Biology subjects

Ishikawa, M.

Publications and source records attributed to Ishikawa, M..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Inferring extrinsic factor-dependent single-cell transcriptome dynamics using a deep generative model

1RNA velocity estimation helps elucidate temporal changes in the single-cell transcriptome. However, current methodologies for inferring single-cell transcriptome dynamics ignore extrinsic factors, such as experimental conditions and neighboring cell. Here, we propose ExDyn--a deep generative model integrated with splicing kinetics for estimating cell state dynamics dependent on extrinsic factors. ExDyn enables the counterfactual inference of cell state dynamics under different conditions. Among the extrinsic factors, ExDyn can extract key features which have large effects on cell state dynamics. ExDyn correctly estimated the difference in dynamics between two conditions and showed better accuracy over existing RNA velocity methods. ExDyn were utilized for unveiling the effect of PERK-knockout on neurosphere differentiation, hematopoietic stem cell differentiation driven by chromatin activity and the dynamics of squamous cell carcinoma cells dependent on colocalized neighboring cells. These results demonstrated that ExDyn is useful for analyzing key features in the dynamic generation of heterogeneous cell populations.

bioinformatics↗

bioRxiv2023

Mapping adipocyte interactome networks by HaloTag enrichment mass-spectrometry

Mapping protein interaction complexes in their natural state in vivo represents the holy grail of protein network analysis. Detection of protein interaction stoichiometry has been an important technical challenge, as few studies have focused this, yet this may be solved by artificial intelligence and proteomics. Here, we describe the development of HaloMS, a high-throughput HaloTag-based affinity purification-mass spectrometry assay for protein interaction discovery. The approach enables the rapid capture of newly expressed proteins, eliminating tedious conventional one-by-one assay. As a proof-of-principle, we used HaloMS to evaluate protein complex interactions of 17 regulatory proteins in human adipocytes. The adipocyte interactome network was validated using an in vitro pull-down assay and artificial intelligence-based prediction tools. The application of HaloMS to probe adipocyte differentiation facilitated the identification of previously unknown transcription factor-protein complexes, revealing proteome-wide human adipocyte transcription factor networks, and shedding light on how different pathways are integrated.

biochemistry↗

bioRxiv2023

Genetic modification strategies for electroporation and CRISPR-Cas-based technologies in the non-competent Gram-negative bacterium Acinetobacter sp. Tol 5

Environmental isolates are promising candidates for new chassis of synthetic biology because of their inherent conversion capabilities and resilience to environmental stresses; however, many remain genetically intractable and unamenable to established genetic tools tailored for model bacteria. Acinetobacter sp. Tol 5 possesses intriguing properties for use in synthetic biology applications. However, genetic manipulation via electroporation is hindered by its low transformation efficiency. This study demonstrated the genetic refinement of the Tol 5 strain, achieving efficient transformation via electroporation. We deleted two genes encoding restriction enzymes. The resulting mutant strain not only exhibited marked efficiency of electrotransformation but also proved receptive to both in vitro and in vivo DNA assembly technologies, thereby facilitating the construction of recombinant DNA. In addition, we successfully adapted a CRISPR-Cas9-based base-editing platform developed for other Acinetobacter species. Our genetic modification strategy allows for the domestication of non-model bacteria, streamlining their utilization in synthetic biology applications.

synthetic biology↗

bioRxiv2023

A Tetravalent Dengue Virus-like Particle Vaccine Induces High levels of Neutralizing Antibodies and Inhibits Dengue Replication in Non-Human Primates

Dengue virus (DENV) represents a significant global health burden, with 50% of the worlds population at risk of infection, and there is an urgent need for next-generation vaccines. Virus-like particle (VLP)-based vaccines, which mimic the antigenic structure of the authentic virus but lack the viral genome, are an attractive approach. Here we describe a dengue VLP (DENVLP) vaccine which generates a robust and long-lasting neutralizing antibody response against all four DENV serotypes in non-human primates. Importantly, DENVLP vaccination produced no ADE response against any of four DENV serotypes. Finally, we demonstrate in a non-human primate challenge model that DENVLP vaccination substantially reduces viral replication. We also transfer the purified IgG from the immunized monkeys into immunodeficient mice, where they protect against subsequent lethal dengue virus challenge, indicating a humoral mechanism of protection. These results indicate that a DENVLP vaccine is a safe and effective vaccine candidate. One Sentence Summary: Immunization of non-human primates with a tetravalent dengue VLP vaccine induces high levels of neutralizing antibodies and reduces the severity of infection for all four dengue serotypes.

microbiology↗

bioRxiv2023

Inference of gene regulatory networks using time-series single-cell RNA-seq data with CRISPR perturbations

Single-cell RNA-seq analysis coupled with CRISPR-based perturbation (scCRISPR) has enabled the inference of gene regulatory networks (GRNs) with causal relationships. However, a snapshot of scCRISPR data may not lead to an accurate inference, since a gene knockout can influence multi-layered downstream over time. Here, we developed RENGE, a new computational method that infers GRNs using a time-series scCRISPR dataset. RENGE models the propagation process of the effects elicited by a gene knockout on its GRN. It can distinguish between direct and indirect regulations, which allows for the inference of regulations by genes that are not knocked out. RENGE therefore outperforms current methods in the accuracy of inferring GRNs. When used on a dataset we derived from human-induced pluripotent stem cells, RENGE yielded a GRN consistent with multiple databases and literature. Accurate inference of GRNs by RENGE would enable the identification of key factors for various biological systems.

bioinformatics↗

bioRxiv2023

Universal pretreatment development for low-input proteomics using Lauryl Maltose Neopentyl Glycol

In recent years, the demand for low-input proteomics, most notably single-cell proteomics (SCP), has increased. In this study, we developed a Lauryl Maltose Neopentyl Glycol (LMNG)-assisted sample preparation (LASP) method that suppresses the loss of proteins and peptides in samples by adding LMNG, a surfactant, to the digested solution and removing the LMNG simply via reversed phase solid-phase extraction. The advantage of removing LMNG during sample preparation for general proteomic analysis is that it prevents mass spectrometry (MS) contamination. When the LASP method was applied to the low-input SP3 method and on-bead digestion in immunoprecipitation-MS, the recovery of the digested peptides was greatly improved. Furthermore, we established a simple and operationally easy sample preparation method for SCP based on the LASP method (scpLASP) and identified a median of 1,175 proteins from a single HEK239F cell using liquid chromatography (LC)-MS/MS with a throughput of 80 samples per day.

biochemistry↗

bioRxiv2023

SiCR: Web application for single-cell repertoire analysis and immune profiling

BackgroundSingle-cell RNA sequencing (scRNA-seq) allows analysis of complete sequences of antigen receptors in individual cells. However, it is a complex technique that requires multiple analyses to obtain accurate results. Although several user-friendly tools for scRNA-seq are available, none are specifically designed for immune profiling. ResultsWe developed a web application called SiCR that is based on the Shiny framework of the R package and specializes in single-cell immune profiling. SiCR allows clustering and cell typing required for both general single-cell and immune profiling analyses, such as predicting whether the chronotype is expanding in each group and the antigen the expanding chronotype targets. These analyses can be performed using a cursor control. SiCR also allows for detailed figure settings, enabling immediate publication of results. ConclusionsSiCR is a comprehensive workbench that can be used by biologists for single-cell immune profiling. Currently, it is the only web application that allows single-cell repertoire analysis using both raw and preprocessed data. Moreover, SiCR significantly reduces the time and effort required to analyze and interpret information in single-cell immune profiling. Therefore, SiCR is a potential reference application for interactive analysis and investigation of biological data, especially for immune profiling.

bioinformatics↗

bioRxiv2023

Comparative analysis of gonadal transcriptomes between turtle and alligator identifies common molecular cues activated during the temperature-sensitive period for sex determination

The mode of sex determination in vertebrates can be categorized as genotypic or environmental. In the case of genotypic sex determination (GSD), the sexual fate of an organism is determined by the chromosome composition with some having dominant genes, named sex-determining genes, that drive the sex phenotypes. By contrast, many reptiles exhibit environmental sex determination (ESD), whereby environmental stimuli drive sex determination, and most notably temperature. To date, temperature-dependent sex determination (TSD) has been found in most turtles, some lizards, and all crocodilians, but commonalities in the controlling processes are not well established. Recent innovative sequencing technology has enabled investigations into gonadal transcriptomic profiles during temperature-sensitive periods (TSP) in various TSD species which can help elucidate the controlling mechanisms. In this study, we conducted a time-course analysis of the gonadal transcriptome during the male-producing temperature (26L) of the Reeves turtle (Chinese three-keeled pond turtle) Mauremys reevesii. We then compared the transcriptome profiles for this turtle species during the TSP with that for the American alligator Alligator mississippiensis to identify conserved reptilian TSD-related genes. Our transcriptome-based findings provide an opportunity to retrieve the candidate molecular cues that are activated during TSP and compare these target responses between TSD and GSD turtle species, and between TSD species. Highlights- A time-course gonadal RNA-seq was conducted using Mauremys reevesii. - Sexual differentiation genes in males were activated at an earlier stage than the ones in females. - Turtle-alligator comparative analysis revealed novel candidate TSD genes.

developmental biology↗

bioRxiv2023

Activation of prefrontal parvalbumin interneurons ameliorates treatment-resistant working memory deficit even under continuous antipsychotic treatment in a mouse model of schizophrenia

BACKGROUNDOne of the critical unmet medical needs in schizophrenia is a remedy for cognitive deficits. However, the neural circuit mechanisms of them remain unresolved. In addition, despite the patients with schizophrenia cannot stop taking antipsychotics due to a high rate of discontinuation-induced relapse, previous studies using animal models of schizophrenia have not considered these clinical situations. METHODSHere, we employ multi-dimensional approaches, including histological analysis in the prelimbic cortex, LC-MS/MS-based in vivo dopamine D2 receptor occupancy analysis for antipsychotic drugs, in vivo calcium imaging and behavioral analyses of mice using chemogenetic manipulation, to investigate neural mechanisms and potential therapeutic interventions for working memory deficit in a mouse model with chronic phencyclidine (PCP) administration that resembles the schizophrenia symptomatology. RESULTSChronic PCP administration led to abnormalities in excitatory and inhibitory synapses, including dendritic spines of pyramidal neurons, vesicular glutamate transporter 1 (VGLUT1) positive terminals, and parvalbumin (PV) positive GABAergic interneurons, in layer 2-3 of the prelimbic cortex. Continuous olanzapine, which achieved a sustained therapeutic window of dopamine D2 receptor occupancy (60-80%) in the striatum, did not affect these synaptic abnormalities and working memory deficit in the PCP-treated mice. We found that the selective prelimbic PV activation, using hM3D(Gq)-DREADD system confirmed by in vivo calcium imaging, restored working memory deficit, even under continuous olanzapine treatment. CONCLUSIONSOur study raises a possibility that intervention in prefrontal PV neurons leads to an add-on therapy to antipsychotics targeting amelioration of treatment-resistant cognitive deficits in schizophrenia.

neuroscience↗

bioRxiv2023

Bioorthogonal chemical labelling of endogenous neurotransmitter receptors in living mouse brains

Neurotransmitter receptors are essential components of synapses for communication between neurons in the brain. Because the spatiotemporal expression profiles and dynamics of neurotransmitter receptors involved in many functions are delicately governed in the brain, in vivo research tools with high spatiotemporal resolution for receptors in intact brains are highly desirable. Covalent chemical labelling of proteins without genetic manipulation is now a powerful method for analyzing receptors in vitro. However, selective target receptor labelling in the brain has not yet been achieved. This study shows that ligand-directed alkoxyacylimidazole (LDAI) chemistry can be used to selectively tether synthetic probes to target endogenous receptors in living mouse brains. The reactive LDAI reagents with negative charges were found to diffuse well over the whole brain and could selectively label target endogenous receptors, including AMPAR, NMDAR, mGlu1, and GABAAR. This simple and robust labelling protocol was then used for various applications: three-dimensional spatial mapping of endogenous receptors in the brains of healthy and disease-model mice; multi-colour receptor imaging; and pulse-chase analysis of the receptor dynamics in postnatal mouse brains. Here, results demonstrated that bioorthogonal receptor modification in living animal brains may provide innovative molecular tools that contribute to the in-depth understanding of complicated brain functions.

biochemistry↗

bioRxiv2022

A novel micro-ECoG recording method for recording multisensory neural activity from the parietal to temporal cortexes in mice

Characterization of inter-regional interactions in brain is essential for understanding the mechanism relevant to normal brain function and neurological disease. The recently developed flexible micro ()-electrocorticography (ECoG) device is one prominent method used to examine large-scale cortical activity across multiple regions. The sheet-shaped ECoG electrodes arrays can be placed on a relatively wide area of cortical surface beneath the skull by inserting the device into the space between skull and brain. Although rats and mice are useful tools for neuroscience, current ECoG recording methods in these animals are limited to the parietal region of cerebral cortex. Recording cortical activity from the temporal region of cortex in mice has proven difficult because of surgical barriers created by the skull and surrounding temporalis muscle anatomy. Here, we developed a sheet-shaped 64-channel ECoG device that allows access to the mouse temporal cortex, and we determined the factor determining the appropriate bending stiffness for the ECoG electrode array. We also established a surgical technique to implant the electrode arrays into the epidural space over a wide area of cerebral cortex covering from the barrel field to olfactory (piriform) cortex, which is the deepest region of the cerebral cortex. Using histology and computed tomography (CT) images, we confirmed that the tip of the ECoG device reached to the most ventral part of cerebral cortex without causing noticeable damage to the brain surface. Moreover, the device simultaneously recorded somatosensory and odor stimulus-evoked neural activity from dorsal and ventral parts of cerebral cortex in awake and anesthetized mice. These data indicate that our ECoG device and surgical techniques enable the recording of large-scale cortical activity from the parietal to temporal cortex in mice, including somatosensory and olfactory cortices. This system will provide more opportunities for the investigation of physiological functions from wider areas of the mouse cerebral cortex than those currently available with existing ECoG techniques.

neuroscience↗

bioRxiv2022

Cryo-EM structures of Na+-pumping NADH-ubiquinone oxidoreductase from Vibrio cholerae

The Na+-pumping NADH-ubiquinone oxidoreductase (Na+-NQR) couples electron transfer from NADH to ubiquinone with Na+-pumping, generating an electrochemical Na+ gradient that is essential for energy-consuming reactions in bacteria. Since Na+-NQR is exclusively found in prokaryotes, it is a promising target for highly selective antibiotics. However, the molecular mechanism of inhibition is not well-understood for lack of the atomic structural information about an inhibitor-bound state. Here we present cryo-electron microscopy structures of Na+- NQR from Vibrio cholerae with or without a bound inhibitor at 2.5- to 3.1-[A] resolution. The structures reveal the arrangement of all six redox cofactors including riboflavin, whose position has been under debate, and a newly assigned 2Fe-2SNqrD/E cluster located between the membrane embedded NqrD and NqrE subunits. A large part of the hydrophilic NqrF near the cytoplasmic membrane surface is barely visible in the density map, suggesting a high degree of flexibility. This flexibility may be responsible to reducing the long distance between the 2Fe- 2S centers in NqrF and NqrD/E, consistent with physiologically relevant electron transfer. Two different types of specific inhibitors (korormicin A and aurachin D-42) bind to the N-terminal region of NqrB, which is disordered in the absence of inhibitors. The current inhibitor-bound structures reasonably explain our previous biochemical findings obtained by different chemistry-based experiments. This study provides a definite foundation for understanding the function of Na+-NQR and the molecular mechanism of its specific inhibitors to support molecular design of new antibiotics targeting the enzyme.

biochemistry↗

bioRxiv2022

Single-cell colocalization analysis using a deep generative model

1Analyzing colocalization of single cells with heterogeneous molecular phenotypes is essential for understanding cell-cell interactions, cellular responses to external stimuli, and their biological functions in diseases and tissues. However, high-throughput methods for identifying spatial proximity at single-cell resolution are practically unavailable. Here, we introduce DeepCOLOR, a computational framework based on a deep generative model that recovers inter-cellular colocalization networks with single cell resolution by the integration of single cell and spatial transcriptomes. It segregates cell populations defined by the colocalization relationships and predicts cell-cell interactions between colocalized single cells. DeepCOLOR could identify plausible cell-cell interaction candidates in mouse brain tissues, human squamous cell carcinoma samples, and human lung tissues infected with SARS-CoV-2 by reconstructing spatial colocalization maps at single-cell resolution. DeepCOLOR is typically applicable to studying cell-cell interactions in any spatial niche. Our newly developed computational framework could help uncover molecular pathways across single cells connected with colocalization networks.

bioinformatics↗

bioRxiv2022

Affective arousal explains infant gaze following under various social context

Gaze following is fundamental to human sociocognitive development, such as language and cultural learning. Previous studies have revealed that infant gaze following is not a reflexive orienting to adults eye movement. Instead, infants adaptively modulate gaze following behaviour depending on social contexts. However, the neurophysiological mechanisms underlying contextual modulation of gaze following remain unclear. We tested whether contextual modulation of infant gaze following is mediated by the infants heart rate, which is hypothesised to indicate the calculation of action value. Forty-one 6- to 9-month-old infants participated in this study. Infants observed either a reliable face, which gazed toward the location of an object, or an unreliable face, which gazed away from the location of an object. Then, the infants watched a video of the same model making eye contact or not showing any ostensive signals, before shifting her gaze toward one of two objects. We revealed that reliability and eye contact independently increased heart rates, which then fully mediate the effect of these social cues on the frequency of infant gaze following. Results suggest that each social cue independently enhances physiological arousal which then accumulatively predicts the likelihood of infant gaze following behaviour.

animal behavior and cognition↗

bioRxiv2021

Revisiting PFA-mediated tissue fixation chemistry: FixEL enables trapping of small molecules in the brain to visualize their distribution dynamics

Various small molecules have been used as functional probes for tissue imaging in medical diagnosis and pharmaceutical drugs for disease treatment. The spatial distribution, target selectivity, and diffusion/extrusion kinetics of small molecules in structurally complicated specimens are critical for function. However, robust methods for precisely evaluating these parameters in the brain have been limited. Herein we report a new method termed "Fixation-driven chemical crosslinking of exogenous ligands (FixEL)" which traps and images exogenously administered molecules-of-interest (MOI) in complex tissues. This method relies on proteins-MOI interactions, and chemical crosslinking of amine-tethered MOI with paraformaldehyde used for perfusion fixation. FixEL is used to obtain images of the distribution of the small molecules and their dynamics, which addresses selective/nonselective binding to proteins, time-dependent localization changes, and diffusion/retention kinetics of MOI such as PET tracer derivatives or drug-like small molecules. Clear imaging of a nanobody distributed in the whole brain was also achieved with high spatial resolution using 2D/3D mode.

neuroscience↗

bioRxiv2021

Role of TLR4 signaling on Porphyromonas gingivalis LPS-induced cardiac dysfunction in mice

Oral infections, particularly periodontitis, are a well-established risk factor for cardiovascular diseases, although the molecular mechanisms involved remain elusive. The aims of the present study were to investigate the effects of lipopolysaccharide derived from Porphyromonas gingivalis (PG-LPS) on cardiac function in mice, and to elucidate the underlying mechanisms. Mice (C57BL/6) were injected with PG-LPS (0.8 mg/kg/day) with or without an inhibitor of Toll-like receptor 4 (TLR4) signaling (TAK-242, 0.8 mg/kg/day) for 4 weeks. Left ventricular ejection function was significantly decreased at 1 week (from 67 {+/-} 0.5 to 58 {+/-} 1.2 %) and remained low at 4 weeks (57 {+/-} 1.0 %). The number of apoptotic myocytes was increased (approximately 7.4-fold), the area of fibrosis was increased (approximately 3.3-fold) and the number of 8-hydroxydeoxyguanosine-positive myocytes, a sensitive indicator of oxidative DNA damage, was increased (approximately 7.6-fold) at 4 weeks in the heart of PG-LPS treated mice. However, levels of various serum pro-inflammatory cytokines in PG-LPS-treated mice were similar to those in control mice. The impairment of cardiac function in PG-LPS-treated mice appears to involve activation of TLR4-NADPH oxidase (NOX) 4 signaling, leading to abundant production of reactive oxygen species and Ca2+ leakage from sarcoplastic reticulumn induced by calmodulin kinase II (CaMKII)-mediated phosphorylation of phospholamban (at Thr-17) and ryanodine receptor 2 (at Ser-2448). Pharmacological inhibition of TLR4 with TAK-242 attenuated the changes in cardiac function in PG-LPS-treated mice. Our results indicate that TLR4-NOX4 signaling may be a new therapeutic target for treatment of cardiovascular diseases in patients with periodontitis.

physiology↗

bioRxiv2021

Endothelial metastasis-associated protein 1 (MTA1) is an essential molecule for angiogenesis

Our previous study revealed that metastasis-associated protein 1 (MTA1), expressed in endothelial cells acts as a factor promoting tube formation. In this study, we aimed to clarify the importance of MTA1 in tube formation using MTA1-knockout endothelial cells (MTA1-KO MSS31 cells). Tube formation was strongly suppressed in MTA1-KO MSS31 cells, whereas MTA1-overexpressed MTA1-KO MSS31 cells regained the ability to form tube-like structures. Furthermore, MTA1-KO MSS31 cells showed higher phosphorylation of non-muscle myosin heavy chain IIa (NMIIa), which resulted in suppression of tube formation; this was attributed to a decrease of MTA1- S100 calcium-binding protein A4 (S100A4) complex formation. Moreover, inhibition of tube formation in MTA1-KO MSS31 cells could not be rescued by stimulation with vascular endothelial growth factor (VEGF). These results demonstrated that MTA1 is an essential molecule for angiogenesis, and that it may be involved in different steps of the angiogenic process compared to the VEGF-VEGF receptor 2 (VEGFR2) pathway. Our findings showed that endothelial MTA1 and its pathway are promising targets for inhibiting tumor angiogenesis, supporting the development of MTA1-based anti-angiogenic therapies.

cell biology↗

bioRxiv2021

Identification of hub molecules of FUS-ALS by Bayesian gene regulatory network analysis of iPSC model: iBRN

Fused in sarcoma/translated in liposarcoma (FUS) is a causative gene of amyotrophic lateral sclerosis (ALS). Mutated FUS causes accumulation of DNA damage and cytosolic stress granule (SG) formation, thereby motor neuron (MN) death. However, key molecular aetiology remains unclear. Here, we applied a novel platform technology, iBRN, "Non-biased" Bayesian gene regulatory network analysis based on induced pluripotent stem cell (iPSC)-derived cell model, to elucidate the molecular aetiology using transcriptome of iPSC-derived MNs harboring FUSH517D. iBRN revealed "hub molecules", which strongly influenced transcriptome network, such as miR-125b-5p-TIMELESS axis and PRKDC for the molecular aetiology. Next, we confirmed miR-125b-5p-TIMELESS axis in FUSH517D MNs such that miR-125b-5p regulated several DNA repair-related genes including TIMELESS. In addition, we validated both introduction of miR-125b-5p and knocking down of TIMELESS caused DNA damage in the cell culture model. Furthermore, PRKDC was strongly associated with FUS mis-localization into SGs by DNA damage under impaired DNA-PK activity. Collectively, our iBRN strategy provides the first compelling evidence to elucidate molecular aetiology in neurodegenerative diseases. HighlightsO_LIA new platform technology, "iBRN", Bayesian gene regulatory network analysis based on iPSC cells C_LIO_LIiBRN identifies hub molecules to strongly influence the gene network in FUS-ALS C_LIO_LIPRKDC specifically acts as a guardian against FUS mis-localization during DNA damage stress C_LIO_LImiR-125b-5p-TIMELESS axis regulates DNA repair-related genes in FUS-ALS. C_LI

neuroscience↗