Biology subjects

Tsuchiya, H.

Publications and source records attributed to Tsuchiya, H..

15 recordsLinked to original sources

bioRxiv2026

Adolescent prodromal phenotypes of psychiatric disorders: insights from a novel DISC1-perturbed monkey model

Neurodevelopmental deficits frequently lead to mild but significant adolescent social and cognitive changes, representing prodromal manifestations of adult-onset psychiatric disorders in a cross-disease manner. Although experimental medicine utilizes animal models to address the mechanisms, rodent brains lack critical structures for human higher brain functions such as the dorsolateral prefrontal cortex. To fill this translational gap, we generated monkeys with an impaired DISC1 gene locus, targeting the multifunctional protein essential for neurodevelopment and involved in transdiagnostic mental health risks. Here we analyze the F0 animals by minimizing mosaicism. At ages equivalent to human adolescence, these monkeys exhibited blunted responsiveness to social stimuli and increased internal drives, mirroring altered social behaviors in the prodromal stage in humans. Resting-state functional MRI data indicated disrupted global functional connectivity and reorganization of network topology, marked by a shift in the dominance of brain hubs from prefrontal regions to sensory areas. This primate model provides a unique platform for studying psychiatric disorders, such as schizophrenia, across the developmental trajectory.

neuroscience↗

bioRxiv2026

Deep learning-mediated detection of accelerated water drinking after aquaresis in V1b vasopressin receptor knockout mice

How water intake is initiated and maintained following V2 vasopressin receptor antagonism remains poorly understood. To elucidate the role of the V1b receptor in managing dehydration stress induced by V2 antagonism, we used deep learning-based computer vision to analyze drinking behavior in V1b knockout (V1bKO) and wild-type (WT) mice. While total water access and intake volume were comparable between genotypes, V1bKO mice exhibited distinct temporal dynamics. Modeling cumulative intake with the Hill equation revealed that the time required to reach 50\% of maximal water access was significantly shorter in V1bKO mice than in WT mice. This accelerated drinking effectively mitigated increases in serum osmolality and body weight loss. A reduced Hill's coefficient in V1bKO mice indicates a reduction of the rapid, cooperative-like water accumulation seen in WT mice. Furthermore, elevated basal hemoglobin levels in V1bKO mice were independent of dehydration, as confirmed via bone marrow transplant. Analysis of movement trajectories revealed that V1bKO mice exhibit a lower proportion of vertical movement (required for nozzle access) despite similar total distances traveled. Collectively, our results demonstrate that the V1b receptor critically regulates water-seeking behavior and osmotic homeostasis.

pharmacology and toxicology↗

bioRxiv2026

A senescent iCAF-like fibroblast state governs therapy resistance in rheumatoid arthritis

ObjectivesRheumatoid arthritis (RA) synovitis frequently persists despite cytokine-targeted therapies, suggesting the existence of pathogenic stromal programs that sustain chronic inflammation independently of canonical immune pathways. Although synovial fibroblasts (SF) are increasingly implicated in treatment resistance, the pathogenic fibroblast states driving refractory disease and their therapeutic vulnerabilities remain poorly defined. MethodsWe integrated multimodal single-cell and spatial profiling of synovial tissue from 54 patients with RA with prospective treatment-response data and functional studies in human fibroblasts and experimental arthritis models. ResultsWe identified a C-X-C motif chemokine 12 (CXCL12)hi Apolipoprotein C1 (APOC1)+ fibroblast population selectively enriched in treatment-refractory synovitis. Spatial analyses demonstrated that these fibroblasts establish CXCL12-dependent plasmablast niches within inflamed synovium, resembling inflammatory cancer-associated fibroblasts (iCAF) that orchestrate immune cell recruitment in the tumor microenvironment. CXCL12hi APOC1+ fibroblasts exhibited a senescence-associated iCAF-like transcriptional program characterized by STAT3-C/EBP activation and APOC1 expression and were associated with poor response to TNF and IL-6 pathway inhibition. Mechanistically, APOC1 knockdown in RA-SF attenuated invasive mesenchymal behavior and disrupted senescence-associated inflammatory programs, identifying APOC1 as a central regulator of pathogenic fibroblast reprogramming. Importantly, genetic or pharmacological elimination of senescent cells ameliorated experimental arthritis and enhanced the efficacy of TNF blockade. ConclusionsThese findings implicate iCAF-like fibroblasts with senescent properties as a mechanistic driver of refractory RA synovitis and highlight stromal senescence programs as preclinically actionable therapeutic targets beyond cytokine inhibition.

immunology↗

bioRxiv2025

High-throughput heterospheroid-based screening identifies drugs that reprogram tumor-associated macrophages

The tumor microenvironment (TME) provides a niche for immune evasion and immunotherapy resistance, in part, by recruiting pro-tumor M2-like macrophages. In the present study, using heterospheroids consisting of cancer cells and macrophages, we identified TAM activators, which are compounds that reprogram M2-like tumor-associated macrophages (TAMs) toward the antitumor M1-like phenotype. THP-1- or human peripheral monocyte-derived macrophages were co-cultured with liver cancer cells in an ultra-low attachment dish to generate heterospheroids. Cell surface marker expression and macrophage infiltration into the heterospheroids were assessed by flow cytometry and fluorescence microscopy, respectively. Lipopolysaccharide (LPS) and interferon-{gamma} (IFN{gamma})-induced M1 marker expression was observed on the macrophages in the homospheroids; however, this induction was suppressed in heterospheroids. Microscopic imaging revealed that macrophage infiltration into the heterospheroids was decreased in the presence of LPS and IFN{gamma}, which prompted us to develop a high-content imaging screen. We identified two compounds [alprostadil (prostaglandin E1) and HX531] with TAM-activating activity. RNA-seq analysis revealed that HX531 modulated the immune and IFN response in cancer cells and cell division in macrophages. Moreover, alprostadil promoted the M1-like polarization of TAMs, increased tumor-infiltrating CD8+ T cells, and enhanced anti-PD-1 antibody therapeutic efficacy in a syngeneic mouse xenograft model. In conclusion, the heterospheroid culture recapitulates the immunosuppressive TME, which prevents the M1 polarization of TAMs. It provides a new platform for screening TAM activators and will enable the development of novel cancer immunotherapeutics when combined with high-content imaging analysis.

pharmacology and toxicology↗

bioRxiv2025

Human fibrosarcoma cells selected for very-high doxorubicin resistance, acquire trabectedin and eribulin cross-resistance, remain sensitive to recombinant methioninase, and have increased c-MYC expression

Doxorubicin is first-line chemotherapy for soft tissue sarcoma; however, the development of drug resistance limits its efficacy. The purpose of the present study was to select very-high doxorubicin-resistant (VHDR) HT1080 fibrosarcoma cells, determine cross-resistance to second-line chemotherapy drugs, determine maintenance of sensitivity to recombinant methioninase (r-METase) alone and in combination with doxorubicin, and measure the level of c-MYC expression. VHDR-HT1080 cells were generated by cultivating HT-1080 cells in a series of step-wise progressively higher concentrations of doxorubicin, ranging from 8 nM to 15 {micro}M, an 1875-fold increase, over a five-month period. The WST-8 reagent was used to assess cell viability. Four groups of in vitro drug-sensitivity tests were conducted, which involved both parental HT1080 and VHDR-HT1080 cells: 1) doxorubicin alone; 2) rMETase alone; 3) a combination of doxorubicin and rMETase; and 4) untreated control. The cross-resistance of VHDR-HT1080 cells to eribulin, trabectedin, gemcitabine and docetaxel was determined. The c-MYC levels in HT1080 and VHDR-HT1080 cells were measured using Western blotting. Doxorubicin had an IC50 of 3.3 {micro}M against HT1080 cells and 38.2 {micro}M against VHDR-HT1080 cells an 11.6-fold increase. The rMETase IC50 value for HT1080 was 0.75 U/ml and 0.59 U/ml for VHDR-HT1080. rMETase sensitized VHDR-HT1080 cells to doxorubicin. VHDR-HT1080 cells were cross-resistant to trabectedin 8.9-fold and cross-resistant to eribulin 1.87-fold compared to parental HT1080 cells. c-MYC expression was 8.4 times higher in VHDR-HT1080 cells compared to HT-1080 cells. The present results suggest rMETase may be used as a future clinical strategy to overcome super-doxorubicin resistance in soft tissue sarcoma.

cancer biology↗

bioRxiv2025

KEAP1 retention in phase-separated p62 bodies drives liver damage in autophagy-deficient conditions

Phase-separated p62 bodies activate NRF2, a key transcription factor for antioxidant response, by sequestering KEAP1, which targets NRF2 for degradation. Although p62 bodies containing KEAP1 are degraded by autophagy, they accumulate in various liver disorders. Their precise disease role remains unclear. We show that excessive KEAP1 retention in p62 bodies and NRF2 activation are major causes of liver damage when autophagy is impaired. In mice with weakened or blocked p62-KEAP1 interactions, KEAP1 retention and NRF2 activation under autophagy-deficient conditions were suppressed. Transcriptome and proteome analyses revealed that NRF2 targets upregulated by autophagy suppression normalized in p62 mutants unable to bind KEAP1. Autophagy deficiency caused organelle accumulation, especially of the ER, regardless of p62 mutation. Liver damage and hepatomegaly from autophagy suppression markedly improved in p62 mutants, particularly those with blocked KEAP1 binding. These findings highlight excessive KEAP1 retention in p62 bodies and defective organelle turnover as key drivers of liver pathology, underscoring the significance of phase separation in vivo.

cell biology↗

bioRxiv2024

Atlas of Cynomolgus Macaque Hematopoiesis

Self-renewal and differentiation are inherent properties of hematopoietic stem cells (HSCs) that are necessary to support hematopoiesis; however, the underlying mechanisms, especially in human, remain unclear. Here, using the cynomolgus macaque as a surrogate model, we develop a new gating strategy to isolate with high purity transplantable cynomolgus HSCs and generated a single-cell transcriptomic map of cynomolgus HSCs and progenitor cells -- that covering gestational periods previously not analyzed in human. We show that hematopoietic cells from the late-1st to early-3rd trimester fetal liver and late-2nd trimester and thereafter bone marrow have repopulating potential, closely mimicking humans. Unexpectedly however, we found unlike in human, cynomolgus HSCs express CD38 but not CD33, indicating that these cellular counterparts are molecularly distinct. Our transcriptomic analysis reveals the presence of a direct differentiation pathway from HSCs to megakaryocyte lineages, lineage-primed multipotent progenitors and also identified putative HSC surface markers. Taken together, our comprehensive dataset highlights not only the utility of cynomolgus monkeys as model systems to study hematopoiesis but also their potential for translational applications.

cell biology↗

bioRxiv2024

Computer vision analysis of mother-infant interaction identified efficient pup retrieval in V1b vasopressin receptor knockout mice

Close contact between lactating rodent mothers and their infants is essential for effective nursing. Whether the mothers effort to retrieve the infants to their nest requires the vasopressin-V1b vasopressin receptor axis has not been fully defined. To address this question, V1b receptor knockout (V1bKO) and control mice were analyzed in pup retrieval test. Because an exploring mother in a new test cage randomly accessed to multiple infants in changing backgrounds over time, a computer vision-based deep learning analysis was applied to continuously calculate the distances between the mother and the infants as a parameter of their relationship. In an open-field, a virgin female V1bKO mice entered fewer times into the center area and moved shorter distances than wild-type (WT). While this behavioral pattern persisted in V1bKO mother, the pup retrieval test demonstrated that total distances between a V1bKO mother and infants came closer in a shorter time than with a WT mother. Moreover, in the medial preoptic area, parts of the V1b receptor transcripts were detected in galanin- and c-fos-positive neurons following maternal stimulation by infants. This research highlights the effectiveness of deep learning analysis in evaluating the mother-infant relationship and the critical role of V1b receptor in pup retrieval during the early lactation phase. Highlights(1) Mother-infant relationship was calculated as distances by computer vision and object detection analysis. (2) V1b knockout female mice tended to avoid entering centerfield in a new cage compared to control female. (3) Lack of V1b vasopressin receptor in mice facilitated the pup retrieval in early infancy, even with shorter distances of total movements.

animal behavior and cognition↗

bioRxiv2022

The developmental origin and the specification of the adrenal cortex in humans and cynomolgus monkeys

Development of the adrenal cortex, a vital endocrine organ, originates in the adrenogonadal primordium, a common progenitor for both the adrenocortical and gonadal lineages in rodents. In contrast, we find that in humans and cynomolgus monkeys, the adrenocortical lineage originates in a temporally and spatially distinct fashion from the gonadal lineage, arising earlier and more anteriorly within the coelomic epithelium. The adrenal primordium arises from adrenogenic coelomic epithelium via an epithelial-to- mesenchymal-like transition, which then progresses into the steroidogenic fetal zone via both direct and indirect routes. Notably, we find that adrenocortical and gonadal lineages exhibit distinct HOX codes, suggesting distinct anterior-posterior regionalization. Together, our assessment of the early divergence of these lineages provides a molecular framework for understanding human adrenal and gonadal disorders. One Sentence SummarySpecification of the adrenal cortex occurs in adrenogenic coelomic epithelium independent of gonadogenesis in humans and cynomolgus monkeys

developmental biology↗

bioRxiv2022

Splicing QTL analysis focusing on coding sequences reveals pathogenicity of disease susceptibility loci.

Splicing QTL (sQTL) are one of the major causal mechanisms in GWAS loci, but their role in disease pathogenesis is poorly understood. One reason is the huge complexity of alternative splicing events producing many unknown isoforms. Here, we proposed two novel approaches, namely integration and selection, for this complexity by focusing on protein-structure of isoforms. First, we integrated isoforms with the same coding sequence (CDS) and identified 369-601 integrated-isoform ratio QTLs (i2-rQTLs), which altered protein-structure, in six immune subsets. Second, we selected CDS incomplete isoforms annotated in GENCODE and identified 175-337 isoform-ratio QTL (i-rQTL). By comprehensive long-read capture RNA-seq among these incomplete isoforms, we revealed 29 full-length isoforms with novel CDSs associated with GWAS traits. Furthermore, we have shown that disease-causal sQTL genes can be identified by evaluating their trans-eQTL effects. Our approaches highlight the understudied role of protein-altering sQTLs and are broadly applicable to other tissues and diseases.

genetics↗

bioRxiv2020

Capturing Human Trophoblast Development with Naive Pluripotent Stem Cells In Vitro

Trophoblast are extra-embryonic cells that are essential to maintain pregnancy. Human trophoblasts arise from the morula as trophectoderm (TE), which, after implantation, differentiates into cytotrophoblast (CT), syncytiotrophoblast (ST), and extravillous trophoblast (EVT) composing the placenta. Here we show that naive, but not primed, human pluripotent stem cells (PSCs) recapitulate trophoblast development. Naive PSC-derived TE and CT (nCT) recreated the human and monkey TE-to-CT transition. nCT self-renewed as CT stem cells and had the characteristics of proliferating villous CT and CT in the cell column of the first trimester. Notably, although primed PSCs differentiated into trophoblast-like cells (pBAP), pBAP were distinct from nCT and human placenta-derived CT stem cells, exhibiting properties consistent of the amnion. Our findings establish an authentic paradigm for human trophoblast development, demonstrating the invaluable properties of naive human PSCs. Our system will provide a platform to study the molecular mechanisms underlying trophoblast development and related diseases.

developmental biology↗

bioRxiv2019

Synovial fibroblasts contribute to the genetic risk of rheumatoid arthritis through the synergistic action of cytokines

ObjectivesSynovial fibroblasts (SFs) produce a variety of pathogenic molecules in the inflamed synovium of rheumatoid arthritis (RA). We aimed to gain insight into the pathogenic mechanisms of SFs through elucidating the genetic contribution to molecular regulatory networks under inflammatory condition. MethodsSFs from RA and osteoarthritis (OA) patients (n=30 each) were stimulated with 8 different cytokines (IFN-, IFN-{gamma}, TNF-, IL-1{beta}, IL-6/sIL-6R, IL-17, TGF-{beta}1, IL-18) or a combination of all 8 (8-mix). Peripheral blood mononuclear cells (PBMCs) from the same patients were fractioned into five major immune cell subsets (CD4+ T cells, CD8+ T cells, B cells, NK cells, monocytes). Integrative analyses including mRNA expression, histone modifications (H3K27ac, H3K4me1, H3K4me3), 3D genome architecture and genetic variations of SNPs were performed. ResultsSFs exposed to synergistically acting cytokines produced markedly higher levels of pathogenic molecules, including CD40 whose expression was significantly affected by a RA risk SNP (rs6074022). Upon chromatin remodeling in activated SFs, RA risk loci were enriched in clusters of enhancers (super-enhancers; SEs) induced by synergistic proinflammatory cytokines. A RA risk SNP (rs28411362), located in a SE under synergistically acting cytokines, formed three-dimensional contact with the promoter of MTF1 gene, whose binding motif showed significant enrichment in stimulation specific-SEs. Consistently, inhibition of MTF1 suppressed cytokine and chemokine production from SFs and ameliorated mice model of arthritis. ConclusionsOur findings established the dynamic landscape of activated SFs, and yielded potential therapeutic targets associated with genetic risk of RA. Key messagesO_ST_ABSWhat is already known about this subject?C_ST_ABSO_LIIn rheumatoid arthritis (RA), a variety of dysregulated molecules from immune cells and mesenchymal cells drive disease progression. Synovial fibroblasts (SFs), the most abundant resident mesenchymal cells in the inflamed synovium, produce a variety of pathogenic molecules including IL-6. C_LIO_LIGenome-wide association studies (GWAS) have identified more than 100 RA susceptibility loci. To gain insight into the pathogenic mechanisms of SFs, understanding the genetic contribution to molecular regulatory networks under inflammatory condition is crucial. C_LI What does this study add?O_LIIntegrated analyses of activated SFs demonstrated that SFs exposed to synergistically acting cytokines produced markedly higher levels of pathogenic molecules. Some of which were significantly affected by RA risk loci in a stimulation-specific manner. C_LIO_LIChromatin remodeling induced by synergistic proinflammatory cytokines were associated with RA heritability. Some transcription factors (MTF1, RUNX1) could be crucial for this structural rearrangement and the formation of inflammatory arthritis. C_LI How might this impact on clinical practice or future developments?O_LIOur findings established the dynamic landscape of activated SFs, and yielded potential therapeutic targets associated with genetic risk of RA. C_LI

immunology↗

bioRxiv2019

Autologous adipose-derived stem cell transplantation enhances healing of wound with exposed bone in a rat model

ObjectivesSoft tissue wounds with exposed bone often require extended healing times and can be associated with severe complications. We describe the ability of artificial dermis with autogenic adipose-derived stem cells (ADSCs) to promote the healing of wounds with exposed bone in a rat model.\n\nMethodsAdipose tissues harvested from the bilateral inguinal regions of Wistar rats were used as ADSCs. Rats were randomly divided into control and ADSC groups to investigate the efficacy of ADSC transplantation for wound healing (n=20 per group). Soft tissue defects were created on the heads of the rats and were covered with artificial dermis with or without the seeded ADSCs. Specimens from these rats were evaluated using digital image analysis, histology, immunohistochemistry, cell labeling, and real-time reverse-transcription polymerase chain reaction (Real-time RT-PCR).\n\nResultsThe average global wound area was significantly smaller in the ADSC group than in the control group on days 3, 7, and 14 after surgery (p<0.05). After 14 days, the blood vessel density in the wound increased by 1.6-fold in the ADSC group compared with that in the control group (p<0.01). Real-time RT-PCR results showed higher Fgfb and Vegf expression levels at all time points, and higher Tgfb1 and Tgfb3 expression levels until 14 days after surgery, in the ADSC group than in the control group (p<0.05).\n\nConclusionsIn wounds with exposed bone, autogenic ADSCs can promote vascularization and wound healing. Use of this cell source has multiple benefits, including convenient clinical application and lack of ethical concerns.

animal behavior and cognition↗

bioRxiv2019

Comparison of muscle activity, strength and balance, before and after a 6-month training using the FIFA11+ program (part 2)

PurposeSports injuries can significantly impact an athletes career, as well as impose a high financial burden on teams. Therefore, the prevention of sports injuries is an essential aspect of sports medicine. To evaluate the effects of a 6-month training period, using part 2 of the FIFA11+, on the activation and strength of core and lower limb muscles, and on static and dynamic balance performance. Materials and MethodsEight college male soccer players, 20.4{+/-}0.5 years old, completed the FIFA11+ program (part 2) at least 3x per week for 6 months. The following variables were measured, before and after the 6-month training program: activity of more than 30 muscles (with a focus on core and lower limb muscles), measured using the standardized uptake values of 18F-fluorodeoxyglucose (FDG) on positron emission tomography (PET-CT); isokinetic strength of the knee flexor and extensor and hip abductor muscles, measured at 60{degrees}/s; static balance over a 60-s period, measured using a Gravicorder; and dynamic balance, measured using the Star Excursion Balance Test. ResultsTraining improved activity levels of core (obliquus externus abdominis and erector spinae) and lower limb (tibialis anterior of the both legs) muscles (p[≤]0.03), corrected the between-limb difference in activation of the semimembranosus and improved dynamic balance, with a greater training effect on the non-dominant limb (p[≤]0.02). Training also improved knee flexor force of the non-dominant lower limb (p=0.02). ConclusionRoutine performance of the FIFA11+ (part 2) program can improve activation of core and lower limb muscles, with a concomitant improvement in dynamic balance.

physiology↗

bioRxiv2019

The synergistic antitumor effect of combined Anti-Human Epidermal Growth Factor Receptor 2 (HER2) antibody and Gamma Interferon therapy to Ab resistant breast cancer cells

Anti-HER2 antibody is molecular targeted antibody for cancer therapy. Approximately 20% of breast cancers are characterized by overexpression of HER2 protein. However, the recurrence rate was 30% and the metastasis rate was 18% one year after treatment of Anti-HER2 antibody for HER2 positive breast cancer. The resistance to antibody treatment is a major problem for patients. We previously reported that Anti-HER2 antibody and Gamma Interferon (IFN-{gamma}) combined therapy showed higher anti-tumor effect than usual therapy in vitro and in vivo mouse experiments. In this study, we evaluated whether anti-HER2 antibody and IFN-{gamma} combined therapy shows good synergistic effect against drug resistant HER2 positive breast cancer cells and higher antitumor effect than conventional clinical treatment. The resistant cell lines were made under the continuous presence of antibody until cell growth was not affected by the drug. We divided the resistant cells into the appropriate number of groups, which we and treated with anti-cancer therapy. We evaluated the antitumor effect for both in vitro study and in vivo mouse xenograft model prepared with the same immunogenicity. And we investigated the differences of immunofluorescence staining of CD8, Gr-1 and PDL-1 in tissues, especially related to immunity system. The combined therapy showed significantly higher anti-tumor effect than other groups in vitro and in vivo experiments. The combined therapy affects anti-tumor immunity in this immunofluorescence experiment. Taken together, we showed the possibility that combined therapy could be an effective treatment option for anti-HER2 antibody resistant breast cancer, helping patients suffering from cancer progression after developing treatment resistance.

cancer biology↗