Biology subjects

Watanabe, S.

Publications and source records attributed to Watanabe, S..

At least 73 records · Page 4Linked to original sources

bioRxiv2022

Exploration of the autonomous replication region and its utilization for expression vectors in cyanobacteria.

Due to their photosynthetic capabilities, cyanobacteria is expected to be an ecologically friendly host for the production of biomaterials. However, compared to other bacteria, there is little information of autonomous replication sequences, and tools for genetic engineering, especially expression vector systems, are limited. In this study, we established an effective screening method, namely AR-seq (Autonomous Replication sequencing), for finding autonomous replication regions in cyanobacteria and utilized the region for constructing expression vector. AR-seq using the genomic library of Synechocystis sp. PCC 6803 revealed that a certain region containing Rep-related protein (here named as Cyanobacterial Rep protein A2: CyRepA2) exhibits high autonomous replication activity in a heterologous host cyanobacterium, Synechococcus elongatus PCC 7942. The reporter assay using GFP showed that the expression vector pYS carrying CyRepA2 can be maintained in a wide range of multiple cyanobacterial species, not only S. 6803 and S. 7942, but also Synechococcus sp. PCC 7002 and Anabaena sp. PCC 7120. In S. 7942, the GFP expression in pYS-based system can be tightly regulated by IPTG, achieving 10-fold higher levels than that of chromosome-based system. Furthermore, pYS can be used together with conventional vector pEX, which was constructed from an endogenous plasmid in 5. 7942. The combination of pYS with other vectors is useful for genetic engineering, such as modifying metabolic pathways, and is expected to improve the performance of cyanobacteria as bioproduction chassis.

synthetic biology↗

bioRxiv2022

The conserved upstream ORF of the Arabidopsis ANAC082 gene mediates translational upregulation in response to nucleolar stress

Perturbations in ribosome biogenesis cause a type of cellular stress called nucleolar or ribosomal stress, which triggers adaptive responses in both animal and plant cells. The Arabidopsis ANAC082 transcription factor has been identified as a key mediator of the plant nucleolar stress response. The 5'-untranslated region (5'-UTR) of ANAC082 mRNA contains an upstream ORF (uORF) encoding an evolutionarily conserved amino acid sequence. Here, we report that this uORF mediates the upregulation of ANAC082 translation in response to nucleolar stress. When transgenic Arabidopsis plants containing a luciferase reporter gene under the control of the ANAC082 promoter and 5'-UTR were treated with reagents that induced nucleolar stress, translation of the reporter gene was enhanced in a uORF sequence-dependent manner. Additionally, we examined the effect of an endoplasmic reticulum (ER) stress-inducing reagent on reporter gene expression because the closest homolog of ANAC082 in Arabidopsis, ANAC103, is involved in the ER stress response. However, the ANAC082 uORF did not respond to ER stress. Interestingly, although ANAC103 has a uORF with an amino acid sequence similar to that of the ANAC082 uORF, the C-terminal sequence critical for regulation is not well conserved among ANAC103 homologs in Brassicaceae. Transient expression assays revealed that unlike the ANAC082 uORF, the ANAC103 uORF does not exert a sequence-dependent regulatory effect. Altogether, our findings suggest that the ANAC082 uORF is important for the nucleolar stress response but not for the ER stress response, and that for this reason, the uORF sequence-dependent translational regulation was lost in ANAC103 during evolution.

plant biology↗

bioRxiv2022

Use of the particle agglutination/particle agglutination-inhibition test for antigenic analysis of SARS-CoV-2

The antigenicity of SARS-CoV-2 is a critical issue for the effectiveness of the vaccine, and thus it should be phenotypically evaluated by serological assays as new field isolates emerge. The hemagglutination/hemagglutination-inhibition (HA/HI) tests are well-known as a representative method for antigenic analysis of influenza viruses, but SARS-CoV-2 is unlikely to agglutinate to human or guinea pig red blood cells. Therefore, the antigenic analysis requires complicated enzyme-linked immunosorbent assay (ELISA) or cell-based assays such as the microneutralization assay. In this study, we developed the particle agglutination/particle agglutination-inhibition (PA/PAI) test to easily and rapidly quantify the virus and antibody using human angiotensin-converting enzyme 2 (hACE2)-bound latex beads. The PA titer was positively correlated with the plaque-forming units. The PAI titer using post-infection Syrian hamster antisera clearly revealed the antigenic difference between the omicron and previous variants. The results show the PAI test is useful for easy and rapid antigenic analysis of SARS-CoV-2.

microbiology↗

bioRxiv2022

The Intestinal Microbiome, Dietary Habits, and Physical and Psychological Resilience in Postpartum Women

The population of postpartum women suffering from mental illness is increasing steadily, particularly under conditions of the COVID-19 pandemic. Identifying factors that contribute to resilience in postpartum women is urgently needed to decrease risks of poor physical and psychological functioning. Studies have linked variations in the intestinal microbiota to depression in clinical samples, but the impacts in postpartum women in a Japanese population are unknown. We conducted two studies to examine the links between intestinal microbiota, physical condition, and psychological state in nonclinical, postpartum Japanese women. Our results show that decreasing Lachnospira and alpha diversity of microbiome is related to high mental health risk (i.e., parenting stress and/or depression). Psychological resilience and physical conditions were associated with relative abundances of genera Blautia, Clostridium, Eggerthella. This study contributes to further understanding of the gut-brain axis mechanisms and supports proposals for interventions to enhance resilience in postpartum women.

microbiology↗

bioRxiv2022

Impact of reinfection with SARS-CoV-2 Omicron variants in previously infected hamsters

The diversity of SARS-CoV-2 mutations raises the possibility of reinfection of individuals previously infected with earlier variants, and this risk is further increased by the emergence of the B.1.1.529 Omicron variant. In this study, we used an in vivo, hamster infection model to assess the potential for individuals previously infected with SARS-CoV-2 to be reinfected with Omicron variant and we also investigated the pathology associated with such infections. Initially, Syrian hamsters were inoculated with a lineage A, B.1.1.7, B.1.351, B.1.617.2 or a subvariant of Omicron, BA.1 strain and then reinfected with the BA.1 strain 5 weeks later. Subsequently, the impact of reinfection with Omicron subvariants (BA.1 and BA.2) in individuals previously infected with the BA.1 strain was examined. Although viral infection and replication were suppressed in both the upper and lower airways, following reinfection, virus-associated RNA was detected in the airways of most hamsters. Viral replication was more strongly suppressed in the lower respiratory tract than in the upper respiratory tract. Consistent amino acid substitutions were observed in the upper respiratory tract of infected hamsters after primary infection with variant BA.1, whereas diverse mutations appeared in hamsters reinfected with the same variant. Histopathology showed no acute pneumonia or disease enhancement in any of the reinfection groups and, in addition, the expression of inflammatory cytokines and chemokines in the airways of reinfected animals was only mildly elevated. These findings are important for understanding the risk of reinfection with new variants of SARS-CoV-2. IMPORTANCEThe emergence of SARS-CoV-2 variants and the widespread use of COVID-19 vaccines has resulted in individual differences in immune status against SARS-CoV-2. A decay in immunity over time and the emergence of variants that partially evade the immune response can also lead to reinfection. In this study, we demonstrated that, in hamsters, immunity acquired following primary infection with previous SARS-CoV-2 variants was effective in preventing the onset of pneumonia after reinfection with the Omicron variant. However, viral infection and multiplication in the upper respiratory tract were still observed after reinfection. We also showed that more diverse nonsynonymous mutations appeared in the upper respiratory tract of reinfected hamsters that had acquired immunity from primary infection. This hamster model reveals the within-host evolution of SARS-CoV-2 and its pathology after reinfection, and provides important information for countermeasures against diversifying SARS-CoV-2 variants.

microbiology↗

bioRxiv2022

Characterization of SARS-CoV-2 Omicron BA.2.75 clinical isolates

The prevalence of the Omicron subvariant BA.2.75 is rapidly increasing in India and Nepal. In addition, BA.2.75 has been detected in at least 34 other countries and is spreading globally. However, the virological features of BA.2.75 are largely unknown. Here, we evaluated the replicative ability and pathogenicity of BA.2.75 clinical isolates in Syrian hamsters. Although we found no substantial differences in weight change among hamsters infected with BA.2, BA.5, or BA.2.75, the replicative ability of BA.2.75 in the lungs was higher than that of BA.2 and BA.5. Of note, BA.2.75 caused focal viral pneumonia in hamsters, characterized by patchy inflammation interspersed in alveolar regions, which was not observed in BA.5-infected hamsters. Moreover, in competition assays, BA.2.75 replicated better than BA.5 in the lungs of hamsters. These results suggest that BA.2.75 can cause more severe respiratory disease than BA.5 and BA.2 and should be closely monitored.

microbiology↗

bioRxiv2022

Imbalance of circuit plasticity and consolidation in autism model marmosets is adjusted by oxytocin administration

Alterations in the experience-dependent and autonomous elaboration of neural circuits are assumed to underlie autism spectrum disorder (ASD), though it is unclear what synaptic traits are responsible. Here, we used a valproic acid-induced ASD marmoset model, which shares common molecular features with idiopathic ASD, to investigate the structural dynamics of tuft dendrites of upper-layer pyramidal neurons and adjacent axons in the dorsomedial prefrontal cortex using two-photon microscopy. In model marmosets, dendritic spine turnover was upregulated, and spines were actively generated in clusters and subsequently survived more often than in control marmosets. Presynaptic boutons in local axons but not in commissural long-range axons showed hyperdynamic turnover in model marmosets, suggesting alterations in projection-specific plasticity. Intriguingly, nasal administration of oxytocin reduced the clustered spine emergence. Enhanced clustered spine generation, possibly unique to certain presynaptic partners, may be associated with ASD and may be a potential therapeutic target.

neuroscience↗

bioRxiv2022

Rapid and reversible optogenetic silencing of synaptic transmission by clustering of synaptic vesicles

Silencing neurons acutely and specifically informs about their functional roles in circuits and behavior. Existing optogenetic silencers include ion pumps or channels, and tools that damage the neurotransmitter release machinery. While the former hyperpolarize the cell and can alter ionic gradients, the latter allow only slow recovery, requiring de novo synthesis. Thus, there is a need for other strategies combining fast activation and reversibility. Here, we use light-evoked homo-oligomerization of cryptochrome CRY2 to silence synaptic transmission, by clustering synaptic vesicles (SVs). We benchmark this tool, optoSynC, by electrophysiology and locomotion in Caenorhabditis elegans. optoSynC clusters SVs within 25 s, causing approximation, observable by electron microscopy. Locomotion silencing is rapid (tauon [~]15 s) and recovers quickly (tauoff [~]10 min) after light-off. Further, optoSynC can inhibit exocytosis for several hours, at very low light intensities. optoSynC is a highly efficient, non-ionic optogenetic silencer that may further allow to manipulate different SV pools.

neuroscience↗

bioRxiv2022

Membrane compression by synaptic vesicle exocytosis triggers ultrafast endocytosis

Compensatory endocytosis keeps the surface area of secretory cells constant following exocytosis. At chemical synapses, clathrin-independent ultrafast endocytosis maintains such homeostasis. This endocytic pathway is temporally and spatially coupled to exocytosis, initiating within 50 ms at the region immediately next to where vesicles fuse: the active zone. How synaptic vesicle exocytosis induces ultrafast endocytosis is unknown. Here, we demonstrate that actin filaments are enriched in the region surrounding active zone at mouse hippocampal synapses and that the membrane area conservation due to this actin corral is necessary for exo-endocytic coupling. Simulations suggest that flattening of fused vesicles exerts lateral membrane pressure in the plasma membrane against the actin corral, resulting in rapid formation of endocytic pits at the border between the active zone and the surrounding actin-enriched region. Consistent with our simulations, ultrafast endocytosis does not initiate when actin organization is disrupted, either pharmacologically or by ablation of the actin-binding protein Epsin1. These data suggest that endocytosis is mechanically coupled to exocytosis at synapses.

cell biology↗

bioRxiv2022

Dynamin forms liquid-like condensates at synapses to support ultrafast endocytosis

Endocytosis at synapses is accelerated by the pre-accumulation of Dynamin 1xA at the endocytic zone by Syndapin 1. However, it is unclear how these proteins support the ultrafast kinetics of endocytosis. Here we report that these proteins phase separate at the presynaptic endocytic zone where ultrafast endocytosis takes place. Specifically, the proline-rich motif of Dynamin 1xA interacts with the Src-Homology 3 domain of Syndapin 1 and forms liquid-like condensates. Single-particle tracking of Dynamin 1xA molecules at synapses shows that their diffusion slows down substantially when they are in the condensates, indicating the presence of molecular crowding and intermolecular interaction. When Dynamin 1xA is mutated to disrupt its interaction with Syndapin 1 the condensates do not form. Thus, the liquid-like assembly of these endocytic proteins provides a catalytic platform for ultrafast endocytosis.

neuroscience↗

bioRxiv2022

CaV1 and CaV2 calcium channels mediate the release of distinct pools of synaptic vesicles

Activation of voltage-gated calcium channels at synapses leads to local increases in calcium and the fusion of synaptic vesicles. However, presynaptic output will be determined by the density of calcium channels, the dynamic properties of the channel, the distance to docked vesicles, and the release probability at the docking site. We demonstrate that at C. elegans neuromuscular junctions two different classes of voltage-gated calcium channels, CaV2 and CaV1, mediate the release of distinct pools of synaptic vesicles. CaV2 channels are concentrated in densely packed clusters [~]300 nm in diameter with the active zone proteins Neurexin, -Liprin, SYDE, ELKS/CAST, RIM-BP, -Catulin, and MAGI1. CaV2 channels mediate the fusion of vesicles docked adjacent to the dense projection and are colocalized with the synaptic vesicle priming protein UNC-13L. By contrast, CaV1 channels are dispersed in the synaptic varicosity and are coupled to internal calcium stores via the ryanodine receptor. CaV1 and ryanodine receptor mediate the fusion of vesicles docked broadly in the synaptic varicosity and are colocalized with the vesicle priming protein UNC-13S. Distinct synaptic vesicle pools, released by different calcium channels, could be used to tune the speed, voltage-dependence, and quantal content of neurotransmitter release.

neuroscience↗

bioRxiv2022

Synaptotagmin 7 docks synaptic vesicles for Doc2α-triggered asynchronous neurotransmitter release

Despite decades of intense study, the molecular basis of asynchronous neurotransmitter release remains enigmatic. Synaptotagmin (syt) 7 and Doc2 have both been proposed as Ca2+ sensors that trigger this mode of exocytosis, but conflicting findings have led to controversy. Here, we demonstrate that at excitatory mouse hippocampal synapses, Doc2 is the major Ca2+ sensor for asynchronous release, while syt7 supports this process through activity-dependent docking of synaptic vesicles. In synapses lacking Doc2, asynchronous release after single action potentials is strongly reduced, while deleting syt7 has no effect. However, in the absence of syt7, docked vesicles cannot be replenished on millisecond timescales. Consequently, both synchronous and asynchronous release depress from the second pulse onward during repetitive activity. By contrast, synapses lacking Doc2 have normal activity-dependent docking, but continue to exhibit decreased asynchronous release after multiple stimuli. Moreover, disruption of both Ca2+ sensors is non-additive. These findings result in a new model whereby syt7 drives activity-dependent docking, thus providing synaptic vesicles for synchronous (syt1) and asynchronous (Doc2 and other unidentified sensors) release during ongoing transmission.

neuroscience↗

bioRxiv2022

Secreted dengue virus NS1 is predominantly dimeric and in complex with high-density lipoprotein

Severe dengue infections are characterized by endothelial dysfunction shown to be associated with the secreted nonstructural protein 1 (sNS1), making it an attractive vaccine antigen and biotherapeutic target. To uncover the biologically relevant structure of sNS1, we obtained infection-derived sNS1 (isNS1) from DENV-infected Vero cells through immunoffinity purification instead of recombinant sNS1 (rsNS1) overexpressed in insect or mammalian cell lines. We found that isNS1 appeared as an approximately 250 kDa complex of NS1 and ApoA1 and further determined the cryoEM structures of isNS1 and its complex with a monoclonal antibody/Fab. Indeed, we found that the major species of isNS1 is a complex of the NS1 dimer partially embedded in a High-Density Lipoprotein (HDL) particle. Cross-linking mass spectrometry (XL-MS) studies confirmed that the isNS1 interacts with the major HDL component ApoA1 through interactions that map to the NS1 wing and hydrophobic domains. Furthermore, our studies demonstrated that the sNS1 in sera from DENV-infected mice and a human patient form a similar complex as isNS1. Our results report the molecular architecture of a biological form of sNS1 which may have implications for the molecular pathogenesis of dengue. SummaryCryoEM structures of secreted dengue virus NS1 protein reveal dimers in complex with high-density lipoprotein.

microbiology↗

bioRxiv2022

Cryo-EM structures of human zinc transporter ZnT7 reveal the mechanism of Zn2+ uptake into the Golgi apparatus

Zinc ions (Zn2+) are vital to most cells, with the intracellular concentrations of Zn2+ being tightly regulated by multiple zinc transporters located at the plasma and organelle membranes. We herein present the 2.8-2.9 [A]-resolution cryo-EM structures of a Golgi-localized human Zn2+/H+ antiporter ZnT7 (hZnT7) in its outward- and inward-facing forms. Cryo-EM analyses showed that hZnT7 exists as a homodimer via tight interactions in both the cytosolic and transmembrane (TM) regions of two protomers, each of which contains a single Zn2+-binding site in its TM domain. hZnT7 undergoes a TM-helix rearrangement to create a negatively charged cytosolic cavity for Zn2+ entry in the inward-facing form and a widened luminal cavity for Zn2+ release in the outward-facing form. An exceptionally long cytosolic histidine-rich loop characteristic of hZnT7 can bind at least two Zn2+ ions, likely facilitating Zn2+ recruitment from the cytosol. Unique mechanisms of hZnT7-mediated Zn2+ uptake into the Golgi are proposed.

molecular biology↗

bioRxiv2022

Different efficacies of neutralizing antibodies and antiviral drugs on SARS-CoV-2 Omicron subvariants, BA.1 and BA.2

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariant BA.2 has spread in many countries, replacing the earlier Omicron subvariant BA.1 and other variants. Here, using a cell culture infection assay, we quantified the intrinsic sensitivity of BA.2 and BA.1 compared with other variants of concern, Alpha, Gamma, and Delta, to five approved-neutralizing antibodies and antiviral drugs. Our assay revealed the diverse sensitivities of these variants to antibodies, including the loss of response of both BA.1 and BA.2 to casirivimab and of BA.1 to imdevimab. In contrast, EIDD-1931 and nirmatrelvir showed a more conserved activities to these variants. The viral response profile combined with mathematical analysis estimated differences in antiviral effects among variants in the clinical concentrations. These analyses provide essential evidence that gives insight into variant emergences impact on choosing optimal drug treatment.

microbiology↗

bioRxiv2022

Feedback regulation of RNase E during UV-stress response in the cyanobacterium Synechocystis sp. PCC 6803

Endoribonucleases govern the maturation and degradation of RNA and are indispensable in the posttranscriptional regulation of gene expression. A key endoribonuclease in many bacteria is RNase E. To ensure an appropriate supply of RNase E, some bacteria, such as E. coli, have evolved tightly functioning feedback regulation of RNase E that is mediated in cis by the rne 5'-untranslated region (5' UTR); however, the mechanisms involved in the control of RNase E in other bacteria largely remain unknown. Cyanobacteria rely on solar light as an energy source for photosynthesis, despite the inherent ultraviolet (UV) irradiation. Here, we investigated the global gene expression response in the cyanobacterium Synechocystis sp. PCC 6803 after exposure to UV light and discovered a unique response of RNase E: a rapidly increasing enzymatic activity, although the stability of the protein was decreased. In parallel, we observed an increased accumulation of full-length rne mRNA that was caused by the stabilization of its 5' UTR and suppression of premature transcriptional termination but not by an increased transcription rate. Mapping of RNA 3' ends and in vitro cleavage assays revealed that RNase E cleaves within a stretch of six consecutive uridine residues within the rne 5' UTR, indicating autoregulation via its own 5' UTR. These observations imply that RNase E in cyanobacteria contributes substantially to reshaping the transcriptome during the UV stress response and that its required activity level is maintained despite enhanced turnover of the protein by posttranscriptional feedback regulation.

microbiology↗

bioRxiv2021

Skeletal muscle releases extracellular vesicles with distinct protein and miRNA signatures that accumulate and function within the muscle microenvironment

Extracellular vesicles (EVs) contain various regulatory molecules and mediate intercellular communications. Although EVs are secreted from various cell types, including skeletal muscle cells, and present in the blood, their identity is poorly characterized in vivo, limiting the identification of their origin in the blood. Since the skeletal muscle is the largest organ in the body, it could substantially contribute to circulating EVs as their source. However, due to the lack of defined markers that distinguish SkM-EVs from others, whether the skeletal muscle releases EVs in vivo and how much the skeletal muscle-derived EVs (SkM-EVs) account for plasma EVs remain poorly understood. In this work, we perform quantitative proteomic analyses on EVs released from C2C12 cells and human iPS cell-derived myocytes and identify potential marker proteins that mark SkM-EVs. These markers we identified apply to in vivo tracking of SkM-EVs. The results show that skeletal muscle makes only a subtle contribution to plasma EVs as their source in both control and exercise conditions in mice. On the other hand, we demonstrate that SkM-EVs are concentrated in the skeletal muscle interstitium. Furthermore, we show that interstitium EVs are highly enriched with the muscle-specific miRNAs and repress the expression of the paired box transcription factor Pax7, a master regulator for myogenesis. Taken together, our findings reveal that the skeletal muscle releases exosome-like small EVs with distinct protein and miRNA profiles in vivo and that SkM-EVs mainly play a role within the muscle microenvironment where they accumulate.

physiology↗

bioRxiv2021

In vivo label-free observation of tumor-related blood vessels in small animals using a newly designed photoacoustic 3D imaging system

IntroductionPhotoacoustic technology can be used for non-invasive imaging of blood vessels. In this paper, we report on our prototype photoacoustic imaging system with a newly designed ultrasound sensor and its visualization performance of microvascular in animal. MethodsWe fabricated an experimental system for animals using a high-frequency sensor. The system has two modes: still image mode by wide scanning and moving image mode by small rotation of sensor array. Optical test target, euthanized mice and rats, and live mice were used as objects. ResultsThe results of optical test target showed that the spatial resolution was about 2 times higher than that of our conventional prototype. The image performance in vivo was evaluated in euthanized healthy mice and rats, allowing visualization of detailed blood vessels in the liver and kidneys. In tumor-bearing mice, different results of vascular induction were shown depending on the type of tumor and the method of transplantation. By utilizing the video imaging function, we were able to observe the movement of blood vessels around the tumor. ConclusionWe have demonstrated the feasibility of the system as a less invasive animal experimental device, as it can acquire vascular images in animals in a non-contrast and non-invasive manner.

cancer biology↗