Biology subjects

Sato, Y.

Publications and source records attributed to Sato, Y..

At least 145 records · Page 8Linked to original sources

bioRxiv2020

Direct evidence of abortive lytic infection-mediated establishment of Epstein-Barr virus latency during B-cell infection

Viral infection induces dynamic changes in transcriptional profiles. Virus-induced and anti-viral responses are intertwined during the infection. Epstein-Barr virus (EBV) is a human gammaherpesvirus that provides a model of herpesvirus latency. To measure the transcriptome changes during the establishment of EBV latency, EBV-negative Akata cells were infected with EBV-EGFP and observed by transcriptome sequencing (RNA-seq) at 0, 2, 4, 7, 10, and 14 days after infection. We found transient downregulation of mitotic division-related genes, reflecting reprograming of cell growth by EBV. Moreover, a burst of viral lytic gene expression was detected in the early phase of infection. Experimental and mathematical investigations demonstrated that infectious virions were not produced in the pre-latent phase, suggesting the presence of an abortive lytic infection. Finally, we conducted fate mapping using recombinant EBV, enabling the noninvasive, continuous observation of infected cells during EBV infection. Our tracking analysis provided direct evidence that the abortive lytic infection in the pre-latent phase converges to latent infection during EBV infection of B-cells, shedding light on novel roles of viral lytic gene(s) in establishing latency. Author summaryViral infection is a complex process that activates both virus-triggered and host anti-viral responses. This process has classically been studied by snapshot analysis such as microarray and RNA-seq at discrete time points as population averages. Snapshot data lead to invaluable findings in host-pathogen interactions. However, these "snapshot" omics, even from a single cell, lack temporal resolution. Because the behavior of infected cells is highly dynamic and heterogenous, continuous analysis is required for deciphering the fate of infected cells during viral infection. Here, we exploited fate mapping techniques with recombinant Epstein-Barr virus (EBV) to track the infected cells and recorded a log of lytic gene expression during EBV infection. Our continuous observation of infected cells revealed that EBV established latency in B-cells via an abortive lytic infection in the pre-latent phase.

microbiology↗

bioRxiv2020

Genetic Diversity and Population Structure in the Yaeyama flying fox

The Ryukyu flying fox (Pteropus dasymallus) is distributed throughout the island chain spanning across southern Japan, Taiwan, and possibly the Philippines. Although P. dasymallus is listed as VU (vulnerable) in the IUCN Red List, only few genetic works have been conducted to support its conservation. In this study we analyzed 19 markers (mtDNA haplotypes and 18 microsatellite markers) to evaluate genetic diversity and investigate the genetic structure of this species. mtDNA analysis was conducted with 142 DNA remote samples, mostly from feces, and wing tissues collected on eight islands (Miyako, Ishigaki, Kohama, Kuroshima, Hateruma, Taketomi, Iriomote, Yonaguni). 39 haplotypes were identified in 526bp of the control region, and haplotype network showed no clear genetic structure. Microsatellite analysis was also conducted with 155 samples collected on six islands (Miyako, Ishigaki, Kohama, Taketomi, Iriomote, Yonaguni). It showed that the Yonaguni population exhibits low genetic diversity, high inbreeding, and clear genetic differentiation from other populations. Gene flow between Ishigaki and Miyako through small stepstone islands might be preventing inbreeding of the Miyako population. We provide for the first time indirect proof of long-distance inter-island dispersal in the Ryukyu flying fox and revealed genetic diversity, gene flow and genetic differentiation among populations of the archipelago. These results will be useful for delineating conservation units and designing specific conservation policies for each island based on metapopulation genetic structure.

genetics↗

bioRxiv2020

Erythropoietin production?by the kidney and the liver in response to severe hypoxia evaluated by Western blotting with deglycosylation

The detection of erythropoietin (Epo) protein by Western blotting has required pre-purification of the sample. We developed a new Western blot method to detect plasma and urinary Epo using deglycosylation. Epo in urine and tissue and erythropoiesis-stimulating agents (ESAs) in urine were directly detected by our Western blotting. Plasma Epo and ESAs were detected by our Western blotting after deglycosylation. The broad bands of Epo and ESAs were shifted to 22 kDa by deglycosylation except PEG-bound epoetin {beta} pegol. The 22 kDa band from anemic patient urine was confirmed by Liquid Chromatography/Mass Spectrometry (LC/MS) to contain human Epo. Sever hypoxia (7% O2, 4 hr) caused a 400-fold increase in deglycosylated Epo expression in rat kidneys, which is consistent with the increases in both Epo gene expression and plasma Epo concentration. Immunohistochemistry showed Epo expression in nephrons but not in interstitial cells under control conditions, and hypoxia increased Epo expression in interstitial cells but not in tubules. These data show that intrinsic Epo and all ESAs can be detected by Western blot either directly in urine or after deglycosylation in blood, and that the kidney is the main and sole site of Epo production in control and severe hypoxia. Our method will completely change Epo doping and detection.

physiology↗

bioRxiv2020

Therapeutic Efficacy and Safety of Lenvatinib for Unresectable Hepatocellular Carcinoma Beyond Progression with Sorafenib

Background & AimsThe efficacy and safety of lenvatinib (LEN) as a second/third-line treatment for unresectable hepatocellular carcinoma (HCC) after sorafenib (SOR) therapy remains unknown. We evaluated the outcomes of second/third-line treatment of LEN, investigated the sensitivity of SOR-resistant HCC cell line (PLC/PRF5-R2) to LEN, and their signal transduction pathway by protein array analysis. MethodsWe retrospectively enrolled 57 unresectable HCC patients. Radiologic responses in 53 patients were evaluated by modified Response Evaluation Criteria in Solid Tumors. Active signal transduction pathways in cells were identified by protein array analysis, including 1205 proteins. ResultsPatients comprised 34 tyrosine kinase inhibitor (TKI)-naive (first-line), nine SOR-intolerant (second-line), and ten resistant to regorafenib (third-line). Objective response rates (ORRs) were 61.8% (21/34) in TKI-naive, 33.3% (3/9) in second-line, and 20.0% (2/10) in third-line groups. The overall survival (OS) and the progression free survival (PFS) in the first-line was significantly longer than those in third-line group (p<0.05). Patients with better liver functional reserve (Child score, ALBI grade) exhibited higher ORR and longer OS. LEN was well-tolerated as second/third-line treatment. The IC50 value of LEN against PLC/PRF5-R2 cells (30 M) was significantly higher than that against PLC/PRF5 cells (6.4 M). LEN inhibited significantly more signal transduction pathways related to FRS2, a crucial FGFR downstream molecule, in PLC/PRF5 than PLC/PRF5-R2 cells. ConclusionsLEN was active and safe as a second/third-line treatment for unresectable HCC. LEN seems to be more effective for HCC patients with better hepatic reserve function or before TKI-resistance is acquired because of partial cross-resistance to SOR.

molecular biology↗

bioRxiv2019

Distinct transcriptional programs of SOX2 in different types of small cell lung cancers

SOX2 is an oncogene in human small cell lung cancer (SCLC), an aggressive neuroendocrine (NE) tumor. However, the roles of SOX2 in SCLC remain unclear, and strategies to selectively target SOX2 in SCLC cells have not yet been established. We herein demonstrated that SOX2 is involved in NE differentiation and tumorigenesis in cooperation with ASCL1, a lineage-specific transcriptional factor, in the classical subtype of SCLC cell lines. ASCL1 recruits SOX2, which promotes INSM1 expression. Precursor SCLC lesions were established in Trp53 (-/-); CCSPrtTA; tetOCre; floxedRb1; floxedHes1 mice, and the NE neoplasms induced were positive for Ascl1, Sox2, and Insm1. In contrast to the ASCL1-SOX2 signaling axis to control the SCLC phenotype in classical subtype SCLC, SOX2 targeted distinct genes, such as those related to the Hippo pathway, in ASCL1-negative, variant subtype SCLC. The present results support the importance of the ASCL1-SOX2 axis as a main subtype of SCLC, and suggest the therapeutic potential of targeting the ASCL1-SOX2 signaling axis and the clinical utility of SOX2 as a biological marker in the classical subtype of SCLC.

cancer biology↗

bioRxiv2019

Neighbor GWAS: incorporating neighbor genotypic identity into genome-wide association studies of field herbivory on Arabidopsis thaliana

An increasing number of field studies have shown that the phenotype of an individual plant depends not only on its genotype but also on those of neighboring plants; however, this fact is not taken into consideration in genome-wide association studies (GWAS). Based on the Ising model of ferromagnetism, we incorporated neighbor genotypic identity into a regression model, named "Neighbor GWAS". Our simulations showed that the effective range of neighbor effects could be estimated using an observed phenotype from when the proportion of phenotypic variation explained (PVE) by neighbor effects peaked. The spatial scale of the first nearest neighbors gave the maximum power to detect the causal variants responsible for neighbor effects, unless their effective range was too broad. However, if the effective range of the neighbor effects was broad and minor allele frequencies were low, there was collinearity between the self and neighbor effects. To suppress the false positive detection of neighbor effects, the fixed effect and variance components involved in the neighbor effects should be tested in comparison with a standard GWAS model. We applied neighbor GWAS to field herbivory data from 199 accessions of Arabidopsis thaliana and found that neighbor effects explained 8% more of the PVE of the observed damage than standard GWAS. The neighbor GWAS method provides a novel tool that could facilitate the analysis of complex traits in spatially structured environments and is available as an R package at CRAN (https://cran.rproject.org/package=rNeighborGWAS).

genetics↗

bioRxiv2019

Classification of clear cell renal cell carcinoma based on PKM alternative splicing

Clear cell renal cell carcinoma (ccRCC) accounts for 70-80% of kidney cancer diagnoses and displays high molecular and histologic heterogeneity. Hence, it is necessary to reveal the underlying molecular mechanisms involved in progression of ccRCC to better stratify the patients and design effective treatment strategies. Here, we analyzed the survival outcome of ccRCC patients as a consequence of the differential expression of four transcript isoforms of the pyruvate kinase muscle type (PKM). We first extracted a classification biomarker consisting of eight gene pairs whose within-sample relative expression orderings (REOs) could be used to robustly classify the patients into two groups with distinct molecular characteristics and survival outcomes. Next, we validated our findings in a validation cohort and an independent Japanese ccRCC cohort. We finally performed drug repositioning analysis based on transcriptomic expression profiles of drug-perturbed cancer cell lines and proposed that paracetamol, nizatidine, dimethadione and conessine can be repurposed to treat the patients in one of the subtype of ccRCC whereas chenodeoxycholic acid, fenoterol and hexylcaine can be repurposed to treat the patients in the other subtype.

systems biology↗

bioRxiv2019

Development of CRISPR-Cas13a-based antimicrobials capable of sequence-specific killing of target bacteria

Emergence of antimicrobial-resistant bacteria is an increasingly serious threat to global health, necessitating the development of innovative antimicrobials. We established a series of CRISPR-Cas13a-based antibacterial nucleocapsid, termed CapsidCas13a(s), capable of sequence-specific killing of carbapenem-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus through promiscuous RNA cleavage after recognizing corresponding antimicrobial resistance genes. CapsidCas13a constructs were generated by packaging CRISPR-Cas13a into a bacteriophage capsid to target antimicrobial resistance genes. Contrary to Cas9-based antimicrobials that lack bacterial killing capacity when the target genes are located on a plasmid, the CapsidCas13a(s) exhibited strong bacterial killing activities upon recognizing target genes regardless of their location. The antimicrobials treatment efficacy was confirmed using a Galleria mellonella larvae model. Further, we demonstrated that the CapsidCas13a(s) can assist in bacterial gene detection without employing nucleic acid amplification and optical devices.

microbiology↗

bioRxiv2019

Attenuated Salmonella typhimurium cancer therapy has direct effects on the tumor epithelium in colorectal cancer

Bacterial cancer therapy (BCT) shows great promise for treatment of solid tumors, yet basic mechanisms of bacterial-induced tumor suppression remain undefined. The intestinal epithelium is the natural route of infection for Salmonella and thus harbors innate immune defenses which protect against infection. Attenuated strains of Salmonella enterica serovar Typhimurium (STm) have commonly been used in mouse models of BCT, largely with the use of xenograft and orthotopic transplant cancer models. We aimed to better understand the tumor epithelium-targeted mechanisms of BCT by using mouse models of intestinal tumorigenesis and tumor organoid cultures to assess the effectiveness and mechanisms of treatment with aromatase A-deficient STm (STm{Delta}aroA). STm{Delta}aroA delivered by oral gavage could significantly reduce tumor burden and tumor load in both a colitis-associated colon cancer model (CAC) and in a spontaneous intestinal cancer model, Apcmin/+ mice. STm{Delta}aroA colonization of tumors caused alterations in transcription of mRNAs associated with epithelial-mesenchymal transition as well as metabolic and cell cycle-related transcripts. Metabolomic analysis of tumors demonstrated alteration in the metabolic environment of STm{Delta}aroA-treated tumors, suggesting STm{Delta}aroA imposes metabolic competition on the tumor. Use of tumor organoid cultures in vitro demonstrated that STm{Delta}aroA can directly affect the tumor epithelium with alterations in transcripts and metabolites similar to in vivo-treated tumors. Thereby, we demonstrate that bacterial cancer therapy is efficacious in autochthonous intestinal cancer models, that BCT imposes metabolic competition, and that BCT has direct effects on the tumor epithelium, which have not previously been appreciated.\n\nOne Sentence SummaryAttenuated Salmonella enterica serovar Typhimurium can home to gastrointestinal tumors and directly affect the tumor epithelium, inducing transcriptional and metabolic changes that lead to reduced tumor burden in mice.

cancer biology↗

bioRxiv2019

Acute feeding disorder after oral RNAi exposure results in efficient pest control

Selecting an appropriate target gene is critical to the success of feeding RNA interference (f-RNAi)-based pest control. Gene targets have been chosen based on their ability to induce lethality. However, lethality induction by f-RNAi is slow-acting and crop damage can progress during this time. Here, we show that f-RNAi of death-associated inhibitor of apoptosis protein 1 (diap1), but not two conventional targets vacuolar ATPase subunit A and E, induces acute feeding cessation in the solanaceous pest, Henosepilachna vigintioctopunctata during 24-48 hours. We also found that the feeding cessation by diap1 f-RNAi has species-specificity and occurs with only 1.6 ng dsRNA. Our results suggest that diap1 is an appropriate target in the context of rapid reduction of crop damage. We propose that acute feeding disorder should be assessed as a novel criterion for selecting appropriate target genes for RNAi-based pest control in addition to the conventional criterion based on lethality.

molecular biology↗

bioRxiv2019

CLEC-2 suppresses calcification in cultured osteoblasts

Podoplanin is the only counter-receptor of platelet CLEC-2 and is expressing on mature osteoblast, but there is no report on the role of podoplanin and CLEC-2 in calcification. This study aimed to investigate the role of podoplanin binding to CLEC-2 in the calcification of osteoblasts carrying homozygously deleted Pdpn alleles (Pdpn{Delta}/{Delta}) by heterozygously expressing collagen type I alpha 1 promoter (Col1a)-driven Cre recombinase. There were no macroscopic abnormalities in the bone and dentin of Col1a11-Cre;Pdpn{Delta}/{Delta} mice but the coccygeal bone medullary cavity was very narrow. In the quantitative analysis for alizarin red-stained products and alkaline phosphatase activities on the cultured calvarial osteoblasts, the amounts of calcified products and alkaline phosphatase activity of calvarial osteoblasts of both Pdpnfl/fl and Col1a11-Cre;Pdpn{Delta}/{Delta} mice were significantly higher in the calcification medium than in the -mem. Both the amounts of calcified products and alkaline phosphatase activity of calvarial osteoblasts from Pdpnfl/fl mice were significantly lower in the calcification medium with CLEC-2 than without CLEC-2 while there were no significant differences in the amounts of calcified products and alkaline phosphatase activities of calvarial osteoblasts from Col1a11-Cre;Pdpn{Delta}/{Delta} mice with CLEC-2. Platelet CLEC-2 may play a role in regulating the calcification via binding to podoplanin on mature osteoblasts expressing podoplanin in the medullary cavity of a part of the bone.

cell biology↗

bioRxiv2019

Genetic risks of schizophrenia identified in a matched case-control study

BackgroundGenetic association studies of schizophrenia may be confounded by the pathological heterogeneity and multifactorial nature of this disease. We demonstrated previously that combinations of the three functional single nucleotide polymorphisms (SNPs) rs10770141 of tyrosine hydroxylase (TH) gene, rs4680 of catechol-O-methyltransferase (COMT) gene, and rs1800497 of dopamine D2 receptor (DRD2) gene may be associated with schizophrenia onset, and we tested those associations herein. Methods: We conducted a secondary study of 2,542 individuals in age- and sex-matched case-control populations. The schizophrenia diagnosis was based on the DSM-IV. To reduce the influence of confounders (age and sex), we performed a propensity score matching analysis. Genotyping and associative analyses of rs10770141, rs4680, and rs1800497 with schizophrenia were performed. Results: We analyzed 1,271 schizophrenics (male/female: 574/698; age 47.4{+/-}13.9 years) and 1,271 matched controls (male/female: 603/669; age 46.5{+/-}13.4 years). The estimated odds ratios (ORs) were 1.245 (p<0.001) for rs4680, 1.727 (p<0.0001) for rs1800497, and 1.788 (p<0.0001) for rs10770141. Double SNP analyses revealed the ORs of 2.010 (p<0.0001) for the combination of rs4680*rs1800497, 1.871 (p<0.001) for rs1800497*rs10770141, and 1.428 (p=0.068) for rs4680*rs1800497. Among the individuals with any of the three double SNP risk combinations (which accounted for 35.8% of the involved patients), the estimated OR was 2.224 (p<0.0001). Conclusions: In this validation study, the combinations of functional polymorphisms related to dopaminergic genes were associated with the development of schizophrenia. Analyzing combinations of functional polymorphisms with the control of possible confounders may provide new insights for association research.

genetics↗

bioRxiv2019

Discovery of functional alternatively spliced PKM transcripts in human cancers

The association of pyruvate kinase muscle type (PKM) with survival of cancer patients is controversial. Here, we focus on different transcripts of PKM and investigate the association between their mRNA expression and the clinical survival of the patients in 25 different cancers. We find that the transcript encoding PKM2, and three other functional transcripts are prognostic in multiple cancers. Our integrative analysis shows that the functions of these four transcripts are highly conservative in different cancers. Next, we validate the prognostic effect of these transcripts in an independent kidney renal clear-cell carcinoma (KIRC) cohort and identify a prognostic signature which could distinguish high- and low-risk KIRC patients. Finally, we reveal the functional role of alternatively spliced PKM transcripts in KIRC, and discover the protein products of different transcripts of PKM. Our analysis demonstrated that alternatively spliced transcripts of not only PKM but also other genes should be considered in cancer studies, since it may enable the discovery and targeting of the right protein product for development of the efficient treatment strategies.

systems biology↗

bioRxiv2019

Quantitative measurements of chromatin modification dynamics during zygotic genome activation

Histone posttranslational modifications (PTMs) are key gene expression regulators, but their rapid dynamics during development remain difficult to capture. We describe an approach that images fluorescent antibody fragments to quantify PTM enrichment and active transcription at defined loci during zygotic genome activation in living zebrafish embryos. The technique reveals a drastic increase in histone H3 Lys27 acetylation (H3K27ac) before genome activation both at a specific locus and globally.

cell biology↗

bioRxiv2019

Transcriptional variation in glucosinolate biosynthetic genes and inducible responses to aphid herbivory on field-grown Arabidopsis thaliana

Recently, increasing attempts have been made to understand how plant genes function in natura studies. To determine whether plant defense genes are activated under multiple biotic stimuli, we combined a high-throughput RNA-Seq with insect survey data on 19 accessions of Arabidopsis thaliana growing on the field site of Switzerland. We found that genes with GO annotations "glucosinolate biosynthetic process" and "response to insects" were the most significantly enriched, exhibiting largely variable expression among plant accessions. Nearly half of the total expression variation in glucosinolate biosynthetic genes, AOPs, ESM1, ESP, and TGG1, was explained by among-accession variance. Combined with the field RNA-Seq data, bioassays confirmed that AOP3 was up-regulated in response to the mustard aphid Lipaphis erysimi. In addition, we also found that the expression of a major cis-jasmone activated gene CYP81D11 was positively correlated with the number of the flea beetles Phyllotreta spp. The combined results from RNA-Seq and insect surveys suggested that plants can activate their defenses even when they are exposed to multiple biotic stimuli in natura.

plant biology↗

bioRxiv2019

Management of family relationship informationfor a three-generation cohort study

A system for inputting and storing family information, named \"BirThree Enrollment,\" was developed to promote a birth and three-generation cohort study (BirThree Cohort Study), and this system was operated successfully. In the study, it was necessary to satisfy many operational demands. Input information is overwritten and changed continuously. Complex kinship information must be quickly and accurately input and corrected, and information on those families not yet recruited must be retrieved. For these purposes, many devices are needed, from an input interface to the internal data structure. In the field of genetic statistics, a simple standard expressive form is used for describing family structure. This form has sufficient information for genetics; however, we developed this form further for our purposes in conducting the BirThree Cohort Study. To provide information about family roles as required in the BirThree Cohort Study, we expanded the data structure, and constructed the system that is able to be used for the daily operation. In our system, family pedigree information is stored along with initial clinical information, and enabled the input of all self-reported information to the data base. Operators are able to input this family information before the day is out. As a result, when recruitment is completed, family information will be completed concurrently. Therefore, it is possible to immediately know a certain persons family structure. By using our system, data correction was improved dramatically, and the system was operated successfully. This study is the first report of the method for storing three generations of family data.

epidemiology↗

bioRxiv2018

Genetic combination risk for schizophrenia

Summary ParagraphSchizophrenia is a highly hereditary mental disease1 related to abnormal dopaminergic activities.2,3 To elucidate the mechanisms underlying schizophrenias development, genomic studies have sought to identify the pathogenic genetic polymorphisms. Large-scale genome-wide association studies (GWAS) have reported potential candidate loci that contribute to schizophrenias development.4,5 The risk genetic profiles are not yet established. Here we show that the combination of three functional single nucleotide polymorphisms (SNPs) related to the key factors of dopaminergic signaling can be used to predict the risk of schizophrenias development, though none of the SNPs is known to be associated by itself. These functional SNPs were reported to demonstrate directional influences in their parent gene activity, perhaps characterizing the integrated properties of dopaminergic signaling. Interestingly, the risk combination presented here included the major genotype as well as the minor polymorphisms, suggesting a possible association of unaffected activities of some dopamine-related genes with the disease development. The phenotype speculated based on the allelic status seemed consistent with the conventional pathophysiological hypotheses, although recently developed predictive methods, such as the polygenic risk score, could miss this potent pathogenic role of carrying a normal genotype by evaluating only minor polymorphisms. Our results demonstrate the presence of a subtype in schizophrenia with the favored genetic background related to dopamine signaling. Our findings indicate the possibility that the combinations could characterize integrated biological functions (including neurotransmission) and therefore identify individuals with a disease risk. The biological microenvironment indicated by the functional SNPs could bring an insight to elucidate the pathogenic mechanisms of developing schizophrenia. Furthermore, we believe that our approach will contribute to the development of innovative means to predict disease risks even for other multi-factorial diseases and then, the following preventive medicine.

neuroscience↗

bioRxiv2018

A genetically encoded probe for imaging HA-tagged protein translation, localization, and dynamics in living cells and animals

To expand the toolbox of imaging in living cells, we have engineered a new single chain variable fragment (scFv) that binds the classic linear HA epitope with high affinity and specificity in vivo. The resulting probe, which we call the HA frankenbody, is capable of lighting up in multiple colors HA-tagged nuclear, cytoplasmic, and membrane proteins in diverse living cell types. The HA frankenbody also enables state-of-the-art single-molecule experiments, which we demonstrate by tracking single mRNA translation dynamics in living U2OS cells and neurons. In combination with the SunTag, we track two mRNA species simultaneously to demonstrate comparative single-molecule studies of translation can now be done with genetically encoded tools alone. Finally, we use the HA frankenbody to precisely quantify the expression of HA tagged proteins in developing zebrafish embryos. The versatility of the HA frankenbody makes it a powerful new tool for imaging protein dynamics in vivo.\n\nOne-sentence summaryA genetically encodable intracellular single-chain variable fragment that selectively binds the HA epitope (YPYDVPDYA) with high affinity in living cells and organisms can be used to quantify HA-tagged protein translation, localization, and dynamics.

cell biology↗