Biology subjects

Takahashi, M.

Publications and source records attributed to Takahashi, M..

At least 55 records · Page 3Linked to original sources

bioRxiv2024

Host-encoded DNA methyltransferases modify the epigenome and host tropism of invading phages

Restriction modification (RM) systems are ubiquitous bacterial defense systems; however, some phages evade RM system and adapt to their bacterial hosts. In such cases, phages are thought to stochastically acquire DNA methylation from host-encoded DNA methyltransferases (MTases), facilitating host adaptation. However, no studies have directly compared the methylomes of host bacteria and their infecting phages. Here, we demonstrate the epigenetic landscape of adapted phages with diverse infection histories, focusing on the broad host-range phage KHP30T as its adapts to three Helicobacter pylori strains. Using a multistage infection system, we observed that the adapted phages displayed significantly high titers against the last infected H. pylori strain, suggesting an attendant change in host tropism. Single-molecule real-time sequencing revealed that methylated motifs were predominantly shared between the adapted phages and their most recent host. Our findings enhance our understanding of epigenetic phage-host interactions, which have significant implications for microbial ecology.

microbiology↗

bioRxiv2024

Kaempferol exerts anti-inflammatory effects by accelerating Treg development via AhR-mediated and PU.1/IRF4-dependent transactivation of the Aldh1a2/RALDH2 gene in dendritic cells

Retinaldehyde dehydrogenase 2 encoded in the Aldh1a2 gene (RALDH2) is expressed in intestinal dendritic cells (DCs) in mesenteric lymph nodes (MLNs) and plays a crucial role in Treg development by producing retinoic acid. In the present study, we screened food ingredients and identified kaempferol, a flavonoid, as the compound that most effectively up-regulated RALDH2 expression in DCs. The development of Foxp3+ cells from OT-II-derived naive CD4+ T cells was enhanced by a co-culture with OVA peptide-pulsed bone marrow-derived DCs (BMDCs) in the presence of kaempferol or by a pre-treatment of BMDCs with kaempferol. The involvement of the aryl hydrocarbon receptor (Ahr) in the effects of kaempferol on Aldh1a2 expression was demonstrated by in vitro experiments using siRNA and various agonistic or antagonistic compounds of AhR. AhR suppressed Aldh1a2 gene expression and kaempferol inhibited the AhR activity by functioning as an antagonist of AhR. The kaempferol treatment also increased the protein levels of PU.1 and IRF4, which are essential for transcription of the Aldh1a2 gene in DCs, and accelerated the subsequent recruitment of PU.1 to the Aldh1a2 gene. The kaempferol-induced increase in PU.1 protein levels occurred in an AhR-dependent and transcription-independent manner, whereas the kaempferol treatment transactivated the Irf4 gene in DCs. The frequency of DCs exhibiting RALDH2 activity in the MLNs of mice was increased by the intraperitoneally (i.p.) administration of an AhR antagonist or by the oral administration of astragalin, a 3-O-glucoside of kaempferol. We also observed an increase in Tregs in the Peyers patches of C57BL/6 mice i.p. administered kaempferol-treated BMDCs. We utilized an OVA-induced food allergy model of Balb/c mice to examine the effects of kaempferol in vivo and confirmed that the rapid decrease in body temperature and allergic diarrhea observed just after the OVA challenge were significantly suppressed in mice administered kaempferol.

immunology↗

bioRxiv2024

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

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

immunology↗

bioRxiv2024

MS-DIAL 5 multimodal mass spectrometry data mining unveils lipidome complexities

Lipidomics and metabolomics communities comprise various informatics tools; however, software programs that can handle multimodal mass spectrometry (MS) data with structural annotations guided by the Lipidomics Standards Initiative are limited. Here, we provide MS-DIAL 5 to facilitate the in-depth structural elucidation of lipids through electron-activated dissociation (EAD)-based tandem MS, as well as determine their molecular localization through MS imaging (MSI) data using a species/tissue-specific lipidome database containing the predicted collision-cross section (CCS) values. With the optimized EAD settings using 14 eV kinetic energy conditions, the program correctly delineated the lipid structures based on EAD-MS/MS data from 96.4% of authentic standards. Our workflow was showcased by annotating the sn- and double-bond positions of eye-specific phosphatidylcholine molecules containing very-long-chain polyunsaturated fatty acids (VLC-PUFAs), characterized as PC n-3-VLC-PUFA/FA. Using MSI data from the eye and HeLa cells supplemented with n-3-VLC-PUFA, we identified glycerol 3-phosphate (G3P) acyltransferase (GPAT) as an enzyme candidate responsible for incorporating n-3 VLC-PUFAs into the sn-1 position of phospholipids in mammalian cells, which was confirmed using recombinant proteins in a cell-free system. Therefore, the MS-DIAL 5 environment, combined with optimized MS data acquisition methods, facilitates a better understanding of lipid structures and their localization, offering novel insights into lipid biology.

bioinformatics↗

bioRxiv2023

Trans-omic analysis reveals opposite metabolic dysregulation between feeding and fasting in liver associated with obesity

Dysregulation of liver metabolism associated with obesity during feeding and fasting leads to the breakdown of metabolic homeostasis. However, the underlying mechanism remains unknown. Here, we measured multi-omics data in the liver of wild-type and leptin-deficient obese (ob/ob) mice at ad libitum feeding, and constructed a differential regulatory trans-omic network of metabolic reactions. We compared the trans-omic network at feeding with that at 16 h-fasting constructed in our previous study. Intermediate metabolites in glycolytic and nucleotide metabolism decreased in ob/ob mice at feeding but increased at fasting. Allosteric regulation reversely shifted between feeding and fasting, generally showing activation at feeding while inhibition at fasting in ob/ob mice. Transcriptional regulation was similar between feeding and fasting, generally showing inhibiting transcription factor regulations, activating enzyme protein regulations in ob/ob mice. The opposite metabolic dysregulation between feeding and fasting characterizes breakdown of metabolic homeostasis associated with obesity.

systems biology↗

bioRxiv2023

Efficient workflow for validating homology-independent targeted integration-mediated gene insertion in rod photoreceptor cells to treat dominant-negative mutations causing retinitis pigmentosa

Among the genome-editing methods for repairing disease-causing mutations resulting in dominant inhibition, homology-independent targeted integration (HITI)-mediated gene insertion of the normal form of the causative gene is useful because it allows the development of mutation-agnostic therapeutic products. For the rapid optimization and validation of highly effective HITI-treatment gene constructs against dominant-negative inheritance of inherited retinal dystrophy, we improved the gene constructs available in both plasmid and adeno-associated virus (AAV) vectors, and established a workflow that uses in vivo electroporation to verify the in vivo efficacy. By targeting the mouse Rhodopsin gene, we derived a construct in which HITI-mediated gene insertion occurs in 80%-90% of transduced mouse rod photoreceptor cells. This construct suppressed degeneration and induced visual restoration in the mutant mice. The HITI-treatment constructs for the rhodopsin gene were shown to be effective in AAV vectors, and this construction is available for the mouse Peripherin 2 gene. These findings suggest that the workflow reported here may be useful for the generation of HITI-treatment constructs for various target genes and for the development of gene therapy products.

molecular biology↗

bioRxiv2023

DrugMap: A quantitative pan-cancer analysis of cysteine ligandability

Cysteine-focused chemical proteomic platforms have accelerated the clinical development of covalent inhibitors of a wide-range of targets in cancer. However, how different oncogenic contexts influence cysteine targeting remains unknown. To address this question, we have developed DrugMap, an atlas of cysteine ligandability compiled across 416 cancer cell lines. We unexpectedly find that cysteine ligandability varies across cancer cell lines, and we attribute this to differences in cellular redox states, protein conformational changes, and genetic mutations. Leveraging these findings, we identify actionable cysteines in NF{kappa}B1 and SOX10 and develop corresponding covalent ligands that block the activity of these transcription factors. We demonstrate that the NF{kappa}B1 probe blocks DNA binding, whereas the SOX10 ligand increases SOX10-SOX10 interactions and disrupts melanoma transcriptional signaling. Our findings reveal heterogeneity in cysteine ligandability across cancers, pinpoint cell-intrinsic features driving cysteine targeting, and illustrate the use of covalent probes to disrupt oncogenic transcription factor activity.

systems biology↗

bioRxiv2023

Using data-dependent and independent hybrid acquisitions for fast liquid chromatography-based untargeted lipidomics

Untargeted lipidomics using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has become an essential technique for large cohort studies. When a fast LC gradient of less than 10 min is used for the rapid screening of lipids, the annotation rate decreases because of the lower coverage of the MS/MS spectra caused by the narrow peak width. We propose a systematic procedure to achieve a high annotation rate in fast LC-based untargeted lipidomics by integrating data-dependent acquisition (DDA), and sequential window acquisition of all theoretical mass spectra data-independent acquisition (SWATH-DIA) techniques with the updated MS-DIAL program. Our strategy uses variable SWATH-DIA methods for quality control (QC) samples, which are a mixture of biological samples analyzed multiple times to correct MS signal drifts. In contrast, biological samples are analyzed using DDA to facilitate the structural elucidation of lipids using the pure spectrum to the maximum extent. We demonstrate our workflow using an 8.6 min LC gradient, where QCs are analyzed using five different SWATH-DIA methods. The results indicated that using both DDA and SWATH-DIA achieves 2.0-fold annotation coverage from publicly available benchmark data obtained by a fast LC-DDA-MS technique and offers 94.5% lipid coverage compared with the benchmark dataset from a 25 min LC gradient. Our study demonstrated that harmonized improvements in the analytical conditions and informatics tools provide a comprehensive lipidome in fast LC-based untargeted lipidomics, not only for large-scale studies but also for small-scale experiments, contributing to both clinical applications and basic biology.

bioinformatics↗

bioRxiv2023

Phylogenomics reveals the demography of reverse colonization in a rail species

AimIslands have recently been recognized as potential sources of biodiversity, challenging the traditional view that their small population sizes and low genetic diversity limit such roles. This raises the question of how insular genetic variation becomes incorporated into continental populations, contrary to expectations of unidirectional colonization. Here, we investigate whether and how island-derived genetic variation has influenced a continental population through population establishment and gene flow in a bird species where frequent trans-ocean dispersal is expected. LocationContinental East Asia (Russian Far East), Japanese Archipelago TaxonSwinhoes Rail (Coturnicops exquisitus) Materials and MethodsWe apply integrative phylogenomics to reconstruct the spatiotemporal history of the species. Colonization sequences and gene flow are inferred by comparing four different phylogenetic reconstruction methods, using mitochondrial sequences obtained by Sanger sequencing and genome-wide data obtained by genotyping by sequencing (MIG-seq). We assess a history of colonization and gene flow based on summary statistics, demographic trajectory inference by Stairway Plot2, demographic modeling by fastsimcoal2, and species distribution modeling. ResultsAnalyses collectively supported asymmetric gene flow from the island to the continental population, following divergence around the Middle Pleistocene. Post-divergence, the island maintained a large and stable population, while the continental population underwent a severe bottleneck, suggesting a significant evolutionary role of the island for the continental population. Additionally, evidence of recent re-establishment of the island by continental individuals indicates dynamic exchange and persistence within a continent-island metapopulation. Main conclusionsThe maintenance of insular genetic variation within a dynamic continent-island metapopulation may have enabled the island to act as a genetic and demographic reservoir for the continental population. Thus, continent-island metapopulation dynamics may be a key evolutionary pathway through which island populations contribute to continental genetic diversity.

evolutionary biology↗

bioRxiv2023

In vitro pigmentation of human iPSC-derived retinal pigment epithelium cells does not indicate their quality for cell transplantation

Retinal pigment epithelium (RPE) cells show heterogeneous level of pigmentation when cultured in vitro. To know whether their color in appearance is correlated with the function of the RPE, we analyzed the color intensities of human-induced pluripotent stem cell-derived RPE cells (iPSC-RPE) together with the gene expression profile at the single cell level. For this purpose, we utilized our recent invention, Automated Live imaging and cell Picking System (ALPS), which enabled photographing each cell before RNA-sequencing analysis to profile the gene expression of each cell. While our iPSC-RPE were categorized in 4 clusters by gene expression, the color intensity of iPSC-RPE did not project any specific gene expression profiles. We reasoned this by less correlation between the actual color and the gene expressions that directly define the level of pigmentation, from which we hypothesized the color of RPE cells may be a temporal condition not strongly indicating the functional characteristics of the RPE.

cell biology↗

bioRxiv2023

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

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

molecular biology↗

bioRxiv2023

Low albumin status accompanies multi-layered immunosuppressive phenotypes in metastatic breast cancer patients

Low albumin status is prevalent in advanced cancer patients, but the pathophysiology associated with this anomaly remains largely unexplored. To address this, we aim to search correlations of albumin levels with the transcriptome against peripheral blood mononuclear cells and the plasma metabolome within the same patients having metastatic breast cancers. We confirm that metastatic breast cancer patients exhibit low albumin levels in varying degrees without prominent systemic inflammation. Our data demonstrate that low albumin levels correlate with transcriptome signatures indicative of "neutrophil activation and T-cell down-regulation," an immunosuppressive phenotype. We also find that immunoregulatory metabolites, such as arginine, are reduced in plasma in an albumin-correlated manner, further corroborating systemic immunosuppression. These results are verified using a mouse model of breast cancer. We conclude that low albumin status in metastatic breast cancer patients accompanies immunosuppressive phenotypes, which is likely unfavorable for anti-cancer immunotherapy and thus can be a cause of unsuccessful treatment outcomes.

cancer biology↗

bioRxiv2023

NFκB nuclear dynamics orchestrate inflammatory aging

Upregulation of nuclear factor {kappa}B (NF{kappa}B) signaling is a hallmark of aging and major cause of age-related chronic inflammation; however, its physiological functions and mechanisms remain unclear. By combining mathematical modeling and experiments, we show that dysfunction of negative feedback regulators of NF{kappa}B, I{kappa}B and A20, alters the NF{kappa}B nuclear dynamics from oscillatory to sustained, promoting cellular senescence by remodeling epigenetic regulation and metabolic landscape. Sustained NF{kappa}B activity by I{kappa}B downregulation enhanced inflammation- and senescence-associated gene expression through increased NF{kappa}B-DNA binding and slowed the cell cycle by upregulating purine catabolism via mTORC2/AKT pathways. Notably, I{kappa}B knockdown combined with A20 overexpression resulted in lower NF{kappa}B amplitude, cytokine expression, and SA-{beta}-gal activity than I{kappa}B knockdown alone. I{kappa}B downregulation is correlated with hypoxanthine phosphoribosyltransferase 1 (HPRT1) expression in the purine salvage pathway in aged mouse hearts. Our study suggests that nuclear NF{kappa}B homeostasis is critical for balancing purine metabolism associated with chronic inflammation and tissue aging.

systems biology↗

bioRxiv2023

DIPA-CRISPR gene editing in the yellow fever mosquito Aedes aegypti (Diptera: Culicidae)

Current methods for gene editing in insects rely on embryonic microinjection, which can be challenging for non-specialist laboratories. Recently, an alternative method known as "direct parental" CRISPR (DIPA-CRISPR) was developed. This method involves injecting commercial Cas9 protein and single-guide RNA into adult females, which can efficiently introduce mutations into developing oocytes. However, its versatility has not been fully explored, particularly in insects that have the most derived, polytrophic meroistic ovaries. In this study, we successfully applied DIPA-CRISPR to the yellow fever mosquito Aedes aegypti, which has polytrophic meroistic ovaries. Following adult injection of Cas9 ribonucleoproteins (Cas9 RNPs) targeting the kynurenine 3-monooxygenase gene, we recovered gene-edited G0 individuals. Injection at 24 h after blood-feeding resulted in the highest gene editing efficiency (3.5%), confirming that a key parameter of DIPA-CRISPR is the stage in which the adult females are injected. Together with our previous study, we demonstrated that DIPA-CRISPR is applicable to all three types of insect ovaries (i.e., panoistic, telotrophic, and polytrophic), which indicates that DIPA-CRISPR is a generalizable approach for insect gene editing.

genetics↗

bioRxiv2023

The genomics and evolution of inter-sexual mimicry and female-limited polymorphisms in damselflies

Sex-limited morphs can provide profound insights into the evolution and genomic architecture of complex phenotypes. Inter-sexual mimicry is one particular type of sex-limited polymorphism in which a novel morph resembles the opposite sex. While inter-sexual mimics are known in both sexes and a diverse range of animals, their evolutionary origin is poorly understood. Here, we investigated the genomic basis of female-limited morphs and male mimicry in the Common Bluetail damselfly. Differential gene expression between morphs has been documented in damselflies, but no causal locus has been previously identified. We found that male-mimicry originated in an ancestrally sexually-dimorphic lineage in association with multiple structural changes, probably driven by transposable element activity. These changes resulted in [~]900 kb of novel genomic content that is partly shared by male mimics in a close relative, indicating that male mimicry is a trans-species polymorphism. More recently, a third morph originated following the translocation of part of the male-mimicry sequence into a genomic position [~]3.5 mb apart. We provide evidence of balancing selection maintaining male-mimicry, in line with previous field population studies. Our results underscore how structural variants affecting a handful of potentially regulatory genes and morph-specific genes, can give rise to novel and complex phenotypic polymorphisms.

evolutionary biology↗

bioRxiv2023

Fate-mapping lymphocyte clones and their progenies from induced antigen-signals identifies temporospatial behaviours of T cells mediating tolerance

Tissue homeostasis is maintained by the behaviours of lymphocyte clones responding to antigenic triggers in the face of pathogen, environmental, and developmental challenges. Current methodologies for tracking the behaviour of specific lymphocytes identify clones of a defined antigen-receptor--antigen binding affinity. However, lymphocytes can receive antigenic signals from undefined or endogenous antigens, and the strength of each signal, even for the same lymphocyte, varies with accessory signalling, across tissues and across time. We present a novel fate-mapping mouse, that, by tracking lymphocyte clones and their progenies from induced antigen signals, overcomes these hurdles and provides novel insights into the maintenance of tissue homeostasis. We demonstrate the systems use by investigating the maintenance of localised T cell tolerance in tumour immunity. In a murine tumour model, our system reveals how Tregs differentiate to a reversible, tolerance inducing state within the tumour, and recirculate, while CD8+ T cells failing to recirculate, differentiate to an increasingly exhausted, tolerant state in the tumour. These contrasting T cell behaviours provide means by which immunity can tolerate a particular anatomical niche while maintaining systemic clonal protection. Our system can thus explore lymphocyte behaviours that cannot be tracked by previous methods and will therefore provide novel insights into the fundamental mechanisms underlying immunitys role in tissue homeostasis.

immunology↗

bioRxiv2023

Identification of chemotherapy targets reveals a nucleus-to-mitochondria ROS sensing pathway

Multiple chemotherapies are proposed to cause cell death in part by increasing the steady-state levels of cellular reactive oxygen species (ROS). However, for most of these drugs exactly how the resultant ROS function and are sensed is poorly understood. In particular, its unclear which proteins the ROS modify and their roles in chemotherapy sensitivity/resistance. To answer these questions, we examined 11 chemotherapies with an integrated proteogenomic approach identifying many unique targets for these drugs but also shared ones including ribosomal components, suggesting one mechanism by which chemotherapies regulate translation. We focus on CHK1 which we find is a nuclear H2O2 sensor that promotes an anti-ROS cellular program. CHK1 acts by phosphorylating the mitochondrial-DNA binding protein SSBP1, preventing its mitochondrial localization, which in turn decreases nuclear H2O2. Our results reveal a druggable nucleus-to-mitochondria ROS sensing pathway required to resolve nuclear H2O2 accumulation, which mediates resistance to platinum-based chemotherapies in ovarian cancers.

molecular biology↗

bioRxiv2023

Kaempferol suppresses the activation of mast cells by modulating the expression of FcϵRI and SHIP1

In the present study, we evaluated the effects of kaempferol on bone marrow-derived mast cells (BMMCs). Kaempferol treatment significantly and dose-dependently inhibited IgE-induced degranulation, and cytokine production of BMMCs under the condition that cell viability was maintained. Kaempferol downregulated the surface expression levels of Fc{varepsilon}RI on BMMCs, but the mRNA levels of Fc{varepsilon}RI, {beta}, and {gamma}-chains were not changed by kaempferol treatment. Furthermore, the kaempferol-mediated downregulation of surface Fc{varepsilon}RI on BMMCs was still observed in the presence of a protein-synthesis inhibitor. These results suggest that kaempferol reduced the surface Fc{varepsilon}RI on BMMCs, independently of transcription and translation. We also found that kaempferol inhibited both LPS- and IL-33-induced IL-6 production from BMMCs, without affecting the expression levels of their receptors, TLR4 and ST2. Although kaempferol treatment increased the protein amount of NRF2, a master transcription factor of antioxidant stress, in BMMCs, inhibition of NRF2 did not alter the suppressive effect of kaempferol on degranulation. Finally, we observed that kaempferol treatment increased the levels of mRNA and protein of a phosphatase SHIP1 in BMMCs. These results indicate that kaempferol inhibited the IgE-induced activation of BMMCs by downregulating Fc{varepsilon}RI and upregulating SHIP1, and the SHIP1 increase is involved in the suppression of various signaling-mediated stimulations of BMMCs, such as those associated with TLR4 and ST2.

immunology↗