Biology subjects

Kobayashi, H.

Publications and source records attributed to Kobayashi, H..

At least 55 records · Page 3Linked to original sources

bioRxiv2023

Context-Dependent Modification of PFKFB3 in Hematopoietic Stem Cells Promotes Anaerobic Glycolysis and Ensures Stress Hematopoiesis

Metabolic pathways are plastic and rapidly change in response to stress or perturbation. Current metabolic profiling techniques require lysis of many cells, complicating the tracking of metabolic changes over time after stress in rare cells such as hematopoietic stem cells (HSCs). Here, we aimed to identify the key metabolic enzymes that define differences in glycolytic metabolism between steady-state and stress conditions in HSCs and elucidate their regulatory mechanisms. Through quantitative 13C metabolic flux analysis of glucose metabolism using high-sensitivity glucose tracing and mathematical modeling, we found that HSCs activate the glycolytic rate-limiting enzyme phosphofructokinase (PFK) during proliferation and oxidative phosphorylation (OXPHOS) inhibition. Real-time measurement of adenosine triphosphate (ATP) levels in single HSCs demonstrated that proliferative stress or OXPHOS inhibition led to accelerated glycolysis via increased activity of PFKFB3, the enzyme regulating an allosteric PFK activator, within seconds to meet ATP requirements. Furthermore, varying stresses differentially activated PFKFB3 via PRMT1-dependent methylation during proliferative stress and via AMPK-dependent phosphorylation during OXPHOS inhibition. Overexpression of Pfkfb3 induced HSC proliferation and promoted differentiated cell production, whereas inhibition or loss of Pfkfb3 suppressed them. This study reveals the flexible and multilayered regulation of HSC glycolytic metabolism to sustain hematopoiesis under stress and provides techniques to better understand the physiological metabolism of rare hematopoietic cells. Key PointsO_LICombined isotope tracing, mathematical modeling, and single cell ATP analysis enable high-resolution evaluation of blood cell metabolism. C_LIO_LIUnder stress, HSCs quickly accelerate glycolysis to meet ATP demands and maintain hematopoiesis via context-dependent PFKFB3 activation. C_LI

cell biology↗

bioRxiv2023

Zebrahub - Multimodal Zebrafish Developmental Atlas Reveals the State Transition Dynamics of Late Vertebrate Pluripotent Axial Progenitors

Elucidating the developmental processes of organisms requires a comprehensive understanding of cellular lineages in the spatial, temporal, and molecular domains. In this study, we introduce Zebrahub, a dynamic atlas of zebrafish embryonic development that integrates single-cell sequencing time course data with lineage reconstructions facilitated by light-sheet microscopy. This atlas offers high-resolution and in-depth molecular insights into zebrafish development, achieved through the sequencing of individual embryos across ten developmental stages, complemented by trajectory reconstructions. Zebrahub also incorporates an interactive tool to navigate the complex cellular flows and lineages derived from light-sheet microscopy data, enabling in silico fate mapping experiments. To demonstrate the versatility of our multi-modal resource, we utilize Zebrahub to provide fresh insights into the pluripotency of Neuro-Mesodermal Progenitors (NMPs). Our publicly accessible web-based platform, Zebrahub, is a foundational resource for studying developmental processes at both transcriptional and spatiotemporal levels, providing researchers with an integrated approach to exploring and analyzing the complexities of cellular lineages during zebrafish embryogenesis.

developmental biology↗

bioRxiv2023

A Prefrontal Neural Circuit for Maternal Behavioural Leaning in Mice

Maternal behaviors, which are crucial for the survival of mammalian infants, require the coordinated operation of multiple brain regions to process infant cues, make decisions, and execute motor plans. Although these processes likely demand higher cognitive functions, the prefrontal areas that regulate limbic parental programs remains poorly understood. Here, we show that the orbitofrontal cortex (OFC) excitatory projection neurons promote alloparental caregiving behaviors in female mice. By chronic microendoscopy, we observed robust yet adaptable representations of pup-directed anticipatory and motor-related activities within the OFC. Some of these plastic responses were significantly overlapped with those related to nonsocial reward signals. The inactivation of OFC output reduced the phasic activities of midbrain dopamine (DA) neurons specifically tied to pup retrieval and impaired the modulation of DA release to the ventral striatum during the acquisition of alloparental behaviors. These findings suggest that the OFC transiently boosts DA activity during the acquisition phase, thereby facilitating the manifestation of alloparental behaviors.

neuroscience↗

bioRxiv2022

Comprehensive Analysis of Peptide-Coding Genes Identifies an LRR-only Microprotein That Regulates Reproductive Organs in Marchantia polymorpha

1In the past two decades, many plant peptides have been found to play crucial roles in various biological events by mediating cell-to-cell communications. However, a large number of small open reading frames (sORFs) or short genes capable of encoding peptides remain uncharacterized. In this study, we examined several candidate genes for peptides conserved between two model plants: Arabidopsis thaliana and Marchantia polymorpha. We examined the expression pattern in M. polymorpha and subcellular localization using a transient assay with Nicotiana benthamiana. We found that one candidate, MpSGF10B, was expressed in meristems, gemma cups, and male reproductive organs called antheridiophores. MpSGF10B has an N-terminal signal peptide followed by two leucine-rich repeat (LRR) domains and was secreted to the extracellular region in N. benthamiana and M. polymorpha. Compared with the wild type, two independent knockout mutants had an increased number of antheridiophores, which emerged from meristems. It was revealed in gene ontology enrichment analysis that MpSGF10B was significantly co-expressed with genes related to cell cycle and development. These results suggest that MpSGF10B may regulate the reproductive development in M. polymorpha. Our research should shed light on the unknown role of LRR-only proteins in land plants.

plant biology↗

bioRxiv2022

Conservation and divergence of canonical and non-canonical imprinting in murids

BackgroundGenomic imprinting affects gene expression in a parent-of-origin manner and has a profound impact on complex traits including growth and behaviour. While the rat is widely used to model human pathophysiology, few imprinted genes have been identified in this murid. To systematically identify imprinted genes and genomic imprints in the rat, we used low input methods for genome-wide analyses of gene expression and DNA methylation to profile embryonic and extra-embryonic tissues at allele-specific resolution. ResultsWe identify 14 and 26 imprinted genes in these tissues, respectively, with 10 of these genes imprinted in both tissues. Comparative analyses with mouse revealed that orthologous imprinted gene expression and associated canonical DNA methylation imprints are conserved in the embryo proper of the Muridae family. However, only 3 paternally expressed imprinted genes are conserved in the extra-embryonic tissue of murids, all of which are associated with non-canonical H3K27me3 imprints. The discovery of 8 novel non-canonical imprinted genes unique to the rat is consistent with more rapid evolution of extra-embryonic imprinting. Meta-analysis of novel imprinted genes revealed multiple mechanisms by which species-specific imprinted expression may be established, including H3K27me3 deposition in the oocyte, the birth of ZFP57 binding motifs and the insertion of endogenous retroviral promoters. ConclusionsIn summary, we provide a comprehensive list of imprinted loci in the rat, reveal the extent of conservation of imprinted gene expression, and identify potential mechanisms responsible for the evolution of species-specific imprinting.

genomics↗

bioRxiv2022

Oral administration of S-217622, a SARS-CoV-2 main protease inhibitor, decreases viral load and accelerates recovery from clinical aspects of COVID-19

In parallel with vaccination, oral antiviral agents are highly anticipated to act as countermeasures for the treatment of the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Oral antiviral medication demands not only high antiviral activity but also target specificity, favorable oral bioavailability, and high metabolic stability. Although a large number of compounds have been identified as potential inhibitors of SARS-CoV-2 infection in vitro, few have proven to be effective in vivo. Here, we show that oral administration of S-217622, a novel inhibitor of SARS-CoV-2 main protease (Mpro, also known as 3C-like protease), decreases viral load and ameliorates the disease severity in SARS-CoV-2-infected hamsters. S-217622 inhibited viral proliferation at low nanomolar to sub-micromolar concentrations in cells. Oral administration of S-217622 demonstrated eminent pharmacokinetic properties and accelerated recovery from acute SARS-CoV-2 infection in hamster recipients. Moreover, S-217622 exerted antiviral activity against SARS-CoV-2 variants of concern (VOCs), including the highly pathogenic Delta variant and the recently emerged Omicron variant. Overall, our study provides evidence that S-217622, an antiviral agent that is under evaluation in a phase II/III clinical trial, possesses remarkable antiviral potency and efficacy against SARS-CoV-2 and is a prospective oral therapeutic option for COVID-19.

microbiology↗

bioRxiv2021

Repeated translocation of a supergene underlying rapid sex chromosome turnover in Takifugu fish

Recent studies have revealed a surprising diversity of sex chromosomes in vertebrates. However, the detailed mechanism of their turnover is still elusive. To understand this process, it is necessary to compare closely related species in terms of sex-determining genes and the chromosomes harboring them. Here, we explored the genus Takifugu, in which one strong candidate sex-determining gene, Amhr2, has been identified. To trace the processes involved in transitions in the sex determination system in this genus, we studied 12 species and found that while the Amhr2 locus likely determines sex in the majority of Takifugu species, three species have acquired sex-determining loci at different chromosomal locations. Nevertheless, the generation of genome assemblies for the three species revealed that they share a portion of the male-specific supergene that contains a candidate sex-determining gene, GsdfY, along with genes that potentially play a role in male fitness. The shared supergene span approximately 100 kb and are flanked by two duplicated regions characterized by CACTA transposable elements. These results suggest that the shared supergene has taken over the role of sex-determining locus from Amhr2 in lineages leading to the three species, and repeated translocations of the supergene underlie the turnover of sex chromosomes in these lineages. These findings highlight the underestimated role of a mobile supergene in the turnover of sex chromosomes in vertebrates. SignificanceAlthough turnover of sex chromosomes is very common in many vertebrate lineages, the transition process is still elusive. We studied the sex-determining region (SDR) of 12 congeneric fish species. We found that while nine species retained their ancestral SDR, three species had acquired derived SDRs. Although the derived SDRs resided in three different chromosomes, they harbored a shared supergene flanked by two putative transposable elements. The results highlight the underestimated role of a mobile supergene in turnover of sex chromosomes in vertebrates.

genomics↗

bioRxiv2021

Eliminating fibroblast activation protein-α expressing cells by photoimmunotheranostics

Photoimmunotherapy (PIT) using an antibody conjugated to a near infrared dye IR700 is achieving significant success in target-specific elimination of cells. Fibroblast activation protein alpha (FAP-) is an important target in cancer because of its expression by cancer associated fibroblasts (CAFs) as well as by some cancer cells. CAFs that express FAP- have protumorigenic and immune suppressive functions. Using immunohistochemistry of human breast cancer tissue microarrays, we identified an increase of FAP-+ CAFs in invasive breast cancer tissue compared to adjacent normal tissue. We found FAP- expression increased in fibroblasts co-cultured with cancer cells. In proof-of-principle studies, we engineered human FAP- overexpressing MDA-MB-231 and HT-1080 cancer cells and murine FAP- overexpressing NIH-3T3 fibroblasts to evaluate several anti-FAP- antibodies and selected AF3715 based on its high binding-affinity with both human and mouse FAP-. After conjugation of AF3715 with the phthalocyanine dye IR700, the resultant antibody conjugate, FAP--IR700, was evaluated in cells and tumors for its specificity and effectiveness in eliminating FAP- expressing cell populations with PIT. FAP--IR700-PIT resulted in effective FAP--specific cell killing in the engineered cancer cells and in two patient-derived CAFs in a dose-dependent manner. Following an intravenous injection, FAP--IR700 retention was three-fold higher than IgG-IR700 in FAP- overexpressing tumors, and two-fold higher compared to wild-type tumors. FAP--IR700-PIT resulted in significant growth inhibition of tumors derived from FAP- overexpressing human cancer cells. A reduction of endogenous FAP-+ murine CAFs was identified at 7 days after FAP--IR700-PIT. FAP--targeted NIR-PIT presents a promising strategy to eliminate FAP-+ CAFs.

cancer biology↗

bioRxiv2021

Malaria parasite evades mosquito immunity by glutaminyl cyclase mediated protein modification

Glutaminyl cyclase (QC) modifies N-terminal glutamine or glutamic acid residues of target proteins into cyclic pyroglutamic acid (pGlu). Here, we report the biochemical and functional analysis of Plasmodium QC. We show that Plasmodium sporozoites of QC-null mutants are recognized by the mosquito immune system and melanized when they reach the hemocoel. Sporozoite numbers in salivary glands are also reduced in mosquitoes infected with QC-null or QC catalytically-dead mutants. This phenotype can be rescued by genetic complementation or by disrupting mosquito hemocytes or melanization immune responses. Mutation of a single QC-target glutamine of the major sporozoite surface protein (CSP) also results in immune recognition of sporozoites. These findings reveal QC-mediated post-translational modification of surface proteins as a major mechanism of mosquito immune evasion by Plasmodium sporozoites.

microbiology↗

bioRxiv2021

Single-molecule imaging of microRNA-mediated gene silencing in cells

MicroRNAs (miRNAs) are small non-coding RNAs, which regulate the expression of thousands of genes; miRNAs silence gene expression from complementary mRNAs through translational repression and mRNA decay. For decades, the function of miRNAs has been studied primarily by ensemble methods, where a bulk collection of molecules is measured outside cells. Thus, the behavior of individual molecules during miRNA-mediated gene silencing, as well as their spatiotemporal regulation inside cells, remains mostly unknown. Here we report single-molecule methods to visualize each step of miRNA-mediated gene silencing in situ inside cells. Simultaneous visualization of single mRNAs, translation, and miRNA-binding revealed that miRNAs preferentially bind to translated mRNAs rather than untranslated mRNAs. Spatiotemporal analysis based on our methods uncovered that miRNAs bind to mRNAs immediately after nuclear export. Subsequently, miRNAs induced translational repression and mRNA decay within 30 and 60 min, respectively, after the binding to mRNAs. This methodology provides a framework for studying mRNA regulation at the single-molecule level with spatiotemporal information inside cells.

molecular biology↗

bioRxiv2021

ZAF -- An Open Source Fully Automated Feeder for Aquatic Facilities

In the past few decades, aquatic animals have become popular model organisms in biology, spurring a growing need for establishing aquatic facilities. Zebrafish are widely studied and relatively easy to culture using commercial systems. However, a challenging aspect of maintaining aquatic facilities is animal feeding, which is both time- and resource-consuming. We have developed an open-source fully automatic daily feeding system, Zebrafish Automatic Feeder (ZAF). ZAF is reliable, provides a standardized amount of food to every tank, is cost-efficient and easy to build. The advanced version, ZAF+, allows for the precise control of food distribution as a function of fish density per tank, and has a user-friendly interface. Both ZAF and ZAF+ are adaptable to any laboratory environment and facilitate the implementation of aquatic colonies. Here we provide all blueprints and instructions for building the mechanics, electronics, fluidics, as well as to setup the control software and its user-friendly graphical interface. Importantly, the design is modular and can be scaled to meet different user needs. Furthermore, our results show that ZAF and ZAF+ do not adversely affect zebrafish culture, enabling fully automatic feeding for any aquatic facility.

developmental biology↗

bioRxiv2021

OpenCell: proteome-scale endogenous tagging enables the cartography of human cellular organization

Elucidating the wiring diagram of the human cell is a central goal of the post-genomic era. We combined genome engineering, confocal live-cell imaging, mass spectrometry and data science to systematically map the localization and interactions of human proteins. Our approach provides a data-driven description of the molecular and spatial networks that organize the proteome. Unsupervised clustering of these networks delineates functional communities that facilitate biological discovery, and uncovers that RNA-binding proteins form a specific sub-group defined by unique interaction and localization properties. Furthermore, we discover that remarkably precise functional information can be derived from protein localization patterns, which often contain enough information to identify molecular interactions. Paired with a fully interactive website opencell.czbiohub.org, we provide a resource for the quantitative cartography of human cellular organization.

cell biology↗

bioRxiv2021

Self-Supervised Deep-Learning Encodes High-Resolution Features of Protein Subcellular Localization

Elucidating the diversity and complexity of protein localization is essential to fully understand cellular architecture. Here, we present cytoself, a deep-learning approach for fully self-supervised protein localization profiling and clustering. cytoself leverages a self-supervised training scheme that does not require pre-existing knowledge, categories, or annotations. Training cytoself on images of 1,311 endogenously labeled proteins from the OpenCell database reveals a highly resolved protein localization atlas that recapitulates major scales of cellular organization, from coarse classes such as nuclear, cytoplasmic and vesicular, to the subtle localization signatures of individual protein complexes. We quantitatively validate cytoselfs ability to cluster proteins into organelles and protein complex clusters using a clustering score, and show that cytoself attains higher scores than previous unsupervised or self-supervised approaches. Finally, to better understand the inner workings of our model, we dissect the emergent features from which our clustering is derived, interpret these features in the context of the fluorescence images, and analyze the performance contributions of the different components of our approach.

cell biology↗

bioRxiv2020

High-Resolution, Large Field-of-View, and Multi-View Single Objective Light-Sheet Microscopy

Recent developments in Oblique Plane Microscopy (OPM) have shown that it can achieve high spatio-temporal resolution. Here we describe a single objective light-sheet microscope based on oblique plane illumination that achieves: (i) large field of view and high-resolution imaging via a custom remote focusing objective; (ii) fast volumetric imaging by means of light-sheet stabilised stage scanning - a novel scanning modality that extends the imaging volume without compromising imaging speed nor quality; (iii) multi-view imaging by alternating the orientation of light-sheet illumination and detection to improve the image quality on large samples; (iv) simpler design and ergonomics by remote placement of coverslips to allow inverted imaging, enabling imaging across scales in a high-throughput format. Overall, we achieved a resolution of 450 nm laterally and 2 m axially and a field of view of 3000 m x 800 m x 300 m. We demonstrate the speed, field of view, resolution and versatility of our novel instrument by imaging various systems, including zebrafish whole brain activity, Drosophila egg chamber development, and zebrafish development - up to nine embryos simultaneously.

developmental biology↗

bioRxiv2020

The BMP antagonist Gremlin1 contributes to the development of cortical excitatory neurons, motor balance and fear responses

Bone morphogenetic protein (BMP) signaling is required for early forebrain development and cortical formation. How the endogenous modulators of BMP signaling regulate the structural and functional maturation of the developing brain remains unclear. Here we show that expression of the BMP antagonist, Grem1, marks a neuroprogenitor that gives rise to layer V and VI glutamatergic neurons in the embryonic mouse brain. Lineage tracing of Grem1-expressing cells in the embryonic brain was examined by administration of tamoxifen to pregnant Grem1creERT Rosa26LSLTdtomato mice at 13.5 days post coitum (dpc), followed by collection of embryos later in gestation. In addition, at 14.5 dpc, bulk mRNA seq analysis of differentially expressed transcripts between FACS sorted Grem1 positive and negative cells was performed. We also generated Emx1-cre mediated Grem1 conditional knockout mice (Emx1-Cre;Grem1flox/flox) in which the Grem1 gene was deleted specifically in the dorsal telencephalon. Grem1Emx1cKO animals had reduced cortical thickness, especially layers V and VI and impaired motor balance and fear sensitivity compared to littermate controls. This study has revealed new roles for Grem1 in the structural and functional maturation of the developing cortex. Summary statementThe BMP antagonist, Grem1, marks neuroprogenitors that give rise to deep layer glutamatergic neurons in the embryonic mouse brain. Grem1 conditional knockout mice display cortical and behavioural abnormalities.

developmental biology↗

bioRxiv2020

Selectivity Landscape of 100 Therapeutically Relevant GPCR Profiled by an Effector Translocation-Based BRET Platform

The recognition that individual GPCRs can activate multiple signaling pathways has raised the possibility of developing drugs selectively targeting therapeutically relevant ones. This requires tools to determine which G proteins and {beta}arrestins are activated by a given receptor. Here, we present a set of BRET sensors monitoring the activation of the 12 G protein subtypes based on the translocation of their effectors to the plasma membrane (EMTA). Unlike most of the existing detection systems, EMTA does not require modification of receptors or G proteins (except for Gs). EMTA was found to be suitable for the detection of constitutive activity, inverse agonism, biased signaling and polypharmacology. Profiling of 100 therapeutically relevant human GPCRs resulted in 1,500 pathway-specific concentration-response curves and revealed a great diversity of coupling profiles ranging from exquisite selectivity to broad promiscuity. Overall, this work describes unique resources for studying the complexities underlying GPCR signaling and pharmacology.

pharmacology and toxicology↗

bioRxiv2020

A flexible target-specific anti-infection therapeutic platform that can be applied to different microbial species

The emergence of new microbial pathogens, including drug-resistant strains, complicates treatment, thereby threatening global health. We demonstrated a photoimmuno-antimicrobial strategy (PIAS) that eliminated antibody-targets using a photo-activated anti-pathogen antibody generating mechanical stress that damaged the targets binding sites. PIAS is effective against many pathogens, including methicillin-resistant Staphylococcus aureus (MRSA), the fungal pathogen Candida albicans, and viral particles irrespective of their species or drug-resistance status. Animal experiments demonstrated that PIAS saved mice from fatal infections; microbiome and histochemical analyses indicated no apparent effect on normal host microflora and tissues in PIAS-treated mice. Resistance to PIAS was not observed during the eight years of this study. As a new type of anti-infection therapy, PIAS may contribute to advances in anti-infection strategies.

microbiology↗

bioRxiv2020

Maternal DNMT3A-dependent de novo methylation of the zygotic paternal genome inhibits gene expression in the early embryo

De novo DNA methylation (DNAme) during mammalian spermatogenesis yields a densely methylated genome, with the exception of CpG islands (CGIs), which are hypomethylated in sperm. Following fertilization, the paternal genome undergoes widespread DNAme loss before the first S-phase. Paradoxically, recent mass spectrometry analysis revealed that a low level of de novo DNAme occurs exclusively on the zygotic paternal genome. However, the loci involved and impact on genic transcription was not addressed. Here, we employ allele-specific analysis of wholegenome bisulphite sequencing (WGBS) data and show that a number of genomic regions, including several dozen CGI promoters, are de novo methylated on the paternal genome in 2-cell embryos. A subset of these promoters maintains DNAme through development to the blastocyst stage. Consistent with zygotic paternal DNAme acquisition (PDA), many of these loci are hypermethylated in androgenetic blastocysts but hypomethylated in parthenogenetic blastocysts. Strikingly, PDA is lost following maternal deletion of Dnmt3a. Furthermore, a subset of promoters showing PDA which are normally transcribed from the paternal allele in blastocysts show premature transcription at the 4-cell stage in maternal Dnmt3a knockout embryos. These observations uncover an unexpected role for maternal DNMT3A activity in postfertilization epigenetic reprogramming and transcriptional silencing of the paternal genome.

developmental biology↗