Carlo simulations to understand the spatial distribution of a polymer molecule confined within a rigid spherical capsule under crowding conditions, via a bead-spring chain model. To adjust the crowding level, the polymer is mixed with spherical crowders. As the interior of the capsule becomes more crowded, chain monomers tend to move to the capsule boundary under the penalty of conformational entropy. By incorporating some attraction between monomers and crowders, the polymer chain moves away from the capsule boundary. The interplay, between the conformational entropy, DNA-protein interaction, and molecular crowding induced depletion force between the chain and capsule boundary, may be essential to elucidate the heterogeneous chromatin structure in nuclei. Furthermore, the effects of chain length and size disparity between the monomers and the crowders are also investigated preliminarily.

*Shio Inagaki, Hiroyuki Ebata and Kenichi Yoshikawa,
Steadily oscillating axial bands of binary granules in a nearly filled coaxial cylinder,
Physical Review E 91, 010201(R)/1-5 (2015).

[Summary] Granular materials often segregate under mechanical agitation such as flowing, shaking, or rotating, in contrast to an expectation of mixing. It is well known that bidisperse mixtures of granular materials in a partially filled rotating cylinder exhibit monotonic coarsening dynamics of segregation. Here we report the steady oscillation of segregated axial bands under the stationary rotation of a nearly filled coaxial cylinder for O(10 3 ) revolutions. The axial bands demonstrate steady back-and-forth motion along the axis of rotation. Experimental findings indicated that these axial band dynamics are driven by global convection throughout the system. The essential features of the spatiotemporal dynamics are reproduced with a simple phenomenological equation that incorporates the effect of global convection.

*Shunsuke F. Shimobayashi and *Masatoshi Ichikawa,
Emergence of DNA-Encapsulating Liposomes from a DNA-Lipid Blend Film,
Journal of Physical Chemistry B 118, 10688-10694 (2014).

[Summary] Spontaneous generation of DNA-enclosing liposomes from a DNA−lipid blend film is investigated. The special properties of the lipid vesicles, namely, micrometer size, unilamellarity, and dense polymer encapsulation acquired by the dehydration−rehydration process, are physicochemically revealed. We found that the formation of giant unilamellar vesicles encapsulating DNAs are governed by micropatterns of the films, such as dots and network patterns. From the results, we proposed a plausible physical mechanism for the dehydration−rehydration process, making it possible to optimize the encapsulation of any agent.

Fumi Takabatake, Kenichi Yoshikawa, and *Masatoshi Ichikawa,
Communication: Mode bifurcation of droplet motion under stationary laser irradiation,
The Journal of Chemical Physics 141, 051103/1-4 (2014).

[Summary] The self-propelled motion of a mm-sized oil droplet floating on water, induced by a local temperature gradient generated by CW laser irradiation is reported. The circular droplet exhibits two types of regular periodic motion, reciprocal and circular, around the laser spot under suitable laser power. With an increase in laser power, a mode bifurcation from rectilinear reciprocal motion to circular motion is caused. The essential aspects of this mode bifurcation are discussed in terms of spontaneous symmetry-breaking under temperature-induced interfacial instability, and are theoretically reproduced with simple coupled differential equations.

*Tatsuaki Tsuruyama,
A model of cell biological signaling predicts the phase transition of signaling and provides mathematical formula of signaling,
PLOS ONE 9, e102911 (2014).

[Summary] A biological signal is transmitted by interactions between signaling molecules in the cell. To date, there have been extensive studies regarding signaling pathways using numerical simulation of kinetic equations that are based on equations of continuity and Fick's law. To obtain a mathematical formulation of cell signaling, we propose a stability kinetic model of cell biological signaling of a simple two-parameter model based on the kinetics of the diffusion-limiting step. In the present model, the signaling is regulated by the binding of a cofactor, such as ATP. Non-linearity of the kinetics is given by the diffusion fluctuation in the interaction between signaling molecules, which is different from previous works that hypothesized autocatalytic reactions. Numerical simulations showed the presence of a critical concentration of the cofactor beyond which the cell signaling molecule concentration is altered in a chaos-like oscillation with frequency, which is similar to a discontinuous phase transition in physics. Notably, we found that the frequency is given by the logarithm function of the difference of the outside cofactor concentration from the critical concentration. This implies that the outside alteration of the cofactor concentration is transformed into the oscillatory alteration of cell inner signaling. Further, mathematical stability kinetic analysis predicted a discontinuous dynamic phase transition in the critical state at which the cofactor concentration is equivalent to the critical concentration. In conclusion, the present model illustrates a unique feature of cell signaling, and the stability analysis may provide an analytical framework of the cell signaling system and a novel formulation of biological signaling.

Tomohiro Yanao and *Kenichi Yoshikawa,
Chiral symmetry breaking of a double-stranded helical chain through bend-writhe coupling,
Physical Review E 89, 062713/1-16 (2014).

[Summary] This paper explores asymmetric elasticity of a double-stranded helical chain, which serves as a minimal model of biopolymers. The model consists of two elastic chains that mutually intertwine in a right-handed manner, forming a double-stranded helix. A simple numerical experiment for structural relaxation, which reduces the total elastic energy of the model monotonically without thermal fluctuations, reveals possible asymmetric elasticity inherent in the helical chain. It is first shown that a short segment of the double-stranded helical chain has a tendency to unwind when it is bent. It is also shown that a short segment of the helical chain has a tendency to writhe in the left direction upon bending. This tendency gives rise to a propensity for a longer segment of the chain to form a left-handed superhelix spontaneously upon bending. Finally, this propensity of the helical chain to form a left-handed superhelix is proposed to be a possible origin of the uniform left-handed wrapping of DNA around nucleosome core particles in nature. The results presented here could provide deeper insights into the roles and significance of helical chirality of biopolymers.

Yutaka Sumino and *Kenichi Yoshikawa,
Amoeba-like motion of an oil droplet Chemical model of self-motile organisms,
The European Physical Journal Special Topics 223, 1345–1352 (2014).

[Summary] In this paper, we demonstrate our recent attempt to construct a chemical model system of amoeboid motion. The system is intended to mimic biological motility based on the generation and collapse of an elastic aggregate; it is composed of oil, water, and surfactants. In this chemical system, the oil–water interface shows extension and retreat of spherical extrusions accompanied by the generation of aggregate on the interface. This instability of the oil–water interface can cause autonomous splitting and motion of a floating oil droplet. The current mathematical model based on the generation of a passive elastic gel is explained, as well as the discrepancy between the model and the experiments. We further describe recently observed microscopic characteristics of the aggregate formation process that might cause the interfacial instability. Finally, we discuss the disadvantage of a chemical model system compared with active colloid and in vitro biological systems, and also mention its potential advantages.

Masa Tsuchiya, Midori Hashimoto, Yoshiko Takenaka, Ikuko N. Motoike and *Kenichi Yoshikawa,
Global Genetic Response in a Cancer Cell: Self-Organized Coherent Expression Dynamics,
PLOS ONE 9(8), e105491 (2014).

[Summary] Understanding the basic mechanism of the spatio-temporal self-control of genome-wide gene expression engaged with the complex epigenetic molecular assembly is one of major challenges in current biological science. In this study, the genomewide dynamical profile of gene expression was analyzed for MCF-7 breast cancer cells induced by two distinct ErbB receptor ligands: epidermal growth factor (EGF) and heregulin (HRG), which drive cell proliferation and differentiation, respectively. We focused our attention to elucidate how global genetic responses emerge and to decipher what is an underlying principle for dynamic self-control of genome-wide gene expression. The whole mRNA expression was classified into about a hundred groups according to the root mean square fluctuation (rmsf). These expression groups showed characteristic time-dependent correlations, indicating the existence of collective behaviors on the ensemble of genes with respect to mRNA expression and also to temporal changes in expression. All-or-none responses were observed for HRG and EGF (biphasic statistics) at around 10–20 min. The emergence of time-dependent collective behaviors of expression occurred through bifurcation of a coherent expression state (CES). In the ensemble of mRNA expression, the self-organized CESs reveals distinct characteristic expression domains for biphasic statistics, which exhibits notably the presence of criticality in the expression profile as a route for genomic transition. In time-dependent changes in the expression domains, the dynamics of CES reveals that the temporal development of the characteristic domains is characterized as autonomous bistable switch, which exhibits dynamic criticality (the temporal development of criticality) in the genome-wide coherent expression dynamics. It is expected that elucidation of the biophysical origin for such critical behavior sheds light on the underlying mechanism of the control of whole genome.

*Anatoly A. Zinchenko, Kanta Tsumoto, Shizuaki Murata and Kenichi Yoshikawa,
Crowding by Anionic Nanoparticles Causes DNA Double-Strand Instability and Compaction,
The Journal of Physical Chemistry B 118, 1256-1262 (2014).

[Summary] Up to the present, DNA structural transitions caused by cationic polymers as well as in concentrated solutions of neutral polymers are well documented, while a little is known about DNA interaction with like-charge species. Herein, changes in the structure of DNA induced by anionic nanoparticles of different sizes (20–130 nm) were investigated by combining single-molecule DNA fluorescent microscopy, to monitor the conformational dynamics of long-chain DNA, with spectroscopic methods, to gain insight into changes in the secondary structure of DNA. The results showed that several percent of negatively charged silica nanoparticles induced DNA compaction from a coil to a globule, and this change was accompanied by a decrease in the melting temperature of the DNA double helix. DNA was compacted into toroidal condensates with reduced diameters of about 20–30 nm. Smaller 20 nm nanoparticles triggered a DNA coil–globule transition at lower concentrations, but the exclusion volume for each type of nanoparticle at the point of complete DNA collapse, as estimated by taking into account the depth of the ionic atmosphere, was found to be almost the same.

*Chwen-Yang Shew, Kenta Kondo and Kenichi Yoshikawa,
Rigidity of a spherical capsule switches the localization of encapsulated particles between inner and peripheral regions under crowding condition: Simple model on cellular architecture,
The Journal of Chemical Physics 140, 024907/1-9 (2014).

[Summary] We have investigated the inhomogeneous interior of confined spherical cavities as capsules containingencapsulated binary hard sphere mixtures for different compositions and cavity wall rigidity. Such a greatly simplified model manifests the effects of macromolecular crowding arising from excluded volume interactions in a tiny cell or a cellular nucleus. By fixing the number of large particles, the level of crowding is adjusted by changing the amount of small hard spheres in the cavity. For a rigid cavity, large spheres tend to pack in liquid-like order apart from the surface to the center of the cavity as the crowding level is increased. Whereas, for a soft cavity, larger spheres tend to blend with small spheres in the peripheral region at near the boundary of the cavity, and are susceptible to be depleted from the interior of the cavity as the cavity becomes more crowded. These results may help future elucidation of the thermodynamic pathways to stabilize the inhomogeneous structure ofmixtures confined in cavities, such as the derepression of genome materials around the interior rim of the nucleus in a cancerous cell.

Rastko Joksimovic, Shun N. Watanabe, Sven Riemer, Michael Gradzielski and *Kenichi Yoshikawa,
Self-organized patterning through thedynamic segregation of DNA and silicananoparticles,
Scientific Reports 4, 3660/1-7 (2014).

[Summary] Exotic pattern formation as a result of drying of an aqueous solution containing DNA and silicananoparticles is reported. The pattern due to segregation was found to critically depend on the relative ratioof nanoparticles and DNA, as revealed by polarization microscopy, scanning electron microscopy, andfluorescence microscopy. The blurred radial pattern that is usually observed in the drying of a colloidalsolution was shown to be vividly sharpened in the presence of DNA. Uniquely curved, crescent-shapedmicrometer-scale domains are generated in regions that are rich in nanoparticles. The characteristicsegregated patterns observed in the present study are interpreted in terms of a large aspect ratio between thepersistence length (~50 nm) and the diameter (~2 nm) of double-stranded DNA, and the relatively smallsilica nanoparticles (radius: 5 nm).

Fumihiko Kono, Tetsuya Honda, Aini Wulamujiang, Hironori Haga, *Tatsuaki Tsuruyama,
IFN-γ/CCR5 expression in invariant NKT cells and CCL5 expression in capillary veins of dermal papillae correlate with development of psoriasis vulgaris,
British Journal of Dermatology 170, 1048-1055 (2013).

[Summary] There have been extensive studies regarding which types of T lymphocytes are involved in psoriasis vulgaris (PV). However, it has remained unclear as to which types of T lymphocytes may directly contribute to psoriasiform epidermal and vascular hyperplasia.To understand the role of TCRVα24+ invariant natural killer T (iNKT) in the development of PV, a total of 17 patients with were enrolled in this study.　In the present study, using biopsy samples of PV plaques, TCRVα24+ invariant natural killer T (iNKT) cells were investigated regarding cytokine production to understand their roles in development of disease.The number of IFN-γ+ iNKT cells correlated with the length of the psoriasiform hyperplasia rete ridge and the psoriasis area and severity index (PASI). IFN-γ+ iNKT cells in psoriatic skin exhibited higher C-C chemokine receptor type 5 (CCR5) expression, and the amount of C-C chemokine ligand 5 (CCL5), a ligand for CCR5, was increased in capillary veins of psoriasis plaques. CCR5+ iNKT cell numbers significantly correlated with the number of capillary vein endothelial cells expressing CCL5 in PV. Furthermore, the number of CCL5+ capillary veins correlated with the maximal rete ridge length.CONCLUSIONS:IFN-γ/CCR5 expression in iNKT cells and CCL5 expression in dermal papillae vessels of dermal papillae are correlated with the development of psoriasiform hyperplasia and microabscess. We propose that these iNKT cells may become useful targets for development of novel therapeutic approaches to PV. This article is protected by copyright. All rights reserved.

Yu Kakimoto, Shinji Ito, Hitoshi Abiru, Hirokazu Kotani, Munetaka Ozeki, Keiji Tamaki, Tatsuaki Tsuruyama*,
Sorbin and SH3 domain-containing protein 2 is released from infarcted heart in the very early phase: proteomic analysis of cardiac tissues from patients,
Journal of American Heart Association 2, e000565 (2013).

[Summary] We applied a novel proteomic approach to formalin-fixed, paraffin-embedded human tissue and aimed to reveal the molecular changes in the very early phase of acute myocardial infarction.　Heart tissue samples were collected from 5 patients who died within 7 hours of myocardial infarction and from 5 age- and sex-matched control cases. Infarcted and control myocardia were histopathologically diagnosed and captured using laser microdissection. Proteins were extracted using an originally established method and analyzed using liquid chromatography-tandem mass spectrometry. The label-free quantification demonstrated that the levels of 21 proteins differed significantly between patients and controls. In addition to known biomarkers, the sarcoplasmic protein sorbin and SH3 domain-containing protein 2 (SORBS2) was greatly reduced in infarcted myocardia. Immunohistochemical analysis of cardiac tissues confirmed the decrease, and Western blot analysis showed a significant increase in serum sorbin and SH3 domain-containing protein 2 in acute myocardial infarction patients (n=10) compared with control cases (n=11).Our advanced comprehensive analysis using patient tissues and serums indicated that sarcoplasmic sorbin and SH3 domain-containing protein 2 is released from damaged cardiac tissue into the bloodstream upon lethal acute myocardial infarction.

*Hiroaki Ito, Toru Yamanaka, Shou Kato, Tsutomu Hamada, Masahiro Takagi, *Masatoshi Ichikawa, *Kenichi Yoshikawa,
Dynamical formation of lipid bilayer vesicles fromlipid-coated droplets across a planar monolayer at an oil/water interface,
Soft Matter 9, 9539–9547 (2013).

[Summary] Recently, the transfer method has been shown to be useful for preparing cell-sized phospholipid bilayervesicles, within which desired substances at desired concentrations can be encapsulated, with a desiredasymmetric lipid composition. Here, we investigated the transfer process of water-in-oil (W/O) dropletscoated by phospholipid monolayers across an oil/water interface by both experimental observation andtheoretical modeling. Real-time experimental observation of the transfer revealed that the transferprocess is characterized by three kinetic regimes: a precontact process (approaching regime), an earlyfast process (entering regime), and a late slow process (relaxation regime). In addition, bigger dropletsrequire much more time to transfer than smaller droplets. We propose a theoretical model to interpretthis kinetic process. Our theoretical model reproduces the essential aspects of the transfer kinetics,including its size-dependence.

Tomo Kurimura, *Masatoshi Ichikawa, Masahiro Takinoue, *Kenichi Yoshikawa,
Back-and-forth micromotion of aqueous droplets in a dc electric field,
Physical Review E 88, 042918/1-5 (2013).

[Summary] Recently, it was reported that an aqueous droplet in an oil phase exhibited rhythmic back-and-forth motion under stationary dc voltage on the order of 100 V. Here, we demonstrate that the threshold voltage for inducing such oscillation is successfully decreased to the order of 10 V through downsizing of the experimental system. Notably, the threshold electric field tends to decrease with a nonlinear scaling relationship accompanied by the downsizing. We derive a simple theoretical model to interpret the system size dependence of the threshold voltage. This model equation suggests the unique effect of additional noise, which is qualitatively characterized as a coherent resonance by an actual experiment as a kind of coherent resonance. Our result would provide insight into the construction of micrometer-sized self-commutating motors and actuators in microfluidic and micromechanical devices.