Biology subjects

Murata, K.

Publications and source records attributed to Murata, K..

At least 55 records · Page 3Linked to original sources

bioRxiv2021

Desiccation-induced fibrous condensation of CAHS protein from an anhydrobiotic tardigrade

Anhydrobiosis is one of the most extensively studied forms of cryptobiosis that is induced in certain organisms as a response to desiccation. Anhydrobiotic species has been hypothesized to produce substances that can protect their biological components and/or cell membranes without water. In extremotolerant tardigrades, highly hydrophilic and heat-soluble protein families, cytosolic abundant heat-soluble (CAHS) proteins, have been identified, which are postulated to be integral parts of the tardigrades response to desiccation. However, the molecular mechanisms underlying these protein functions remain to be fully elucidated. In this study, we perfomed in vitro and in vivo characterizations of the self-assembling property of CAHS1 protein, a major isoform of CAHS proteins from Ramazzottius varieornatus, using a series of spectroscopic and microscopic techniques. Our in vitro observations showed that CAHS1 proteins homo-oligomerized via the C-terminal -helical region and formed a hydrogel as their concentration increased, and that these molecular assembling processes were reversible. Furthermore, our in vivo observations demonstrated that the overexpressed CAHS1 proteins formed condensates under desiccation-mimicking conditions. These data strongly suggested that, upon drying, the CAHS1 proteins form oligomers and eventually underwent sol-gel transition in tardigrade cytosols. Thus, it is proposed that the CAHS1 proteins form the cytosolic fibrous condensates, which presumably have variable mechanisms for the desiccation tolerance of tardigrades. These findings provide insights into the protective mechanisms involved in the anhydrobiosis of tardigrades.

biochemistry↗

bioRxiv2021

Effect of differences in mechanical stress in vivo on the onset and progression of knee osteoarthritis

ObjectiveThe effect of the type of mechanical stress on OA onset has not been clarified. The aim of this study was to establish a new model that reproduces the type and increase and decrease of mechanical stress in vivo and to clarify the differences in the mechanism of knee OA onset and progression among the models. DesignTo reproduce the difference in mechanical stress, we used the anterior cruciate ligament transection (ACL-T) model and the destabilization of the medial meniscus (DMM) model. In addition, we created a controlled abnormal tibial translation (CATT) model and a controlled abnormal tibial rotation (CATR) model that suppressed the joint instability of the ACL-T and DMM model, respectively. These four models reproduced the increase and decrease in shear force due to joint instability and compressive stress due to meniscal dysfunction. We performed joint instability analysis with soft X-ray, micro computed tomography analysis, histological analysis, and immunohistological analysis in 4 and 6 weeks. ResultsJoint instability decreased in the CATT and CATR groups. The meniscus deviated in the DMM and CATR groups. Chondrocyte hypertrophy increased in the ACL-T and DMM groups with joint instability. In the subchondral bone, bone resorption was promoted in the ACL-T and CATT groups, and bone formation was promoted in the DMM and CATR groups. ConclusionsIncreased shear force causes articular cartilage degeneration and osteoclast activation in the subchondral bone. In contrast, increased compressive stress promotes bone formation in the subchondral bone earlier than articular cartilage degeneration occurs.

pathology↗

bioRxiv2021

Effect of Suppression of Rotational Joint Instability on Cartilage and Meniscus Degeneration in Mouse Osteoarthritis Model

ObjectiveJoint instability and meniscal dysfunction contribute to the onset and progression of knee osteoarthritis (OA). In the destabilization of the medial meniscus (DMM) model, secondary OA occurs due to the rotational instability and increases compressive stress resulting from the meniscal dysfunction. We created a new controlled abnormal tibial rotation (CATR) model that reduces the rotational instability that occurs in the DMM model. So, we aimed to investigate whether rotational instability affects articular cartilage degeneration using the DMM and CATR models, as confirmed using histology and immunohistochemistry. DesignTwelve-week-old male mice were randomized into 3 groups: DMM group, CATR group, and INTACT group (right knee of the DMM group). After 8 and 12 weeks, we performed the tibial rotational test, safranin-O/fast green staining, and immunohistochemical staining for TNF- and MMP-13. ResultsThe rotational instability in the DMM group was significantly higher than that of the other groups. And articular cartilage degeneration was higher in the DMM group than in the other groups. However, meniscal degeneration was observed in both DMM and CATR groups. The TNF- and MMP-13 positive cell rates in the articular cartilage of the CATR group were lower than those in the DMM group. ConclusionsWe found that the articular cartilage degeneration was effectively suppressed by controlling the rotational instability caused by meniscal dysfunction. These findings suggest that suppression of rotational instability in the knee joint is an effective therapeutic measure for preventing OA progression.

pathology↗

bioRxiv2021

Nasal delivery of single-domain antibodies improve symptoms of SARS-CoV-2 infection in an animal model

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the disease COVID-19 can lead to serious symptoms, such as severe pneumonia, in the elderly and those with underlying medical conditions. While vaccines are now available, they do not work for everyone and therapeutic drugs are still needed particularly for treating life-threatening conditions. Here, we showed nasal delivery of a new, unmodified camelid single-domain antibody (VHH), termed K-874A, effectively inhibited SARS-CoV-2 titers in infected lungs of Syrian hamsters without causing weight loss and cytokine induction. In vitro studies demonstrated that K-874A neutralized SARS-CoV-2 in both VeroE6/TMPRSS2 and human lung-derived alveolar organoid cells. Unlike other drug candidates, K-874A blocks viral membrane fusion rather than viral attachment. Cryo-electron microscopy revealed K-874A bound between the receptor binding domain and N-terminal domain of the virus S protein. Further, infected cells treated with K-874A produced fewer virus progeny that were less infective. We propose that direct administration of K-874A to the lung via a nebulizer could be a new treatment for preventing the reinfection of amplified virus in COVID-19 patients. Author summaryVaccines for COVID-19 are now available but therapeutic drugs are still needed to treat life-threatening cases and those who cannot be vaccinated. We discovered a new heavy-chain single-domain antibody that can effectively neutralize the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes COVID-19. Unlike other drug candidates, which prevent the virus from attaching to the receptor on the host cell, this new antibody acts by blocking the virus membrane from fusing with the host cell membrane. We studied the behavior of the new antibody in vitro using VeroE6/TMPRSS2 cells and human lung organoids. When delivered through the nose to infected Syrian hamsters, we found that this antibody could prevent the typical symptoms caused by SARS-CoV-2. Our results are significant because delivering simple drugs directly to infected lungs using a nebulizer could increase the potency of the drugs while reducing the risk of immune reaction that could occur if the drugs escape or are delivered through the blood stream.

microbiology↗

bioRxiv2021

Structural basis of LhcbM5-mediated state transitions in green algae

In green algae and plants, state transitions serve as a short-term light acclimation process to regulate light harvesting capacity of photosystems I and II (PSI and PSII). During the process, a portion of the light-harvesting complexes II (LHCII) are phosphorylated, dissociate from PSII and bind PSI to form PSI-LHCI-LHCII supercomplex. Here we report high-resolution structures of PSI-LHCI-LHCII supercomplex from Chlamydomonas reinhardtii, revealing the mechanism of assembly between PSI-LHCI complex and two phosphorylated LHCII trimers containing all four types of LhcbM proteins. Two specific LhcbM isoforms, namely LhcbM1 and LhcbM5, directly interact with the PSI core through their phosphorylated amino-terminal regions. Furthermore, biochemical and functional studies on mutant strains lacking either LhcbM1 or LhcbM5 indicate that only LhcbM5 is indispensable in the supercomplex formation. The results unraveled the specific interactions and potential excitation energy transfer routes between green algal PSI and two phosphorylated LHCIIs.

plant biology↗

bioRxiv2021

Atomic structure of human sapovirus capsid by single particle cryo-electron microscopy

Sapovirus is a cause of acute gastroenteritis in humans and animals. Infants and younger children have the greatest disease burden. Although it shares many similarities with norovirus, the lack of detailed structural information has hampered the development of vaccines and therapeutics. Here, we investigated the human sapovirus VLP by single particle cryo-electron microscopy and are the first to report the atomic structure of the capsid at 2.9 [A] resolution. The atomic model revealed the domain interactions of the capsid protein and functionally important amino acid residues. The extended loop from the P1 subdomain was involved in interactions in the P2 domain, forming unique arch-like dimeric protrusions of capsid proteins. All hypervariable regions that are important candidates for immune response or receptor binding, formed a large cluster at the top of the P domain. These results pave the way for developing vaccines, antiviral drugs, and diagnostic systems for this infectious disease.

microbiology↗

bioRxiv2021

A novel capsid protein network allows the characteristic inner membrane structure of Marseilleviridae giant viruses

Marseilleviridae is a family of the new order of giant viruses, which exhibit a characteristic inner membrane. Here, we investigated the entire structure of tokyovirus, a species of Marseillevirus at 7.7 [A] resolution using 1 MV high-voltage cryo-EM and single particle analysis. The minor capsid lattice formed by five proteins, shows a novel structure compared to other icosahedral giant viruses. Under the minor capsid proteins, scaffold proteins connect two five-fold vertices and interact with the inner membrane. Previously reported giant viruses utilise "tape measure" proteins, proposed to control its capsid size, which could not be identified in tokyovirus, but scaffold proteins appear to perform a similar role. A density on top of the major capsid protein was identified, which suggested to be a 14kDa glycoprotein. Our observations suggest that the icosahedral particle of Marseilleviridae is constructed with a novel capsid protein network, which allows the characteristic inner membrane structure.

microbiology↗

bioRxiv2020

DAJIN-assisted multiplex genotyping to validate the outcomes of CRISPR-Cas-based genome editing

Genome editing can introduce designed mutations into a target genomic site. Recent research has revealed that it can also induce various unintended events such as structural variations, small indels, and substitutions at, and in some cases, away from the target site. These rearrangements may result in confounding phenotypes in biomedical research samples and cause a concern in clinical or agricultural applications. However, current genotyping methods do not allow a comprehensive analysis of diverse mutations for phasing and mosaic variant detection. Here, we developed a genotyping method with an on-target site analysis software named Determine Allele mutations and Judge Intended genotype by Nanopore sequencer (DAJIN) that can automatically identify and classify both intended and unintended diverse mutations, including point mutations, deletions, inversions, and cis double knock-in at single-nucleotide resolution. Our approach with DAJIN can handle approximately 100 samples under different editing conditions in a single run. With its high versatility, scalability, and convenience, DAJIN-assisted multiplex genotyping may become a new standard for validating genome editing outcomes.

genomics↗

bioRxiv2020

CUBIC-Cloud: An Integrative Computational Framework Towards Community-Driven Whole-Mouse-Brain Mapping

Recent advancements in tissue clearing technologies have offered unparalleled opportunities for researchers to explore the whole mouse brain at cellular resolution. With the expansion of this experimental technique, however, a scalable and easy-to-use computational tool is in demand to effectively analyze and integrate whole-brain mapping datasets. To that end, here we present CUBIC-Cloud, a cloud-based framework to quantify, visualize and integrate whole mouse brain data. CUBIC-Cloud is a fully automated system where users can upload their whole-brain data, run analysis and publish the results. We demonstrate the generality of CUBIC-Cloud by a variety of applications. First, we investigated brain-wide distribution of PV, Sst, ChAT, Th and Iba1 expressing cells. Second, A{beta} plaque deposition in AD model mouse brains were quantified. Third, we reconstructed neuronal activity profile under LPS-induced inflammation by c-Fos immunostaining. Last, we show brain-wide connectivity mapping by pseudo-typed Rabies virus. Together, CUBIC-Cloud provides an integrative platform to advance scalable and collaborative whole-brain mapping.

neuroscience↗

bioRxiv2020

EXOC1 regulates cell morphology of spermatogonia and spermatocytes in mice

Spermatogenesis requires high regulation of germ cell morphology. The spermatogonia regulates its differentiation state by its own migration. The male germ cells differentiate and mature with the formation of syncytia, failure of forming the appropriate syncytia results in the arrest of spermatogenesis at the spermatocyte stage. However, the detailed molecular mechanisms of male germ cell morphological regulation are unknown. Here, we found that EXOC1 is important for the pseudopod formation of spermatogonia and spermatocyte syncytia in mice. We found that while EXOC1 contributes to the inactivation of Rac1 in the pseudopod formation of spermatogonia, in spermatocyte syncytium formation, EXOC1 and SNAP23 cooperate with STX2. Our results showed that EXOC1 functions in concert with various cell morphology regulators in spermatogenesis. Since EXOC1 is known to bind to several cell morphogenesis factors, this study is expected to be the starting point for the discovery of many morphological regulators of male germ cells.

developmental biology↗

bioRxiv2020

Brain/MINDS Beyond Human Brain MRI Study: Multi-Site Harmonization for Brain Disorders Throughout the Lifespan

Psychiatric and neurological disorders are afflictions of the brain that can affect individuals throughout their lifespan. Many brain magnetic resonance imaging (MRI) studies have been conducted; however, imaging-based biomarkers are not yet well established for diagnostic and therapeutic use. This article describes an outline of the planned study, the Brain/MINDS Beyond human brain MRI project (FY2018 [~] FY2023), which aims to establish clinically-relevant imaging biomarkers with multi-site harmonization by collecting data from healthy traveling subjects (TS) at 13 research sites. Collection of data in psychiatric and neurological disorders across the lifespan is also scheduled at 13 sites, whereas designing measurement procedures, developing and analyzing neuroimaging protocols, and databasing are done at three research sites. The Harmonization protocol (HARP) was established for five high-quality 3T scanners to obtain multimodal brain images including T1 and T2-weighted, resting state and task functional and diffusion-weighted MRI. Data are preprocessed and analyzed using approaches developed by the Human Connectome Project. Preliminary results in 30 TS demonstrated cortical thickness, myelin, functional connectivity measures are comparable across 5 scanners, providing high reproducibility and sensitivity to subject-specific connectome. A total of 75 TS, as well as patients with various psychiatric and neurological disorders, are scheduled to participate in the project, allowing a mixed model statistical harmonization. The HARP protocols are publicly available online, and all the imaging, demographic and clinical information, harmonizing database will also be made available by 2024. To the best of our knowledge, this is the first project to implement a rigorous, prospective harmonization protocol with multi-site TS data. It explores intractable brain disorders across the lifespan and may help to identify the disease-specific pathophysiology and imaging biomarkers for clinical practice.

neuroscience↗

bioRxiv2020

Characterization of amyloid β fibril formation under microgravity conditions

Amyloid fibrils are self-assembled and ordered proteinaceous supramolecules structurally characterized by the cross-{beta} spine. Amyloid formation is known to be related to various diseases typified by neurogenerative disorders and involved in a variety of functional roles. Whereas common mechanisms for amyloid formation have been postulated across diverse systems, the mesoscopic morphology of the fibrils is significantly affected by the type of solution condition in which it grows. Amyloid formation is also thought to share a phenomenological similarity with protein crystallization. While many studies have demonstrated the effect of gravity on protein crystallization, its effect on amyloid formation has not been reported. In this study, we conducted an experiment at the International Space Station (ISS) to characterize fibril formation of 40-residue amyloid {beta} (A{beta}(1-40)) under microgravity conditions. Our comparative analyses revealed that the A{beta}(1-40) fibrilization progresses much more slowly on the ISS than on the ground, similarly to protein crystallization. Furthermore, microgravity promoted the formation of distinct morphologies of A{beta}(1-40) fibrils. Our findings demonstrate that the ISS provides an ideal experimental environment for detailed investigations of amyloid formation mechanisms by eliminating the conventionally uncontrollable factors derived from gravity.

biophysics↗

bioRxiv2020

Sub-3 A resolution structure of apoferritin using a multi-purpose TEM with a side-entry cryo-holder

The structural analysis of protein complexes by cryo-electron microscopy (cryo-EM) single particle analysis (SPA) has had great impact as a biophysical method in recent years. Many results of cryo-EM SPA are based on state-of-the-art cryo-electron microscopes customized for SPA. These are currently only available in limited locations around the world, where securing machine time is highly competitive. One potential solution for this time-competitive situation is to reuse existing multi-purpose equipment. Here, we used a multi-purpose TEM with a side entry cryo-holder at our facility to evaluate the potential of high-resolution SPA. We report a 3 [A] resolution map of apoferritin with local resolution extending to 2.6 [A]. The map clearly showed two positions of an aromatic side chain. We also verified the optimal imaging conditions depending on different electron microscope and camera combinations. This study demonstrates the possibilities of more widely available and established electron microscopes, and their applications for cryo-EM SPA.

molecular biology↗

bioRxiv2019

Dynamic rotation of the protruding domain enhances the infectivity of norovirus

Norovirus is the major cause of epidemic nonbacterial gastroenteritis worldwide. Lack of structural information on infection and replication mechanisms hampers the development of effective vaccines and remedies. Here, using cryo-electron microscopy, we show that the capsid structure of murine noroviruses changes in response to aqueous conditions. By twisting the flexible hinge connecting two domains, the protruding (P) domain reversibly rises off the shell (S) domain in solutions of higher pH, but rests on the S domain in solutions of lower pH. Metal ions help to stabilize the latter conformation in this process. Furthermore, in the resting conformation, the cellular receptor CD300lf is readily accessible, and thus infection efficiency is significantly enhanced. Two P domain conformations were also found in the human norovirus GII.3 capsid. These results provide new insights into the infection mechanisms of the non-envelope viruses that function in dramatic environmental changes such as the digestive tract. Significance StatementThe capsid structures of caliciviruses have been reported to be classified into two different types, according to species and genotype. One is the rising P domain type as shown in human norovirus GII.10 and rabbit hemorrhagic disease virus, where the P domain rises from the S domain surface. The other is the resting P domain type as shown in human norovirus GI.1, sapovirus and San Miguel sea lion virus, where the P domain rests upon the S domain. Here, we demonstrate that the P domain of the murine norovirus infectious particles changes reversibly between the rising and resting P domain types in response to aqueous conditions. Our findings provide new insights into the mechanisms of viral infection of caliciviruses.

pathology↗

bioRxiv2019

Cables2 Is a novel Smad2-regulatory factor essential for early embryonic development in mice

CDK5 and Abl enzyme substrate 2 (Cables2), a member of the Cables family that has a C-terminal cyclin box-like domain, is widely expressed in adult mouse tissues. However, the physiological role of Cables2 in vivo is unknown. We show here that Cables2-deficiency causes post-gastrulation embryonic lethality in mice. The mutant embryos progress to gastrulation, but then arrest, and fail to grow. Analysis of gene expression patterns reveals that formation of the anterior visceral endoderm and the primitive streak is impaired in Cables2-deficient embryos. Tetraploid complementation analyses support the critical requirement of Cables2 in both the epiblast and visceral endoderm for progression of embryogenesis. In addition, we show that Cables2 physically interacts with a key mediator of the canonical Nodal pathway, Smad2, and augments its transcriptional activity. These findings provide novel insights into the essential role of Cables2 in the early embryonic development in mice.

developmental biology↗

bioRxiv2019

Isolation of an archaeon at the prokaryote-eukaryote interface

The origin of eukaryotes remains enigmatic. Current data suggests that eukaryotes may have risen from an archaeal lineage known as \"Asgard archaea\". Despite the eukaryote-like genomic features found in these archaea, the evolutionary transition from archaea to eukaryotes remains unclear due to the lack of cultured representatives and corresponding physiological insight. Here we report the decade-long isolation of a Lokiarchaeota-related Asgard archaeon from deep marine sediment. The archaeon, \"Candidatus Prometheoarchaeum syntrophicum strain MK-D1\", is an anaerobic, extremely slow-growing, small cocci ([~]550 nm), that degrades amino acids through syntrophy. Although eukaryote-like intracellular complexities have been proposed for Asgard archaea, the isolate has no visible organella-like structure. Ca. P. syntrophicum instead displays morphological complexity - unique long, and often, branching protrusions. Based on cultivation and genomics, we propose an \"Entangle-Engulf-Enslave (E3) model\" for eukaryogenesis through archaea-alphaproteobacteria symbiosis mediated by the physical complexities and metabolic dependency of the hosting archaeon.

microbiology↗

bioRxiv2019

Towards HCP-Style Macaque Connectomes: 24-Channel 3T Multi-Array Coil, MRI Sequences and Preprocessing

Macaque monkeys are an important model species for understanding cortical organization of primates, yet tools and methods for noninvasive image acquisition (e.g. MRI RF coils and pulse sequence protocols) and image data preprocessing have lagged behind those developed for humans. To resolve the structural and functional characteristics of the relatively thin macaque cortex, high spatial, temporal, and angular resolutions are required while maintaining high signal-to-noise ratio to ensure good image quality. To address these challenges, we developed a macaque 24-channel receive coil for 3-T MRI with parallel imaging capabilities. This coil enabled adaptation of the Human Connectome Project (HCP) image acquisition protocols to the macaque brain. We also adapted HCP preprocessing methods optimized for the macaque brain, including spatial minimal preprocessing of structural, functional MRI (fMRI), and diffusion MRI (dMRI). The coil provided high signal-to-noise ratio and high efficiency in data acquisition, allowing four- and five-fold acceleration for dMRI and fMRI, respectively. Automated parcellation of cortex, reconstruction of cortical surface, removal of artefacts and nuisance signals in fMRI, and distortion correction of dMRI performed well, and the overall quality of basic neurobiological measures was comparable with those for the HCP. The resulting HCP-style in vivo macaque MRI data show considerable promise for analyzing cortical architecture and functional and structural connectivity using advanced methods that have previously only been available for humans.\n\nHighlights[tpltrtarr] 24-channel 3T MR receive coil designed for the smaller macaque brain.\n[tpltrtarr]In vivo macaque imaging protocols adapted according to guidelines from the HCP.\n[tpltrtarr]Parallel imaging yields five- and four-fold acceleration in fMRI and dMRI sampling.\n[tpltrtarr]HCPs minimal preprocessing and denoising pipelines adapted for macaques.\n[tpltrtarr]The multi-modal MRI data show considerable promise for HCP-style analyses.

neuroscience↗

bioRxiv2018

Streptococcal phosphotransferase system imports unsaturated hyaluronan disaccharide derived from host extracellular matrices

Certain bacterial species target the polysaccharide glycosaminoglycans (GAGs) of animal extracellular matrices for colonization and/or infection. GAGs such as hyaluronan and chondroitin sulfate consist of repeating disaccharide units of uronate and amino sugar residues, and are depolymerized to unsaturated disaccharides by bacterial extracellular or cell-surface polysaccharide lyase. The disaccharides are degraded and metabolized by cytoplasmic enzymes such as unsaturated glucuronyl hydrolase, isomerase, and reductase. The genes encoding these enzymes are assembled to form a GAG genetic cluster. Here, we demonstrate the Streptococcus agalactiae phosphotransferase system (PTS) for import of unsaturated hyaluronan disaccharide. S. agalactiae NEM316 was found to depolymerize and assimilate hyaluronan, whereas its mutant with a disruption in PTS genes included in the GAG cluster was unable to grow on hyaluronan, while retaining the ability to depolymerize hyaluronan. Using toluene-treated wild-type cells, the PTS import activity of unsaturated hyaluronan disaccharide was significantly higher than that observed in the absence of the substrate. In contrast, the PTS mutant was unable to import unsaturated hyaluronan disaccharide, indicating that the corresponding PTS is the only importer of fragmented hyaluronan, which is suitable for PTS to phosphorylate the substrate at the C-6 position. The three-dimensional structure of streptococcal EIIA, one of the PTS components, was found to contain a Rossman-fold motif by X-ray crystallization. Docking of EIIA with another component EIIB by modeling provided structural insights into the phosphate transfer mechanism. This study is the first to identify the substrate (unsaturated hyaluronan disaccharide) recognized and imported by the streptococcal PTS.\n\nIMPORTANCE (118/120 words)The PTS identified in this work imports sulfate group-free unsaturated hyaluronan disaccharide as a result of the phosphorylation of the substrate at the C-6 position. S. agalactiae can be indigenous to animal hyaluronan-rich tissues owing to the bacterial molecular system for fragmentation, import, degradation, and metabolism of hyaluronan. Distinct from hyaluronan, most GAGs, which are sulfated at the C-6 position, are unsuitable for PTS due to its inability to phosphorylate the substrate. More recently, we have identified a solute-binding protein-dependent ABC transporter in a pathogenic Streptobacillus moniliformis as an importer of sulfated and non-sulfated fragmented GAGs without any substrate modification. Our findings regarding PTS and ABC transporter shed light on bacterial clever colonization/infection system targeting various animal GAGs.

microbiology↗