Biology subjects

Asahi, T.

Publications and source records attributed to Asahi, T..

12 recordsLinked to original sources

bioRxiv2026

Repetitive sequence material shapes the earliest stages of de novo gene evolution in insects

A fundamental unresolved question in molecular evolution is how novel genes arise from noncoding DNA and become fixed within stable gene repertoires. Here, we performed comparative genomic analyses across evolutionary timescales in insects using chromosome-scale genome assemblies of two cricket species, Teleogryllus occipitalis and Tarbinskiellus portentosus. Using conservative criteria, we identified 41 de novo gene candidates derived from intergenic regions in the Te. occipitalis lineage. These genes are simple and compact, exhibit hallmarks of evolutionarily young genes, and frequently contain fragments of transposable elements and simple sequence repeats. Across insects, such repetitive sequence fragments show positional homology but lack sequence conservation in older genes, suggesting that they serve as sequence material for gene emergence during early stages of gene evolution. In contrast, insertions after gene establishment are strongly constrained. We propose a model in which stages of gene evolution are characterized by shifts in selective pressure on the incorporation of sequence material.

evolutionary biology↗

bioRxiv2026

Osteopontin Upregulation Defines a Pre-Rupture State in Thoracic Aortic Aneurysms in Mice and Humans

BackgroundThoracic aortic aneurysm (TAA) is a life-threatening condition with an unpredictable lisk of rupture. Current clinical parameters have limited ability to accurately predict imminent rupture. Osteopontin (OPN) has been implicated in aortic aneurysm pathology, however, it role as a marker of imminent rupture remains. unclear. We investigated the dynamics of OPN expression dynamics in a mouse model with predictable rupture timing and validated our findings in human TAA. MethodsOne-month-old fibrillin-1 hypomorphic (Fbn1mgR/mgR) mice were used as a TAA model; with wild-type (WT) mice served as controls. Angiotensin II (AngII) was administered to Fbn1mgR/mgR to induce acute aortic rupture. Single-section transcriptome analysis and immunofluorescence staining were performed on ascending aortic tissue at 24 and 72 hours after AngII infusion, with pre-treatment Fbn1mgR/mgR and WT mice serving as controls. To determine conservation in human disease, we reanalyzed publicly available single-cell RNA sequencing data from ascending thoracic aortic aneurysm (ATAA) patients. ResultsAngII infusion induced progressive mortality beginning at 24 hours, with approximately 60% survival at 72 hours and nearly no survival by 8 days in Fbn1mgR/mgR mice. At this pre-rupture time point, OPN showed prominent upregulation at both mRNA and protein levels in ascending aortic tissues compared to controls. Immunofluorescence staining revealed increased OPN expression in the aortic wall, particularly in regions exhibiting structural deterioration. Reanalysis of human ATAA single-cell data showed elevated OPN expression compared to controls, with enrichment in immune cell populations, especially macrophages. Within the macrophage compartment, subcluster analysis identified a stress-responsive subpopulation (MC1) that was markedly expanded and almost exclusively composed of ATAA-derived cells, representing the primary source of OPN upregulation. ConclusionsOPN upregulation represents a conserved molecular signature of the pre-rupture state in TAA across mice and humans. Our mode, which enables predictable rupture timing, allowed the capture of acute pre-rupture molecular changes, suggesting OPN as a potential biomarker for predicting imminent aortic rupture.

molecular biology↗

bioRxiv2025

Praja1 protects cells from DNA damage through direct DNA binding

Praja1 is known as an E3 ubiquitin ligase that regulates multiple functions through protein degradation. It acquired nuclear localization signal after gene duplication and although studies have shown some significant roles of nuclear Praja1, comprehensive analysis still lacks. In this study, we performed comparative proteomics and biochemical analyses to elucidate the functions of Praja1 in the nucleus. First, proteomics analysis applied to nuclear localization deficient Praja1 exhibited signs of DNA damage response fluctuation. Subsequent comet assay revealed Praja1 protecting cells from various DNA damage sources. Similarly, cells lacking Praja1 became more sensitive to DNA damage-induced cell death, while E. coli expressing Praja1 exhibited resistance to DNA damage. To further elucidate the molecular basis of DNA protection, gel shift assay showed direct binding of Praja1 to DNA through electrostatic interactions within its intrinsically disordered region. Further in vitro damaging assay suggested that Praja1 may induce structural changes that enhance DNA repair efficiency upon binding to DNA. Together, these results provide insights into the evolutionarily novel role of nuclear Praja1 in protecting against DNA damage.

molecular biology↗

bioRxiv2025

Chromosome-scale genome assembly and annotation of the two-spotted cricket Gryllus bimaculatus (Orthoptera: Gryllidae)

The two-spotted cricket, Gryllus bimaculatus, is a key hemimetabolous model organism for developmental biology, neuroscience, and regeneration. The existing reference genome is, however, highly fragmented into 47,877 scaffolds, hampering chromosome-scale analyses for these fields. Here, we report a high-quality, chromosome-scale genome assembly for the white-eyed mutant strain of this cricket, generated using a combination of Nanopore and PacBio HiFi long reads, integrated with Hi-C data. The final 1.62 Gbp assembly achieves a scaffold N50 of 107.4 Mbp, a significant improvement in contiguity over the previous 6.3 Mbp N50. We anchored 94.45% of the assembly into 15 pseudomolecules, consistent with the known karyotype (n = 15). The genome completeness (BUSCO v6.0.0 insecta_odb12) reached 98.1%. We also updated the annotation, identifying 14,964 protein-coding genes. This gene set shows markedly improved completeness (BUSCO v6.0.0 insecta_odb12: 95.7%) compared with the previous annotation (81.2%) and successfully recovers all nine essential neuropeptide genes previously reported as missing from the draft assembly. This chromosome-scale genomic resource provides an essential foundation for comparative and functional genomics in G. bimaculatus.

genomics↗

bioRxiv2025

Temporal orchestration of transcriptional and epigenomic programming underlying maternal embryonic diapause in a cricket model

Maternal perception of environmental conditions can direct offspring developmental trajectories, providing adaptive flexibility across taxa. In the band-legged ground cricket Dianemobius nigrofasciatus, maternal exposure to short days induces embryonic diapause at the cellular blastoderm stage in offspring. Here, we investigate molecular mechanisms underlying this transgenerational adaptation through genome assembly (1.45 Gbp) and time-series transcriptomic analyses of diapause and non-diapause eggs from 12 to 72 hours post-oviposition. Despite morphological similarity, diapause-destined eggs show early upregulation of ATP-dependent chromatin remodeling genes at 24 hours. ATAC-seq reveals reduced chromatin accessibility at neural and cell cycle-related genes. Time-series clustering identifies precocious shifts in RNA processing machinery (peaking at 24 versus 40 hours in non-diapause eggs), followed by metabolic regulation toward amino acid catabolism and gluconeogenesis sustaining long-term survival during developmental arrest. Our findings reveal diapause as actively coordinated molecular programming involving epigenetic, transcriptional, and metabolic remodeling, providing insights into transgenerational environmental adaptation.

developmental biology↗

bioRxiv2024

Mitochondrial Dysfunction and ER Stress in CB1 Receptor Antagonist-Induced Apoptosis in Human Neuroblastoma SH-SY5Y Cells

Cannabinoid receptor type 1 (CB1R) is the key modulator of neuronal viability. In particular, CB1R antagonists provide neuroprotective effects on neurotoxicity caused by e.g. neuronal injury. However, the underlying mechanisms and potential limitations of CB1R antagonism remain unclear. Here we investigated the impact of environmental conditions on CB1R antagonist effects. Unexpectedly, we have found that cell-permeable CB1R antagonists, rimonabant and AM251, induced significant cell death in human neuroblastoma SH-SY5Y cells under serum-free conditions. Mitochondrial morphological analysis revealed mitochondrial swelling characterized by their network fragmentation and cristae reduction. Additionally, phosphoproteomics analysis showed an upregulation of phosphorylated EIF2AK3 (also known as PERK), leading to the activation of the eIF2/ATF4/CHOP pathway, indicative of endoplasmic reticulum (ER) stress. Rimonabant and AM251 treatment also triggered caspase-dependent apoptosis, evidenced by the detection of cleaved caspase 3 and cleaved PARP. These results suggest that cell-permeable CB1R antagonists promote apoptosis via mitochondrial dysfunction and ER stress under serum-free conditions in SH-SY5Y cells. Our findings indicate that while CB1R antagonists may be neuroprotective in certain conditions, they may also pose a neurotoxic risk in environments characterized by cellular stress or nutrient deprivation.

pharmacology and toxicology↗

bioRxiv2024

E3 ligase Praja1 mediates ubiquitination and degradation of microtubule-associated protein Tau

RING-H2 type E3 ligase Praja family is composed of Praja1 and Praja2, which promote the degradation of substrates through the ubiquitin-proteasome system. Both paralogs contribute to neuronal maturation and differentiation, indicating a significant role in the nervous system. Aggregation-prone proteins associated with neurodegenerative diseases, including TDP-43 and -synuclein, are degraded and/or suppressed by Praja1. Furthermore, the expression level of the MAPT gene, which is frequently mutated in Alzheimers, is regulated by Praja2. While the Praja family has been shown to recognize various aggregation-prone proteins as substrates, it has not been determined whether Tau, a key protein that aggregates in tauopathies, is also recognized by Praja proteins. In this study, we show that Praja1, but not Praja2, recognizes Tau as a candidate substrate. We observed that Tau expression in human neuroblastoma SH-SY5Y cells decreased depending on the E3 ligase activity of Praja1. Furthermore, Praja1 polyubiquitinated and interacted with Tau, indicating that it is a target substrate. Next, by combining ancestral sequence reconstruction and mutational analysis, we revealed that the Praja1-Tau interaction began via deletion of the N- and C-terminal regions of Praja1, occurring just after the duplication of the Praja family in the common ancestor of placentals. Lastly, to test whether this interaction is disrupted under pathological conditions, P301L Tau was introduced, resulting in a degradation similar to that of wild-type Tau. These results reveal an unidentified mechanism of Tau proteostasis by Praja1 and may provide insight into the pathogenesis of neurodegenerative diseases, including tauopathy.

biochemistry↗

bioRxiv2024

Cannabinoid CB2 receptors enhance high-fat diet evoked peripheral neuroinflammation

It is known that cannabinoid type 2 (CB2) receptor has anti-inflammatory role, therefore animals without CB2 receptors show enhanced inflammation and pain in the model of chronic pain e.g. neuropathic pain. We previously proposed the upregulated leptin signaling at the peripheral nerve as one of the underlying molecular mechanisms of pain exacerbation in nerve-injured CB2 knockouts, as they displayed robust upregulation of leptin receptors and leptin signaling in peripheral nerve. Due to these past results we hypothesized that CB2 receptor deficiency might also modify the peripheral neuroinflammation lead by chronic exposure to high fat diet (HFD). Interestingly, CB2 knockout animals showed the significant resistance to the HFD-induced neuroinflammation. Namely, 5-week feeding of HFD induced substantial hypersensitivity in WT animals, while tactile sensitivity of HFD-fed CB2 knockouts remained intact. HFD-fed WT animals also displayed the robust upregulation of chemokine CXCR4 expression with increased macrophage infiltration, which was never observed in HFD-fed CB2 knockout mice. Moreover, 5-week HFD-exposure lead significant increase of CD11b+Ly6G-Ly6Chigh cells and decrease of CD11b+Ly6G+Ly6Clow cells in the spleen of WT animals, which was also not found in either HFD-fed CB2 knockouts or standard diet-fed WT and CB2 animals. Together with past report, these results suggest that CB2 receptors might have the double-sided regulatory role in context of the inflammation development, or more widely, immune system regulation. We propose that CB2 signaling is not always anti-inflammatory and could take pro-inflammatory role depending on the cause of the inflammation.

pathology↗

bioRxiv2023

DeepLabCut-based Behavioural and Posture Analysis in a Cricket

Circadian rhythms are indispensable intrinsic programs that regulate the daily rhythmicity of physiological processes, such as feeding and sleep. The cricket has been employed as a model organism for understanding the neural mechanisms underlying circadian rhythms in insects. However, previous studies measuring rhythm-controlled behaviours only analysed locomotive activity using seesaw-type and infrared sensor-based actometers. Meanwhile, advances in deep learning techniques have made it possible to analyse animal behaviour and posture using software that is devoid of human bias and does not require physical tagging of individual animals. Here, we present a system that can simultaneously quantify multiple behaviours in individual crickets, such as locomotor activity, feeding, and sleep-like states, in the long-term, using DeepLabCut, a supervised machine learning-based software for body keypoints labeling. Our system successfully labelled the six body parts of a single cricket with a high level of confidence and produced reliable data showing the diurnal rhythms of multiple behaviours. Our system also enabled the estimation of sleep-like states by focusing on posture, instead of immobility time, which is a conventional parameter. We anticipate that this system will provide an opportunity for simultaneous and automatic prediction of cricket behaviour and posture, facilitating the study of circadian rhythms.

animal behavior and cognition↗

bioRxiv2023

Integrative analysis of in vitro strategies to induce human vascularized cerebral organoids using single-cell RNA sequencing data

Cerebral organoids are three-dimensional in vitro cultured brains that mimic the function and structure of the human brain. One of the major challenges for cerebral organoids is the lack of functional vasculature. Without perfusable vessels, oxygen and nutrient supplies may be insufficient for long-term culture, hindering the investigation of the neurovascular interactions. Recently, several strategies for the vascularization of human cerebral organoids have been reported. However, the generalizable trends and variability among different strategies are unclear due to the lack of a comprehensive characterization and comparison of these vascularization strategies. In this study, we aimed to explore the effect of different vascularization strategies on the nervous system and vasculature in human cerebral organoids. We integrated single-cell RNA sequencing data of multiple vascularized and vascular organoids and fetal brains from publicly available datasets and assessed the protocol-dependent and culture-day-dependent effects on the cell composition and transcriptomic profiles in neuronal and vascular cells. We revealed the similarities and uniqueness of multiple vascularization strategies and demonstrated the transcriptomic effects of vascular induction on neuronal and mesodermal-like cell populations. Moreover, our data suggested that the interaction between neurons and mesodermal-like cell populations is important for the cerebrovascular-specific profile of endothelial-like cells. This study highlights the current challenges to vascularization strategies in human cerebral organoids and offers a benchmark for the future fabrication of vascularized organoids.

neuroscience↗

bioRxiv2022

Liver type 1 innate lymphoid cells lacking IL-7 receptor are a native killer cell subset fostered by parenchymal niches

Group 1 innate lymphoid cells (G1-ILCs), including circulating natural killer (NK) cells and tissue-resident type 1 ILCs (ILC1s), are innate immune sentinels critical for responses against infection and cancer. In contrast to relatively uniform NK cells through the body, diverse ILC1 subsets have been characterized across and within tissues in mice, but their developmental and functional heterogeneity remain unsolved. Here, using multimodal in vivo approaches including fate-mapping and targeting of the interleukin 15 (IL-15)-producing microenvironment, we demonstrate that liver parenchymal niches support the development of a cytotoxic ILC1 subset lacking IL-7 receptor (7R- ILC1s). During ontogeny, fetal liver (FL) G1-ILCs arise perivascularly and then differentiate into 7R- ILC1s within sinusoids. Hepatocyte-derived IL-15 supports parenchymal development of FL G1-ILCs to maintain adult pool of 7R- ILC1s. IL-7R+ (7R+) ILC1s in the liver, candidate precursors for 7R- ILC1s, are not essential for 7R- ILC1 development in physiological conditions. Functionally, 7R- ILC1s exhibit killing activity at steady state through granzyme B expression, which is underpinned by constitutive mTOR activity, unlike NK cells with exogenous stimulation-dependent cytotoxicity. Our study reveals the unique ontogeny and functions of liver-specific ILC1s, providing a detailed interpretation of ILC1 heterogeneity.

immunology↗

bioRxiv2020

Immunohistochemical Characterization of Phosphorylated Ubiquitin in the Mouse Hippocampus

Mitochondrial autophagy (mitophagy) is an essential and evolutionarily conserved process that maintains mitochondrial integrity via the removal of damaged or superfluous mitochondria in eukaryotic cells. Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1) and Parkin promote mitophagy and function in a common signaling pathway. PINK1-mediated ubiquitin phosphorylation at Serine 65 (Ser65-pUb) is a key event in the efficient execution of PINK1/Parkin-dependent mitophagy. However, few studies have used immunohistochemistry to analyze Ser65-pUb in the mouse. Here, we examined the immunohistochemical characteristics of Ser65-pUb in the mouse hippocampus. Some hippocampal cells were Ser65-pUb positive, whereas the remaining cells expressed no or low levels of Ser65-pUb. PINK1 deficiency resulted in a decrease in the density of Ser65-pUb-positive cells, consistent with a previous hypothesis based on in vitro research. Interestingly, Ser65-pUb-positive cells were detected in hippocampi lacking PINK1 expression. The CA3 pyramidal cell layer and the dentate gyrus (DG) granule cell layer exhibited significant reductions in the density of Ser65-pUb-positive cells in PINK1-deficient mice. Moreover, Ser65-pUb immunoreactivity colocalized predominantly with neuronal markers. These findings suggest that Ser65-pUb may serve as a biomarker of in situ PINK1 signaling in the mouse hippocampus; however, the results should be interpreted with caution, as PINK1 deficiency downregulated Ser65-pUb only partially.

neuroscience↗