Details of a Researcher - FUNAHASHI Akira

Papers - FUNAHASHI Akira

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人工知能および高速レーザー照射システムの統合による高品質ヒト心筋細胞の作製と臨床応用

遠山周吾, 舟橋啓

福田記念医療技術振興財団情報 ((公財)福田記念医療技術振興財団) ( 35 ) 181 - 186 2022.12

Joint Work, Accepted

自動高速レーザー照射システムにより目的外細胞を除去し、ロット差の極めて少ない心筋細胞を作製するための前段階として、AIによる機械学習を用いて目的細胞と目的外細胞を識別する手法を開発することを目的とした。自動化心筋細胞拍動評価システムを用いて培養皿上で拍動する心筋細胞の動画を効率よく撮影し、拍動方向のベクトル解析により心筋領域を同定することで非心筋細胞との領域の判別が可能となった。セグメンテーション精度と心筋領域の割合の関係を定性的に確認すると、心筋領域の割合が高い画像のセグメンテーション精度は約80%に達したが、心筋領域の割合が低い画像のセグメンテーションは約1%と非常に低いことが確認された。

Intercellular interaction mechanisms promote diversity in intracellular ATP concentration in Escherichia coli populations

RJ Nakatani, M Itabashi, TG Yamada, NF Hiroi, A Funahashi

Scientific reports 12 (1), 17946 (Scientific Reports) 12 ( 1 ) 17946 2022.12

Joint Work, Accepted

In fluctuating environments, many microorganisms acquire phenotypic heterogeneity as a survival tactic to increase the likelihood of survival of the overall population. One example of this interindividual heterogeneity is the diversity of ATP concentration among members of Escherichia coli populations under glucose deprivation. Despite the importance of such environmentally driven phenotypic heterogeneity, how the differences in intracellular ATP concentration emerge among individual E. coli organisms is unknown. In this study, we focused on the mechanism through which individual E. coli achieve high intracellular ATP concentrations. First, we measured the ATP retained by E. coli over time when cultured at low (0.1 mM) and control (22.2 mM) concentrations of glucose and obtained the chronological change in ATP concentrations. Then, by comparing these chronological change of ATP concentrations and analyzing whether stochastic state transitions, periodic oscillations, cellular age, and intercellular communication—which have been reported as molecular biological mechanisms for generating interindividual heterogeneity—are involved, we showed that the appearance of high ATP-holding individuals observed among E. coli can be explained only by intercellular transmission. By performing metabolomic analysis of post-culture medium, we revealed a significant increase in the ATP, especially at low glucose, and that the number of E. coli that retain significantly higher ATP can be controlled by adding large amounts of ATP to the medium, even in populations cultured under control glucose concentrations. These results reveal for the first time that ATP-mediated intercellular transmission enables some individuals in E. coli populations grown at low glucose to retain large amounts of ATP.

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An explainable deep learning-based algorithm with an attention mechanism for predicting the live birth potential of mouse embryos

Y Tokuoka, TG Yamada, D Mashiko, Z Ikeda, TJ Kobayashi, K Yamagata, ...

Artificial Intelligence in Medicine 134, 102432 (Artificial Intelligence in Medicine) 134 102432 2022.12

Joint Work, Accepted, ISSN 09333657

In assisted reproductive technology (ART), embryos produced by in vitro fertilization (IVF) are graded according to their live birth potential, and high-grade embryos are preferentially transplanted. However, rates of live birth following clinical ART remain low worldwide. Grading is based on the embryo shape at a limited number of stages and does not consider the shape of embryos and intracellular structures, e.g., nuclei, at various stages important for normal embryogenesis. Here, we developed a Normalized Multi-View Attention Network (NVAN) that directly predicts live birth potential from the nuclear structure in live-cell fluorescence images of mouse embryos from zygote to across a wide range of stages. The input is morphological features of cell nuclei, which were extracted as multivariate time-series data by using the segmentation algorithm for mouse embryos. The classification accuracy of our method (83.87%) greatly exceeded that of existing machine-learning methods and that of visual inspection by embryo culture specialists. Our method also has a new attention mechanism that allows us to determine which values of multivariate time-series data, used to describe nuclear morphology, were the basis for the prediction. By visualizing the features that contributed most to the prediction of live birth potential, we found that the size and shape of the nucleus at the morula stage and at the time of cell division were important for live birth prediction. We anticipate that our method will help ART and developmental engineering as a new basic technology for IVF embryo selection.

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High quality genome assembly of the anhydrobiotic midge provides insights on a single chromosome-based emergence of extreme desiccation tolerance

Y Yoshida, N Shaikhutdinov, O Kozlova, M Itoh, M Tagami, M Murata, ...

NAR Genomics and Bioinformatics 4 (2), lqac029 (NAR Genomics and Bioinformatics) 4 ( 2 ) lqac029 2022.06

Joint Work, Accepted

Non-biting midges (Chironomidae) are known to inhabit a wide range of environments, and certain species can tolerate extreme conditions, where the rest of insects cannot survive. In particular, the sleeping chironomid Polypedilum vanderplanki is known for the remarkable ability of its larvae to withstand almost complete desiccation by entering a state called anhydrobiosis. Chromosome numbers in chironomids are higher than in other dipterans and this extra genomic resource might facilitate rapid adaptation to novel environments. We used improved sequencing strategies to assemble a chromosome-level genome sequence for P. vanderplanki for deep comparative analysis of genomic location of genes associated with desiccation tolerance. Using whole genome-based cross-species and intra-species analysis, we provide evidence for the unique functional specialization of Chromosome 4 through extensive acquisition of novel genes. In contrast to other insect genomes, in the sleeping chironomid a uniquely high degree of subfunctionalization in paralogous anhydrobiosis genes occurs in this chromosome, as well as pseudogenization in a highly duplicated gene family. Our findings suggest that the Chromosome 4 in Polypedilum is a site of high genetic turnover, allowing it to act as a 'sandbox' for evolutionary experiments, thus facilitating the rapid adaptation of midges to harsh environments.

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Temperature elevation detection in migrating cells

T Nakamura, J Sakamoto, K Okabe, A Taniguchi, TG Yamada, S Nonaka, ...

Optics Continuum 1 (5), 1085-1097 (OSA Continuum) 1 ( 5 ) 1085 - 1097 2022.05

Joint Work, Accepted

Active and dynamic migration and deformation of cells are universal research targets in cell biology. The leader cells that develop at the wound edge are required to actively reorganize their structure for migration and deformation. Such active reorganization of cellular morphology possibly affects the temperature inside the cells via biochemical reactions. On the other hand, it is also possible that changes of intracellular temperature may first trigger and induce active migration and cellular deformation. Recent development of temperature sensors for cells enables the display of temperature difference between two adjacent cell populations such as the leader and follower cells around a wound. We used two different temperature measurement methods to check the temperature of leader and follower cells in a wound healing assay system and found that leader cells were 10° ∼ 101°C warmer than follower cells. We also confirmed that, when the cells were artificially warmed with an infrared laser, they started moving, and when the laser was stopped, the cells also stopped moving. These results suggest that the warmth of the leader cells is not simply a result of their active movement, but also can play the role of a trigger for cell migration and deformation. Our results shed light on the possibility that various observed cases of intracellular temperature increase may trigger associated biological phenomena.

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Analyzing the submodule structure in deep learning models to reduce the search space of Neural Architecture Search

TOMIYAMA Mamoru, HIKI Yusuke, SHIBUYA Yusuke, TOKUOKA Yuta, YAMADA Takahiro G., FUNAHASHI Akira

Proceedings of the Annual Conference of JSAI (The Japanese Society for Artificial Intelligence) JSAI2022 ( 0 ) 3Yin204 - 3Yin204 2022

Joint Work, Accepted

Neural Architecture Search (NAS) is a method to automatically design architectures of deep learning. In NAS, the optimal architecture is determined from search space, which is a set of architecture substructures. However, the search space of the existing NAS is limited due to the huge amount of computation, so there is a possibility of missing high-performance architectures. Therefore, we hypothesized architectures with task-specific substructures are high-performance, and proposed a method to identify task-specific substructures that contribute to design efficient search space in NAS. We developed a method based on Graph Convolutional Network outputs task specificity for each substructure. We confirmed the correlation between task specificity and task accuracy for each substructure, so our method can identify task-specific substructures that contribute to accuracy. Our method is expected to contribute to NAS development that determines high-performance architectures with reduced computational complexity.

Cas9-mediated genome editing reveals a significant contribution of calcium signaling pathways to anhydrobiosis in Pv11 cells

Yugo Miyata, Hiroto Fuse, Shoko Tokumoto, Yusuke Hiki, Ruslan Deviatiiarov, Yuki Yoshida, Takahiro G. Yamada, Richard Cornette, Oleg Gusev, Elena Shagimardanova, Akira Funahashi, Takahiro Kikawada

Scientific Reports (Springer Science and Business Media LLC) 11 ( 1 ) 19698 2021.12

Joint Work, Accepted, ISSN 2045-2322

<title>Abstract</title>Pv11 is an insect cell line established from the midge <italic>Polypedilum vanderplanki</italic>, whose larval form exhibits an extreme desiccation tolerance known as anhydrobiosis. Pv11 itself is also capable of anhydrobiosis, which is induced by trehalose treatment. Here we report the successful construction of a genome editing system for Pv11 cells and its application to the identification of signaling pathways involved in anhydrobiosis. Using the Cas9-mediated gene knock-in system, we established Pv11 cells that stably expressed GCaMP3 to monitor intracellular Ca2+ mobilization. Intriguingly, trehalose treatment evoked a transient increase in cytosolic Ca2+ concentration, and further experiments revealed that the calmodulin–calcineurin–NFAT pathway contributes to tolerance of trehalose treatment as well as desiccation tolerance, while the calmodulin–calmodulin kinase–CREB pathway conferred only desiccation tolerance on Pv11 cells. Thus, our results show a critical contribution of the trehalose-induced Ca2+ surge to anhydrobiosis and demonstrate temporally different roles for each signaling pathway.

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Sbmlwebapp: Web-based simulation, steady-state analysis, and parameter estimation of systems biology models

Yamada T.G., Ii K., König M., Feierabend M., Dräger A., Funahashi A.

Processes (Processes) 9 ( 10 ) 2021.10

Joint Work, Accepted

In systems biology, biological phenomena are often modeled by Ordinary Differential Equations (ODEs) and distributed in the de facto standard file format SBML. The primary analyses performed with such models are dynamic simulation, steady-state analysis, and parameter estimation. These methodologies are mathematically formalized, and libraries for such analyses have been published. Several tools exist to create, simulate, or visualize models encoded in SBML. However, setting up and establishing analysis environments is a crucial hurdle for non-modelers. Therefore, easy access to perform fundamental analyses of ODE models is a significant challenge. We developed SBMLWebApp, a web-based service to execute SBML-based simulation, steady-state analysis, and parameter estimation directly in the browser without the need for any setup or prior knowledge to address this issue. SBMLWebApp visualizes the result and numerical table of each analysis and provides a download of the results. SBMLWebApp allows users to select and analyze SBML models directly from the BioModels Database. Taken together, SBMLWebApp provides barrier-free access to an SBML analysis environment for simulation, steady-state analysis, and parameter estimation for SBML models. SBMLWebApp is implemented in Java™ based on an Apache Tomcat® web server using COPASI, the Systems Biology Simulation Core Library (SBSCL), and LibSBMLSim as simulation engines. SBMLWebApp is licensed under MIT with source code freely available. At the end of this article, the Data Availability Statement gives the internet links to the two websites to find the source code and run the program online.

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Genome-Wide Role of HSF1 in Transcriptional Regulation of Desiccation Tolerance in the Anhydrobiotic Cell Line, Pv11.

Shoko Tokumoto, Yugo Miyata, Ruslan Deviatiiarov, Takahiro G Yamada, Yusuke Hiki, Olga Kozlova, Yuki Yoshida, Richard Cornette, Akira Funahashi, Elena Shagimardanova, Oleg Gusev, Takahiro Kikawada

International journal of molecular sciences (International Journal of Molecular Sciences) 22 ( 11 ) 2021.06

Joint Work, Accepted, ISSN 16616596

The Pv11, an insect cell line established from the midge Polypedilum vanderplanki, is capable of extreme hypometabolic desiccation tolerance, so-called anhydrobiosis. We previously discovered that heat shock factor 1 (HSF1) contributes to the acquisition of desiccation tolerance by Pv11 cells, but the mechanistic details have yet to be elucidated. Here, by analyzing the gene expression profiles of newly established HSF1-knockout and -rescue cell lines, we show that HSF1 has a genome-wide effect on gene regulation in Pv11. The HSF1-knockout cells exhibit a reduced desiccation survival rate, but this is completely restored in HSF1-rescue cells. By comparing mRNA profiles of the two cell lines, we reveal that HSF1 induces anhydrobiosis-related genes, especially genes encoding late embryogenesis abundant proteins and thioredoxins, but represses a group of genes involved in basal cellular processes, thus promoting an extreme hypometabolism state in the cell. In addition, HSF1 binding motifs are enriched in the promoters of anhydrobiosis-related genes and we demonstrate binding of HSF1 to these promoters by ChIP-qPCR. Thus, HSF1 directly regulates the transcription of anhydrobiosis-related genes and consequently plays a pivotal role in the induction of anhydrobiotic ability in Pv11 cells.

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Electrical Field Chamber with Dinamically Adaptable Stimuli for Myocyte Orientation During Cultivation

SRM Ramirez, T Yamada, A Funahashi, H Onoe

2021 IEEE 34th International Conference on Micro Electro Mechanical Systems … (Proceedings of the IEEE International Conference on Micro Electro Mechanical Systems (MEMS)) 2021-January 498 - 501 2021.01

Joint Work, Accepted, ISSN 10846999

Tissue engineering is a promising technology for both its possible applications in the fields of regenerative medicine and medical research. One of its challenges is to create an environment which allows the cell cultures to develop correctly. This environment is far from static and normally is modified by the cells themselves. To this end, adaptable systems have to be developed to ensure a successful development of the in-vitro tissue. This research focuses on a system which is able to modify an external electrical stimulus on a culture chamber, during its cultivation. This system, through a variation of the stimulus direction, successfully improved the orientation of the developing myocytes.

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Control and measurement of nano/micro-space temperature that changes cellular behavior

T Nakamura, T Saito, R Tanimoto, T Yamada, A Funahashi, A Taniguchi, ...

JSAP-OSA Joint Symposia, 10p_N404_14 (Optics InfoBase Conference Papers) 2021

Joint Work, Accepted

COVID-19 Disease Map, building a computational repository of SARS-CoV-2 virus-host interaction mechanisms

M Ostaszewski, A Mazein, ME Gillespie, I Kuperstein, A Niarakis, ...

Scientific data 7 (1), 136 7 ( 1 ) 136 2020.05

Joint Work, Accepted, ISSN 2052-4463

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Neural differentiation dynamics controlled by multiple feedback loops in a comprehensive molecular interaction network

T Iwasaki, R Takiguchi, T Hiraiwa, TG Yamada, K Yamazaki, NF Hiroi, ...

Processes 8 (2), 166 (Processes) 8 ( 2 ) 2020.02

Research paper (scientific journal), Joint Work, Accepted

© 2020 by the authors. Mathematical model simulation is a useful method for understanding the complex behavior of a living system. The construction of mathematical models using comprehensive information is one of the techniques of model construction. Such a comprehensive knowledge-based network tends to become a large-scale network. As a result, the variation of analyses is limited to a particular kind of analysis because of the size and complexity of the model. To analyze a large-scale regulatory network of neural differentiation, we propose a contractive method that preserves the dynamic behavior of a large network. The method consists of the following two steps: comprehensive network building and network reduction. The reduction phase can extract network loop structures from a large-scale regulatory network, and the subnetworks were combined to preserve the dynamics of the original large-scale network. We confirmed that the extracted loop combination reproduced the known dynamics of HES1 and ASCL1 before and after differentiation, including oscillation and equilibrium of their concentrations. The model also reproduced the effects of the overexpression and knockdown of the Id2 gene. Our model suggests that the characteristic change in HES1 and ASCL1 expression in the large-scale regulatory network is controlled by a combination of four feedback loops, including a large loop, which has not been focused on. The model extracted by our method has the potential to reveal the critical mechanisms of neural differentiation. The method is applicable to other biological events.

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Direct cell counting using macro-scale smartphone images of cell aggregates

C Imashiro, Y Tokuoka, K Kikuhara, TG Yamada, K Takemura, ...

IEEE Access 8, 170033-170043 (Institute of Electrical and Electronics Engineers ({IEEE})) 8 170033 - 170043 2020

Joint Work, Accepted, ISSN 2169-3536

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Deep Learning for Non-Invasive Determination of the Differentiation Status of Human Neuronal Cells by Using Phase-Contrast Photomicrographs

M Ooka, Y Tokuoka, S Nishimoto, NF Hiroi, TG Yamada, A Funahashi

Applied Sciences 9 (24), 5503 (Applied Sciences (Switzerland)) 9 ( 24 ) 2019.12

Research paper (scientific journal), Joint Work, Accepted

© 2019 by the authors. Regenerative medicine using neural stem cells (NSCs), which self-renew and have pluripotency, has recently attracted a lot of interest. Much research has focused on the transplantation of differentiated NSCs to damaged tissues for the treatment of various neurodegenerative diseases and spinal cord injuries. However, current approaches for distinguishing differentiated from non-differentiated NSCs at the single-cell level have low reproducibility or are invasive to the cells. Here, we developed a fully automated, non-invasive convolutional neural network-based model to determine the differentiation status of human NSCs at the single-cell level from phase-contrast photomicrographs; after training, our model showed an accuracy of identification greater than 94%. To understand how our model distinguished between differentiated and non-differentiated NSCs, we evaluated the informative features it learned for the two cell types and found that it had learned several biologically relevant features related to NSC shape during differentiation. We also used our model to examine the differentiation of NSCs over time; the findings confirmed our model's ability to distinguish between non-differentiated and differentiated NSCs. Thus, our model was able to non-invasively and quantitatively identify differentiated NSCs with high accuracy and reproducibility, and, therefore, could be an ideal means of identifying differentiated NSCs in the clinic.

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Xitosbml: A modeling tool for creating spatial systems biology markup language models from microscopic images

K Ii, K Mashimo, M Ozeki, TG Yamada, N Hiroi, A Funahashi

Frontiers in genetics 10, 1027 (Frontiers in Genetics) 10 1027 2019.10

Research paper (scientific journal), Joint Work, Accepted, ISSN 1664-8021

© Copyright © 2019 Ii, Mashimo, Ozeki, Yamada, Hiroi and Funahashi. XitoSBML is a software tool designed to create an SBML (Systems Biology Markup Language) Level 3 Version 1 document from microscopic cellular images. It is implemented as an ImageJ plug-in and is designed to create spatial models that reflect the three-dimensional cellular geometry. With XitoSBML, users can perform spatial model simulations based on realistic cellular geometry by using SBML-supported software tools, including simulators such as Virtual Cell and Spatial Simulator. XitoSBML is open-source and is available at https://github.com/spatialsimulator/XitoSBML/. XitoSBML is confirmed to run on most 32/64-bit operating systems: Windows, MacOS, and Linux.

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Activation of cell migration via morphological changes in focal adhesions depends on shear stress in MYCN-amplified neuroblastoma cells

Hiraiwa T., Yamada T., Miki N., Funahashi A., Hiroi N.

Journal of the Royal Society, Interface (Journal of the Royal Society, Interface) 16 ( 152 ) 20180934 2019.03

Research paper (scientific journal), Joint Work, Accepted, ISSN 17425689

Neuroblastoma is the most common solid tumour of childhood, and it metastasizes to distant organs. However, the mechanism of metastasis, which generally depends on the cell motility of the neuroblastoma, remains unclear. In many solid tumours, it has been reported that shear stress promotes metastasis. Here, we investigated the relationship between shear stress and cell motility in the MYCN-amplified human neuroblastoma cell line IMR32, using a microfluidic device. We confirmed that most of the cells migrated downstream, and cell motility increased dramatically when the cells were exposed to a shear stress of 0.4 Pa, equivalent to that expected in vivo. We observed that the morphological features of focal adhesion were changed under a shear stress of 0.4 Pa. We also investigated the relationship between malignancy and the motility of IMR32 cells under shear stress. Decreasing the expression of MYCN in IMR32 cells via siRNA transfection inhibited cell motility by a shear stress of 0.4 Pa. These results suggest that MYCN-amplified neuroblastoma cells under high shear stress migrate to distant organs due to high cell motility, allowing cell migration to lymphatic vessels and venules.

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ネムリユスリカの培養細胞の乾燥耐性に迫る

Kikawada Takahiro

バイオサイエンスとインダストリー(B&I) ((一般財団法人)バイオインダストリー協会(JBA)) 77 ( 4 ) 310 - 311 2019

Joint Work

画像解析と深層学習を用いた診断の基礎

舟橋啓

病理と臨床 37 631 - 635 2019

Joint Work, Accepted

Quantitative analysis of sensitivity to a Wnt3a gradient in determination of the pole-to-pole axis of mitotic cells by using a microfluidic device

Hiraiwa T., Nakai Y., Yamada T., Tanimoto R., Kimura H., Matsumoto Y., Miki N., Hiroi N., Funahashi A.

FEBS Open Bio (FEBS Open Bio) 8 ( 12 ) 1920 - 1935 2018.12

Research paper (scientific journal), Joint Work, Accepted, ISSN 22115463

© 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd. Proper determination of the cell division axis is essential during development. Wnt3a is a known regulator of the cell division axis; however, the sensitivity of cells to Wnt3a signalling and its role in determining the cell division axis have not been measured to date. To address this gap, we took advantage of the asymmetric distribution of outer dense fibre 2 (ODF2/cenexin) proteins on centrosomes in dividing cells. To precisely quantify the sensitivity of cells to Wnt3a signalling, we developed a microfluidic cell culture device, which can produce a quantitative gradient of signalling molecules. We confirmed that mitotic SH-SY5Y neuroblastoma cells could detect a 2.5 ~ 5 × 10−3 nm·μm−1 Wnt3a concentration gradient and demonstrated that this gradient is sufficient to affect the determination of the pole-to-pole axis of cell division during the later stages of mitosis.

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