Biology subjects

Yamanaka, M.

Publications and source records attributed to Yamanaka, M..

8 recordsLinked to original sources

bioRxiv2025

Phase-targeting rapid cryofixation of the beating heart and histological analysis unveil contractile state-dependent sarcomere dynamics

The heart is a functional syncytium consisting of numerous cardiomyocytes that repetitively exhibit coordinated contractions/relaxations. However, the extent to which myocyte sarcomere arrangements in the heart differ across beats is unknown. To examine this, we conducted cardiac phase-targeting rapid cryofixation of Langendorff-perfused rat hearts. We adjusted the timepoint of cryogen exposure to the electrically paced heart and observed phase-dependent differences in the sarcomere length (SL) of subepicardial myocytes by -actinin immunohistochemistry, namely a significantly shorter SL during systole than during diastole. We detected spatially inhomogeneous SL distributions by generating a heatmap of the myocardium. For peak systole the SL heatmap exhibited nearly uniform SL shortening within and among the individual myocytes with some myocardia exhibiting nonuniform SLs. During diastole, the heart showed predominant SL elongation, which was also accompanied by patchy distributions of locally short-SL regions, reflecting inhomogeneous SLs. This SL inhomogeneity was attenuated by pharmacological relaxation by 2,3-butanedione monoxime. The heatmap of the rapidly-frozen heart during ventricular fibrillation also revealed inhomogeneous SLs within and among individual myocytes. Overall, cardiac phase-targeting cryofixation unveiled in-depth behaviors on SL in the heart. Our cryofixation strategy will open a new horizon to clarify precise spatiotemporal changes in sarcomere structures and understand cardiac functions.

biophysics↗

bioRxiv2024

Development of a novel microphysiological system for neurotoxicity prediction using human iPSC-derived neurons with morphological deep learning

Microphysiological system (MPS) is an in vitro culture technology that reproduces the physiological microenvironment and functionality of humans, and is expected to be applied for drug screening. In this study, we developed a MPS for structured culture of human iPSC-derived neurons, then predict drug-induced neurotoxicity by morphological deep learning. For human iPSC-derived cortical neurons, after administration of three different amyloid {beta} (A{beta}) peptides, neurotoxicity effects were evaluated by a deep learning image analysis, training two artificial intelligence (AI) models on neurites and PSD-95 images. The combined results indicated that A{beta} 1-42 and 1-40, but not 1-28, induced synaptic degeneration, which is close to clinical reports. For human iPSC-derived sensory neurons, after administration of representative CIPN-related anti-cancer drugs, the toxic effects on soma and axons were evaluated by AI model using neurites images. Significant toxicity was detected in positive drugs and could be classified by different effects on soma or axon, suggesting that the current method provides an effective evaluation of chemotherapy-induced peripheral neuropathy. Taken together, it suggests that the present MPS combined with morphological deep learning is a useful platform for in vitro neurotoxicity assessment.

pharmacology and toxicology↗

bioRxiv2023

Raman microscopy of cryofixed biological specimens for high-resolution and high-sensitivity chemical imaging

Raman microscopy is an emerging molecular imaging technology, yet its signal-to-noise-ratio (SNR) in measurements of biological specimens is severely limited due to the small cross-section of Raman scattering. Here, we present Raman imaging techniques of cryofixed specimens to overcome SNR limitations by enabling long exposure of specimens under highly stabilized low-temperature conditions. The observation of frozen specimens in a cryostat at a constant low temperature immediately after rapid freezing enabled the improvement of SNR and significantly enhanced spatial and spectral resolution. We also confirmed that the cryofixation can preserve physicochemical states of specimens by observing alkyne-labeled coenzyme Q in cytosol and hemeproteins in acute ischemic myocardium, which cannot be done by fixation using chemical reagents. Finally, we applied the technique for multiplex Raman imaging of label-free endogenous molecules and alkyne-tagged molecules in cryofixed HeLa cells, demonstrating its capability of high-content imaging of complex biological phenomena while maintaining physiological conditions.

biophysics↗

bioRxiv2023

Time-deterministic cryo-optical microscopy

We developed a technique to rapidly freeze biological systems during observation under an optical microscope, which allows for detailed optical observation at a precise time in the biological dynamics at high spatial resolution and quantifiability under low-temperature conditions. Our study further found that ion distributions and molecular conformations can be fixed in space and time, demonstrated by visualization of calcium waves rapid-frozen at an instant using fluorescent ion indicators. These results show that time-deterministic cryo-optical microscopy can suspend cellular dynamics while maintaining molecular and ionic states, allowing us to view cellular dynamics in a way that has not been possible before. One-Sentence SummaryRapid freezing of dynamic biological systems during optical observation presents time information to the fixed samples

biophysics↗

bioRxiv2022

An in vitro microfluidic culture device for peripheral neurotoxicity prediction at low concentration based on deep learning

In this study, a microfluidic culture device was developed including related evaluation methods using deep learning for the purpose of constructing a rapid assessment platform for peripheral neuropathy caused by compounds. Primary rodent dorsal root ganglion was cultured in the microfluidic culture device that could separate the cell body and neurites, and the neurites morphological changes were analyzed by immunofluorescence images. Separated neurites successful culture in the microfluidic device for more than 1 month indicated that a series of test processes from culture to drug stimulation and fluorescence observation is possible. Additionally, cultured samples were treated with several anticancer drugs known to cause peripheral neurotoxicity (i.e., vincristine, oxaliplatin, and paclitaxel) and analyzed the neurites morphological changes by deep learning for image analysis. After training, artificial intelligence (AI) could identify neurite morphological changes caused by each compound and precisely predicted the toxicity positivity, even at low concentrations. For testing compounds, AI could also precisely detect toxicity negative and positive based on neurite images, even at low concentrations. Therefore, this microfluidic culture system is supposed to be useful for in vitro toxicity assessment.

pharmacology and toxicology↗

bioRxiv2022

EPI-589, a redox-active neuroprotectant, potently protects cultured cells from oxidative stress and alleviates symptomatic and pathological progression of motor neuron disease in the wobbler mouse

Oxidative stress is believed to play a significant role in the pathophysiology of amyotrophic lateral sclerosis (ALS), the most common form of motor neuron disease. The present study aims to firstly investigate the antioxidant activities of EPI-589, a small-molecule quinone derivative, under cell-free or cell culture conditions, and explore the in vivo efficacy of EPI-589 in the wobbler mouse model of human motor neuron disease. The reduced form of EPI-589 showed hydroxyl radical scavenging activities, whereas the oxidized form i.e. EPI-589 did not. In cellular models utilizing ALS patient-derived fibroblasts carrying mutations in the fused in sarcoma (FUS) gene or superoxide dismutase 1 (SOD1) gene, EPI-589 potently protected cells from oxidative stress induced by buthionine sulfoximine and ferric citrate. Protective effect of EPI-589 was also observed in culture of mouse immortalized striatal STHdHQ7/Q7 cells with cystine deprivation. In wobbler mice, oral administration of dietary EPI-589 provided long-lasting amelioration of both of deterioration of the rotarod walking performance and progression of forelimb deformity in wobbler mice throughout the treatment. In separate studies, we found that EPI-589 significantly suppressed changes of pathophysiological markers such as plasma phosphorylated neurofilament heavy chain, urinary 8-hydroxy-2-deoxyguanosine, and cervical N-acetylaspartate in untreated wobbler mice. Thus, the present study firstly demonstrates that EPI-589 is a highly potent, redox-active neuroprotectant and robustly delays the symptomatic and pathophysiological progression of motor neuron disease in the wobbler mouse, and these findings strongly encourage further exploration of the therapeutic potential of EPI-589 for the treatment of ALS.

pharmacology and toxicology↗

bioRxiv2020

In vitro nonalcoholic fatty liver disease model with cyclo-olefin-polymer-based microphysiological systems

Nonalcoholic fatty liver disease (NAFLD) is one of the common chronic liver conditions, whose treatment involves curing patients without liver transplantation. Understanding the mechanism of NAFLD initiation and progression would enable development of new diagnostic tools and drugs; however, until now, the underlying mechanisms of this condition remain largely unknown owing to the lack of experimental settings that can simplify the complicated NAFLD process in vitro. Microphysiological systems (MPSs) have long been used to recapture human pathophysiological conditions in vitro for applications in drug discovery. However, polydimethylsiloxane (PDMS) has been used in most of these MPSs as the structural material; it absorbs hydrophobic molecules, such as free fatty acids (FFAs), which are the key components that initiate NAFLD. Therefore, the current PDMS-based MPSs cannot be directly applied to in vitro NALFD modeling. In this work, we present an in vitro NAFLD model with an MPS made of cyclo-olefin polymer (COP), namely COP-MPS, to prevent absorption of FFAs. We demonstrated induction of the NAFLD-like phenotype in HepaRG hepatocyte-like cells cultured in the COP-MPS by introducing FFAs. The FFAs induced lipid accumulation in the HepaRG cells, resulting in inactivation of the apoptotic cells. We believe that the proposed COP-MPS can contribute toward investigations of NAFLD mechanisms and identification of new drugs to prevent the progression of liver disease and avoid liver transplantation.

bioengineering↗

bioRxiv2020

Cyclo olefin polymer-based solvent-free mass-productive microphysiological systems

A microphysiological system (MPS) holds a great promise for drug screening and toxicological testing as an alternative to animal models. However, this platform has several issues in terms of the materials used (e.g., polydimethylsiloxane), such as the absorbance of tested drug candidates and fluorescent dyes by the material, as well as the effect on cultured cellular status, thus misleading the results obtained from cell assays and fabrication processes. Hence, to eliminate the issues mentioned above, we developed a cyclo olefin polymer (COP)-based MPS via photobonding process using vacuum ultraviolet (VUV), named COP-VUV-MPS. COP-VUV-MPS showed better chemical resistance and avoided molecule absorption. COP-VUV-MPS could maintain the stemness of environmentally sensitive human-induced pluripotent stem cells without causing undesired cellular phenotypes and gene expression. These results suggested that COP-VUV-MPS might be broadly used for the advancement of MPS and applications in drug development and in vitro toxicological testing.Competing Interest StatementM.Y. is an employee of Ushio Inc. The part of this project was financially supported by Ushio Inc. Kyoto University (K.K.) and Ushio INC. (M.Y.) filed a Japanese patent application based on the research presented herein. The remaining authors declare no competing interests.View Full Text

bioengineering↗