Biology subjects

Takei, Y.

Publications and source records attributed to Takei, Y..

15 recordsLinked to original sources

bioRxiv2026

Hippocampal interferon-response, unfolded-protein-response and synaptic transcriptional signatures in RORγt-transgenic mice

Aim: ROR{gamma}t-transgenic mice provide a model of sustained T helper 17-cell bias, but the associated hippocampal transcriptional profile is incompletely characterized. We investigated gene-level and gene-set expression differences in this model. Methods: Bilateral hippocampal samples from 10-week-old male ROR{gamma}t-transgenic mice and wild-type littermates (n = 3 per genotype) underwent bulk RNA sequencing. Gene-level differential expression was evaluated using the Empirical Analysis of DGE tool in CLC Genomics Workbench. Preranked gene set enrichment analysis examined coordinated expression differences after expression filtering and exclusion of Rorc (13,813 genes). Results: Sixteen genes met a false discovery rate < 0.05 (nine higher and seven lower in transgenic mice), including Rorc, C4b, Xbp1, Nptx2 and Mt2. Of 212 candidates selected using unadjusted P < 0.05 and a twofold-change threshold, 155 (73.1%) had a mean expression below 0.5 reads per kilobase of transcript per million mapped reads. Hallmark interferon--response, interferon-{gamma}-response, and unfolded-protein-response gene sets showed positive enrichment. Thirteen Gene Ontology biological-process sets met a false discovery rate < 0.05, including negatively enriched sets related to excitatory postsynaptic potential, glutamatergic transmission, and calcineurin-mediated signaling. Il17a and Il17f had zero reported counts, and none of the displayed leukocyte or central nervous system cell-marker genes met false discovery rate < 0.05. Conclusion: This exploratory dataset identifies interferon-response, unfolded-protein-response and synaptic transcriptional signatures associated with the ROR{gamma}t-transgenic genotype. The cellular sources, causal mediators and functional consequences remain unresolved.

neuroscience↗

bioRxiv2026

Male RORγt-transgenic mice exhibit lower body weight and an altered locomotor response to a novel environment

ROR{gamma}t, encoded by Rorc, is a key transcriptional regulator of T helper 17 (Th17) cell differentiation, and IL-17A signaling has been implicated in neural development and behavior. We examined behavioral phenotypes in male ROR{gamma}t-transgenic mice with T-cell-biased ROR{gamma}t overexpression and their wild-type littermates. Mice were assessed using the resident-intruder paradigm and the light-dark box test, followed by body-weight measurement. ROR{gamma}t-transgenic mice had significantly lower body weight than wild-type mice. No genotype differences were detected in attack latency, attack frequency, light-compartment occupancy, light-dark transitions, or transition latency. In contrast, analysis of locomotor activity in 1-min bins revealed a significant genotype x time interaction. Transgenic mice showed nominally reduced locomotor activity during the first minute of the light-dark box test, although this single-bin comparison did not remain significant after correction for multiple comparisons. These findings indicate that T-cell-biased ROR{gamma}t overexpression is associated with altered temporal dynamics of locomotor activity in a novel environment rather than a generalized anxiety-like or aggression-related phenotype.

animal behavior and cognition↗

bioRxiv2025

Synaptic MEMOIR: mapping individual synapses of neurons with protein barcodes

Obtaining wiring diagrams of brains has been a major achievement for neuroscience. However, an underlying challenge in connectomics is the fundamental tradeoff between the imaging resolution needed to resolve synapses and the volume of the brain that can be imaged. For example, electron microscopy (EM) visualizes synaptic sites with ~5 nm resolution, but is difficult to scale beyond volumes of 1 mm3. Here, we present Synaptic MEMOIR (Memory with Engineered Mutagenesis with Optical in situ Readout) that enables imaging of neuronal projections in animal brains with single-synapse resolution. Synaptic MEMOIR is built around three key design features. First, protein barcodes are transported to synapses to allow matching of synaptic barcodes to cell body barcodes without high resolution imaging and the error-prone process of tracing neuronal processes across long distances. Second, Synaptic MEMOIR uses continuous mutagenesis to generate a large diversity of barcodes to uniquely label cell bodies and synapses. Last, the timing of recombination and transport can be tuned to record synaptic age or projection information. Combining these features, we demonstrated projection mapping of 113 neurons in the Drosophila melanogaster optic lobe in a volume of 9.5 million m3. Because synapses are identified by transported barcodes with optical microscopy at 300 nm resolution, this approach can potentially scale to much larger volumes, similar at least to those imaged in recent mouse brain transcriptomics atlases. In addition, synaptic MEMOIR can match barcodes across brain sections, and does not require tissue clearing to track long-range projections. Together, these results provide the foundation for a scalable optical image-based system for reconstructing the neural wiring diagrams of brains across different developmental stages, genetic backgrounds and perturbations. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=46 SRC="FIGDIR/small/690442v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@8363cdorg.highwire.dtl.DTLVardef@d3240org.highwire.dtl.DTLVardef@5e2793org.highwire.dtl.DTLVardef@1784f2a_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2025

Genome-wide chromatin recording resolves dynamic cell state changes

Understanding how the chromatin state of a cell influences its future behavior is a major challenge throughout biology. However, most chromatin profiling methods are limited to endpoint assays. Here, we present LagTag, a method for recovery of earlier and endpoint chromatin states in the same mammalian cells. In this approach, transient expression of bacterial adenine methyltransferase fusions records the DNA binding profiles of chromatin-associated proteins of interest at earlier timepoints. Subsequent tagmentation and sequencing recovers the earlier chromatin profile from adenine methylation profiles, alongside endpoint profiles of endogenous chromatin-associated proteins. We verified that LagTag profiles aligned with those from established methods in mouse and human cells. We then applied LagTag to record and recover dynamic chromatin state transitions during mouse embryonic stem cell differentiation, capturing transcriptional signatures from pre- and post-differentiation timepoints within the same cell population. LagTag thus provides a foundation for temporally resolved chromatin profiling.

genomics↗

bioRxiv2025

Preclinical Prediction of Resistance and Optimization of Sequential Therapy for ALK-positive Lung Cancer Using Next-generation ALK Inhibitors

BackgroundAnaplastic lymphoma kinase (ALK) gene rearrangements occur in approximately 5% of non-small cell lung cancers (NSCLCs). Although ALK tyrosine kinase inhibitors provide substantial clinical benefits, acquired resistance-conferring mutations frequently emerge, leading to disease progression. Preclinical prediction of these mutations might help guide the development of more effective sequential treatment strategies prior to clinical application. ObjectiveTo predict the emergence of resistance mutations to the investigational ALK inhibitors zotizalkib (TPX-0131), gilteritinib (ASP2215), and neladalkib (NVL-655) following resistance to first-line alectinib and assess the potential of these drugs as second-line therapies. MethodsA polymerase chain reaction (PCR)-based mutagenesis system was used to introduce random mutations into ALK cDNA harboring representative alectinib-resistant mutations. Mutant libraries were expressed in Ba/F3 cells, which were exposed to each inhibitor. Drug-resistant clones were isolated, sequenced, and evaluated for drug sensitivity using viability assays and immunoblotting. ResultsSeveral resistance mutations against zotizalkib, gilteritinib, and neladalkib were identified. Sequential use of these agents effectively suppressed all predicted resistance patterns with G1202R or I1171N. ConclusionsThis PCR-based platform provides a valuable approach for anticipating resistance mutations and guiding the design of optimized sequential therapies. Zotizalkib, gilteritinib, and neladalkib might represent promising alternatives to lorlatinib as second-line treatments for ALK-positive NSCLC. Key PointsO_LIPCR-based mutation prediction system was successfully applied to fourth-generation ALK inhibitors. C_LIO_LIshowed efficacy against G1202R-positive relapses with minimal evidence of secondary resistance mutations. C_LIO_LIcombinations of gilteritinib with either neladalkib or ensartinib may sustain efficacy and delay resistance in I1171N-positive relapses. C_LI

cancer biology↗

bioRxiv2024

Reconstructing cell histories in space with image-readable base editor recording

Knowing the ancestral states and lineage relationships of individual cells could unravel the dynamic programs underlying development. Engineering cells to actively record information within their own genomic DNA could reveal these histories, but existing recording systems have limited information capacity or disrupt spatial context. Here, we introduce baseMEMOIR, which combines base editing, sequential hybridization imaging, and Bayesian inference to allow reconstruction of high-resolution cell lineage trees and cell state dynamics while preserving spatial organization. BaseMEMOIR stochastically and irreversibly edits engineered dinucleotides to one of three alternative image-readable states. By genomically integrating arrays of editable dinucleotides, we constructed an embryonic stem cell line with 792 bits of recordable, image-readable memory, a 50-fold increase over the state of the art. Simulations showed that this memory size was sufficient for accurate reconstruction of deep lineage trees. Experimentally, baseMEMOIR allowed precise reconstruction of lineage trees 6 or more generations deep in embryonic stem cell colonies. Further, it also allowed inference of ancestral cell states and their quantitative cell state transition rates, all from endpoint images. baseMEMOIR thus provides a scalable framework for reconstructing single cell histories in spatially organized multicellular systems.

synthetic biology↗

bioRxiv2023

High-resolution spatial multi-omics reveals cell-type specific nuclear compartments

The mammalian nucleus is compartmentalized by diverse subnuclear structures. These subnuclear structures, marked by nuclear bodies and histone modifications, are often cell-type specific and affect gene regulation and 3D genome organization1-3. Understanding nuclear organization requires identifying the molecular constituents of subnuclear structures and mapping their associations with specific genomic loci in individual cells, within complex tissues. Here, we introduce two-layer DNA seqFISH+, which allows simultaneous mapping of 100,049 genomic loci, together with nascent transcriptome for 17,856 genes and a diverse set of immunofluorescently labeled subnuclear structures all in single cells in cell lines and adult mouse cerebellum. Using these multi-omics datasets, we showed that repressive chromatin compartments are more variable by cell type than active compartments. We also discovered a single exception to this rule: an RNA polymerase II (RNAPII)-enriched compartment was associated with long, cell-type specific genes (> 200kb), in a manner distinct from nuclear speckles. Further, our analysis revealed that cell-type specific facultative and constitutive heterochromatin compartments marked by H3K27me3 and H4K20me3 are enriched at specific genes and gene clusters, respectively, and shape radial chromosomal positioning and inter-chromosomal interactions in neurons and glial cells. Together, our results provide a single-cell high-resolution multi-omics view of subnuclear compartments, associated genomic loci, and their impacts on gene regulation, directly within complex tissues.

genomics↗

bioRxiv2023

3D genome organization around nuclear speckles drives mRNA splicing efficiency

The nucleus is highly organized such that factors involved in transcription and processing of distinct classes of RNA are organized within specific nuclear bodies. One such nuclear body is the nuclear speckle, which is defined by high concentrations of protein and non-coding RNA regulators of pre-mRNA splicing. What functional role, if any, speckles might play in the process of mRNA splicing remains unknown. Here we show that genes localized near nuclear speckles display higher spliceosome concentrations, increased spliceosome binding to their pre-mRNAs, and higher co-transcriptional splicing levels relative to genes that are located farther from nuclear speckles. We show that directed recruitment of a pre-mRNA to nuclear speckles is sufficient to drive increased mRNA splicing levels. Finally, we show that gene organization around nuclear speckles is highly dynamic with differential localization between cell types corresponding to differences in Pol II occupancy. Together, our results integrate the longstanding observations of nuclear speckles with the biochemistry of mRNA splicing and demonstrate a critical role for dynamic 3D spatial organization of genomic DNA in driving spliceosome concentrations and controlling the efficiency of mRNA splicing

molecular biology↗

bioRxiv2022

How to select the best model from AlphaFold2 structures?

Among the methods for protein structure prediction, which is important in biological research, AlphaFold2 has demonstrated astonishing accuracy in the 14th Community Wide Experiment on the Critical Assessment of Techniques for Protein Structure Prediction (CASP14). The accuracy is close to the level of experimental structure determination. Furthermore, AlphaFold2 predicts three-dimensional structures and estimates the accuracy of the predicted structures. AlphaFold2 outputs two model accuracy estimation scores, pLDDT, and pTM, enabling the user to judge the reliability of the predicted structures. Original research of AlphaFold2 showed that those scores had good correlations to actual prediction accuracy. However, it was unclear whether we could select a structure close to the native structure when multiple structures are predicted for a single protein. In this study, we generated several hundred structures with different combinations of parameters for 500 proteins and verified the performance of the accuracy estimation scores of AlphaFold2. In addition, we compared those scores with existing accuracy estimation methods. As a result, pLDDT and pTM showed better performance than the existing accuracy estimation methods for AlphaFold2 structures. However, the estimation performance of relative accuracy of the scores was still insufficient, and the improvement would be needed for further utilization of AlphaFold2.

bioinformatics↗

bioRxiv2021

Elevated METTL9 is associated with peritoneal dissemination in human scirrhous gastric cancers

Methylation, the most common chemical modification of cellular components such as DNA, RNA, and proteins, impacts biological processes including transcription, RNA processing, and protein dynamics. Although abnormal expression of methyltransferase can lead to various diseases including cancers, little is known about the relationship between methyltransferase and cancers. Here we aimed to understand the role of methyltransferase in cancer metastasis. We found that elevated methyltransferase-like 9 (METTL9) is closely associated with the acquisition of metastatic activity in human scirrhous gastric cancers. The stable knockdown of METTL9 via an shRNA vector technique in our original metastatic cells from scirrhous gastric cancer patients significantly inhibited migration and invasion. In metastatic cells, METTL9 protein is predominantly localized in mitochondria, and the METTL9 knockdown significantly reduced mitochondrial Complex I activity. METTL9 can be a promising molecular target to inhibit peritoneal dissemination of scirrhous gastric cancers. This report is the first to describe the relationship between METTL9 and cancer metastasis. HighlightsO_LIElevated METTL9 correlates with metastasis in human scirrhous gastric cancer. C_LIO_LIThis is the first report on the biological relationship between METTL9 and metastasis. C_LIO_LIMETTL9 protein localizes mainly in mitochondria in metastatic scirrhous gastric cancer. C_LIO_LIMETTL9 knockdown reduces mitochondrial Complex I activity to decrease cell migration and invasion in metastatic scirrhous gastric cancer. C_LIO_LIMETTL9 holds promise against peritoneal dissemination of scirrhous gastric cancer. C_LI

cancer biology↗

bioRxiv2021

Integrated spatial genomics in tissues reveals invariant and cell type dependent nuclear architecture

Nuclear architecture in tissues can arise from cell-type specific organization of nuclear bodies, chromatin states and chromosome structures. However, the lack of genome-wide measurements to interrelate such modalities within single cells limits our overall understanding of nuclear architecture. Here, we demonstrate integrated spatial genomics in the mouse brain cortex, imaging thousands of genomic loci along with RNAs and subnuclear markers simultaneously in individual cells. We revealed chromatin fixed points, combined with cell-type specific organization of nuclear bodies, arrange the interchromosomal organization and radial positioning of chromosomes in diverse cell types. At the sub-megabase level, we uncovered a collection of single-cell chromosome domain structures, including those for the active and inactive X chromosomes. These results advance our understanding of single-cell nuclear architecture in complex tissues.

systems biology↗

bioRxiv2020

Global architecture of the nucleus in single cells by DNA seqFISH+ and multiplexed immunofluorescence

Identifying the relationships between chromosome structures, chromatin states, and gene expression is an overarching goal of nuclear organization studies. Because individual cells are highly variable at all three levels, it is essential to map all three modalities in the same single cell, a task that has been difficult to accomplish with existing tools. Here, we report the direct super-resolution imaging of over 3,660 chromosomal loci in single mouse embryonic stem cells (mESCs) by DNA seqFISH+, along with 17 chromatin marks by sequential immunofluorescence (IF) and the expression profile of 70 RNAs, in the same cells. We discovered that the nucleus is separated into zones defined by distinct combinatorial chromatin marks. DNA loci and nascent transcripts are enriched at the interfaces between specific nuclear zones, and the level of gene expression correlates with an association between active or nuclear speckle zones. Our analysis also uncovered several distinct mESCs subpopulations with characteristic combinatorial chromatin states that extend beyond known transcriptional states, suggesting that the metastable states of mESCs are more complex than previously appreciated. Using clonal analysis, we show that the global levels of some chromatin marks, such as H3K27me3 and macroH2A1 (mH2A1), are heritable over at least 3-4 generations, whereas other marks fluctuate on a faster time scale. The long-lived chromatin states may represent "hidden variables" that explain the observed functional heterogeneity in differentiation decisions in single mESCs. Our integrated spatial genomics approach can be used to further explore the existence and biological relevance of molecular heterogeneity within cell populations in diverse biological systems.

systems biology↗

bioRxiv2019

Molecular mechanisms for intestinal HCO3- secretion and its regulation by guanylin in seawater-acclimated eels

The intestine of marine teleosts secretes HCO3- into the lumen and precipitates Ca2+ and Mg2+ in the imbibed seawater as carbonates to decrease luminal fluid osmolality and facilitate water absorption. However, reports on studies on the hormonal regulation of HCO3- secretion are just emerging. Here, we showed that guanylin (GN) applied to the mucosal side of intestinal epithelia increased HCO3- secretion in seawater-acclimated eels. The effect of GN on HCO3- secretion was slower than that on the short-circuit current, and the time-course of the GN effect was similar to that of bumetanide. Mucosal bumetanide and serosal 4,4-dinitrostilbene-2,2-disulfonic acid (DNDS) inhibited the GN effect, suggesting an involvement of apical Na+-K+-2Cl- cotransporter (NKCC2) and basolateral Cl-/HCO3- exchanger (AE)/Na+-HCO3- cotransporter (NBC) in the GN effect. However, mucosal DNDS and diphenylamine-2-carboxylic acid (DPC) failed to inhibit the GN effect, showing that apical AE and Cl- channel are not involved. To identify molecular species of possible transporters involved in the GN effect, we performed RNA-seq analyses followed by quantitative real-time PCR after transfer of eels to seawater. Among the genes upregulated after seawater transfer, those of Slc26a3a, b (DRAa, b) and Slc26a6a, c (Pat-1a, c) on the apical membrane of the intestinal epithelial cells, and those of Sls4a4a (NBCe1a), Slc4a7 (NBCn1), Slc4a10a (NBCn2a) and Slc26a1 (Sat-1) on the basolateral membrane were candidate transporters involved in HCO3- secretion. Judging from the slow effect of GN, we suggest that GN inhibits NKCC2b on the apical membrane and decreases cytosolic Cl- and Na+, which then activates apical DNDS-insensitive DRAa, b and basolateral DNDS-sensitive NBCela, n1, n2a to enhance transcellular HCO3- flux across the intestinal epithelia of seawater-acclimated eels.

physiology↗

bioRxiv2018

Dynamics and spatial genomics of the nascent transcriptome in single mESCs by intron seqFISH

Recent single cell experiments have revealed significant heterogeneities at the levels of transcription, DNA methylation and chromosome organization in individual cells. However, existing method of profiling mRNAs effectively averages transcriptional dynamics over many hours due to hours-long life time of most mRNAs. To capture the instantaneous activity of the transcriptome that reflects the rapid regulatory changes in cells, we imaged up to 10,421 nascent transcription active sites (TAS) in single mouse embryonic stem cells using seqFISH followed by multiple rounds of single molecule FISH and immunofluorescence. We observed that nascent transcription active sites appear to be distributed on the surface of individual chromosome territories and are dispersed throughout the nucleus. In addition, there are significant variability in the number of active transcription sites in single cells, representing globally more active to quiescent states. These states interconverted on the time scale of 2 hours as determined by a single cell pulse-chase experiment. Thus, transcriptome level seqFISH experiments provide an unprecedented spatial and dynamic view of chromosome organization and global nascent transcription activity in single cells.

systems biology↗

bioRxiv2017

Multiplexed dynamic imaging of genomic loci in single cells by combined CRISPR imaging and DNA sequential FISH

Visualization of chromosome dynamics allows the investigation of spatiotemporal chromatin organization and its role in gene regulation and other cellular processes. However, current approaches to label multiple genomic loci in live cells have a fundamental limitation in the number of loci that can be labelled and uniquely identified. Here we describe an approach we call \"track first and identify later\" for multiplexed visualization of chromosome dynamics by combining two techniques: CRISPR labeling and DNA sequential fluorescence in situ hybridization (DNA seqFISH). Our approach first labels and tracks chromosomal loci in live cells with the CRISPR system, then barcodes those loci by DNA seqFISH in fixed cells and resolves their identities. We demonstrate our approach by tracking telomere dynamics, identifying 12 unique subtelomeric regions with variable detection efficiencies, and tracking back the telomere dynamics of respective chromosomes in mouse embryonic stem cells.

systems biology↗