Biology subjects

Shimizu, Y.

Publications and source records attributed to Shimizu, Y..

34 records · Page 2Linked to original sources

bioRxiv2023

Rapid Vacuolar Sorting of the Borate Transporter BOR1 Requires the Adaptor Protein Complex AP-4 in Arabidopsis

Plants maintain nutrient homeostasis by controlling the activities and abundance of nutrient transporters. In Arabidopsis thaliana, the borate (B) transporter BOR1 plays a role in the efficient translocation of B under low-B conditions. BOR1 undergoes polyubiquitination in the presence of sufficient B and is then transported to the vacuole via multivesicular bodies (MVBs) to prevent B accumulation in tissues at a toxic level. A previous study indicated that BOR1 physically interacts with subunits of adaptor protein complexes AP-3 and AP-4, both involved in vacuolar sorting pathways. In this study, we investigated the roles of AP-3 and AP-4 subunits in BOR1 trafficking in Arabidopsis. The lack of AP-3 subunits did not affect either vacuolar sorting or polar localization of BOR1-GFP, whereas the absence of AP-4 subunits resulted in a delay in high-B-induced vacuolar sorting without affecting polar localization. Super-resolution microscopy revealed a rapid sorting of BOR1-GFP into AP-4-positive spots in the trans-Golgi network (TGN) upon high-B supply. These results indicate that AP-4 is involved in sequestration of ubiquitinated BOR1 into a TGN-specific subdomain "vacuolar-trafficking zone," and is required for efficient sorting to MVB and vacuole. Our findings elucidate the rapid vacuolar sorting process facilitated by AP-4 in plant nutrient transporters. Subject areas(7) Membrane and transport

plant biology↗

bioRxiv2023

Overcoming antibody-resistant SARS-CoV-2 variants with bispecific antibodies constructed using non-neutralizing antibodies

A current challenge is the emergence of SARS-CoV-2 variants, such as BQ.1.1 and XBB.1.5, that can evade immune defenses, thereby limiting antibody drug effectiveness. Emergency-use antibody drugs, including the widely effective bebtelovimab, are losing their benefits. One potential approach to address this issue are bispecific antibodies which combine the targeting abilities of two antibodies with distinct epitopes. We engineered neutralizing bispecific antibodies in the IgG-scFv format from two initially non-neutralizing antibodies, CvMab-6 (which binds to the receptor-binding domain [RBD]) and CvMab-62 (targeting a spike protein S2 subunit epitope adjacent to the known anti-S2 antibody epitope). Furthermore, we created a bispecific antibody by incorporating the scFv of bebtelovimab with our anti-S2 antibody, demonstrating significant restoration of effectiveness against bebtelovimab-resistant BQ.1.1 variants. This study highlights the potential of neutralizing bispecific antibodies, which combine existing less effective anti-RBD antibodies with anti-S2 antibodies, to revive the effectiveness of antibody therapeutics compromised by immune-evading variants.

microbiology↗

bioRxiv2023

Identification of key virus--prokaryote infection pairs that contribute to viral shunt in a freshwater lake

Viruses infect and kill productive prokaryotes in a density-or frequency-dependent manner and affect carbon cycling. However, the effects of the stratification transition, including the stratified and destratified periods, on the changes in prokaryotic/viral communities and the interactions among them remain unclear. We conducted a monthly survey of the surface and deep layers of a large and deep freshwater lake (Lake Biwa, Japan) for a year and analyzed the prokaryotic production and prokaryotic/viral metagenome. Our analysis (including 1 608 prokaryotes and 13 761 viruses) revealed that 19 prokaryotic species, accounting for [~]40% of total abundance, might be suppressed by viruses when prokaryotic production is higher. This suggests that a small proportion of prokaryotes contribute to a large amount of prokaryotic abundance, and these prokaryotes are infected and lysed by viruses, driving the viral shunt in the freshwater lake. Furthermore, we found that annual vertical mixing might yield a similar rate of community change between the surface and deep layers. This finding might be valuable in understanding how the communities change when the stratification of freshwater lakes is affected by global warming in the future.

microbiology↗

bioRxiv2023

Physicochemical properties of the vacuolar membrane and cellular factors determine formation of vacuolar invaginations

Vacuoles change their morphology in response to stress. In yeast exposed to chronically high temperatures, vacuolar membranes get deformed and invaginations are formed. We show that phase-separation of vacuolar membrane occurred after heat stress leading to the formation of the invagination. In addition, Hfl1, a vacuolar membrane-localized Atg8-binding protein, was found to suppress the excess vacuolar invaginations after heat stress. At that time, Hfl1 formed foci at the neck of the invaginations in wild-type cells, whereas it was efficiently degraded in the vacuole in the atg8{Delta} mutant. Genetic analysis showed that the endosomal sorting complex required for transport (ESCRT) machinery was necessary to form the invaginations irrespective of Atg8 or Hfl1. In contrast, a combined mutation with the vacuole BAR domain protein Ivy1 led to vacuoles in hfl1{Delta}ivy1{Delta} and atg8{Delta}ivy1{Delta} mutants having constitutively invaginated structures; moreover, these mutants showed stress-sensitive phenotypes. Our findings suggest that vacuolar invaginations result from the combination of changes in the physiochemical properties of the vacuolar membrane and other cellular factors. Summary statementVacuolar invaginations occur through a combination of the changes of physiochemical properties of the vacuolar membrane and other cellular factors in yeast. Particularly, Hfl1 suppresses the excess invaginations.

cell biology↗

bioRxiv2022

SARS-CoV-2-neutralizing humoral IgA response occurs earlier but modest and diminishes faster compared to IgG response.

Secretory immunoglobulin A (IgA) plays a crucial role in the mucosal immunity for preventing the invasion of the exogenous antigens, however, little has been understood about the neutralizing activity of serum IgA. Here, to examine the role of IgA antibodies against COVID-19 illnesses, we determined the neutralizing activity of serum/plasma IgG and IgA purified from previously SARS-CoV-2-infected and COVID-19 mRNA-vaccine-receiving individuals. We found that serum/plasma IgA possesses substantial but rather modest neutralizing activity against SARS-CoV-2 compared to IgG with no significant correlation with the disease severity. Neutralizing IgA and IgG antibodies achieved the greatest activity at approximately 25 and 35 days after symptom onset, respectively. However, neutralizing IgA activity quickly diminished and went down below the detection limit approximately 70 days after onset, while substantial IgG activity was observed till 200 days after onset. The total neutralizing activity in sera/plasmas of those with COVID-19 largely correlated with that in purified-IgG and purified-IgA and levels of anti-SARS-CoV-2-S1-binding IgG and anti-SARS-CoV-2-S1-binding IgA. In individuals who were previously infected with SARS-CoV-2 but had no detectable neutralizing IgA activity, a single dose of BNT162b2 or mRNA-1273 elicited potent serum/plasma neutralizing IgA activity but the second dose did not further strengthen the neutralization antibody response. The present data show that the systemic immune stimulation with natural infection and COVID-19 mRNA-vaccines elicit both SARS-CoV-2-specific neutralizing IgG and IgA response in serum, but the IgA response is modest and diminishes faster compared to IgG response. Author SummaryImmunoglobulin A (IgA) is the most abundant type of antibody in the body mostly located on mucosal surfaces as a dimeric secretory IgA. Such secretory IgA plays an important role in preventing the adherence and invasions of foreign objects by its neutralizing activity, while monomeric serum IgA is thought to relate to the phagocytic immune system activation. Here, we report that individuals with the novel coronavirus disease (COVID-19) developed both systemic neutralizing IgG and IgA active against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although the neutralizing IgA response was quick and reached the highest activity 25 days post-symptom-onset, compared to 35 days for IgG response, neutralizing IgA activity was modest and diminished faster than neutralizing IgG response. In individuals, who recovered from COVID-19 but had no detectable neutralizing IgA activity, a single dose of COVID-19 mRNA-vaccine elicited potent neutralizing IgA activity but the second dose did not further strengthen the antibody response. Our study provides novel insights into the role and the kinetics of serum IgA against the viral pathogen both in naturally-infected and COVID-19 mRNA-vaccine-receiving COVID-19-convalescent individuals.

microbiology↗

bioRxiv2022

Genetic architectures of postmating isolation and morphology of two highly diverged rockfishes (genus Sebastes)

Postmating isolation is thought to be an important driver of the late stages of speciation. However, relatively little is empirically known about the process compared to other isolating mechanisms that drive the early stages of speciation, especially in non-model organisms. We characterized the genetic architecture of postmating isolation between two rockfishes, Sebastes schlegelii and S. trivittatus, whose reproductive isolation is complete. We examined transmission ratio distortion (TRD) patterns of genetic markers in two reciprocal backcross populations. Markers showing either of the two types of TRD was widespread across the genome, with some of the distorted markers forming extensive clusters around the recombination coldspots. These suggest that the postmating isolation effectively prevents gene flow across the genome and the recombination landscape contributes to the genetic architecture. Comparisons between two backcross families and two developmental stages showed little similarity in the distorted markers, suggesting asymmetry and stage-specificity of the isolation. This may be due to hybrid incompatibility involving maternal factors or extrinsic selection. The lack of sex-ratio distortion in the mapping families suggested that Haldanes rule in terms of hybrid inviability does not hold. Additionally, QTL mapping detected significant QTLs for sex and the morphological traits relevant to speciation and convergence of rockfishes, including body coloration. Genes in the melanocortin system, including agouti-signaling protein 1 (asip1) and melanocortin 1 receptor (mc1r), might underlie the horizontal and vertical color patterns on the body, respectively. These findings constitute an essential step towards a comprehensive understanding of speciation and morphological diversification of rockfishes.

evolutionary biology↗

bioRxiv2022

Evolutionarily conserved effects of Notch signaling drive intestinal graft-versus-host disease in mice and non-human primates

Notch signaling promotes T-cell pathogenicity and graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (allo-HCT) in mice, with a dominant role for the Delta-like ligand DLL4. To assess if Notchs effects are evolutionarily conserved and identify key mechanisms, we studied antibody-mediated DLL4 blockade in a non-human primate model similar to human allo-HCT. Short-term DLL4 blockade improved post-transplant survival with striking, durable protection from gastrointestinal GVHD, out of proportion to other disease sites. Unlike prior immunosuppressive strategies, anti-DLL4 interfered with a T-cell transcriptional program associated with intestinal infiltration. In cross-species investigations, Notch inhibition decreased surface abundance of the gut-homing integrin a4b7 in conventional T-cells via b1 competition for a4 binding, while preserving a4b7 in regulatory T-cells. Thereby, DLL4/Notch blockade decreased effector T-cell infiltration into the gut, with increased regulatory to conventional T-cell ratios early after allo-HCT. Our results identify a conserved, biologically unique and targetable role of DLL4/Notch signaling in GVHD. One Sentence SummaryNotch signaling promotes pathogenic effector T cell infiltration of the intestine during acute graft-versus-host disease.

immunology↗

bioRxiv2021

NR4A3 is oxidative stress responsible transcription factor through HMOX1, and also controls cell cycle through CCNE1 and CDK2 in pancreatic islet derived 1.1B4 cells

The mechanism of antioxidant defense system is still controversial. As islet {beta}-cell is weak in oxidative condition, that causes diabetes mellitus, therefore, antioxidant defense system of human pancreatic islet derived 1.1B4 cell was analyzed. Cells were exposed to H2O2 and comprehensive gene expression was analyzed by Agilent human microarray. HMOX1 and NR4A3, member of orphan receptor, were up-regulated. Therefore, NR4A3 was knocked down with siRNA, then analyzed gene expression by microarray, and found that the knocked down cells were weak in oxidative stress. HMOX1 expression was strongly inhibited by siRNA of NR4A3, and NR4A3 responsible sequence of aaggtca was found near the HMOX1 gene, suggesting NR4A3 is oxidative stress responsible transcription factor through HMOX1 expression. The expression of CCNE1 and CDK2 was also inhibited by knocked down of NR4A3, it is suggested NR4A3 is also important transcription factor for cell growth regulation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/457070v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@18d0371org.highwire.dtl.DTLVardef@dd1eacorg.highwire.dtl.DTLVardef@108b1b9org.highwire.dtl.DTLVardef@1cbf899_HPS_FORMAT_FIGEXP M_FIG Hydrogen peroxide induces NR4A3 and binds to aaggtca sequence of HMOX1, and increased transcription of HMOX1. Resulting heme oxygenase produces biliverdin, antioxidants, from heme. NR4A3 also bind to aaggtca sequence of CDK2 and CCNE1, resulting CDK2 and Cyclin E. CDK2 bind to cyclin E and cell goes from G1 to S phase. C_FIG

genomics↗

bioRxiv2021

Versatile and multiplexed mass spectrometry-based absolute quantification with cell-free-synthesized internal standard peptides

Preparation of stable isotope-labeled internal standard peptides is crucial for mass spectrometry (MS)-based targeted proteomics. Herein, we developed versatile and multiplexed absolute protein quantification method using MS. A previously developed method based on the cell-free peptide synthesis system, termed MS-based quantification by isotope-labeled cell-free products (MS-QBiC), was improved for multiple peptide synthesis in one-pot reaction. We pluralized the quantification tags used for the quantification of synthesized peptides and thus, made it possible to use cell-free synthesized isotope-labeled peptides as mixtures for the absolute quantification. The improved multiplexed MS-QBiC method was proved to be applied to clarify ribosomal proteins stoichiometry in the ribosomal subunit, one of the largest cellular complexes. The study demonstrates that the developed method enables the preparation of several dozens and even several hundreds of internal standard peptides within a few days for quantification of multiple proteins with only a single-run of MS analysis.

synthetic biology↗

bioRxiv2021

Induction of antioxidant and detoxifying enzymes by oriental bezoar

ObjectivesOriental bezoar, a gallstone formed in the gall sac of Bos taurus Linne var. domesticus Gmelin (Bovidae), has been used as a crude drug in oriental medicine for stroke, convulsion, epilepsy, swelling and pain in the throat, and high fever. Despite reports on the antioxidative effect of oriental bezoar-containing drugs, in vitro-cultured calculus bovis (a substitute for oriental bezoar), and its components (namely, bilirubin and biliverdin), the intracellular mechanism of the antioxidative action of oriental bezoar remains unclear. Therefore, we studied the effect of oriental bezoar on the nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway, which transcriptionally regulates genes encoding antioxidant and detoxifying enzymes, such as heme oxygenase-1 (HO-1), glutathione-S-transferase alpha 1 (GSTA1), and quinone oxidoreductase 1 (NQO1), to elucidate its antioxidative effect. MethodsARE promoter activity and mRNA expression (HO-1, GSTA1, and NQO1) in HepG2 cells supplemented with test drugs were examined in vitro by luciferase reporter assay and real-time PCR, respectively. ResultsOriental bezoar induced ARE promoter activity and increased the mRNA expression level of HO-1, GSTA1, and NQO1. Its components, namely, bilirubin and biliverdin, also increased the ARE promoter activity. ConclusionThese results suggest that oriental bezoar can induce antioxidative and detoxification effects via the Nrf2-ARE pathway.

pharmacology and toxicology↗

bioRxiv2020

Protective effects of saffron and its constituent crocetin to motor symptoms, short life span and rough-eyed phenotypes in the fly models of Parkinson's disease in vivo

Parkinsons disease (PD) is a common neurodegenerative disorder with motor symptoms linked to the loss of dopaminergic neurons in the brain. -Synuclein is an aggregation-prone neural protein that plays a role in the pathogenesis of PD. In our previous paper, we found that saffron; the stigma of Crocus sativus Linne (Iridaceae), and its constituents (crocin and crocetin) suppressed aggregation of -synuclein and promoted the dissociation of -synuclein fibrils in vitro. In this study, we investigated the effect of dietary saffron and its constituent, crocetin, in vivo on a fly PD model overexpressing several mutant -synuclein in a tissue-specific manner. Saffron and crocetin significantly suppressed the decrease of climbing ability in the Drosophila overexpressing A30P (A30P fly PD model) or G51D (G51D fly PD model) mutated -synuclein in neurons. Saffron and crocetin extended the life span in the G51D fly PD model. Saffron suppressed the rough-eyed phenotype and the dispersion of the size histogram of the ocular long axis in A30P fly PD model in eye. Saffron had a cytoprotective effect on a human neuronal cell line with -synuclein fibrils. These data showed that saffron and its constituent crocetin have protective effects on the progression of PD disease in animals in vivo and suggest that saffron and crocetin can be used to treat PD.

pharmacology and toxicology↗

bioRxiv2020

Long term sterile immunity induced by an adjuvant-containing live-attenuated AIDS virus

Antigen 85B (Ag85B) is one of the most dominant proteins secreted from most mycobacterial species, and it induces Th1-type immune responses as an adjuvant. We genetically constructed a live attenuated simian human immunodeficiency virus to express the adjuvant molecule Ag85B (SHIV-Ag85B). SHIV-Ag85B could not be detected 4 weeks after injection in cynomolgus macaques, and strong SHIV-specific T cell responses were induced in these macaques. When these macaques in which SHIV-Ag85B had become undetectable were challenged with pathogenic SHIV89.6P at 37 weeks after SHIV-Ag85B became undetectable, SHIV89.6P could not be detected after the challenge. Eradication of SHIV89.6P was confirmed by adoptive transfer experiments and CD8-depletion studies. The SHIV-Ag85B-inoculated macaques showed enhancement of Gag-specific monofunctional and polyfunctional CD8+ T cells in the acute phase of pathogenic SHIV challenge. The results suggest that SHIV-Ag85B elicited strong sterile immune responses against pathogenic SHIV and that it may lead to the development of a vaccine for AIDS virus infection. ImportanceDevelopment of an effective HIV vaccine has been a major priority to control the worldwide AIDS epidemic. The moderately attenuated prototypic vaccine strain SIVmac239{Delta}nef has been used in various studies; however, it does not provide sufficient effects to prevent infection. The use of adjuvant in vaccination is thought to be useful for enhancing the immune responses to various pathogens. In the present study, we constructed a live attenuated SHIV virus expressing adjuvant molecule Ag85B and assessed vaccine effects in cynomolgus macaques. The present study shows that live-attenuated SHIV expressing Ag85B elicits viral antigen-specific polyfunctional CD8+ T cell responses against pathogenic SHIV and provide the possibility of eradicating a pathogenic lentivirus from infected animals.

immunology↗

bioRxiv2020

Visuospatial n-back test for early detection of mild cognitive impairment in patients with Parkinsons Disease : an fMRI study

BackgroundCognitive impairment is a common symptom in the patients with Parkinsons disease (PD). In delineating a therapeutic plan, the early diagnosis of mild cognitive impairment in PD (PD-MCI) is important. Patients with PD-MCI have severe impairment in frontal executive function and/or visuospatial recognition. However, the clinical assessment of these functions is not routinely performed. MethodIn this study, we aimed to clarify the advantage of visuospatial version of the n-back test as a tool for the early detection of neuropsychological change in the patients with PD-MCI. The score of 0-back test reflects visuospatial recognition, and the scores of 1-back and 2-back reflect visuospatial working memory. PD-MCI was classified according to the criteria provided by the Movement Disorder Society Task Force for mild cognitive impairment in PD. We recruited 13 patients with PD-MCI, and 15 patients with cognitive normal PD. Using functional MRI (fMRI), we also aimed to clarify the specific brain regions associated with the impairment of visuospatial working memory. ResultWe demonstrated that the correct answer rate of patients with PD-MCI was lower in the 2-back test than patients with PD-CN. However, we did not find statistical difference in the 0-back test. These results indicate the preservation of visuospatial recognition and the impairment of visuospatial working memory in the patients with PD-MCI. We revealed the reduced activation within the middle frontal gyrus (MFG) and the inferior parietal lobule (IPL) during the 2-back test in the patients with PD-MCI. It may be associated with the severity of cortico-striatal dysfunction in the dopaminergic neural network which is associated with Lewy body pathology. ConclusionThe visuospatial n-back test has advantages for use in rapid and early detection of impaired visual recognition and working memory. The combination of functional neuroimaging and neuropsychological tests may provide markers for the increased risk of dementia before the development of an irreversible disease-specific pathology.

neuroscience↗

bioRxiv2019

Genome-wide analysis of transcriptional bursting-induced noise in mammalian cells

Transcriptional bursting is stochastic activation and inactivation of promoters, leading to discontinuous production of mRNA, and is considered to be a contributing factor to cell-to-cell heterogeneity in gene expression. However, it remains elusive how the kinetic properties of transcriptional bursting (e.g., burst size, burst frequency, and noise induced by transcriptional bursting) are regulated in mammalian cells. In this study, we performed a genome-wide analysis of transcriptional bursting in mouse embryonic stem cells (mESCs) using single-cell RNA-sequencing. We found that the kinetics of transcriptional bursting was determined by a combination of promoter and gene body binding proteins, including polycomb repressive complex 2 and transcription elongation-related factors. Furthermore, large-scale CRISPR-Cas9-based screening and functional analysis revealed that the Akt/MAPK signaling pathway regulated bursting kinetics by modulating transcription elongation efficiency. These results uncover key molecular mechanisms underlying transcriptional bursting and cell-to-cell gene expression noise in mammalian cells.

systems biology↗

bioRxiv2018

Genome-wide association meta-analysis identifies novel GP2 gene risk variants for pancreatic cancer in the Japanese population

The etiology of pancreatic cancer remains largely unknown. Here, we report the results of a meta-analysis of three genome-wide association studies (GWASs) comprising 2,039 pancreatic cancer cases and 32,592 controls, the largest sample size in the Japanese population. We identified 3 (13q12.2, 13q22.1, and 16p12.3) genome-wide significant loci (P<5.0x10-8) and 4 suggestive loci (P<1.0x10-6) for pancreatic cancer. Of these risk loci, 16p12.3 is novel; the lead SNP maps to rs78193826 (odds ratio (OR)=1.46, 95% CI=1.29-1.66, P=4.28x10-9), an Asian-specific, nonsynonymous glycoprotein 2 (GP2) gene variant predicted to be highly deleterious. Additionally, the gene-based GWAS identified a novel gene, KRT8, which is linked to exocrine pancreatic and liver diseases. The identified GP2 gene variants were pleiotropic for multiple traits, including type 2 diabetes, hemoglobin A1c (HbA1c) levels, and pancreatic cancer. Mendelian randomization analyses corroborated causality between HbA1c and pancreatic cancer. These findings suggest that GP2 gene variants are associated with pancreatic cancer susceptibility in the Japanese population, prompting further functional characterization of this locus.

genetics↗

bioRxiv2018

Quantitative characterization of translational riboregulators using an in vitro transcription-translation system

Riboregulators are short RNA sequences that, upon binding to a ligand, change their secondary structure and influence the expression rate of a downstream gene. They constitute an attractive alternative to transcription factors for building synthetic gene regulatory networks because they can be engineered de novo and they have a fast turnover and a low metabolic burden. However, riboregulators are generally designed in silico and tested in vivo, which only provides a yes/no evaluation of their performances, thus hindering the improvement of design algorithms. Here we show that a cell-free transcription-translation (TX-TL) system provides valuable quantitative information about the performances of in silico designed riboregulators. In particular, we use the ribosome as an exquisite molecular machine that detects functional riboregulators, precisely measures their concentration and linearly amplifies the signal by generating a fluorescent protein. We apply this method to characterize two types of translational riboregulators composed of a cis-repressed (cr) and a trans-activating (ta) strand. At the DNA level we demonstrate that high concentrations of taDNA poisoned the activator until total shut off. At the RNA level, we show that this approach provides a fast and simple way to measure dissociation constants of functional riboregulators, in contrast to standard mobility-shift assays. Our method opens the route for using cell-free TX-TL systems for the quantitative characterization of functional riboregulators in order to improve their design in silico.

synthetic biology↗