Biology subjects

Kinoshita, K.

Publications and source records attributed to Kinoshita, K..

At least 19 recordsLinked to original sources

bioRxiv2026

Reprogramming protein interfaces between adenylation and carrier protein domains in nonribosomal peptide synthetases

Nonribosomal peptide synthetases (NRPSs) assemble structurally diverse bioactive natural products through selective communication between adenylation (A) and carrier protein (CP) domains. Although rational rewiring of these protein-protein interactions could enable customized NRPS design, this remains challenging due to the dynamic nature of protein interfaces. Here we show that structure-guided interface reprogramming enables productive non-cognate A- CP pairings across enterobactin, vibriobactin, pyochelin, and vicenistatin biosynthetic systems. Engineered interactions between the A domains EntE, VibE, or PchD and the non-cognate CPs VinL or EntB were validated by biochemical, kinetic, and structural analyses. Reconstituted pathways containing engineered VibE, EntB, and EntF restored enterobactin production and increased yield to 2.2-fold that of the native EntE-EntB-EntF system. Interface-engineered PchD also enhanced production of a non-native salicylic acid-norspermidine conjugate. An X-ray structure and molecular dynamics simulations of an engineered non-cognate A-CP complex reveal recognition principles for programmable NRPS interface design.

biochemistry↗

bioRxiv2026

Unsupervised identification of low-frequency antigen-specific TCRs using distance-based anomaly scoring

Identifying antigen-specific T cell receptors (TCRs) within the diverse human repertoire remains challenging due to their extremely low frequencies, often as rare as one per million cells. Here, we propose a novel unsupervised approach that detects low-frequency antigen-specific TCRs through distance-based anomaly detection in TCR sequence space. Our method is based on the observation that antigen-specific TCRs preferentially localize at the periphery of V gene clusters rather than cluster centers. Using TCRdist3 to quantify sequence distances, we identify query TCRs that are anomalous compared to reference repertoires within their V-J gene combinations. We validated this approach across three immunological contexts: COVID-19 infection, influenza vaccination, and yellow fever vaccination. For SARS-CoV-2-specific TCR detection in a COVID-19 patient, our method demonstrated 34.3% accuracy, significantly outperforming similarity-based (ALICE: 8.0%) and frequency-based methods (edgeR: 5.8%, the Pogorelyy method: 6.3%), and uniquely detected low-frequency antigen-specific TCRs at clone count one. The minimal overlap with conventional approaches ([≤]6.7%) indicates our method captures distinct TCR clones overlooked by existing analyses. This spatial distribution-based paradigm provides a complementary strategy for TCR specificity detection, particularly valuable for identifying rare antigen-specific clones essential for understanding immune responses.

bioinformatics↗

bioRxiv2026

Monoaminergic variation, cortical structure, and disaster trauma interact to shape emotional vulnerability

Emotional vulnerability arises from the interplay of genetic variation, cortical network capacity, and subjective processing of adverse experiences, yet these components are rarely examined together in population-scale datasets. Monoaminergic signaling offers an opportunity for understanding such interactions because variation in related genes shapes sensitivity to environmental inputs. Among them, the vesicular monoamine transporter 1 (VMAT1) Thr136Ile variant is notable for its functional effects and its long-term maintenance at intermediate frequencies, a pattern consistent with context-dependent selection. These properties make it an informative marker for investigating how genetic sensitivity interacts with neurobiological and experiential factors to shape affective functioning. Using data from up to 9,625 participants in the Tohoku Medical Megabank Project, a cohort established after the 2011 Great East Japan Earthquake, we investigated how monoaminergic variation, cortical morphology, and traumatic memory shape affective functioning. We found associations between the Thr136Ile variant and negative affectivity and depressive symptoms, consistent with prior reports of heightened emotional reactivity associated with the 136Thr allele. However, magnitude of these effects was substantially amplified in specific experiential and neurobiological contexts. Subjective trauma sensitivity, capturing the discrepancy between earthquake disruption and current traumatic memory, displayed a genotype-dependent association with depressive symptoms, and this relationship was strongest when parietal or insular cortical surface area was smaller. Together, these results identify a multilevel pathway through which monoaminergic variation contributes to emotional vulnerability by interacting with both cortical network capacity and trauma processing. The context-dependent influence of monoaminergic variation may further contribute to maintenance of affective diversity in human populations.

neuroscience↗

bioRxiv2026

Tourism impacts on fecal glucocorticoid metabolites of free-ranging mountain gorillas in Bwindi Impenetrable National Park, Uganda

Mountain gorilla tourism plays a vital role in their conservation; however, increasing tourism intensity may elevate gorilla stress levels. Despite this concern, knowledge regarding its physiological effects remains limited. This study non-invasively assessed the impact of tourism on gorilla stress by measuring fecal glucocorticoid metabolites (fGCMs). Fecal samples were collected from habituated gorilla groups in Bwindi Impenetrable National Park, Uganda, between 2017-2018. Concentrations of fGCMs were analyzed using a field-friendly extraction method and enzyme immunoassay. To assess the specific impacts of tourism, we focused on the Rushegura group, which is frequently visited by tourists, and used Bayesian linear mixed-effects models to examine associations between log-transformed fGCMs (log-fGCMs) and tourism intensity (daily tourist numbers and visit frequency). Additionally, we performed a inter-group comparison with the Mubare group, which was visited less frequently but underwent more dynamic social changes, to evaluate how log-fGCMs levels differed across different group contexts. In the Rushegura group, the number of tourists and visits frequently exceeded the maximum limits established by the tourism guidelines (i.e., one visit of eight people per day). In this group, log-fGCMs increased with the number of tourists and were higher during days with multiple visits. The model incorporating visit frequency demonstrated better predictive performance. On the other hand, Mubare gorillas showed higher log-fGCMs than Rushegura gorillas, likely reflecting the influence of social instability. These findings underscore the importance of adhering to the tourism guidelines--specifically regarding group size and visit frequency--to minimize physiological stress and associated risks, such as disease transmission. Although this pilot study lacks site-specific biological validation for this subspecies, the results provide preliminary insights into the physiological stress responses of Bwindi mountain gorillas to tourism. Further long-term, large-scale studies involving multiple gorilla groups are necessary to better understand how tourism and other factors (e.g., habituation status and social factors) influence their stress levels.

ecology↗

bioRxiv2025

ibSLS: A Biobank for Democratizing Access to Multi-Omics Data and Biospecimens from Spaceflight Research

During spaceflight, astronauts are exposed to extreme conditions such as microgravity, cosmic radiation, and confinement, which can cause a wide range of health problems. To elucidate the molecular mechanisms underlying these issues and to develop intervention strategies for maintaining physiological homeostasis during space missions, space life science research using mouse models is actively conducted on the International Space Station (ISS). However, because of the high cost and technical complexity of spaceflight experiments, it is critical to maximize the scientific value of each mission by ensuring broad accessibility to both data and biospecimens. To this end, we developed the integrated biobank for Space Life Sciences (ibSLS; https://ibsls.megabank.tohoku.ac.jp), a data-visualization and sample-sharing platform that provides access to transcriptomic and metabolomic datasets generated from JAXAs Mouse Habitat Unit (MHU) missions. The platform features a user-friendly interface, tools for cross-mission analysis, and integration with human multi-omics databases, enabling cross-species comparisons. In addition, ibSLS facilitates biospecimen requests to support downstream research. By promoting open access to spaceflight-derived data and biological resources, ibSLS encourages the participation of researchers from diverse fields in space life science. We believe that ibSLS will make a valuable contribution to both biomedical research on spaceflight-related health issues and the study of diseases on Earth.

bioinformatics↗

bioRxiv2025

A chromosome-level genome resource for the lethal tapeworm Sparganum proliferum, with candidate genomic safe harbours for functional genetics

Sparganum proliferum is an enigmatic and highly proliferative cestode known for its ability to form invasive larval infections in mammalian hosts. Here, we present the first chromosome-level genome assembly for a member of the order Diphyllobothriidea, generated using a hybrid sequencing approach that integrates Oxford Nanopore/PacBio long-read sequencing, Illumina short-read sequencing, and Hi-C scaffolding. The final genome assembly spanned approximately 681 megabases (Mb) across nine chromosomes. BUSCO analysis revealed 81.9% completeness, whereas repeat annotation identified 55.8% of the genome as repetitive elements. Gene annotation uncovered approximately 29,000 protein-coding genes including [~]6200 transposon associated genes, highlighting the complex genomic landscape underlying parasitic lifestyles. Synteny analysis with other cestode linages including Echinococcus and Hymenolepis provided insights into the structural organisation and evolutionary trajectory of S. proliferum. In addition, we identified candidate genomic safe harbour (GSH) loci and promoters from housekeeping genes, offering potential for stable transgene integration. This high-quality genome serves as a critical resource for studying parasite evolution, host adaptation mechanisms, and the molecular basis of invasive proliferation in cestodes.

genomics↗

bioRxiv2025

High-Throughput Centrifuge Force Microscopy Reveals Dynamic Immune-Cell Avidity at the Single-Cell Level

Cell-cell binding, mediated by the physical interactions of receptors and their ligands, plays a fundamental role in immune processes such as immune surveillance and T-cell activation. However, current approaches for measuring cell avidity often lack either throughput or quantitative precision. Here, we introduce a high-throughput approach for quantifying cell binding lifetimes and strength using a centrifuge force microscope (CFM)--a compact microscope operating within a standard benchtop centrifuge. The CFM enables live monitoring of single-cell interactions under force, conducting thousands of force experiments in parallel. To facilitate the real-time study of live cell interactions, we developed a next-generation CFM with multichannel fluorescence imaging capabilities. This system accommodates measurements in two modes: cell-protein binding and cell-cell avidity assays. Using this system, we investigated immune-cell binding mediated by Bispecific T-cell Engager (BiTE) molecules, a novel immunotherapy designed to enhance immune-cell targeting of cancer cells. In cell-protein assays, we quantified T- and B-cell unbinding from BiTE-functionalized surfaces, revealing receptor-specific relationships between ligand concentration and binding strength. In cell-cell assays, we examined BiTE-mediated binding of T-cells to Nalm6 B-cells, a precursor leukemia cell line, uncovering a strong, time-dependent increase in BiTE-mediated immune-cell avidity. By integrating high-throughput and quantitative single-cell force analysis, the CFM provides new insights into the dynamic nature of immunological interactions under force, with broad implications for immunotherapy and cellular mechanics.

biophysics↗

bioRxiv2025

Functional interplay between condensin I and topoisomerase IIα in single-molecule DNA compaction

Condensin I and topoisomerase II (topo II) are chromosomal ATPases essential for mitotic chromosome assembly. Mechanistically how the two ATPases cooperate to assemble mitotic chromosomes remains unknown. Here we use total internal reflection fluorescence microscopy to analyze the interplay between condensin I and topo II at single-molecule resolution. As observed in previous studies, condensin I alone predominantly forms DNA loops in an ATP-dependent manner. However, when topo II is included in the reaction, condensin I forms stable compact structures (termed "lumps") in a manner dependent on the C-terminal domain of topo II. Each of the stable lumps contains a single condensin I complex and a single topo II dimer. Remarkably, we find that topo II, when catalytically active, renders the lumps resistant to protease treatment. Several lines of evidence show that the protease-resistant lumps contain knotted DNA. A mutant condensin I complex defective in ATP hydrolysis, together with topo II, forms smaller lumps in which the probability of DNA knotting is greatly reduced. Our results demonstrate how topo II-mediated strand passage is coupled with condensin I-mediated loop extrusion to generate a compact DNA structure. Together with recent studies, we discuss the functional implications of these observations in mitotic chromosome assembly and stabilization.

molecular biology↗

bioRxiv2024

JG2: an updated version of the Japanese population-specific reference genome

This study presents the construction of JG2, an updated population-specific reference genome for the Japanese population. Utilizing data from three individuals previously employed in the construction of JG1, several methodologies were employed to enhance genomic coverage and assembly quality. Hi-C sequencing technology facilitated phase-aware assembly, generating two haploid assemblies per individual and enabling improved representation of genetic variation. A meta-assembly strategy and a majority decision approach further refined assembly quality by combining the best sequences from multiple assemblies and minimizing the inclusion of rare variants. The resulting JG2 genome comprises chromosome-level sequences, mitochondrial chromosomes, and unplaced scaffolds, offering more comprehensive coverage of the Japanese genome. Comparative analyses with other reference genomes demonstrated the accuracy and representativeness of JG2, highlighting its utility for genetic research involving the Japanese population. Overall, by adopting the phased assembly technique, JG2 represents a significant advancement over the collapsed assembly-based JG1, providing researchers with a more precise and comprehensive resource for understanding the genetic landscape of the Japanese population. The sequences and annotations are available on the jMorp website (https://jmorp.megabank.tohoku.ac.jp/).

genomics↗

bioRxiv2024

Next-generation sequencing analysis with a population-specific reference genome

Next-generation sequencing (NGS) has become widely available and is routinely used in basic research and clinical practice. The reference genome sequence is an essential resource for NGS analysis, and several population-specific reference genomes have recently been constructed to provide a choice to deal with the vast genetic diversity of human samples. However, resources supporting population-specific references are insufficient, and it is burdensome to perform analysis using these reference genomes. Here, we constructed a set of resources to support NGS analysis using the Japanese reference genome sequence, JG. We created resources for variant calling, gene and repeat element annotations, variant-effect prediction, read mappability, and RNA-seq analysis. We also provide a resource for reference coordinate conversion for further annotation enrichment. We then provide a variant calling protocol using JG-based resources. Our resources provide a guide to prepare sufficient resources for the use of population-specific reference genomes and can facilitate the migration of reference genomes.

bioinformatics↗

bioRxiv2023

Multi-omics profiling reveals phenotypic and functional heterogeneity of neutrophils in COVID-19

BackgroundAccumulating evidence has revealed unexpected phenotypic heterogeneity and diverse functions of neutrophils in several diseases. Coronavirus disease (COVID-19) can alter the leukocyte phenotype based on disease severity, including neutrophil activation in severe cases. However, the plasticity of neutrophil phenotypes and their relative impact on COVID-19 pathogenesis has not been well addressed. This study aimed to identify and validate the heterogeneity of neutrophils in COVID-19 and evaluate the phenotypic alterations for each subpopulation. MethodsWe analyzed public single-cell RNA-seq, bulk RNA-seq, and human plasma proteome data from healthy donors and patients with COVID-19 to investigate neutrophil subpopulations and their response to disease pathogenesis. ResultsWe identified eight neutrophil subtypes, namely C1-C8, and found that they exhibited distinct features, including activation signatures and multiple enriched pathways. The neutrophil subtype C4 (DEFA1/1B/3+) associated with severe and fatal disease. Bulk RNA-seq and proteome dataset analyses using a cellular deconvolution approach validated the relative abundances of neutrophil subtypes and the expansion of C4 (DEFA1/1B/3+) in severe COVID-19 patients. Cell- cell communication analysis revealed representative ligand-receptor interactions among the identified neutrophil subtypes. Notably, the C4 (DEFA1/1B/3+) fraction showed transmembrane receptor expression of CD45 and CAP1 as well as the secretion of pro-platelet basic protein (PPBP). We further demonstrated the clinical potential of PPBP as a novel diagnostic biomarker for severe COVID-19. ConclusionOur work has great value in terms of both clinical and public health as it furthers our understanding of the phenotypic and functional heterogeneity of neutrophils and other cell populations in multiple diseases.

immunology↗

bioRxiv2023

Elevator motion of chloride ion transporter SLC26A9 induced by STAS domain

The anion exchanger SLC26A9, consisting of the transmembrane (TM) domain and the cytoplasmic STAS domain, plays an essential role in regulating chloride transport across cell membranes. Recent studies have indicated that C-terminal helices block the entrance of the putative ion transport pathway. However, the precise functions of the STAS domain and C-terminal helix, as well as the underlying molecular mechanisms governing the transport process, remain poorly understood. In this study, we performed molecular dynamics simulations of three distinct models of human SLC26A9: full-length (FL), STAS domain removal ({Delta}STAS), and C-terminus removal ({Delta}C), to investigate their conformational dynamics and ion binding properties. Stable binding of ions to the binding sites was exclusively observed in the {Delta}C model in these simulations. Comparing the FL and {Delta}C simulations, the {Delta}C model displayed enhanced motion of the STAS domain. Furthermore, comparing the {Delta}STAS and {Delta}C simulations, the {Delta}STAS simulation failed to exhibit stable ion bindings to the sites despite the absence of the C-terminus blocking the ion transmission pathway in both systems. These results suggest that the removal of the C-terminus not only unblocks the access of ions to the permeation pathway but also triggers STAS domain motion, gating the TM domain to promote ions entry into their binding site. Further analysis revealed that the asymmetric motion of STAS domain leads to the expansion of the ion permeation pathway within the TM domain, resulting in the stiffening of the flexible TM12 helix near the ion binding site. This structural change in the TM12 helix stabilizes chloride ion binding, which is essential for SLC26A9 elevator motion. Overall, our study provides new insights into the molecular mechanisms of SLC26A9 transport and may pave the way for the development of novel treatments for diseases associated with dysregulated ion transport. SIGNIFICANCEWe explored the mechanism by which the human protein SLC26A9 transports chloride in the cell. SLC26A9 is a potential therapeutic target for patients with cystic fibrosis, as by targeting drugs to it, it may be possible to restore chloride ion transport in epithelial cells. To design therapeutic drugs, it is essential to understand how the protein works. Our findings support an elevator-type mechanism, in which chloride ions bind to SLC26A9 inside the cell and are then released by the protein to the extracellular environment. We find that the STAS domain of SLC26A9 has critical roles in binding chloride and induces conformational changes in the transmembrane domain that facilitate chloride transport.

biophysics↗

bioRxiv2023

Downregulation of CYP7B1 caused by lipotoxicity associates with the progression of non-alcoholic steatohepatitis

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide, with an incidence of >25% of the adult population. NAFLD ranges from benign simple steatosis to non-alcoholic steatohepatitis (NASH). However, its transition mechanisms underlying the pathogenesis remain to be clarified. The expression of Cyp7b1 gene is downregulated in the liver of leptin-deficient mice and methionine and choline-deficient diet-fed mice based on previous microarray data. Thus, in this study, we investigated the effect of CYP7B1 restoration on the progression of NASH in mice fed MCD diet and its association with oxidative and lipid stresses. Our results suggest that restoration of CYP7B1 expression attenuates hepatitis and fibrosis and that lipid and oxidative stresses observed in the early stage of NASH suppresses Cyp7b1 transcription in hepatocytes.

biochemistry↗

bioRxiv2022

Identification of variation in Fibromelanosis region on chromosome 20 for determining the purity of Indonesian Cemani chicken

Ayam Cemani is a local Indonesian chicken with heavy pigmentation in plumage colour, skin, eyes, and inner body organs. This trait with dermal hyperpigmentation is identical to Fibromelanosis (Fm) mutation in a Silkie chicken. The causal mutation of the Fm trait is due to an inverted duplication and junction of two genomic regions involving the Endothelin3 (EDN3) gene on chromosome 20. There are two duplication boundaries; one is specific to the Fm allele, the other is common for both Fm and fm+ allele. Determining birds that are homozygous or heterozygous at this locus is useful for unifying the Fm trait of Cemani populations. This study develops a method for determining the presence or absence of Fm mutation by PCR amplification using the inverted sequences specific to the Fm allele. Further, it develops the restriction fragment length polymorphism (RFLP) method in regions common to the Fm and wild-type fm+ allele. We aim to establish a simple method for detecting homozygous (Fm/Fm) and heterozygous (Fm/fm+) individuals with Fm mutation and to clarify the degree of fixation of the Fm trait in the Ayam Cemani populations and the association between the phenotype and genotype. The result showed that mostly, the phenotype for Cemani with Fm/ fm+ genotype is reddish black in their comb; meanwhile, the Cemani with (Fm/Fm) genotype showed heavy black pigmentation. Our study concluded that using the PCR-RFLP method. We can discriminate between Fm homozygous and heterozygous birds in the Cemani population. Thus, this briefly genotyping method effectively maintains and protects the pure line of Cemani chicken.

genetics↗

bioRxiv2022

Elasticity control of entangled chromosomes: crosstalk between condensin complexes and nucleosomes

Condensin-mediated loop extrusion is now considered as the main driving force of mitotic chromosome assembly. Recent experiments have shown, however, that a class of mutant condensin complexes deficient in loop extrusion can assemble chromosome-like structures in Xenopus egg extracts, although these structures are somewhat different from those assembled by wild-type condensin complexes. In the absence of topoisomerase II (topo II), the mutant condensin complexes produce an unusual round-shaped structure termed a bean, which consists of a DNA-dense central core surrounded by a DNA-sparse halo. The mutant condensin complexes accumulate in the core whereas histones are more concentrated in the halo than in the core. We consider that this peculiar structure serves as a model system to study how DNA entanglements, nucleosomes, and condensin functionally crosstalk with each other. To gain insight into how the bean structure is formed, here we construct a theoretical model. Our theory predicts that the core is formed by attractive interactions between mutant condensin complexes whereas the halo is stabilized by the energy reduction through the selective accumulation of nucleosomes. The formation of the halo increases the elastic free energy due to the DNA entanglement in the core, but the latter free energy is compensated by condensin complexes that suppress the assembly of nucleosomes.

biophysics↗

bioRxiv2022

PNPO-PLP Axis Senses Prolonged Hypoxia by Regulating Lysosomal Activity

Oxygen is critical for all metazoan organisms on the earth and impacts various biological processes in physiological and pathological conditions. While oxygen sensing systems inducing acute hypoxic response, including HIF pathway, have been identified, those operating in prolonged hypoxia remain to be elucidated. Here, we show that pyridoxine 5-phosphate oxidase (PNPO) that catalyzes bioactivation of vitamin B6 serves as an oxygen sensor and regulates lysosomal activity in macrophages. Decline of PNPO activity under prolonged hypoxia reduced an active form of vitamin B6, pyridoxal 5-phosphate (PLP), and inhibited lysosomal activity, leading to the augmentation of inflammatory response of macrophages. The PNPO-PLP axis creates a distinct layer of oxygen sensing, which gradually turns down and up the PLP-dependent metabolism according to prolonged changes in oxygen availability.

biochemistry↗

bioRxiv2022

Cell cycle-specific loading of condensin I is regulated by the N-terminal tail of its kleisin subunit

Condensin I is a pentameric protein complex that plays an essential role in mitotic chromosome assembly in eukaryotic cells. Although it has been shown that condensin I loading is mitosis-specific, it remains poorly understood how the robust cell cycle regulation of condensin I is achieved. Here we set up a panel of in vitro assays to demonstrate that cell cycle-specific loading of condensin I is regulated by the N-terminal tail (N-tail) of its kleisin subunit CAP-H. Deletion of the N-tail accelerates condensin I loading and chromosome assembly in Xenopus egg mitotic extracts. Phosphorylation-deficient and phosphorylation-mimetic mutations in the CAP-H N-tail decelerate and accelerate condensin I loading, respectively. Remarkably, deletion of the N-tail enables condensin I to assemble mitotic chromosome-like structures even in interphase extracts. Together with other extract-free functional assays in vitro, our results uncover one of the multilayered mechanisms that ensure cell cycle-specific loading of condensin I onto chromosomes.

cell biology↗

bioRxiv2022

Molecular dissection of condensin II-mediated chromosome assembly using in vitro assays

In vertebrates, condensin I and condensin II cooperate to assemble rod-shaped chromosomes during mitosis. Despite recent progress in our understanding of the mechanism of action of condensin I, how condensin II might work has remained largely unexplored. By introducing recombinant condensin II complexes into Xenopus egg extracts, we dissect the roles of its individual subunits in chromosome assembly. We show that two HEAT subunits, CAP-D3 and CAP-G2, play positive and negative roles, respectively, in condensin II-mediated chromosome assembly. The SMC ATPase and the basic amino acid clusters of the kleisin subunit are essential for this process. Deletion of the C-terminal tail of CAP-D3 increases the ability of condensin II to assemble chromosomes and further exposes a hidden function of CAP-G2 in the lateral compaction of chromosomes. Taken together, our results uncover a multilayered regulatory mechanism unique to condensin II, and provide profound implications in the evolution of condensin II.

cell biology↗