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Yamashita, T.

Publications and source records attributed to Yamashita, T..

At least 19 recordsLinked to original sources

A chromosome-level genome assembly of the classical model nematode Panagrellus redivivus for comparative studies of parasitism evolution

Panagrellus redivivus is a free-living nematode that served as an early model system for nematode biology prior to Caenorhabditis elegans. As a member of Clade IV, a clade harboring major plant-parasitic (e.g., Bursaphelenchus xylophilus) and animal-parasitic (e.g., Strongyloides spp.) species, P. redivivus represents a crucial, phylogenetically proximate non-parasitic reference for investigating the evolution of parasitism. Here, we present a chromosome-level genome assembly of P. redivivus constructed using Oxford Nanopore long reads, Illumina short reads, and Hi-C scaffolding. The assembly is resolved into five chromosome-scale scaffolds, with completeness validated by Hi-C contact maps and the identification of a non-canonical telomeric repeat motif. The assembly demonstrates high gene-space completeness (97.1% BUSCO) and contains 17,839 predicted protein-coding genes. Comparative orthology analysis revealed extensive orthogroup sharing between P. redivivus and Clade IV parasites, enabling comparative evolutionary studies with reduced confounding effects from deep lineage divergence. This high-quality genome provides a key resource for investigating the genetic basis of distinctive biological traits, including ovoviviparity, as well as gene family dynamics and parasite evolution.

genomics↗

Extraocular Photoreception Controls nocturnal hatching via a neuroendocrine pathway in False Clownfish

Hatching is the transition from embryo to larva in oviparous animals. Birds, reptiles, and insects rupture the eggshell mechanically, whereas fish and amphibians digest the egg envelope using hatching enzymes. When and where larvae emerge influences their fitness. Many species regulate hatching timing using environmental cues, such as light, oxygen availability, and physical stimuli. In fish, hatching is driven by a neuroendocrine event that triggers hatching enzyme secretion; however, the mechanisms controlling this event are unknown. Using the false clownfish Amphiprion ocellaris, which hatches synchronously within 1-2 h after sunset, we showed that the role of thyrotropin-releasing hormone (TRH) as a hatching hormone, previously established in zebrafish and medaka, extended to clownfish, where TRH activated Ca2+; signaling in hatching gland cells through its receptor trhra, reinforcing the TRH-Ca2+-hatching enzyme axis as a conserved core module of teleost hatching. In clownfish, this module was additionally placed under upstream control by extraocular photoreception because dark-induced hatching persisted in eyeless embryos. This photoreceptive system was most sensitive to green light (550 nm), and several opsins with absorbance maxima in the corresponding middle- to long-wavelength range were expressed outside the eyes of pre-hatching embryos. Coupling extraocular photoreception to a conserved neuroendocrine module placed a critical life-history transition under stringent photic control, releasing larvae only in complete darkness, an adaptation that may have underpinned the success of this lineage in predator-rich coral reefs.

zoology↗

Sensory neuron dysfunction and hyperexcitability in dorsal root ganglia at disease onset in the SOD1G93A mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder traditionally characterized by motor neuron degeneration, but emerging evidence indicates sensory system involvement. Despite reports of sensory abnormalities in some patients, the molecular and functional alterations in dorsal root ganglion (DRG) neurons remain insufficiently characterized. We investigated DRG pathology at disease onset in 12-week-old SOD1G93A mice using an integrated transcriptomic, morphological, and electrophysiological approach. RNA sequencing of lumbar DRG identified 35 differentially expressed genes, predominantly upregulated, enriched in oxidative stress-related and phagosome pathways. Comparative analysis with motor neuron transcriptomes revealed distinct gene expression profiles, indicating sensory neuron-specific molecular responses. Immunohistochemistry demonstrated reduced soma diameter in both A- and C-fiber DRG neurons. Nav channel colocalization increased for Nav1.7 in A fibers and Nav1.8 in both fiber types, whereas Nav1.6 was unchanged. Whole-cell patch-clamp recordings showed depolarized resting membrane potential, increased spike amplitude, and enhanced repetitive firing in A-fiber neurons, consistent with hyperexcitability, while C fibers showed no significant functional changes. These findings demonstrate early molecular, structural, and functional alterations in primary sensory neurons in ALS, supporting pathology beyond motor neurons and identifying sensory neuron excitability as a potential therapeutic target.

neuroscience↗

KK-LC-1 reveals evolutionary divergence in the regulation of sperm motility between humans and mice

CT83 (KK-LC-1) is a cancer-testis antigen originally identified in human lung cancer cells and has recently attracted attention as a potential target for cancer therapy. Although KK-LC-1 orthologs have been identified in up to 160 animal species, a murine homolog had not previously been identified, hindering in vivo analysis of its physiological function. In this study, we identified the mouse homolog of KK-LC-1 and performed a comparative analysis of its properties in humans and mice, together with an investigation of its biological function using gene knockout (KO) mice. The murine Kk-lc-1 gene is located on the X chromosome and, like its human counterpart, contains an N-terminal transmembrane domain. In both humans and mice, KK-LC-1 is expressed specifically in the testis and localizes to the head and tail regions of sperm. Analysis of Kk-lc-1-deficient mice revealed normal spermatogenesis, and both male and female KO mice were fertile. However, sperm from Kk-lc-1-deficient males exhibited reduced motility caused by decreased flexibility of the midpiece and failed to penetrate the oocyte zona pellucida in vitro. This defect was rescued by artificial insemination using epididymal sperm, suggesting that maternal factors in vivo may compensate for reduced sperm motility. Although impaired sperm motility during in vitro fertilization (IVF) was rescued by murine Kk-lc-1, functional rescue by human KK-LC-1 was not observed. These findings indicate that KK-LC-1 contributes to sperm motility but is not essential for fertility. Moreover, species-specific differences in KK-LC-1-mediated regulation of sperm motility suggest functional divergence during evolution. The role of KK-LC-1 in sperm motility should therefore be considered in the clinical development of cancer therapies targeting KK-LC-1.

developmental biology↗

Self-regenerating opsin from reef-building coral

Opsins underlie diverse physiological responses to light in animals. In the dark, most opsins bind the chromophore 11-cis retinal, which isomerizes to all-trans form upon light absorption, representing the initial key step in signaling. Maintenance of opsin function therefore requires continuous regeneration of the inactive, 11-cis-retinal-bound state. Here, we report a novel type of opsin, AtAntho2c, from a reef-building coral, whose active form, bound to all-trans retinal, can thermally revert to the initial dark state bound to 11-cis retinal. A cysteine residue in extracellular loop 2 region plays a key role in the self-regeneration ability. Using time-resolved and low-temperature spectroscopies, we identify two spectrally distinct photointermediates prior to the all-trans to 11-cis isomerization in AtAntho2c, whose formation rates and yields are found to vary depending on temperature and pH conditions. The active form of AtAntho2c activates Gi/o G protein, resulting in a transient and repeatable decrease in cellular cAMP levels upon repeated light stimulations, even in the absence of exogenous retinal in cultured cells. Furthermore, we confirm that cells expressing AtAntho2c exhibit membrane hyperpolarization via GIRK channel activation light-dependently. These properties highlight the potential of AtAntho2c as a versatile optogenetic actuator capable of repeatedly modulate Gi/o signaling without retinal supplementation. Significance StatementLight-sensitive proteins, opsins, form active states upon light absorption, leading to intracellular G protein signaling and various cellular outputs. The active states require specific enzymatic machinery or another photon absorption to regenerate inactive opsins ready to respond to repeated light stimuli and maintain continuous responsiveness. In our study, we identify and analyze a coral opsin of which the active state rapidly and autonomously reverts to the inactive state in the dark through thermal isomerization of the retinal chromophore within the opsin. This regeneration mechanism allows the opsin to respond to repeated light stimuli at high temporal resolution and maintain large signal amplitude without the need for exogenous retinal making this opsin potentially useful for developing versatile optogenetic tools.

biophysics↗

Single-cell RNA sequencing reveals collagen interactions between detached osteoclasts and activated fibroblasts in granulation tissue surrounding sequestra in medication-related osteonecrosis of the jaw

ObjectivesMedication-related osteonecrosis of the jaw (MRONJ) is a rare but serious complication of anti-resorptive agents used for osteoporosis and bone metastasis. As the global population ages, the number of patients with MRONJ continues to rise. Resorptive osteoclasts firmly attached to bone surfaces cannot be detected using single-cell RNA-Seq (scRNA-Seq); however, we hypothesized that multinucleated giant and detached osteoclasts (DetOCs) can be detected by this method. This study aimed to characterize DetOCs with scRNA-seq. Materials and MethodsscRNA-Seq and immunohistochemistry staining was performed on granulation tissues surrounding sequestra from four patients with MRONJ and radicular cysts, a common odontogenic infectious disease with normal bone metabolism. ResultsscRNA-Seq and immunohistochemistry staining revealed that DetOCs were detected exclusively in the granulation tissues of MRONJ. These DetOCs expressed well-known osteoclast markers as well as COL27A1. Subcluster analysis revealed that SPP1+ TREM2+ macrophages differentiated into DetOCs and expressed several types of collagens. Immunostaining confirmed the expression of COL27A1 not only in DetOCs in MRONJ but also in osteoclasts in mandibular cancer. CellChat analysis revealed interactions mediated by collagen signaling pathways between DetOCs, myeloid cells, and activated fibroblasts. ConclusionsWe successfully characterized DetOCs and identified a distinctive microenvironment involving DetOCs and activated fibroblasts in the granulation tissues surrounding sequestra in MRONJ. Clinical RelevanceIdentification of DetOCs and their unique interaction with activated fibroblasts in MRONJ lesions provides novel insight into the pathophysiology of sequestrum formation, suggesting potential therapeutic targets to ameliorate MRONJ.

immunology↗

MDA5 multimerization on LINE RNA drives pathogenic extracellular immune complexes in autoimmunity

Autoantibodies are hallmarks of many autoimmune diseases, but their potential pathogenic roles, particularly for those targeting intracellular proteins, remain unclear. Anti-MDA5-positive dermatomyositis (anti-MDA5 DM) is characterized by autoantibodies against the intracellular protein MDA51,2, a conserved innate immune receptor that recognizes viral dsRNA by forming filaments3. Here, using four patient-derived monoclonal autoantibodies (mAbs), we reconstitute and define the molecular architecture, biogenesis, and immunological activity of pathogenic MDA5 immune complexes. Our cryo-EM analysis revealed that these mAbs bind dsRNA-scaffolded MDA5 filaments in at least two distinct binding modes, each exhibiting striking epitope convergence using germline-encoded residues. Extracellular immune complexes formed between mAbs and filamentous, but not monomeric, MDA5 potently activate multiple innate immune pathways, with the magnitude of activation determined by antibody binding mode and immune complex stoichiometry. Antibody bivalency further crosslinks MDA5 filaments into higher-order aggregates with heightened immunostimulatory activity, demonstrating an active role of autoantibodies in shaping immune complex architecture. Analysis of patient plasma reveals elevated levels of extracellular MDA5 filaments and identifies LINE retroelement-derived dsRNA as a structural scaffold. Notably, MDA5 immune complexes induce endogenous LINE dsRNA expression, likely promoting additional MDA5 filament formation and extracellular release through inflammatory cell death. These data thus support a self-amplifying inflammatory cycle as a pathogenic mechanism for anti-MDA5 DM. Collectively, our study defines a broadly applicable architectural principle, in which higher-order organization and binding modes of autoantibodies--beyond antibody affinity or nucleic acid presence alone--govern innate immune activation.

biochemistry↗

Capturing self-renewing multipotent neural crest stem cells from human pluripotent stem cells

During embryonic development, neural crest cells (NCCs) represent a multipotent population characterized by an inherently transient nature, rapidly differentiating into various lineages. This instability has presented a fundamental challenge, as it is exceedingly difficult to maintain these cells in a stable, multipotent state in vitro. In this study, we report a robust culture system dependent on Wnt and FGF signaling that enables the long-term (>6 months) expansion of human iPSC-derived neural crest stem cells (NCSCs). These NCSCs retain their self-renewal and differentiation capacity, validated at the single-cell clonal level. ATAC-seq analysis indicated that posterior NCSCs maintain a more permissive chromatin structure at neuronal gene loci. Furthermore, ChIP-seq analysis revealed that the key transcription factor SOX10 binds to the regulatory regions of genes involved in both maintenance and differentiation. This system provides a stable source of human NCSCs, offering a valuable platform for developmental biology, disease modeling, and regenerative medicine.

developmental biology↗

Paracrine Factor Local Gradient-Generating System for Engineering Perfusable Vascularized Hepatocyte Tissues with Perfusion-Induced Proliferation

Donor organ shortages drive the urgent need for engineered hepatocyte tissues, yet functional vascular integration remains a major bottleneck in liver tissue engineering. Current vascularization strategies struggle to achieve perfusable microvessels that penetrate hepatocyte tissue. Furthermore, in vitro recapitulation of hepatic regeneration remains a major challenge. This study presents a paracrine factor local gradient (PFLG)-generating system that constructs vascularized, perfusable hepatocyte tissues and recapitulates the perfusion-mediated proliferative capacity of primary hepatocytes. The PFLG-generating platform integrates fibroblast-loaded cryogels with a microfluidic device to direct angiogenesis prior to hepatocyte seeding, thereby enabling microvessels to penetrate three-dimensional hepatocyte tissue. Within the vascularized constructs, microvessels directly penetrated the hepatocyte parenchyma, recapitulating intimate hepatocyte-microvessel contact in vivo. These constructs enhanced hepatocyte polarity and functional bile canaliculi. Importantly, under perfusion culture, robust hepatocyte proliferation was induced, with Ki67-positive hepatocytes increasing significantly, including mitotic cells while preserving polarity. By contrast, this proliferative response was minimal under static conditions. Time-lapse imaging and functional assays confirmed perfusion through penetrating microvessels. These findings demonstrate that perfusion-mediated cues are essential for inducing hepatocyte proliferation while maintaining functional polarity. This modular and programmable culture platform lays the foundation for scale-up toward transplantable liver tissues.

bioengineering↗

How opsins diversified after the teleost whole-genome duplication: Insights from two parietopsins of the red piranha, Pygocentrus nattereri

The teleost whole-genome duplication (TGD) contributed to functional diversification of opsins. Some TGD paralogs, including those of parapinopsin (PP), Vertebrate Ancient (VA) opsin, and long wavelength-sensitive (LWS) opsin, show different absorption spectra and/or expression patterns. However, our knowledge of detailed evolutionary processes and mechanisms by which TGD contributed to opsin diversification is still limited. Here, we report that TGD paralogs of parietopsin (PT1 and PT2) are retained in extant species. While most species, including the zebrafish, have only PT1, several characins, including the red piranha (Pygocentrus nattereri), retain both PT1 and PT2. The Mexican tetra (Astyanax mexicanus) and catfishes have only PT2. To assess the degree of functional diversification between PT1 and PT2, we characterized spectral properties and expression patterns. Maximum absorption spectra differ slightly among PTs. Those of red piranha PT1, PT2, Mexican tetra PT2, and Japanese catfish (Silurus asotus) PT2 were located at 517 nm, 528 nm, 517 nm, and ~535 nm, respectively. Fluorescence in situ hybridization showed that (1) piranha PT1 and PT2 are expressed in the same pineal cells, and (2) they are also co-expressed with PP1. Based on these findings, functional diversification of opsins after gene duplication is discussed.

evolutionary biology↗

Kidney-specific lipid metabolism underlies stable chemical individuality in domestic cats and other felids

Domestic cats uniquely accumulate lipid droplets (LDs) in renal proximal tubular epithelial cells, but their chemical composition and biological functions remain unknown. Here, we identify a distinctive renal specialization in felids: lipid droplets enriched in branched-chain fatty acids (BFAs). These BFAs accumulate as triglyceride components and are excreted in urine as free fatty acids with stable and individually distinctive profiles. Behavioral assays show that cats discriminate urine samples based on BFA composition, even against complex volatile backgrounds, suggesting BFAs as robust, metabolically encoded olfactory signatures for individual recognition. Comparative analyses of non-domesticated felids reveal that BFA production is broadly conserved but exhibits both species-specific and individual variation. These findings demonstrate how a core metabolic pathway has been evolutionarily co-opted into a chemical signature system. This discovery highlights an unusual integration of renal physiology with social communication and expand our understanding of lipid metabolism, organ-level adaptation, and the evolution of chemical individuality in mammals. Significance statementUnique lipid droplets in felid kidneys reveal how organ-specific metabolism can be adapted for social communication.

zoology↗

Flow-driven construction of capillary-scale vessels with predefined geometries in natural hydrogels

Forming capillary networks with predefined geometries is a critical challenge in engineering complex three-dimensional tissues in vitro. While bioprinting and microfluidic technologies have enabled vascular tissue fabrication, precise control over capillary-scale vascularization remains limited. In this study, we investigated vascular formation process along hydrogel microchannels to elucidate mechanisms governing capillary-scale lumen formation. Microchannels were fabricated by femtosecond laser ablation in collagen and fibrin hydrogels. We optimized multiphoton lithography parameters to fabricate microchannels within these hydrogels and analyzed vascular formation along the channels. Luminal vascular structures formed readily in 50-m channels, while vascular formation failed in 20-m channels under static conditions, suggesting a significant shift in endothelial organization at the cellular scale. Flow stimulation significantly promoted vessel formation through collective endothelial cell migration and adhesion, whereas static conditions induced endothelial-to-mesenchymal transition. These findings provide key insights into capillary-scale vascularization and contribute to the development of more complex architectures with predefined shapes.

bioengineering↗

A key spectral tuning site of UV-sensitive vertebrate non-visual opsin Opn5

Opsins are photoreceptive proteins responsible for visual and non-visual photoreceptions in animals. In general, vertebrates have multiple visual and non-visual opsins whose spectral sensitivities range from the UV to the red region. Among these opsins, Opn5 has been widely identified in vertebrates from fishes to primates and functions as a non-visual opsin in various tissues including the retina and brain. Vertebrate Opn5 has been characterized as a UV-sensitive bistable opsin. Thus, Opn5 provides one of the molecular mechanisms determining the short wavelength limit that vertebrates can detect. In this study, we searched for the amino acid residue responsible for the UV light sensitivity of Opn5. Our mutational analysis revealed that Opn5 acquired visible light sensitivity by the substitution of Lys91 with an amino acid other than arginine or tyrosine residue. In addition, the mutations at Lys91 altered the preferential binding of the retinal isomers in Opn5. Therefore, the conservation of Lys91 among vertebrate Opn5 proteins would be necessary to enable Opn5 to work as the shortest wavelength sensor in various tissues.

biochemistry↗

FcγR- and CD9-dependent synapse engulfing microglia in the thalamus drives cognitive impairment following cortical brain injury

Various states of microglia appear in neuroinflammation, but their impact on brain function and behavior is not fully understood. Here we report that synapse engulfing microglia in the thalamus are crucial for cognitive impairment after cortical brain injury. Region-specific manipulations of reactive microglia in the chronic phase of injuries showed that microglial changes in the thalamus, but not in the hippocampus, impaired recognition memory. Single-cell RNA-sequencing analysis revealed the enrichment of synapse engulfing microglia in the thalamus, which developed in a CD9-dependent manner and caused synaptic loss and recognition memory deficits. In the thalamus, the blood-brain barrier was disrupted, and extravasated {gamma}-immunoglobulins (IgG) co-localized with synapse engulfing microglia. Fc{gamma} receptor III blockade in the thalamus reduced synapse engulfing microglia, synapse loss, and recognition memory deficits. These findings demonstrate that the induction of synapse engulfing microglia in the thalamus by extravasated IgG/Fc{gamma}RIII and CD9 signals causes recognition memory deficits after cortical brain injury.

immunology↗

Mouse-Geneformer: A Deep Learning Model for Mouse Single-Cell Transcriptome and Its Cross-Species Utility

Deep learning techniques are increasingly utilized to analyze large-scale single-cell RNA sequencing (scRNA-seq) data, offering valuable insights from complex transcriptome datasets. Geneformer, a pre-trained model using a Transformer Encoder architecture and human scRNA-seq datasets, has demonstrated remarkable success in human transcriptome analysis. However, given the prominence of the mouse, Mus musculus, as a primary mammalian model in biological and medical research, there is an acute need for a mouse-specific version of Geneformer. In this study, we developed a mouse-specific Geneformer (mouse-Geneformer) by constructing a large transcriptome dataset consisting of 21 million mouse scRNA-seq profiles and pre-training Geneformer on this dataset. The mouse-Geneformer effectively models the mouse transcriptome and, upon fine-tuning for downstream tasks, enhances the accuracy of cell type classification. In silico perturbation experiments using mouse-Geneformer successfully identified disease-causing genes that have been validated in in vivo experiments. These results demonstrate the feasibility of analyzing mouse data with mouse-Geneformer and highlight the robustness of the Geneformer architecture, applicable to any species with large-scale transcriptome data available. Furthermore, we found that mouse-Geneformer can analyze human transcriptome data in a cross-species manner. After the ortholog-based gene name conversion, the analysis of human scRNA-seq data using mouse-Geneformer, followed by fine-tuning with human data, achieved cell type classification accuracy comparable to that obtained using the original human Geneformer. In in silico simulation experiments using human disease models, we obtained results similar to human-Geneformer for the myocardial infarction model but only partially consistent results for the COVID-19 model, a trait unique to humans (laboratory mice are not susceptible to SARS-CoV-2). These findings suggest the potential for cross-species application of the Geneformer model while emphasizing the importance of species-specific models for capturing the full complexity of disease mechanisms. Despite the existence of the original Geneformer tailored for humans, human research could benefit from mouse-Geneformer due to its inclusion of samples that are ethically or technically inaccessible for humans, such as embryonic tissues and certain disease models. Additionally, this cross-species approach indicates potential use for non-model organisms, where obtaining large-scale single-cell transcriptome data is challenging. Author SummaryResearchers have developed Geneformer, a powerful tool that utilizes advanced deep learning techniques and large-scale single-cell transcriptome data to analyze human cell genetic activity. However, given the extensive use of mice (Mus musculus) in medical and biology research, there is a need for a similar tool tailored to this model organism. To address this gap, we developed mouse-Geneformer, an adaptation of Geneformer trained on a large dataset of mouse single-cell RNA sequencing data obtained from 20 million cells. Mouse-Geneformer demonstrates high accuracy in identifying distinct cell types and predicting disease-causing genes in gene manipulation simulation experiments. Moreover, mouse-Geneformer exhibited comparable accuracy to the original human Geneformer, even when applied to human cell data, suggesting its potential for cross-species use. For instance, it performed well in studying heart disease but was less consistent with COVID-19, likely due to the differences between species in how they react to the virus. Overall, mouse-Geneformer could be a valuable resource for studying not only mice but also other animals, especially when large-scale data are challenging to obtain. Furthermore, this cross-species approach may probe beneficial in human research, especially for tissues that are difficult to access, such as embryonic samples.

bioinformatics↗

Ex vivo SIM-AFM measurements reveal the spatial correlation of stiffness and molecular distributions in 3D living tissue

Living tissues each exhibit a distinct stiffness, which provides cells with key environmental cues that regulate their behaviors. Despite this significance, our understanding of the spatiotemporal dynamics and the biological roles of stiffness in three-dimensional tissues is currently limited due to a lack of appropriate measurement techniques. To address this issue, we propose a new method combining upright structured illumination microscopy (USIM) and atomic force microscopy (AFM) to obtain precisely coordinated stiffness maps and biomolecular fluorescence images of thick living tissue slices. Using mouse embryonic skin as a representative tissue with mechanically heterogeneous structures inside, we validate the measurement principle of USIM-AFM. Live measurement of tissue stiffness distributions revealed the highly heterogeneous mechanical nature of embryonic skin as well as the role of collagens in maintaining its integrity. Furthermore, quantitative comparisons of stiffness distributions of preserved tissue samples unveiled the distinct impacts of conventional tissue preservation techniques on the tissue stiffness pattern. This series of experiments highlights the importance of live mechanical testing of tissue-scale samples. Our USIM-AFM technique provides a new methodology to reveal the dynamic nature of tissue stiffness and its correlation with biomolecular distributions in live tissues and thus could serve as a technical basis for exploring tissue-scale mechanobiology. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/595975v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@8a32f5org.highwire.dtl.DTLVardef@117a350org.highwire.dtl.DTLVardef@e2fb99org.highwire.dtl.DTLVardef@1f41d24_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

A large pocket structure surrounding the catalytic center in the BCG protein from Mycobacterium tuberculosis

The widespread prevalence of tuberculosis and the need for extensive chemotherapy in its treatment are caused by the ability of tuberculosis (TB) bacteria to enter a dormant state. Therefore, it is crucial to develop effective compounds that can inhibit the growth of TB bacteria in both the active and dormant states. Previous research has identified agelasine D (marine sponge-derived diterpene alkaloid) capable of resisting dormant mycobacteria. Additionally, BCG3185c has been designated as a target mycobacterial protein. In this study, the crystal structure of BCG3185c was determined at a resolution of 1.86 [A]. Analysis of this crystal structure revealed a large pocket toward the catalytic center of BCG3185c, which is potentially adequate for agelasine D binding. Based on the results of the interaction analysis between agelasine D and Bacillus Calmette-Guerin (BCG) proteins, it was inferred that the binding of agelasine D to this large pocket is crucial for suppressing the growth of Mycobacterium tuberculosis.

biochemistry↗

NMDAR Phosphoproteome Controls Synaptic Growth and Learning

In the mammalian brain, NMDA receptors (NMDARs) activation triggers a calcium-dependent signal transduction cascade resulting in postsynaptic remodeling and behavioral learning. However, the phosphoprotein signal flow through this transduction network is poorly understood. Here, we show that NMDAR-dependent phosphorylation drives the assembly of protein signaling complexes that regulate synaptic morphology and behavior. We performed large-scale phosphoproteomic analyses of protein kinase target proteins in successive layers of the signaling network in mouse striatal/accumbal slices. NMDARs activation resulted in the phosphorylation of 194 proteins, including Rho GTPase regulators. CaMKII-mediated phosphorylation of ARHGEF2 increased its RhoGEF activity, thereby activating the RhoA-Rho-kinase pathway. Subsequent phosphoproteomics of Rho-kinase revealed 221 protein targets, including SHANK3. Experimental validation revealed a pathway from NMDAR-dependent calcium influx through CaMKII, ARHGEF2, Rho-kinase, and SHANK3 to coordinate assembly of an actin-tethered postsynaptic complex of SHANK3/NMDAR/PSD95/DLGAP3 for spine growth and aversive learning. These findings show that NMDARs initiate metabolic phosphorylation for learning.

neuroscience↗