Biology subjects

Yamada, T.

Publications and source records attributed to Yamada, T..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Asymmetry and Heterogeneity in the Plasma Membrane

Plasma membranes (PMs) exhibit asymmetry between their two leaflets in terms of phospholipid headgroups, unsaturation, and resulting membrane properties such as packing and fluidity. Lateral heterogeneity, including the formation of lipid domains, is another crucial aspect of PMs with significant biological implications. However, the nature and even the existence of lipid domains in the two leaflets of PMs remain elusive, hindering a complete understanding of the significance of lipid asymmetry. Using coarse-grained molecular dynamics simulation of the asymmetric PM, we find that the outer leaflet predominantly adopts a liquid-ordered state, whereas the inner leaflet separates into nanoscale ({approx}10 nm) liquid-ordered and liquid-disordered domains, exhibiting highly dynamic fusion and fission events. This structural asymmetry is further reinforced by asymmetric lateral stress resulting from a cholesterol bias toward the outer leaflet. These findings suggest distinct functional roles for the two leaflets, modulated by asym-metric lateral stress. Additionally, comparing the phase behavior of asymmetric and fully scrambled PMs reveals a key determinant of domain size: intact PMs maintain nanoscale domains, while cell-derived giant PM vesicles, which have lost the strict lipid asymmetry, exhibit microscale domains.

biophysics↗

bioRxiv2025

A conserved mechanism of LRRC8 channel inhibition by distinct drugs

We employed a Leucine Rich Repeat Containing 8 (LRRC8) channel chimera, termed 8C-8A(IL125), to investigate the molecular mechanism of action of the novel volume-sensitive anion channel (VRAC) inhibitor, zafirlukast. 8C-8A(IL125) comprises LRRC8C (8C) and 25 residues from LRRC8A (8A) intracellular loop 1 (IL1) and forms volume-sensitive, structurally defined heptameric channels with normal pharmacological properties. In silico docking and modeling with AlphaFold3 identified a putative zafirlukast binding site comprising the amino (N)-terminal domain (NTD) and inter-subunit fenestrae between transmembrane (TM) helices 1 and 2. Consistent with this model, mutations in NTD, TM1, and TM2 alter 8C-8A(IL125) and heteromeric 8A/8C sensitivity to zafirlukast and the structurally distinct drug pranlukast. Inhibition is not mediated by extracellular pore block or the so-called lipid gate. Mutations or low pH conditions that enhance voltage-dependent inactivation also increase zafirlukast sensitivity. We propose zafirlukast and pranlukast promote channel inactivation through destabilization of the pore.

pharmacology and toxicology↗

bioRxiv2024

Automated Harmonization and Large-Scale Integration of Heterogeneous Biomedical Sample Metadata Using Large Language Models

The exponential growth of biomedical data has created an urgent need for efficient integration and analysis of heterogeneous sample metadata across studies. However, current methods for harmonizing and standardizing these metadata are largely manual, time-consuming, and prone to inconsistencies. Here, we present a novel computational framework that leverages large language models (LLMs) to automate the harmonization and large-scale integration of diverse biomedical sample metadata. Our approach combines semantic clustering techniques with LLM-driven natural language processing to extract, interpret, and standardize metadata from various sources, including research papers, supplementary tables, and text data from public databases. We demonstrate the efficacy of our framework by applying it to thousands of human gut microbiome papers, successfully extracting and integrating metadata from over 400,000 samples. Our method achieved a 50% recovery rate of manually curated metadata, significantly outperforming traditional rule-based methods. Furthermore, our framework enabled the creation of a unified, searchable database of standardized metadata, facilitating cross-study analyses and revealing previously obscured patterns in microbiome composition across diverse populations and conditions. The scalability and adaptability of our approach suggest its potential applicability to a wide range of biomedical fields, potentially accelerating meta-analyses and fostering new insights from existing data. This work represents a significant advancement in biomedical data integration, offering a powerful tool for researchers to unlock the full potential of accumulated scientific knowledge.

bioinformatics↗

bioRxiv2024

Step-by-Step Maturation Mechanism of Binary Toxin Pore Revealed by Cryo-EM Analysis

Membrane pore-forming proteins (PFPs) form ring-shaped membrane-translocating oligomers on membranes, contributing to infection, immunity, and cell death functions. Binary toxins produced by some bacteria consist of an enzymatic component that acts as a toxin and a membrane-binding component that forms a pore that delivers the enzymatic component into target cells. Cryo-electron microscopy (cryo-EM) has advanced our understanding of these translocation mechanisms by revealing several binary toxin complexes structures. However, the mechanisms underlying the initial pore formation remain unclear. We determined the structures of several oligomeric forms of the membrane-binding component Ib of the Iota toxin from Clostridium perfringens at various stages of pore formation. Structural comparisons revealed how the symmetrically arranged soluble oligomer (prepore) asymmetrically mature into a transmembrane oligomer (pore). These findings enhance our understanding of mechanisms of PFP and provide a structural basis for developing nanodevices using membrane pores. Significance StatementAlthough the mode of action of pore-forming proteins (PFPs) has been increasingly elucidated, the structural mechanisms underlying their maturation into membrane-spanning pores remain elusive. One of the key challenges is the difficulty in capturing the intermediate states between the soluble prepore and the mature pore, and its structural analysis. In this study, we successfully prepared the super-complex formed by a bacterial toxin PFP. The super-complex inhibited the constituting oligomers to maturate into the pore, which enabled structural determination of multiple intermediate state. Comparative structural analysis revealed that the transition from prepore to pore occurs through stepwise, domino-like conformational changes. The analysis workflow and insights from this study are expected to advance the field of PFP research.

molecular biology↗

bioRxiv2024

DeepES: Deep learning-based enzyme screening to identify orphan enzyme genes

MotivationProgress in sequencing technology has led to determination of large numbers of protein sequences, and large enzyme databases are now available. Although many computational tools for enzyme annotation were developed, sequence information is unavailable for many enzymes, known as orphan enzymes. These orphan enzymes hinder sequence similarity-based functional annotation, leading gaps in understanding the association between sequences and enzymatic reactions. ResultsTherefore, we developed DeepES, a deep learning-based tool for enzyme screening to identify orphan enzyme genes, focusing on biosynthetic gene clusters and reaction class. DeepES uses protein sequences as inputs and evaluates whether the input genes contain biosynthetic gene clusters of interest by integrating the outputs of the binary classifier for each reaction class. The validation results suggested that DeepES can capture functional similarity between protein sequences, and it can be implemented to explore orphan enzyme genes. By applying DeepES to 4744 metagenome-assembled genomes, we identified candidate genes for 236 orphan enzymes, including those involved in short-chain fatty acid production as a characteristic pathway in human gut bacteria. Availability and implementationDeepES is available at https://github.com/yamada-lab/DeepES. Model weights and the candidate genes are available at Zenodo (https://doi.org/10.5281/zenodo.11123900).

bioinformatics↗

bioRxiv2024

Genetic tuning of intrinsically photosensitive retinal ganglion cell subtype identity to drive visual behavior

The melanopsin-expressing, intrinsically photosensitive retinal ganglion cells (ipRGCs) comprise a subset of the [~]40 retinal ganglion cell types in the mouse retina and drive a diverse array of light-evoked behaviors from circadian photoentrainment to pupil constriction to contrast sensitivity for visual perception. Central to the ability of ipRGCs to control this diverse array of behaviors is the distinct complement of morphophysiological features and gene expression patterns found in the M1-M6 ipRGC subtypes. However, the genetic regulatory programs that give rise to subtypes of ipRGCs are unknown. Here, we identify the transcription factor Brn3b (Pou4f2) as a key genetic regulator that shapes the unique functions of ipRGC subtypes and their diverse downstream visual behaviors.

neuroscience↗

bioRxiv2024

An efficient gene targeting system using Δku80 and functional analysis of Cyp51A in Trichophyton rubrum.

Trichophyton rubrum is one of the most frequently isolated fungi in patients with dermatophytosis. Despite its clinical significance, the molecular mechanisms of drug resistance and pathogenicity of T. rubrum remain to be elucidated because of the lack of genetic tools, such as efficient gene targeting systems. In this study, we generated a T. rubrum strain that lacks the nonhomologous end-joining-related gene ku80 ({Delta}ku80) and then developed a highly efficient genetic recombination system with gene targeting efficiency that was 46 times higher than that using the wild-type strain. Cyp51A and Cyp51B are 14--lanosterol demethylase isozymes in T. rubrum that promote ergosterol biosynthesis and are the targets of azole antifungal drugs. The expression of cyp51A mRNA was induced by the addition of the azole antifungal drug efinaconazole, whereas no such induction was detected for cyp51B, suggesting that Cyp51A functions as an azole-responsive Cyp51 isozyme. To explore the contribution of Cyp51A to susceptibility to azole drugs, the neomycin phosphotransferase (nptII) gene cassette was inserted into the cyp51A 3'-UTR region of {Delta}ku80 to destabilize the mRNA of cyp51A. In this mutant, although the expression level of cyp51A mRNA was comparable to that of the parent strain, the induction of cyp51A mRNA expression by efinaconazole was diminished. The minimum inhibitory concentration for several azole drugs of this strain was reduced, suggesting that dermatophyte Cyp51A contributes to the tolerance for azole drugs. These findings suggest that an efficient gene targeting system using {Delta}ku80 in T. rubrum is applicable for analyzing genes encoding drug targets. Key PointsO_LIA novel gene targeting system using {Delta}ku80 strain was established in T. rubrum C_LIO_LICyp51A in T. rubrum responds to the azole antifungal drug efinaconazole C_LIO_LICyp51A contributes to azole drug tolerance in T. rubrum C_LI

microbiology↗

bioRxiv2024

DNA-free and genotype-independent CRISPR/Cas9 system in soybean

The CRISPR/Cas9 system has revolutionized the field of plant genetic engineering. Here we report a smart genome editing system of soybean by using iPB-RNP method without introducing foreign DNA and requiring traditional tissue culture processes such as embryogenesis and organogenesis. Shoot apical meristem (SAM) of embryonic axes was used the target tissue for genome editing, because the SAM in soybean mature seeds has stem cells and specific cell layer developing germ cells during reproductive growth stage. In the iPB-RNP method, the complex of ribonucleoprotein (RNP) and Cas9 protein was directly delivered into SAM stem cells via particle bombardment and genome-edited plants were generated from these SAMs. Soybean allergenic gene Gly m Bd 30K, which we previously generated genome-editing soybean by using Agrobacterium-mediated transformation and particle bombardment in our previous studies, was targeted in this study. Many E0 (the first generation of genome-edited) plants in this experiment harbored mutant alleles at the targeted locus. Editing frequency of inducing mutations transmissible to the E1 generation was approximately 0.4 to 4.6 % of all E0 plants utilized in various soybean varieties. Furthermore, Gly m Bd 30K protein in mature seeds was not detected by western blot analysis due to flame-sift mutations. Our results offer a practical approach for both plant regeneration- and DNA-free genome editing achieved by delivering RNP into the SAM of dicotyledonous plants.

plant biology↗

bioRxiv2024

Targeting dermatophyte Cdc42 and Rac GTPase signaling to hinder hyphal elongation and virulence

The identification of novel molecular targets for antifungal drugs is critical due to limited treatment options and drug-resistance threats. We screened inhibitors of small GTPases, molecular switches in signal transduction, in Trichophyton rubrum, the primary cause of dermatophytosis. Our study found that chemical and genetic inhibition of Cdc42 and Rac GTPases, which are involved in cellular morphological changes, significantly impair hyphal formation, and are crucial for pathogenic fungal growth and virulence. Genetic repression of Cdc24, a guanine nucleotide exchange factor of Cdc42 and Rac, led to hyphal growth defects, abnormal cell morphology, and cell death. Chemical screening identified EHop-016 as an inhibitor of Cdc24 activity, which improved outcomes in in vitro nail infection and invertible infection models of T. rubrum. Our results suggest the Cdc24-Cdc42/Rac pathway as a promising therapeutic target for antifungal agent development, with EHop-016 as a potential lead compound.

microbiology↗

bioRxiv2024

Discovering and exploring the hidden diversity of human gut viruses using highly enriched virome samples

Viruses are an abundant and crucial component of the human microbiome, but accurately discovering them via metagenomics is still challenging. Currently, the available viral reference genomes poorly represent the diversity in microbiome samples, and expanding such a set of viral references is difficult. As a result, many viruses are still undetectable through metagenomics even when considering the power of de novo metagenomic assembly and binning, as viruses lack universal markers. Here, we describe a novel approach to catalog new viral members of the human gut microbiome and show how the resulting resource improves metagenomic analyses. We retrieved >3,000 viral-like particles (VLP) enriched metagenomic samples (viromes), evaluated the efficiency of the enrichment in each sample to leverage the viromes of highest purity, and applied multiple analysis steps involving assembly and comparison with hundreds of thousands of metagenome-assembled genomes to discover new viral genomes. We reported over 162,000 viral sequences passing quality control from thousands of gut metagenomes and viromes. The great majority of the retrieved viral sequences ([~]94.4%) were of unknown origin, most had a CRISPR spacer matching host bacteria, and four of them could be detected in >50% of a set of 18,756 gut metagenomes we surveyed. We included the obtained collection of sequences in a new MetaPhlAn 4.1 release, which can quantify reads within a metagenome matching the known and newly uncovered viral diversity. Additionally, we released the viral database for further virome and metagenomic studies of the human microbiome.

microbiology↗

bioRxiv2024

First-night effect reduces the beneficial effects of sleep on visual plasticity and modifies the underlying neurochemical processes

Individuals experience difficulty falling asleep in a new environment, termed the first night effect (FNE). However, the impact of the FNE on sleep-induced brain plasticity remains unclear. Here, using a within-subject design, we found that the FNE significantly reduces visual plasticity during sleep in young adults. Sleep-onset latency (SOL), an indicator of the FNE, was significantly longer during the first sleep session than the second session, confirming the FNE. We assessed performance gains in visual perceptual learning after sleep and increases in the excitatory-to-inhibitory neurotransmitter (E/I) ratio in early visual areas during sleep using magnetic resonance spectroscopy and polysomnography. These parameters were significantly smaller in sleep with the FNE than in sleep without the FNE; however, these parameters were not correlated with SOL. These results suggest that while the neural mechanisms of the FNE and brain plasticity are independent, sleep disturbances temporarily block the neurochemical process fundamental for brain plasticity.

neuroscience↗

bioRxiv2024

Tcf12 controls dynamic calvarial bone growth and motor learning in mice

Heterozygous loss-of-function mutations of TCF12 and TWIST1 can each cause craniosynostosis and neurodevelopmental delay in humans. Twist1-Tcf12 interaction plays an important role in regulating suture development. Although the molecular and cellular mechanisms underlying craniosynostosis and neurocognitive dysfunctions in Twist1+/- mice have been studied, less information on the role of Tcf12 in these defects is available. To investigate the functional mechanism of Tcf12 in regulating skull and brain development, we analyzed the skull shape of Wnt1-Cre;Mesp1-Cre;Tcf12fl/fl mice and found that, despite mild coronal synostosis, their skull shape appears to be similar to that of controls. We also found evidence of impaired motor learning ability in Tcf12 mutant mice. Furthermore, loss of Tcf12 in neural crest lineage leads to upregulated Runx2 expression in the calvarial mesenchyme and posterior expansion of the frontal bone in Wnt1-Cre;Tcf12fl/fl mice. Mechanistically, we show that Lmx1b is a direct downstream target of Tcf12 for the regulation of osteogenic differentiation in the calvarial mesenchyme during embryonic development. Importantly, overexpression of Lmx1b inhibits osteogenic differentiation in the calvarial mesenchyme of Wnt1-Cre;Tcf12fl/fl mice, indicating Tcf12s regulation of Lmx1b expression is crucial for controlling osteogenesis during calvarial bone development. Our study suggests that Tcf12 expression in the brain is crucial for motor learning. Moreover, this study establishes a new molecular mechanism underlying regulation of calvarial bone formation. Author SummaryCraniosynostosis is characterized by premature fusion of cranial sutures and associated with abnormal skull growth, delayed brain development, and often impaired brain functions. Loss-of-function mutation of TCF12 can cause coronal synostosis and neurodevelopmental delay in humans. In developing mouse sutures, Tcf12 is essential for maintaining the boundary between sutural and osteogenic cells. However, roles of Tcf12 in skull formation and brain development have not been fully investigated. In this study, we show that loss of Tcf12 leads to brain abnormalities even in the absence of coronal synostosis and that frontal bone expansion results from upregulated osteogenic differentiation in the calvarial mesenchyme in mice. Furthermore, we identify Lmx1b as a downstream target of Tcf12 for the regulation of osteogenic differentiation in the calvarial mesenchyme during frontal bone development. Our findings highlight the role of Tcf12 in the development of calvarial bones and provide new insight into molecular mechanisms for regulation of calvarial bone formation.

developmental biology↗

bioRxiv2023

The Ptk2-Pma1 pathway enhances tolerance to terbinafine in Trichophyton rubrum

The increasing prevalence of dermatophyte resistance to terbinafine, a key drug in the treatment of dermatophytosis, represents a significant obstacle to treatment. Trichophyton rubrum is the most commonly isolated fungus in dermatophytosis. In T. rubrum, we identified TERG_07844, a gene encoding a previously uncharacterized putative protein kinase, as an ortholog of budding yeast Saccharomyces cerevisiae polyamine transport kinase 2 (Ptk2) and found that T. rubrum Ptk2 (TrPtk2) is involved in terbinafine tolerance. In both T. rubrum and S. cerevisiae, Ptk2 knockout strains were more sensitive to terbinafine compared to the wild types, suggesting that promotion of terbinafine tolerance is a conserved function of fungal Ptk2. The T. rubrum Ptk2 knockout strain ({Delta}TrPtk2) was sensitive to omeprazole, an inhibitor of plasma membrane proton pump Pma1, which is activated through phosphorylation by Ptk2 in S. cerevisiae. Overexpression of T. rubrum Pma1 (TrPma1) in {Delta}TrPtk2 suppressed terbinafine sensitivity, suggesting that the induction of terbinafine tolerance by TrPtk2 is mediated by TrPma1. Furthermore, omeprazole increased the terbinafine sensitivity of clinically isolated terbinafine-resistant strains. These findings suggest that, in dermatophytes, the TrPtk2-TrPma1 pathway plays a key role in promoting intrinsic terbinafine tolerance and may serve as a potential target for combinational antifungal therapy against terbinafine- resistant dermatophytes.

microbiology↗

bioRxiv2023

Neonics and mites can interfere with extending the bee-colony longevity during wintering

A mathematical model that can estimate the apparent longevity of bee-colony proposed by the authors to clarify that the longevity normally changes with seasons as follows: The longevity maintains almost constant from at 20 to 30 days from late spring to late autumn, thereafter, it continues to extend till late spring up to 160 to 200 days. Immediately after overwintering, the longevity is shortened almost vertically from one sixth to one tenth. Such normal seasonal changes in longevity are shown when a pesticide-free food is administered to the bee-colony and when pesticide-containing sugar syrup is. However, abnormal seasonal changes in longevity, which does not extend even if winter approaches, are shown for the bee-colony ingesting neonicotinoid-containing pollen and for the colony infested with Varroa mites. Judging from the fact that pollen is the main food for the bee-brood, that mites parasitize larvae and pupae, and that the vital functions and organs of honeybees are created during the larval and pupal stages, it can be inferred that a neonicotinoid-containing pollen paste and parasitic mites cause serious damage to the bee ability to detect the arrival of winter. Such dysfunction in the larval and pupal stages probably interferes with extending the longevity of adult bees even as winter approaches.

zoology↗

bioRxiv2023

Decoupling of the Onset of Anharmonicity between a Protein and Its Surface Water around 200 K

The protein dynamical transition at [~] 200 K, where the biomolecule transforms from a harmonic, non-functional form to an anharmonic, functional state, has been thought to be slaved to the thermal activation of dynamics in its surface hydration water. Here, by selectively probing the dynamics of protein and hydration water using elastic neutron scattering and isotopic labelling, we found that the onset of anharmonicity in the two components around 200 K are decoupled. The one in protein is an intrinsic transition, whose characteristic temperature is independent of the instrumental resolution time, but varies with the biomolecular structure and the amount of hydration, while the one of water is merely a resolution effect.

biophysics↗

bioRxiv2023

Improved Protein Model in SPICA Force Field

The previous version of the SPICA coarse-grained (CG) force field (FF) protein model focused primarily on membrane proteins and successfully reproduced the dimerization free energies of several transmembrane helices and stable structures of various membrane protein assemblies. However, that model had limited accuracy when applied to other proteins, such as intrinsically disordered proteins (IDPs) and peripheral proteins, because the dimensions of the IDPs in an aqueous solution were too compact, and protein binding on the lipid membrane surface was over-stabilized. To improve the accuracy of the SPICA FF model for the simulation of such systems, in this study we introduce protein secondary structure-dependent nonbonded interaction parameters to the backbone segments and re-optimize almost all nonbonded parameters for amino acids. The improved FF proposed here successfully reproduces the radius of gyration of various IDPs, the binding sensitivity of several peripheral membrane proteins, and the dimerization free energies of several transmembrane helices. The new model also shows improved agreement with experiments on the free energy of peptide association in water. In addition, an extensive library of nonbonded interactions between proteins and lipids, including various glycerophospholipids, sphingolipids, and cholesterol, allows the study of specific interactions between lipids and peripheral and transmembrane proteins. Hence, the new SPICA FF (version 2) proposed herein is applicable with high accuracy for simulating a wide range of protein systems. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/557852v2_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@8b026dorg.highwire.dtl.DTLVardef@4c622forg.highwire.dtl.DTLVardef@c2081forg.highwire.dtl.DTLVardef@a34b69_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

Evaluation of the appropriateness of pesticide administration concentrations in long-term field experiments and modest proposal

For investigating the effects of pesticides on bee colonies in actual apiaries, field experiments in an open environment are effective. It is natural to assume that the total food or pesticide intake taken by a honey bee colony includes their intakes from nature in addition to each administered in the field experiment. Therefore, in field experiments, setting the administration-concentration of pesticides for honey bee colonies is one of the most difficult tasks. The validity of pesticide concentrations administered to bee colonies was evaluated by various intakes of pesticides and foods obtained from five long-term field experiments conducted in Midwest Japan with distinct changes of four seasons and Maui, U.S.A. without cold winter. The author found a relationship, logPBD=logCP-2.4, between pesticide concentration (CP) and the average daily intake of pesticide ingested by one bee (PBD), and found that their ratio PBD/CP was about 1/250, which was significantly lower than the LD50 value. This fact supports that the concentration of pesticide administration in long-term field experiments conducted so far was not too high, but rather reasonable. In addition, the average daily intakes of sugar syrup and pollen paste ingested by one bee was found to be 3.1 mg/bee/day and 0.8 mg/bee/day, respectively, and notwithstanding their wide variation, be roughly constant regardless of the concentration of pesticides. I believe that this paper will help provide new guidelines for setting pesticide dosing concentrations, which have not been clear so far, in field experiments on the administration of pesticides to bee colonies.

ecology↗

bioRxiv2023

Mega-Enhancer Bodies Organize Neuronal Long Genes in the Cerebellum

Exceptionally long genes and cis-regulatory enhancers are selectively activated in mammalian brain neurons, and these loci are mutation hotspots in neurological disorders. However, the organization of these large genomic elements at the level of chromosome folding, beyond local enhancer-promoter interactions, remains poorly understood. Here, we report the discovery of a nuclear subcompartment in the mouse cerebellum formed by near-megabase long enhancers and their associated long genes encoding synaptic or signaling proteins. Genomic regions within this subcompartment reside in the outer-half of the nucleus, separated from other transcriptionally active structures. Using an in vivo CRISPR genetic mini-screen, we uncover a specific role for the transcription factor Etv1 in coupling the compartmentalization of neuronal long genes with their expression. Together, our study defines mechanisms that organize transcriptionally active genes across chromosomes in the mammalian brain.

neuroscience↗