Biology subjects

Watanabe, T.

Publications and source records attributed to Watanabe, T..

At least 55 records · Page 3Linked to original sources

bioRxiv2023

Characterizing Human KIF1Bβ Motor Activity by Single-Molecule Motility Assays and Caenorhabtidis elegans Genetics.

The axonal transport of synaptic vesicle precursors relies on KIF1A and UNC-104 ortholog motors. In mammals, KIF1B{beta} is also responsible for the axonal transport of synaptic vesicle precursors. Mutations in KIF1A and KIF1B{beta} lead to a wide range of neuropathies. While previous studies have revealed the biochemical, biophysical and cell biological properties of KIF1A, and its defects in neurological disorders, the fundamental properties of KIF1B{beta} remain elusive. In this study, we determined the motile parameters of KIF1B{beta} through single-molecule motility assays. Additionally, we established simple methods for testing the axonal transport activity of human KIF1B{beta} using Caenorhabditis elegans genetics. Taking advantage of these methods, we demonstrated that these assays enable the detection of reduced KIF1B{beta} activities both in vitro and in vivo, that is caused by a disease-associated mutation.

cell biology↗

bioRxiv2023

Atf3 controls transitioning in female mitochondrial cardiomyopathy as identified by single-cell transcriptomics

Oxidative phosphorylation defects results in mitochondrial diseases, with cardiac involvement markedly impacting prognosis. However, the mechanisms underlying the transition from compensation to dysfunction in response to metabolic deficiency remain unclear, impeding the development of effective treatments. Here, we employed single-nucleus RNA sequencing (snRNA-seq) on hearts from mitochondrial cardiomyopathy (MCM) mice with cardiac-specific Ndufs6 knockdown (FS6KD). Pseudotime trajectory analysis of cardiomyocytes from early stage of female FS6KD hearts revealed dynamic cellular state transitioning from compensation to severe compromise, coincided with transient upregulation of a critical transcription factor, activating transcription factor 3 (Atf3). Genetic ablation or adeno-associated virus-mediated Atf3 knockdown in FS6KD mice effectively delayed cardiomyopathy progression in a female-specific manner. Notably, human MCM snRNA-seq revealed a similar transition, including the dynamic expression of ATF3. In conclusion, our findings highlight a fate-determining role of Atf3 in female MCM progression, providing a promising therapeutic candidate for the currently intractable disease.

cell biology↗

bioRxiv2023

Kaposis sarcoma-associated herpesvirus ORF67.5 functions as a component of the terminase complex

Kaposis sarcoma-associated herpesvirus (KSHV) is a double-stranded DNA gammaherpesvirus, with a poorly characterized lytic replication cycle. However, the lytic replication cycle of the alpha- and betaherpesviruses are well characterized. During lytic infection of alpha- and betaherpesviruses, the viral genome is replicated as a precursor form, which contains tandem genomes linked via terminal repeats (TRs). One genomic unit of the precursor form is packaged into a capsid and is cleaved at the TR by the terminase complex. While the alpha- and betaherpesvirus terminases are well characterized, the KSHV terminase remains poorly understood. KSHV ORF7, ORF29, and ORF67.5 are presumed to be components of the terminase complex based on their homology to other terminase proteins. We previously reported that ORF7-deficient KSHV formed numerous immature soccer ball-like capsids and failed to cleave the TRs. ORF7 interacted with ORF29 and ORF67.5, however ORF29 and ORF67.5 did not interact with each other. While these results suggested that ORF7 is important for KSHV terminase function and capsid formation, the function of ORF67.5 was completely unknown. Therefore, to analyze the function of ORF67.5, we constructed ORF67.5-deficient BAC16. ORF67.5-deficient KSHV failed to produce infectious virus, failed to cleave the TRs, and numerous soccer ball-like capsids were observed in ORF67.5-deficient KSHV-harboring cells. Furthermore, ORF67.5 promoted the interaction between ORF7 and ORF29, and ORF29 increased the interaction between ORF67.5 and ORF7. Thus, our data indicated that ORF67.5 functions as a component of the KSHV terminase complex by contributing to TR cleavage, terminase complex formation, capsid formation, and virus production. IMPORTANCEAlthough the formation and function of the alpha- and betaherpesvirus terminase complexes are well understood, the KSHV terminase complex is still largely uncharacterized. This complex presumably contains KSHV ORF7, ORF29, and ORF67.5. We were the first to report the presence of soccer ball-like capsids in ORF7-deficient KSHV-harboring lytic-induced cells. Here, we demonstrated that ORF67.5-deficient KSHV also formed the soccer ball-like capsids in lytic-induced cells. Moreover, ORF67.5 was required for TR cleavage, infectious virus production, and enhancement of the interaction between ORF7 and ORF29. ORF67.5 has several highly conserved regions among its human herpesviral homologs. These regions were necessary for virus production and for the interaction of ORF67.5 with ORF7, which was supported by the AI-predicted structure model. Importantly, our results provide the first evidence showing that ORF67.5 is essential for terminase complex formation and TR cleavage.

microbiology↗

bioRxiv2023

Conserved cysteine residues in Kaposis sarcoma herpesvirus ORF34 are necessary for viral production and viral pre-initiation complex formation.

Kaposis sarcoma herpesvirus (KSHV) ORF34 plays a significant role as a component of the viral pre-initiation complex (vPIC), which is indispensable for late gene expression across beta and gamma herpesviruses. Although the key role of ORF34 within the vPIC and its function as a hub protein have been recognized, further clarification regarding its specific contribution to vPIC functionality and interactions with other components is required. This study employed a deep-learning algorithm-assisted structural model of ORF34, revealing highly conserved amino acid residues across human beta- and gamma-herpesviruses localized in structured domains. Thus, we engineered ORF34 alanine-scanning mutants by substituting conserved residues with alanine. These mutants were evaluated for their ability to interact with other vPIC factors and restore viral production in cells harboring the ORF34-deficient KSHV-BAC. Our experimental results highlight the crucial role of the 4 cysteine residues conserved in ORF34: a tetrahedral arrangement consisting of a pair of C-Xn-C consensus motifs. This suggests the potential incorporation of metal cations in interacting with ORF24 and ORF66 vPIC components, facilitating late gene transcription, and promoting overall virus production by capturing metal cations. In summary, our findings underline the essential role of conserved cysteines in KSHV ORF34 for effective vPIC assembly and viral replication, thereby enhancing our understanding of the complex interplay between the vPIC components. IMPORTANCEThe initiation of late gene transcription is universally conserved across the gamma- and beta-herpesvirus families. This process employs a viral pre-initiation complex (vPIC), which is analogous to a cellular PIC. Although KSHV ORF34 is a critical factor for viral replication and is a component of the vPIC, the specifics of vPIC formation and the essential domains crucial for its function remain unclear. Structural predictions suggest that the 4 conserved cysteines (C170, C175, C256, and C259) form a tetrahedron that coordinates the metal cation. We further investigated the role of these conserved amino acids in interactions with other vPIC components, late gene expression, and virus production, to demonstrate for the first time that these cysteines are pivotal for these functions. This discovery not only deepens our comprehensive understanding of ORF34 and vPIC dynamics but also lays the groundwork for more detailed studies on herpesvirus replication mechanisms in future research.

microbiology↗

bioRxiv2023

Taxonomic difference in marine bloom-forming phytoplanktonic species affects dynamics of both bloom-responding prokaryotes and prokaryotic viruses

The production of dissolved organic matter during phytoplankton blooms and consumption by heterotrophic prokaryotes promotes marine carbon biogeochemical cycling. Although prokaryotic viruses are crucial biological entities, their dynamics during such blooms are not fully understood. Here, we investigated the dynamics of coastal prokaryotic communities and viruses during blooms in a microcosm experiment using dissolved intracellular fractions of taxonomically distinct phytoplankton, the diatom Chaetoceros sp. (CIF) and the raphidophycean alga Heterosigma akashiwo (HIF). Ribosomal RNA gene amplicon and viral metagenomic analyses revealed that particular prokaryotes and prokaryotic viruses specifically increased in either CIF and HIF, indicating that different phytoplankton intracellular fractions promote distinct dynamics of not only prokaryotic community but also prokaryotic viruses. Our microcosm experiments and environmental data mining identified both known and novel possible host-virus pairs. In particular, a growth of phytoplanktonic organic matter-associated prokaryotes, such as Bacteroidetes Polaribacter and NS9 marine group, Vibrio spp., and Rhodobacteriales Nereida and Planktomarina, was accompanied by an increase in viruses predicted to infect Bacteroidetes, Vibrio, and Rhodobacteriales, respectively. Collectively, our findings suggest that elucidating tripartite relationships among phytoplankton, prokaryotes, and prokaryotic viruses would further our understanding of coastal microbial ecosystems. We state that -All the data underlying the study are available at the DNA Data Bank of Japan (DDBJ) under project number PRJDB14359 and accession number DRA014887. -This study was supported by Grants-in-Aid for Scientific Research (No. 16H06429, No. 17H03850, No. 21H05057, and No. 21J14854) from the Japan Society for the Promotion of Science (JSPS). -We have no potential conflicts of interest to declare. -We have read and understood your journals policies, and we believe that neither the manuscript nor the study violates any of these. -We do not use any clinical data, human subjects, or laboratory animals. -None of the materials have been published or are under consideration for publication elsewhere. CRediTHiroaki Takebe: Funding acquisition, conceptualization, investigation, formal analysis, visualization, and writing (original draft). Kento Tominaga: Conceptualization, investigation, and writing (review and editing). Tatsuhiro Isozaki: investigation. Tetsuhiro Watanabe: Resources. Keigo Yamamoto: Resources. Ryoma Kamikawa: Conceptualization, supervision, and writing (review and editing). Takashi Yoshida: Funding acquisition, project administration, conceptualization, supervision, and writing (review and editing).

microbiology↗

bioRxiv2022

oskar acts with the transcription factor Creb to regulate long-term memory in crickets

Novel genes have the potential to drive the evolution of new biological mechanisms, or to integrate into pre-existing regulatory circuits and contribute to the regulation of older, conserved biological functions. One such gene, the novel insect-specific gene oskar, was first identified based on its role in establishing the Drosophila melanogaster germ line. We previously showed that this gene likely arose through an unusual domain transfer event involving bacterial endosymbionts, and played a somatic role before evolving its well-known germ line function. Here, we provide empirical support for this hypothesis in the form of evidence for a novel neural role for oskar. We show that oskar is expressed in the adult neural stem cells of a hemimetabolous insect, the cricket Gryllus bimaculatus. In these stem cells, called neuroblasts, oskar is required together with the ancient animal transcription factor Creb to regulate long-term (but not short-term) olfactory memory. We provide evidence that oskar positively regulates Creb, which plays a conserved role in long-term memory across animals, and that oskar in turn may be a direct target of Creb. Together with previous reports of a role for oskar in nervous system development and function in crickets and flies, our results are consistent with the hypothesis that oskars original somatic role may have been in the insect nervous system. Moreover, its co-localization and functional cooperation with the conserved pluripotency gene piwi in the nervous system may have facilitated oskars later co-option to the germ line in holometabolous insects.

animal behavior and cognition↗

bioRxiv2022

mRNA-based generation of marmoset PGCLCs capable of differentiation into gonocyte-like cells

Primate germ cell development remains largely unexplored due to limitations in sample collection and the long duration of development. In mice, primordial germ cell-like cells (PGCLCs) derived from pluripotent stem cells (PSCs) can develop into functional gametes by in vitro culture or in vivo transplantation. Such PGCLC-mediated induction of mature gametes in primates is highly useful for understanding human germ cell development. Since marmosets generate functional sperm earlier than other species, recapitulating the whole male germ cell development process is technically more feasible. Here, we induced the differentiation of iPSCs into gonocyte-like cells via PGCLCs in marmosets. First, we developed an mRNA transfection-based method to efficiently generate PGCLC. Subsequently, to promote PGCLC differentiation, xenoreconstituted testes (xrtestes) were generated in the mouse kidney capsule. PGCLCs show progressive DNA demethylation and stepwise expression of developmental marker genes. This study provides an efficient platform for the study of marmoset germ cell development. HighlightsO_LIAn mRNA transfection-based PGCLC induction method is developed C_LIO_LIMarmoset PGCLCs are efficiently induced from iPSCs C_LIO_LIMarmoset PGCLCs differentiate into gonocyte-like cells in mouse kidneys C_LIO_LIDevelopmentally regulated expression and demethylation are recapitulated C_LI eTOC blurbWatanabe et al. efficiently induced marmoset primordial germ cell-like cells (PGCLCs) using an mRNA transfection-based approach. PGCLCs further develop into gonocyte-like cells in the xenoreconstituted testes constructed under mouse kidney capsules. This system faithfully reproduced in vivo developmental processes (e.g., stage-specific expression of developmentally regulated genes and DNA demethylation).

developmental biology↗

bioRxiv2022

A unique case in which Kimoto-style fermentation was completed with Leuconostoc as the dominant genus without transitioning to Lactobacillus.

The Kimoto-style fermentation starter is a traditional preparation method of sake brewing. In this process, specific microbial transition patterns have been observed within nitrate-reducing bacteria and lactic acid bacteria during the production process of the fermentation starter. We have characterized phylogenetic compositions and diversity of the bacterial community in a sake brewery performing the Kimoto-style fermentation. Comparing the time-series changes with other sake breweries previously reported, we found a novel type of Kimoto-style fermentation in which the microbial transition differed significantly from other breweries during the fermentation step. Specifically, the lactic acid bacteria, Leuconostoc spp. was a predominant species in the late stage in the preparation process of fermentation starter, on the other hand, Lactobacillus spp., which plays a pivotal role in other breweries, was not detected in this analysis. The discovery of this new variation of microbiome transition in Kimoto-style fermentation has further deepened our understanding of the diversity of sake brewing.

microbiology↗

bioRxiv2022

A comprehensive list of the replication promoters of Bunyavirales reveals a unique promoter structure in Nairoviridae differing from other virus families

Bunyaviruses belong to the order Bunyavirales, the largest group of RNA viruses. They infect a wide variety of host species around the world, including plants, animals and humans, and pose a major threat to public health. Major families in the order Bunyavirales have tri-segmented negative-sense RNA genomes, the 5 and 3 ends of which form complementary strands that serve as a replication promoter. Elucidation of the mechanisms by which viral RNA-dependent RNA polymerase recognizes the promoter to initiates RNA synthesis is important for understanding viral replication and pathogenesis, and for developing antivirals. A list of replication promoter configuration patterns may provide details on the differences in the replication mechanisms among bunyaviruses. Here, by using public sequence data of all known bunyavirus species, we constructed a comprehensive list of the replication promoters comprising 40 nucleotides in both the 5 and 3 ends of the genome that form a specific complementary strand. We showed that among tri-segmented bunyaviruses, viruses belonging to the family Nairoviridae, including the highly pathogenic Crimean-Congo hemorrhagic fever virus, have evolved a GC-rich promoter structure that differs from that of other bunyaviruses. The unique promoter structure might be related to the large genome size of the family Nairoviridae among bunyaviruses. It is possible that the large genome architecture confers a pathogenic advantage. The promoter list provided in this report is expected to be useful for predicting virus family-specific replication mechanisms of segmented negative-sense RNA viruses.

microbiology↗

bioRxiv2022

Actin nano-architecture of phagocytic podosomes

Podosomes are actin-enriched adhesion structures important for multiple cellular processes, including migration, bone remodeling, and phagocytosis. Here, we characterized the structure and organization of phagocytic podosomes using interferometric photoactivated localization microscopy (iPALM), a super-resolution microscopy technique capable of 15-20 nm resolution, together with structured illumination microscopy (SIM) and localization-based superresolution microscopy. Phagocytic podosomes were observed during frustrated phagocytosis, a model in which cells attempt to engulf micro-patterned IgG antibodies. For circular patterns, this resulted in regular arrays of podosomes with well-defined geometry. Using persistent homology, we developed a pipeline for semi-automatic identification and measurement of podosome features. These studies revealed an "hourglass" shape of the podosome actin core, a protruding "knob" at the bottom of the core, and two actin networks extending from the core. Additionally, the distributions of paxillin, talin, myosin II, -actinin, cortactin, and microtubules relative to actin were characterized.

cell biology↗

bioRxiv2022

The contribution of Kaposis sarcoma-associated herpesvirus ORF7 and its zinc-finger motif to viral genome cleavage and capsid formation

Kaposis sarcoma-associated herpesvirus (KSHV) is the causative agent of endothelial and B cell malignancies. During KSHV lytic infection, lytic-related proteins are synthesized, viral genomes are replicated as a tandemly repeated form, and subsequently, capsids are assembled. The herpesvirus terminase complex is proposed to package an appropriate genome unit into an immature capsid, by cleavage of terminal repeats (TRs) flanking tandemly linked viral genomes. Although the mechanism of capsid formation in - and {beta}-herpesviruses are well-studied, in KSHV, it remains largely unknown. It has been proposed that KSHV ORF7 is a terminase subunit, and ORF7 harbors a zinc-finger motif, which is conserved among other herpesviral terminases. However, the biological significance of ORF7 is unknown. We previously reported that KSHV ORF17 is essential for the cleavage of inner scaffold proteins in capsid maturation, and ORF17 knockout (KO) induced capsid formation arrest between the procapsid and B-capsid stages. However, it remains unknown if ORF7-mediated viral DNA cleavage occurs before or after ORF17-mediated scaffold collapse. We analyzed the role of ORF7 during capsid formation using ORF7-KO-, ORF7&17-double-KO (DKO)-, and ORF7-zinc-finger motif mutant-KSHVs. We found that ORF7 acted after ORF17 in the capsid formation process, and ORF7-KO-KSHV produced incomplete capsids harboring non-spherical internal structures, which resembled soccer balls. This soccer ball-like capsid was formed after ORF17-mediated B-capsid formation. Moreover, ORF7-KO- and zinc-finger motif KO-KSHV failed to appropriately cleave the TR on replicated genome and had a defect in virion production. Thus, our data revealed that ORF7 contributes to terminase-mediated viral genome cleavage and capsid formation. IMPORTANCEIn herpesviral capsid formation, the viral terminase complex cleaves the TR sites on newly synthesized tandemly repeating genomes and inserts an appropriate genomic unit into an immature capsid. Herpes simplex virus 1 (HSV-1) UL28 is a subunit of the terminase complex that cleaves the replicated viral genome. However, the physiological importance of the UL28 homolog, KSHV ORF7, remains poorly understood. Here, using several ORF7-deficent KSHVs, we found that ORF7 acted after ORF17-mediated scaffold collapse in the capsid maturation process. Moreover, ORF7 and its zinc-finger motif were essential for both cleavage of TR sites on the KSHV genome and virus production. ORF7-deficient KSHVs produced incomplete capsids that resembled a soccer ball. To our knowledge, this is the first report showing ORF7-KO-induced soccer ball-like capsids production and ORF7 function in the KSHV capsid assembly process. Our findings provide insights into the role of ORF7 in KSHV capsid formation.

microbiology↗

bioRxiv2022

Avian H7N9 influenza viruses are evolutionarily constrained by stochastic processes during replication and transmission in mammals

H7N9 avian influenza viruses (AIV) have caused over 1,500 documented human infections since emerging in 2013. Although wild type H7N9 AIV can transmit by respiratory droplets in ferrets, they have not yet caused widespread outbreaks in humans. Previous studies have revealed molecular determinants of H7N9 AIV virus host-switching, but little is known about potential evolutionary constraints on this process. Here we compare patterns of sequence evolution for H7N9 AIV and mammalian H1N1 viruses during replication and transmission in ferrets. We show that three main factors - purifying selection, stochasticity, and very narrow transmission bottlenecks - combine to severely constrain the ability of H7N9 AIV to effectively adapt to mammalian hosts in isolated, acute spillover events. We find rare evidence of natural selection favoring new or mammalian-adapting mutations within ferrets, but no evidence of natural selection acting during transmission. We conclude that human-adapted H7N9 viruses are unlikely to emerge during typical spillover infections. Our findings are instead consistent with a model in which the emergence of a human-transmissible virus would be a rare and unpredictable, though highly consequential, "jackpot" event. Strategies to limit the total number of spillover infections will limit opportunities for the virus to win this evolutionary lottery.

microbiology↗

bioRxiv2022

Spatial Models of Pattern Formation During Phagocytosis

Phagocytosis, the biological process in which cells ingest large particles such as bacteria, is a key component of the innate immune response. Fc{gamma} receptor (Fc{gamma}R)-mediated phagocytosis is initiated when these receptors are activated after binding immunoglobulin G (IgG). Receptor activation initiates a signaling cascade that leads to the formation of the phagocytic cup and culminates with ingestion of the foreign particle. In the experimental system termed "frustrated phagocytosis", cells attempt to internalize micropatterned disks of IgG. Cells that engage in frustrated phagocytosis form "rosettes" of actin-enriched structures called podosomes around the IgG disk. The mechanism that generates the rosette pattern is unknown. We present data that supports the involvement of Cdc42, a member of the Rho family of GTPases, in pattern formation. Cdc42 acts downstream of receptor activation, upstream of actin polymerization, and is known to play a role in polarity establishment. Reaction-diffusion models for GTPase spatiotemporal dynamics exist. We demonstrate how the addition of negative feedback and minor changes to these models can generate the experimentally observed rosette pattern of podosomes. We show that this pattern formation can occur through two general mechanisms. In the first mechanism, an intermediate species forms a ring of high activity around the IgG disk, which then promotes rosette organization. The second mechanism does not require initial ring formation but relies on spatial gradients of intermediate chemical species that are selectively activated over the IgG patch. Finally, we analyze the models to suggest experiments to test their validity. Author SummaryPhagocytosis, the process by which cells ingest foreign bodies, plays an important role in innate immunity. Phagocytosis is initiated when antibodies coating the surface of a foreign body are recognized by immune cells, such as macrophages. To study early events in phagocytosis, we used "frustrated phagocytosis", an experimental system in which antibodies are micropatterned in disks on a cover slip. The cytoskeleton of cells attempting to phagocytose these disks organizes into "rosette" patterns around the disks. To investigate mechanisms that underlie rosette formation we turned to mathematical modeling based on reaction-diffusion equations. Building on existing models for polarity establishment, our analysis revealed two mechanisms for rosette formation. In the first scenario an initial ring of an intermediate signaling molecule forms around the disk, while in the second scenario rosette formation is driven by gradients of positive and negative pathway regulators that are activated over the disk. Finally, we analyze our models to suggest experiments for testing these mechanisms.

systems biology↗

bioRxiv2022

Frequency of a nucleotide overhang at the 5' end of hemorrhagic fever mammarenavirus genomes in public sequence data

Mammarenaviruses, such as Lassa virus and South American hemorrhagic fever (SAHF) virus, cause severe hemorrhagic fevers in humans, and pose major threats to public health. Mammarenaviruses consist of a bi-segmented negative-sense RNA genome in which the 5 and 3 ends form complementary strands that serve as a replication promoter. Some mammarenaviruses have a nucleotide overhang at the 5 genome end. By examining the complementarity of 5 and 3 genome ends using public mammarenavirus genome sequences, we found that the 5 guanine overhang (5-G overhang) was present more frequently in Lassa and SAHF viruses than in other viruses. The 5-G overhang in the Lassa and SAHF virus sequences was found to be restricted to the L and S segments, respectively. If the genome end sequence data in the public database are accurate, the 5-G overhang may be related to the high pathogenicity of mammarenaviruses in humans.

microbiology↗

bioRxiv2022

Efficient learning in children with rapid GABA boosting after training

It is unclear why and how children learn more efficiently than adults, although inhibitory systems, which play an important role in stabilizing learning, are immature in children. Here, we found that despite a lower baseline concentration of {gamma}-aminobutyric acid (GABA) in early visual cortical areas in children (8 to 11 years old) than adults (18 to 35 years old), children exhibited a rapid boost of GABA immediately after visual training, whereas the concentration of GABA in adults remained unchanged after training. Moreover, behavioral experiments showed that children stabilized visual learning much faster than adults, showing rapid development of resilience to retrograde interference. These results together suggest that inhibitory systems in childrens brains are more dynamic and adapt more quickly to stabilize learning than in adults. One Sentence SummaryChildren learn more efficiently than adults due to faster stabilization of learning with rapid GABA boosting after training.

neuroscience↗

bioRxiv2021

Morphological variation and phylogeny of Karenia selliformis (Gymnodiniales, Dinophyceae) in an intensive cold-water algal bloom in eastern Hokkaido, Japan in September-November 2021

Harmful algal blooms responsible for mass mortalities of marine organisms have so far been rare in Hokkaido, northern Japan, although fish killing blooms have been frequently reported from western Japanese coasts. In September-November 2021, a huge and prolonged cold-water bloom occurred along the Pacific coast of eastern Hokkaido, Japan, and was associated with intensive mortalities of sea urchin, fish, octopus, shellfish, etc. In this study, morphology and phylogeny of the dominant and co-occurred unarmored dinoflagellates of the Kareniaceae in the bloom were examined by using light microscopy, scanning electron microscopy and molecular phylogeny inferred from ITS and LSU rDNA (D1-D3) sequences. Morphological observation and molecular phylogeny showed that the dominant species was Karenia selliformis, with co-occurrences of other kareniacean dinoflagellates, Kr. longicanalis, Kr. mikimotoi, Karlodinium sp., Takayama cf. acrotrocha, Takayama tuberculata and Takayama sp. The typical cell forms of K. selliformis in the bloom were discoid, dorsoventrally flattened, and larger than the cell sizes in previous reports, 35.3-43.6 (39.4{+/-}2.1) m in length. Transparent cells of Kr. selliformis lacking or having several shrunken chloroplasts and oil droplets were also found. Cells of Kr. selliformis had morphological variation, but the species could be distinguished from other co-occurred Karenia species by its numerous (46-105) and small granular (2.9-4.6 m in diameter) chloroplasts and the nucleus positioned in the hypocone. Cell density of Kr. selliformis exceeding 100 cells/mL was recorded in the range of temperature 9.8-17.6{degrees}C. The rDNA sequences determined from Kr. selliformis in the blooms of Hokkaido, Japan in 2021 were identical to those from another bloom in Kamchatka, Russia in 2020. HighlightsO_LIA marine fauna-destructive harmful algal bloom in the Pacific coast of eastern Hokkaido, Japan in September-November 2021 was dominated by Karenia selliformis. C_LIO_LICells of Karenia selliformis typical in the bloom were discoid and possessing numerous small chloroplasts, approximately 70 in number. C_LIO_LICells of Karenia selliformis showed morphological variation in size and shape, and transparent motile cells lacking or having degraded chloroplasts were also present. C_LIO_LICo-occurred kareniaceans in the bloom were Karenia longicanalis, Karenia mikimotoi, Karlodinium sp. and Takayama spp. C_LIO_LIrDNA sequences of Karenia selliformis in the blooms of Hokkaido in 2021 and Kamchatka in 2020 were identical, which belong to the group I of Kr. selliformis. C_LI

ecology↗

bioRxiv2021

Dynamics of peripheral blood flow across sleep stages

Study ObjectivesPulse rate variability (PRV) derived from peripheral blood flow has been reported as a surrogate parameter for heart rate variability (HRV). However, there are currently no studies reporting systematic comparisons of PRV with HRV in a normal sleep state. Whether PRV can provide similar information regarding sleep stages remains unclear. Peripheral blood flow may also be modulated differently across sleep stages. Thus, we aimed to investigate blood flow dynamics and compare PRV with HRV across sleep stages to see if blood flow can provide further information about sleep stages. MethodsWe performed electrocardiography and simultaneously measured blood flow from the right index finger and ear concha of 45 healthy participants (13 women; mean age, 22.5 {+/-} 3.4 years) during one night of sleep. Time-domain, frequency-domain, and non-linear indices of PRV/HRV, and time- and frequencydomain blood flow parameters were calculated. ResultsFinger-PRV results showed similar patterns to HRV results for most parameters. Finger-blood flow parameters in the time and frequency domains also showed information about the different sleep stages. Further, both finger- and ear-blood flow results showed 0.2-0.3 Hz oscillations that varied with sleep stages, with a significant increase in N3, suggesting a modulation (of respiration) within this frequency band. ConclusionsThese results suggest that PRV can provide as much information as HRV for different sleep stages. Furthermore, the results show that blood flow + PRV could be more advantageous than HRV alone in the assessment of the sleep state and related autonomic nervous activity. Statement of SignificanceThe research provides comprehensive information on peripheral blood flow (BF) activity across sleep stages for the first time, as the major novelty of our work. The second contribution is a systematic study comparing BF-derived pulse rate variability (PRV) with heart rate variability across sleep stages in a normal sleep state. We believe that our work makes a significant contribution to the literature because it provides comprehensive information on the potential of BF+PRV as a new biomarker for assessing the sleep state. Further, this study contributes to developing a more convenient method of assessing the sleep state in the clinical and home/work setting.

physiology↗

bioRxiv2021

Aberrant localization of CDC42 C-terminal variants to the Golgi apparatus drives pyrin inflammasome-dependent autoinflammation

Mutations in the C-terminal region of the CDC42 gene cause severe neonatal-onset autoinflammation. Elevated levels of serum IL-18 in patients and effectiveness of IL-1{beta}-blocking therapy indicate that the pathology involves abnormal inflammasome activation; however, the mechanism underlying autoinflammation remains to be elucidated. Using induced-pluripotent stem cells established from patients carrying CDC42R186C, we found that patient-derived cells secreted larger amounts of IL-1{beta} in response to pyrin-activating stimuli. Aberrant palmitoylation and localization of CDC42R186C protein to the Golgi apparatus promoted pyrin inflammasome assembly downstream of pyrin dephosphorylation. Aberrant subcellular localization was the common pathological feature shared by CDC42 C-terminal variants with inflammatory phenotypes, including CDC42*192C*24 that also localizes to the Golgi apparatus. Furthermore, the level of pyrin inflammasome overactivation paralleled that of mutant protein accumulation in the Golgi apparatus, but no that of the mutant GTPase activity. These results reveal an unexpected association between CDC42 subcellular localization and pyrin inflammasome activation that could pave way for elucidating the mechanism of pyrin inflammasome formation.

immunology↗