Biology subjects

Otsuka, Y.

Publications and source records attributed to Otsuka, Y..

14 recordsLinked to original sources

bioRxiv2025

A SARS-CoV-2 entry inhibitor trimerizes to lock the spike protein in a closed conformation

The SARS-CoV-2 spike protein binds its receptor ACE2 to initiate target-cell infection. To engage ACE2, at least one of the three receptor-binding domains (RBDs) of the spike must adopt the up orientation. Here we describe S22, a potent, bioavailable, and non-toxic inhibitor of BA.2 and all subsequent Omicron variants. Cryo-EM analyses showed that S22 assembled as a trimer in a previously uncharacterized pocket of the spike apex, stabilizing all three RBDs in the down orientation, thereby preventing ACE2 association. Binding studies, especially those using mixed S22-sensitive and -resistant spikes, imply a cooperative assembly of three S22 molecules with three RBDs, resulting in an unusually slow S22 off-rate. Consistent with its slow dissociation and favorable pharmacokinetics, S22 suppressed viral replication 100-fold in the lungs of XBB.1.5-infected mice. Thus, S22 potently inhibits Omicron entry through a distinct mechanism whereby a small compound assembles cooperatively as a trimer to stabilize spike in an inactive conformation.

microbiology↗

bioRxiv2025

Discovery of a potent anti-Zika virus benzamide series targeting the viral protein NS4B

1.Zika virus (ZIKV), a member of the Flaviviridae family, causes significant public health concerns through congenital Zika syndrome and Guillain-Barre syndrome, yet no effective anti-ZIKV drugs or vaccines are available. To address this critical need, we conducted phenotypic, cytopathic effect-based, high-throughput screening followed by medicinal chemistry optimization and discovered novel benzamide anti-ZIKV leads. Current best compounds demonstrated superior potency (EC50 values 40-400 nM, CC50 > 50 {micro}M) compared to NITD-008, the most potent known anti-ZIKV agent. Time-of-addition assays, resistant virus selection studies, and biophysical binding experiments confirmed that NS4B interference constitutes the primary antiviral mechanism. Notably, resistance mutations mapped to the C-terminus of NS4B, distinct from other flavivirus NS4B inhibitors targeting dengue or yellow fever viruses, revealing novel insights into a critical function of the region. These findings establish NS4B as an Achilles heel for flaviviruses and support the development of pan-flavivirus therapeutics targeting this essential viral protein. 2. AUTHOR SUMMARYSince its pandemic spread in 2015-2016, Zika virus infection remains a significant public health threat worldwide. The virus can cause severe brain damage in developing babies and serious neurological complications like Guillain-Barre syndrome in adults. Despite these devastating consequences, we currently lack effective medicines or vaccines to prevent the virus from spreading through communities or from mothers to their unborn children. To address this critical gap, we conducted a large-scale screening of chemical compounds and discovered a promising new class of molecules that can effectively stop Zika virus from replication. Using medicinal chemistry techniques, we were able to make these compounds even more potent against the virus. In follow-up studies, we found that our compounds work by interfering with a specific viral protein called NS4B, which the virus needs to replicate its genome within the cell. Remarkably, other research teams studying related other flaviviruses (e.g., dengue and yellow fever virus) have independently discovered that this same protein is a vulnerable target. Our findings suggest that NS4B represents a universal weakness across the entire flavivirus family, making it an attractive target for developing broad-spectrum antiviral treatments.

microbiology↗

bioRxiv2024

Development of a Novel Japanese Eel Myoblast Cell Line for Application in Cultured Meat Production

The present study investigates the isolation, analysis, and characterization of primary cultured cells derived from the muscle tissue of Japanese eel (Anguilla japonica), culminating in establishing a spontaneously immortalized myoblast cell line, JEM1129. We isolated satellite cells from eel muscle tissue to establish a foundation for cultured eel meat production. While initial cell cultures contained myoblasts, continued passaging led to a decline in myoblast characteristics and an increase in fibroblast-like cells. RNA-Seq and RT-qPCR analyses showed significant downregulation of well-established markers for satellite cells and myoblasts, such as pax7a and myoD, over successive passages, highlighting a loss of myoblastic traits. Single-cell cloning was employed to overcome this challenge and maintain myoblast purity, leading to the successful creation of the JEM1129 cell line. These JEM1129 cells demonstrated enhanced expression of myoblast marker genes, exceeding the initial primary culture cell population. The cells showed strong myotube formation, particularly when cultured in a differentiation medium, indicating their robust potential for muscle development. The JEM1129 cell line represents a significant advancement in the cultivation of eel muscle cells, offering a promising avenue for cultured meat production. The findings contribute to a deeper understanding of muscle cell biology and provide valuable insights into using fish-derived myoblasts for cultured meat production. HighlightsSingle-cell cloning established a stable Japanese eel myoblast line, JEM1129. Single-cell cloning aids in establishing spontaneously immortalized fish myoblasts. JEM1129 demonstrates robust myogenic differentiation, crucial for cultured meat. Potential for sustainable eel meat production reducing wild eel depletion.

cell biology↗

bioRxiv2023

Establishing an AI-based evaluation system that quantifies social/pathophysiological behaviors of common marmosets

Nonhuman primates (NHPs) are indispensable animal models by virtue of the continuity of behavioral repertoires across primates, including humans. However, behavioral assessment at the laboratory level has so far been limited. By applying multiple deep neural networks trained with large-scale datasets, we established an evaluation system that could reconstruct and estimate three-dimensional (3D) poses of common marmosets, a small NHP that is suitable for analyzing complex natural behaviors in laboratory setups. We further developed downstream analytic methodologies to quantify a variety of behavioral parameters beyond simple motion kinematics, such as social interactions and the internal state behind actions, obtained solely from 3D pose data. Moreover, a fully unsupervised approach enabled us to detect progressively-appearing symptomatic behaviors over a year in a Parkinsons disease model. The high-throughput and versatile nature of our analytic pipeline will open a new avenue for neuroscience research dealing with big-data analyses of social/pathophysiological behaviors in NHPs.

neuroscience↗

bioRxiv2022

Bacterial catabolic system of acetovanillone and acetosyringone useful for upgrading aromatic compounds obtained through chemical lignin depolymerization

Acetovanillone is a major aromatic monomer produced in oxidative/base-catalyzed lignin depolymerization. However, the production of chemical products from acetovanillone has not been explored due to the lack of information on the microbial acetovanillone catabolic system. Here acvABCDEF was identified as specifically induced genes during the growth of Sphingobium sp. strain SYK-6 cells with acetovanillone and these genes were essential for SYK-6 growth on acetovanillone and acetosyringone (a syringyl-type acetophenone derivative). AcvAB and AcvF produced in Escherichia coli phosphorylated acetovanillone/acetosyringone and dephosphorylated the phosphorylated acetovanillone/acetosyringone, respectively. AcvCDE produced in Sphingobium japonicum UT26S converted the dephosphorylated phosphorylated acetovanillone/acetosyringone intermediate into vanilloyl acetic acid/3- (4-hydroxy-3,5-dimethoxyphenyl)-3-oxopropanoic acid through carboxylation. To demonstrate the feasibility of producing cis,cis-muconic acid from acetovanillone, a metabolic modification on a mutant of Pseudomonas sp. strain NGC7 that accumulates cis,cis-muconic acid from catechol was performed. The resulting strain expressing vceA and vceB required for converting vanilloyl acetic acid to vanillic acid and aroY encoding protocatechuic acid decarboxylase in addition to acvABCDEF successfully converted 1.2 mM acetovanillone to approximate equimolar cis,cis-muconic acid. Our results are expected to help improve the yield and purity of value-added chemical production from lignin through biological funneling. IMPORTANCEIn the alkaline oxidation of lignin, aromatic aldehydes (vanillin, syringaldehyde, and p-hydroxybenzaldehyde), aromatic acids (vanillic acid, syringic acid, and p- hydroxybenzoic acid), and acetophenone-related compounds (acetovanillone, acetosyringone, and 4-hydroxyacetophenone) are produced as major aromatic monomers. Also, base-catalyzed depolymerization of guaiacyl lignin resulted in vanillin, vanillic acid, guaiacol, and acetovanillone as primary aromatic monomers. To date, microbial catabolic systems of vanillin, vanillic acid, and guaiacol have been well characterized, and the production of value-added chemicals from them has also been explored. However, due to the lack of information on the microbial acetovanillone and acetosyringone catabolic system, chemical production from acetovanillone and acetosyringone has not been achieved. This is the first study to elucidate the acetovanillone/acetosyringone catabolic system, and to demonstrate the potential of using these genes for value-added chemicals production from these compounds.

microbiology↗

bioRxiv2021

Positive Feedback Regulation of fzd7 Expression Robustly Shapes Wnt Signaling Range in Early Heart Development

Secreted molecules called morphogens govern tissue patterning in a concentration-dependent manner. However, it is still unclear how reproducible patterning can be achieved with diffusing molecules, especially when patterning differentiation of a thin region. Wnt is a morphogen that organizes cardiac development; especially Wnt6 patterns the cardiogenic mesoderm to induce differentiation of a thin pericardium in Xenopus. It is, however, unclear how Wnt6 can pattern such a thin tissue. In this study, we reveal that a Wnt receptor, frizzled7, is expressed in a Wnt-dependent manner in the prospective heart region, and that this receptor-feedback is essential for shaping a steep gradient of Wnt. In addition, the feedback imparts robustness against fluctuations of Wnt ligand production and allows the system to reach a steady state quickly. We also found a Wnt antagonist sFRP1, which is expressed at the opposite side of Wnt source, accumulates on a novel type of heparan sulfate (HS), N-acetyl-rich HS, which is highly presented in the outer of cardiogenic mesoderm, achieving local inhibition of Wnt signaling by restricting sFRP1 spreading. These two intricate regulatory systems restrict Wnt signaling and ensure reproducible patterning of a thin pericardium tissue.

developmental biology↗

bioRxiv2021

Development of affinity beads-based in vitro metal-ligand binding assay reveals dominant cadmium affinity of thiol-rich small peptides phytochelatins beyond glutathione

For a better understanding of metal-ligand interaction and its function in cells, we developed an easy, sensitive, and high-throughput method to quantify ligand-metal(loid) binding affinity under physiological conditions by combining ligand-attached affinity beads and inductively coupled plasma-optical emission spectrometry (ICP-OES). Glutathione (GSH) and two phytochelatins (PC2 and PC3, small peptides with different numbers of free thiols) were employed as model ligands and attached to hydrophilic beads. The principle of the assay resembles that of affinity purification of proteins in biochemistry: metals binding to the ligand on the beads and the rest in the buffer are separated by a spin-column and quantified by ICP-OES. The binding assay using the GSH-attached beads and various metal(loid)s suggested the different affinity of the metal-GSH interactions, in accordance with the order of the Irving-Williams series and the reported stability constants. The binding assay using PC2 or PC3-attached beads suggested positive binding between PCs and Ni(II), Cu(II), Zn(II), Cd(II), and As(III) in accordance with the number of thiols in PC2 and PC3. We then conducted the competition assay using Cd(II), Mn(II), Fe(II), Cu(II), and Zn(II) and the results suggested a better binding affinity of PC2 with Cd(II) than with the essential metals. Another competition assay using PC2 and GSH suggested a robust binding affinity between PCs and Cd(II) compared to GSH and Cd(II). These results suggested the dominance of PC-Cd complex formation in vitro, supporting the physiological importance of PCs for the detoxification of cadmium in vivo. We also discuss the potential application of the assay.

biochemistry↗

bioRxiv2021

NR4A3 is oxidative stress responsible transcription factor through HMOX1, and also controls cell cycle through CCNE1 and CDK2 in pancreatic islet derived 1.1B4 cells

The mechanism of antioxidant defense system is still controversial. As islet {beta}-cell is weak in oxidative condition, that causes diabetes mellitus, therefore, antioxidant defense system of human pancreatic islet derived 1.1B4 cell was analyzed. Cells were exposed to H2O2 and comprehensive gene expression was analyzed by Agilent human microarray. HMOX1 and NR4A3, member of orphan receptor, were up-regulated. Therefore, NR4A3 was knocked down with siRNA, then analyzed gene expression by microarray, and found that the knocked down cells were weak in oxidative stress. HMOX1 expression was strongly inhibited by siRNA of NR4A3, and NR4A3 responsible sequence of aaggtca was found near the HMOX1 gene, suggesting NR4A3 is oxidative stress responsible transcription factor through HMOX1 expression. The expression of CCNE1 and CDK2 was also inhibited by knocked down of NR4A3, it is suggested NR4A3 is also important transcription factor for cell growth regulation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/457070v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@18d0371org.highwire.dtl.DTLVardef@dd1eacorg.highwire.dtl.DTLVardef@108b1b9org.highwire.dtl.DTLVardef@1cbf899_HPS_FORMAT_FIGEXP M_FIG Hydrogen peroxide induces NR4A3 and binds to aaggtca sequence of HMOX1, and increased transcription of HMOX1. Resulting heme oxygenase produces biliverdin, antioxidants, from heme. NR4A3 also bind to aaggtca sequence of CDK2 and CCNE1, resulting CDK2 and Cyclin E. CDK2 bind to cyclin E and cell goes from G1 to S phase. C_FIG

genomics↗

bioRxiv2021

Identification of Potent Small Molecule Inhibitors of SARS-CoV-2 Entry

The severe acute respiratory syndrome coronavirus 2 responsible for COVID-19 remains a persistent threat to mankind, especially for the immunocompromised and elderly for which the vaccine may have limited effectiveness. Entry of SARS-CoV-2 requires a high affinity interaction of the viral spike protein with the cellular receptor angiotensin-converting enzyme 2. Novel mutations on the spike protein correlate with the high transmissibility of new variants of SARS-CoV-2, highlighting the need for small molecule inhibitors of virus entry into target cells. We report the identification of such inhibitors through a robust high-throughput screen testing 15,000 small molecules from unique libraries. Several leads were validated in a suite of mechanistic assays, including whole cell SARS-CoV-2 infectivity assays. The main lead compound, Calpeptin, was further characterized using SARS-CoV-1 and the novel SARS-CoV-2 variant entry assays, SARS-CoV-2 protease assays and molecular docking. This study reveals Calpeptin as a potent and specific inhibitor of SARS-CoV-2 and some variants.

microbiology↗

bioRxiv2021

Scn1a-GFP transgenic mouse revealed Nav1.1 expression in neocortical pyramidal tract projection neurons

Expressions of voltage-gated sodium channels Nav1.1 and Nav1.2, encoded by SCN1A and SCN2A genes respectively, have been reported to be mutually exclusive in most brain regions. In juvenile and adult neocortex, Nav1.1 is predominantly expressed in inhibitory neurons while Nav1.2 is in excitatory neurons. Although a distinct subpopulation of layer V (L5) neocortical excitatory neurons were also reported to express Nav1.1, their nature has been uncharacterized. In hippocampus, Nav1.1 has been proposed to be expressed only in inhibitory neurons. By using newly-generated transgenic mouse lines expressing Scn1a promoter-driven green fluorescent protein (GFP), here we confirm the mutually-exclusive expressions of Nav1.1 and Nav1.2 and the absence of Nav1.1 in hippocampal excitatory neurons. We also show that Nav1.1 is expressed in inhibitory and a subpopulation of excitatory neurons not only in L5 but all layers of neocortex. By using neocortical excitatory projection neuron markers including FEZF2 for L5 pyramidal tract (PT) and TBR1 for layer VI (L6) cortico-thalamic (CT) projection neurons, we further show that most L5 PT neurons and a minor subpopulation of layer II/III (L2/3) cortico-cortical (CC) neurons express Nav1.1 while the majority of L6 CT, L5/6 cortico-striatal (CS) and L2/3 CC neurons express Nav1.2. These observations now contribute to the elucidation of pathological neural circuits for diseases such as epilepsies and neurodevelopmental disorders caused by SCN1A and SCN2A mutations.

neuroscience↗

bioRxiv2020

Sphingobium sp. SYK-6 syringate O-demethylase gene is regulated by DesX, unlike other vanillate and syringate catabolic genes regulated by DesR

Syringate and vanillate are the major metabolites of lignin biodegradation. In Sphingobium sp. strain SYK-6, syringate is O demethylated to gallate by consecutive reactions catalyzed by DesA and LigM, and vanillate is O demethylated to protocatechuate by a reaction catalyzed by LigM. The gallate ring is cleaved by DesB, and protocatechuate is catabolized via the protocatechuate 4,5-cleavage pathway. The transcriptions of desA, ligM, and desB are induced by syringate and vanillate, while that of ligM and desB are negatively regulated by the MarR-type transcriptional regulator DesR, which is not involved in desA regulation. Here we clarified the regulatory system for desA transcription by analyzing the IclR-type transcriptional regulator desX, located downstream of desA. Quantitative reverse transcription (RT)-PCR analyses of a desX mutant indicates that the transcription of desA was negatively regulated by DesX. In contrast, DesX was not involved in the regulation of ligM and desB. The ferulate catabolic genes (ferBA) under the control of a MarR-type transcriptional regulator FerC are located upstream of desA. RT-PCR analyses suggest that the ferB-ferA-SLG_25010-desA gene cluster consists of the ferBA operon and the SLG_25010-desA operon. Promoter assays reveal that a syringate- and vanillate-inducible promoter is located upstream of SLG_25010. Purified DesX bound to this promoter region, which overlaps with an 18-bp-inverted repeat sequence that appears to be essential for the DNA binding of DesX. Syringate and vanillate inhibited the DNA binding of DesX, indicating that these compounds are effector molecules of DesX. IMPORTANCESyringate is a major degradation product in the microbial and chemical degradation of syringyl lignin. Along with other low-molecular-weight aromatic compounds, syringate is produced by chemical lignin depolymerization. Converting this mixture into value-added chemicals using bacterial metabolism (i.e., biological funneling) is a promising option for lignin valorization. To construct an efficient microbial lignin conversion system, it is necessary to identify and characterize the genes involved in the uptake and catabolism of lignin-derived aromatic compounds and elucidate their transcriptional regulation. In this study, we found that the transcription of desA, encoding syringate O-demethylase in SYK-6, is regulated by an IclR-type of transcriptional regulator, DesX. The findings of this study, combined with our previous results on desR (a MarR transcriptional regulator that controls the transcription of ligM and desB), provide an overall picture of the transcriptional regulatory systems for syringate and vanillate catabolism in SYK-6.

microbiology↗

bioRxiv2019

An unbiased high-throughput drug screen reveals a potential therapeutic vulnerability in the most lethal molecular subtype of pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is predicted to become the second leading cause of cancer-related deaths in the United States by 2020, due in part to innate resistance to widely used chemotherapeutic agents and limited knowledge about key molecular factors that drive tumor aggression. We previously reported a novel negative prognostic biomarker, keratin 17 (K17), whose overexpression in cancer results in shortened patient survival. In this study, we aimed to determine the predictive value of K17 and explore the therapeutic vulnerability in K17-expressing PDAC, using an unbiased high-throughput drug screen. Patient-derived data analysis showed that K17 expression correlates with resistance to Gemcitabine (Gem). In multiple in vitro models of PDAC, spanning human and murine PDAC cells, we determined that the expression of K17 results in a more than two-fold increase in resistance to Gem and 5-fluorouracil, key components of current standard-of-care chemotherapeutic regimens. Furthermore, through an unbiased drug screen, we discovered that Podophyllotoxin (PPT), a microtubule inhibitor, showed at least two-fold higher sensitivity in K17-expressing compared to K17-negative PDAC cells. In the clinic, another microtubule inhibitor, Paclitaxel (PTX), is used in combination with Gem as a first line chemotherapeutic regimen for pancreatic, breast, lung, and ovarian cancer. Surprisingly, we found that when combined with Gem, PPT but not PTX, was synergistic in inhibiting the viability of K17-expressing PDAC cells. This provides evidence that PPT or its derivatives could potentially be combined with Gem to enhance treatment efficacy for the approximately 50% of PDACs that express high levels of K17. In summary, we reported that K17 is a novel target for developing a biomarker-based personalized treatment for PDAC.

cancer biology↗

bioRxiv2019

A Rab11-GEF Parcas recruits Rab11 onto recycling endosomes for rhodopsin transport in Drosophila photoreceptors

Rab11 and its effectors dRip11 and MyoV are essential for polarized post-Golgi vesicle trafficking to photosensitive membrane rhabdomeres in Drosophila photoreceptors. Here, we found that Parcas (Pcs), recently shown to have guanine-nucleotide-exchange (GEF) activity toward Rab11, co-localizes with Rab11 on the trans-side of Golgi units and post-Golgi vesicles at the base of the rhabdomeres in pupal photoreceptors. Pcs fused with the EM-tag APEX2 localizes on 150-300 nm vesicles at the trans-side of Golgi units, which are presumably fly recycling endosomes (RE). Loss of Pcs impairs Rab11 localization on the trans-side of Golgi units and induces the cytoplasmic accumulation of post-Golgi vesicles bearing rhabdomere proteins, as observed in Rab11-deficiency. In contrast, loss of the specific subunits of TRAPPII, another known Rab11-GEF, does not cause any defects on the eye development nor the transport of rhabdomere proteins, however, simultaneous loss of TRAPPII and Pcs shows severe defects on eye development. These results indicated that in pupal photoreceptors, Pcs is the predominant Rab11-GEF, and TRAPPII performs a function that is redundant but subsidiary to that of Pcs. Summary statementParcas, a Rab11-GEF, recruits Rab11 onto Golgi-associated RE and post-Golgi vesicles in fly photoreceptors. Loss of Parcas results in rhodopsin accumulation within the cytoplasm, similar to that observed under Rab11-deficiency.

cell biology↗

bioRxiv2018

mitoNEET Regulates Mitochondrial Iron Homeostasis Interacting with Transferrin Receptor

Iron is an essential trace element for regulation of redox and mitochondrial function, and then mitochondrial iron content is tightly regulated in mammals. We focused on a novel protein localized at the outer mitochondrial membrane. Immunoelectron microscopy revealed transferrin receptor (TfR) displayed an intimate relationship with the mitochondria, and mass spectrometry analysis also revealed mitoNEET interacted with TfR in vitro. Moreover, mitoNEET was endogenously coprecipitated with TfR in the heart, which indicates that mitoNEET also interacts with TfR in vivo. We generated mice with cardiac-specific deletion of mitoNEET (mitoNEET-knockout). Iron contents in isolated mitochondria were significantly increased in mitoNEET-knockout mice compared to control mice. Mitochondrial reactive oxygen species (ROS) were higher, and mitochondrial maximal capacity and reserve capacity were significantly decreased in mitoNEET-knockout mice, which was consistent with cardiac dysfunction evaluated by echocardiography. The complex formation of mitoNEET with TfR may regulate mitochondrial iron contents via an influx of iron. A disruption of mitoNEET could thus be involved in mitochondrial ROS production by iron overload in the heart.

cell biology↗