Biology subjects

Kojima, M.

Publications and source records attributed to Kojima, M..

At least 19 recordsLinked to original sources

bioRxiv2026

SPATNIC enables H&E-grounded cancer-cell identification across spatial and single-cell transcriptomic modalities in colorectal cancer

Tumors contain diverse non-cancer cells that constitute the tumor microenvironment; thus, identifying which cells are cancerous is a foundational and critical first step in cancer research. However, to our knowledge, no transcriptome-based classifier has been trained using pathologist-defined annotations from matched H&E images at single-cell resolution. Moreover, few tools can be shared across diverse platforms. Here, we present SPATNIC, a variational autoencoder that classifies individual cells as cancer or normal, trained on Xenium cells labeled by a pathologist review of matched H&E images. It matches or exceeds the performance of existing tools in primary colorectal tumors and liver and lung metastases. SPATNIC generalizes zero-shot across Visium, Visium HD, CosMx, Xenium, MERSCOPE, and single-cell RNA sequencing. In public single-cell atlases, it identified a rare POU2F3 tuft-like cancer stem cell population missed by existing annotations and associated with worse survival. In rectal cancer profiled before and after chemoradiotherapy, it separated cancer from normal epithelium in tissue, thus enabling per-patient screening of antibody-drug conjugate (ADC) targets.

cancer biology↗

bioRxiv2026

VLCFA-mediated inter-cell layer communication controls cellular pluripotency in Arabidopsis callus

Plants have remarkable capacity to reconstruct entire organ systems from tissue explants. In Arabidopsis two-step tissue culture system, pluripotency regulators are specifically expressed in the middle-cell layer of the stratified callus tissue. However, regulatory mechanisms underlying the radial patterning of callus remained unclear. Here, we found that very-long-chain fatty acids (VLCFAs) synthesized in the epidermis-like outermost layer are essential for pluripotency acquisition and successful shoot regeneration. Our genetic and transcriptomic analyses revealed that the regulatory roles of VLCFAs on pluripotency acquisition involve inter-cell layer signaling in callus tissue, while they are at least partly independent of ATML1/PDF2 functions and cuticular wax synthesis in the outermost layer. VLCFAs spatially restrict procambium cell identity by non-cell-autonomously suppressing cytokinin signaling, thereby allowing for establishment of the middle-cell layer. We propose that the inhibitory relationships between layer-specific regulators underlie the intricate balance of cellular fate determination in pluripotent callus.

plant biology↗

bioRxiv2026

An Open Benchmark for Systems Vaccinology: Insights from the CMI-PB Challenges

Systems vaccinology approaches have identified factors affecting vaccine responses in multiple studies, but the ability of computational models to generalize these findings to unseen data remains unclear. We established a community resource to create and compare models predicting B. pertussis booster vaccination responses and put such modeling approaches to the test. We compiled multi-modal experimental training data from three independent cohorts (n=117 individuals), and asked investigators to predict vaccine responses in a cohort of 54 newly recruited individuals using only their pre-booster vaccination data. We benchmarked a total of 107 computational models. Top-performing models were characterized by workflows that prioritized rigorous data preprocessing, robust imputation of missing data, and the use of multi-omics integration or non-linear machine learning. We identified pre-existing antigen-specific antibody titers and baseline monocyte frequencies as the most consistent predictors of post-vaccination immunity, highlighting the dominant role of individual immune setpoints. We established the resulting datasets and evaluation framework as a community resource to advance predictive immunology and facilitate personalized vaccination strategies.

immunology↗

bioRxiv2026

Single-cell spatial multiomics identifies POSTN+ CAFs mediating chemoradiotherapy resistance in rectal cancer

Neoadjuvant chemoradiotherapy (CRT) is standard for locally advanced rectal cancer (LARC), yet many patients retain residual disease. To resolve CRT-associated remodeling of the tumor microenvironment, we generated a multimodal spatial atlas from serial sections of paired pretreatment and post-treatment specimens from 24 patients using Xenium single-cell spatial transcriptomics and PhenoCycler multiplex proteomics, profiling 2.8 million cells; matched Visium HD datasets were generated on adjacent serial sections. Resistance was most strongly associated with fibroblast and myeloid programs adjacent to residual tumor. We identify a periostin (POSTN)-expressing CAF subset selectively enriched around residual tumor cells in non-responders, displaying a myofibroblastic phenotype and activating extracellular matrix remodeling, noncanonical WNT signaling, and immunosuppressive pathways. Tumor cells neighboring POSTN+ CAFs show consistent epithelial-mesenchymal transition signatures. Together, this atlas enables interrogation of CRT-induced spatial remodeling and nominates POSTN+ CAFs as key mediators and targets of CRT resistance, with direct relevance to CRT-based combination strategies.

cancer biology↗

bioRxiv2026

Homoeo-alleles of wheat GNI2 fuel grain yields across input environments

Grain yield in wheat is frequently driven by pre-anthesis growth and how carbon is directed to reproductive parts, determining the potential size of the grain-producing sink. However, the genetic mechanisms controlling this carbon allocation remain unclear. In this study we discovered a series of loci in tetraploid and hexaploid wheats that, when combined, confer an average grain yield advantage in hexaploid winter wheat of approx. 5-7% under high-input farming while approx. 10-15% under low-input conditions. The responsible gene is GRAIN NUMBER INCREASE 2 (GNI2), ancestral to its duplicate GNI1, and represented by the homoeo-allelic series GNI-A2, GNI-B2, and GNI-D2. GNI1 and all GNI2 copies additively affect floral growth and fertility by modulating reproductive allocation and harvest index. Herewith, we describe a series of grain yield-relevant GNI2 homoeo-alleles with a proven track record for being beneficial in both high- and low-input environments. Their deployment offers a sustainable pathway to raise global grain yields in the future.

plant biology↗

bioRxiv2026

The submergence-induced drastic morphological plasticity of root in the amphibious plant Callitriche palustris

Amphibious plants can thrive in both terrestrial and submerged environments, which are fundamentally distinct. Although morphological plasticity of leaf known as heterophylly has been well investigated, the morphological plasticity of root in amphibious plants remains poorly understood. In this study, we discovered that an amphibious plant Callitriche palustris (Plantaginaceae), which has significant heterophylly, has a remarkable morphological plasticity also in root in response to submergence. This species develops thin roots with abundant root hairs, fewer cortical and epidermal cells, and smaller aerenchyma in the terrestrial condition. On the other hand, it develops thicker roots with few root hairs, more cortical and epidermal cells, and larger aerenchyma in the submerged condition. We call this morphological plasticity of root as "heterorhizy". Phytohormone perturbation experiments revealed that abscisic acid (ABA) and gibberellin regulate root hair development and root cell division respectively. We also found the possibility that heterorhizy was acquired in the genus Callitriche. Additionally, a similar form of root hair plasticity was also observed in the phylogenetically distinct amphibious species Ludwigia arcuata (Onagraceae). Furthermore, the absence of root hair development underwater and the similar structure of aerenchyma to C. palustris were broadly seen across diverse aquatic plants. This study provides new insights into the root morphological responses to submerged environments in aquatic plants.

plant biology↗

bioRxiv2025

Human Interleukin-4-Dependent Facilitation of Human IgG Production in PBL-NOG-hIL-4-Tg mice

Immune-humanized mice provide valuable experimental models for evaluating immune-based therapies, yet the induction of human antigen-specific IgG production remains limited due to species-specific incompatibilities. Our previous work demonstrated that NOG-hIL-4-Tg mice, which express human interleukin-4 (IL-4), support human T and B cell maintenance and enable antigen-specific IgG production following transplantation of human peripheral blood mononuclear cells (PBMCs). In this study, we investigated how IL-4 enhances antibody responses in this model. Flow cytometry and histological analysis revealed that human B cell maintenance was associated with specific plasma hIL-4 concentration ranges at supraphysiological levels. T and B cell receptor repertoire analysis using next-generation sequencing showed that clonal diversity remained largely conserved for one month and decreased to three months post-engraftment. Immunoglobulin repertoire profiling confirmed IgG class switching in an IL-4-concentration-dependent manner. Among the IgG subclasses, IgG3 increased during the first and second months and then decreased thereafter. In contrast, IgG1 tended to increase over time; however, the proportion of IgG subclass varied among individual donors. Following immunization with two distinct peptide antigens, the mice produced enhanced levels of antigen-reactive IgG. However, many B cell clones also exhibited weak responses to unrelated third-party antigens, likely reflecting insufficient affinity maturation. Histological evaluation showed tertiary lymphoid structure (TLS)-like accumulations of B and T cells in the spleen, although fully developed germinal centers were absent. Taken together, these findings demonstrate that NOG-hIL-4-Tg mice maintain human B cells within a regulated IL-4 environment, promote early formation of splenic tertiary lymphoid structures, and support IgG production characterized by clonal expansion, class switching, and somatic hypermutation. These results confirm that human IL-4 expression supports human antibody responses in the PBL-NOG-hIL-4-Tg mouse system. (278)

immunology↗

bioRxiv2025

Cytokinin receptor AHK3 modulates root-to-shoot transport of trans-zeatin type cytokinin

Trans-zeatin (tZ)-type cytokinins (CKs) are synthesized in roots in response to nitrate, transported to shoot via xylem, and coordinate diverse physiological processes in aerial organs. Within this mechanism, the regulation of CK biosynthesis by nitrate signaling via NIN-like protein 7 as well as the loading of tZ-type CKs into xylem by ATP-binding cassette transporter G14 have been well studied. However, the roles of other components remain unclear. Here, we show that CK perception and degradation in roots, as mediated by Arabidopsis histidine kinase 3 (AHK3) and CK oxidase/dehydrogenase 4 (CKX4), modulate xylem tZ-type CKs transport and leaf CK status. Grafting experiments demonstrated that root-specific AHK3 deficiency systemically increased leaf blade area through long-distance signals of root-derived tZ-type CK, perceived by shoot-expressed AHK3. Transcriptome and hormonome analyses revealed that root-specific AHK3 deficiency reduced CKX4 expression in roots, elevating tZ-type CK levels in roots and xylem sap and thereby enhancing leaf CK response. Transfer experiments manipulating root nitrate levels showed that root-specific AHK3 deficiency promoted leaf blade area in a manner dependent on both nitrate and root-derived tZ-type CK signaling. Moreover, both nitrate signals and root-expressed AHK3 are required for maximal CKX4 induction in roots, and root-specific CKX4 deficiency enhanced leaf blade area in a nitrate-dependent manner. These findings reveal a novel mechanism in which an AHK3-CKX4 module governs xylem transport of tZ-type CKs, fine-tuning leaf size according to nitrogen availability in roots. SIGNIFICANCE STATEMENTTo clarify a mechanism that attenuates trans-zeatin-type cytokinin transport from roots to shoots in response to nitrate signaling, this study examined the root-specific role of cytokinin receptors. Our results show that cytokinin perception and degradation, as mediated by Arabidopsis histidine kinase 3 and cytokinin oxidase/dehydrogenase 4, modulate xylem trans-zeatin-type cytokinin transport, thereby fine-tuning leaf cytokinin status and growth in a nitrate-dependent manner, providing new insights into long-distance cytokinin transport according to nitrogen availability.

plant biology↗

bioRxiv2025

Chromatin organizer ASCL1 governs gene programs in thymic epithelial cells, defining immunological self

Immunological self-tolerance depends on medullary thymic epithelial cells (mTECs), which express a broad repertoire of self-antigens to support negative selection of autoreactive T cells and the development of regulatory T cells. Although the autoimmune regulator AIRE is essential for this process, additional factors are required to establish the full mTEC gene expression program. Because thymoma is frequently associated with autoimmunity, implicating defective thymic tolerance, we performed single-cell RNA sequencing of TECs from thymoma patients and identified the transcription factor ASCL1 as selectively downregulated in tumor mTECs. Deletion of Ascl1 in mouse TECs resulted in spontaneous autoimmunity without inducing thymic tumorigenesis. Transcriptomic and chromatin accessibility analyses revealed that ASCL1 influences mTEC gene expression programs and is associated with corresponding changes in chromatin accessibility. Genetic interaction analyses further suggested that ASCL1 activity in immature mTECs modulates the AIRE dependency of gene expression in mature mTECs. Together, these findings identify ASCL1 as a key regulator of mTEC function and central tolerance, providing insight into mechanisms that safeguard immune homeostasis and whose disruption may contribute to autoimmune disease.

immunology↗

bioRxiv2025

Cell-Free Protein Crystallization Enables Rapid Structure Determination of Disaccharides and Trisaccharides Using Galectin-10 Crystals

It is critical to understand the conformational selection and dynamics of flexible saccharides via protein-ligand interactions in efforts to elucidate their biofunctional roles. Protein crystals can serve as scaffolds to immobilize small molecules, enabling structural and dynamic analysis of saccharides that are difficult to study by conventional approaches. However, constructing versatile scaffold crystals for high-throughput structural analysis remains challenging because this work involves laborious protein production and crystallization workflows. Here, we report rapid crystallization and structural analysis of saccharide-bound scaffolds by applying cell-free protein crystallization (CFPC) to galectin-10 (Gal-10), a lectin known to crystallize spontaneously in vivo. Using CFPC-generated Gal-10 crystals, we obtained the first atomic-resolution structures of melezitose, one of the trisaccharide, bound to the protein scaffold, revealing binding modes inaccessible by conventional approaches. Normalized B-factor analysis combined with molecular dynamics simulations reveals how the binding-site architecture modulates saccharide flexibility and immobilization. This platform can be extended to other flexible ligands and fragment-based screening.

biophysics↗

bioRxiv2025

MAX2-dependent signaling regulates the transition from 2D to 3D growth by suppressing cytokinin accumulation in Physcomitrium patens

Plants undergo distinct developmental phases throughout their life cycle. Proper regulation of the timing of phase transition in response to internal and external cues is crucial to optimize proliferation and reproduction. In Physcomitrium patens (P. patens), a moss, the transition from growth of filamentous tissue growth called protonema to gametophore bud formation represents a shift from two-dimensional (2D) to three-demensional (3D) growrh, ultimately leading to reproductive development. MORE AXILLARY GROWTH2 (MAX2), an F-box protein, targets repressor proteins in the KARRIKIN INSENSITIVE2 (KAI2)-dependent signaling pathway for degradation via the 26S proteasome-dependent pathway. KAI2 perceives an unidentified ligand known as KAI2 ligand (KL) as well as karrikins. The four SMAX1-LIKE (SMXL) family proteins work as suppressors in the MAX2-dependent signaling in P. patens. Gametophore bud formation was accelerated when MAX2-dependent signaling was disrupted, while it was delayed in Ppsmxls, loss of function mutants of the PpSMXLs. These results indicate that MAX2-dependent signaling suppresses gametophore bud formation in P. patens, which has not been previously described. We demonstrate that cytokinin activity and contents are increased in Ppmax2 mutants, and cytokinin application rescued the delay in bud formation in Ppsmxl mutants, suggesting that MAX2-dependent signaling suppresses cytokinin accumulation. The four SMXL genes redundantly act to promote gametophore bud formation; PpSMXLC and PpSMXLD, which contain the conserved degron sequence, act as the repressor of MAX2-dependent signaling, while PpSMXLA and PpSMXLB function independent of proteolysis-dependent signaling. Based on these findings, we propose that modulating cytokinin activity to control developmental phase transition may be a conserved role of MAX2-dependent signaling in bryophytes. Significance StatementPlant hormones, both individually and in combination, regulate almost all aspects of plant growth and development. Gametophore bud formation in Physcomitrium patens, a moss, marks the transition from two-dimensional (2D) filamentous growth to three-dimensional (3D) shoot growth. We demonstrate that gametophore bud formation is controlled by the sequential action of two plant hormones, KL, an unidentified hormone that is transduced through MAX2-dependent signaling, and cytokinin. Four SMXL genes, repressors in MAX2-dependent signaling, redundantly promote bud formation: two function within the signaling pathway, while the other two operate independently of it. Our findings uncover novel mechanisms that directly link MAX2-dependent signaling to developmental phase transition by regulating stem cell activity, achieved through the regulation of cytokinin accumulation in Physcomitrium patens.

plant biology↗

bioRxiv2025

Systematic evaluation of single-cell multimodal data integration for comprehensive human reference atlas.

The integration of multimodal single-cell data enables comprehensive organ reference atlases, yet its impact remains largely unexplored, particularly in complex tissues. We generated a benchmarking dataset for the renal cortex by integrating 3 and 5 scRNA-seq with joint snRNA-seq and snATAC-seq, profiling 119,744 high-quality nuclei/cells from 19 donors. To align cell identities and enable consistent comparisons, we developed the interpretable machine learning tool scOMM (single-cell Omics Multimodal Mapping) and systematically assessed integration strategies. "Horizontal" integration of scRNA and snRNA-seq improved cell-type identification, while "vertical" integration of snRNA-seq and snATAC-seq had an additive effect, enhancing resolution in homogeneous populations and difficult-to-identify states. Global integration was especially effective in identifying adaptive states and rare cell types, including WFDC2-expressing Thick Ascending Limb and Norn cells, previously undetected in kidney atlases. Our work establishes a robust framework for multimodal reference atlas generation, advancing single-cell analysis and extending its applicability to diverse tissues.

genomics↗

bioRxiv2025

The Knock-In Atlas: A web resource for targeted protein trap by CRISPR/Cas9 in human and mouse cell lines

Various cell engineering techniques have been developed by leveraging the CRISPR-Cas9 technology, but large-scale resources for targeted gene knock-in are still limited. Here we introduce the Knock-in Atlas, a web resource for gene tagging by fluorescent proteins by inserting artificial exons in target gene introns. To produce knock-in cells efficiently and reproducibly, we carefully chose and catalogued guide RNAs (gRNAs) for targeting genes in the human and mouse genomes by taking the gRNA efficacy scores and protein structures around the insertion sites into account. As of August 2025, we have characterized knock-in cell lines for 350 proteins, with a focus on RNA binding proteins, by flow cytometry and confocal microscopy. The transfection and flow cytometry protocols were optimized for several cell lines including HEK293T, eHAP1, HeLa, THP-1, Neuro2a, mouse embryonic fibroblast (MEF) and mouse embryonic stem cell (mESC). A website has been launched to organize the results of initial characterization including flow cytometry data after transfection, confocal microscopy, and western blot results for the genes for which knock-in HEK293T cell lines were already made. The site also provides a database to organize the information of pre-designed gRNAs for the human and mouse genomes. .

molecular biology↗

bioRxiv2024

Shoot nitrate status regulates Arabidopsis shoot growth and systemic transcriptional responses via shoot adenylate isopentenyltransferase 3

Plants systemically regulate growth and gene expression according to their internal nitrate status. Our previous study reported that shoot nitrate accumulation increases shoot expression of adenosine phosphate-ISOPENTENYLTRANSFERASE 3 (IPT3) and shoot levels of N6-({Delta}2-isopentenyl) adenine (iP)-type cytokinins (CKs). IPT3 expression is localized in the phloem, and iP-type CKs, which are synthesized by IPT3, are phloem-mobile. As CKs are a class of phytohormones that control growth and genome-wide gene expression, shoot-expressed IPT3 may mediate the systemic regulation of growth and transcriptomic responses to shoot nitrate status. To examine this, we developed a novel system to manipulate nitrate levels and IPT3 expression in a shoot-specific manner and performed growth analysis, CK determination, and RNA-sequencing. Our results demonstrated that shoot nitrate accumulation significantly promoted shoot growth and elevated shoot concentrations of iP ribotides, iP-7-N-glucoside, and iP-9-N-glucoside through the action of shoot IPT3. Transcriptomic responses to shoot nitrate accumulation were largely tuned by shoot IPT3, with opposite effects in shoots and roots. Shoot IPT3 amplified shoot responses of nitrate-inducible genes and immune response genes to shoot nitrate accumulation, while it dampened root responses of nitrate transport/assimilation genes. This transcriptomic modulation via shoot IPT3 was accompanied by coherent transcriptional changes in genes encoding mobile peptides and transcriptional repressors. Here we present a novel scheme integrating shoot nitrate status responses with CK signaling.

plant biology↗

bioRxiv2024

Putting computational models of immunity to the test - an invited challenge to predict B. pertussis vaccination outcomes

Systems vaccinology studies have been used to build computational models that predict individual vaccine responses and identify the factors contributing to differences in outcome. Comparing such models is challenging due to variability in study designs. To address this, we established a community resource to compare models predicting B. pertussis booster responses and generate experimental data for the explicit purpose of model evaluation. We here describe our second computational prediction challenge using this resource, where we benchmarked 49 algorithms from 53 scientists. We found that the most successful models stood out in their handling of nonlinearities, reducing large feature sets to representative subsets, and advanced data preprocessing. In contrast, we found that models adopted from literature that were developed to predict vaccine antibody responses in other settings performed poorly, reinforcing the need for purpose-built models. Overall, this demonstrates the value of purpose-generated datasets for rigorous and open model evaluations to identify features that improve the reliability and applicability of computational models in vaccine response prediction.

immunology↗

bioRxiv2024

KAI2-dependent signaling controls vegetative reproduction in Marchantia polymorpha through activation of LOG-mediated cytokinin synthesis

Marchantia polymorpha reproduces vegetatively (asexually) by producing propagules known as gemmae within gemma cups and sexually through spores. We previously reported that KARRIKIN INSENSITIVE 2 (KAI2)-dependent signaling promotes gemma cup and gemma formation. KAI2A perceives unidentified endogenous ligand(s), tentatively referred to as KAI2 ligands (KL). Perception of KL by KAI2 triggers MAX2-dependent proteolysis of MpSMXL. In this study, we identified genes working downstream of KAI2-dependent signaling in M. polymorpha. We found that KAI2-dependent signaling positively controls the expression of MpLONLEY GUY (MpLOG), encoding a cytokinin biosynthesis enzyme. Disruption of the MpLOG function decreased endogenous cytokinin levels and caused defects similar to KAI2-dependent signaling mutants. Moreover, supplying exogenous cytokinins rescued the defects of Mplog and KAI2-dependent signaling mutants, implying that cytokinins work downstream of KAI2-dependent signaling. Activation of MpLOG by KAI2-dependent signaling occurs in a highly cell-type-specific manner, leading to cell-specific induction of GEMMA CUP-ASSOCIATED MYB1 (GCAM1), the master regulator of vegetative reproduction of M. polymorpha. We propose a genetic cascade, starting from KAI2-dependent signaling, that promotes vegetative reproduction through the induction of MpLOG and GCAM1. The interaction between KAI2-dependent signaling and cytokinin in M. polymorpha provides a novel insight into the function and evolution of KAI2-dependent signaling.

plant biology↗

bioRxiv2024

Th1 polarization in Bordetella pertussis vaccine responses is maintained through a positive feedback loop

Outbreaks of Bordetella pertussis (BP), the causative agent of whooping cough, continue despite broad vaccination coverage and have been increasing since vaccination switched from whole-BP (wP) to acellular BP (aP) vaccines. wP vaccination has been associated with more durable protective immunity and an induced Th1 polarized memory T cell response. Here, a multi-omics approach was applied to profile the immune response of 30 wP and 31 aP-primed individuals and identify correlates of T cell polarization before and after Tdap booster vaccination. We found that transcriptional changes indicating an interferon response on day 1 post-booster along with elevated plasma concentrations of IFN-{gamma} and interferon-induced chemokines that peaked at day 1-3 post-booster correlated best with the Th1 polarization of the vaccine-induced memory T cell response on day 28. Our studies suggest that wP-primed individuals maintain their Th1 polarization through this early memory interferon response. This suggests that stimulating the interferon pathway during vaccination could be an effective strategy to elicit a predominant Th1 response in aP-primed individuals that protects better against infection.

immunology↗

bioRxiv2024

Multiomics-based assessment of the impact of airflow on diverse plant callus cultures

Plant cell culture has multiple applications in biotechnology and horticulture, from plant propagation to the production of high-value biomolecules. However, the interplay between cellular diversity and ambient conditions influences the metabolism of cultured tissues; understanding these factors in detail will inform efforts to optimize culture conditions. This study presents multiomics datasets from callus cultures of tobacco (Nicotiana tabacum), rice (Oryza sativa), and two bamboo species (Phyllostachys nigra and P. bambusoides). Over four weeks, calli were cultured under continuous moisture without airflow or gradually reduced ambient moisture with airflow. For each sample, gene expression was profiled with high-throughput RNA sequencing, 442 metabolites were measured using liquid chromatography (LC) with triple-quadrupole mass spectrometry (LC-QqQMS), and 31 phytohormones were quantified using ultra-performance LC coupled with a tandem quadrupole mass spectrometer equipped with an electrospray interface (UPLC-ESI-qMS/MS) and ultra-high-performance LC- orbitrap MS (UHPLC-Orbitrap MS). These datasets highlight the impact of airflow on callus cultures, revealing differences between and within species, and provide a comprehensive resource to explore the physiology of callus growth.

plant biology↗