Search bioRxiv⌕ Search

Biology subjects

Ito, T.

Publications and source records attributed to Ito, T..

At least 55 records · Page 3Linked to original sources

Savings in visuomotor learning is associated with connectivity changes within a cerebello-thalamo-cortical network encoding movement errors

Savings refers to faster relearning upon re-exposure to a previously experienced movement perturbation. One theory suggests that the brain recognizes past errors and is therefore more able to learn from them. If true, there should be a modification of the neural response to errors during re-exposure to a perturbation. To test this idea, we imaged the brains of participants who underwent two sessions (1 day apart) of adaptation to a visuomotor perturbation and investigated brain responses to movement errors. The magnitude of movement error was entered into different types of GLMs to study error-related activation and coactivation (or functional connectivity). We identified a cerebello-thalamo-cortical network involved in the processing of movement errors during adaptation. We found that connectivity between regions of this network (i.e., between the cerebellum and the thalamus, and between the primary somatosensory cortex and the anterior cingulate cortex) became stronger during re-adaptation. Importantly, participants with the largest increases in connectivity strength were those who demonstrated the largest amounts of savings. These results establish a relationship between the ability of the brain to represent errors and the phenomenon of savings.

neuroscience↗

Strategic targeting of Cas9 nickase expands tandem gene arrays

Expanding tandem gene arrays facilitates adaptation through dosage effects and gene family formation via sequence diversification. However, experimental induction of such expansions remains challenging. Here we introduce a method termed break-induced replication (BIR)-mediated tandem repeat expansion (BITREx) to address this challenge. BITREx strategically places Cas9 nickase adjacent to a tandem gene array to break the replication fork that has replicated the array, forming a single-end double-strand break. This break is subsequently end-resected to become single-stranded. Since there is no repeat unit downstream of the break, the single-stranded DNA often invades an upstream unit to initiate ectopic BIR, resulting in array expansion. BITREx has successfully expanded gene arrays in budding yeast, with the CUP1 array reaching [~]1 Mb. Furthermore, appropriate splint DNA allows BITREx to generate tandem arrays de novo from single-copy genes. We have also demonstrated BITREx in mammalian cells. Therefore, BITREx will find various unique applications in genome engineering.

genomics↗

Frame-shift mutation of InCO might cause early flowering of Japanese morning glory and might have contributed to northward expansion

Japanese morning glory (Ipomoea nil), a short day plant, has been used for studying flowering times. Here, quantitative trait loci (QTL) analysis for days from sowing to flowering (DTF) of F2 between I. nil var. Tokyo Kokei Standard (TKS) and I. hederacea line var. Q65, an early flowering variety, revealed four QTLs: QTL Ipomoea Flowering 1-4 (qIF1-4). The position of qIF3, which had the most significant effect among the four QTLs, corresponds with that of I. nil (or I. hederacea) CONSTANS (InCO/IhCO) in the linkage map. There is a single-base In/Del in the coding sequence of InCO/IhCO. The single-base deletion (SBD) causes a frame-shift mutation and loss of function in TKS allele (inco-1). I. nil accessions bearing inco-1 tend to flower early, similarly to rice varieties bearing the loss of function allele of CO ortholog, hd1. Consequently, inco-1 was inferred to reduce DTF. This inferred effect of inco-1 corresponds with the effect of the TKS allele of qIF3. Because inco-1 is found exclusively in Asian accessions, the SBD in inco-1 might have played an important role in the expansion of Japanese morning glories, originally native to the tropical regions of the Americas, into temperate Asia.

plant biology↗

Sense codon-misassociated eRF1 elicits widespread ribosome stalling and induction of quality control

The rescue pathway of stalled ribosomes and ribosome-associated quality control (RQC) serves as a surveillance system for aberrant translation that senses ribosome collisions. Although the molecular mechanism has been extensively studied, the endogenous contexts of ribosome collision rescued by the system are poorly understood. Here, beginning with a study of the codon specificity of the eukaryotic translation termination factor eRF1, we show that transient binding of eRF1 to the UUA sense codon leads to ribosome collision and provides a constitutive source of rescue substrates in humans. eRF1-selective Monosome-Seq and Disome-Seq revealed that eRF1 was recruited not only to stop codons but also to near-cognate sense codons, including the UUA codon. The eRF1 on UUA codons delays translation elongation but does not trigger the termination reaction. Remarkably, Disome-Seq with the depletion of ASCC3 and 4EHP, key factors for the ribosome rescue and translation initiation repression, showed that ribosomes stalled at UUA codons consist of a subpopulation suppressed by those factors. Failure of ribosome collision repression by ASCC3-4EHP triggers stress response, such as expression of the stress-induced transcription factor ATF3. This study highlights the impact of sense codon misrecognition by the termination factor on translation homeostasis in human cells.

molecular biology↗

Transplantation of bioengineered lung using decellularized mouse lungs and primary human endothelial cells.

Lung transplantation is a critical treatment for patients with end-stage lung diseases like idiopathic pulmonary fibrosis, but challenges such as donor shortages and post-transplant complications persist. Bioengineered lungs, integrating patient-specific cells into decellularized animal scaffolds, present a promising alternative. Despite progress in using bioengineered lungs in animal models, functionality and structure remain immature. This protocol addresses a critical barrier in organ bioengineering: the need for a cost-effective experimental platform. By using mouse models instead of larger animals like rats or swine, researchers can significantly reduce the resources required for each experiment, accelerating research progress. The protocol outlines a detailed procedure for lung bioengineering using mouse heart-lung blocks and human primary cells, focusing on isolation strategy for the mouse heart-lung block, decellularization, bioreactor setup, perfusion-based organ culture, and orthotopic transplantation of bioengineered lungs. This mouse-scale platform not only reduces experimental costs but also provides a viable framework for optimizing cell types and numbers for recellularization, testing different cell types using histological and molecular methods, and ensuring blood flow post-transplantation. The method holds potential for broad applications, including studying cell interactions in three-dimensional culture conditions, cell-matrix interactions, and ex vivo cancer modeling, thereby advancing the field of organ bioengineering. SUMMARYThis paper describes how to create bioengineered mouse lungs using the decellularization and recellularization methods. It also describes subsequent orthotopic lung transplantation in detail.

bioengineering↗

DNA methylation protects cancer cells against non-canonical senescence

Inhibitors of DNA methylation such as 5-aza-deoxycytidine are widely used in experimental and clinical settings. However, their mechanism of action is such that DNA damage inevitably co-occurs with loss of DNA methylation, making it challenging to discern their respective effects. Here we deconvolute the effects of decreased DNA methylation and DNA damage on cancer cells, by using degron alleles of key DNA methylation regulators. We report that cancer cells with decreased DNA methylation --but no DNA damage-- enter cellular senescence, with G1 arrest, SASP expression, and SA-{beta}-gal positivity. This senescence is independent of p53 and Rb, but involves p21, which is cytoplasmic and inhibits apoptosis, and cGAS, playing a STING-independent role in the nucleus. Xenograft experiments show that tumor cells can be made senescent in vivo by decreasing DNA methylation. These findings reveal the intrinsic effects of loss of DNA methylation in cancer cells and have practical implications for future therapeutic approaches.

molecular biology↗

Temporal Single Cell Analysis of Leukemia Microenvironment Identifies Taurine-Taurine Transporter Axis as a Key Regulator of Myeloid Leukemia

Signals from the microenvironment are known to be critical for development, sustaining adult stem cells, and for oncogenic progression. While candidate niche-driven signals that can promote cancer progression have been identified1-6, concerted efforts to comprehensively map microenvironmental ligands for cancer stem cell specific surface receptors have been lacking. Here, we use temporal single cell RNA-sequencing to identify molecular cues from the bone marrow stromal niche that engage leukemia stem cells (LSC) during oncogenic progression. We integrate these data with our RNA-seq analysis of human LSCs from distinct aggressive myeloid cancer subtypes and our CRISPR based in vivo LSC dependency map7 to develop a temporal receptor-ligand interactome essential for disease progression. These analyses identify the taurine transporter (TauT)-taurine axis as a critical dependency of myeloid malignancies. We show that taurine production is restricted to the osteolineage population during cancer initiation and expansion. Inhibiting taurine synthesis in osteolineage cells impairs LSC growth and survival. Our experiments with the TauT genetic loss of function murine model indicate that its loss significantly impairs the progression of aggressive myeloid leukemias in vivo by downregulating glycolysis. Further, TauT inhibition using a small molecule strongly impairs the growth and survival of patient derived myeloid leukemia cells. Finally, we show that TauT inhibition can synergize with the clinically approved oxidative phosphorylation inhibitor venetoclax8, 9 to block the growth of primary human leukemia cells. Given that aggressive myeloid leukemias continue to be refractory to current therapies and have poor prognosis, our work indicates targeting the taurine transporter may be of therapeutic significance. Collectively, our data establishes a temporal landscape of stromal signals during cancer progression and identifies taurine-taurine transporter signaling as an important new regulator of myeloid malignancies.

cancer biology↗

ILC2s navigate tissue redistribution during infection using stage-specific S1P receptors

Lymphocytes can circulate as well as take residence within tissues. While the mechanisms by which circulating populations are recruited to infection sites have been extensively characterized, the molecular basis for the recirculation of tissue-resident cells is less understood. Here, we show that helminth infection- or IL-25-induced redistribution of intestinal group 2 innate lymphoid cells (ILC2s) requires access to the lymphatic vessel network. Although the secondary lymphoid structure is an essential signal hub for adaptive lymphocyte differentiation and dispatch, it is redundant for ILC2 migration and effector function. Upon IL-25 stimulation, a dramatic change in epigenetic landscape occurs in intestinal ILC2s, leading to the expression of sphingosine-1-phosphate receptors (S1PRs). Among the various S1PRs, we found that S1PR5 is critical for ILC2 exit from intestinal tissue to lymph. By contrast, S1PR1 plays a dominant role in ILC2 egress from mesenteric lymph nodes to blood circulation and then to distal tissues including the lung where the redistributed ILC2s contribute to tissue repair. The requirement of two S1PRs for ILC2 migration is largely due to the dynamic expression of the tissue-retention marker CD69, which mediates S1PR1 internalization. Thus, our study demonstrates a stage-specific requirement of different S1P receptors for ILC2 redistribution during infection. We therefore propose a fundamental paradigm that innate and adaptive lymphocytes utilize a shared vascular network frame and specialized navigation cues for migration.

immunology↗

Roles of C/EBPα, GATA2, TGF-β-signaling, and epigenetic regulation in the expression of basophil-specific protease genes

In the present study, we analyzed the transcriptional regulation of genes encoding basophil-specific proteases Mcpt8 and Mcpt11 to clarify the molecular mechanisms by which the commitment between basophil and mast cell (MC) is determined. We used bone marrow-derived (BM) cells maintained in the presence of IL-3, in which basophil-like cells and MC-like cells exist. Knock down (KD) and overexpression of a transcription factor C/EBP showed that Cebpa mRNA levels were associated with those of Mcpt8 and Prss34 (a gene symbol of Mcpt11). Treatment with trichostatin A (TSA), a histone deacetylase inhibitor, decreased mRNA levels of Cebpa, Mcpt8, and Prss34 in BM cells, whereas these mRNA levels were increased by the treatment with an inhibitor of DNA methyltransferase. TSA treatment upregulated mRNA levels of Gata1 and Mitf in BM cells, and Mitf KD but not Gata1 KD increased mRNA levels of Cebpa and Prss34. Furthermore, Gata2 KD significantly decreased mRNA levels of Mcpt8 and Prss34 without affecting Cebpa mRNA level. In addition, mRNA levels of Gata1 and Mitf were increased in BM cells transfected with Cebpa siRNA, and decreased in Cebpa-overexpressing BM cells. We also found the TGF-{beta} treatment increased mRNA levels of Mcpt8, Prss34, along with upregulation of Gata2 and Cebpa transcription. Taken together, we conclude that the transcription of basophil-specific protease genes was positively regulated by C/EBP, GATA2, and TGF-{beta} signaling with modification of epigenetic regulation.

immunology↗

Effects of foam rolling intervention on lactate clearance and cognitive function after high-intensity exercise with incremental load

PurposeThe effects foam rolling (FR) intervention on lactate clearance and the impaired executive function associated with fatigue after high-intensity exercise remain unclear. This study examined whether FR is an effective tool for fatigue recovery. MethodsEighteen healthy adults (10 males, 8 females; 21.8{+/-}1.7 years) without consistent exercise habits participated. The participants performed high-intensity exercises using an ergometer with a progressive load. Post-exercise FR intervention was compared with the control condition. Measurements included lactate, vigor/fatigue by the Profile of Mood States 2nd edition, cognitive function (cognitive task performance), and leg and body rating of perceived exertion (RPE) pre-and post-exercise, and post-intervention (5 min post-exercise). ResultsThe blood lactate concentration post-FR intervention (-7.3{+/-}3.0 mmol/L) was significantly reduced. Increased lactate clearance by FR correlated with faster recovery of executive function for those with greater lactate clearance, although cognitive fatigue was not observed after high-intensity exercise (P = .086, r = 0.41). Lactate clearance was not significantly correlated with RPE in the FR condition, whereas RPE decreased with increased lactate clearance for those with greater lactate clearance in the control condition (leg; r = 0.778, body; r = 0.669). ConclusionsThese results indicated FR intervention may be an effective recovery tool for exhausting exercises.

physiology↗

NR4A3 deficiency ameliorates contact hypersensitivity and exacerbates psoriasis by regulating gene expression in dendritic cells

NR4A3 is a transcription factor that belongs to the nuclear receptor superfamily. To reveal the roles of NR4A3 in skin diseases, we constructed contact hypersensitivity (CHS)- and imiquimod-induced psoriasis models in Nr4a3-/- mice. In the CHS induced Nr4a3-/- mice, ear swelling was significantly reduced, accompanied by suppressed migration of dendritic cells (DCs), in which CCR7 expression was reduced. In contrast, ear swelling in psoriasis model Nr4a3-/- mice was enhanced. The expression levels of inflammatory cytokines in psoriasis skin lesions and in TLR7-ligand-stimulated DCs were increased by NR4A3 deficiency. Subcutaneous (s.c.) injection of hapten-treated Nr4a3+/+ DCs induced ear swelling in Nr4a3-/- mice, and s.c. injection of TLR7-ligand-stimulated Nr4a3-/- DCs caused significant ear swelling. Compared with Nr4a3+/+ DCs, Nr4a3-/- DCs expressed lower levels of CCR7 and IRF4, and higher levels of TLR7, IRF7, IFN-{beta}, and PU.1. Taken together, NR4A3 deficiency ameliorates CHS and exacerbates psoriasis by regulating the expression of CCR7, TLR7, and related transcription factors in DCs.

immunology↗

IGSF3 is a homophilic cell adhesion molecule that drives lung metastasis of melanoma by promoting adhesion to vascular endothelium

The immunoglobulin superfamily (IgSF) is one of the largest families of cell-surface molecules involved in various cell-cell interactions, including cancer-stromal interactions. In this study, we conducted a comprehensive RT-PCR-based screening for IgSF molecules that promote experimental lung metastasis in mice. By comparing the expression of 325 genes encoding cell-surface IgSF molecules between mouse melanoma B16 cells and its highly metastatic subline, B16F10 cells, we found that expression of the Immunoglobulin superfamily member 3 (Igsf3) was significantly enhanced in B16F10 cells than in B16 cells. Knockdown of Igsf3 in B16F10 cells significantly reduced lung metastasis following intravenous injection into C57BL/6 mice. IGSF3 promoted adhesion of B16F10 cells to vascular endothelial cells and functioned as a homophilic cell adhesion molecule between B16F10 cells and vascular endothelial cells. Notably, the knockdown of IGSF3 in either B16F10 cells or vascular endothelial cells suppressed the transendothelial migration of B16F10 cells. Moreover, IGSF3 knockdown suppressed the extravasation of B16F10 cells into the lungs after intravenous injection. These results suggest that IGSF3 promotes the metastatic potential of B16F10 cells in the lungs by facilitating their adhesion to vascular endothelial cells.

cancer biology↗

Pateamine A mediates RNA sequence-selective translation repression by anchoring eIF4A and DDX3 to GNG motifs

Small-molecule compounds that elicit mRNA-selective translation repression have attracted interest due to their potential for expansion of druggable space. However, only limited examples have been reported to date. Here, we show that pateamine A (PatA) represses translation in an mRNA-selective manner by clamping eIF4A, a DEAD-box RNA-binding protein, on GNG motifs. Through a systematic comparison of multiple eIF4A inhibitors by ribosome profiling, we found that PatA has unique mRNA selectivity in translation repression. Unbiased Bind-n-Seq revealed that PatA-targeted eIF4A exhibits a sequence preference for GNG motifs in an ATP-independent manner. This unusual RNA binding sterically hinders scanning by 40S ribosomes. In silico simulation, combination of classical molecular dynamics simulation and quantum chemical calculation, and the subsequent development of an inactive PatA derivative revealed that the positive charge of the tertiary amine on the trienyl arm induces G selectivity. Moreover, we identified DDX3, another DEAD-box protein, as an alternative target of PatA, showing the same effect as on eIF4A. Our results provide an example of the sequence-selective anchoring of RNA-binding proteins and mRNA-selective inhibition of protein synthesis by small-molecule compounds.

molecular biology↗

Sperm competition influences sperm quality and seminal plasma protein expression among territorial males of the courtship cichlid

Sperm competition drives the plasticity of sperm quality and seminal fluid. However, how environmental and behavioral factors influence sperm competition and its outcome on the plasticity of sperm quality are unknown. In this study, we show males can asses the sperm competition level, and their fertilization-promoting sperm traits changes according to intensity. The promiscuous Tanganyikan cichlids Ophthalmotilapia ventralis perform their fertilization in the female mouth cavity. Sperm competition occurs in the female mouth as sperm is collected from multiple males. Behavioral (i.e., courtship success rates [CSR]) and environmental (density of spawning sites [DS]) factors are related to sperm collection. Seminal plasma glycoprotein 120 (SPP120) aids in sperm immobilization and aggregation, and sperm quality (longevity and velocity) could increase the probability of siring offspring. Based on an assumption of the involvement of DS and CSR on sperm collection, DS and CSR could influence the expression of SPP120 and sperm quality. We examined how sperm quality and SPP120 affect sperm competition-associated CSR and DS. Sperm longevity and velocity were positively correlated with DS. SPP120 expression was also positively correlated with CSR and DS. The plasticity of sperm quality and amount of SPP120 result in high fertilization competency according to sperm competition level.

evolutionary biology↗

Transcription factor-mediated direct cellular reprogramming yields cell-type specific DNA methylation signature

Direct reprogramming is a technique for inducing the conversion of one type of somatic cell into another by the forced expression of defined transcription factors. Cell differentiation is generally determined by specific gene expression profiles based on distinct genome-wide epigenetic signatures. Although the CpG methylation of genomic DNA is an essential epigenetic factor that affects the transcriptional state of genes, little is known about how DNA methylation changes and what roles it plays in direct reprogramming. Here, we performed comparative genome-wide DNA methylation analyses of mouse embryonic fibroblasts (MEFs) and cells composing organoids formed by intestinal stem cells (ISCs) or induced ISCs (iISCs) that were directly induced from MEFs to investigate the impact of DNA methylation dynamics on direct reprogramming. We found that the methylation state of CpG was similar between cells forming ISC organoids and iISC organoids, while they differed widely from those in MEFs. Moreover, genomic regions that were differentially methylated between ISC organoid- and iISC organoid-forming cells did not significantly affect gene expression. These results demonstrate the accuracy and safety of iISC induction, as they show that the DNA methylation state transitions to a state close to that of ISCs during direct reprogramming from MEFs to iISCs.

cell biology↗

Non-canonical functions of UHRF1 maintain DNA methylation homeostasis in cancer cells

DNA methylation is an essential epigenetic chromatin modification, and its maintenance in mammals requires the protein UHRF1. It is yet unclear if UHRF1 functions solely by stimulating DNA methylation maintenance by DNMT1, or if it has important additional functions. Using degron alleles, we show that UHRF1 depletion causes a much greater loss of DNA methylation than DNMT1 depletion. This is not caused by passive demethylation as UHRF1-depleted cells proliferate more slowly than DNMT1-depleted cells. Instead, bioinformatics, proteomics and genetics experiments establish that UHRF1, besides activating DNMT1, interacts with DNMT3A and DNMT3B and promotes their activity. In addition, we show that UHRF1 antagonizes active DNA demethylation by TET2. Therefore, UHRF1 has non-canonical roles that contribute importantly to DNA methylation homeostasis; these findings have practical implications for epigenetics in health and disease.

molecular biology↗

Bioinformatic and fine-scale chromosomal mapping uncover the essence and evolution of eliminated chromosomes in the Japanese hagfish, Eptatretus burgeri, through repetitive DNA family analysis

In the Japanese hagfish, Eptatretus burgeri, approximately 21% of the genomic DNA in germ cells (2n=52) consists of 16 chromosomes (eliminated [E]-chromosomes) that are eliminated from presumptive somatic cells (2n=36). To uncover the eliminated genome (E-genome), we have identified 16 eliminated repetitive DNA families from eight hagfish species, with 11 of these repeats being selectively amplified in the germline genome of E. burgeri. Furthermore, we have demonstrated that six of these sequences, namely EEEb1-6, are exclusively localized on all 16 E-chromosomes. This has led to the hypothesis that the eight pairs of E-chromosomes are derived from one pair of ancestral chromosomes via multiple duplication events over a prolonged evolutionary period. NGS analysis has recently facilitated the re-assembly of two distinct draft genomes of E. burgeri, derived from the testis and liver. This advancement allows for the prediction of not only nonrepetitive eliminated sequences but also over 100 repetitive and eliminated sequences, accomplished through K-mer-based analysis. In this study, we report four novel eliminated repetitive DNA sequences (designated as EEEb7-10) and confirm the relative chromosomal localization of all eliminated repeats (EEEb1-10) by fluorescence in situ hybridization (FISH). With the exception of EEEb10, all sequences were exclusively detected on EEEb1-positive chromosomes. Surprisingly, EEEb10 was detected as an intense signal on EEEb1-positive chromosomes and as a scattered signal on other chromosomes in germ cells. The study further divided the eight pairs of E-chromosomes into six groups based on the signal distribution of each DNA family, and fiber-FISH experiments showed that the EEEb2-10 family was dispersed in the EEEb1-positive extended chromatin fiber. These findings provide new insights into the mechanisms underlying chromosome elimination and the evolution of E-chromosomes, supporting our previous hypothesis.

molecular biology↗

Hybridization and its impact on the ontogenetic allometry of skulls in macaques

The role of hybridization in morphological diversification is a fundamental topic in evolutionary biology. However, despite the accumulated knowledge on adult hybrid variation, how hybridization affects ontogenetic allometry is less well understood. Here, we investigated the effects of hybridization on postnatal ontogenetic allometry in the skulls of a putative hybrid population of introduced Taiwanese macaques (Macaca cyclopis) and native Japanese macaques (M. fuscata). Genomic analyses indicated that the population consisted of individuals with varying degrees of admixture, formed by male migration from Japanese to Taiwanese macaques. For overall skull shape, ontogenetic trajectories were shifted by hybridization in a nearly additive manner, with moderate transgressive variation observed throughout development. In contrast, for the maxillary sinus (hollow space in the face), hybrids grew as fast as Taiwanese macaques, diverging from Japanese macaques, which showed slow growth. Consequently, adult hybrids showed a mosaic pattern, i.e., the maxillary sinus is as large as that of Taiwanese macaques, while the overall skull shape is intermediate. Our findings suggest that the transgressive variation can be caused by prenatal shape modification and non-additive inheritance on regional growth rates, highlighting the complex genetic and ontogenetic bases underlying hybridization-induced morphological diversification.

evolutionary biology↗