Biology subjects

Sato, H.

Publications and source records attributed to Sato, H..

At least 55 records · Page 3Linked to original sources

bioRxiv2022

A red fluorescent genetically encoded biosensor for extracellular L-lactate

L-Lactate, traditionally recognized as a waste product of metabolism, is now appreciated as a key intercellular energy currency in mammals. To enable investigations of intercellular shuttling of L-lactate, we have previously reported eLACCO1.1, a green fluorescent genetically encoded biosensor for extracellular L-lactate. eLACCO1.1 enables cellular resolution imaging of extracellular L-lactate in cultured mammalian cells and brain tissue. However, eLACCO1.1 spectrally overlaps with commonly used optical biosensors and actuators, limiting its application for multiplexed imaging or combined use with optogenetic actuators. Here, we report a red fluorescent extracellular L-lactate biosensor, designated R-eLACCO2. R-eLACCO2 is the end-product of extensive directed evolution and exhibits a large fluorescence response to L-lactate with high molecular specificity in vitro. We demonstrate that R-eLACCO2 with optimized leader and anchor sequences shows a large fluorescence change in response to extracellular L-lactate on the membrane of live mammalian cells. R-eLACCO2 should enable multicolor imaging of extracellular L-lactate in combination with other fluorescent probes and optogenetic actuators.

bioengineering↗

bioRxiv2022

Analysis of genetic relatedness between gastric and oral H. pylori in patients with early gastric cancer using multilocus sequence typing

Although oral cavity is the second most colonized site after the stomach, the association between gastric and oral Helicobacter pylori remains unclear. This study aimed to compare the genetic relatedness between gastric and oral H. pylori in Japanese patients with early gastric cancer by multilocus sequence typing (MLST) analysis using seven housekeeping genes. Gastric biopsy specimens and oral samples, including saliva, supragingival dental biofilm, and superficial layers of the tongue, were collected from 21 patients positive for H. pylori by a fecal antigen test. The number of H. pylori allelic profiles of seven loci obtained from oral and gastric samples ranged from zero to seven since the yield of DNA was small even when the nested PCR was performed. The alleles of seven loci from both collection sites were determined from only one patient, and two out of seven alleles matched between oral and gastric samples. MLST analysis revealed that only one sample had a matching oral and gastric H. pylori genotype, suggesting that different genotypes of H. pylori inhabit the oral cavity and gastric mucosa. The phylogenetic analysis showed that oral H. pylori in two patients was markedly similar to gastric H. pylori, implying that the the origins of two strains may be the same, and the stomach and the oral cavity may be infected at the same time. In brief, although different genotypes of H. pylori exist in the oral cavity independently of H. pylori present in the stomach, there are rare cases in which the same H. pylori is present in the stomach and the oral cavity. It is necessary to establish a culture method for oral H. pylori for elucidating whether the oral cavity will act as the source of the gastric infection, as our analysis was based on a limited number of allele sequences. Author summaryHericobacter pylori is a clinically important pathogen that causes chronic gastritis and peptic ulcers, which are associated with gastric carcinoma. Oral H. pylori DNA has also been detected in a range of oral specimens, including saliva, supra- or sub-gingival biofilm, dentin caries, dental pulp, infected root canal, and coating of the tongue. Thus, researchers speculate that the main route of transmission of gastric H. pylori infections is via the oral cavity, however, there is inadequate evidence to support this suggestion. This study aimed to investigate if the oral and gastric H. pylori have the same origin using multilocus sequence typing. The results of this study provided some insight into the role of oral cavity as the source of H. pylori infections in stomach.

microbiology↗

bioRxiv2022

Structural basis for activation of DNMT1

DNMT1 is an essential enzyme that maintains genomic DNA methylation, and its function is regulated by mechanisms that are not yet fully understood. Here, we report the cryo-EM structure of human DNMT1 bound to its two natural activators: hemimethylated DNA and ubiquitinated histone H3. We find that a hitherto unstudied linker, between the RFTS and CXXC domains, plays a key role for activation. It contains a conserved -helix which engages a crucial "Toggle" pocket, displacing a previously described inhibitory linker, and allowing the DNA recognition helix to spring into the active conformation. This is accompanied by large-scale reorganization of the inhibitory RFTS and CXXC domains, allowing the enzyme to gain full activity. Our results therefore provide a mechanistic basis for the activation of DNMT1, with consequences for basic research and drug design.

biochemistry↗

bioRxiv2022

Transcription factors that specify cell fate activate cell cycle regulator genes to determine cell numbers in ascidian larval tissues

In animal development, most cell types stop dividing before terminal differentiation; thus, cell cycle control is tightly linked to cell differentiation programs. Although cell cycle control in animal development has been studied extensively, such links are not well understood. In ascidian embryos, cell lineages do not vary among individuals, and rounds of the cell cycle are determined according to cell lineages. In the present study, we first showed that maternal factors promote approximately 10 rounds of cell division without zygotic gene expression. Notochord and muscle cells stop dividing after fewer than 10 rounds of cell division, and we show that a Cdk inhibitor (Cdkn1.b) is responsible. Cdkn1.b is also necessary for epidermal cells to stop dividing. In contrast, mesenchymal and endodermal cells divided more than 10 times, and Myc, which encodes a proto-oncogenic transcription factor, is responsible for maintaining cell cycle progression in these tissues. Expression of Cdkn1.b in notochord and muscle is controlled by the same developmental programs that specify the developmental fate of notochord and muscle. Likewise, expression of Myc in mesenchyme and endoderm was under control of the same developmental programs that specify the developmental fate of mesenchyme and endoderm. Because these transcription factors that regulate Cdkn1.b and Myc are essential factors for fate specification of these tissues, cell fate specification and cell cycle control are linked by those transcription factors. In other words, ectodermal, mesodermal, and endodermal tissues in ascidian embryos control the cell cycle through Cdkn1.b and Myc, which are under the control of transcription factors that specify cell fate.

developmental biology↗

bioRxiv2022

An improved MS2-MCP imaging system with minimal perturbation of mRNA stability

The MS2-MCP imaging system is widely used to study the mRNA spatial distribution in living cells. Here, we report that the MS2-MCP system may destabilize the tagged mRNA by targeting it to the nonsense-mediated mRNA decay pathway. We introduce an improved version, which has minimal perturbation of the mRNA stability.

cell biology↗

bioRxiv2021

Large-scale mapping of positional changes of hypoxia-responsive genes upon activation

Chromosome structure and nuclear organization are important factors in the regulation of gene expression. Transcription of a gene is influenced by local and global chromosome features such as condensation status and histone modifications. The relationship between the position of a gene in the cell nucleus and its activity is less clear. Here, we used high-throughput imaging to perform a large-scale analysis of the spatial location of a set of nearly 100 hypoxia-inducible genes to determine whether their location within the nucleus is correlated with their activity state upon stimulation. Radial distance analysis demonstrated that the majority of HIF- and CREB-inducible hypoxia responsive genes are located in the intermediate region of the nucleus. Radial position of numerous responsive genes changed upon hypoxic treatment. Analysis of the relative distances amongst a subset of HIF target gene groups revealed that some gene pairs also altered their relative location to each other upon hypoxic treatment, suggesting higher order chromatin rearrangements. While these changes in location occurred in response to hypoxic activation of the target genes, they did not correlate with the extent of their activation. These results suggest that induction of the hypoxia-responsive gene expression program is accompanied by spatial alterations of the genome, but that radial and relative gene positions are not directly related to gene activity.

cell biology↗

bioRxiv2021

Cellular variability of nonsense-mediated mRNA decay

Nonsense-mediated mRNA decay (NMD) is an mRNA degradation pathway that eliminates transcripts containing premature termination codons (PTCs). Half-lives of the mRNAs containing PTCs demonstrate that a small percent escape surveillance and do not degrade. It is not known whether this escape represents variable mRNA degradation within cells or, alternatively cells within the population are resistant. Here we demonstrate a single-cell approach with a bi-directional reporter, which expresses two {beta}-globin genes with or without a PTC in the same cell, to characterize the efficiency of NMD in individual cells. We found a broad range of NMD efficiency in the population; some cells degraded essentially all of the mRNAs, while others escaped NMD almost completely. Characterization of NMD efficiency together with NMD regulators in single cells showed cell-to-cell variability of NMD reflects the differential level of surveillance factors, SMG1 and phosphorylated UPF1. A single-cell fluorescent reporter system that enabled detection of NMD using flow cytometry revealed that this escape occurred either by translational readthrough at the PTC or by a failure of mRNA degradation after successful translation termination at the PTC.

cell biology↗

bioRxiv2021

Thalamocortical axons control the cytoarchitecture of neocortical layers by area-specific supply of secretory proteins

Neuronal abundance and thickness of each cortical layer is specific to each area, but how this fundamental feature arises during development remains poorly understood. While some of area-specific features are controlled by intrinsic cues such as morphogens and transcription factors, the exact influence and mechanisms of action by extrinsic cues, in particular the thalamic axons, have not been fully established. Here we identify a thalamus-derived factor, VGF, which is indispensable for thalamocortical axons to maintain the proper amount of layer 4 neurons in the mouse sensory cortices. This process is prerequisite for further maturation of the primary somatosensory area, such as barrel field formation instructed by a neuronal activity-dependent mechanism. Our results also provide an insight into regionalization of brain in that highly site-specific axon projection anterogradely confers further regional complexity upon the target field through locally secreting signaling molecules from axon terminals.

neuroscience↗

bioRxiv2020

Combinations of maternal-specific repressive epigenetic marks in the endosperm control seed dormancy

Polycomb Repressive Complex 2 (PRC2)-mediated trimethylation of histone H3 on lysine 27 (H3K27me3) and methylation of histone 3 on lysine 9 (H3K9me) are two repressive epigenetic modifications that are typically localized in distinct regions of the genome. For reasons unknown, however, they co-occur in some organisms and special tissue types. In this study, we show that maternal alleles marked by H3K27me3 in the Arabidopsis endosperm were targeted by the H3K27me3 demethylase REF6 and became activated during germination. In contrast, maternal alleles marked by H3K27me3, H3K9me2, and CHGm were protected from REF6 targeting and remained silenced. Our study unveils that combinations of different repressive epigenetic modifications time a key adaptive trait by modulating access of REF6.

molecular biology↗

bioRxiv2020

Effects of species traits and ecosystem characteristics on species detection by eDNA metabarcoding in lake fish communities

Although environmental DNA (eDNA) metabarcoding is acknowledged to be an exceptionally useful and powerful tool for monitoring surveys, it has limited applicability, particularly for nationwide surveys. To evaluate the performance of eDNA metabarcoding in broad-scale monitoring, we examined the effects of species ecological/biological traits and ecosystem characteristics on species detection rates and the consequences for community analysis. We conducted eDNA metabarcoding on fish communities in 18 Japanese lakes on a country-wide scale. By comparing species records, we found that certain species traits, including body size, body shape, saltwater tolerance, and habitat preferences, influenced eDNA detection. We also found that the proportion of species detected decreased significantly with an increase in lake surface area, owing to an ecosystem-size effect on species detection. We conclude that species traits, including habitat preferences and body size, and ecosystem size should be taken into consideration when assessing the performance of eDNA metabarcoding in broad-scale monitoring.

ecology↗

bioRxiv2020

Roles of the ClC chloride channel CLH-1 in food-associated salt chemotaxis behavior of C. elegans

The ability of animals to process dynamic sensory information facilitates foraging in an ever changing environment. However, molecular and neural mechanisms underlying such ability remain elusive. The ClC anion channels/transporters play a pivotal role in cellular ion homeostasis across all phlya. Here we find a ClC chloride channel is involved in salt concentration chemotaxis of C. elegans. Genetic screening identified two altered-function mutations of clh-1 that disrupt experience-dependent salt chemotaxis. Using genetically encoded fluorescent sensors, we demonstrate that CLH-1 contributes to regulation of chloride and calcium dynamics of salt-sensing neuron, ASER. The mutant CLH-1 reduced responsiveness of ASER to salt stimuli in terms of both temporal resolution and intensity, which could disrupt navigation strategies for approaching preferred salt concentration. Furthermore, other ClC genes appeared to act redundantly in salt chemotaxis. These findings provide insights into the regulatory mechanism of neuronal responsivity by ClCs that contribute to modulation of navigation behavior.

genomics↗

bioRxiv2019

A cellular stress response induced by the CRISPR/dCas9 activation system is not heritable through cell divisions

The CRISPR/Cas9 system can be modified to perform epigenetic editing by utilizing the catalytically-inactive (dead) Cas9 (dCas9) to recruit regulatory proteins to specific genomic locations. In prior studies, epigenetic editing with multimers of the transactivator VP16 and guide RNAs (gRNAs) was found to cause adverse cellular responses. These side effects may confound studies inducing new cellular properties, especially if the cellular responses are maintained through cell divisions - an epigenetic regulatory property. Here we show how distinct components of this CRISPR/dCas9 activation system, particularly untargeted gRNAs, upregulate genes associated with transcriptional stress, defense response, and regulation of cell death. Our results highlight a previously undetected acute stress response to CRISPR/dCas9 components in human cells, which is transient and not maintained through cell divisions.

genetics↗

bioRxiv2019

Quantitative Analysis of Effects of a Single 60Co Gamma Ray Point Exposure on Time-Dependent Change in Locomotor Activity in Rats

Fatigue is one of the earliest nonspecific symptoms of radiation exposure in humans, but its etiology, mechanism, and dose dependency remain unexplained. Investigating initial behavioral changes caused by irradiation of animals might provide important information to aid understanding of early health effects of radiation exposure and clinical features of radiation injury. Although previous studies in rodents suggested that radiation exposure leads to reduced activity, detailed properties of the effects were unrevealed due to a lack of proper statistical analysis, which is needed to better elucidate details of changes in locomotor activity. Ten-week-old male Wistar rats were subjected to single point external whole-body irradiation with 60Co gamma rays at 0, 2.0, 3.5, and 5.0 Gy (4 rats per group). Infrared sensors were used to continuously record locomotor activity of each rat. Cumulative number of movements during the night was defined as "activity" for each day. A non-linear mixed effects model accounting for individual differences and daily fluctuation of activity was applied to analyze the rats longitudinal locomotor data. Despite a small number of animals per group, our statistical method successfully revealed characteristics of the changes in locomotor activity after radiation exposure, showing that 1) reduction in activity occurred immediately--and in a dose-dependent manner--after irradiation and 2) recovery to pre-irradiation levels required almost one week, with the same recovery rate in each dose group. In addition to improving our understanding of radiation effects on locomotor activity, this statistical framework should be useful to analyze other data with similar structure.

pharmacology and toxicology↗

bioRxiv2019

Population-specific and transethnic genome-wide analyses reveal distinct and shared genetic risks of coronary artery disease

To elucidate the genetics of coronary artery disease (CAD) in the Japanese population, we conducted a large-scale genome-wide association study (GWAS) of 168,228 Japanese (25,892 cases and 142,336 controls) with genotype imputation using a newly developed reference panel of Japanese haplotypes including 1,782 CAD cases and 3,148 controls. We detected 9 novel disease-susceptibility loci and Japanese-specific rare variants contributing to disease severity and increased cardiovascular mortality. We then conducted a transethnic meta-analysis and discovered 37 additional novel loci. Using the result of the meta-analysis, we derived a polygenic risk score (PRS) for CAD, which outperformed those derived from either Japanese or European GWAS. The PRS prioritized risk factors among various clinical parameters and segregated individuals with increased risk of long-term cardiovascular mortality. Our data improves clinical characterization of CAD genetics and suggests the utility of transethnic meta-analysis for PRS derivation in non-European populations.

genetics↗

bioRxiv2019

Environmental DNA analysis shows high potential as a tool for estimating intraspecific genetic diversity in a wild fish population

Environmental DNA (eDNA) analysis has recently been used as a new tool for estimating intraspecific diversity. However, whether known haplotypes contained in a sample can be detected correctly using eDNA-based methods has been examined only by an aquarium experiment. Here, we tested whether the haplotypes of Ayu fish (Plecoglossus altivelis altivelis) detected in a capture survey could also be detected from an eDNA sample derived from the field that contained various haplotypes with low concentrations and foreign substances. A water sample and Ayu specimens collected from a river on the same day were analysed by eDNA analysis and Sanger sequencing, respectively. The 10 L water sample was divided into 20 filters for each of which 15 PCR replications were performed. After high-throughput sequencing, denoising was performed using two of the most widely used denoising packages, UNOISE3 and DADA2. Of the 42 haplotypes obtained from the Sanger sequencing of 96 specimens, 38 (UNOISE3) and 41 (DADA2) haplotypes were detected by eDNA analysis. When DADA2 was used, except for one haplotype, haplotypes owned by at least two specimens were detected from all the filter replications. This study showed that the eDNA analysis for evaluating intraspecific genetic diversity provides comparable results for large-scale capture-based conventional methods, suggesting that it could become a more efficient survey method for investigating intraspecific genetic diversity in the field.

molecular biology↗

bioRxiv2019

An annotation dataset facilitates automatic annotation of whole-brain activity imaging of C. elegans

Annotation of cell identity is an essential process in neuroscience that allows for comparing neural activities across different animals. In C. elegans, although unique identities have been assigned to all neurons, the number of annotatable neurons in an intact animal is limited in practice and comprehensive methods for cell annotation are required. Here we propose an efficient annotation method that can be integrated with the whole-brain imaging technique. We systematically identified neurons in the head region of 311 adult worms using 35 cell-specific promoters and created a dataset of the expression patterns and the positions of the neurons. The large positional variations illustrated the difficulty of the annotation task. We investigated multiple combinations of cell-specific promoters to tackle this problem. We also developed an automatic annotation method with human interaction functionality that facilitates annotation for whole-brain imaging.

neuroscience↗

bioRxiv2019

CAT1/SLC7A1 acts as a cellular receptor for bovine leukemia virus infection

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis, the most common neoplastic disease of cattle, which is closely related to human T-cell leukemia viruses. BLV has spread worldwide and causes a serious problem for the cattle industry. The cellular receptor specifically binds with viral envelope glycoprotein (Env) and this attachment mediates cell fusion to lead virus entry. BLV Env reportedly binds to cationic amino acid transporter 1 (CAT1)/SLC7A1, but whether the CAT1/SLC7A1 is an actual receptor for BLV remains unknown. Here, we showed that CAT1 functioned as an infection receptor, interacting with BLV particles. Cells expressing undetectable CAT1 levels were resistant to BLV infection but became highly susceptible upon CAT1 overexpression. CAT1 exhibited specific binding to BLV particles on the cell surface and co-localized with the Env in endomembrane compartments and membrane. Knockdown of CAT1 in permissive cells significantly reduced binding to BLV particles and BLV infection. In addition, bovine serum with neutralizing activity from a BLV-infected cattle inhibited BLV particles. Expression of CAT1 from various species demonstrated no species-specificity for BLV infection, implicating CAT1 as a functional BLV receptor responsible for its broad host range. These findings provide insights for BLV infection and for developing new strategies for treating BLV and preventing its spread.\n\nAuthor SummaryBovine leukemia virus (BLV), which can infect a variety of animal species and induce lymphoma in cattle, is a member of the family Retroviridae. BLV induces huge economic losses by not only lymphoma but also subclinical forms of the disease. In addition, BLV is frequently used as an animal model of human T-cell leukemia virus (HTLV), as BLV has many similar characteristics to HTLV. Thus, understanding BLV pathogenesis contribute to resolve not only BLV-but also HTLV-induced problems. Retroviral envelope glycoprotein (Env) is specifically recognized by the cellular receptor at cell surface, which induces a conformational changes between viral and cell membrane to entry. Thus, the elucidation of cellular receptor for BLV infection is very important for virus entry. However, the BLV receptor has not been identified yet. In the current study, we found that BLV Env protein binds to cationic amino acid transporter 1 (CAT1)/SLC7A1 at cell surface, artificial expression of CAT1 in CAT1-negative cells confers the cells susceptible to BLV infection, and CAT1-silencing significantly reduces BLV infection, concluding that CAT1 is the BLV receptor. These findings will have far reaching great advantages of insights in the retrovirus study.

microbiology↗

bioRxiv2019

Benchmarking principal component analysis for large-scale single-cell RNA-sequencing

Principal component analysis (PCA) is an essential method for analyzing single-cell RNA-seq (scRNA-seq) datasets, but large-scale scRNA-seq datasets require long computational times and a large memory capacity.\n\nIn this work, we review 21 fast and memory-efficient PCA implementations (10 algorithms) and evaluate their application using 4 real and 18 synthetic datasets. Our benchmarking showed that some PCA algorithms are faster, more memory efficient, and more accurate than others. In consideration of the differences in the computational environments of users and developers, we have also developed guidelines to assist with selection of appropriate PCA implementations.

bioinformatics↗