Biology subjects
Publications and source records attributed to Tanaka, K..
We report the complete genome sequences of eight Fusobacterium watanabei clinical isolates, ranging from 1.95 to 2.09 Mbp. Analysis against the Genome Taxonomy Database (GTDB) indicates that Fusobacterium watanabei genomes are part of the "Fusobacterium nucleatum_J" group, which also encompasses the previously published FNU strain and Fna C1 isolates.
Sustained labeling of fluids is crucial for their investigation in animal models. Here, we introduce a mouse line (Alb-mSc-ST), where blood and interstitial fluid are labeled with the red fluorescent protein mScarlet and SpyTag. The SpyTag-SpyCatcher technology is exploited to monitor circulating fluid properties by biosensors or detect blood-brain barrier disruption. This approach represents a valuable tool for studying vascular structure, permeability and microenvironment in body organs in vivo.
The small GTPase RAS is a signalling hub activating multiple pathways, which may respond differently to a constitutively active RAS mutation. We explored this issue using fission yeast, where RAS-mediated pheromone signalling (PS) activates the MAPKSpk1 and Cdc42 pathways. We observed that in cells with the yeast RAS mutant, ras1.G17V, the MAPKSpk1 activation was attenuated similarly to that in wildtype cells, whereas Cdc42 activation was prolonged. We built a mathematical model implementing PS negative-feedback circuits and competition between the two Ras1 effectors, MAPKKKByr2 and Cdc42-GEFScd1. The model robustly predicted the MAPKSpk1 activation dynamics of an additional 20 PS mutants. In support of the model, a recombinant Cdc42-GEFScd1 competed with MAPKKKByr2 for Ras1 binding and overexpression of the Ras binding domain of either Cdc42-GEFScd1 or MAPKKKByr2 inhibited both downstream pathways. Our study has established that the constitutively active RAS signalling propagates differently to downstream pathways where the system prevents MAPK overactivation.
Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) provide therapeutic benefit in EGFR mutation-positive non-small cell lung cancer, but some individuals develop early resistance. We performed spatial transcriptomics analysis of pre- and posttreatment tumor samples from the same patients to explore the underlying mechanisms of such early resistance. The proportion and activation of fibroblasts increased in association with the development of early resistance, whereas a distinct tumor cell cluster showed activation of tumor necrosis factor- signaling via the NF-{kappa}B pathway even before treatment. Also in the early resistance sample, specific tumor cell clusters interacted with immune and stromal cells. Immature tertiary lymphoid structures (TLSs) were enriched in the early resistance sample, whereas mature TLSs were observed in the long-term response sample. These findings implicate tumor heterogeneity and an inflammatory tumor microenvironment in early EGFR-TKI resistance, providing insight into potential therapeutic strategies to improve treatment outcomes.
Anomer-retaining glycoside hydrolases (GHs) generally catalyze a double displacement reaction via a covalent intermediate; however, neutron crystallography of glycoside ligand-bound states has not been performed. In this study, we investigated {beta}-glucosidase Td2F2 from GH family 1 as a model enzyme for anomer-retaining GHs. We determined joint X-ray/neutron structures of Td2F2 in ligand-free form, covalent intermediate with a 2-deoxy-2-fluoro glucoside (2F-Glc) inhibitor, and glucose product complex using hydrogen/deuterium-exchanged crystals at room temperature, with neutron diffraction resolutions of 1.80-1.70 [A]. Extensive hydrogen bonds recognizing the hydroxy groups of 2F-Glc were identified, along with the positions of deuterium atoms. The acid/base catalyst residue Glu166 was anchored by a hydrogen bond network pivoted by Asn293. Tyr295 forms a hydrogen bond with the catalytic nucleophile residue Glu352 in the ligand-free and glucose complex forms, while the active center undergoes significant reorganization, including side chain displacements of Glu352 and Tyr295, as well as the incorporation of a water molecule. An alternative conformation of Tyr295 was observed in the 2F-Glc structure at room temperature, suggesting its role in positioning the nucleophilic water during the deglycosylation step. The tyrosine hydrogen bonded to the nucleophile is also conserved in many other anomer-retaining GH families, underscoring its importance in catalysis. Based on the deuterium/hydrogen positions determined from neutron structures, we proposed a detailed reaction mechanism for Td2F2. Significance StatementGlycoside hydrolases perform diverse functions in organisms, with over 180 known enzyme families. Although hydrogen bonds and proton transfer play important roles in enzymatic reactions, hydrogen atoms are generally invisible in macromolecular X-ray crystallography. In anomer-retaining glycoside hydrolases, general acid/base catalysis and the formation and hydrolysis of a covalent glycosyl-enzyme intermediate have been postulated. Here, we report neutron crystal structures of a {beta}-glucosidase, where hydrogen and deuterium atoms were visualized at high resolution. An intricate hydrogen-bonding network and remarkable remodeling at the catalytic center were observed during covalent intermediate formation, revealing a detailed catalytic mechanism. The enzyme belongs to glycoside hydrolase family 1 and represents numerous enzymes employing the anomer-retaining mechanism.
Responding to stress caused by various environmental changes is essential for living organisms. In cyanobacteria that perform oxygenic photosynthesis, the highly conserved histidine kinase Hik33/NblS homologs respond to diverse stressors such as high light, low temperature, high salts, high osmolarity, and reactive oxygen species. However, how this unique protein kinase responds to such divergent stresses remains unknown. This study has focused on the underlying stress sensing mechanism of NblS in Synechococcus elongatus PCC 7942. First, the sensory response by NblS was analyzed in vivo by monitoring the NblS-regulated hliA transcript accumulation with treatment of various benzoquinone reagents known as photosystem II (PSII) electron acceptors. It was found that molecular responses induced by various stresses were diminished in the presence of 2,6-dichloro-1,4-benzoquinone, which accepts electrons specifically from the PSII-bound plastoquinone QB. Cell fractionation analysis indicated that NblS was localized in the thylakoid membrane, which was consistent with its predicted membrane-spanning structure. In the thylakoid membrane, NblS was found in approximately 400 kDa and 800 kDa unknown complexes in clear native PAGE (CN-PAGE). Immunoprecipitation analysis of the cross-linked thylakoid membrane revealed that NblS is associated with D2 and CP47 proteins but not with CP43 protein, and thus it was suggested that dimeric NblS is associated with RC47-like complex, an assembly intermediate complex of PSII, as [RC47like-NblS2] (370 kDa) or [RC47like-NblS2]2 (740 kDa). We propose that the redox status of an RC47-bound plastoquinone molecule is a cue for the NblS response.
Differentiated muscle cells contain myofibrils and well-organized organelles, enabling powerful contractions. Muscle cell reorganization occurs in response to various physiological stimuli; however, the mechanisms behind this remodeling remain enigmatic due to the lack of a genetically trackable system. Previously, we reported that a subset of larval muscle cells is remodeled into adult abdominal muscle through an autophagy-dependent mechanism in Drosophila. To unveil the underlying mechanisms of this remodeling, we performed a comparative time-course RNA-seq analysis of isolated muscle cells with or without autophagy. It revealed both transcriptional dynamics independent of autophagy and highlighted the significance of BNIP3-mediated mitophagy in muscle remodeling. Mechanistically, we found that BNIP3 recruits autophagic machinery to mitochondria through its LC3-interacting (LIR) motif and minimal essential region (MER), which interact with Atg8a and Atg18a, respectively. Loss of BNIP3 leads to a substantial accumulation of larval mitochondria, ultimately impairing muscle remodeling. In summary, this study demonstrates that BNIP3-dependent mitophagy is critical for orchestrating the dynamic process of muscle remodeling. Impact statementA time-course RNA-seq analysis of muscle remodeling reveals transcriptional dynamics independent of autophagy and highlights the significance of BNIP3 in mitochondrial degradation in vivo.
The fermentative production of valuable chemicals from lignocellulosic feedstocks has attracted considerable attention. Although Saccharomyces cerevisiae is a promising microbial host, it lacks the ability to efficiently metabolize xylose, a major component of lignocellulosic feedstocks. The xylose oxidative pathway offers advantages such as simplified metabolic regulation and fewer enzymatic steps. Specifically, the pathway involves the conversion of xylose into 2-keto-3-deoxy-xylonate, which can be channeled into two distinct pathways, the Dahms pathway and the Weimberg pathway. However, the growth of yeast on xylose as the sole carbon source through the xylose oxidative pathway has not been achieved, limiting its utilization. We successfully engineered S. cerevisiae to metabolize xylose as its sole carbon source via the xylose oxidative pathways, achieved by enhancing enzyme activities through iron metabolism engineering and rational enzyme selection. We found that increasing the supply of the iron-sulfur cluster to activate the bottleneck enzyme XylD by BOL2 disruption and tTYW1 overexpression facilitated the growth on xylose and the production of ethylene glycol at 1.5 g/L via the Dahms pathway. Furthermore, phylogenetic analysis of xylonate dehydratases led to the identification of a highly active homologous enzyme. A strain possessing the Dahms pathway with this highly active enzyme exhibited reduced xylonate accumulation. Furthermore, the introduction of enzymes based on phylogenetic tree analysis allowed for the utilization of xylose as the sole carbon source through the Weimberg pathway. This study highlights the potential of iron metabolism engineering and phylogenetic enzyme selection for the development of non-native metabolic pathways in yeast. Key pointsO_LI1.5 g/L ethylene glycol was produced via Dahms pathway in S. cerevisiae. C_LIO_LIEnzyme activation enabled growth on xylose via both the Dahms and Weimberg pathways. C_LIO_LITested enzymes in this study may expand application of xylose oxidative pathway. C_LI
Cholinergic interneurons of the dorsomedial striatum may play a role in social hierarchy dynamics. A social hierarchy is an organization of individuals by rank that occurs in social animals. Establishing a new social hierarchy involves flexible behavior in deciding whether to be a winner or loser, experience of winning or losing, and stabilization of rank. The neural circuits underlying such flexible behavior have yet to be fully understood, but previous research indicates that cholinergic interneurons in the dorsomedial striatum play a role in behavioral flexibility. We used the dominance tube test to measure ranking within group housed mice, before and after between-cage competitions using the same test. We found that the experience of winning or losing against mice from different cages not only contributes to new social hierarchies among the competitors, but also causally influences the subsequent social hierarchy among their cage mates in the home cage - supporting the hypothesis of winner-loser effects on later social ranking. To test the hypothesis that cholinergic interneurons contribute to social hierarchy dynamics, we made a selective lesion of cholinergic interneurons in the dorsomedial striatum. The lesion did not prevent social hierarchy formation among pairs of similarly ranked individuals from different cages. However, it reduced the loser effect of external competition on the subsequent home-cage rankings in dominant mice. In light of these results we suggest that cholinergic interneurons in dorsomedial striatum increase the flexibility of social hierarchy dynamics. Significance statementThe effect of winning or losing a competition on subsequent ranking in mouse home cage social hierarchies was examined using the dominance tube test. We found that losing, when dominant mice were defeated by equally ranked mice from another cage, led to decreased social rank in their home cage. Conversely, winning by initially subordinate mice led to increased rank in the home cage social hierarchy. The loser effect on subsequent behavior in dominant mice was reduced after selective lesions of the cholinergic interneurons of the dorsomedial striatum. We suggest that losing might produce these effects by altering the activity of cholinergic interneurons, and thus modulating synaptic plasticity in neural circuits involved in flexible decision making and positive reinforcement.
Extracellular ATP (eATP) signaling in Arabidopsis thaliana is mediated by the purinoceptor P2K1. Previous studies have clarified that the downstream transcriptional responses to eATP involve jasmonate (JA)-based signaling components such as the JA receptor (COI1) and JA-responsive bHLH transcription factors (MYCs). However, the specific contributions of JA signaling itself on eATP signaling are unexplored. Here, we report that JA primes plant responses to eATP through P2K1. Our findings show that JA treatment significantly upregulates P2K1 transcription, corroborating our observation that JA facilitates eATP-induced cytosolic calcium elevation and transcriptional reprogramming in a JA signaling-dependent manner. Additionally, we find that salicylic acid pretreatment represses eATP-induced plant response. These results suggest that JA accumulation during biotic or abiotic stresses may potentiate eATP signaling, enabling plants to better cope with subsequent stress events. SIGNIFICANCE STATEMENTPlant hormone jasmonate (JA) enhances plant responses to extracellular ATP (eATP) in Arabidopsis thaliana through a mechanism dependent on the JA receptor COI1 and the eATP receptor P2K1. The reciprocal amplification of these signals provides a mechanistic explanation for how plants effectively respond to different stress events.
Although placental mammals are at high risk of maternal-fetal immune conflict, it is unclear how the semi-allogeneic fetus avoids rejection. Cholesterol sulfate (CS) is a bioactive metabolite that inhibits leukocyte migration and activation. This study investigated the roles and cells associated with CS in pregnant mice and humans. CS was sequentially produced by maternal-derived endometrial cells and fetal-derived placental trophoblasts before and after placentation. When mated with allogeneic males, CS-deficient mice showed increased fetal resorption rates under induced placental inflammation. This phenotype disappeared when the activity of the CS-producing enzyme was restored in the placenta. Placental CS levels were reduced in patients with "villitis of unknown etiology." Thus, we uncovered the spatiotemporal control of CS production and its relevance to local immunosuppression during pregnancy.
ParMs generally exist on low copy number plasmids where they contribute to plasmid segregation and stable inheritance. We carried out bioinformatics analysis, which indicated that ParM genes are not only confined to plasmids but are also occasionally found on genomes. Here we report the discovery and characterization of two chromosome encoded ParMs (cParMs) from the genomes of Desulfitobacterium hafniense (Dh-cParM1) and Clostridium botulinum (Cb-cParM). Both cParMs form filaments, exhibit nucleotide hydrolysis, and possess characteristic ParM subunit structures. Dh-cParM1 forms single and tightly coupled double filaments and is highly conserved on the chromosomes of five of six Desulfitobacterium species. Interestingly, these bacteria have not been reported to harbour plasmids. Cb-cParM possesses unique properties. Its filaments were stable after nucleotide hydrolysis and Pi release, and its ParR, (Cb-cParR) did not affect the initial stage of Cb-cParM polymerization but displayed properties of a depolymerization factor for mature filaments. These results indicate functional, polymerizing ParMs can be encoded on genomes, suggesting that ParM roles may extend to other functions beyond plasmid segregation.
The Auxin Response Factors (ARFs) family of transcription factors are the central mediators of auxin-triggered transcriptional regulation. Functionally different classes of extant ARFs operate as antagonistic auxin-dependent and -independent regulators. While part of the evolutionary trajectory to the present auxin response functions has been reconstructed, it is unclear how ARFs emerged, and how early diversification led to functionally different proteins. Here, we use in silico and in vivo analyses to revisit the molecular events that led to the origin and subsequent evolution of the ARFs. We reveal the shared origin of ARFs from preexisting domains, uncovering a protein fold homologous to the ARF DNA-binding fold in a conserved eukaryotic chromatin regulator. Building on this, we reconstruct the complete evolutionary history of ARFs, including the divergence events leading to the appearance of the ARF classes and defining the main molecular targets for their functional diversification. We derive a complete evolutionary trajectory that led to the emergence of the nuclear auxin signalling pathway.
The p16-3MR mouse model has previously been reported to effectively visualize and eliminate senescent cells in vivo. However, we now report significant issues with this model. Noninvasive bioluminescence imaging without shaving the black fur detected almost no luminescence signals, but they became visible after shaving. However, these signals were very faint and attributed to the auto-luminescence of coelenterazine-h, rather than reporter gene expression. Further experiments showed no significant bioluminescent changes with aging, doxorubicin treatment, or during wound healing, contradicting earlier reports. Comparative analysis between p16-3MR mice from different sources revealed no differences, suggesting functional issues with Renilla luciferase (Rluc) and herpes simplex virus 1 thymidine kinase (HSV-TK) expression in this mouse model. Our findings underscore the necessity of using white-furred mice or ensuring proper fur shaving for accurate in vivo bioluminescence imaging. We recommend the reevaluation of previously published studies that used p16-3MR mice without proper controls to ensure data accuracy. These results provide critical insights for researchers using p16-3MR mice in senescence studies.
Endothelial cell responses to fluid shear stress from blood flow are crucial for vascular development, function and disease. A complex of PECAM-1, VE-cadherin, VEGF receptors (VEGFRs) and PlexinD1 located at cell-cell junctions mediates many of these events. But available evidence suggests that another mechanosensor upstream of PECAM-1 initiates signaling. Hypothesizing that GPCR and G proteins may serve this role, we performed siRNA screening of G subunits and found that Gi2 and Gq/11 are required for activation of the junctional complex. We then developed a new activation assay, which showed that these G proteins are activated by flow. We next mapped the G residues required for activation and developed an affinity purification method that used this information to identify latrophilin-2 (Lphn-2/ADGRL2) as the upstream GPCR. Latrophilin-2 is required for all PECAM-1 downstream events tested. In both mice and zebrafish, latrophilin-2 is required for flow-dependent angiogenesis and artery remodeling. Furthermore, endothelial specific knockout demonstrates that latrophilin plays a role in flow-dependent artery remodeling. Human genetic data reveal a correlation between the latrophilin-2-encoding Adgrl2 gene and cardiovascular disease. Together, these results define a pathway that connects latrophilin-dependent G protein activation to subsequent endothelial signaling, vascular physiology and disease.
Histones bind directly to DNA and play a role in regulating gene expression mediated by chromatin structure. DNA sequences of these histone genes are quite similar, which has hindered individual analyses. The exact function of the 13 different isoforms of histone H2B remains unclear. In this report, a comprehensive gene expression analysis of the H2B isoforms, focusing on tissue specificity, was conducted. We generated mice lacking the H2bc27 gene, which exhibited brain-specific expression of E14.5, and proceeded to characterize the brain tissue. The phenotype of H2bc27 knockout brains was similar to that of wild-type brains, yet transcriptome analysis indicated that H2bc27 is associated with regulating the expression of several functional genes involved in mouse brain development. The methods used in this study may serve to facilitate comprehensive H2B isoform analysis.
The cucumber (Cucumis sativus) is an economically important vegetable crop cultivated and consumed worldwide. Despite its popularity, the manner in which cucumbers were dispersed from their origin in South Asia to the rest of the world, particularly to the east, remains a mystery due to the lack of written records. In this study, we performed genotyping-by-sequencing (GBS) on 723 worldwide cucumber accessions, mainly deposited in the Japanese National Agriculture and Food Research Organization (NARO) Genebank, to characterize their genetic diversity, relationships, and population structure. Analyses based on over 60,000 genome-wide single-nucleotide polymorphisms identified by GBS revealed clear genetic differentiation between Southeast and East Asian populations, suggesting that they reached their respective region independently, not progressively. A deeper investigation of the East Asian population identified two subpopulations with different fruit characteristics, supporting the traditional classification of East Asian cucumbers into two types thought to have been introduced by independent routes. Finally, we developed a core collection of 100 accessions representing at least 93.2% of the genetic diversity present in the entire collection. The genetic relationships and population structure, their associations with geographic distribution and phenotypic traits, and the core collection presented in this study are valuable resources for elucidating the dispersal history and promoting the efficient use and management of genetic resources for research and breeding in cucumber. Key messageGenotyping-by-sequencing of 723 worldwide cucumber genetic resources revealed that cucumbers were dispersed eastward via at least three distinct routes, one to Southeast Asia and two from different directions to East Asia.
SpinX, an AI-guided spindle tracking software, allows the 3-dimensional (3D) tracking of metaphase spindle movements in mammalian cells. Using over 900 images of dividing cells, we create the Multi-SpinX framework to significantly expand SpinXs applications: a) to track spindles and cell cortex in multicellular environments, b) to combine two object tracking (spindle with kinetochores marked by centromeric probes) and c) to extend spindle tracking beyond metaphase to prometaphase and anaphase stages where spindle morphology is different. We have used a human-in-the-loop approach to assess our optimisation steps, to manually identify challenges and to build a robust computational pipeline for segmenting kinetochore pairs and spindles. Spindles of both H1299 and RPE1 cells have been assessed and validated for use through Multi-SpinX, and we expect the tool to be versatile in enabling quantitative studies of mitotic subcellular dynamics.