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Watanabe, R.

Publications and source records attributed to Watanabe, R..

At least 19 recordsLinked to original sources

HPV-Associated Tonsillar Intraepithelial Neoplasia and Oral High-Risk HPV mRNA as a Candidate Biomarker

Early detection of human papillomavirus (HPV)-associated oropharyngeal cancer is an unmet need, as its premalignant lesions remain unestablished. We hypothesized that such lesions exist in individuals with oral high-risk HPV (HR-HPV) mRNA. We preoperatively screened 1,180 individuals undergoing tonsillectomy due to benign conditions and detected five with oral HR-HPV mRNA. Serial sectioning of the entire excised tonsils identified HPV-associated intraepithelial lesions in three of these five individuals. A similar lesion was identified in an additional individual with oral HPV16 infection with unknown oral HR-HPV mRNA status. Intraepithelial lesions in two individuals were HPV16-associated and showed moderate-to-severe atypia, whereas those in the other two individuals were associated with other HR-HPV and showed minimal-to-mild atypia. Based on HPV life cycle, the former were classified as transforming lesions and the latter as productive-to-intermediate lesions. Spatial transcriptomic analysis revealed molecular profiles intermediate between those of normal epithelium and HPV-associated oropharyngeal cancer. These findings establish the first evidence for HPV-associated tonsillar premalignant lesions and suggest oral HR-HPV mRNA as a candidate biomarker.

cancer biology↗

Unsupervised alignment reveals shared but locally refined colour qualia structure from toddlerhood to adulthood

Whether subjective colour experience (e.g., experience of 'red') is shared across children and adults remains a foundational question in developmental psychology and consciousness research. To test this, we compared the relational structures of their colour experiences and identified the mapping that best preserved these structures. Using pairwise similarity judgements for 13 basic colours, we applied Gromov Wasserstein optimal transport (GWOT), an unsupervised method requiring no prespecified stimulus experience correspondence to compare toddlers, preschool children, and adults. Preschoolers' and adults' group-averaged structures perfectly aligned, whereas toddlers' structure did not fully align with either older group. However, removing specific colours, particularly those with relatively late acquired linguistic boundaries, such as light blue-restored near-perfect alignment. Moreover, toddlers with greater colour-word knowledge showed closer correspondence with the adult similarity structure. These findings suggest that the global relational geometry of colour experience is largely established by toddlerhood, with limited local differences linked to colour-language acquisition.

neuroscience↗

Structural heterogeneity of CTE Type I tau filaments with an interface-shifted polymorph

Tau filament types can differ in inter-protofilament packing while sharing the same protofilament fold. Motivated by our recent findings in vacuolar tauopathy, we analyzed cryo-electron microscopy (cryo-EM) data from brain-derived chronic traumatic encephalopathy (CTE) tau filaments deposited in EMPIAR-10313. We resolved the canonical CTE Type I structure and a previously unreported CTE Type I-like structure with a shifted protofilament interface at 2.78 and 2.96 A, respectively. Mapping particle-state assignments back onto the original micrographs showed that the CTE Type I and CTE Type I-like packing states can occupy locally contiguous regions within the same fibrils. These findings identify an interface-shifted CTE Type I-like structure and support its local coexistence with CTE Type I within individual fibrils.

molecular biology↗

Heterotypic interactions and sequence features modulate cellular reflectin condensate dynamics

Reflectin proteins drive dynamic structural coloration in cephalopods by organizing into dense intracellular lamellar structures that dictate local refractive index. While reconstituted reflectins readily undergo liquid-liquid phase separation in vitro, these assemblies frequently undergo dynamic arrest, vitrifying into non-dynamic condensates. Here, we investigate how primary sequence features, post-translational modifications, and heterotypic interactions regulate the material properties of reflectin condensates within the environment of mammalian HeLa cells. Using confocal microscopy and fluorescence recovery after photobleaching (FRAP), we demonstrate that canonical block copolymeric A-type reflectins readily form dynamically arrested condensates, with the linker blocks primarily responsible for the observed arrest. In contrast, non-canonical B/C reflectin variants exhibit significantly greater fluidity and rapid recovery kinetics. We show that phosphomimetic substitutions progressively fluidize some reflectin condensates. Lastly, we find that heterotypic condensates composed of canonical and non-canonical reflectins in combinations associated with reversible iridescence in squid substantially enhance canonical mobility. Our findings establish a biophysical framework in which phosphorylation and heterotypic mixing cooperatively suppress dynamic arrest, enabling the reversible material transitions required for active cephalopod camouflage and communication.

biophysics↗

Dissociable pupil-linked arousal during overt and inner speech

Inner (covert) speech, the silent production of language in ones mind, plays a central role in human cognition and is known to activate speech-related cortical regions while also requiring executive and attentional resources. However, the conventional focus on cortical similarity or dissimilarity between inner and overt speech leaves unaddressed the question of whether arousal systems sustain cortical language processing. Here, using pupillometry across three controlled counting experiments, we show a dissociation between inner and overt speech. Overt speech consistently led to pupil dilation across all conditions. Inner speech, by contrast, produced a strikingly different profile: pupil constriction during simple counting, no deviation from baseline during noun-phrase counting, and clear dilation only under a high cognitive load, but with dilation remaining significantly less than that for overt speech in every condition. Covert verbal production thus proceeds at a reduced arousal cost, yet this cost scales flexibly with task demands rather than being fixed. This pupillometric dissociation reveals that inner and overt speech occupy fundamentally distinct arousal states, even when their cortical substrates partially overlap. These findings open a non-invasive window for tracking inner speech physiology across development, individual differences, and clinical populations in whom inner speech is altered.

neuroscience↗

Platform of single-molecule enzyme activity-based liquid biopsy for detection of pancreatic adenocarcinoma at early stages

Liquid biopsy analyzes biomolecules in biofluids to provide critical information for the early detection of diseases and the development of more effective treatment strategies. Among these biomolecules, the functional states of proteins offer particularly valuable insights due to their direct correlation with phenotypic changes. However, current approaches for assessing protein function in liquid biopsies are often limited by low detection sensitivity. Here, we present a liquid biopsy platform that analyzes single-molecule protease/peptidase activity to detect pathological alterations in enzyme function in blood samples. The platform demonstrates potential for identifying patients with early-stage (stage I-II) pancreatic ductal adenocarcinoma (PDAC).

biochemistry↗

Extracellular stiffness regulates cell fate determination and drives the emergence of evolutionary novelty in teleost heart

The diversification of life is driven by the acquisition of new phenotypic traits, known as evolutionary novelties. While some genetic mechanisms underlying such traits have been identified, the role of physical properties of cellular and extracellular components remains largely unexplored. Here, we show that the evolution and development of the bulbus arteriosus (BA)-a teleost-specific, smooth muscle-rich heart outflow tract-is regulated by the mechanical properties of its extracellular environment. Specifically, we demonstrate that the teleost-specific extracellular matrix gene elastin b confers the uniquely low stiffness of the BA. In contrast to the BA, the homologous organ in non-teleost actinopterygians, the conus arteriosus (CA), and the ventricle are predominantly composed of cardiomyocytes, and exhibit higher extracellular stiffness. Loss of elastin b function by knockdown or knockout leads to ectopic cardiomyocyte formation in the BA, accompanied by increased extracellular stiffness comparable to that of the ventricle. Furthermore, artificial stiffening of the BA extracellular environment is sufficient to induce ectopic cardiomyocyte differentiation. Taken together, these findings demonstrate that extracellular stiffness governs cell fate determination and highlight its role in the emergence of evolutionary novelties in the teleost heart. Significant StatementThe acquisition of new phenotypic traits is a key driver for a diversification of life. While genetic underpinnings of such traits have been well studied, the contribution of physical properties remains elusive. Here, we show that extracellular stiffness plays a key role in the evolution and development of the bulbus arteriosus (BA), a smooth muscle-based cardiac outflow tract unique to teleost hearts. We demonstrate that elastin b, a teleost-specific gene, confers characteristically low stiffness to the BA, which is required for proper cell fate determination. Notably, such low stiffness is absent in non-teleost fishes and in teleosts with impaired elastin b function, and artificial stiffening alters cell fate. Our findings highlight the importance of physical properties in driving evolutionary novelties.

developmental biology↗

Repositioning of polyubiquitin alters the pathologic tau filament structure

Structurally diverse tau filaments form proteinaceous aggregates in a heterogeneous group of neurodegenerative diseases called tauopathies1. The factors extrinsic to the highly ordered core structure that influence tau filament stability are not well understood. Here, we found that polyubiquitinated tau filaments from Alzheimers disease and vacuolar tauopathy human brain tissue exhibit distinct seeding patterns in mice, in association with differences in tau filament ultrastructure determined by cryo-electron microscopy. Interestingly, chemical modulation of the polarity of polyubiquitin adjacent to the tau core with the small molecule ubistatin B resulted in the repositioning of poorly structured densities towards positively charged residues on the highly structured core filament, leading to shifting of the protofilament-protofilament interface of certain vacuolar tauopathy tau filaments. These results suggest that the structure of tau filaments that are associated with different seeding activities in vivo can be influenced by post-translational modifications.

neuroscience↗

Robust CENP-A incorporation in human cells is independent of transcription and cohesin components

Centromeres are essential chromosomal components that ensure proper cell division by serving as assembly sites for kinetochores, which connect chromosomes to spindle microtubules. Centromeres are marked by the evolutionarily conserved centromere-specific histone H3 variant, CENP-A, which is deposited into centromere nucleosomes during G1 in human cells. Centromeres retain cohesin, a ring-like protein complex during mitosis, protecting sister chromatid cohesion and centromere transcription to prevent chromosome missegregation. Previous work in Drosophila has suggested that centromere transcription and centromeric RNAs are important for CENP-A deposition in chromatin. During mitosis centromeric cohesin is critical for centromere transcription. However, it is not clear how or if centromeric transcription and cohesin contribute to CENP-A deposition in G1 in human cells. To address these questions, we combined a cell synchronization strategy with the Auxin Inducible Degron technology and transcription inhibition in human cells. In contrast to Drosophila cells, our results demonstrated that neither centromeric transcription nor cohesin is required for CENP-A deposition in human cells. Our data demonstrate clear differences in the CENP-A deposition mechanism between human and Drosophila cells. These findings provide deeper insights into the plasticity underlying centromere maintenance and highlight evolutionary divergence in centromere maintenance systems across species.

cell biology↗

Downregulation of Semaphorin 4A in keratinocytes reflects the features of non-lesional psoriasis

AbstractPsoriasis is a multifactorial disorder mediated by IL-17-producing T cells, involving immune cells and skin-constituting cells. Semaphorin 4A (Sema4A), an immune semaphorin, is known to take part in T helper type 1/17 differentiation and activation. However, Sema4A is also crucial for maintaining peripheral tissue homeostasis and its involvement in skin remains unknown. Here, we revealed that while Sema4A expression was pronounced in psoriatic blood lymphocytes and monocytes, it was downregulated in the keratinocytes of both psoriatic lesions and non-lesions compared to controls. Imiquimod application induced more severe dermatitis in Sema4A knockout (KO) mice compared to wild-type (WT) mice. The naive skin of Sema4AKO mice showed increased T cell infiltration and IL-17A expression along with thicker epidermis and distinct cytokeratin expression compared to WT mice, which are hallmarks of psoriatic non-lesions. Analysis of bone marrow chimeric mice suggested that Sema4A expression in keratinocytes plays a regulatory role in imiquimod-induced dermatitis. The epidermis of psoriatic non-lesion and Sema4AKO mice demonstrated mTOR complex 1 upregulation, and the application of mTOR inhibitors reversed the skewed expression of cytokeratins in Sema4AKO mice. Conclusively, Sema4A- mediated signaling cascades can be triggers for psoriasis and targets in the treatment and prevention of psoriasis.

immunology↗

Subnanometer structure of medusavirus capsid during maturation using cryo-electron microscopy

Medusavirus is a giant virus classified into an independent family of Mamonoviridae. Medusavirus-infected amoebae release immature particles in addition to the virions. These particles were suggested to exhibit the maturation process of this virus, but the structure of these capsids during maturation remains unknown. Here, we applied a block-based reconstruction method in cryo-electron microscopy (cryo-EM) single particle analysis to these viral capsids, extending these resolutions to 7-10 [A]. The map revealed a novel network consisted of minor capsid proteins (mCPs), supporting major capsid proteins (MCPs). A predicted molecular model of the MCP fitted into the cryo-EM map clarified the boundaries between the MCP and the underlining mCPs and between the MCP and the outer spikes, identifying the molecular interactions between the MCP and these components. Several structural changes of mCPs were observed beneath the 5-fold vertices of immature particles, depending on the presence or absence of the underlying internal membrane. Furthermore, the lower part of penton proteins on the 5-fold vertices were also lost in the mature virions. These dynamic structural changes of mCPs exhibit important functions in the maturation process of medusavirus. ImportanceStructural changes in giant virus capsids during maturation have not been known. Medusavirus is a unique giant virus, where amoebae infected with the virus release immature particles in addition to mature virions. In this study, immature and mature medusavirus particles were investigated using cryo-electron microscopy, and firstly reported the structural changes in the viral capsid during maturation. In DNA empty particles, the conformation of the minor capsid proteins dynamically changed depending on the presence or absence of the underlying internal membranes. In DNA full particles, the lower part of the penton proteins was lost. These are the first report of the structural changes in the viral capsid during giant virus maturation.

molecular biology↗

Intracellular Ebola Virus nucleocapsid assembly revealed by in situ cryo-electron tomography.

Filoviruses, including Ebola and Marburg viruses, cause hemorrhagic fevers with up to 90% lethality. The viral nucleocapsid is assembled by polymerization of the nucleoprotein (NP) along the viral genome, together with the viral proteins VP24 and VP35. We employed cryo-electron tomography of cells transfected with viral proteins and infected with model Ebola virus, to illuminate assembly intermediates as well as a 9[A] map of the complete intracellular assembly. This structure reveals a previously unresolved, third, and outer layer of NP complexed with VP35. The intrinsically-disordered-region together with the C-terminal domain of this outer layer of NP provides the constant-width between intracellular nucleocapsid bundles and likely functions as a flexible tether to the viral matrix protein in virion. A comparison of intracellular nucleocapsid with prior in-virion nucleocapsid structures reveals the nucleocapsid further condenses vertically in-virion. The interfaces responsible for nucleocapsid assembly are highly conserved and offer targets for broadly effective antivirals.

microbiology↗

The cryo-EM structure of the CENP-A nucleosome in complex with ggKNL2

Centromere protein A (CENP-A) nucleosome is an epigenetic marker that specifies centromere position. The Mis18 complex is a licensing factor for new CENP-A deposition via the CENP-A chaperone, Holliday junction recognition protein (HJURP) on the centromere chromatin. Chicken KINETOCHORE NULL2 (KNL2) (ggKNL2), a Mis18 complex component, has a CENP-C-like motif, and our previous study suggested that ggKNL2 directly binds to the CENP-A nucleosome to recruit HJURP/CENP-A to the centromere. However, the molecular basis for CENP-A nucleosome recognition by ggKNK2 remains unclear. Here, we present the cryo-EM structure of the chicken CENP-A nucleosome in complex with a ggKNL2 fragment containing a CENP-C-like motif. Chicken KNL2 distinguishes between CENP-A and histone H3 in the nucleosome using the CENP-C-like motif and its downstream region. Both the C-terminal tail and RG-loop of CENP-A are simultaneously recognized as CENP-A characteristics. The CENP-A nucleosome-ggKNL2 interaction is thus essential for CENP-A deposition. Furthermore, our structural, biochemical, and cell biology data indicate that ggKNL2 alters its binding partner at the centromere during chicken cell cycle progression.

biochemistry↗

Particle morphology of medusavirus inside and outside the cells reveals a new maturation process of giant viruses

Medusavirus, a giant virus, is phylogenetically closer to eukaryotes than the other giant viruses and has been recently classified as an independent species. However, details of its morphology and maturation process in host cells remain unclear. Here, we investigated the particle morphology of medusavirus inside and outside infected cells using conventional transmission electron microscopy (C-TEM) and cryo-electron microscopy (cryo-EM). The C-TEM of amoeba infected with the medusavirus showed four types of particles: empty, DNA-full, and the corresponding intermediates. Time-dependent changes in the proportion and following intracellular localization of these particles suggested a new maturation process for the medusavirus. Empty particles and viral DNAs were produced independently in the cytoplasm and nucleus, respectively, and only empty particles located near the nucleus incorporated the viral DNA into the capsid. All four types of particles were also found outside the cells. The cryo-EM of these particles showed that the intact capsid structure, covered with three different types of spikes, was conserved among all particle types, although with minor size-related differences. The internal membrane exhibited a structural array similar to that of the capsid, interacted closely with the capsid, and displayed open membrane structures in the empty and empty-intermediate particles. This result suggests that the open structures in the internal membrane are used for an exchange of scaffold proteins and viral DNA during the maturation process. This new model of the maturation process of medusavirus provides insight into the structural and behavioral diversity of giant viruses. ImportanceGiant viruses exhibit diverse morphologies and maturation processes. In the present study, medusavirus showed four types of particle morphologies both inside and outside the infected cells, when propagated in the laboratory using amoeba culture. Time-course analysis of the medusavirus particles in the infected cells reveals a new maturation process. Empty viral particles and viral DNAs were produced independently in the cytoplasm and nucleus, and only the empty particles located near the nucleus incorporated the viral DNA. Consequently, many immature particles, along with mature virions, were released from the host cells. Except for showing a small change in size, the capsid structures were well preserved during the maturation process. The empty viral particles and corresponding intermediates showed open membrane structures, which are presumably used for exchanging scaffold proteins and viral DNAs.

microbiology↗

Pain burden, sensory profile and inflammatory cytokines of dogs with naturally-occurring neuropathic pain treated with gabapentin alone or with meloxicam

Canine neuropathic pain (NeuP) has been poorly investigated. This study aimed to evaluate the pain burden, sensory profile and inflammatory cytokines in dogs with naturally-occurring NeuP. Twenty-nine client-owned dogs with NeuP were included in a prospective, partially masked, randomized crossover clinical trial, and treated with gabapentin/placebo/gabapentin-meloxicam or gabapentin-meloxicam/placebo/gabapentin (each treatment block of 7 days; total 21 days). Pain scores, mechanical (MNT) and electrical (ENT) nociceptive thresholds and descending noxious inhibitory controls (DNIC) were assessed at baseline, days 7, 14, and 21. DNIC was evaluated using {Delta}MNT (after-before conditioning stimulus). Positive or negative {Delta}MNT corresponded to inhibitory or facilitatory pain profiles, respectively. Data from baseline were compared to those of sixteen healthy controls. {Delta}MNT, but not MNT and ENT, was significantly larger in controls (2.3 {+/-} 0.9 N) than in NeuP (-0.2 {+/-} 0.7 N). The percentage of dogs with facilitatory sensory profile was similar at baseline and after placebo (61.5-63%), and between controls and after gabapentin (33.3-34.6%). Pain scores were lower than baseline after gabapentin or gabapentin-meloxicam. Cytokine levels were not different between groups or treatments. Dogs with NeuP have deficient inhibitory pain mechanisms. Pain burden was reduced after gabapentin and gabapentin-meloxicam depending on the pain scoring instrument used.

neuroscience↗

PEA15 loss of function and defective cerebral development in the domestic cat

Cerebral cortical size and organization are critical features of neurodevelopment and human evolution, for which genetic investigation in model organisms can provide insight into developmental mechanisms and the causes of cerebral malformations. However, some abnormalities in cerebral cortical proliferation and folding are challenging to study in laboratory mice due to the absence of gyri and sulci in rodents. We report an autosomal recessive allele in domestic cats associated with impaired cerebral cortical expansion and folding, giving rise to a smooth, lissencephalic brain, and that appears to be caused by homozygosity for a frameshift in PEA15 (phosphoprotein expressed in astrocytes-15). Notably, previous studies of a Pea15 targeted mutation in mice did not reveal structural brain abnormalities. Affected cats, however, present with a non-progressive hypermetric gait and tremors, develop dissociative behavioral defects and aggression with age, and exhibit profound malformation of the cerebrum, with a 45% average decrease in overall brain weight, and reduction or absence of the ectosylvian, sylvian and anterior cingulate gyrus. Histologically, the cerebral cortical layers are disorganized, there is substantial loss of white matter in tracts such as the corona radiata and internal capsule, but the cerebellum is relatively spared. RNA-seq and immunohistochemical analysis reveal astrocytosis. Fibroblasts cultured from affected cats exhibit increased TNF-mediated apoptosis, and increased FGFb-induced proliferation, consistent with previous studies implicating PEA15 as an intracellular adapter protein, and suggesting an underlying pathophysiology in which increased death of neurons accompanied by increased proliferation of astrocytes gives rise to abnormal organization of neuronal layers and loss of white matter. Taken together, our work points to a new role for PEA15 in development of a complex cerebral cortex that is only apparent in gyrencephalic species. SummaryGyrification is the neurodevelopmental process in certain mammalian species during which the cerebral cortex expands and folds resulting in the classic wrinkled appearance of the brain. Abnormalities in this process underlie many congenital malformations of the brain. However, unlike many other human malformations, genetic insight into gyrification is not possible in laboratory mice because rodents have a lissencephalic or smooth cerebral cortex. We identified a mutation in domestic cats that likely causes failure of the cerebral cortex to expand and fold properly, and discovered that the mutation impairs production of a protein, PEA15 (phosphoprotein expressed in astrocytes-15), involved in intracellular signaling. Affected cats have profound abnormalities in brain development, with minimal changes in their superficial behavior and neurologic function. Additional studies of tissue and cultured cells from affected animals suggest a pathophysiologic mechanism in which increased death of neurons accompanied by increased cell division of astrocytes gives rise to abnormal organization of neuronal layers and loss of white matter. These results provide new insight into a developmental process that is unique to animals with gyrencephalic brains.

genetics↗

Parkinson's Disease-linked LRRK2 structure and model for microtubule interaction

Leucine Rich Repeat Kinase 2 (LRRK2) is the most commonly mutated gene in familial Parkinsons disease. LRRK2 is proposed to function in membrane trafficking and co-localizes with microtubules. We report the 3.5[A] structure of the catalytic half of LRRK2, and an atomic model of microtubule-associated LRRK2 built using a reported 14[A] cryo-electron tomography in situ structure. We propose that the conformation of LRRK2s kinase domain regulates its microtubule interaction, with a closed conformation favoring binding. We show that the catalytic half of LRRK2 is sufficient for microtubule binding and blocks the motility of the microtubule-based motors kinesin and dynein in vitro. Kinase inhibitors that stabilize an open conformation relieve this interference and reduce LRRK2 filament formation in cells, while those that stabilize a closed conformation do not. Our findings suggest that LRRK2 is a roadblock for microtubule-based motors and have implications for the design of therapeutic LRRK2 kinase inhibitors.

biophysics↗

The in situ structure of Parkinson’s disease-linked LRRK2

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of familial Parkinsons disease. LRRK2 is a multi-domain protein containing a kinase and GTPase. Using in situ cryo-electron tomography and subtomogram averaging, we reveal a 14-[A] structure of LRRK2 bearing a pathogenic mutation that oligomerizes as a right-handed double-helix around microtubules, which are left-handed. Using integrative modeling, we determine the architecture of LRRK2, showing that the GTPase points towards the microtubule, while the kinase is exposed to the cytoplasm. We identify two oligomerization interfaces mediated by non-catalytic domains. Mutation of one of these abolishes LRRK2 microtubule-association. Our work demonstrates the power of cryo-electron tomography to obtain structures of previously unsolved proteins in their cellular environment and provides insights into LRRK2 function and pathogenicity.

biophysics↗