Biology subjects

Onodera, A.

Publications and source records attributed to Onodera, A..

4 recordsLinked to original sources

bioRxiv2025

A Novel MiRNA Detection System Combining TWJ-SDA,Multistep L-TEAM, and CRISPR-Cas3

MicroRNAs (miRNAs) serve critical regulatory roles in gene expression and are valuable biomarkers for early disease detection. However, their inherent low concentration in biological fluids poses significant detection challenges. Although traditional methods like real-time quantitative PCR (RT-qPCR) are highly sensitive, they require thermal cycling, limiting their application in point-of-care testing (POCT). Here, we present an isothermal amplification-based miRNA detection system integrating Three-Way Junction (TWJ) formation, Multistep Low-Temperature Amplification (L-TEAM), and CRISPR-Cas3-mediated signal amplification. The integration of the Multistep L-TEAM with the TWJ method achieves high sensitivity, detecting miRNA at concentrations as low as 10 femtomolar within 50 minutes, and effectively distinguishes single-nucleotide mismatches. When CRISPR-Cas3-mediated reaction was integrated, it still proved effective for confirming the presence of the target, but its quantitative reliability requires further optimization. We developed a predictive model using machine learning to facilitate rational optimization of experimental conditions through contribution analysis and to establish a methodology for designing more favorable sequences. The modular nature of our method permits adaptation to diverse miRNA targets without modifications to the fundamental amplification mechanism.

molecular biology↗

bioRxiv2025

Loss of TET function in T regulatory cells yields ex-Treg cells biased toward T follicular helper cells, causingautoimmune diseases through autoantibody production

T regulatory cells (Treg cells) express the transcription factor FOXP3 and maintain immune homeostasis by attenuating effector responses. Treg cells are prone to lose FOXP3 and convert to pathological ex-Treg cells under conditions of strong or chronic inflammation. One mechanism for loss of FOXP3 expression involves increased DNA methylation of intronic enhancers CNS1 and CNS2 in the Foxp3 locus; these enhancers are maintained in a demethylated state by TET enzymes, 5-methylcytosine (5mC) dioxygenases that generate 5-hydroxymethylcytosine (5hmC) and other oxidized methylcytosines that are essential intermediates in all pathways of DNA demethylation. We previously showed that FOXP3+ Treg cells from Tet2/3-deficient (Tet2/3 DKO) mice displayed increased methylation of CNS1 and CNS2 and converted to FOXP3-negative ex-Treg cells considerably more efficiently than WT Treg cells. Here we extend our previous analysis of Foxp3-Cre Tet2/3fl/fl mice. We classified the mice as DKO- moderate or DKO-severe based on the total number of leukocytes in the spleen and peripheral lymph nodes and investigated the phenotypic and molecular basis for the progressive inflammation occurring in these mice. RNA-seq as well as histological and immunocytochemical analyses showed a striking expansion of T follicular helper (Tfh) cells and plasma cells in Tet2/3 DKO-severe mice. RNA-seq analyses also revealed increased induction of interferon-stimulated genes (ISGs) in CD4+ FOXP3- T cells from these mice, and single-cell (sc) RNA-seq analyses suggested strongly that this was due to skewed differentiation of both Tet2/3 DKO FOXP3+ Treg cells and Tet2/3 DKO FOXP3- ex-Treg cells into Tfh-like cells. Base-resolution "6-base" sequencing showed the expected loss of 5hmC and increased 5mC in Tfh cells purified from Tet2/3 DKO-severe mice, and suggested that the observed bias in gene expression patterns could arise both from a direct increase in methylation of essential enhancers stemming from TET deficiency, or because methylation interfered with binding of methylation-sensitive transcriptional regulators including CTCF.

immunology↗

bioRxiv2024

Glutamate, GABA, and dense-core vesicle secretion regulate predatory feeding in the nematode Pristionchus pacificus

Nematodes are one of the most diverse groups of organisms, found in various environments, and exhibit various feeding behaviours. Pristionchus pacificus displays two types of feeding behaviours: predatory and bacterial feeding. Previously, we showed that the neurotransmitter serotonin plays important roles in regulating predatory feeding in P. pacificus. However, the role of other neurotransmitters in predatory feeding remains unclear. Using the CRISPR/Cas9 system, we generated mutants of the dense-core vesicle (DCV)-based neuropeptide secretion system and genes related to seven neurotransmitters. Predatory assays revealed that the vesicular glutamate transporter Ppa-EAT-4, glutamic acid decarboxylase Ppa-UNC-25 which is involved in GABA synthesis, and the calcium-dependent activator protein for DCV secretion Ppa-UNC-31, play roles in predatory feeding behaviour. We assessed the pharyngeal movement necessary for predation as well as locomotion rate in these mutants. While the Ppa-eat-4 and Ppa-unc-31 mutants decreased predation movement, the Ppa-unc-25 mutant showed a reduction only in bacterial events compared to wild type animals. Additionally, Ppa-unc-25 and Ppa-unc-31 decreased in motor movement, potentially reducing predation efficiency. Together, these results suggest that glutamate, GABA and DCV secretion modulate feeding behaviours in P. pacificus. Our mutant collection of neurotransmitter-related genes will be useful for future analysis of neurobiology and behavioural evolution.

animal behavior and cognition↗

bioRxiv2024

Stimulatory and inhibitory G-protein signaling relays drive cAMP accumulation for timely metamorphosis in the chordate Ciona

Larvae of the ascidian Ciona initiate metamorphosis tens of minutes after adhesion to a substratum via their adhesive organ. The gap between adhesion and metamorphosis initiation is suggested to ensure the rigidity of adhesion, allowing Ciona to maintain settlement after losing locomotive activity through metamorphosis. The mechanism producing the gap is unknown. Here, by combining gene functional analyses, pharmacological analyses, and live imaging, we propose that the gap represents the time required for sufficient cAMP accumulation to trigger metamorphosis. Not only the Gs pathway but also the Gi and Gq pathways are involved in the initiation of metamorphosis in the downstream signaling cascade of the neurotransmitter GABA, the known initiator of Ciona metamorphosis. The mutual crosstalk of stimulatory and inhibitory G-proteins functions as the accelerator and brake for cAMP production, ensuring the faithful initiation of metamorphosis at an appropriate time and in the right situation.

developmental biology↗