Biology subjects

Nakagawa, S.

Publications and source records attributed to Nakagawa, S..

At least 73 records · Page 4Linked to original sources

bioRxiv2021

An emerging SARS-CoV-2 mutant evading cellular immunity and increasing viral infectivity

During the current SARS-CoV-2 pandemic that is devastating the modern societies worldwide, many variants that naturally acquire multiple mutations have emerged. Emerging mutations can affect viral properties such as infectivity and immune resistance. Although the sensitivity of naturally occurring SARS-CoV-2 variants to humoral immunity has recently been investigated, that to human leukocyte antigen (HLA)-restricted cellular immunity remains unaddressed. Here we demonstrate that two recently emerging mutants in the receptor binding domain of the SARS-CoV-2 spike protein, L452R (in B.1.427/429) and Y453F (in B.1.298), can escape from the HLA-24-restricted cellular immunity. These mutations reinforce the affinity to viral receptor ACE2, and notably, the L452R mutation increases protein stability, viral infectivity, and potentially promotes viral replication. Our data suggest that the HLA-restricted cellular immunity potentially affects the evolution of viral phenotypes, and the escape from cellular immunity can be a further threat of the SARS-CoV-2 pandemic. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/438288v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@153428forg.highwire.dtl.DTLVardef@136ca5aorg.highwire.dtl.DTLVardef@1ee490org.highwire.dtl.DTLVardef@2fe478_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2021

An ancient retroviral RNA element hidden in mammalian genomes and its involvement in co-opted retroviral gene regulation

Retroviruses utilize multiple unique RNA elements to control several aspects of RNA processing, such as splicing, subcellular export, and translation. However, it is mostly unclear whether such functional RNA elements are present in endogenous retroviruses (ERVs), many of which were inserted into the host genomes millions of years ago. Previously, in human ERV-derived syncytin-1 gene, we found a cis-acting RNA element named SPRE that enhances its protein expression. In this study, we found a 17-nt common sequence in SPRE of syncytin-1 and another ERV-derived gene, syncytin-2, and the sequence is confirmed to be essential for the expression of the proteins. We detected the sequences of SPRE-like elements in 41 ERV families. Though the SPRE-like elements were not found in currently prevailing (i.e. exogenous) viral sequences, more than thousands of copies of the elements were found in several mammalian genomes, suggesting the ancient integration and propagation of the SPRE-harboring retroviruses in mammalian lineages. Indeed, other mammalian ERV-derived genes: mac-syncytin-3 of macaque, syncytin-Ten1 of tenrec, and syncytin-Car1 of Carnivora contain the SPRE-like elements, and we validated their function for efficient protein expression by in vitro assays. A reporter assay revealed that the enhancement of gene expression by SPRE depended on reporter genes. Moreover, the mutation in SPRE did not affect the gene expression in codon-optimized syncytin-2. However, the same mutation in SPRE impaired the gene expression in wild-type syncytin-2, suggesting that the SPRE dependency of Syncytin-2 expression is due to the negative factors such as inefficient codon frequency or repressive elements within the coding sequence. These results provide new implications that ERVs harbor unique RNA elements involved in the regulation of ERV-derived genes.

molecular biology↗

bioRxiv2021

MinION™, a portable long-read sequencer, enables rapid vaginal microbiota analysis in a clinical setting

It has been suggested that the local microbiota in the reproductive organs is relevant to womens health and may also affect pregnancy outcomes. Analysis of partial 16S ribosomal RNA (rRNA) gene sequences generated by short-read sequencers has been used to identify vaginal and endometrial microbiota, but it requires a long time to obtain the results, making it unsuitable for the rapid analysis of small samples in a clinical context. We demonstrated a simple workflow using the nanopore sequencer MinION that allows high-resolution and rapid differentiation of vaginal microbiota. Vaginal samples collected from 18 participants were subjected to DNA extraction and full-length 16S rRNA gene sequencing with MinION. The principal coordinate analysis showed no differences in the bacterial compositions regardless of the sample collection method. The vaginal microbiota results could be reported within 2 days of specimen receipt. Although bacterial vaginosis (BV) was not diagnosed by the Nugent score in any cases, groups with both healthy and BV-like vaginal microbiota were clearly characterized by MinION sequencing. We conclude that full-length 16S rRNA gene sequencing analysis with MinION provides a rapid means for identifying vaginal bacteria with higher resolution. Species-level profiling of human vaginal microbiota by MinION sequencing can allow the analysis of associations with conditions such as genital infections, endometritis, and threatened miscarriage.

microbiology↗

bioRxiv2020

Genomic diversity of SARS-CoV-2 can be accelerated by a mutation in the nsp14 gene

Coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), encode a proofreading exonuclease, nonstructural protein 14 (nsp14), that helps ensure replication competence at a low evolutionary rate compared with other RNA viruses. In the current pandemic, SARS-CoV-2 has accumulated diverse genomic mutations including in nsp14. Here, to clarify whether amino acid substitutions in nsp14 affect the genomic diversity and evolution of SARS-CoV-2, we searched for amino acid substitutions in nature that may interfere with nsp14 function. We found that viruses carrying a proline-to-leucine change at position 203 (P203L) have a high evolutionary rate and that a recombinant SARS-CoV-2 virus with the P203L mutation acquired more diverse genomic mutations than wild-type virus during its replication in hamsters. Our findings suggest that substitutions, such as P203L, in nsp14 may accelerate the genomic diversity of SARS-CoV-2, contributing to virus evolution during the pandemic.

microbiology↗

bioRxiv2020

Paternal hypoxia exposure primes offspring for increased hypoxia resistance

In a time of rapid environmental change, understanding how the challenges experienced by one generation can influence the fitness of future generations is critically needed. Using tolerance assays, transcriptomic and methylome approaches, we use zebrafish as a model to investigate transgenerational acclimation to hypoxia. We show that short-term paternal exposure to hypoxia endows offspring with greater tolerance to acute hypoxia. We detected two hemoglobin genes that are significantly upregulated by more than 7-fold in the offspring of hypoxia exposed males. Moreover, the offspring which maintained equilibrium the longest showed greatest upregulation in hemoglobin expression. We did not detect differential methylation at any of the differentially expressed genes, suggesting that another epigenetic mechanism is responsible for alterations in gene expression. Overall, our findings suggest that a memory of past hypoxia exposure is maintained and that this environmentally induced information is transferred to subsequent generations, pre-acclimating progeny to cope with hypoxic conditions.

evolutionary biology↗

bioRxiv2020

Low Repeatability of Aversive Learning in Zebrafish (Danio rerio)

Aversive learning - avoiding certain situations based on negative experiences - can profoundly increase fitness in animal species. The extent to which this cognitive mechanism could evolve depends upon individual differences in aversive learning being stable through time, and heritable across generations, yet no published study has quantified the stability of individual differences in aversive learning using the repeatability statistic, R (also known as the intra-class correlation). We assessed the repeatability of aversive learning by conditioning approximately 100 zebrafish (Danio rerio) to avoid a colour cue associated with a mild electric shock. Across eight different colour conditions zebrafish did not show consistent individual differences in aversive learning (R = 0.04). Within conditions, when zebrafish were twice conditioned to the same colour, blue conditioning was more repeatable than green conditioning (R = 0.15 and R = 0.02). In contrast to the low repeatability estimates for aversive learning, zebrafish showed moderately consistent individual differences in colour preference during the baseline period (i.e. prior to aversive conditioning; R ~ 0.45). Overall, aversive learning responses of zebrafish were weak and variable (difference in time spent near the aversive cue <6 seconds per minute), but individual differences in learning ability did not explain substantial variability. We speculate that either the effect of aversive learning was too weak to quantify consistent individual differences, or directional selection might have eroded additive genetic variance. Finally, we discuss how confounded repeatability assays and publication bias could have inflated average estimates of repeatability in animal behaviour publications. Summary StatementZebrafish exhibit low repeatability (intra-class correlation) in an aversive learning assay possibly due to past selection pressure exhausting genetic variance in this learning trait.

animal behavior and cognition↗

bioRxiv2020

Paraspeckles are constructed as block copolymer micelles through microphase separation

Paraspeckles are constructed by NEAT1_2 architectural long noncoding RNAs and possess characteristic cylindrical shapes with highly ordered internal organization, distinct from typical liquid-liquid phase-separated condensates. We experimentally and theoretically investigated how the shape and organization of paraspeckles are determined. We identified the NEAT1_2 RNA domains responsible for shell localization of the NEAT1_2 ends, which determine the characteristic internal organization. We then applied a theoretical framework using soft matter physics to understand the principles that determine the NEAT1_2 organization, shape, number, and size of paraspeckles. By treating paraspeckles as amphipathic block copolymer micelles, we could explain and predict the experimentally observed behaviors of paraspeckles upon NEAT1_2 domain deletions or transcriptional modulation. Thus, we propose that paraspeckles are block copolymer micelles assembled through microphase separation. This work provides an experimentally-based theoretical framework for the concept that ribonucleoprotein complexes (RNPs) can act as block copolymers to form RNA-scaffolding microphase-separated condensates in cells.

molecular biology↗

bioRxiv2020

Embrace heterogeneity to improve reproducibility: A perspective from meta-analysis of variation in preclinical research

The reproducibility of research results has been a cause of increasing concern to the scientific community. The long-held belief that experimental standardization begets reproducibility has also been recently challenged, with the observation that the reduction of variability within studies can lead to idiosyncratic, lab-specific results that are irreproducible. An alternative approach is to, instead, deliberately introduce heterogeneity; known as "heterogenization" of experimental design. Here, we explore a novel perspective in the heterogenization program in a meta-analysis of variability in observed phenotypic outcomes in both control and experimental animal models of ischaemic stroke. First, by quantifying inter-individual variability across control groups we illustrate that the samount of heterogeneity in disease-state (infarct volume) differs according to methodological approach, for example, in disease-induction methods and disease models. We argue that such methods may improve reproducibility by creating diverse and representative distribution of baseline disease-state in the reference group, against which treatment efficacy is assessed. Second, we illustrate how meta-analysis can be used to simultaneously assess efficacy and stability (i.e., mean effect and among-individual variability). We identify treatments that have efficacy and are generalizable to the population level (i.e. low inter-individual variability), as well as those where there is high inter-individual variability in response; for these latter treatments translation to a clinical setting may require nuance. We argue that by embracing rather than seeking to minimise variability in phenotypic outcomes, we can motivate the shift towards heterogenization and improve both the reproducibility and generalizability of preclinical research.

neuroscience↗

bioRxiv2020

Rapid profiling of drug-resistant bacteria using DNA-binding dyes and a nanopore-based DNA sequencer

Spread of drug-resistant bacteria is a serious problem worldwide. We thus designed a new sequence-based protocol that can quickly identify bacterial compositions of clinical samples and their drug-resistance profiles simultaneously. Here we utilized propidium monoazide (PMA) that prohibits DNA amplifications from dead bacteria, and subjected the original and antibiotics-treated samples to 16S rRNA metagenome sequencing. We tested our protocol on bacterial mixtures, and observed that sequencing reads derived from drug-resistant bacteria were significantly increased compared with those from drug-sensitive bacteria when samples were treated by antibiotics. Our protocol is scalable and will be useful for quickly profiling drug-resistant bacteria.

microbiology↗

bioRxiv2020

Expression of ERV3-1 in Leukocytes of Acute Myelogenous Leukemia Patients

Acute myelogenous leukemia (AML) is one of the major hematological malignancies. In the human genome, several have been found to originate from retroviruses, and some of which are involved in progression of various cancers. Hence, to investigate whether retroviral-like genes are associated with the development of AML, we conducted a transcriptome sequencing analysis of 12 retroviral-like genes of 150 AML patients using The Cancer Genome Atlas database. We found high expression of ERV3-1, an envelope gene of endogenous retrovirus group 3 member 1. In particular, blood and bone marrow cells of the myeloid lineage in AML patients, exhibited higher expression of ERV3-1 than those of the monocytic AML lineage. We also examined the protein expression of ERV3-1 by immunohistochemical analysis and found expression of ERV3-1 protein in 7 out of 12 AML patients, with a particular concentration observed at the membrane of some leukemic cells. Transcriptome analysis further suggested that upregulated ERV3-1 expression may be associated with chromosome 8 trisomy as anomaly was found to be more common among the high expression group compared to the low expression group. However, this finding was not corroborated by the immunohistochemical data. This discrepancy may have been caused, in part, by the small number of samples analyzed in this study. Although the precise associated molecular mechanisms remain unclear, our results suggest that ERV3-1 may be involved in AML development. HighlightsO_LIExpression of 12 retroviral-like genes in the human genome were analyzed using transcriptome data of 150 acute myelogenous leukemia (AML) patients. C_LIO_LIERV3-1, an envelope gene of endogenous retrovirus group 3 member 1, was found to uniquely show high expression level. C_LIO_LIMorphologic characteristics and chromosomal abnormalities are found to be related with the expression of ERV3-1. C_LI

molecular biology↗

bioRxiv2020

Nanopore direct RNA sequencing detects DUX4-activated repeats and isoforms in human muscle cells

Facioscapulohumeral muscular dystrophy (FSHD) is an inherited muscle disease caused by misexpression of the DUX4 gene in skeletal muscle. DUX4 is a transcription factor which is normally expressed in the cleavage-stage embryo and regulates gene expression involved in early embryonic development. Recent studies revealed that DUX4 also activates the transcription of repetitive elements such as endogenous retroviruses (ERVs), mammalian apparent LTR-retrotransposons (MaLRs), and pericentromeric satellite repeats (HSATII). DUX4-bound ERV sequences also create alternative promoters for genes or long non-coding RNAs (lncRNAs), producing fusion transcripts. To further understand transcriptional regulation by DUX4, we performed nanopore long-read direct RNA sequencing (dRNA-seq) of human muscle cells induced by DUX4, because long reads show whole isoforms with greater confidence. We successfully detected differential expression of known DUX4-induced genes, and discovered 61 differentially-expressed repeat loci, which are near DUX4-ChIP peaks. We also identified 247 gene-ERV fusion transcripts, of which 216 were not reported previously. In addition, long-read dRNA-seq clearly shows that RNA splicing is a common event in DUX4-activated ERV transcripts. Long-read analysis showed non-LTR transposons including Alu are also transcribed from LTRs. Our findings revealed further complexity of DUX4-induced ERV transcripts. This catalogue of DUX4-activated repetitive elements may provide useful information to elucidate the pathology of FSHD. Also, our results indicate that nanopore dRNA-seq has complementary strengths to conventional short read cDNA sequencing.

genomics↗

bioRxiv2020

partR2: Partitioning R2 in generalized linear mixed models

The coefficient of determination R2 quantifies the amount of variance explained by regression coefficients in a linear model. It can be seen as the fixed-effects complement to the repeatability R (intra-class correlation) for the variance explained by random effects and thus as a tool for variance decomposition. The R2 of a model can be further partitioned into the variance explained by a particular predictor or a combination of predictors using semi-partial (part) R2 and structure coefficients, but this is rarely done due to a lack of software implementing these statistics. Here, we introduce partR2, an R package that quantifies part R2 for fixed effect predictors based on (generalized) linear mixed-effect model fits. The package iteratively removes predictors of interest from the model and monitors the change in the variance of the linear predictor. The difference to the full model gives a measure of the amount of variance explained uniquely by a particular predictor or a set of predictors. partR2 also estimates structure coefficients as the correlation between a predictor and fitted values, which provide an estimate of the total contribution of a fixed effect to the overall prediction, independent of other predictors. Structure coefficients can be converted to the total variance explained by a predictor, here called inclusive R2, as the square of the structure coefficients times total R2. Furthermore, the package reports beta weights (standardized regression coefficients). Finally,partR2 implements parametric bootstrapping to quantify confidence intervals for each estimate. We illustrate the use of partR2 with real example datasets for Gaussian and binomial GLMMs and discuss interactions, which pose a specific challenge for partitioning the explained variance among predictors.

bioinformatics↗

bioRxiv2020

A novel saline-soluble, rapidly-metabolized RyR1 inhibitor rescues volatile anesthesia-induced death and environmental heat stroke in a mouse model relevant to malignant hyperthermia

Mutations in the type 1 ryanodine receptor (RyR1), a Ca2+ release channel in skeletal muscle, hyperactivate the channel to cause malignant hyperthermia (MH) and are implicated in severe heat stroke. Dantrolene, the only approved drug for MH, has the disadvantages of having very poor water solubility and long plasma half-life. We show here that a novel RyR1-selective inhibitor, 6,7-(methylenedioxy)-1-octyl-4-quinolone-3-carboxylic acid (Compound 1, Cpd1), effectively prevents and treats MH and heat stroke in several mouse models relevant to MH. Cpd1 reduced resting intracellular Ca2+, inhibited halothane- and isoflurane-induced Ca2+ release, suppressed caffeine-induced contracture in skeletal muscle, reduced sarcolemmal cation influx, and prevented or reversed the fulminant MH crisis induced by isoflurane anesthesia and rescued animals from heat stroke caused by environmental heat stress. Notably, Cpd1 has great advantages of better water solubility and rapid clearance in vivo over dantrolene. Cpd1 has the potential to be a promising new candidate for effective treatment of patients carrying RyR1 mutations.

pharmacology and toxicology↗

bioRxiv2020

Activity-specificity trade-off gives PI5P4Kβ a nucleotide preference to function as a GTP-sensing kinase.

Most kinases function with ATP. However, contrary to the prevailing dogma, phosphatidylinositol 5-phosphate 4-kinase {beta} (PI5P4K{beta}) utilizes GTP as a primary phosphate donor with a unique binding mode for GTP. Although PI5P4K{beta} is evolved from a primordial ATP-utilizing enzyme, PI4P5K, how PI5P4K{beta} evolutionarily acquired the GTP preference to function as a cellular GTP sensor remains unclear. In this study, we show that the short nucleotide base-recognition motif, TRNVF, is responsible for the GTP binding of PI5P4K{beta}, and also confers onto PI5P4K{beta} an unexpected specificity that extends to inosine triphosphate (ITP) and xanthosine triphosphate (XTP). A mutational study with GTP analogues suggests that the extended specificity is an obligatory consequence to the acquisition of GTP-dependent activity. However, as the cellular concentrations of ITP and XTP are typically negligible, PI5P4K{beta} can still function as a GTP sensor, suggesting that the cellular physiological conditions leave room for the functional evolution of PI5P4K{beta}.

biochemistry↗

bioRxiv2020

Sex and Power: sexual dimorphism in trait variability and its eco-evolutionary and statistical implications

Biomedical and clinical sciences are experiencing a renewed interest in the fact that males and females differ in many anatomic, physiological, and behavioral traits. Sex differences in trait variability, however, are yet to receive similar recognition. In medical science, mammalian females are assumed to have higher trait variability due to estrous cycles (the estrus-mediated variability hypothesis); historically in biomedical research, females have been excluded for this reason. Contrastingly, evolutionary theory and associated data support the greater male variability hypothesis. Here, we test these competing hypotheses in 218 traits measured in >26,900 mice, using meta-analysis methods. Neither hypothesis could universally explain patterns in trait variability. Sex-bias in variability was trait-dependent. While greater male variability was found in morphological traits, females were much more variable in immunological traits. Sex-specific variability has eco-evolutionary ramifications including sex-dependent responses to climate change, as well as statistical implications including power analysis considering sex difference in variance.

evolutionary biology↗

bioRxiv2020

SARS-CoV-2 ORF3b is a potent interferon antagonist whose activity is further increased by a naturally occurring elongation variant

One of the features distinguishing SARS-CoV-2 from its more pathogenic counterpart SARS-CoV is the presence of premature stop codons in its ORF3b gene. Here, we show that SARS-CoV-2 ORF3b is a potent interferon antagonist, suppressing the induction of type I interferon more efficiently than its SARS-CoV ortholog. Phylogenetic analyses and functional assays revealed that SARS-CoV-2-related viruses from bats and pangolins also encode truncated ORF3b gene products with strong anti-interferon activity. Furthermore, analyses of more than 15,000 SARS-CoV-2 sequences identified a natural variant, in which a longer ORF3b reading frame was reconstituted. This variant was isolated from two patients with severe disease and further increased the ability of ORF3b to suppress interferon induction. Thus, our findings not only help to explain the poor interferon response in COVID-19 patients, but also describe a possibility of the emergence of natural SARS-CoV-2 quasispecies with extended ORF3b that may exacerbate COVID-19 symptoms. HighlightsO_LIORF3b of SARS-CoV-2 and related bat and pangolin viruses is a potent IFN antagonist C_LIO_LISARS-CoV-2 ORF3b suppresses IFN induction more efficiently than SARS-CoV ortholog C_LIO_LIThe anti-IFN activity of ORF3b depends on the length of its C-terminus C_LIO_LIAn ORF3b with increased IFN antagonism was isolated from two severe COVID-19 cases C_LI

microbiology↗

bioRxiv2020

Full-length 16S rRNA gene amplicon analysis of human gut microbiota using MinION™ nanopore sequencing confers species-level resolution

BackgroundSpecies-level genetic characterization of complex bacterial communities has important clinical applications in both diagnosis and treatment. Amplicon sequencing of the 16S ribosomal RNA (rRNA) gene has proven to be a powerful strategy for the taxonomic classification of bacteria. This study aims to improve the method for full-length 16S rRNA gene analysis using the nanopore long-read sequencer MinION. We compared it to the conventional short-read sequencing method in both a mock bacterial community and human fecal samples. ResultsWe modified our existing protocol for full-length 16S amplicon sequencing by MinION. A new strategy for library construction with an optimized primer set overcame PCR-associated bias and enabled taxonomic classification across a broad range of bacterial species. We compared the performance of full-length and short-read 16S amplicon sequencing for the characterization of human gut microbiota with a complex bacterial composition. The relative abundance of dominant bacterial genera was highly similar between full-length and short-read sequencing. At the species level, MinION long-read sequencing had better resolution for discriminating between members of particular taxa such as Bifidobacterium, allowing an accurate representation of the sample bacterial composition. ConclusionsOur present microbiome study, comparing the discriminatory power of full-length and short-read sequencing, clearly illustrated the analytical advantage of sequencing the full-length 16S rRNA gene, which provided the requisite species-level resolution and accuracy in clinical settings.

microbiology↗

bioRxiv2020

Plasmidome analysis of carbapenem-resistant Enterobacteriaceae isolated in Vietnam

Carbapenem-resistant Enterobacterales (CRE) represent a serious threat to public health due to the lack of treatment and high mortality. The rate of antimicrobial resistance of Enterobacterales isolates to major antimicrobials, including carbapenems, is much higher in Vietnam than in Western countries, but the reasons remain unknown due to the lack of genomic epidemiology research. A previous study suggested that carbapenem resistance genes, such as the carbapenemase gene blaNDM, spread via plasmids among Enterobacterales in Vietnam. In this study, we characterized blaNDM-carrying plasmids in Enterobacterales isolated in Vietnam, and identified several possible cases of horizontal transfer of plasmids both within and among species of bacteria. Twenty-five carbapenem-nonsusceptible isolates from a medical institution in Hanoi were sequenced on Illumina short-read sequencers, and 13 blaNDM-positive isolates, including isolates of Klebsiella pneumoniae, Escherichia coli, Citrobacter freundii, Morganella morganii, and Proteus mirabilis, were further sequenced on an Oxford Nanopore Technologies long-read sequencer to obtain complete plasmid sequences. Almost identical 73 kb IncFII(pSE11)::IncN hybrid plasmids carrying blaNDM-1 were found in a P. mirabilis isolate and an M. morganii isolate. A 112 kb IncFII(pRSB107)::IncN hybrid plasmid carrying blaNDM-1 in an E. coli isolate had partially identical sequences with a 39 kb IncR plasmid carrying blaNDM-1 and an 88 kb IncFII(pHN7A8)::IncN hybrid plasmid in a C. freundii isolate. 148-149 kb IncFIA(Hl1)::IncA/C2 plasmids and 75-76 kb IncFII(Yp) plasmids, both carrying blaNDM-1 were shared among three sequence type 11 (ST11) isolates and three ST395 isolates of K. pneumoniae, respectively. Most of the plasmids co-carried genes conferring resistance to clinically relevant antimicrobials, including third-generation cephalosporins, aminoglycosides, and fluoroquinolones, in addition to blaNDM-1. These results provide insight into the genetic basis of CRE in Vietnam, and could help control nosocomial infections.

microbiology↗