業 績  

  • *Hiroki Kojima, Tom Froese, Mizuki Oka, Hiroyuki Iizuka andTakashi Ikegami,
    T. A Sensorimotor Signature of the Transition to Conscious Social Perception: Co-regulation of Active and Passive Touch.,
    Frontiers in psychology 8, 1778 (2017).
    It is not yet well understood how we become conscious of the presence of other people as being other subjects in their own right. Developmental and phenomenological approaches are converging on a relational hypothesis: my perception of a “you” is primarily constituted by another subject’s attention being directed toward “me.” This is particularly the case when my body is being physically explored in an intentional manner. We set out to characterize the sensorimotor signature of the transition to being aware of the other by re-analyzing time series of embodied interactions between pairs of adults (recorded during a “perceptual crossing” experiment). Measures of turn-taking and movement synchrony were used to quantify social coordination, and transfer entropy was used to quantify direction of influence. We found that the transition leading to one’s conscious perception of the other’s presence was indeed characterized by a significant increase in one’s passive reception of the other’s tactile stimulations. Unexpectedly, one’s clear experience of such passive touch was consistently followed by a switch to active touching of the other, while the other correspondingly became more passive, which suggests that this intersubjective experience was reciprocally co-regulated by both participants.
  • Sinapayen, L., & Ikegami, T.,
    Online fitting of computational cost to environmental complexity: Predictive coding with the ε-network,
    Proceedings of the 14th European Conference on Artificial Life 2017 14, 380-387 (2017).
    We propose the Epsilon Network (ε-network), a network that automatically adjusts its size to the complexity of a stream of data while performing online learning. The network optimises its topology during training, simultaneously adding and removing neurons and weights: it adds neurons where they can raise performance, and removes redundant neurons while preserving performance. The network is a neural realisation of the ε-machine devised by Crutchfield and al. (Crutchfield and Young (1989)). In this paper our network is trained to predict video frames; we evaluate it on simple, complex, and noisy videos and show that the final number of neurons is a good indicator of the complexity and predictability of the data stream.
  • Doi, I., Ikegami, T., Masumori, A., Kojima, H., Ogawa K., & Ishiguro, H.,
    A new design principle for an autonomous robot,
    Proceedings of the 14th European Conference on Artificial Life 2017 14, 490-466 (2017).
    Based on the principle of artificial life, we developed an upper-body android called "Alter." Alter is human-like in appearance, receives sensory information from the outside via an autonomous sensor system located around the android, and moves spontaneously using two autonomous systems of internal dynamics. Its body and arms contain a central pattern generator with seven degrees of freedom and hundreds of plastic artificial neurons. We investigated Alter’s environmental adaptability and the spontaneity of is behavioral patterns. In addition, we discuss the conditions under which a robot can become lifelike.
  • Masumori, A., Sinapayen, L., & Ikegami, T.,
    Learning by Stimulation Avoidance Scale to Large Neural Networks,
    Proceedings of the 14th European Conference on Artificial Life 2017 14, 275-282 (2017).
    Spiking neural networks with spike-timing dependent plasticity (STDP) can learn to avoid the external stimulations spontaneously. This principle is called "Learning by Stimulation Avoidance" (LSA) and can be used to reproduce learning experiments on cultured biological neural networks. LSA has promising potential, but its application and limitations have not be studied extensively. This paper focuses on the scalability of LSA for large networks and shows that LSA works well in small networks (100 neurons) and can be scaled to networks up to approximately 3,000 neurons.
  • Masumori, A., Maruyama, N., Mita, T., Bakkum, D., Frey, U., Takahashi, H.,& Ikegami, T.,
    Learning by Stimulation Avoidance in Cultured Neuronal Cells,
    The 2nd International Symposium on Swarm Behavior and Bio-Inspired Robotics(SWARM 2017) , 275-282 (2017).
    Cultured neural systems are much simpler than real brain systems, however, they have essential properties, including spontaneous activity, various types and distribution of cells, high connectivity, and rich and complex controllability. Previous works show that dissociated cultured neurons can learn a simple task as if it behaves to avoid a stimulation from outside. By simulating a simple model neural network, we show that spiking neural networks with spike- timing dependent plasticity (STDP) can reproduce such learning results. Recently, we extended the phenomenon to larger networks. We name this principle ”learning by stimulation avoidance” (LSA). The efficiency of LSA is proved by computer simulation, and the next step is to see whether the same mechanism actually works in a biological neural network or not. In this study, we conducted learning experiments using smaller-sized cultured neuronal cells than previous works, and this revealed that such learning results can be scaled to small network in vitro like the results in silico. In addition, the results of neural dynamics analysis suggest that cultured neurons change the network structure with synaptic plasticity to achieve a learning task rather than merely increase whole firing rates. It also suggest that LSA based on a synaptic plasticity can actually work in biological neural networks.
  • Maruyama, N., Hashimoto, Y., Mototake, Y., Saito, D., & Ikegami, T.,
    Revisiting Classification of Large Scale Flocking,
    The 2nd International Symposium on Swarm Behavior and Bio-Inspired Robotics(SWARM 2017) , 307-310 (2017).
    Simulation of a million flocks is studied by using a simple boid model. It is modeled with a differential equation on the 3D space. Flocking pattern in general is a function of both space and time scales, and it is not a mere aggregation process in a physical space. We also calculate the fluctuation of speed and the local number for each flock size and report the characteristics of fluctuation dependent on flock size. This paper can clarify the hierarchical nature of flocking.
  • Hashimoto, Y., Ikegami, T.,
    Novelty production in tagging crowds,
    The 2nd International Symposium on Swarm Behavior and Bio-Inspired Robotics(SWARM 2017), 311-312 (2017).
    In social tagging systems, service users create a number of tags, each of which is reused according to people's preferences. As a result, the diversity of tag vocabulary continues to increase as they are exposed to selection pressure from cultural preferences of the time. This is analogous to living ecosystems in nature. We address the question how new words arise and get accepted by crowds in terms of community structure of the network defined by the vocabulary usage of users. The result suggests that a new word, or novelty, that would be accepted by other users tend to come from the peripheral part of the network.
  • Yugi K., Kuroda S.,
    Metabolism as a signal generator across trans-omic networks at distinct time scales,
    Curr. Opin. Sys. Biol. 8, 59-66 (2018).
    Metabolism not only involves outcomes of signal transduction, gene expression, and chromatin/DNA modification but also serves as a signal generator across omic layers. Metabolic signals emerge from interplay of biochemical interactions across different omic layers denoted the trans-omic network. Importantly, metabolic signals selectively use distinct time scales inherent to each omic layer and coordinately control cellular homeostasis. Recent advances in comprehensive measurement technologies allow identification of metabolism-centric trans-omic networks. Here we review the roles of metabolism as a generator of signals that circulates trans-omic network in distinct time scales, thereby maintaining cellular homeostasis in higher organisms.
  • Ueno A, Omori Y, Sugita Y, Watanabe S, Chaya T, Kozuka T, Kon T, Yoshida S, Matsushita K, Kuwahara R, Kajimura N, Okada Y, *Furukawa T.,
    Lrit1, a retinal transmembrane protein, regulates selective synapse formation in cone photoreceptor cells and visual acuity.,
    Cell Reports 22, 3562-3573 (2018).
    In the vertebrate retina, cone photoreceptors play crucial roles in photopic vision by transmitting light-evoked signals to ON- and/or OFF-bipolar cells. However, the mechanisms underlying selective synapse formation in the cone photoreceptor pathway remain poorly understood. Here, we found that Lrit1, a leucine-rich transmembrane protein, localizes to the photoreceptor synaptic terminal and regulates the synaptic connection between cone photoreceptors and cone ON-bipolar cells. Lrit1-deficient retinas exhibit an aberrant morphology of cone photoreceptor pedicles, as well as an impairment of signal transmission from cone photoreceptors to cone ON-bipolar cells. Furthermore, we demonstrated that Lrit1 interacts with Frmpd2, a photoreceptor scaffold protein, and with mGluR6, an ON-bipolar cell-specific glutamate receptor. Additionally, Lrit1-null mice showed visual acuity impairments in their optokinetic responses. These results suggest that the Frmpd2-Lrit1-mGluR6 axis regulates selective synapse formation in cone photoreceptors and is essential for normal visual function.
  • Ohashi, K., Fujii, M., Uda, S., Kubota, H., Komada, H., Sakaguchi, K., Ogawa, W., Kuroda, S.,
    Increase in hepatic and decrease in peripheral insulin clearance characterize abnormal temporal patterns of serum insulin in diabetic subjects,
    NPJ Syst. Biol. 4, 14 (2018).
    Insulin plays a central role in glucose homeostasis, and impairment of insulin action causes glucose intolerance and leads to type 2 diabetes mellitus (T2DM). A decrease in the transient peak and sustained increase of circulating insulin following an infusion of glucose accompany T2DM pathogenesis. However, the mechanism underlying this abnormal temporal pattern of circulating insulin concentration remains unknown. Here we show that changes in opposite direction of hepatic and peripheral insulin clearance characterize this abnormal temporal pattern of circulating insulin concentration observed in T2DM. We developed a mathematical model using a hyperglycemic and hyperinsulinemic-euglycemic clamp in 111 subjects, including healthy normoglycemic and diabetic subjects. The hepatic and peripheral insulin clearance significantly increase and decrease, respectively, from healthy to borderline type and T2DM. The increased hepatic insulin clearance reduces the amplitude of circulating insulin concentration, whereas the decreased peripheral insulin clearance changes the temporal patterns of circulating insulin concentration from transient to sustained. These results provide further insight into the pathogenesis of T2DM, and thus may contribute to develop better treatment of this condition.
  • Seohyun Lee, Hyuno Kim, Hideo Higuchi,
    Focus Stabilization by Axial Position Feedback in Biomedical Imaging Microscopy,
    IEEE Sensors Applications Symposium 2018, 309-314 (2018).
  • Motoshi Kaya, Yoshiaki Tani, Takumi Washio, Toshiaki Hisada and Hideo Higuchi,
    Coordinated force generation of skeletal myosins in myofilaments through motor coupling,
    Nature Communications 8, 16036 (2017).
    In contrast to processive molecular motors, skeletal myosins form a large motor ensemble for contraction of muscles against high loads. Despite numerous information on the molecular properties of skeletal myosin, its ensemble effects on collective force generation have not been rigorously clarified. Here we show 4 nm stepwise actin displacements generated by synthetic myofilaments beyond a load of 30 pN, implying that steps cannot be driven exclusively by single myosins, but potentially by coordinated force generations among multiple myosins. The simulation model shows that stepwise actin displacements are primarily caused by coordinated force generation among myosin molecules. Moreover, the probability of coordinated force generation can be enhanced against high loads by utilizing three factors: strain-dependent kinetics between force-generating states; multiple power stroke steps; and high ATP concentrations. Compared with other molecular motors, our findings reveal how the properties of skeletal myosin are tuned to perform cooperative force generation for efficient muscle contraction.
  • Akira Adachi, Satoshi Senmatsu, Ryuta Asada, Takuya Abe, Charles S. Hoffman, Kunihiro Ohta, *Kouji Hirota,
    Interplay between chromatin modulators and histone acetylation regulates the formation of accessible chromatin in the upstream regulatory region of fission yeast fbp1.,
    Genes & Genetic Systems , in press (2018).
    Numerous noncoding RNA transcripts are detected in eukaryotic cells. Noncoding RNAs transcribed across gene promoters are involved in the regulation of mRNA transcription via chromatin modulation. This function of noncoding RNA transcription was first demonstrated for the fission yeast fbp1 gene, where a cascade of noncoding RNA transcription events induces chromatin remodeling to facilitate transcription factor binding. We recently demonstrated that the noncoding RNAs from the fbp1 upstream region facilitate binding of the transcription activator Atf1 and thereby promote histone acetylation. Histone acetylation by histone acetyl transferases (HATs) and ATP-dependent chromatin remodelers (ADCRs) are implicated in chromatin remodeling, but the interplay between HATs and ADCRs in this process has not been fully elucidated. Here, we examine the roles played by two distinct ADCRs, Snf22 and Hrp3, and by the HAT Gcn5 in the transcriptional activation of fbp1. Snf22 and Hrp3 redundantly promote disassembly of chromatin in the fbp1 upstream region. Gcn5 critically contributes to nucleosome eviction in the absence of either Snf22 or Hrp3, presumably by recruiting Hrp3 in snf22∆ cells and Snf22 in hrp3∆ cells. Conversely, Gcn5-dependent histone H3 acetylation is impaired in snf22∆/hrp3∆ cells, suggesting that both redundant ADCRs induce recruitment of Gcn5 to the chromatin array in the fbp1 upstream region. These results reveal a previously unappreciated interplay between ADCRs and histone acetylation in which histone acetylation facilitates recruitment of ADCRs, while ADCRs are required for histone acetylation
  • Atsushi Kamimura, *Kunihiko Kaneko,
    Exponential growth for self-reproduction in a catalytic reaction network: relevance of a minority molecular species and crowdedness,
    New Journal of Physics 20, 035001 (2018).
    Explanation of exponential growth in self-reproduction is an important step toward elucidation of the origins of life because optimization of the growth potential across rounds of selection is necessary for Darwinian evolution. To produce another copy with approximately the same composition, the exponential growth rates for all components have to be equal. How such balanced growth is achieved, however, is not a trivial question, because this kind of growth requires orchestrated replication of the components in stochastic and nonlinear catalytic reactions. By considering a mutually catalyzing reaction in two- and three-dimensional lattices, as represented by a cellular automaton model, we show that self-reproduction with exponential growth is possible only when the replication and degradation of one molecular species is much slower than those of the others, i.e., when there is a minority molecule. Here, the synergetic effect of molecular discreteness and crowding is necessary to produce the exponential growth. Otherwise, the growth curves show superexponential growth because of nonlinearity of the catalytic reactions or subexponential growth due to replication inhibition by overcrowding of molecules. Our study emphasizes that the minority molecular species in a catalytic reaction network is necessary to acquire evolvability at the primitive stage of life.
  • K. Tokuyama, Y. Toya, T. Horinouchi, C. Furusawa, F. Matsuda, *H. Shimizu,
    Application of adaptive laboratory evolution to overcome a flux limitation in an Escherichia coli production strain.,
    Biotechnology and Bioengineering, 1-10 (2018).
    Gene deletion strategies using flux balance analysis (FBA) have improved the growth-coupled production of various compounds. However, the productivities were often below the expectation because the cells failed to adapt to these genetic perturbations. Here, we demonstrate the productivity of the succinate of the designed gene deletion strain was improved by adaptive laboratory evolution (ALE). Although FBA predicted deletions of adhE-pykAF-gldA-pflB lead to produce succinate from glycerol with a yield of 0.45 C-mol/C-mol, the knockout mutant did not produce only 0.08 C-mol/Cmol, experimentally. After the ALE experiments, the highest succinate yield of an evolved strain reached to the expected value. Genome sequencing analysis revealed all evolved strains possessed novel mutations in ppc of I829S or R849S. In vitro enzymatic assay and metabolic profiling analysis revealed that these mutations desensitizing an allosteric inhibition by L-aspartate and improved the flux through Ppc, while the activity of Ppc in the unevolved strain was tightly regulated by L-aspartate. These result demonstrated that the evolved strains achieved the improvement of succinate production by expanding the flux space of Ppc, realizing the predicted metabolic state by FBA.
  • C. Furusawa, T. Horinouchi, T. Maeda,,
    Toward prediction and control of antibiotic-resistance evolution,
    Current Opinion in Biotechnology 54, 45-59 (2018).
    The emergence of antibiotic-resistant bacteria is a serious public concern. To deal with this problem, recent advances in technology and the use of laboratory evolution experiments have provided valuable information on the phenotypic and genotypic changes that occur during the evolution of resistance. These studies have demonstrated the existence of evolutionary constraints on the development of drug-resistance, which suggests predictability in its evolution. In this review, we focus on the possibility to predict and control the evolution of antibiotic resistance, based on quantitative analysis of phenotypic and genotypic changes observed in bacterial laboratory evolution. We emphasize the key challenges in evolutionary biology that will contribute to the development of appropriate treatment strategies for preventing resistance evolution.
  • *Takaki Yamamoto and Masaki Sano,
    Theoretical model of chirality-induced helical self-propulsion,
    Phys. Rev. E 97, 012607/1-11 (2018).
    We recently reported the experimental realization of a chiral artificial microswimmer exhibiting helical self-propulsion [T. Yamamoto and M. Sano, Soft Matter 13, 3328 (2017)]. In the experiment, cholesteric liquid crystal (CLC) droplets dispersed in surfactant solutions swam spontaneously, driven by the Marangoni flow, in helical paths whose handedness is determined by the chirality of the component molecules of CLC. To study the mechanism of the emergence of the helical self-propelled motion, we propose a phenomenological model of the self-propelled helical motion of the CLC droplets. Our model is constructed by symmetry argument in chiral systems, and it describes the dynamics of CLC droplets with coupled time-evolution equations in terms of a velocity, an angular velocity, and a tensor variable representing the symmetry of the helical director field of the droplet. We found that helical motions as well as other chiral motions appear in our model. By investigating bifurcation behaviors between each chiral motion, we found that the chiral coupling terms between the velocity and the angular velocity, the structural anisotropy of the CLC droplet, and the nonlinearity of model equations play a crucial role in the emergence of the helical motion of the CLC droplet.
  • T. Hirasawa, M. Saito, K. Yoshikawa, C. Furusawa,,
    Integrated Analysis of the Transcriptome and Metabolome of Corynebacterium glutamicum during Penicillin-Induced Glutamic Acid Production,
    Biotechnology Journal 1700612, 1-11 (2018).
    Corynebacterium glutamicum is known for its ability to produce glutamic acid and has been utilized for the fermentative production of various amino acids. Glutamic acid production in C. glutamicum is induced by penicillin. In this study, we analyzed the transcriptome and metabolome of C. glutamicum to understand the mechanism of penicillin‐induced glutamic acid production. Transcriptomic analysis with DNA microarray revealed that expression of some glycolysis‐ and TCA cycle‐related genes, which include those encoding the enzymes involved in conversion of glucose to 2‐oxoglutaric acid, was upregulated after penicillin addition. Meanwhile, expression of some TCA cycle‐related genes, encoding the enzymes for conversion of 2‐oxoglutaric acid to oxaloacetic acid, and the anaplerotic reactions decreased. In addition, expression of NCgl1221 and odhI, encoding proteins involved in glutamic acid excretion and inhibition of the 2‐oxoglutarate dehydrogenase, respectively, was upregulated. Functional category enrichment analysis of genes upregulated and downregulated after penicillin addition revealed that genes for signal transduction systems were enriched among upregulated genes, whereas those for energy production and carbohydrate and amino acid metabolisms were enriched among the downregulated genes. As for the metabolomic analysis using capillary electrophoresis time‐of‐flight mass spectrometry, the intracellular content of most metabolites of the glycolysis and the TCA cycle decreased dramatically after penicillin addition. Overall, these results indicate that the cellular metabolism and glutamic acid excretion are mainly optimized at the transcription level during penicillin‐induced glutamic acid production by C. glutamicum.
  • Daiki Nishiguchi, Junichiro Iwasawa, Hong-Ren Jiang and Masaki Sano,
    Flagellar dynamics of chains of active Janus particles fueled by an AC electric field,
    New Journal of Physics 20, 015002/1-14 (2018).
    We study the active dynamics of self-propelled asymmetrical colloidal particles (Janus particles) fueled by an AC electric field. Both the speed and direction of the self-propulsion, and the strength of the attractive interaction between particles can be controlled by tuning the frequency of the applied electric field and the ion concentration of the solution. The strong attractive force at high ion concentration gives rise to chain formation of the Janus particles, which can be explained by the quadrupolar charge distribution on the particles. Chain formation is observed irrespective of the direction of the self-propulsion of the particles. When both the position and the orientation of the heads of the chains are fixed, they exhibit beating behavior reminiscent of eukaryotic flagella. The beating frequency of the chains of Janus particles depends on the applied voltage and thus on the self-propulsive force. The scaling relation between the beating frequency and the self-propulsive force deviates from theoretical predictions made previously on active filaments. However, this discrepancy is resolved by assuming that the attractive interaction between the particles is mediated by the quadrupolar distribution of the induced charges, which gives indirect but convincing evidence on the mechanisms of the Janus particles. This signifies that the dependence between the propulsion mechanism and the interaction mechanism, which had been dismissed previously, can modify the dispersion relations of beating behaviors. In addition, hydrodynamic interaction within the chain, and its effect on propulsion speed, are discussed. These provide new insights into active filaments, such as optimal flagellar design for biological functions.

  • Eng Kuan Moo, Daniel R.Peterson, Timothy R. Leonard, Motoshi Kaya and Walter Herzog,
    In vivo muscle force and muscle power during near-maximal frog jumps.,
    PloS one (2017).
    Frogs’ outstanding jumping ability has been associated with a high power output from the leg extensor muscles. Two main theories have emerged to explain the high power output of the frog leg extensor muscles, either (i) the contractile conditions of all leg extensor muscles are optimized in terms of muscle length and speed of shortening, or (ii) maximal power is achieved through a dynamic catch mechanism that uncouples fibre shortening from the corresponding muscle-tendon unit shortening. As in vivo instantaneous power generation in frog hind limb muscles during jumping has never been measured directly, it is hard to distinguish between the two theories. In this study, we determined the instantaneous variable power output of the plantaris longus (PL) of Lithobates pipiens (also known as Rana pipiens), by directly measuring the in vivo force, length change, and speed of muscle and fibre shortening in near maximal jumps. Fifteen near maximal jumps (> 50cm in horizontal distance) were analyzed. High instantaneous peak power in PL (536 ± 47 W/kg) was achieved by optimizing the contractile conditions in terms of the force-length but not the force-velocity relationship, and by a dynamic catch mechanism that decouples fascicle shortening from muscle-tendon unit shortening. We also found that the extra-muscular free tendon likely amplifies the peak power output of the PL by modulating fascicle shortening length and shortening velocity for optimum power output, but not by releasing stored energy through recoiling as the tendon only started recoiling after peak PL power had been achieved.
  • Lana Sinapayen, Atsushi Masumori, Takashi Ikegami,
    Learning by Stimulation Avoidance: A Principle to Control Spiking Neural Networks Dynamics.,
    PLoS One 12(2), e0170388 (2017).
    Learning based on networks of real neurons, and learning based on biologically inspired models of neural networks, have yet to find general learning rules leading to widespread applications. In this paper, we argue for the existence of a principle allowing to steer the dynamics of a biologically inspired neural network. Using carefully timed external stimulation, the network can be driven towards a desired dynamical state. We term this principle "Learning by Stimulation Avoidance" (LSA). We demonstrate through simulation that the minimal sufficient conditions leading to LSA in artificial networks are also sufficient to reproduce learning results similar to those obtained in biological neurons by Shahaf and Marom, and in addition explains synaptic pruning. We examined the underlying mechanism by simulating a small network of 3 neurons, then scaled it up to a hundred neurons. We show that LSA has a higher explanatory power than existing hypotheses about the response of biological neural networks to external simulation, and can be used as a learning rule for an embodied application: learning of wall avoidance by a simulated robot. In other works, reinforcement learning with spiking networks can be obtained through global reward signals akin simulating the dopamine system; we believe that this is the first project demonstrating sensory- motor learning with random spiking networks through Hebbian learning relying on environmental conditions without a separate reward system.
  • Takumi Washio, Toshiaki Hisada, Seine A. Shintani and Hideo Higuchi,
    Analysis of spontaneous oscillations for a three state power stroke model,
    Physical Review E. 95, 022411 (2017).
    Our study considers the mechanism of the spontaneous oscillations of molecular motors that are driven by the power stroke principle by applying linear stability analysis around the stationary solution. By representing the coupling equation of microscopic molecular motor dynamics and mesoscopic sarcomeric dynamics by a rank-1 updated matrix system, we derived the analytical representations of the eigenmodes of the Jacobian matrix that cause the oscillation. Based on these analytical representations, we successfully derived the essential conditions for the oscillation in terms of the rate constants of the power stroke and the reversal stroke transitions of the molecular motor. Unlike the two-state model, in which the dependence of the detachment rates on the motor coordinates or the applied forces on the motors plays a key role for the oscillation, our three-state power stroke model demonstrates that the dependence of the rate constants of the power and reversal strokes on the strains in the elastic elements in the motor molecules plays a key role, where these rate constants are rationally determined from the free energy available for the power stroke, the stiffness of the elastic element in the molecular motor, and the working stroke size. By applying the experimentally confirmed values to the free energy, the stiffness, and the working stroke size, our numerical model reproduces well the experimentally observed oscillatory behavior. Furthermore, our analysis shows that two eigenmodes with real positive eigenvalues characterize the oscillatory behavior, where the eigenmode with the larger eigenvalue indicates the transient of the system of the quick sarcomeric lengthening induced by the collective reversal strokes, and the smaller eigenvalue correlates with the speed of sarcomeric shortening, which is much slower than lengthening. Applying the perturbation analyses with primal physical parameters, we find that these two real eigenvalues occur on two branches derived from a merge point of a pair of complex-conjugate eigenvalues generated by Hopf bifurcation.
  • Takaho Tsuchiya, Masashi Fujii, Naoki Matsuda, Katsuyuki Kunida, Shinsuke Uda, Hiroyuki Kubota, Katsumi Konishi, *Shinya Kuroda,
    System identification of signaling dependent gene expression with different time-scale data,
    PLOS Computational Biology 13(12), e1005913 (2017).
    Author summary The key points of this study are two-fold: The first point is the decoding mechanism for cell differentiation. We previously demonstrated the encoding mechanism of cell fate decision information by transient and sustained ERK activation in PC12 cells, and also identified the decoding genes essential for cell differentiation in PC12 cells, including Metrnl, Dclk1, and Serpinb1a, denoted as LP (latent process) genes, which are the decoders of neurite length information. Importantly, the expression levels of the LP genes, but not the phosphorylation level of ERK, correlate with neurite length. Thus, the decoding mechanism of signaling activities by LP gene expression is a key issue for understanding the mechanism of cell differentiation. Here we identified a selective NGF- and PACAP-signaling decoding system by LP gene expression for neurite extension by developing a system identification method. The second point is the modeling. Cells decode information of signaling activation at a scale of tens of minutes by downstream gene expression with a scale of hours to days, leading to cell fate decisions such as cell differentiation. However, no system identification method with such different time scales exists. Here we developed a signal recovery technique in the field of compressed sensing originally developed for image analysis to biological sparse data of different time scales of signaling and gene expression.
  • Napo K. M. Cheung, Ryohei Nakamura, Ayako Uno, Masahiko Kumagai, Hiroto S. Fukushima, Shinichi Morishita & Hiroyuki Takeda*,
    Unlinking the methylome pattern from uncleotide sequence, revealed by large-scale in vivo genome engineering and methylome editing in medaka fish,
    PloS Genetics 13, e1007123 (2017).
  • Suguru Takagi, Benjamin T. Cocanougher, Sawako Niki, Dohjin Miyamoto, Hiroshi Kohsaka, Hokuto Kazama, Richard D. Fetter, James W. Truman, Marta Zlatic, Albert Cardona and *Akinao Nose,
    Divergent connectivity of homologous command-like neurons mediates segment-specific touch responses in Drosophila,
    Neuron 96(6), 1373-1387 (2017).
    Animals adaptively respond to a tactile stimulus by choosing an ethologically relevant behavior depending on the location of the stimuli. Here, we investigate how somatosensory inputs on different body segments are linked to distinct motor outputs in Drosophila larvae. Larvae escape by backward locomotion when touched on the head, while they crawl forward when touched on the tail. We identify a class of segmentally repeated second-order somatosensory interneurons, that we named Wave, whose activation in anterior and posterior segments elicit backward and forward locomotion, respectively. Anterior and posterior Wave neurons extend their dendrites in opposite directions to receive somatosensory inputs from the head and tail, respectively. Downstream of anterior Wave neurons, we identify premotor circuits including the neuron A03a5, which together with Wave, is necessary for the backward locomotion touch response. Thus, Wave neurons match their receptive field to appropriate motor programs by participating in different circuits in different segments.
  • James R. Krycer, Katsuyuki Yugi, Akiyoshi Hirayama, Daniel J. Fazakerley, Lake-Ee Quek, Richard Scalzo, Satoshi Ohno, Mark P. Hodson, Satsuki Ikeda, Futaba Shoji, Kumi Suzuki, Westa Domanova, Benjamin L. Parker, Marin E. Nelson, Sean J. Humphrey, Nigel Turner, Kyle L. Hoehn, Gregory J. Cooney, Tomoyoshi Soga, *Shinya Kuroda and *David E. James,
    Dynamic metabolomics reveals that insulin primes the adipocyte for glucose metabolism,
    Cell Reports 21(12), 3536-3547 (2017).
    Insulin triggers an extensive signaling cascade to coordinate adipocyte glucose metabolism. It is considered that the major role of insulin is to provide anabolic substrates by activating GLUT4-dependent glucose uptake. However, insulin stimulates phosphorylation of many metabolic proteins. To examine the implications of this on glucose metabolism, we performed dynamic tracer metabolomics in cultured adipocytes treated with insulin. Temporal analysis of metabolite concentrations and tracer labeling revealed rapid and distinct changes in glucose metabolism, favoring specific glycolytic branch points and pyruvate anaplerosis. Integrating dynamic metabolomics and phosphoproteomics data revealed that insulin-dependent phosphorylation of anabolic enzymes occurred prior to substrate accumulation. Indeed, glycogen synthesis was activated independently of glucose supply. We refer to this phenomenon as metabolic priming, whereby insulin signaling creates a demand-driven system to "pull" glucose into specific anabolic pathways. This complements the supply-driven regulation of anabolism by substrate accumulation and highlights an additional role for insulin action in adipocyte glucose metabolism.
  • Takayuki Katoh, Yoshihiko Iwane, *Hiroaki Suga,
    Logical engineering of D-arm and T-stem of tRNA that enhances d-amino acid incorporation.,
    Nucleic Acids Research 45, 12601–12610 (2017).
  • 古澤力, 前田智也, 堀之内貴明,
    実験医学別冊「あなたのラボにAI×ロボットがやってくる(夏目徹 編) (2017).
  • Ryan Kniewel, Hajime Murakami, Yan Liu, Masaru Ito, Kunihiro Ohta, Nancy M. Hollingsworth and *Scott Keeney,
    Histone H3 threonine 11 phosphorylation is catalyzed directly by the meiosis-specific kinase Mek1 and provides a molecular readout of Mek1 activity in vivo. ,
    GENETICS 207, 1313-1333 (2017).
    Saccharomyces cerevisiae Mek1 is a CHK2/Rad53-family kinase that regulates meiotic recombination and progression upon its activation in response to DNA double-strand breaks (DSBs). The full catalog of direct Mek1 phosphorylation targets remains unknown. Here, we show that phosphorylation of histone H3 on threonine 11 (H3 T11ph) is induced by meiotic DSBs in S. cerevisiae and Schizosaccharomyces pombe. Molecular genetic experiments in S. cerevisiae confirmed that Mek1 is required for H3 T11ph and revealed that phosphorylation is rapidly reversed when Mek1 kinase is no longer active. Reconstituting histone phosphorylation in vitro with recombinant proteins demonstrated that Mek1 directly catalyzes H3 T11 phosphorylation. Mutating H3 T11 to nonphosphorylatable residues conferred no detectable defects in otherwise unperturbed meiosis, although the mutations modestly reduced spore viability in certain strains where Rad51 is used for strand exchange in place of Dmc1. H3 T11ph is therefore mostly dispensable for Mek1 function. However, H3 T11ph provides an excellent marker of ongoing Mek1 kinase activity in vivo. Anti-H3 T11ph chromatin immunoprecipitation followed by deep sequencing demonstrated that H3 T11ph was highly enriched at presumed sites of attachment of chromatin to chromosome axes, gave a more modest signal along chromatin loops, and was present at still lower levels immediately adjacent to DSB hotspots. These localization patterns closely tracked the distribution of Red1 and Hop1, axis proteins required for Mek1 activation. These findings provide insight into the spatial disposition of Mek1 kinase activity and the higher order organization of recombining meiotic chromosomes.
  • Chiba K, Chien K, Sobu Y, Hata S, Kato S, Nakaya T, Okada Y, Nairn AC, Kinjo M, Taru H, Wang R, Suzuki T.,
    Phosphorylation of KLC1 modifies interaction with JIP1 and abolishes the enhanced fast velocity of APP transport by kinesin-1 ,
    MOLECULAR BIOLOGY OF THE CELL 28, 3857-3869 (2017).
    In neurons, amyloid beta-protein precursor (APP) is transported by binding to kinesin-1, mediated by JNK-interacting protein 1b (JIP1b), which generates the enhanced fast velocity (EFV) and efficient high frequency (EHF) of APP anterograde transport. Previously, we showed that EFV requires conventional interaction between the JIP1b C-terminal region and the kinesin light chain 1 (KLC1) tetratricopeptide repeat, whereas EHF requires a novel interaction between the central region of JIP1b and the coiled-coil domain of KLC1. We found that phosphorylatable Thr466 of KLC1 regulates the conventional interaction with JIP1b. Substitution of Glu for Thr466 abolished this interaction and EFV, but did not impair the novel interaction responsible for EHF. Phosphorylation of KLC1 at Thr466 increased in aged brains, and JIP1 binding to kinesin-1 decreased, suggesting that APP transport is impaired by aging. We conclude that phosphorylation of KLC1 at Thr466 regulates the velocity of transport of APP by kinesin-1 by modulating its interaction with JIP1b.
  • Kazuki Ichikawa, Shingo Tomioka, Yuta Suzuki, Ryohei Nakamura, Koichiro Doi, Jun Yoshimura, Masahiko Kumagai, Yusuke Inoue, Yui Uchida, Naoki Irie, Hiroyuki Takeda* & Shinich Morishita*,
    Centromere evolution and CpG methylation during vertebrate speciation ,
    Nature Communications 1833, 1-11 (2017).
    Centromeres and large-scale structural variants evolve and contribute to genome diversityduring vertebrate speciation. Here, we perform de novo long-read genome assembly of threeinbred medaka strains that are derived from geographically isolated subpopulations andundergo speciation. Using single-molecule real-time (SMRT) sequencing, we obtain threechromosome-mapped genomes of length ~734, ~678, and ~744Mbp with a resource oftwenty-two centromeric regions of length 20–345kbp. Centromeres are positionally conservedamong the three strains and even between four pairs of chromosomes that wereduplicated by the teleost-specific whole-genome duplication 320–350 million years ago. Thecentromeres do not all evolve at a similar pace; rather, centromeric monomers in nonacrocentricchromosomes evolve significantly faster than those in acrocentric chromosomes.Using methylation sensitive SMRT reads, we uncover centromeres are mostly hypermethylatedbut have hypomethylated sub-regions that acquire unique sequence compositionsindependently. These findings reveal the potential of non-acrocentric centromere evolution tocontribute to speciation.
  • Tsukasa Kusakizako,Yoshiki Tanaka, Christopher J. Hipolito, *Hiroaki Suga, Osamu Nureki,
    Crystallographic analysis of MATE-type multidrug exporter with its inhibitors.,
    Methods Mol Biol. 1700, 7-57 (2017).
    Multidrug exporters expressed in pathogens efflux substrate drugs such as antibiotics, and thus, the development of inhibitors against them has eagerly been anticipated. Furthermore, the crystal structures of multidrug exporters with their inhibitors provide novel insights into the inhibitory mechanism and the development of more specific and effective inhibitors. We previously reported the complex structures of the Multidrug And Toxic compound Extrusion (MATE)-type multidrug exporter with the macrocyclic peptides, which inhibit the efflux of substrates by the MATE-type multidrug exporter (Tanaka et al., Nature 496:247–251, 2013). In this chapter, we describe methodologies of the screening and synthesis of macrocyclic peptides as inhibitors, as well as the purification, crystallization, and structure determination of the complexes of the MATE-type multidrug exporter with its inhibitors.
  • *Nobuto Takeuchi, Paulien Hogeweg, Kunihiko Kaneko,
    Conceptualizing the Origin of Life in terms of Evolution,
    Philosophical Transactions of the Royal Society A 375, 20160346 (2017).
    In this opinion piece, we discuss how to place evolution in the context of origin-of-life research. Our discussion starts with a popular definition: ‘life is a self-sustained chemical system capable of undergoing Darwinian evolution’. According to this definition, the origin of life is the same as the origin of evolution: evolution is the ‘end’ of the origin of life. This perspective, however, has a limitation, in that the ability of evolution in and of itself is insufficient to explain the origin of life as we know it, as indicated by Spiegelman’s and Lincoln and Joyce’s experiments. This limitation provokes a crucial question: What conditions are required for replicating systems to evolve into life? From this perspective, the origin of life includes the emergence of life through evolution: evolution is a ‘means’ of the origin of life. After reviewing Eigen’s pioneering work on this question, we mention our ongoing work suggesting that a key condition might be conflicting multi-level evolution. Taken together, there are thus two questions regarding the origin of life: how evolution gets started, and how evolution produces life. Evolution is, therefore, at the centre of the origin of life, where the two lines of enquiry must meet.This article is part of the themed issue ‘Reconceptualizing the origins of life’.
  • Atsushi Shibai, Yusuke Takahashi, Yuka Ishizawa, Daisuke Motooka, Shota Nakamura, Bei-Wen Ying, and *Saburo Tsuru,
    Mutation accumulation under UV radiation in Escherichia coli,
    Scientific Reports 7(14531), 1-12 (2017).
  • Jo Marie Bacusmo, Alexandra B. Kuzmishin, William A. Cantara, Yuki Goto, *Hiroaki Suga & Karin Musier-Forsyth,
    Quality control by trans-editing factor prevents global mistranslation of non-protein amino acid α-aminobutyrate,
    RNA Biol., 1-10 (2017).
  • *石原秀至、澤井哲,
    パリティ 32, 22-25 (2017).
  • Ryo Yokota, Yuki Kaminaga, Tetsuya J. Kobayashi,
    Quantification of Inter-Sample Differences in T-Cell Receptor Repertoires Using Sequence-Based Information.,
    Frontier Immunology 8, 1500 (2017).
    Inter-sample comparisons of T-cell receptor (TCR) repertoires are crucial for gaining a better understanding of the immunological states determined by different collections of T cells from different donor sites, cell types, and genetic and pathological backgrounds. For quantitative comparison, most previous studies utilized conventional methods in ecology, which focus on TCR sequences that overlap between pairwise samples. Some recent studies attempted another approach that is categorized into Poisson abundance models using the abundance distribution of observed TCR sequences. However, these methods ignore the details of the measured sequences and are consequently unable to identify sub-repertoires that might have important contributions to the observed inter-sample differences. Moreover, the sparsity of sequence data due to the huge diversity of repertoires hampers the performance of these methods, especially when few overlapping sequences exist. In this paper, we propose a new approach for REpertoire COmparison in Low Dimensions (RECOLD) based on TCR sequence information, which can estimate the low-dimensional structure by embedding the pairwise sequence dissimilarities in high-dimensional sequence space. The inter-sample differences between repertoires are then quantified by information-theoretic measures among the distributions of data estimated in the embedded space. Using datasets of mouse and human TCR repertoires, we demonstrate that RECOLD can accurately identify the inter-sample hierarchical structures, which have a good correspondence with our intuitive understanding about sample conditions. Moreover, for the dataset of transgenic mice that have strong restrictions on the diversity of their repertoires, our estimated inter-sample structure was consistent with the structure estimated by previous methods based on abundance or overlapping sequence information. For the dataset of human healthy donors and Sezary syndrome patients, our method also showed robust estimation performance even under the condition of high sparsity in TCR sequences, while previous studies failed to estimate the structure. In addition, we identified the sequences that contribute to the pairwise-sample differences between the repertoires with the different genetic backgrounds of mice. Such identification of the sequences contributing to variation in immune cell repertoires may provide substantial insight for the development of new immunotherapies and vaccines.
  • T. S. Hatakeyama, *C. Furusawa, ,
    Metabolic dynamics restricted by conserved carriers: Jamming and feedback,
    PLOS COMPUTATIONAL BIOLOGY 13-11, e1005847 (2017).
    To uncover the processes and mechanisms of cellular physiology, it first necessary to gain an understanding of the underlying metabolic dynamics. Recent studies using a constraint-based approach succeeded in predicting the steady states of cellular metabolic systems by utilizing conserved quantities in the metabolic networks such as carriers such as ATP/ADP as an energy carrier or NADH/NAD(+) as a hydrogen carrier. Although such conservation quantities restrict not only the steady state but also the dynamics themselves, the latter aspect has not yet been completely understood. Here, to study the dynamics of metabolic systems, we propose adopting a carrier cycling cascade (CCC), which includes the dynamics of both substrates and carriers, a commonly observed motif in metabolic systems such as the glycolytic and fermentation pathways. We demonstrate that the conservation laws lead to the jamming of the flux and feedback. The CCC can show slow relaxation, with a longer timescale than that of elementary reactions, and is accompanied by both robustness against small environmental fluctuations and responsiveness against large environmental changes. Moreover, the CCC demonstrates robustness against internal fluctuations due to the feedback based on the moiety conservation. We identified the key parameters underlying the robustness of this model against external and internal fluctuations and estimated it in several metabolic systems.
  • K. Tanabe, N. Sakata, H. Shimizu, *C. Furusawa,,
    Prediction of Cross-resistance and Collateral Sensitivity by Gene Expression profiles and Genomic Mutations,
    SCIENTIFIC REPORTS 7, 14009 (2017).
    In adaptive evolution, an increase in fitness to an environment is frequently accompanied by changes in fitness to other environmental conditions, called cross-resistance and sensitivity. Although the networks between fitness changes affect the course of evolution substantially, the mechanisms underlying such fitness changes are yet to be fully elucidated. Herein, we performed high-throughput laboratory evolution of Escherichia coli under various stress conditions using an automated culture system, and quantified how the acquisition of resistance to one stressor alters the resistance to other stressors. We demonstrated that resistance changes could be quantitatively predicted based on changes in the transcriptome of the resistant strains. We also identified several genes and gene functions, for which mutations were commonly fixed in the strains resistant to the same stress, which could partially explain the observed cross-resistance and collateral sensitivity. The integration of transcriptome and genome data enabled us to clarify the bacterial stress resistance mechanisms.
  • Sanki Tashiro Yuki Nishihara Kazuto Kugou Kunihiro Ohta *Junko Kanoh,
    Subtelomeres constitute a safeguard for gene expression and chromosome homeostasis,
    Nucleic Acids Research 45, 10333-10349 (2017).
    The subtelomere, a telomere-adjacent chromosomal domain, contains species-specific homologous DNA sequences, in addition to various genes. However, the functions of subtelomeres, particularly subtelomeric homologous (SH) sequences, remain elusive. Here, we report the first comprehensive analyses of the cellular functions of SH sequences in the fission yeast, Schizosaccharomyces pombe. Complete removal of SH sequences from the genome revealed that they are dispensable for mitosis, meiosis and telomere length control. However, when telomeres are lost, SH sequences prevent deleterious inter-chromosomal end fusion by facilitating intra-chromosomal circularization. Surprisingly, SH-deleted cells sometimes survive telomere loss through inter-chromosomal end fusions via homologous loci such as LTRs, accompanied by centromere inactivation of either chromosome. Moreover, SH sequences function as a buffer region against the spreading of subtelomeric heterochromatin into the neighboring gene-rich regions. Furthermore, we found a nucleosome-free region at the subtelomeric border, which may be a second barrier that blocks heterochromatin spreading into the subtelomere-adjacent euchromatin. Thus, our results demonstrate multiple defense functions of subtelomeres in chromosome homeostasis and gene expression.
  • 岡田康志,
    生体の科学 68(5), 378-379 (2017).
  • 小田有沙,平田祥人,太田邦史,合原一幸,
    実験医学 最強のステップUPシリーズ シングルセル解析プロトコール 別冊, 313-318 (2017).
  • Haiyang Hu, Masahiro Uesaka, Song Guo, Kotaro Shimai, Tsai-Ming Lu, Fang Li, Satoko Fujimoto, Masato Ishikawa, Shiping Liu, Yohei Sasagawa, Guojie Zhang, Shigeru Kuratani, Jr-Kai Yu, Takehiro G. Kusakabe, Philipp Khaitovich, Naoki Irie*; the EXPANDE Consortium,
    Constrained vertebrate evolution by pleiotropic genes,
    Nature Ecology & Evolution 1, 1722-1730 (2017).
    Despite morphological diversification of chordates over 550 million years of evolution, their shared basic anatomical pattern (or ‘bodyplan’) remains conserved by unknown mechanisms. The developmental hourglass model attributes this to phylum-wide conserved, constrained organogenesis stages that pattern the bodyplan (the phylotype hypothesis); however, there has been no quantitative testing of this idea with a phylum-wide comparison of species. Here, based on data from early-to-late embryonic transcriptomes collected from eight chordates, we suggest that the phylotype hypothesis would be better applied to vertebrates than chordates. Furthermore, we found that vertebrates’ conserved mid-embryonic developmental programmes are intensively recruited to other developmental processes, and the degree of the recruitment positively correlates with their evolutionary conservation and essentiality for normal development. Thus, we propose that the intensively recruited genetic system during vertebrates’ organogenesis period imposed constraints on its diversification through pleiotropic constraints, which ultimately led to the common anatomical pattern observed in vertebrates.
  • Ryohei Nakamura, Ayako Uno, Masahiko Kumagai, Shinichi Morishita&Hiroyuki Takeda*,
    Hypomethylated domain-enriched DNA motifs prepattern the accessible nucleosome organization in teleosts ,
    Epigenetics & Chromatin 110, 44 (2017).
  • Ryuta Asada Miki Umeda Akira Adachi Satoshi Senmatsu Takuya Abe Hiroshi Iwasaki Kunihiro Ohta Charles S. Hoffman *Kouji Hirota,
    Recruitment and delivery of the fission yeast Rst2 transcription factor via a local genome structure counteracts repression by Tup1-family corepressors. ,
    Nucleic Acids Res. 45, 9361-9371 (2017).
    Transcription factors (TFs) determine the transcription activity of target genes and play a central role in controlling the transcription in response to various environmental stresses. Three dimensional genome structures such as local loops play a fundamental role in the regulation of transcription, although the link between such structures and the regulation of TF binding to cis-regulatory elements remains to be elucidated. Here, we show that during transcriptional activation of the fission yeast fbp1 gene, binding of Rst2 (a critical C2H2 zinc-finger TF) is mediated by a local loop structure. During fbp1 activation, Rst2 is first recruited to upstream-activating sequence 1 (UAS1), then it subsequently binds to UAS2 (a critical cis-regulatory site located approximately 600 base pairs downstream of UAS1) through a loop structure that brings UAS1 and UAS2 into spatially close proximity. Tup11/12 (the Tup-family corepressors) suppress direct binding of Rst2 to UAS2, but this suppression is counteracted by the recruitment of Rst2 at UAS1 and following delivery to UAS2 through a loop structure. These data demonstrate a previously unappreciated mechanism for the recruitment and expansion of TF-DNA interactions within a promoter mediated by local three-dimensional genome structures and for timely TF-binding via counteractive regulation by the Tup-family corepressors.
  • Takayuki Katoh, Yoshihiko Iwane & *Hiroaki Suga,
    tRNA engineering for manipulating genetic code.,
    RNA Biol. 2017 Jul 19, 1-8 (2017).
  • Yusuke Inoue, Tomonori Saga, Takumi Aikawa, Masahiko Kumagai, Atsuko Shimada, Yasushi Kawaguchi, Kiyoshi Naruse, Shinichi Morishita, Akihiko Koga & Hiroyuki Takeda*,
    Complete fusion of a transposon and herpesvirus created the Teratorn mobile element in medaka fish ,
    Nature Communications 15, 551 (2017).
  • *Shuji Ishihara, Philippe Marcq, and Kaoru Sugimura,
    From cells to tissue: A continuum model of epithelial mechanics,
    Physical Review E 96, 022418/1-14 (2017).
    A two-dimensional continuum model of epithelial tissue mechanics was formulated using cellular-levelmechanical ingredients and cell morphogenetic processes, including cellular shape changes and cellularrearrangements. This model incorporates stress and deformation tensors, which can be compared withexperimental data. Focusing on the interplay between cell shape changes and cell rearrangements, we elucidateddynamical behavior underlying passive relaxation, active contraction-elongation, and tissue shear flow, includinga mechanism for contraction-elongation, whereby tissue flows perpendicularly to the axis of cell elongation. Thisstudy provides an integrated scheme for the understanding of the orchestration of morphogenetic processes inindividual cells to achieve epithelial tissue morphogenesis.
  • Byung-Sik Shin, Takayuki Katoh, Erik Gutierrez, Joo-Ran Kim, *Hiroaki Suga, Thomas E. Dever,
    Amino acid substrates impose polyamine, eIF5A, or hypusine requirement for peptide synthesis,
    Nucleic Acids Res. 45(14), 8392-8402 (2017).
  • Masahiko Ueda, Nobuto Takeuchi, *Kunihiko Kaneko,
    Stronger selection can slow down evolution driven by recombination on a smooth fitness landscape,
    PLoS ONE 12(8), e0183120 (2017).
    Stronger selection implies faster evolution—that is, the greater the force, the faster the change. This apparently self-evident proposition, however, is derived under the assumption that genetic variation within a population is primarily supplied by mutation (i.e. mutation-driven evolution). Here, we show that this proposition does not actually hold for recombination-driven evolution, i.e. evolution in which genetic variation is primarily created by recombination rather than mutation. By numerically investigating population genetics models of recombination, migration and selection, we demonstrate that stronger selection can slow down evolution on a perfectly smooth fitness landscape. Through simple analytical calculation, this apparently counter-intuitive result is shown to stem from two opposing effects of natural selection on the rate of evolution. On the one hand, natural selection tends to increase the rate of evolution by increasing the fixation probability of fitter genotypes. On the other hand, natural selection tends to decrease the rate of evolution by decreasing the chance of recombination between immigrants and resident individuals. As a consequence of these opposing effects, there is a finite selection pressure maximizing the rate of evolution. Hence, stronger selection can imply slower evolution if genetic variation is primarily supplied by recombination.
  • *Nobuto Takeuchi, Paulien Hogeweg, Kunihiko Kaneko,
    The origin of a primordial genome through spontaneous symmetry breaking,
    Nature Communications 8, 250 (2017).
    The heredity of a cell is provided by a small number of non-catalytic templates—the genome. How did genomes originate? Here, we demonstrate the possibility that genome-like molecules arise from symmetry breaking between complementary strands of self-replicating molecules. Our model assumes a population of protocells, each containing a population of self-replicating catalytic molecules. The protocells evolve towards maximising the catalytic activities of the molecules to increase their growth rates. Conversely, the molecules evolve towards minimising their catalytic activities to increase their intracellular relative fitness. These conflicting tendencies induce the symmetry breaking, whereby one strand of the molecules remains catalytic and increases its copy number (enzyme-like molecules), whereas the other becomes non-catalytic and decreases its copy number (genome-like molecules). This asymmetry increases the equilibrium cellular fitness by decreasing mutation pressure and increasing intracellular genetic drift. These results implicate conflicting multilevel evolution as a key cause of the origin of genetic complexity.
  • Kwesi Teye, Koji Hashimoto, Sanae Numata, Kunihiro Ohta, Marek Haftek & *Takashi Hashimoto,
    Altmetric: 1More detailArticle | OPENMultimerization is required for antigen binding activity of an engineered IgM/IgG chimeric antibody recognizing a skin-related antigen,
    Scientific Reports 7, 8212 (2017).
    Monoclonal antibodies offer great tools for research. We encountered a potentially useful mouse IgM monoclonal antibody whose antigen is expressed in normal skin but lost in human skin cancer. Because IgM is difficult to work with and the antigen was unknown, we decided to convert the IgM (µ) to IgG (γ) version. After cDNA for the antibody was obtained by RACE PCR, we made a series of molecules with different combinations of IgM and IgG domains. Whereas VH-Cµ1-Cµ2-Cγ3 and VH-Cµ1-Cµ2-Hinge-Cγ2-Cγ3 functionally bound to the antigen, VH-Cγ1-Hinge-Cγ2-Cγ3, VH-Cµ1-Hinge-Cγ2-Cγ3, and VH-Cµ1-Cµ2-Cγ2-Cγ3 did not. Gel filtration analyses revealed that the functional molecules tend to form multimers and the multimeric forms retained antigen binding activity. Furthermore, the mutation of amino acid residue p.309Q > C of mouse IgG and addition of IgM tailpiece to the C-terminus of the molecules induced multimer formation, dramatically enhanced antibody functionality and all non-functional molecules became strongly functional. The functional molecules could be bound by protein A/protein G and other IgG specific reagents and therefore should be useful for further characterization of the antigen. Our study revealed that multimerization of converted IgM is functionally important for antigen binding activity of engineered IgM/IgG chimeric antibodies.
  • T. Horinouchi, A. Sakai, H. Kotani, K Tanabe, *C. Furusawa,
    Improvement of isopropanol tolerance of Escherichia coli using adaptive laboratory evolution and omics technologies,
    JOURNAL OF BIOTECHNOLOGY 255, 47-56 (2017).
    Isopropanol (IPA) is the secondary alcohol that can be dehydrated to yield propylene. To produce IPA using microorganisms, a significant issue is that the toxicity of IPA causes retardation or inhibition of cell growth, decreasing the yield. One possible strategy to overcome this problem is to improve IPA tolerance of production organisms. For the understanding of tolerance to IPA, we performed parallel adaptive laboratory evolution (ALE) of Escherichia coli under IPA stress. To identify the genotypic change during ALE, we performed genome re-sequencing analyses of obtained tolerant strains. To verify which mutations were contributed to IPA tolerance, we constructed the mutant strains and quantify the IPA tolerance of the constructed mutants. From these analyses, we found that five mutations (relA, marC, proQ, yfgO, and rraA) provided the increase of IPA tolerance. To understand the phenotypic change during ALE, we performed transcriptome analysis of tolerant strains. From transcriptome analysis, we found that expression levels of genes related to biosynthetic pathways of amino acids, iron ion homeostasis, and energy metabolisms were changed in the tolerant strains. Results from these experiments provide fundamental bases for designing IPA tolerant strains for industrial purposes.
  • Yu Sakurai , Wataru Mizumura, Manami Murata, Tomoya Hada, Shoshiro Yamamoto, Kenichiro Ito, Kazuhiro Iwasaki, Takayuki Katoh, Yuki Goto, Asako Takagi, Michinori Kohara, *Hiroaki Suga, and Hideyoshi Harashima,
    Efficient siRNA delivery by lipid nanoparticles modified with a nonstandard macrocyclic peptide for EpCAM-targeting,
    Mol. Pharmaceutics 14 (10), 3290–3298 (2017).
  • Louise J. Walport, Richard Obexer, *HiroakiSuga,
    Strategies for transitioning macrocyclic peptides to cell-permeable drug leads,
    Curr Opin Biotechnol. 48, 242-250 (2017).
  • Eric M. Danhart, Marina Bakhtina, William A. Cantara, Alexandra B. Kuzmishin, Xiao Ma, Brianne L. Sanford, Oscar Vargas-Rodriguez, Marija Košutić, Yuki Goto, *Hiroaki Suga, Kotaro Nakanishi, Ronald Micura, Mark P. Foster and Karin Musier-Forsyth,
    Conformational and chemical selection by a trans-acting editing domain,
    Proc Natl Acad Sci U S A 114(33), E6774-E6783 (2017).
  • *Hirotaka Matsumoto, Hisanori Kiryu, Chikara Furusawa, Minoru S. H. Ko, Shigeru B. H. Ko, Norio Gouda, Tetsutaro Hayashi, Itoshi Nikaido,
    SCODE: an efficient regulatory network inference algorithm from single-cell RNA-Seq during differentiation,
    BIOINFORMATICS 33-15, 2314-2321 (2017).
    Motivation: The analysis of RNA-Seq data from individual differentiating cells enables us to reconstruct the differentiation process and the degree of differentiation (in pseudo-time) of each cell. Such analyses can reveal detailed expression dynamics and functional relationships for differentiation. To further elucidate differentiation processes, more insight into gene regulatory networks is required. The pseudo-time can be regarded as time information and, therefore, single-cell RNA-Seq data are time-course data with high time resolution. Although time-course data are useful for inferring networks, conventional inference algorithms for such data suffer from high time complexity when the number of samples and genes is large. Therefore, a novel algorithm is necessary to infer networks from single-cell RNA-Seq during differentiation. Results: In this study, we developed the novel and efficient algorithm SCODE to infer regulatory networks, based on ordinary differential equations. We applied SCODE to three single-cell RNA-Seq datasets and confirmed that SCODE can reconstruct observed expression dynamics. We evaluated SCODE by comparing its inferred networks with use of a DNaseI-footprint based network. The performance of SCODE was best for two of the datasets and nearly best for the remaining dataset. We also compared the runtimes and showed that the runtimes for SCODE are significantly shorter than for alternatives. Thus, our algorithm provides a promising approach for further single-cell differentiation analyses.
  • *畠山哲央, 金子邦彦,
    生物物理 57, 186-190 (2017).
    Robustness and plasticity are important characteristics common to a variety of biological systems and have attracted much attention not only from biologists but also from physicists interested in biosystems. Whereas robustness concerns insensitivity to perturbations against external changes, plasticity concerns changeability upon external inputs. How these two properties are compatible with each other is an important question to be addressed. Recently we have uncovered universal reciprocity relationship between the robustness of period and plasticity of phase in biochemical oscillators such as circadian clocks. We review this relationship and discuss its theoretical origin and biological relevance.
  • Tadasu Nozaki, Ryosuke Imai, Mai Tanbo, Ryosuke Nagashima, Sachiko Tamura, Tomomi Tani, Yasumasa Joti, Masaru Tomita, Kayo Hibino, Masato T. Kanemaki, Kerstin S. Wendt, Yasushi Okada, Takeharu Nagai, Kazuhiro Maeshima,
    Dynamic Organization of Chromatin Domains Revealed by Super-Resolution Live-Cell Imaging,
    MOLECULAR CELL 67, 282-293 (2017).
    Dynamic Organization of Chromatin Domains Revealed by Super-Resolution Live-Cell Imaging
  • Tetsuya Hiraiwa, Erina Kuranaga and *Tatsuo Shibata,
    Wave propagation of junctional remodeling in collective cell movement of epithelial tissue: Numerical simulation study,
    frontiers in Cell and Developmental Biology 5, 66/1-15 (2017).
    During animal development, epithelial cells forming a monolayer sheet move collectively to achieve the morphogenesis of epithelial tissues. One driving mechanism of such collective cell movement is junctional remodeling, which is found in the process of clockwise rotation of Drosophila male terminalia during metamorphosis. However, it still remains unknown how the motions of cells are spatiotemporally organized for collective movement by this mechanism. Since these moving cells undergo elastic deformations, the influence of junctional remodeling may mechanically propagate among them, leading to spatiotemporal pattern formations. Here, using a numerical cellular vertex model, we found that the junctional remodeling in collective cell movement exhibits spatiotemporal self-organization without requiring spatial patterns of molecular signaling activity. The junctional remodeling propagates as a wave in a specific direction with a much faster speed than that of cell movement. Such propagation occurs in both the absence and presence of fluctuations in the contraction of cell boundaries.
  • Yuma Fujimoto, Takahiro Sagawa, *Kunihiko Kaneko,
    Hierarchical prisoner’s dilemma in hierarchical game for resource competition,
    New Journal of Physics 19, 073008 (2017).
    Dilemmas in cooperation are one of the major concerns in game theory. In a public goods game, each individual cooperates by paying a cost or defecting without paying it, and receives a reward from the group out of the collected cost. Thus, defecting is beneficial for each individual, while cooperation is beneficial for the group. Now, groups (say, countries) consisting of individuals also play games. To study such a multi-level game, we introduce a hierarchical game in which multiple groups compete for limited resources by utilizing the collected cost in each group, where the power to appropriate resources increases with the population of the group. Analyzing this hierarchical game, we found a hierarchical prisoner's dilemma, in which groups choose the defecting policy (say, armament) as a Nash strategy to optimize each group's benefit, while cooperation optimizes the total benefit. On the other hand, for each individual, refusing to pay the cost (say, tax) is a Nash strategy, which turns out to be a cooperation policy for the group, thus leading to a hierarchical dilemma. Here the group reward increases with the group size. However, we find that there exists an optimal group size that maximizes the individual payoff. Furthermore, when the population asymmetry between two groups is large, the smaller group will choose a cooperation policy (say, disarmament) to avoid excessive response from the larger group, and the prisoner's dilemma between the groups is resolved. Accordingly, the relevance of this hierarchical game on policy selection in society and the optimal size of human or animal groups are discussed.
  • 小澤高嶺 太田邦史,
    「昆虫の形態の多様性とエピゲノム」—子供のころの疑問を明らかに ,
    生物の科学 遺伝 4, 314-318 (2017).
  • Tetsuya J. Kobayashi, Yuki Sughiyama ,
    Information thermodynamic structure in population dynamics in fluctuating environment,
    Physical Review E 96, 012402 (2017).
    Adaptation in a fluctuating environment is a process of fueling environmental information to gain fitness. Living systems have gradually developed strategies for adaptation from random and passive diversification of the phenotype to more proactive decision making, in which environmental information is sensed and exploited more actively and effectively. Understanding the fundamental relation between fitness and information is therefore crucial to clarify the limits and universal properties of adaptation. In this work, we elucidate the underlying stochastic and information-thermodynamic structure in this process, by deriving causal fluctuation relations (FRs) of fitness and information. Combined with a duality between phenotypic and environmental dynamics, the FRs reveal the limit of fitness gain, the relation of time reversibility with the achievability of the limit, and the possibility and condition for gaining excess fitness due to environmental fluctuation. The loss of fitness due to causal constraints and the limited capacity of real organisms is shown to be the difference between time-forward and time-backward path probabilities of phenotypic and environmental dynamics. Furthermore, the FRs generalize the concept of the evolutionary stable state (ESS) for fluctuating environment by giving the probability that the optimal strategy on average can be invaded by a suboptimal one owing to rare environmental fluctuation. These results clarify the information- thermodynamic structures in adaptation and evolution.
  • Masashi K. Kajita, Kazuyuki Aihara, Tetsuya J. Kobayashi,
    Balancing specificity, sensitivity, and speed of ligand discrimination by zero-order ultraspecificity,
    Physical Review E 96, 012405 (2017).
    Specific interactions between receptors and their target ligands in the presence of nontarget ligands are crucial for biological processes such as T cell ligand discrimination. To discriminate between the target and nontarget ligands, cells have to increase specificity to the target ligands by amplifying the small differences in affinity among ligands. In addition, sensitivity to the ligand concentration and quick discrimination are also important to detect low amounts of target ligands and facilitate fast cellular decision making after ligand recognition. In this work we propose a mechanism for nonlinear specificity amplification (ultraspecificity) based on zero-order saturating reactions, whichwas originally proposed to explain nonlinear sensitivity amplification (ultrasensitivity) to the ligand concentration. In contrast to the previously proposed proofreading mechanisms that amplify the specificity by a multistep reaction, our model can produce an optimal balance of specificity, sensitivity, and quick discrimination. Furthermore, we show that a model for insensitivity to a large number of nontarget ligands can be naturally derived from a model with the zero-order ultraspecificity. The zero-order ultraspecificity, therefore, may provide an alternative way to understand ligand discrimination from the viewpoint of nonlinear properties in biochemical reactions.
  • Nozaki T, Imai R, Tanbo M, Nagashima R, Tamura S, Tani T, Joti Y, Tomita M, Hibino K, Kanemaki MT, Wendt KS, Okada Y, Nagai T, Maeshima K,
    Dynamic Organization of Chromatin Domains Revealed by Super-Resolution Live-Cell Imaging,
    MOLECULAR CELL 67, 282-293 (2017).
    The eukaryotic genome is organized within cells as chromatin. For proper information output, higher-order chromatin structures can be regulated dynamically. How such structures form and behave in various cellular processes remains unclear. Here, by combining super-resolution imaging (photoactivated localization microscopy [PALM]) and single-nucleosome tracking, we developed a nuclear imaging system to visualize the higher-order structures along with their dynamics in live mammalian cells. We demonstrated that nucleosomes form compact domains with a peak diameter of similar to 160 nm and move coherently in live cells. The heterochromatin-rich regions showed more domains and less movement. With cell differentiation, the domains became more apparent, with reduced dynamics. Furthermore, various perturbation experiments indicated that they are organized by a combination of factors, including cohesin and nucleosome-nucleosome interactions. Notably, we observed the domains during mitosis, suggesting that they act as building blocks of chromosomes and may serve as information units throughout the cell cycle.
  • 岡田康志,
    医学の歩み 262(5), 573-579 (2017).
  • Kazuki Harada, Tetsuya Kitaguchi, Taichi Kamiya, Kyaw Htet Aung, Kazuaki Nakamura, Kunihiro Ohta and *Takashi Tsuboi,
    Lysophosphatidylinositol-induced activation of the cation channel TRPV2 triggers glucagon-like peptide-1 secretion in enteroendocrine L cells. ,
    J. Biol. Chem. 292, 10855-10864 (2017).
    The lysophosphatidylinositol (LPI) has crucial roles in multiple physiological processes, including insulin exocytosis from pancreatic islets. However, the role of LPI in secretion of glucagon-like peptide-1 (GLP-1), a hormone that enhances glucose-induced insulin secretion, is unclear. Here, we used the murine enteroendocrine L cell line GLUTag and primary murine small intestinal cells to elucidate the mechanism of LPI-induced GLP-1 secretion. Exogenous LPI addition increased intracellular Ca2+ concentrations ([Ca2+]i) in GLUTag cells and induced GLP-1 secretion from both GLUTag and acutely prepared primary intestinal cells. The [Ca2+]i increase was suppressed by an antagonist for G protein-coupled receptor 55 (GPR55) and by silencing of GPR55 expression, indicating involvement of Gq and G12/13 signaling pathways in the LPI-induced increased [Ca2+]i levels and GLP-1 secretion. However, GPR55 agonists did not mimic many of the effects of LPI. We also found that phospholipase C inhibitor and Rho-associated kinase inhibitor suppressed the [Ca2+]i increase and that LPI increased the number of focal adhesions, indicating actin reorganization. Of note, blockage or silencing of transient receptor potential cation channel subfamily V member 2 (TRPV2) channels suppressed both the LPI-induced [Ca2+]i increase and GLP-1 secretion. Furthermore, LPI accelerated TRPV2 translocation to the plasma membrane, which was significantly suppressed by a GPR55 antagonist. These findings suggest that TRPV2 activation via actin reorganization induced by Gq and G12/13 signaling is involved in LPI-stimulated GLP-1 secretion in enteroendocrine L cells. Because GPR55 agonists largely failed to mimic the effects of LPI, its actions on L cells are at least partially independent of GPR55 activation.
  • Yusuke Himeoka, *Kunihiko Kaneko,
    Theory for transitions between log and stationary phases: universal laws for lag time,
    Physical Review X 7, 021049 (2017).
    The quantitative characterization of bacterial growth has attracted substantial attention since Monod’s pioneering study. Theoretical and experimental works have uncovered several laws for describing the exponential growth phase, in which the number of cells grows exponentially. However, microorganism growth also exhibits lag, stationary, and death phases under starvation conditions, in which cell growth is highly suppressed, for which quantitative laws or theories are markedly underdeveloped. In fact, the models commonly adopted for the exponential phase that consist of autocatalytic chemical components, including ribosomes, can only show exponential growth or decay in a population; thus, phases that halt growth are not realized. Here, we propose a simple, coarse-grained cell model that includes an extra class of macromolecular components in addition to the autocatalytic active components that facilitate cellular growth. These extra components form a complex with the active components to inhibit the catalytic process. Depending on the nutrient condition, the model exhibits typical transitions among the lag, exponential, stationary, and death phases. Furthermore, the lag time needed for growth recovery after starvation follows the square root of the starvation time and is inversely related to the maximal growth rate. This is in agreement with experimental observations, in which the length of time of cell starvation is memorized in the slow accumulation of molecules. Moreover, the lag time distributed among cells is skewed with a long time tail. If the starvation time is longer, an exponential tail appears, which is also consistent with experimental data. Our theory further predicts a strong dependence of lag time on the speed of substrate depletion, which can be tested experimentally. The present model and theoretical analysis provide universal growth laws beyond the exponential phase, offering insight into how cells halt growth without entering the death phase.
  • Hidenori Nakaoka, *Yuichi Wakamoto,
    Aging, mortality, and the fast growth trade-off of Schizosaccharomyces pombe,
    PLoS Biology 15, e2001109 (2017).
    Replicative aging has been demonstrated in asymmetrically dividing unicellular organisms, seemingly caused by unequal damage partitioning. Although asymmetric segregation and inheritance of potential aging factors also occur in symmetrically dividing species, it nevertheless remains controversial whether this results in aging. Based on large-scale single-cell lineage data obtained by time-lapse microscopy with a microfluidic device, in this report, we demonstrate the absence of replicative aging in old-pole cell lineages of Schizosaccharomyces pombe cultured under constant favorable conditions. By monitoring more than 1,500 cell lineages in 7 different culture conditions, we showed that both cell division and death rates are remarkably constant for at least 50-80 generations. Our measurements revealed that the death rate per cellular generation increases with the division rate, pointing to a physiological trade-off with fast growth under balanced growth conditions. We also observed the formation and inheritance of Hsp104-associated protein aggregates, which are a potential aging factor in old-pole cell lineages, and found that these aggregates exhibited a tendency to preferentially remain at the old poles for several generations. However, the aggregates were eventually segregated from old-pole cells upon cell division and probabilistically allocated to new-pole cells. We found that cell deaths were typically preceded by sudden acceleration of protein aggregation; thus, a relatively large amount of protein aggregates existed at the very ends of the dead cell lineages. Our lineage tracking analyses, however, revealed that the quantity and inheritance of protein aggregates increased neither cellular generation time nor cell death initiation rates. Furthermore, our results demonstrated that unusually large amounts of protein aggregates induced by oxidative stress exposure did not result in aging; old-pole cells resumed normal growth upon stress removal, despite the fact that most of them inherited significant quantities of aggregates. These results collectively indicate that protein aggregates are not a major determinant of triggering cell death in S. pombe and thus cannot be an appropriate molecular marker or index for replicative aging under both favorable and stressful environmental conditions.
  • M. Yoshida, S. G. Reyes, S. Tsuda, T. Horinouchi, C. Furusawa,*L. Cronin, ,
    Time-programmable drug dosing allows the manipulation, suppression and reversal of antibiotic drug resistance in vitro,
    NATURE COMMUNICATIONS 8, 15589 (2017).
    Multi-drug strategies have been attempted to prolong the efficacy of existing antibiotics, but with limited success. Here we show that the evolution of multi-drug-resistant Escherichia coli can be manipulated in vitro by administering pairs of antibiotics and switching between them in ON/OFF manner. Using a multiplexed cell culture system, we find that switching between certain combinations of antibiotics completely suppresses the development of resistance to one of the antibiotics. Using this data, we develop a simple deterministic model, which allows us to predict the fate of multi-drug evolution in this system. Furthermore, we are able to reverse established drug resistance based on the model prediction by modulating antibiotic selection stresses. Our results support the idea that the development of antibiotic resistance may be potentially controlled via continuous switching of drugs.
  • A. Germond, V. Kumar, T. Ichimura, J. Moreau, C. Furusawa, H. Fujita,,
    Raman spectroscopy as a tool for ecology and evolution,
    Scientists are always on the lookout for new modalities of information which could reveal new biological features that are useful for deciphering the complexity of biological systems. Here, we introduce Raman spectroscopy as a prime candidate for ecology and evolution. To encourage the integration of this microscopy technique in the field of ecology and evolution, it is crucial to discuss first how Raman spectroscopy fits within the conceptual, technical and pragmatic considerations of ecology and evolution. In this paper, we show that the spectral information holds reliable indicators of intra-and interspecies variations, which can be related to the environment, selective pressures and fitness. Moreover, we show how the technical and pragmatic aspects of this modality (non-destructive, non-labelling, speed, relative low cost, etc.) enable it to be combined with more conventional methodologies. With this paper, we hope to open new avenues of research and extend the scope of available methodologies used in ecology and evolution.
  • Keita Kamino, Yohei Kondo, Akihiko Nakajima, Mai Honda-Kitahara, Kunihiko Kaneko, and *Satoshi Sawai,
    Fold-change detection and scale-invariance of cell-cell signaling in social amoeba,
    Proceedings of the National Academy of Sciences of the United States of America 114(21), E4149-E4157 (2017).
    Cell–cell signaling is subject to variability in the extracellular volume, cell number, and dilution that potentially increase uncertainty in the absolute concentrations of the extracellular signaling molecules. To direct cell aggregation, the social amoebae Dictyostelium discoideum collectively give rise to oscillations and waves of cyclic adenosine 3′,5′-monophosphate (cAMP) under a wide range of cell density. To date, the systems-level mechanism underlying the robustness is unclear. By using quantitative live-cell imaging, here we show that the magnitude of the cAMP relay response of individual cells is determined by fold change in the extracellular cAMP concentrations. The range of cell density and exogenous cAMP concentrations that support oscillations at the population level agrees well with conditions that support a large fold-change–dependent response at the singlecell level. Mathematical analysis suggests that invariance of the oscillations to density transformation is a natural outcome of combining secrete-and-sense systems with a fold-change detection mechanism.
  • Xiao Song, Lu-yi Lu, Toby Passioura, *Hiroaki Suga,
    Macrocyclic peptide inhibitors for the protein-protein interaction of Zaire Ebola virus protein 24 and karyopherin alpha 5,
    Org Biomol Chem 15(24), 5155-5160 (2017).
  • S. Suzuki, T. Horinouchi, *C. Furusawa,,
    Acceleration and suppression of resistance development by antibiotic combinations,
    BMC GENOMICS 18, 328 (2017).
    Background: The emergence and spread of antibiotic resistance in bacteria is becoming a global public health problem. Combination therapy, i.e., the simultaneous use of multiple antibiotics, is used for long-term treatment to suppress the emergence of resistant strains. However, the effect of the combinatorial use of multiple drugs on the development of resistance remains elusive, especially in a quantitative assessment. Results: To understand the evolutionary dynamics under combination therapy, we performed laboratory evolution of Escherichia coli under simultaneous addition of two-drug combinations. We demonstrated that simultaneous addition of a certain combinations of two drugs with collateral sensitivity to each other could suppress the acquisition of resistance to both drugs. Furthermore, we found that the combinatorial use of enoxacin, a DNA replication inhibitor, with Chloramphenicol can accelerate acquisition of resistance to Chloramphenicol. Genome resequencing analyses of the evolved strains suggested that the acceleration of resistance acquisition was caused by an increase of mutation frequency when enoxacin was added. Conclusions: Integration of laboratory evolution and whole-genome sequencing enabled us to characterize the development of resistance in bacteria under combination therapy. These results provide a basis for rational selection of antibiotic combinations that suppress resistance development effectively.
  • *Kyogo Kawaguchi, Ryoichiro Kageyama & *Masaki Sano,
    Topological defects control collective dynamics in neural progenitor cell cultures,
    Nature 545, 327–331 (2017).
    Cultured stem cells have become a standard platform not only for regenerative medicine and developmental biology but also for biophysical studies. Yet, the characterization of cultured stem cells at the level of morphology and of the macroscopic patterns resulting from cell-to-cell interactions remains largely qualitative. Here we report on the collective dynamics of cultured murine neural progenitor cells (NPCs), which are multipotent stem cells that give rise to cells in the central nervous system1. At low densities, NPCs moved randomly in an amoeba-like fashion. However, NPCs at high density elongated and aligned their shapes with one another, gliding at relatively high velocities. Although the direction of motion of individual cells reversed stochastically along the axes of alignment, the cells were capable of forming an aligned pattern up to length scales similar to that of the migratory stream observed in the adult brain2. The two-dimensional order of alignment within the culture showed a liquid-crystalline pattern containing interspersed topological defects with winding numbers of +1/2 and −1/2 (half-integer due to the nematic feature that arises from the head–tail symmetry of cell-to-cell interaction). We identified rapid cell accumulation at +1/2 defects and the formation of three-dimensional mounds. Imaging at the single-cell level around the defects allowed us to quantify the velocity field and the evolving cell density; cells not only concentrate at +1/2 defects, but also escape from −1/2 defects. We propose a generic mechanism for the instability in cell density around the defects that arises from the interplay between the anisotropic friction and the active force field.
  • Shintaro Yamada, Mika Okamura, Arisa Oda, Hiroshi Murakami, Kunihiro Ohta and *Takatomi Yamada,
    Correlation of meiotic DSB formation and transcription initiation around fission yeast recombination hotspots,
    GENETICS 206, 801-809 (2017).
    Meiotic homologous recombination, a critical event for ensuring faithful chromosome segregation and creating genetic diversity, is initiated by programed DNA double strand breaks (DSBs) formed at recombination hotspots. Meiotic DSB formation is likely to be influenced by other DNA-templated processes including transcription, but how DSB formation and transcription interact with each other has not been understood well. In this study, we used fission yeast to investigate a possible interplay of these two events. A group of hotspots in fission yeast are associated with sequences similar to cyclic AMP response element (CRE) and activated by the ATF/CREB family transcription factor dimer Atf1-Pcr1. We first focused on one of those hotspots, ade6-3049, and Atf1. Our results showed that multiple transcripts, shorter than the ade6 full length mRNA, emanate from a region surrounding the ade6-3049 hotspot. Interestingly, we found that the previously-known recombination activation region of Atf1 is also a transactivation domain, whose deletion affected DSB formation and short transcripts production at ade6-3049. These results point to a possibility that the two events may be related to each other at ade6-3049. In fact, comparison of published datasets of meiotic transcriptome and hotspot map suggested that hotspots are very often located close to meiotically transcribed regions. These observations therefore propose that meiotic DSB formation in fission yeast may be connected to transcription of surrounding regions.
  • Akane Kawamura, Martin Münzel, Tatsuya Kojima, Clarence Yapp, Bhaskar Bhushan, Yuki Goto, Anthony Tumber, Takayuki Katoh, Oliver N. F. King, Toby Passioura, Louise J. Walport, Stephanie B. Hatch, Sarah Madden, Susanne Müller, Paul E. Brennan, Rasheduzzaman Chowdhury, Richard J. Hopkinson, *Hiroaki Suga & Christopher J. Schofield,
    Highly selective inhibition of histone demethylases by de novo macrocyclic peptides,
    Nat Commun. 8, 14773 (2017).
  • *Takaki Yamamoto and Masaki Sano,
    Chirality-induced helical self-propulsion of cholesteric liquid crystal droplets,
    Soft Matter 13, 3328-3333 (2017).
    We report the first experimental realization of a chiral artificial microswimmer exhibiting helical motionwithout any external fields. We discovered that a cholesteric liquid crystal (CLC) droplet with a helicaldirector field swims in a helical path driven by the Marangoni flow in an aqueous surfactant solution. Wealso showed that the handedness of the helical path is reversed when that of the CLC droplet isreversed by replacing the chiral dopant with the enantiomer. In contrast, nematic liquid crystal (NLC)droplets exhibited ballistic motions. These results suggest that the helical motion of the CLC droplets isdriven by chiral couplings between the Marangoni flow and rotational motion via the helical directorfield of CLC droplets.
  • Hao Yu, Patricia Dranchak, Zhiru Li, Ryan MacArthur, Matthew S. Munson, Nurjahan Mehzabeen, Nathan J. Baird, Kevin P. Battalie, David Ross, Scott Lovell, Clotilde K. S. Carlow, *Hiroaki Suga & James Inglese,
    Macrocycle peptides delineate locked-open inhibition mechanism for microorganism phosphoglycerate mutases,
    Nat Commun 8, 14932 (2017).
  • Takashi Amoh, Keiji Murakami, Reiko Kariyama, Kenji Hori, Yasuhiko Irie, Darija Viducic, Katsuhiko Hirota, Jun Igarashi, *Hiroaki Suga, Hiromi Kumon, Yoichiro Miyake,
    A pseudomonas aeruginosa quorum-sensing autoinducer analog enhances the activity of antibiotics against resistant strains,
    J Med Invest 64(1.2), 101-109 (2017).
  • Robustness of spatial patterns in buffered reaction-diffusion systems and its reciprocity with phase plasticity,
    Tetsuhiro S. Hatakeyama, *Kunihiko Kaneko,
    Physical Review E, Rapid Communication 95, 030201(R) (2017).
    The robustness of spatial patterns against perturbations is an indispensable property of developmental processes for organisms, which need to adapt to changing environments. Although specific mechanisms for this robustness have been extensively investigated, little is known about a general mechanism for achieving robustness in reaction-diffusion systems. Here, we propose a buffered reaction-diffusion system, in which active states of chemicals mediated by buffer molecules contribute to reactions, and demonstrate that robustness of the pattern wavelength is achieved by the dynamics of the buffer molecule. This robustness is analytically explained as a result of the scaling properties of the buffered system, which also lead to a reciprocal relationship between the wavelength's robustness and the plasticity of the spatial phase upon external perturbations. Finally, we explore the relevance of this reciprocity to biological systems.
  • Richard Obexer, Louise J. Walport, *Hiroaki Suga,
    Exploring sequence space: harnessing chemical and biological diversity towards new peptide leads,
    Curr Opin Chem Biol. 38, 52-61 (2017).
  • Yui Uchida*, Masahiro Uesaka, Takayoshi Yamamoto, Hiroyuki Takeda and Naoki Irie*,
    Embryonic lethality is not sufficient to explain hourglass-like conservation of vertebrate embryos,
    EvoDevo 9:7, 1-11 (2017).
    Background: Understanding the general trends in developmental changes during animal evolution, which are often associated with morphological diversification, has long been a central issue in evolutionary developmental biology. Recent comparative transcriptomic studies revealed that mid-embryonic period tend to be more evolutionarily conserved than those in earlier or later periods (hourglass model). While the model has been demonstrated in a variety of animal groups, the exact evolutionary mechanism remains to be clarified. Here, we tested a possibility that the mid-embryonic period (pharyngula period in vertebrates) is highly prone to embryonic lethality, and the resulting negative selections lead to evolutionary conservation of this phase. Results: By measuring the rate of lethal phenotypes of three different species of vertebrate embryos subjected to two kinds of perturbations: transient perturbations and genetic mutations, we found that the early stages showed the highest rate of lethal phenotypes in all three species. Conclusion: Our results consistently showed that the "mid-embryonic lethality hypothesis" may not be supported, suggesting that negative selection by embryonic lethality could not explain hourglass-like conservation of animal embryos. This highlights the potential contribution of alternative mechanisms such as developmental constraints.
  • Jonathan T.Young, Tetsuhiro S. Hatakeyama, *Kunihiko Kaneko,
    Dynamics Robustness of Linear Signaling Cascades,
    PLOS Computational Biology 13, e1005434 (2017).
    A most important property of biochemical systems is robustness. Static robustness, e.g., homeostasis, is the insensitivity of a state against perturbations, whereas dynamics robustness, e.g., homeorhesis, is the insensitivity of a dynamic process. In contrast to the extensively studied static robustness, dynamics robustness, i.e., how a system creates an invariant temporal profile against perturbations, is little explored despite transient dynamics being crucial for cellular fates and are reported to be robust experimentally. For example, the duration of a stimulus elicits different phenotypic responses, and signaling networks process and encode temporal information. Hence, robustness in time courses will be necessary for functional biochemical networks. Based on dynamical systems theory, we uncovered a general mechanism to achieve dynamics robustness. Using a three-stage linear signaling cascade as an example, we found that the temporal profiles and response duration post-stimulus is robust to perturbations against certain parameters. Then analyzing the linearized model, we elucidated the criteria of when signaling cascades will display dynamics robustness. We found that changes in the upstream modules are masked in the cascade, and that the response duration is mainly controlled by the rate-limiting module and organization of the cascade’s kinetics. Specifically, we found two necessary conditions for dynamics robustness in signaling cascades: 1) Constraint on the rate-limiting process: The phosphatase activity in the perturbed module is not the slowest. 2) Constraints on the initial conditions: The kinase activity needs to be fast enough such that each module is saturated even with fast phosphatase activity and upstream changes are attenuated. We discussed the relevance of such robustness to several biological examples and the validity of the above conditions therein. Given the applicability of dynamics robustness to a variety of systems, it will provide a general basis for how biological systems function dynamically.
  • *Nen Saito, Kunihiko Kaneko,
    Embedding dual function into molecular motors through collective motion,
    Scientific Reports 7, 44288 (2017).
    Protein motors, such as kinesins and dyneins, bind to a microtubule and travel along it in a specific direction. Previously, it was thought that the directionality for a given motor was constant in the absence of an external force. However, the directionality of the kinesin-5 Cin8 was recently found to change as the number of motors that bind to the same microtubule is increased. Here, we introduce a simple mechanical model of a microtubule-sliding assay in which multiple motors interact with the filament. We show that, due to the collective phenomenon, the directionality of the motor changes (e.g., from minus- to plus- end directionality), depending on the number of motors. This is induced by a large diffusive component in the directional walk and by the subsequent frustrated motor configuration, in which multiple motors pull the filament in opposite directions, similar to a game of tug-of-war. A possible role of the dual-directional motors for the mitotic spindle formation is also discussed. Our framework provides a general mechanism to embed two conflicting tasks into a single molecular machine, which works context-dependently.
  • Seino A.K. Jongkees, Sami Caner, Christina Tysoe, Gary D. Brayer, Stephen G. Withers, *Hiroaki Suga,
    Rapid discovery of potent and selective glycosidase-inhibiting de novo peptides,
    Cell Chem Biol 24(3), 381-390 (2017).
  • 大橋郁、藤井雅史、小川渉、*黒田真也,
    実験医学 35(5), 830-833 (2017).
  • Masashi Fujii, Kaoru Ohashi, Yasuaki Karasawa, Minori Hikichi and *Shinya Kuroda,
    Small-Volume effect enables robust, sensitive, and efficient information transfer in the spine,
    Biophysical Journal 112(4), 813-826 (2017).
    Why is the spine of a neuron so small that it can contain only small numbers of molecules and reactions inevitably become stochastic? We previously showed that, despite such noisy conditions, the spine exhibits robust, sensitive, and efficient features of information transfer using the probability of Ca2+ increase; however, the mechanisms are unknown. In this study, we show that the small volume effect enables robust, sensitive, and efficient information transfer in the spine volume, but not in the cell volume. In the spine volume, the intrinsic noise in reactions becomes larger than the extrinsic noise of input, resulting in robust information transfer despite input fluctuation. In the spine volume, stochasticity makes the Ca2+ increase occur with a lower intensity of input, causing higher sensitivity to lower intensity of input. The volume-dependency of information transfer increases its efficiency in the spine volume. Thus, we propose that the small-volume effect is the functional reason why the spine has to be so small.
  • *Yohei Murakami, Masanori Koyama, Shigeyuki Oba, Shinya Kuroda and Shin Ishii,
    Model-based control of the temporal patterns of intracellular signaling in silico,
    Biophysics and Physicobiology 14, 29-40 (2017).
    The functions of intracellular signal transduction systems are determined by the temporal behavior of intracellular molecules and their interactions. Of the many dynamical properties of the system, the relationship between the dynamics of upstream molecules and downstream molecules is particularly important. A useful tool in understanding this relationship is a methodology to control the dynamics of intracellular molecules with an extracellular stimulus. However, this is a difficult task because the relationship between the levels of upstream molecules and those of downstream molecules is often not only stochastic, but also time-inhomogeneous, nonlinear, and not one-to-one. In this paper, we present an easy-to-implement model-based control method that makes the target downstream molecule to trace a desired time course by changing the concentration of a controllable upstream molecule. Our method uses predictions from Monte Carlo simulations of the model to decide the strength of the stimulus, while using a particle-based approach to make inferences regarding unobservable states. We applied our method to in silico control problems of insulin-dependent AKT pathway model and EGF-dependent Akt pathway model with system noise. We show that our method can robustly control the dynamics of the intracellular molecules against unknown system noise of various strengths, even in the absence of complete knowledge of the true model of the target system.
  • 岡田康志,
    O plus E 39(2), 74-178 (2017).
  • Katsuyuki Yugi and Shinya Kuroda ,
    Metabolism-Centric Trans-Omics,
    Cell Systems 4(1), 19-20 (2017).
    Two recent studies in Cell and Science demonstrate the reconstruction of global mechanistic networks and identification of regulatory principles from multi-omics data.
  • H. Shimizu, C. Furusawa, T. Hirasawa, K. Yoshikawa, Y. Toya, T. Shirai, F. Matsuda,,
    Omics-Integrated Approach for Metabolic State Analysis of Microbial Processes: Innovationsand Future Directions,
    Applied Bioengineering -, 213-236 (2017).
    Optimization of metabolic pathways is important in the development of bio-processes that facilitate the production of chemicals and fuels by microbial cells. Predictions of metabolic pathways by genome-scale metabolic (GSM) reaction models and evaluations of the performance of developed cell factories by experimental ¹³C metabolic flux analysis can be used to elucidate the state of the cell. Flux balance analysis (FBA) is a simple approach to analyze metabolic flux distributions using linear programming (LP) and GSM reaction models. Using the appropriate GSM and objective function, FBA can predict the relationships among genotype, environmental condition, and production yields at steady states, and these can be used to improve microbial production. This chapter discusses using GSM and experimental evaluations for in silico metabolic simulations of the state of metabolism. It describes the integration of omics data for the development of bioproduction processes.
  • Toby Passioura, *Hiroaki Suga,
    A RaPID way to discover nonstandard macrocyclic peptide modulators of drug targets,
    Chem Commun (Camb) 53(12), 1931-1940 (2017).
  • Yuki Sughiyama, Tetsuya J. Kobayashi,
    Steady-state thermodynamics for population growth in fluctuating environments,
    Physical Review E 95, 012131 (2017).
    We report that population dynamics in fluctuating environments is characterized by a mathematically equivalent structure to steady-state thermodynamics. By employing the structure, population growth in fluctuating environments is decomposed into housekeeping and excess parts. The housekeeping part represents the integral of the stationary growth rate for each condition during a history of the environmental change. The excess part accounts for the excess growth induced by environmental fluctuations. Focusing on the excess growth, we obtain a Clausius inequality, which gives the upper bound of the excess growth. The equality is shown to be achieved in quasistatic environmental changes. We also clarify that this bound can be evaluated by the "lineage fitness", which is an experimentally observable quantity.
  • oichi Kosodo, Taeko Suetsugu, Tetsuya J. Kobayashi, Fumio Matsuzaki,
    Systematic time-dependent visualization and quantitation of the neurogenic rate in brain organoids,
    Biochemical and Biophysical Research Communications 483, 94-100 (2017).
    Organoids mimicking the formation of the brain cortex have been demonstrated to be powerful tools for developmental studies as well as pathological investigations of brain malformations. Here, we report an integrated approach for the quantification of temporal neural production (neurogenic rate) in organoids derived from embryonic brains. Spherical tissue fragments with polarized cytoarchitectures were incubated in multiple cavities arranged in a polymethylmethacrylate chip. The time-dependent neurogenic rate in the organoids was monitored by the level of EGFP under the promoter of Tbr2, a transcription factor that is transiently expressed in neural fate-committed progenitors during corticogenesis. Importantly, our monitoring system exhibited a quick response to DAPT, a drug that promotes neural differentiation. Furthermore, we successfully quantified the temporal neurogenic rate in a large number of organoids by applying image processing that semi-automatically recognized the positions of organoids and measured their signal intensities from sequential images. Taken together, we provide a strategy to quantitate the neurogenic rate in brain organoids in a time-dependent manner, which will also be a potent method for monitoring organoid formation and drug activity in other tissue types. (C) 2017 Elsevier Inc. All rights reserved.
  • *Taro Toyota, Taisuke Banno, Juan M. Castro, and Masayuki Imai,
    Locomotion and transformation of underwater micrometer-sized molecular aggregates under chemical stimuli,
    J. Phys. Soc. Jpn. 86, 101006 (2017).
    In this review paper, we introduce the mobility and transformation of micrometer-sized molecular aggregates (i.e., oil droplets, liquid crystalline droplets and tubes, and giant vesicles) in water under chemical stimuli. These molecular aggregates comprising lipophilic and/or amphiphilic molecules have drawn much attention as plausible prebiotic cellular structures and dynamic microreactors related to the origins of life. Here, we highlight recent advances and issues concerning the construction of such systems and discuss the implications of these findings to our understanding of protocellular systems.
  • *能瀬聡直、高坂洋史、伏木彬,
    生体の科学 68(5), 478-479 (2017).
  • 岡田康志,
    顕微鏡 52(2), 62-66 (2017).
    生きた細胞内の微細構造が動く様子を高分解能で観察するためには,空間分解能と比例して時間分解能を向上させる必要がある.しかし,従来の超解像顕微鏡法においては,時間分解能を犠牲にして空間分解能を向上させるものが多く,生きた細胞の観察には適していなかった.筆者らは,超解像顕微鏡法の一つである構造化照明法の原理が共焦点顕微鏡と類似していることに基づき,共焦点顕微鏡の光学系を用いることで構造化照明法を高速化することに成功した.生細胞観察に広く用いられているスピニングディスク式の共焦点顕微鏡の光学系を利用することで,時間分解能10 ms(100フレーム毎秒),空間分解能約100 nm(回折限界の2倍)が達成され,生きた細胞内での細胞内小器官の微細動態が観察可能となった.
  • 岡田康志,
    顕微鏡 52(2), 61 (2017).
  • S. Suzuki, T. Horinouchi, *C. Furusawa, ,
    Expression profiling of antibiotic resistant bacteria obtained by laboratory evolution,
    Methods Mol Biol. 1520, 263-279 (2017).
    To elucidate the mechanisms of antibiotic resistance, integrating phenotypic and genotypic features in resistant strains is important. Here, we describe the expression profiling of antibiotic-resistant Escherichia coli strains obtained by laboratory evolution, and a method for extracting a small number of genes whose expression changes can contribute to the acquisition of resistance.
  • C. Furusawa,
    Analysis of Drug Resistance Using Experimental Evolution,
    Yakugaku Zasshi 137, 373-376 (2017).
    The emergence of drug-resistant bacteria is a growing concern for global public health. One possible strategy to deal with the problem of resistant bacteria is to understand the dynamics of adaptive evolution under antibiotics and then develop methods to suppress such adaptive evolution. For this purpose, we performed experimental evolution of Escherichia coli under various antibiotics and obtained resistant strains. The phenotypic changes in these resistant strains were quantified by transcriptome analysis, and the genomic changes were analyzed using next-generation sequencers. The results demonstrated that the resistance could be quantitatively predicted by changes in the expression of a small number of genes. Several candidate mutations contributing to the resistance were identified, while phenotype-genotype mapping was suggested to be complex and included various mutations that caused similar phenotypic changes. We also found that combinatorial use of appropriate pairs of antibiotics can suppress the emergence of resistant strains. In the presentation, I discussed how the integration of multi-omics data in experimentally obtained resistant strains enables us to develop methods to suppress the adaptive evolution of antibiotic resistance.
  • Shingo Suzuki, Takaaki Horinouchi, *Chikara Furusawa,
    Expression profiling of antibiotic resistant bacteria obtained by laboratory evolution,
    Methods in Molecular Biology 1520, 263-279 (2017).
    To elucidate the mechanisms of antibiotic resistance, integrating phenotypic and genotypic features in resistant strains is important. Here, we describe the expression profiling of antibiotic resistant Escherichia coli strains obtained by laboratory evolution, and a method for extracting a small number of genes whose expression changes can contribute to the acquisition of resistance.
  • Takahiro Kohsokabe, Kunihiko Kaneko,
    Boundary-Induced Pattern Formation from Temporal Oscillation: Spatial Map Analysis,
    Europhysics Letters 116, 48005/1-6 (2017).
  • Takumi Washio, Seine A. Shintani, Hideo Higuchi and Toshiaki Hisada,
    Analysis of spontaneous oscillations for a three state power stroke model,
    Physical Review E 95, 022411 (2017).
  • 樋口秀男,
    理学部ニュース 48, 08 (2017).
  • 中岡秀憲, 梅谷実樹, *若本祐一,
    化学と生物, in press.
  • Ryohei Seki, Cai Li, Qi Fang, Shinichi Hayashi, Shiro Egawa, Jiang Hu, Luohao Xu, Hailin Pan, Mao Kondo, Tomohiko Sato, Haruka Matsubara, Namiko Kamiyama, Keiichi Kitajima, Daisuke Saito, Yang Liu, M. Thomas P. Gilbert, Qi Zhou, Xing Xu, Toshihiko Shiroishi, Naoki Irie*, Koji Tamura* & Guojie Zhang*,
    Functional roles of aves class-specific cis-regulatory elements on macroevolution of bird-specific features,
    Nature Communications 8, 14229 (2017).
    Unlike microevolutionary processes, little is known about the genetic basis of macroevolutionary processes. One of these magnificent examples is the transition from non-avian dinosaurs to birds that has created numerous evolutionary innovations such as self-powered flight and its associated wings with flight feathers. By analysing 48 bird genomes, we identified millions of avian-specific highly conserved elements (ASHCEs) that predominantly (>99%) reside in non-coding regions. Many ASHCEs show differential histone modifications that may participate in regulation of limb development. Comparative embryonic gene expression analyses across tetrapod species suggest ASHCE-associated genes have unique roles in developing avian limbs. In particular, we demonstrate how the ASHCE driven avian-specific expression of gene Sim1 driven by ASHCE may be associated with the evolution and development of flight feathers. Together, these findings demonstrate regulatory roles of ASHCEs in the creation of avian-specific traits, and further highlight the importance of cis-regulatory rewiring during macroevolutionary changes.
  • Taro Ozaki, Kona Yamashita, Yuki Goto, Morito Shimomura, Shohei Hayashi, Shumpei Asamizu, Yoshinori Sugai, Haruo Ikeda, Hiroaki Suga, Hiroyasu Onaka,
    Dissection of goadsporin biosynthesis by in vitro reconstitution leading to designer analoges expressed in vivo,
    Nature Communications 8, 14207 (2017).
    Goadsporin (GS) is a member of ribosomally synthesized and post-translationally modified peptides (RiPPs), containing an N-terminal acetyl moiety, six azoles and two dehydroalanines in the peptidic main chain. Although the enzymes involved in GS biosynthesis have been defined, the principle of how the respective enzymes control the specific modifications remains elusive. Here we report a one-pot synthesis of GS using the enzymes reconstituted in the 'flexible' in vitro translation system, referred to as the FIT-GS system. This system allows us to readily prepare not only the precursor peptide from its synthetic DNA template but also 52 mutants, enabling us to dissect the modification determinants of GodA for each enzyme. The in vitro knowledge has also led us to successfully produce designer GS analogues in vivo. The methodology demonstrated in this work is also applicable to other RiPP biosynthesis, allowing us to rapidly investigate the principle of modification events with great ease.
  • S. A. K. Jongkees, S. Umemoto, Hiroaki Suga,
    Linker-free incorporation of carbohydrates into in vitro displayed macrocyclic peptides,
    Chemical Science 8, 1474-1481 (2017).
  • Takayuki Katoh, Kenya Tajima, Hiroaki Suga,
    Consecutive Elongation of D-Amino Acids in Translation,
    Cell Chemical Biology 24, 46-54 (2017).
    Recent progress in the field of genetic code reprogramming using a reconstituted cell-free translation system has made it possible to incorporate a wide array of non-proteinogenic amino acids, including N-methyl-amino acids and D-amino acids. Despite the fact that up to ten N-methyl-amino acid residues can be continuously elongated, the successive incorporation of even two D-amino acids into a nascent peptide chain remains a formidable challenge, thus far being nearly impossible. Here we report achievement of continuous D-amino acid elongation by the use of engineered tRNAs and optimized concentrations of translation factors, enabling us to incorporate up to ten consecutive D-Ser residues into a nascent peptide chain. We have also expressed macrocyclic peptides consisting of four or five consecutive D-amino acids consisting of D-Phe, D-Ser, D-Ala, or D-Cys closed by either a disulfide bond or a thioether bond
  • *豊田太郎, 風山祐輝, 大崎寿久, 竹内昌治,
    分析化学 65, 715-727 (2017).
  • Tomoyuki Mano, *Jean-Baptiste Delfau, Junichiro Iwasawa, and Masaki Sano,
    Optimal run-and-tumble based transportation of a Janus particle with active steering,
    Proceedings of the National Academy of Sciences 114(13), E2580-E2589 (2017).
    Commanding the swimming of micrometric objects subjected to thermal agitation is always challenging both for artificial and living systems. Now, artificial swimmers can be designed whose self-propelling force can be tuned at will. However, orienting such small particles to an arbitrary direction requires counterbalancing the random rotational diffusion. Here, we introduce a simple concept to reorient artificial swimmers, granting them a motion similar to the run-and-tumbling behavior of Escherichia coli. We demonstrate it using Janus particles with asymmetric surface coating and moving under an AC electric field. Moreover, we determine the optimal strategy and compare it with biological swimmers. Our results encourage further investigation into dynamical behavior of colloidal particles, as well as application to microscopic devices.
  • *Daiki Nishiguchi, Ken H. Nagai, Hugues Chate, and Masaki Sano,
    Long-range nematic order and anomalous fluctuations in suspensions of swimming filamentous bacteria,
    Physical Review E 95, 020601(R) /1-6 (2017).
    We study the collective dynamics of elongated swimmers in a very thin fluid layer by devising long, filamentous, non-tumbling bacteria. The strong confinement induces weak nematic alignment upon collision, which, for large enough density of cells, gives rise to global nematic order. This homogeneous but fluctuating phase, observed on the largest experimentally-accessible scale of millimeters,  exhibits the properties predicted by standard models for flocking such as the Vicsek-style model of polar particles  with nematic alignment: true long-range nematic order and non-trivial giant number fluctuations.
  • *J.-B. Delfau, John J. Molina and M. Sano,
    Collective behavior of strongly confined suspensions of squirmers,
    Europhysics Letters 114, 24001/1-5 (2016).
    We run numerical simulations of strongly confined suspensions of model spherical swimmers called “squirmers”. Because of the confinement, the Stokeslet dipoles generated by the particles are quickly screened and the far-field flow is dominated by the source dipole for all the different kinds of squirmers. However, we show that the collective behavior of the suspension still depends on the self-propelling mechanism of the swimmers as polar states can only be observed for neutral squirmers. We demonstrate that the near-field hydrodynamic interactions play a crucial role in the alignment of the orientation vectors of spherical particles. Moreover, we point out thatthe enstrophy and the fluid fluctuations of an active suspension also depend on the nature of the squirmers.
  • Masashi Fujii, Kaoru Ohashi, Yasuaki Karasawa, Minori Hikichi, *Shinya Kuroda,
    Small-volume effect enables robust, sensitive, and efficient information transfer in the spine,
    Biophysical Journal 112, 813-826 (2017).
    Why is the spine of a neuron so small that it can contain only small numbers of molecules and reactions inevitably become stochastic? We previously showed that, despite such noisy conditions, the spine exhibits robust, sensitive, and efficient features of information transfer using the probability of Ca2+ increase; however, the mechanisms are unknown. In this study, we show that the small volume effect enables robust, sensitive, and efficient information transfer in the spine volume, but not in the cell volume. In the spine volume, the intrinsic noise in reactions becomes larger than the extrinsic noise of input, resulting in robust information transfer despite input fluctuation. In the spine volume, stochasticity makes the Ca2+ increase occur with a lower intensity of input, causing higher sensitivity to lower intensity of input. The volume-dependency of information transfer increases its efficiency in the spine volume. Thus, we propose that the small-volume effect is the functional reason why the spine has to be so small.
  • Hideki Terajima, *Hikari Yoshitane, Haruka Ozaki, Yutaka Suzuki,Shigeki Shimba, Shinya Kuroda, Wataru Iwasaki and *Yoshitaka Fukada,
    ADARB1 catalyzes circadian A-to-I editing and regulates RNA rhythm,
    Nature Genetics 49, 146-151 (2017).
    It has been proposed that the CLOCK-ARNTL (BMAL1) complex drives circadian transcription of thousands of genes, including Per and Cry family genes that encode suppressors of CLOCK-ARNTL-dependent transcription. However, recent studies demonstrated that 70-80% of circadian-oscillating mRNAs have no obvious rhythms in their de novo transcription, indicating the potential importance of post-transcriptional regulation. Our CLOCK-ChIP-seq analysis identified rhythmic expression of adenosine deaminase, RNA-specific, B1 (Adarb1, also known as Adar2), an adenosine-to-inosine (A-to-I) RNA-editing enzyme. RNA-seq showed circadian rhythms of ADARB1-mediated A-to-I editing in a variety of transcripts. In Adarb1-knockout mice, rhythms of large populations of mRNA were attenuated, indicating a profound impact of ADARB1-mediated A-to-I editing on RNA rhythms. Furthermore, Adarb1-knockout mice exhibited short-period rhythms in locomotor activity and gene expression. These phenotypes were associated with abnormal accumulation of CRY2. The present study identifies A-to-I RNA editing as a key mechanism of post-transcriptional regulation in the circadian clockwork.
  • *Katsuyuki Yugi,*Shinya Kuroda ,
    Metabolism-Centric Trans-Omics,
    Cell Systems 4, 19–20 (2017).
    Two recent studies in Cell and Science demonstrate the reconstruction of global mechanistic networks and identification of regulatory principles from multi-omics data.
  • Teruyuki Matsunaga, Hiroshi Kohsaka, and *Akinao Nose,
    Gap junction-mediated signaling from motor neurons regulates motor generation in the central circuits of larval Drosophila,
    The Jounal of Neuroscience 37(8), 2045-2060 (2017).
    In this study, we used the peristaltic crawling of Drosophila larvae as a model to study how motor patterns are regulated by central circuits. We built an experimental system that allows simultaneous application of optogenetics and calcium imaging to the isolated ventral nerve cord (VNC). We then investigated the effects of manipulating local activity of motor neurons (MNs) on fictive locomotion observed as waves of MN activity propagating along neuromeres. Optical inhibition of MNs with halorhodopsin3 in a middle segment (A4, A5, or A6), but not other segments, dramatically decreased the frequency of the motor waves. Conversely, local activation of MNs with channelrhodopsin2 in a posterior segment (A6 or A7) increased the frequency of the motor waves. Since peripheral nerves mediating sensory feedback were severed in the VNC preparation, these results indicate that MNs send signals to the central circuits to regulate motor pattern generation. Our results also indicate segmental specificity in the roles of MNs in motor control. The effects of the local MN activity manipulation were lost in shaking-B2 (shakB2) or ogre2, gap-junction mutations in Drosophila, or upon acute application of the gap junction blocker carbenoxolone, implicating electrical synapses in the signaling from MNs. Cell-type-specific RNAi suggested shakB and ogre function in MNs and interneurons, respectively, during the signaling. Our results not only reveal an unexpected role for MNs in motor pattern regulation, but also introduce a powerful experimental system that enables examination of the input–output relationship among the component neurons in this system.SIGNIFICANCE STATEMENT Motor neurons are generally considered passive players in motor pattern generation, simply relaying information from upstream interneuronal circuits to the target muscles. This study shows instead that MNs play active roles in the control of motor generation by conveying information via gap junctions to the central pattern-generating circuits in larval Drosophila, providing novel insights into motor circuit control. The experimental system introduced in this study also presents a new approach for studying intersegmentally coordinated locomotion. Unlike traditional electrophysiology methods, this system enables the simultaneous recording and manipulation of populations of neurons that are genetically specified and span multiple segments.
  • Lana Sinapayen, Atsushi Masumori, Takashi Ikegami,
    Learning by Stimulation Avoidance: A Principle to Control Spiking Neural Networks Dynamics,
    PLoS One 12(2), e0170388 (2017).
  • *Yoichi Kosodo, Taeko Suetsugu, Tetsuya J. Kobayashi, Fumio Matsuzaki,
    Systematic time-dependent visualization and quantitation of the neurogenic rate in brain organoids,
    Biochemical and Biophysical Research Communications 483, 94-100 (2017).
  • *Yuki Sughiyama, Tetsuya J. Kobayaashi,
    Steady-state thermodynamics for population growth in fluctuating environments,
    Physical Review E 95, 012131 (2017).

  • Aleksandr Drozd  Olaf Witkowski Satoshi Matsuoka*Takashi Ikegami,
    Critical Mass in the Emergence of Collective Intelligence: a Parallelized Simulation of Swarms in Noisy Environments. ,
    Artificial Life and Robotics 21, 317-323 (2016).
    We extend an abstract agent-based swarming model based on the evolution of neural network controllers, to explore further the emergence of swarming. Our model is grounded in the ecological situation, in which agents canaccess some information from the environment about theresource location, but through a noisy channel. Swarmingcritically improves the efficiency of group foraging, byallowing agents to reach resource areas much more easilyby correcting individual mistakes in group dynamics. Ashigh levels of noise may make the emergence of collectivebehavior depend on a critical mass of agents, it is crucialto reach sufficient computing power to allow for the evolution of the whole set of dynamics in simulation. Sincesimulating neural controllers and information exchangesbetween agents are computationally intensive, to scale upsimulations to model critical masses of individuals, the implementation requires careful optimization. We applytechniques from astrophysics known as treecodes to compute the signal propagation, and efficiently parallelizefor multi-core architectures. Our results open up futureresearch on signal-based emergent collective behavior asa valid collective strategy for uninformed search over adomain space.
  • *Taylor, T., Bedau, M., Channon, A., Ackley, D., Banzhaf, W., Beslon, G., Dolson, E., Froese, T., Hickinbotham, S., Ikegami, T., McMullin, B., Packard, N., Rasmussen, S., Virgo, N., Agmon, E., Clark, E., McGregor, S., Ofria, C., Ropella, G., Spector, L., Stanley K. O., Stanton, A., Timperley, C., Vostinar, A., Wiser, M.,
    Open-Ended Evolution: Perspectives from the OEE Workshop in York,
    Artificial Life 22, 408–423 (2016).
    We describe the content and outcomes of the First Workshop on Open-Ended Evolution: Recent Progress and Future Milestones (OEE1), held during the ECAL 2015 conference at the University of York, UK, in July 2015. We briefly summarize the content of the workshopʼs talks, and identify the main themes that emerged from the open discussions. Two important conclusions from the discussions are: (1) the idea of pluralism about OEE—itseems clear that there is more than one interesting and important kind of OEE; and (2) the importance of distinguishing observable behavioral hallmarks of systems undergoing OEE from hypothesized underlying mechanisms that explain why a system exhibits those hallmarks. We summarize the different hallmarks and mechanismsdiscussed during the workshop, and list the specific systems that were highlighted with respect to particular hallmarks and mechanisms. We conclude by identifying some of the most important open research questions about OEE that are apparent in light of the discussions.The York workshop provides a foundation for a follow-up OEE2 workshop taking place at the ALIFE XV conference in Cancún, Mexico, in July 2016. Additional materials from the York workshop, including talk abstracts, presentation slides, and videos of each talk, are available at http://alife.org/ws/oee1.
  • *Olaf Witkowski, Takashi Ikegami,
    Emergence of Swarming Behavior: Foraging Agents Evolve Collective Motion Based on Signaling,
    PLOS ONE 11, 1-26 (2016).
    Swarming behavior is common in biology, from cell colonies to insect swarms and bird flocks. However, the conditions leading to the emergence of such behavior are still subject to research. Since Reynolds' boids, many artificial models have reproduced swarming behavior, focusing on details ranging from obstacle avoidance to the introduction of fixed leaders. This paper presents a model of evolved artificial agents, able to develop swarming using only their ability to listen to each other's signals. The model simulates a population of agents looking for a vital resource they cannot directly detect, in a 3D environment. Instead of a centralized algorithm, each agent is controlled by an artificial neural network, whose weights are encoded in a genotype and adapted by an original asynchronous genetic algorithm. The results demonstrate that agents progressively evolve the ability to use the information exchanged between each other via signaling to establish temporary leader-follower relations. These relations allow agents to form swarming patterns, emerging as a transient behavior that improves the agents' ability to forage for the resource. Once they have acquired the ability to swarm, the individuals are able to outperform the non-swarmers at finding the resource. The population hence reaches a neutral evolutionary space which leads to a genetic drift of the genotypes. This reductionist approach to signal-based swarming not only contributes to shed light on the minimal conditions for the evolution of a swarming behavior, but also more generally it exemplifies the effect communication can have on optimal search patterns in collective groups of individuals.
  • Takashi Ikegami, Naoto Horibe, M Hanczyc,
    Potential Memory Effects in Self-Moving Oil Droplets,
    A series of chemical experiments have been designed to investigate the emergence of spontaneous self-movement in a simple chemical system consisting of oil droplets using oleic anhydride as fuel. Using spatial and temporal droplet tracking, we analyzed in detail the motion of single droplets in an aqueous environment. In particular we note that the variation in a single droplet's behavior, analyzed as the stop-go interval, displays a power law distribution indicating bias in behavior. In addition, the rate of movement of the droplet in comparison to the diffusion rate of chemical reaction products in the system may effect the movement of the droplet resulting in long time scale memory effects. These discoveries illustrate that coupling a chemical reaction (hydrolysis of the anhydride) to a physical body (the oil droplet) can result in an instability that affects many aspects of the droplet system, from reaction rate to convective flow patterns to overall shape to macroscopic behavior.
  • Do Won Hwang, Namryeong Bahng, Kenichiro Ito, Seunggyun Ha, Mee Young Kim, Eunji Lee, *Hiroaki Suga, Dong Soo Lee,
    In vivo targeting of c-Met using a non-standard macrocyclic peptide in gastric carcinoma,
    Cancer Lett 385, 144-149 (2016).
  • Tsukasa Kusakizako,Yoshiki Tanaka, Christopher J. Hipolito, *Hiroaki Suga, Osamu Nureki,
    Crystallographic analysis of MATE-type multidrug exporter with its inhibitors.,
    Methods Mol Biol. 1700, 7-57 (--).
    Multidrug exporters expressed in pathogens efflux substrate drugs such as antibiotics, and thus, the development of inhibitors against them has eagerly been anticipated. Furthermore, the crystal structures of multidrug exporters with their inhibitors provide novel insights into the inhibitory mechanism and the development of more specific and effective inhibitors. We previously reported the complex structures of the Multidrug And Toxic compound Extrusion (MATE)-type multidrug exporter with the macrocyclic peptides, which inhibit the efflux of substrates by the MATE-type multidrug exporter (Tanaka et al., Nature 496:247–251, 2013). In this chapter, we describe methodologies of the screening and synthesis of macrocyclic peptides as inhibitors, as well as the purification, crystallization, and structure determination of the complexes of the MATE-type multidrug exporter with its inhibitors.
  • *Chikara Furusawa, Tomoyuki Yamaguchi,
    Robust and accurate discrimination of self/non-self antigen presentations by regulatory T cell suppression,
    PLoS One 11(9), e0163134 (2016).
    The immune response by T cells usually discriminates self and non-self antigens, even though the negative selection of self-reactive T cells is imperfect and a certain fraction of T cells can respond to self-antigens. In this study, we construct a simple mathematical model of T cell populations to analyze how such self/non-self discrimination is possible. The results demonstrate that the control of the immune response by regulatory T cells enables a robust and accurate discrimination of self and non-self antigens, even when there is a significant overlap between the affinity distribution of T cells to self and non-self antigens. Here, the number of regulatory T cells in the system acts as a global variable controlling the T cell population dynamics. The present study provides a basis for the development of a quantitative theory for self and non-self discrimination in the immune system and a possible strategy for its experimental verification.
  • Shingo Suzuki, Takaaki Horinouchi, *Chikara Furusawa,
    Phenotypic changes associated with the fitness cost in antibiotic resistant Escherichia coli strains,
    Molecular Biosystems 12(2), 414-20 (2016).
    The acquisition of antibiotic resistance in bacterial cells is often accompanied with a reduction of fitness in the absence of antibiotics, known as the “fitness cost.” The magnitude of this fitness cost is an important biological parameter that influences the degree to which antibiotic resistant strains become widespread. However, the relationship between the fitness cost and comprehensive phenotypic and genotypic changes remains unclear. Here, we quantified the fitness cost of resistant strains obtained by experimental evolution in the presence of various antibiotics, and analyzed how the cost correlated to phenotypic and genotypic changes in the resistant strains.
  • *古澤力, 鈴木真吾, 堀之内貴明,
    バイオサイエンスとインダストリー 74(2), 138-140 (2016).
  • Nen Saito and Kunihiko Kaneko,
    Collective motion switches motor direction along filament,
    Scientific reports, in press.
  • Kenji Shinoda and Kunihiko Kaneko,
    Chaotic Griffiths Phase with Anomalous Lyapunov Spectra in Coupled Map Networks,
    Physical Review Letters 117, 254101/1-6 (2016).
  • Jumpei F Yamagishi, Nen Saito, Kunihiko Kaneko,
    Symbiotic Cell Differentiation and Cooperative Growth in Multicellular Aggregates,
    PLOS Computational Biology 12(10), e1005042/1-17 (2016).
    As cells grow and divide under a given environment, they become crowded and resourcesare limited, as seen in bacterial biofilms and multicellular aggregates. These cells oftenshow strong interactions through exchanging chemicals, as evident in quorum sensing, toachieve mutualism and division of labor. Here, to achieve stable division of labor, threecharacteristics are required. First, isogenous cells differentiate into several types. Second,this aggregate of distinct cell types shows better growth than that of isolated cells withoutinteraction and differentiation, by achieving division of labor. Third, this cell aggregate isrobust with respect to the number distribution of differentiated cell types. Indeed, theoreticalstudies have thus far considered how such cooperation is achieved when the ability of celldifferentiation is presumed. Here, we address how cells acquire the ability of cell differentiationand division of labor simultaneously, which is also connected with the robustness of acell society. For this purpose, we developed a dynamical-systems model of cells consistingof chemical components with intracellular catalytic reaction dynamics. The reactions convertexternal nutrients into internal components for cellular growth, and the divided cellsinteract through chemical diffusion. We found that cells sharing an identical catalytic networkspontaneously differentiate via induction from cell-cell interactions, and then achievedivision of labor, enabling a higher growth rate than that in the unicellular case. This symbioticdifferentiation emerged for a class of reaction networks under the condition of nutrientlimitation and strong cell-cell interactions. Then, robustness in the cell type distribution wasachieved, while instability of collective growth could emerge even among the cooperativecells when the internal reserves of products were dominant. The present mechanism issimple and general as a natural consequence of interacting cells with limited resources,and is consistent with the observed behaviors and forms of several aggregates of unicellularorganisms.
  • Tomoki Kurikawa, Kunihiko Kaneko,
    Dynamic Organization of Hierarchical Memories,
    PLoS ONE 11, e0162640/1-19 (2016).
    In the brain, external objects are categorized in a hierarchical way. Although it is widelyaccepted that objects are represented as static attractors in neural state space, this viewdoes not take account interaction between intrinsic neural dynamics and external input,which is essential to understand how neural system responds to inputs. Indeed, structuredspontaneous neural activity without external inputs is known to exist, and its relationshipwith evoked activities is discussed. Then, how categorical representation is embedded intothe spontaneous and evoked activities has to be uncovered. To address this question, westudied bifurcation process with increasing input after hierarchically clustered associativememories are learned. We found a “dynamic categorization”; neural activity without inputwanders globally over the state space including all memories. Then with the increase ofinput strength, diffuse representation of higher category exhibits transitions to focused onesspecific to each object. The hierarchy of memories is embedded in the transition probabilityfrom one memory to another during the spontaneous dynamics. With increased inputstrength, neural activity wanders over a narrower state space including a smaller set ofmemories, showing more specific category or memory corresponding to the applied input.Moreover, such coarse-to-fine transitions are also observed temporally during transient processunder constant input, which agrees with experimental findings in the temporal cortex.These results suggest the hierarchy emerging through interaction with an external inputunderlies hierarchy during transient process, as well as in the spontaneous activity.
  • Nen Saito, Yuki Sughiyama, and Kunihiko Kaneko,
    Motif analysis for small-number effects in chemical reaction dynamics,
    The Journal of Chemical Physics 145, 094111/1-7 (2016).
    The number of molecules involved in a cell or subcellular structure is sometimes rather small.In this situation, ordinary macroscopic-level fluctuations can be overwhelmed by non-negligible largefluctuations, which results in drastic changes in chemical-reaction dynamics and statistics comparedto those observed under a macroscopic system (i.e., with a large number of molecules). In order tounderstand how salient changes emerge from fluctuations in molecular number, we here quantitativelydefine small-number effect by focusing on a “mesoscopic” level, in which the concentration distributionis distinguishable both from micro- and macroscopic ones and propose a criterion for determiningwhether or not such an effect can emerge in a given chemical reaction network. Using the proposedcriterion, we systematically derive a list of motifs of chemical reaction networks that can showsmall-number effects, which includes motifs showing emergence of the power law and the bimodaldistribution observable in a mesoscopic regime with respect to molecule number. The list of motifsprovided herein is helpful in the search for candidates of biochemical reactions with a small-numbereffect for possible biological functions, as well as for designing a reaction system whose behavior canchange drastically depending on molecule number, rather than concentration.
  • Yohei Saito, Yuki Sughiyama, Kunihiko Kaneko, and Tetsuya J. Kobayashi,
    Discreteness-induced transitions in multibody reaction systems,
    Physical Review E 94, 022140/1-8 (2016).
  • Atsushi Kamimura, Kunihiko Kaneko,
    Negative scaling relationship between molecular diversity and resource abundances,
    Physical Review E 93, 062419/1-13 (2016).
    Cell reproduction involves replication of diverse molecule species, in contrast to a simple replication system with fewer components. To address this question of diversity, we study theoretically a cell system with catalytic reaction dynamics that grows by uptake of environmental resources. It is shown that limited resources lead to increased diversity of components within the system, and the number of coexisting species increases with a negative power of the resource uptake. The relationship is explained from the optimum growth speed of the cell, determined by a tradeoff between the utility of diverse resources and the concentration onto fewer components to increase the reaction rate.
  • Benjamin Pfeuty and Kunihiko Kaneko,
    Requirements for efficient cell-type proportioning: regulatory timescales, stochasticity and lateral inhibition,
    Physical Biology 13, 026007/1-9 (2016).
    The proper functioning of multicellular organisms requires the robust establishment of preciseproportions between distinct cell types. This developmental differentiation process typically involvesintracellular regulatory and stochastic mechanisms to generate cell-fate diversity as well as intercellularsignaling mechanisms to coordinate cell-fate decisions at tissue level. We thus surmise that keyinsights about the developmental regulation of cell-type proportion can be captured by the modelingstudy of clustering dynamics in population of inhibitory-coupled noisy bistable systems. This generalclass of dynamical system is shown to exhibit a very stable two-cluster state, but also metastability,collective oscillations or noise-induced state hopping, which can prevent from timely and reliablyreaching a robust and well-proportioned clustered state. To circumvent these obstacles or to avoidfine-tuning, we highlight a general strategy based on dual-time positive feedback loops, such asmediated through transcriptional versus epigenetic mechanisms, which improves proportionregulation by coordinating early and flexible lineage priming with late and firm commitment. Thisresult sheds new light on the respective and cooperative roles of multiple regulatory feedback,stochasticity and lateral inhibition in developmental dynamics.
  • Nobuto Takeuchi, Kunihiko Kaneko, Paulien Hogeweg,
    Evolutionarily stable disequilibrium: endless dynamics of evolution in a stationary population,
    Proceedings of the Royal Society B 283, 20153109 (2016).
    Evolution is often conceived as changes in the properties of a population over generations. Does this notion exhaust the possible dynamics of evolution? Life is hierarchically organized, and evolution can operate at multiple levels with conflicting tendencies. Using a minimal model of such conflicting multilevel evolution, we demonstrate the possibility of a novel mode of evolution that challenges the above notion: individuals ceaselessly modify their genetically inherited phenotype and fitness along their lines of descent, without involving apparent changes in the properties of the population. The model assumes a population of primitive cells (protocells, for short), each containing a population of replicating catalytic molecules. Protocells are selected towards maximizing the catalytic activity of internal molecules, whereas molecules tend to evolve towards minimizing it in order to maximize their relative fitness within a protocell. These conflicting evolutionary tendencies at different levels and genetic drift drive the lineages of protocells to oscillate endlessly between high and low intracellular catalytic activity, i.e. high and low fitness, along their lines of descent. This oscillation, however, occurs independently in different lineages, so that the population as a whole appears stationary. Therefore, ongoing evolution can be hidden behind an apparently stationary population owing to conflicting multilevel evolution.
  • Yusuke Himeoka and Kunihiko Kaneko,
    Enzyme oscillation can enhance the thermodynamic efficiency of cellular metabolism:consequence of anti-phase coupling between reaction flux and affinity,
    Physical Biology 13, 026002/1-12 (2016).
    Cells generally convert nutrient resources to products via energy transduction. Accordingly, thethermodynamic efficiency of this conversion process is one of the most essential characteristics ofliving organisms. However, although these processes occur under conditions of dynamicmetabolism, most studies of cellular thermodynamic efficiency have been restricted to examiningsteady states; thus, the relevance of dynamics to this efficiency has not yet been elucidated. Here, wedevelop a simple model of metabolic reactions with anabolism–catabolism coupling catalyzed byenzymes. Through application of external oscillation in the enzyme abundances, the thermodynamicefficiency of metabolism was found to be improved. This result is in strong contrast withthat observed in the oscillatory input, in which the efficiency always decreased with oscillation. Thisimprovement was effectively achieved by separating the anabolic and catabolic reactions, whichtend to disequilibrate each other, and taking advantage of the temporal oscillations so that each ofthe antagonistic reactions could progress near equilibrium. In this case, anti-phase oscillationbetween the reaction flux and chemical affinity through oscillation of enzyme abundances isessential. This improvement was also confirmed in a model capable of generating autonomousoscillations in enzyme abundances. Finally, the possible relevance of the improvement inthermodynamic efficiency is discussed with respect to the potential for manipulation of metabolicoscillations in microorganisms
  • 高井啓, 岡田康志,
    Journal of Japanese Biochemical Society 88(5), 669-673 (2016).
  • 岡田康志,
    レーザー研究 44(10), 643-647 (2016).
  • 岡田康志,
    Clinical Neuroscience 34(6), 638-642 (2016).
  • Morito Sakuma, Sayaka Kita, and Hideo Higuchi,
    Quantitative evaluation of malignant gliomas damage induced by photoactivation of IR700 dye,
    Science and Technology of Advanced Materials 22, 473-482 (2016).
  • Gaigai Yu, Hiroyuki Onodera, Yuki Aono, Fuun Kawano, Yoshibumi Ueda, Akihiro Furuya, Hideyuki Suzuki, and *Moritoshi Sato,
    Optical manipulation of the alpha subunits of heterotrimeric G proteins using photoswitchable dimerization systems,
    Scientific Reports 6, 35777 (2016).
    Alpha subunits of heterotrimeric G proteins (Gα) are involved in a variety of cellular functions. Herewe report an optogenetic strategy to spatially and temporally manipulate Gα in living cells. More speci cally, we applied the blue light-induced dimerization system, known as the Magnet system, and an alternative red light-induced dimerization system consisting of Arabidopsis thaliana phytochrome B (PhyB) and phytochrome-interacting factor 6 (PIF6) to optically control the activation of two di erent classes of Gα (Gαq and Gαs). By utilizing this strategy, we demonstrate successful regulation of Ca2+ and cAMP using light in mammalian cells. The present strategy is generally applicable to di erent kinds of Gα and could contribute to expanding possibilities of spatiotemporal regulation of Gα in mammalian cells.
  • Fuun Kawano, Risako Okazaki, Masayuki Yazawa, and *Moritoshi Sato,
    A photoactivatable Cre–loxP recombination system for optogenetic genome engineering,
    Nature Chemical Biology 12, 1059-1064 (2016).
    Genome engineering techniques represented by the Cre–loxP recombination system have been used extensively for biomedical research. However, powerful and useful techniques for genome engineering that have high spatiotemporal precision remain elusive. Here we develop a highly efficient photoactivatable Cre recombinase (PA-Cre) to optogenetically control genome engi- neering in vivo. PA-Cre is based on the reassembly of split Cre fragments by light-inducible dimerization of the Magnet system. PA-Cre enables sharp induction (up to 320-fold) of DNA recombination and is efficiently activated even by low-intensity illu- mination (~0.04 W m−2) or short periods of pulsed illumination (~30 s). We demonstrate that PA-Cre allows for efficient DNA recombination in an internal organ of living mice through noninvasive external illumination using a LED light source. The pres- ent PA-Cre provides a powerful tool to greatly facilitate optogenetic genome engineering in vivo.
  • Keiji Fushimi, Takahiro Nakajima, Yuki Aono, Tatsuro Yamamoto, Ni-Ni-Win, Masahiko Ikeuchi, Moritoshi Sato and *Rei Narikawa,
    Photoconversion and fluorescence properties of a red/green-type cyanobacteriochrome AM1_C0023g2 that binds not only phycocyanobilin but also biliverdin,
    Frontiers in Microbiology 7, 588 (2016).
    Cyanobacteriochromes (CBCRs) are distantly related to the red/far-red responsive phytochromes. Red/green-type CBCRs are widely distributed among various cyanobacteria. The red/green-type CBCRs covalently bind phycocyanobilin (PCB) and show red/green reversible photoconversion. Recent studies revealed that some red/green-type CBCRs from chlorophyll d-bearing cyanobacterium Acaryochloris marina covalently bind not only PCB but also biliverdin (BV). The BV-binding CBCRs show far-red/orange reversible photoconversion. Here, we identified another CBCR (AM1_C0023g2) from A. marina that also covalently binds not only PCB but also BV with high binding efficiencies, although BV chromophore is unstable in the presence of urea. Replacement of Ser334 with Gly resulted in significant improvement in the yield of the BV-binding holoprotein, thereby ensuring that the mutant protein is a fine platform for future development of optogenetic switches. We also succeeded in detecting near- infrared fluorescence from mammalian cells harboring PCB-binding AM1_C0023g2 whose fluorescence quantum yield is 3.0%. Here the PCB-binding holoprotein is shown as a platform for future development of fluorescent probes.
  • *佐藤守俊,
    実験医学増刊号「All about ゲノム編集」 34, 47-52 (2016).
    光を使って生命現象を光で「操る」ことができるとしたら,ライフサイエンスや医療はどうなるだろう?例えば,細胞内シグナル伝達を光で操作できるようになれば,代謝,分泌,細胞増殖,細胞分化,細胞死等の生命機能を自由自在にコントロールできるようになるかもしれない.ゲノムの塩基配列を光で自由自在に書き換えたり,遺伝子のはたらきを自由自在に制御できるようになったらどうだろう?光が得意とする高い時間・空間制御能をもってすれば,狙ったtime windowのみで,狙った生体部位のみで,様々な生命機能や疾患をコントロールできるかもしれない.本稿では,このような未来の実現を目指した筆者らの研究の一端を紹介する.
  • Takasho Nozoe, Edo Kussell, and *Yuichi Wakamoto,
    Inferring fitness landscapes and selection on phenotypic states from single-cell genealogical data,
    PLoS Genetics ad(3), e1006653 (2017).
    Recent advances in single-cell time-lapse microscopy have revealed non-genetic heterogeneity and temporal fluctuations of cellular phenotypes. While different phenotypic traits such as abundance of growth-related proteins in single cells may have differential effects on the reproductive success of cells, rigorous experimental quantification of this process has remained elusive due to the complexity of single cell physiology within the context of a proliferating population. We introduce and apply a practical empirical method to quantify the fitness landscapes of arbitrary phenotypic traits, using genealogical data in the form of population lineage trees which can include phenotypic data of various kinds. Our inference methodology for fitness landscapes determines how reproductivity is correlated to cellular phenotypes, and provides a natural generalization of bulk growth rate measures for single-cell histories. Using this technique, we quantify the strength of selection acting on different cellular phenotypic traits within populations, which allows us to determine whether a change in population growth is caused by individual cells' response, selection within a population, or by a mixture of these two processes. By applying these methods to single-cell time-lapse data of growing bacterial populations that express a resistance-conferring protein under antibiotic stress, we show how the distributions, fitness landscapes, and selection strength of single-cell phenotypes are affected by the drug. Our work provides a unified and practical framework for quantitative measurements of fitness landscapes and selection strength for any statistical quantities definable on lineages, and thus elucidates the adaptive significance of phenotypic states in time series data. The method is applicable in diverse fields, from single cell biology to stem cell differentiation and viral evolution.
  • Mikihiro Hashimoto, Takashi Nozoe, Hidenori Nakaoka, Reiko Okura, Sayo Akiyoshi, Kunihiko Kaneko, Edo Kussell, and *Yuichi Wakamoto,
    Noise-driven growth rate gain in clonal cellular populations,
    PNAS 113, 3251-3256 (2016).
    Cellular populations in both nature and the lab are comprised of phenotypically heterogeneous individuals that compete with each other resulting in complex population dynamics. Predicting population growth characteristics based on knowledge of heterogeneous single-cell dynamics remains challenging. By observing groups of cells for hundreds of generations at single-cell resolution, we reveal that growth noise causes clonal populations of Escherichia coli to double faster than the mean doubling time of their constituent single cells across a broad set of balanced-growth conditions. We show that the population-level growth rate gain as well as age structures of populations and of cell lineages in competition are predictable. Furthermore, we theoretically reveal that the growth rate gain can be linked with the relative entropy of lineage generation time distributions. Unexpectedly, we find an empirical linear relation between the means and the variances of generation times across conditions, which provides a general constraint on maximal growth rates. Together, these results demonstrate a fundamental benefit of noise for population growth, and identify a growth law that sets a ‘speed limit’ for proliferation.
  • Maroš Pleška, Long Qian, Reiko Okura, Tobias Bergmiller, Yuichi Wakamoto, Edo Kussell, and *Călin C. Guet,
    Bacterial autoimmunity due to a restriction-modification system,
    Current Biology 26, 404-409 (2016).
    Restriction-modification (RM) systems represent a minimal and ubiquitous biological system of self/non-self discrimination in prokaryotes, which protects hosts from exogenous DNA. The mechanism is based on the balance between methyltransferase (M) and cognate restriction endonuclease (R). M tags endogenous DNA as self by methylating short specific DNA sequences called restriction sites, whereas R recognizes unmethylated restriction sites as non-self and introduces a double-stranded DNA break. Restriction sites are significantly underrepresented in prokaryotic genomes, suggesting that the discrimination mechanism is imperfect and occasionally leads to autoimmunity due to self-DNA cleavage (self-restriction). Furthermore, RM systems can promote DNA recombination and contribute to genetic variation in microbial populations, thus facilitating adaptive evolution. However, cleavage of self-DNA by RM systems as elements shaping prokaryotic genomes has not been directly detected, and its cause, frequency, and outcome are unknown. We quantify self-restriction caused by two RM systems of Escherichia coli and find that, in agreement with levels of restriction site avoidance, EcoRI, but not EcoRV, cleaves self-DNA at a measurable rate. Self-restriction is a stochastic process, which temporarily induces the SOS response, and is followed by DNA repair, maintaining cell viability. We find that RM systems with higher restriction efficiency against bacteriophage infections exhibit a higher rate of self-restriction, and that this rate can be further increased by stochastic imbalance between R and M. Our results identify molecular noise in RM systems as a factor shaping prokaryotic genomes.
  • Ayako Uno, Ryohei Nakamura, Tatsuya Tsukahara, Wei Qu, Sumio Sugano, Yutaka Suzuki, Shinichi Morishita, and Hiroyuki Takeda,
    Comparative analysis of genome and epigenome in the closely related medaka species identifies conserved sequence preferences for DNA hypomethylated domains,
    Zoological Science 33, 368-365 (2016).
  • Takane Ozawa, Tomoko Mizuhara, Masataka Arata, Masakazu Shimada, Teruyuki Niimi, Kensuke Okada, *Yasukazu Okada, and *Kunihiro Ohta,
    Histone deacetylases control module-specific phenotypic plasticity in beetle weapons,
    PNAS 113, 15042–15047 (2016).
    Nutritional conditions during early development influence the plastic expression of adult phenotypes. Among several body modules of animals, the development of sexually selected exaggerated traits exhibits striking nutrition sensitivity, resulting in positive allometry and hypervariability distinct from other traits. Using de novo RNA sequencing and comprehensive RNA interference (RNAi) for epigenetic modifying factors, we found that histone deacetylases (HDACs) and polycomb group (PcG) proteins preferentially influence the size of mandibles (exaggerated male weapon) and demonstrate nutrition-dependent hypervariability in the broad-horned flour beetle, Gnatocerus cornutus. RNAi-mediated HDAC1 knockdown (KD) in G. cornutus larvae caused specific curtailment of mandibles in adults, whereas HDAC3 KD led to hypertrophy. Notably, these KDs conferred opposite effects on wing size, but little effect on the size of the core body and genital modules. PcG RNAi also reduced adult mandible size. These results suggest that the plastic development of exaggerated traits is controlled in a module-specific manner by HDACs.
  • Koji Hashimoto, Kohei Kurosawa, Akiho Murayama, *Hidetaka Seo, and Kunihiro Ohta,
    B Cell-Based Seamless Engineering of Antibody Fc Domains,
    PLoS One 11, e0167232 (2016).
    Engineering of monoclonal antibodies (mAbs) enables us to obtain mAbs with additional functions. In particular, modifications in antibody’s Fc (fragment, crystallizable) region can provide multiple benefits such as added toxicity by drug conjugation, higher affinity to Fc receptors on immunocytes, or the addition of functional modules. However, the generation of recombinant antibodies requires multiple laborious bioengineering steps. We previously developed a technology that enables rapid in vitro screening and isolation of specific mAb-expressing cells from the libraries constructed with chicken B-cell line DT40 (referred to as the ‘ADLib system’). To upgrade this ADLib system with the ability to generate customized mAbs, we developed a novel and rapid engineering technology that enables seamless exchanges of mAbs’ Fc domains after initial selections of mAb-producing clones by the ADLib system, using a gene-replacement unit for recombinase-mediated cassette exchange (RMCE). In this system, Cre-recombinase recognition sites were inserted into the Fc region of the active DT40 IgM allele, allowing the replacement of the Fc domain by the sequences of interest upon co-transfection of a Cre recombinase and a donor DNA, enabling the rapid exchange of Fc regions. Combining this method with the ADLib system, we demonstrate rapid Fc engineering to generate fluorescent antibodies and to enhance affinity to Fc receptors.
  • Naomichi Takemata and *Kunihiro Ohta,
    Role of non-coding RNA transcription around gene regulatory elements in transcription factor recruitment,
    RNA Biology 14, 1-5 (2016).
    Eukaryotic cells produce a variety of non-coding RNAs (ncRNAs), many of which have been shown to play pivotal roles in biological processes such as differentiation, maintenance of pluripotency of stem cells, and cellular response to various stresses. Genome-wide analyses have revealed that many ncRNAs are transcribed around regulatory DNA elements located proximal or distal to gene promoters, but their biological functions are largely unknown. Recently, it has been demonstrated in yeast and mouse that ncRNA transcription around gene promoters and enhancers facilitates DNA binding of transcription factors to their target sites. These results suggest universal roles of promoter/enhancer-associated ncRNAs in the recruitment of transcription factors to their binding sites.
  • *Yoshito Hirata, *Arisa Oda, Kunihiro Ohta, and Kazuyuki Aihara,
    Three-dimensional reconstruction of single-cell chromosome structure using recurrence plots,
    Scientific Reports 6, 34982 (2016).
    Single-cell analysis of the three-dimensional (3D) chromosome structure can reveal cell-to-cell variability in genome activities. Here, we propose to apply recurrence plots, a mathematical method of nonlinear time series analysis, to reconstruct the 3D chromosome structure of a single cell based on information of chromosomal contacts from genome-wide chromosome conformation capture (Hi-C) data. This recurrence plot-based reconstruction (RPR) method enables rapid reconstruction of a unique structure in single cells, even from incomplete Hi-C information.
  • Atsuko Miki, Josephine Galipon, Satoshi Sawai, Toshifumi Inada, and *Kunihiro Ohta,
    RNA decay systems enhance reciprocal switching of sense and antisense transcripts in response to glucose starvation,
    Genes to Cells 21, 1276-1289 (2016).
    Antisense RNA has emerged as a crucial regulator of opposite-strand protein-coding genes in the long noncoding RNA (lncRNA) category, but little is known about their dynamics and decay process in the context of a stress response. Antisense transcripts from the fission yeast fbp1 locus (fbp1-as) are expressed in glucose-rich conditions and anticorrelated with transcription of metabolic stress-induced lncRNA (mlonRNA) and mRNA on the sense strand during glucose starvation. Here, we investigate the localization and decay of antisense RNAs at fbp1 and other loci, and propose a model to explain the rapid switch between antisense and sense mlonRNA/mRNA transcription triggered by glucose starvation. We show that fbp1-as shares many features with mRNAs, such as a 5′-cap and poly(A)-tail, and that its decay partially depends upon Rrp6, a cofactor of the nuclear exosome complex involved in 3′–5′ degradation of RNA. Fluorescence in situ hybridization and polysome fractionation show that the majority of remaining fbp1-as localizes to the cytoplasm and binds to polyribosomes in glucose-rich conditions. Furthermore, fbp1-as and antisense RNA at other stress-responsive loci are promptly degraded via the cotranslational nonsense-mediated decay (NMD) pathway. These results suggest NMD may potentiate the swift disappearance of antisense RNAs in response to cellular stress.
  • Naoyoshi Kumakura, Hiroka Otsuki, Masaru Ito, Mika Nomoto, Yasuomi Tada, Kunihiro Ohta, and *Yuichiro Watanabe,
    Arabidopsis AtRRP44 has ribonuclease activity that is required for cell viability,
    Plant Biotechnology 33, 77–85 (2016).
    The RNA exosome is a multiprotein complex responsible for 3′ to 5′ degradation and processing of various classes of RNAs in eukaryotes. Rrp44/Dis3 is the catalytic center of the RNA exosome in yeast and human. Previously, we identified Arabidopsis thaliana RRP44 (AtRRP44) as a single functional homolog of Rrp44/Dis3. Although AtRRP44 is potentially a catalytic center of the plant RNA exosome, the ribonuclease activity of AtRRP44 has not been tested. Here, we show that AtRRP44 has ribonuclease activity using in vitro translated recombinant proteins. Mutation of the aspartic acid residue D489 of AtRRP44 to asparagine (D489N) resulted in loss of ribonuclease activity, indicating that aspartic acid is at the active site. The wild-type AtRRP44 protein rescued the growth defect of Saccharomyces cerevisiae rrp44 mutants, but the D489N mutated AtRRP44 did not. This finding suggests that the ribonuclease activity of wild-type AtRRP44 is required for yeast cell viability. We also showed that AtRRP44 was highly expressed in organs experiencing active cell turnover, such as shoot apical meristem, root apical meristem, and lateral root primordium. Along with previous studies showing that loss of RRP44 in Arabidopsis is lethal, our results suggest that AtRRP44 has ribonuclease activity that is related to plant development.
  • Risa Mitsumori, Tomoe Ohashi, Kazuto Kugou, Ayako Ichino ,Kei Taniguchi, Kunihiro Ohta, Hiroyuki Uchida, and *Masaya Oki,
    Analysis of novel Sir3 binding regions in Saccharomyces cerevisiae,
    The Journal of Biochemistry 160, 11-17 (2016).
    In Saccharomyces cerevisiae, the HMR, HML, telomere and rDNA regions are silenced. Silencing at the rDNA region requires Sir2, and silencing at the HMR, HML and telomere regions requires binding of a protein complex, consisting of Sir2, Sir3 and Sir4, that mediates repression of gene expression. Here, several novel Sir3 binding domains, termed CN domains (Chromosomal Novel Sir3 binding region), were identified using chromatin immunoprecipitation (ChIP) on chip analysis of S. cerevisiae chromosomes. Furthermore, analysis of G1-arrested cells demonstrated that Sir3 binding was elevated in G1-arrested cells compared with logarithmically growing asynchronous cells, and that Sir3 binding varied with the cell cycle. In addition to 14 CN regions identified from analysis of logarithmically growing asynchronous cells (CN1-14), 11 CN regions were identified from G1-arrested cells (CN15-25). Gene expression at some CN regions did not differ between WT and sir3Δ strains. Sir3 at conventional heterochromatic regions is thought to be recruited to chromosomes by Sir2 and Sir4; however, in this study, Sir3 binding occurred at some CN regions even in sir2Δ and sir4Δ backgrounds. Taken together, our results suggest that Sir3 exhibits novel binding parameters and gene regulatory functions at the CN binding domains.
  • Naomichi Takemata, Arisa Oda, Takatomi Yamada, Josephine Galipon, Tomoichiro Miyoshi, Yutaka Suzuki, Sumio Sugano, Charles S. Hoffman, Kouji Hirota,and *Kunihiro Ohta,
    Local potentiation of stress-responsive genes by upstream noncoding transcription,
    Nucleic Acids Research 44 , 5174-5189 (2016).
    It has been postulated that a myriad of long noncoding RNAs (lncRNAs) contribute to gene regulation. In fission yeast, glucose starvation triggers lncRNA transcription across promoter regions of stress-responsive genes including fbp1 (fructose-1,6-bisphosphatase1). At the fbp1 promoter, this transcription promotes chromatin remodeling and fbp1 mRNA expression. Here, we demonstrate that such upstream noncoding transcription facilitates promoter association of the stress-responsive transcriptional activator Atf1 at the sites of transcription, leading to activation of the downstream stress genes. Genome-wide analyses revealed that ∼50 Atf1-binding sites show marked decrease in Atf1 occupancy when cells are treated with a transcription inhibitor. Most of these transcription-enhanced Atf1-binding sites are associated with stress-dependent induction of the adjacent mRNAs or lncRNAs, as observed in fbp1 These Atf1-binding sites exhibit low Atf1 occupancy and high histone density in glucose-rich conditions, and undergo dramatic changes in chromatin status after glucose depletion: enhanced Atf1 binding, histone eviction, and histone H3 acetylation. We also found that upstream transcripts bind to the Groucho-Tup1 type transcriptional corepressors Tup11 and Tup12, and locally antagonize their repressive functions on Atf1 binding. These results reveal a new mechanism in which upstream noncoding transcription locally magnifies the specific activation of stress-inducible genes via counteraction of corepressors.
  • Sanki Tashiro, Tetsuya Handa, Atsushi Matsuda, Takuto Ban, Toru Takigawa, Kazumi Miyasato, Kojiro Ishii, Kazuto Kugou, Kunihiro Ohta, Yasushi Hiraoka, Hisao Masukata and *Junko Kanoh,
    Shugoshin forms a specialized chromatin domain at subtelomeres that regulates transcription and replication timing,
    Nature Communications 7, 10393 (2016).
    A chromosome is composed of structurally and functionally distinct domains. However, the molecular mechanisms underlying the formation of chromatin structure and the function of subtelomeres, the telomere-adjacent regions, remain obscure. Here we report the roles of the conserved centromeric protein Shugoshin 2 (Sgo2) in defining chromatin structure and functions of the subtelomeres in the fission yeast Schizosaccharomyces pombe. We show that Sgo2 localizes at the subtelomeres preferentially during G2 phase and is essential for the formation of a highly condensed subtelomeric chromatin body ‘knob’. Furthermore, the absence of Sgo2 leads to the derepression of the subtelomeric genes and premature DNA replication at the subtelomeric late origins. Thus, the subtelomeric specialized chromatin domain organized by Sgo2 represses both transcription and replication to ensure proper gene expression and replication timing.
  • Atsuko Miki, Josephine Galipon, Satoshi Sawai, Toshifumi Inada, and Kunihiro Ohta,
    RNA decay systems enhance reciprocal switching of sense and antisense transcripts in response to glucose starvation. Genes to Cells,
    Genes to Cells 21, 1276-1289 (2016).
    Antisense RNA has emerged as a crucial regulator of opposite-strand protein-coding genes in the long noncoding RNA (lncRNA) category, but little is known about their dynamics and decay process in the context of a stress response. Antisense transcripts from the fission yeast fbp1 locus (fbp1-as) are expressed in glucose-rich conditions and anticorrelated with transcription of metabolic stress-induced lncRNA (mlonRNA) and mRNA on the sense strand during glucose starvation. Here, we investigate the localization and decay of antisense RNAs at fbp1 and other loci, and propose a model to explain the rapid switch between antisense and sense mlonRNA/mRNA transcription triggered by glucose starvation. We show that fbp1-as shares many features with mRNAs, such as a 5'-cap and poly(A)-tail, and that its decay partially depends upon Rrp6, a cofactor of the nuclear exosome complex involved in 3'-5' degradation of RNA. Fluorescence in situ hybridization and polysome fractionation show that the majority of remaining fbp1-as localizes to the cytoplasm and binds to polyribosomes in glucose-rich conditions. Furthermore, fbp1-as and antisense RNA at other stress-responsive loci are promptly degraded via the cotranslational nonsense-mediated decay (NMD) pathway. These results suggest NMD may potentiate the swift disappearance of antisense RNAs in response to cellular stress.
  • Akihiko Nakajima, Motohiko Ishida, Taihei Fujimori, Yuichi Wakamoto, and Satoshi Sawai,
    The Microfluidic lighthouse: an omnidirectional gradient generator,
    Lab on a Chip 16, 4382-4394 (2016).
    Studies of chemotactic cell migration rely heavily on various assay systems designed to evaluate the ability of cells to move in response to attractant molecules. In particular, the development of microfluidics-based devices in recent years has made it possible to spatially distribute attractant molecules in graded profiles that are sufficiently stable and precise to test theoretical predictions regarding the accuracy and efficiency of chemotaxis and the underlying mechanism of stimulus perception. However, because the gradient is fixed in a direction orthogonal to the laminar flow and thus the chamber geometry, conventional devices are limited for the study of cell re-orientation to gradients that move or change directions. Here, we describe the development of a simple radially symmetric microfluidics device that can deliver laminar flow in 360°. A stimulant introduced either from the central inlet or by photo uncaging is focused into the laminar flow in a direction determined by the relative rate of regulated flow from multiple side channels. Schemes for flow regulation and an extended duplexed device were designed to generate and move gradients in desired orientations and speed, and then tested to steer cell migration of Dictyostelium and neutrophil-like HL60 cells. The device provided a high degree of freedom in the positioning and orientation of attractant gradients, and thus may serve as a versatile platform for studying cell migration, re-orientation, and steering.
  • Satoshi Kuwana, Hiroshi Senoo, Satoshi Sawai, and Masashi Fukuzawa,
    A novel, lineage-primed prestalk cell subtype involved in the morphogenesis of D. discoideum,
    Developmental Biology 416, 286-299 (2016).
    Dictyostelium morphogenesis requires the tip, which acts as an organizer and conducts orchestrated cell movement and cell differentiation. At the slug stage the tip region contains prestalk A (pstA) cells, which are usually recognized by their expression of reporter constructs that utilize a fragment of the promoter of the ecmA gene. Here, using the promoter region of the o-methyl transferase 12 gene (omt12) to drive reporter expression, we demonstrate the presence, also within the pstA region, of a novel prestalk cell subtype: the pstVA cells. Surprisingly, a sub-population of the vegetative cells express a pstVA: GFP marker and, sort out to the tip, both when developing alone and when co-developed with an excess of unmarked cells. The development of such a purified GFP-marked population is greatly accelerated: by precocious cell aggregation and tip formation with accompanying precocious elevation of developmental gene transcription. We therefore suggest that the tip contains at least two prestalk cell subtypes: the developmentally-specified pstA cells and the lineage-primed pstVA cells. It is presumably the pstVA cells that play the dominant role in morphogenesis during the earlier stages of development. The basis for the lineage priming is, however, unclear because we can find no correlation between pstVA differentiation and nutrient status during growth or cell cycle position at the time of starvation, the two known determinants of probable cell fate.
  • Fumihito Fukujin, Akihiko Nakajima, Nao Shimada, and Satoshi Sawai,
    Self-organization of chemoattractant waves in Dictyostelium depends on F-actin and cell–substrate adhesion,
    Journal of The Royal Society Interface 13 (2016).
    In the social amoeba Dictyostelium discoideum, travelling waves of extracellular cyclic adenosine monophosphate (cAMP) self-organize in cell populations and direct aggregation of individual cells to form multicellular fruiting bodies. In contrast to the large body of studies that addressed how movement of cells is determined by spatial and temporal cues encoded in the dynamic cAMP gradients, how cell mechanics affect the formation of a self-generated chemoattractant field has received less attention. Here, we show, by live cell imaging analysis, that the periodicity of the synchronized cAMP waves increases in cells treated with the actin inhibitor latrunculin. Detail analysis of the extracellular cAMP-induced transients of cytosolic cAMP (cAMP relay response) in well-isolated cells demonstrated that their amplitude and duration were markedly reduced in latrunculin-treated cells. Similarly, in cells strongly adhered to a poly-l-lysine-coated surface, the response was suppressed, and the periodicity of the population-level oscillations was markedly lengthened. Our results suggest that cortical F-actin is dispensable for the basic low amplitude relay response but essential for its full amplification and that this enhanced response is necessary to establish high-frequency signalling centres. The observed F-actin dependence may prevent aggregation centres from establishing in microenvironments that are incompatible with cell migration.
  • Akihiko Nakajima, Satoshi Sawai,
    Dissecting Spatial and Temporal Sensing in Dictyostelium Chemotaxis Using a Wave Gradient Generator,
    Methods in Molecular Biology 1407, 107-122 (2016).
    External cues that dictate the direction of cell migration are likely dynamic during many biological processes such as embryonic development and wound healing. Until recently, how cells integrate spatial and temporal information to determine the direction of migration has remained elusive. In Dictyostelium discoideum, the chemoattractant cAMP that directs cell aggregation propagates as periodic waves. In light of the fact that any temporally evolving complex signals, in principle, can be expressed as a sum of sinusoidal functions with various frequencies, the Dictyostelium system serves as a minimal example, where the dynamic signal is in the simplest form of near sinusoidal wave with one dominant frequency. Here, we describe a method to emulate the traveling waves in a fluidics device. The text provides step-by-step instructions on the device setup and describes ways to analyze the acquired data. These include quantification of membrane translocation of fluorescently labeled proteins in individual Dictyostelium cells and estimation of exogenous cAMP profiles. The described approach has already helped decipher spatial and temporal aspects of chemotactic sensing in Dictyostelium. More specifically, it allowed one to discriminate the temporal and the spatial sensing aspects of directional sensing. With some modifications, one should be able to implement similar analysis in other cell types.
  • Naohiro Terasaka, Kazuki Futai, Takayuki Katoh, Hiroaki Suga,
    A human microRNA precursor binding to folic acid discovered by small RNA transcriptomic SELEX,
    RNA 22(12), 1918-1928 (2016).
    RNA aptamers are structured motifs that bind to specific molecules. A growing number of RNAs bearing aptamer elements, whose functions are modulated by direct binding of metabolites, have been found in living cells. Recent studies have suggested that more small RNAs binding to metabolites likely exist and may be involved in diverse cellular processes. However, conventional methods are not necessarily suitable for the discovery of such RNA aptamer elements in small RNAs with lengths ranging from 50 to 200 nucleotides, due to the far more abundant tRNAs in this size range. Here, we describe a new in vitro selection method to uncover naturally occurring small RNAs capable of binding to a ligand of interest, referred to as small RNA transcriptomic SELEX (smaRt-SELEX). By means of this method, we identified a motif in human precursor microRNA 125a (hsa-pre-miR-125a) that interacts with folic acid. Mutation studies revealed that the terminal loop region of hsa-pre-miR-125a is important for this binding interaction. This method has potential for the discovery of new RNA aptamer elements or catalytic motifs in biological small RNA fractions.
  • Yukiko Matsunaga, Nasir K. Bashiruddin, Yu Kitago, Junichi Takagi, Hiroaki Suga ,
    Allosteric inhibition of a semaphorin 4D receptor plexin B1 by a high affinity macrocyclic peptide,
    Cell Chemical Biology 23, 1341-1350 (2016).
  • Yuki Goto, Hiroaki Suga,
    A post-translational cyclodehydratase, PatD, tolerates sequence variation in the C-terminal region of the substrate peptides,
    Chemistry Letters 45, 1247-1249 (2016).
  • Kazuki Futai, Naohiro Terasaka, Takayuki Katoh, Hiroaki Suga ,
    tRid, an enabling method to isolate previously inaccessible small RNA fractions,
    Methods 106, 105-111 (2016).
    Detection of rare small RNA species whose sizes are overlapping with tRNAs often suffers from insufficient sensitivity due to the overwhelming abundance of tRNAs. We here report a method, named tRid (tRNA rid), for removing abundant tRNAs from small RNA fractions regardless of tRNA sequence species. By means of tRid, we are able to selectively enrich small RNAs which have been previously difficult to access due to mass existence of tRNAs in such fractions. A flexible tRNA-acylation ribozyme, known as flexizyme, is a key tool where the total tRNAs are aminoacylated with N-biotinylated phenylalanine regardless of tRNA sequences, and therefore the biotin-tagged tRNAs could be readily removed from the small RNA fractions by the use of streptavidin-immobilized magnetic beads. Next generation sequencing of the isolated small RNA fraction revealed that small RNAs with less than 200 nt were effectively enriched, allowing us to identify previously unknown small RNAs in HeLa and E. coll.
  • Takayuki Katoh, Ingo Wohlgemuth, Masanobu Nagano, Marina V. Rodnina, and Hiroaki Suga,
    Essential structural elements in tRNA(Pro) for EF-P-mediated alleviation of translation stalling,
    Nature Communications 7, 11657 (2016).
    The ribosome stalls on translation of polyproline sequences due to inefficient peptide bond formation between consecutive prolines. The translation factor EF-P is able to alleviate this stalling by accelerating Pro-Pro formation. However, the mechanism by which EF-P recognizes the stalled complexes and accelerates peptide bond formation is not known. Here, we use genetic code reprogramming through a flexible in-vitro translation (FIT) system to investigate how mutations in tRNA(Pro) affect EF-P function. We show that the 9-nt D-loop closed by the stable D-stem sequence in tRNA(Pro) is a crucial recognition determinant for EF-P. Such D-arm structures are shared only among the tRNA(Pro) isoacceptors and tRNA(fMet) in Escherichia coli, and the D-arm of tRNA(fMet) is essential for EF-P-induced acceleration of fMet-puromycin formation. Thus, the activity of EF-P is controlled by recognition elements in the tRNA D-arm
  • Yoshihiko Iwane, Azusa Hitomi, Hiroshi Murakami, Takayuki Katoh, Yuki Goto, and Hiroaki Suga,
    Expanding the amino acid repertoire of ribosomal polypeptide synthesis via the artificial division of codon boxes,
    Nature Chemistry 8(4), 317-325 (2016).
  • *Taisuke Banno, Yuki Tanaka, Kouichi Asakura, *Taro Toyota,
    Self-propelled oil droplets and their morphological change to giant vesicles induced by a surfactant solution at low pH,
    Langmuir 32, 9591-9597 (2016).
  • Taisuke Banno, Arisa Asami, Naoko Ueno, Hiroyuki Kitahata, Yuki Koyano, Kouichi Asakura, *Taro Toyota,
    Deformable self-propelled micro-object comprising underwater oil droplets,
    Scientific Reports 6, 31292 (2016).
  • *Yuno Natsume, Taro Toyota,
    Appearance of crystalline pattern for colloidal particles encapsulated in giant vesicles,
    Transactions of the Materials Research Society of Japan 41, 147-149 (2016).
  • Masahito Hayashi, Masayoshi Nishiyama, Yuki Kazayama, Taro Toyota, Yoshie Harada, *Kingo Takiguchi,
    Reversible morphological control of tubulin-encapsulating giant liposomes by hydrostatic pressure,
    Langmuir 32, 3794-3802 (2016).
  • Yuki Kazayama, Tetsuhiko Teshima, Toshihisa Osaki, *Shoji Takeuchi, *Taro Toyota,
    Integrated microfluidic system for size-based selection and trapping of giant vesicles,
    Analytical Chemistry 88, 1111-1116 (2016).
  • Kazuma Gotoh, Tomonori Nomoto, Taro Toyota, *Masanori Fujinami,
    Effects of halide ions on the acceptor phase in spontaneous chemical oscillations in donor/membrane/acceptor systems,
    Journal of Colloid and Interface Science 462, 351-358 (2016).
  • Yuno Natsume, *Taro Toyota,
    Asymmetrical polyhedral configuration of giant vesicles induced by orderly array of encapsulated colloidal particles,
    PLOS ONE 11, e0146683 (2016).
  • *豊田太郎, 本多智,
    生物物理 56, 165-167 (2016).
  • Tetsuya Hiraiwa, and Guillaume Salbreux,
    Role of Turnover in Active Stress Generation in a Filament Network,
    Physical Review Letters 116, 188101 (2016).
    We study the effect of turnover of cross-linkers, motors, and filaments on the generation of a contractile stress in a network of filaments connected by passive cross-linkers and subjected to the forces exerted by molecular motors. We perform numerical simulations where filaments are treated as rigid rods and molecular motors move fast compared to the time scale of an exchange of cross-linkers. We show that molecular motors create a contractile stress above a critical number of cross-linkers. When passive cross-linkers are allowed to turn over, the stress exerted by the network vanishes due to the formation of clusters. When both filaments and passive cross-linkers turn over, clustering is prevented and the network reaches a dynamic contractile steady state. A maximum stress is reached for an optimum ratio of the filament and cross-linker turnover rates. Taken together, our work reveals conditions for stress generation by molecular motors in a fluid isotropic network of rearranging filaments.
  • *Takao Ohta, Mitsusuke Tarama and Masaki Sano,
    Simple model of cell crawling,
    Physica D 318-319, 3-11 (2016).
    Based on symmetry consideration of migration and shape deformations, we formulate phenomenologically the dynamics of cell crawling in two dimensions. Forces are introduced to change the cell shape. The shape deformations induce migration of the cell on a substrate. For time-independent forces we show that not only a stationary motion but also a limit cycle oscillation of the migration velocity and the shape occurs as a result of nonlinear coupling between different deformation modes. Time-dependent forces are generated in a stochastic manner by utilizing the so-called coherence resonance of an excitable system. The present coarse-grained model has a flexibility that it can be applied, e.g., both to keratocyte cells and to View the MathML source cells, which exhibit quite different dynamics from each other. The key factors for the motile behavior inherent in each cell type are identified in our model.
  • *John J. Molina, Kotaro Otomura, Hayato Shiba, Hideki Kobayashi, Masaki Sano, and Ryoichi Yamamoto,
    Rheological evaluation of colloidal dispersions using the smoothed profile method: formulation and applications,
    Journal of Fluid Mechanics 792, 590-619 (2016).
    The smoothed profile method is extended to study the rheological behaviour of colloidal dispersions under shear flow by using the Lees–Edwards boundary conditions. We start with a reformulation of the smoothed profile method, a direct numerical simulation method for colloidal dispersions, so that it can be used with the Lees–Edwards boundary condition, under steady or oscillatory-shear flow. By this reformulation, all the resultant physical quantities, including local and total shear stresses, become available through direct calculation. Three simple rheological simulations are then performed for (1) a spherical particle, (2) a rigid bead chain and (3) a collision of two spherical particles under shear flow. Quantitative validity of these simulations is examined by comparing the viscosity with that obtained from theory and Stokesian dynamics calculations. Finally, we consider the shear-thinning behaviour of concentrated colloidal dispersions.
  • *Masaki Sano and Keiichi Tamai,
    A Universal Transition to Turbulence in Channel Flow,
    Nature Physics 12, 249-253 (2016).
    Transition from laminar to turbulent flow drastically changes the mixing, transport, and drag properties of fluids, yet when and how turbulence emerges is elusive even for simple flow within pipes and rectangular channels1,2. Unlike the onset of temporal disorder, which is identified as the universal route to chaos in confined flows3,4, characterization of the onset of spatio-temporal disorder has been an outstanding challenge because turbulent domains irregularly decay or spread as they propagate downstream. Here, through extensive experimental investigation of channel flow, we identify a distinctive transition with critical behavior. Turbulent domains continuously injected from an inlet ultimately decayed, or in contrast, spread depending on flow rates. Near a transition point, critical behavior was observed. We investigate both spatial and temporal dynamics of turbulent clusters, measuring four critical exponents, a universal scaling function and a scaling relation, all in agreement with the (2+1) dimensional directed percolation universality class.
  • *佐野雅己,
    数理科学 631, 1-4 (2016).
    「数理モデルと普遍性」- モデル化で捉える自然現象の本質 -と題する特集を企画し、統計物理のモデルを中心に単純な数理モデルが自然現象の普遍性をみごとに表す場合があることを延べ、各記事の概要を紹介した。
  • Takanori Sano, Kentaro Kawata, Satoshi Ohono, Katsuyuki Yugi, Hiroaki Kakuda,Hiroyuki Kubota, Shinsuke Uda, Masashi Fujii, Katsuyuki Kunida, Daisuke Hoshino,Atsushi Hatano, Yuki Ito, Miharu Sato, Yutaka Suzuki, Shinya Kuroda,
    Selective control of up-regulated and down-regulated genes by temporal patterns and doses of insulin,
    Science Signaling 9, ra112 (2016).
    Secretion of insulin transiently increases after eating, resulting in a high circulating concentration. Fasting limits insulin secretion, resulting in a low concentration of insulin in the circulation. We analyzed transcriptional responses to different temporal patterns and doses of insulin in the hepatoma FAO cells and identified 13 up-regulated and 16 down-regulated insulin-responsive genes (IRGs). The up-regulated IRGs responded more rapidly than did the down-regulated IRGs to transient stepwise or pulsatile increases in insulin concentration, whereas the down-regulated IRGs were repressed at lower concentrations of insulin than those required to stimulate the up-regulated IRGs. Mathematical modeling of the insulin response as two stages-(i) insulin signaling to transcription and (ii)transcription and mRNA stability-indicated that the first stage was the more rapid stage for the down-regulated IRGs, whereas the second stage of transcription was the more rapid stage for the up-regulated IRGs. A subset of the IRGs that were up-regulated or down-regulated in the FAO cells was similarly regulated in the livers of rats injected with a single dose of insulin. Thus, not only can cells respond to insulin but they can also interpret the intensity and pattern of signal to produce distinct transcriptional responses. These results provide insight that may be useful in treating obesity and type 2 diabetes associated with aberrant insulin production or tissue responsiveness.
  • Takamasa Kudo, *Shinsuke Uda, Takaho Tsuchiya, Takumi Wada, Yasuaki Karasawa, Masashi Fujii, Takeshi H. Saito, *Shinya Kuroda,
    Laguerre filter analysis with partial least square regression reveals a priming effect of ERK and CREB on c-FOS induction,
    PLoS ONE 11, e0160548 (2016).
    Signaling networks are made up of limited numbers of molecules and yet can code information that controls different cellular states through temporal patterns and a combination of signaling molecules. In this study, we used a data-driven modeling approach, the Laguerre filter with partial least square regression, to describe how temporal and combinatorial patterns of signaling molecules are decoded by their downstream targets. The Laguerre filter is a time series model used to represent a nonlinear system based on Volterra series expansion. Furthermore, with this approach, each component of the Volterra series expansion is expanded by Laguerre basis functions. We combined two approaches, application of a Laguerre filter and partial least squares (PLS) regression, and applied the combined approach to analysis of a signal transduction network. We applied the Laguerre filter with PLS regression to identify input and output (IO) relationships between MAP kinases and the products of immediate early genes (IEGs). We found that Laguerre filter with PLS regression performs better than Laguerre filter with ordinary regression for the reproduction of a time series of IEGs. Analysis of the nonlinear characteristics extracted using the Laguerre filter revealed a priming effect of ERK and CREB on c-FOS induction. Specifically, we found that the effects of a first pulse of ERK enhance the subsequent effects on c-FOS induction of treatment with a second pulse of ERK, a finding consistent with prior molecular biological knowledge. The variable importance of projections and output loadings in PLS regression predicted the upstream dependency of each IEG. Thus, a Laguerre filter with partial least square regression approach appears to be a powerful method to find the processing mechanism of temporal patterns and combination of signaling molecules by their downstream gene expression.
  • Sarah Filippi, Chris P. Barnes, Paul D.W. Kirk, Takamasa Kudo, Katsuyuki Kunida, Siobhan S. McMahon, Takaho Tsuchiya, Takumi Wada, Shinya Kuroda, and *Michael P.H. Stumpf ,
    Robustness of MEK-ERK dynamics and origins of Cell-to-Cell variability in MAPK signaling,
    Cell Reports 15, 2524-2535 (2016).
    Cellular signaling processes can exhibit pronounced cell-to-cell variability in genetically identical cells. This affects how individual cells respond differentially to the same environmental stimulus. However, the origins of cell-to-cell variability in cellular signaling systems remain poorly understood. Here, we measure the dynamics of phosphorylated MEK and ERK across cell populations and quantify the levels of population heterogeneity over time using high-throughput image cytometry. We use a statistical modeling framework to show that extrinsic noise, particularly that from upstream MEK, is the dominant factor causing cell-to-cell variability in ERK phosphorylation, rather than stochasticity in the phosphorylation/dephosphorylation of ERK. We furthermore show that without extrinsic noise in the core module, variable (including noisy) signals would be faithfully reproduced downstream, but the within-module extrinsic variability distorts these signals and leads to a drastic reduction in the mutual information between incoming signal and ERK activity.
  • Eri Hasegawa, James W. Truman, and *Akinao Nose,
    Identification of excitatory premotor interneurons which regulate local muscle contraction during Drosophila larval locomotion,
    Scientific Reports 6, 30806 (2016).
    We use Drosophila larval locomotion as a model to elucidate the working principles of motor circuits. Larval locomotion is generated by rhythmic and sequential contractions of body-wall muscles from the posterior to anterior segments, which in turn are regulated by motor neurons present in the corresponding neuromeres. Motor neurons are known to receive both excitatory and inhibitory inputs, combined action of which likely regulates patterned motor activity during locomotion. Although recent studies identified candidate inhibitory premotor interneurons, the identity of premotor interneurons that provide excitatory drive to motor neurons during locomotion remains unknown. In this study, we searched for and identified two putative excitatory premotor interneurons in this system, termed CLI1 and CLI2 (cholinergic lateral interneuron 1 and 2). These neurons were segmentally arrayed and activated sequentially from the posterior to anterior segments during peristalsis. Consistent with their being excitatory premotor interneurons, the CLIs formed GRASP- and ChAT-positive putative synapses with motoneurons and were active just prior to motoneuronal firing in each segment. Moreover, local activation of CLI1s induced contraction of muscles in the corresponding body segments. Taken together, our results suggest that the CLIs directly activate motoneurons sequentially along the segments during larval locomotion.
  • Aleksandr Drozd, Olaf Witkowski, Satoshi Matsuoka, Takashi Ikegami,
    Critical Mass in the Emergence of Collective Intelligence: a Parallelized Simulation of Swarms in Noisy Environments,
    Artificial Life and Robotics 21, 317-323 (2016).
  • Tim Taylor, Mark Bedau, Alastair Channon, David Ackley, Wolfgang Banzhaf, Guillaume Beslon, Emily Dolson, Tom Froese, Simon Hickinbotham, Takashi Ikegami, Barry McMullin, Norman Packard, Steen Rasmussen, Nathaniel Virgo, Eran Agmon, Edward Clark, Simon McGregor,Charles Ofria, Glen Ropella, Lee Spector, Kenneth O. Stanley, Adam Stanton, Christopher Timperley, Anya Vostinar, and Michael Wiser,
    Open-Ended Evolution: Perspectives from the OEE Workshop in York,
    Artificial Life 22, 408-423 (2016).
  • Olaf Witkowski, Takashi Ikegami,
    Emergence of Swarming Behavior: Foraging Agents Evolve Collective Motion Based on Signaling,
    PLoS ONE 11(4), e0152756 (2016).
  • Julien Hubert and Takashi Ikegami,
    How long did it last? Memorizing interval timings in a simple robotic task,
    Proceedings of the Artificial Life Conference 2016, 406-407 (2016).
  • Martin Biehl, Takashi Ikegami, Daniel Polani,
    Towards information based spatiotemporal patterns as a foundation for agent representation in dynamical systems,
    Proceedings of the Artificial Life Conference 2016, 722-729 (2016).
  • *Yohei Saito, Yuki Sughiyama, Kunihiko Kaneko, Tetsuya J. Kobayashi,
    Discreteness-induced transitions in multibody reaction systems,
    Physical Review E 94, 22140 (2016).
  • *Yukinobu Arata, Michio Hiroshima, Chan-Gi Pack, Ravikrishna Ramanujam, Fumio Motegi, Kenichi Nakazato, Hitoshi Sawa, Tetsuya J. Kobayashi, Tatsuo Shibata, and Yasushi Sako,
    Cortical Polarity of the RING Protein PAR-2 Is Maintained by Exchange Rate Kinetics at the Cortical-Cytoplasmic Boundary,
    Cell Reports 16, 2156-2168 (2016).
  • *Tetsuya J. Kobayashi, Ryo Yokota, Kazuyuki Aihara,
    Feedback Regulation and its Efficiency in Biochemical Networks,
    Journal of Statistical Physics 162, 1425-1449 (2016).
  • *秋山泰身, 小林徹也,
    医学の歩み 259, 839-842 (2016).