Biology subjects

Yamamoto, T.

Publications and source records attributed to Yamamoto, T..

At least 91 records · Page 5Linked to original sources

bioRxiv2022

Hit-and-run silencing of endogenous DUX4 by targeting DNA hypomethylation on D4Z4 repeats in facioscapulohumeral muscular dystrophy

Facioscapulohumeral muscular dystrophy (FSHD), a progressive skeletal muscle disorder, is epigenetically characterized by DNA hypomethylation of the D4Z4 repeats in the 4q35 region, which enables aberrant DUX4 expression. Sustainable DUX4 suppression is thus a promising therapeutic strategy by which to prevent disease progression, but most of the supposed methods to achieve this depend on the expression of a mediator biochemical entity that would potentially narrow the quality of life of individuals with FSHD in the clinical context. In this study, we report that by applying hit-and-run silencing with dCas9-mediated epigenetic editing targeting DNA hypomethylation on D4Z4 repeats, we could achieve the suppression of endogenous DUX4 in our FSHD patient-derived iPSC model. Notably, DNA methylation was significantly upregulated in FSHD cells and suppression effects were observed for at least two weeks after intervention, which was not the case with transient treatments of typical dCas9-KRAB alone. Off-target analysis showed that despite the potential genome-wide risk for DNA methylation, the impact on the transcriptome was limited. We propose that hit-and-run silencing could be a promising option to prevent disease progression with minimum intervention for individuals with FSHD, motivating further study for clinical development. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/487997v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@19e85dforg.highwire.dtl.DTLVardef@672798org.highwire.dtl.DTLVardef@58717dorg.highwire.dtl.DTLVardef@83044b_HPS_FORMAT_FIGEXP M_FIG C_FIG

pathology↗

bioRxiv2022

Application of B cell immortalization for the isolation of antibodies and B cell clones from vaccine and infection settings

The isolation and characterization of neutralizing antibodies from infection and vaccine settings will inform future vaccine design, and methodologies that streamline the isolation of antibodies and the generation of B cell clones are of great interest. Retroviral transduction to express Bcl-6 and Bcl-xL in primary B cells has been shown to promote long-term B cell survival and antibody secretion in vitro, and can be used to isolate antibodies from memory B cells. The application of this methodology to B cell subsets from tissues and to B cells from individuals with chronic infection has not been extensively characterized. Here, we characterize Bcl-6/Bcl-xL B cell immortalization across multiple tissue types and B cell subsets in healthy and HIV-1 infected individuals, as well as individuals recovering from malaria. In HIV-1- and malaria-uninfected donors, naive and memory B cell subsets from PBMC and tonsil tissue transformed with similar efficiencies, and displayed similar characteristics after transformation with respect to their longevity and immunoglobulin secretion. In HIV-1-viremic individuals or in individuals after malaria infection, the CD27-CD21- memory B cell subsets transformed with lower efficiencies compared to the CD27+CD21+ populations, but following transformation B cells expanded and secreted IgG with similar efficiency. Using B cells from HIV-1-infected individuals, we combined Bcl-6/Bcl-xL B cell immortalization with a HIV-1 microneutralization assay to isolate broadly neutralizing antibodies related to VRC13 and VRC38.01. Overall, Bcl-6/Bcl-xL B cell immortalization can be used to isolate antibodies and generate B cell clones from multiple different B cell populations, albeit with different efficiencies.

immunology↗

bioRxiv2022

GBScleanR: Robust genotyping error correction using hidden Markov model with error pattern recognition.

Reduced-representation sequencing (RRS) provides cost-effective and time-saving genotyping platforms. Although the outstanding advantage of RRS in throughput, the obtained genotype data usually contains a large number of errors. Several error correction methods employing hidden Morkov model (HMM) have been developed to overcome these issues. Those methods assume that markers have a uniform error rate with no bias in the allele read ratio. However, bias does occur because of uneven amplification of genomic fragments and read mismapping. In this paper we introduce an error correction tool, GBScleanR, which enables robust and precise error correction for noisy RRS-based genotype data by incorporating marker-specific error rates into the HMM. The results indicate that GBScleanR improves the accuracy by more than 25 percentage points at maximum as compared to the existing tools in simulation datasets and achieves the most reliable genotype estimation in real data even with error prone markers.

bioinformatics↗

bioRxiv2022

Loop extrusion driven volume phase transition of entangled chromosomes

Mitotic chromosomes without nucleosomes have been reconstituted in recent experiments. When topo II is depleted from the reconstituted chromosomes, these chromosomes are entangled and form sparklers, where DNA is condensed in the core with linker histone H1.8 and condensin is localized at the periphery. To understand the mechanism of the assembly of sparklers, we here take into account the loop extrusion by condensin in an extension of the theory of entangled polymer gels. The loop extrusion stiffens an entangled DNA network because DNA segments in the elastically effective chains are translocated to loops, which are elastically ineffective. Our theory predicts that the loop extrusion by condensin drives the volume phase transition that collapses a swollen entangled DNA gel as the stiffening of the network destabilizes the swollen phase. This is an important element to understand the mechanism of the assembly of the reconstituted chromosomes.

biophysics↗

bioRxiv2022

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

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

microbiology↗

bioRxiv2022

STREAMING-tag system reveals spatiotemporal relationships between transcriptional regulatory factors and transcriptional activity

Transcription is a dynamic process that stochastically switches between the ON and OFF states. To detect the dynamic relationship among protein clusters of RNA polymerase II (RNAPII) and coactivators, gene loci, and transcriptional activity, we inserted an MS2 repeat, a TetO repeat, and inteins with a selection marker just downstream of the transcription start site (TSS). By optimizing the individual elements, we have developed the Spliced TetO REpeAt, MS2 repeat, and INtein sandwiched reporter Gene tag (STREAMING-tag) system. Clusters of RNAPII and BRD4 were observed proximally to the TSS of Nanog when the gene was transcribed in mouse embryonic stem cells. In contrast, clusters of MED19 and MED22 Mediator subunits were constitutively located near the TSS. Thus, the STREAMING-tag system revealed the spatiotemporal relationships between transcriptional activity and protein clusters near the gene. This powerful tool is useful for quantitatively understanding dynamic transcriptional regulation in living cells.

molecular biology↗

bioRxiv2021

The establishment of multiple knockout mutants of Colletotrichum orbiculare by CRISPR/Cas9 and Cre/loxP systems

Phytopathogenic fungi belonging to the Colletotrichum genus cause devastating damage for many plant species. Among them, Colletotrichum orbiculare is employed as a model fungus to analyze molecular aspects of plant-fungus interactions. Although gene disruption via homologous recombination (HR) was established for C. orbiculare, this approach is laborious due to its low efficiency. Here we developed methods to efficiently generate multiple knockout mutants of C. orbiculare. We first found that CRISPR/Cas9 system massively promoted gene-targeting efficiency. By transiently introducing a CRISPR/Cas9 vector, more than 90 % of obtained transformants were knockout mutants. Furthermore, we optimized a self-excision Cre/loxP marker recycling system for C. orbiculare because limited availability of desired selective markers hampers sequential gene disruption. In this system, integrated selective marker is removable from the genome via Cre recombinase driven by a xylose-inducible promoter, enabling reuse of the same selective marker for the next transformation. Using our CRISPR/Cas9 and Cre/loxP systems, we attempted to identify functional sugar transporters in C. orbiculare. Multiple disruptions of putative quinate transporter genes restrict fungal growth on media containing quinate as a sole carbon source, confirming their functionality as quinate transporters. Our analyses revealed that quinate acquisition is dispensable during fungal infection because this mutant displayed normal virulence to host plants. In addition, we successfully built mutations of 17 cellobiose transporter genes in a strain. From the data of knockout mutants established in this study, we inferred that repetitive rounds of gene disruption using CRISPR/Cas9 and Cre/loxP systems do not cause negative effects for fungal virulence and growth. Therefore, these systems will be powerful tools to perform systematic gene targeting approach for C. orbiculare.

plant biology↗

bioRxiv2021

Resistance to chemical carcinogenesis induction via a dampened inflammatory response in naked mole-rats

Naked mole-rats (NMRs) have a very low spontaneous carcinogenesis rate, which has prompted scientists to study their cancer resistance mechanisms in order to provide clues for human cancer prevention. Although cancer resistance in NMRs has been intensively investigated at the cellular level, it is still unknown how strongly resistant NMR individuals are to carcinogenesis and how NMR tissues respond to experimental carcinogenesis induction. Here, we show that NMRs exhibit extraordinary resistance against potent chemical carcinogenesis induction through a dampened inflammatory response. Although carcinogenic insults damaged skin cells of both NMRs and mice, NMR skin showed markedly lower immune cell infiltration and reduced induction of inflammatory genes. NMRs harbor loss-of-function mutations in receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL) genes, which are essential for necroptosis, a type of necrotic cell death that activates strong inflammation. A necroptosis-inducing stimulus did not increase death of NMR cells. After carcinogenic insults, leakage of the HMGB1, a marker of necrotic cell death, was not increased in NMR skin. In mice, inhibition or knockout of RIPK3 reduced immune cell infiltration and delayed the onset of chemical carcinogenesis. Therefore, necroptosis deficiency may serve as a cancer resistance mechanism via attenuating the inflammatory response in NMRs. Our study sheds light on the importance of a dampened inflammatory response as a non-cell-autonomous cancer resistance mechanism in NMRs. Further in vivo study of the unusual tissue immune system and carcinogenesis resistance of NMRs may lead to the development of new strategies to prevent carcinogenesis in humans. Significance StatementIn contrast with intensive studies of cancer resistance mechanisms in naked mole-rats (NMRs) at the cellular level, little is known about how NMR individuals respond to carcinogenesis induction, despite the fact that cell-to-cell interactions in tissues regulate carcinogenesis in vivo. Here, we demonstrate that NMRs are remarkably resistant to chemical carcinogenesis induction and characteristically have attenuated tissue inflammatory responses to carcinogenic insults. NMRs have loss-of-function mutations in RIPK3 and MLKL genes and thus cannot activate necroptosis, a type of inflammation-inducing cell death. RIPK3 inhibition in mice reduced immune cell infiltration in response to carcinogenic insults and delayed the onset of chemical-induced carcinogenesis. Our results highlight the importance of studies on dampened tissue inflammatory responses to understand cancer resistance of NMRs.

cancer biology↗

bioRxiv2021

Post-transcriptionally regulated genes are essential for pluripotent stem cell survival

The effects of transcription factors on the maintenance and differentiation of pluripotent stem cells (PSCs) have been well studied. However, the importance of post-transcriptional regulatory mechanisms, which cause the quantitative dissociation of mRNA and protein expression, has not been explored in detail. Here, by combining transcriptome and proteome profiling, we identified 228 post-transcriptionally regulated genes with strict upregulation of the protein level in PSCs. Among them, we found that 84 genes were vital for the survival of PSCs and HDFs, including 20 genes that were specifically necessary for the survival of PSCs. These 20 proteins were upregulated only in PSCs and not in differentiated cells derived from the three germ layers. Subcellular fractionation of the mRNA showed that the expression of most of the 20 proteins was regulated at the mRNA localization stage from the nucleus to the cytoplasm, but their translation efficiency was constant. Together, these results revealed that post-transcriptionally regulated genes have a crucial role in PSC survival.

molecular biology↗

bioRxiv2021

Antibacterial 3D-printed PMMA/ceramic composites

Following the rising interested on 3D-printed technologies, this research explores the possibility to use stereo-lithography to 3D print PMMA resins reinforced with up to 15% in weight of antibacterial ceramic powders. Three different reinforcements were tested, following previous literature data: aluminum nitride, titanium oxide and barium titanate. Between the three powders, the most uniform dispersion was achieved using aluminum nitride. Initial screenings with mixed and cured composite resins showed that between the three composite materials, only aluminum nitride or barium titanate PMMA showed a clear antibacterial effect when compared to the pristine reference, with aluminum nitride being the most effective against E. coli. When 3D printed using stereo-lithography, the composite containing aluminum nitride showed an even higher degree of dispersion and comparable antibacterial effects. Moreover, aluminum nitride reinforced PMMA resins showed good mechanical properties, comparable to the basic resin, and could be further strengthened by a standard post-curing process.

bioengineering↗

bioRxiv2021

Positive Feedback Regulation of fzd7 Expression Robustly Shapes Wnt Signaling Range in Early Heart Development

Secreted molecules called morphogens govern tissue patterning in a concentration-dependent manner. However, it is still unclear how reproducible patterning can be achieved with diffusing molecules, especially when patterning differentiation of a thin region. Wnt is a morphogen that organizes cardiac development; especially Wnt6 patterns the cardiogenic mesoderm to induce differentiation of a thin pericardium in Xenopus. It is, however, unclear how Wnt6 can pattern such a thin tissue. In this study, we reveal that a Wnt receptor, frizzled7, is expressed in a Wnt-dependent manner in the prospective heart region, and that this receptor-feedback is essential for shaping a steep gradient of Wnt. In addition, the feedback imparts robustness against fluctuations of Wnt ligand production and allows the system to reach a steady state quickly. We also found a Wnt antagonist sFRP1, which is expressed at the opposite side of Wnt source, accumulates on a novel type of heparan sulfate (HS), N-acetyl-rich HS, which is highly presented in the outer of cardiogenic mesoderm, achieving local inhibition of Wnt signaling by restricting sFRP1 spreading. These two intricate regulatory systems restrict Wnt signaling and ensure reproducible patterning of a thin pericardium tissue.

developmental biology↗

bioRxiv2021

Nascent ribosomal RNA acts as surfactant that suppresses growth of fibrillar centers in nucleolus

Liquid-liquid phase separation (LLPS) has been thought to be the biophysical principle governing the assembly of the multiphase structures of nucleoli, the site of ribosomal biogenesis. Condensates assembled through LLPS increase their sizes to minimize the surface energy as far as their components are available. However, multiple microphases, fibrillar centers (FCs), dispersed in a nucleolus are stable and their sizes do not grow unless the transcription of pre-ribosomal RNA (pre-rRNA) is inhibited. To understand the mechanism of the suppression of the FC growth, we here construct a minimal theoretical model by taking into account the nascent pre-rRNAs tethered to the FC surfaces by RNA polymerase I. Our theory predicts that nascent pre-rRNAs generate the lateral osmotic pressure that counteracts the surface tension of the FCs and this suppresses the growth of the FCs over the stable size. The stable FC size decreases with increasing the transcription rate and decreasing the RNA processing rate. This prediction is supported by our experiments showing that RNA polymerase inhibitors increase the FC size in a dose-dependent manner. This theory may provide insight into the general mechanism of the size control of nuclear bodies. Significance statementThe nucleolus, a site of pre-ribosomal RNA (pre-rRNA) production, has a characteristic multiphase structure, which has been thought to be assembled through liquid-liquid phase separation (LLPS). Although condensates assembled through LLPS grow by coarsening or coalescence as far as the components are available, the multiple inner phases, fibrillar centers (FCs), are dispersed in a nucleolus. To investigate the underlying mechanism, we constructed a minimal theoretical model by considering nascent pre-rRNAs tethered to RNA polymerase I at the FC surface. This model is supported by our experiments and explains previous experimental observations. This work shed light on the role of nascent RNAs to control the size of nuclear bodies.

biophysics↗

bioRxiv2021

H+/K+ ion pump enhances cytoskeletal polarity to drive gastrulation in sea urchin embryo

Gastrulation is a universal process in the morphogenesis of many animal embryos. Although morphological and molecular events in gastrulation have been well studied, the mechanical driving forces and underlying regulatory mechanisms are not fully understood. Here, we investigated the gastrulation of embryos of a sea urchin, Hemicentrotus pulcherrimus, which involves the invagination of a single-layered vegetal plate into the blastocoel. We observed that omeprazole, a proton pump inhibitor capable of perturbing the left-right asymmetry of sea urchin embryo, induced "partial exogastrulation" where the secondary invagination proceeds outward. During early gastrulation, intracellular apical-basal polarity of F-actin distribution in vegetal half were higher than those in animal half, while omeprazole treatment disturbed the apical-basal polarity of F-actin distribution in vegetal half. Furthermore, gastrulation stopped and even partial exogastrulation did not occur when F-actin polymerization or degradation in whole embryo was partially inhibited via RhoA or YAP1 knockout. A mathematical model of the early gastrulation reproduced the shapes of both normal and exogastrulating embryos using cell-dependent cytoskeletal features based on F-actin. Additionally, such cell position-dependent intracellular F-actin distributions might be regulated by intracellular pH distributions. Therefore, apical-basal polarity of F-actin distribution disrupted by omeprazole may induce the partial exogastrulation via anomalous secondary invagination.

developmental biology↗

bioRxiv2021

Synaptotagmin 2 is ectopically overexpressed in excitatory presynapses of a widely used CaMK2a-Cre mouse line

The CaMKII-Cre mouse line, one of the earliest established Cre driver lines, has resulted in over 800 papers to date. Here, we demonstrate that the second most widely used CaMKII-Cre line, Tg(Camk2a-cre)2Gsc (or CamiCre), shows ectopic overexpression of synaptotagmin 2, the most efficient Ca2+ sensor for fast synchronous neurotransmitter release, in excitatory presynapses of Cre+ brains. Moreover, RNA-seq analysis showed aberrant expression in Cre+ hippocampus, including upregulation of immediate early genes, such as Arc and Fos, and genes presumably derived from bacterial artificial chromosome transgene, such as Slc6a7. Most importantly, CamiCre+ mice showed functional phenotypes, such as hyperactivity and enhanced associative learning, suggesting neural activities are affected. These unexpected results suggest difficulties in interpreting results from studies using the CamiCre line and raise awareness of potential pitfalls in the use of Cre driver lines in general.

genetics↗

bioRxiv2021

Graph-based machine learning reveals rules of spatiotemporal cell interactions in tissues

Robustness in developing and homeostatic tissues is supported by various types of spatiotemporal cell-to-cell interactions. Although live imaging and cell tracking are powerful in providing direct evidence of cell coordination rules, extracting and comparing these rules across many tissues with potentially different length and timescales of coordination requires a versatile framework of analysis. Here we demonstrate that graph neural network (GNN) models are suited for this purpose, by showing how they can be applied to predict cell fate in tissues and utilized to infer the cell interactions governing the multicellular dynamics. Analyzing the live mammalian epidermis data, where spatiotemporal graphs constructed from cell tracks and cell contacts are given as inputs, GNN discovers distinct neighbor cell fate coordination rules that depend on the region of the body. This approach demonstrates how the GNN framework is powerful in inferring general cell interaction rules from live data without prior knowledge of the signaling involved.

cell biology↗

bioRxiv2021

3D matrix adhesion composition facilitates nuclear force coupling to drive invasive cell migration

Cell invasion and metastasis is a multi-step process, initialised through the acquisition of a migratory phenotype and the ability to move through differing and complex 3D extracellular environments. In this study we set out to identify the parameters required for invasive cell migration in 3D environments. Cells interact with the extracellular matrix via transmembrane-spanning integrin adhesion complexes, which are well characterised in cells plated on 2D surfaces, yet much less is known about them in cells embedded in 3D matrices. We establish a technique to determine the composition of cell matrix adhesion complexes of invasive breast cancer cells in 3D matrices and on 2D surfaces and we identify an interaction complex enriched in 3D adhesive sites required for 3D invasive migration. Depletion of {beta}-PIX-Myosin18A (Myo18A) abolishes cancer cell invasion, without negatively affecting matrix degradation, Rho GTPase signalling, or protrusion formation in collagen matrices. Instead, in a mechanism only seen in cells moving through 3D matrix, {beta}-PIX and Myo18A drive the polarised recruitment of non-muscle Myosin 2A (NM2A) to the tips of protrusions. This recruitment of NM2A is required for the creation of an NM2A-NM2B isoform gradient, which ranges from the protrusion to the nucleus. We observe a requirement for active force transmission to the nucleus during invasive migration that is needed to pull the nucleus forward. We postulate that the establishment of the NM2A-NM2B actomyosin gradient facilitates the coupling of cell-matrix interactions at the protrusive cell front with nuclear movement, enabling effective invasive migration and front-rear cell polarity.

cell biology↗

bioRxiv2021

Extracellular adenosine induces hypersecretion of IL-17A by T-helper 17 cells through the adenosine A2a receptor to promote neutrophilic inflammation

Extracellular adenosine, produced from ATP secreted by neuronal or immune cells, may play a role in endogenous regulation of inflammatory responses. However, the underlying molecular mechanisms are largely unknown. Here, we show that adenosine primes hypersecretion of interleukin (IL)-17A by CD4+ T cells via T cell receptor activation. This hypersecretion was also induced by an adenosine A2a receptor (A2aR) agonist, PSB0777. In addition, an A2aR antagonist (Istradefylline), and inhibitors of adenyl cyclase, protein kinase A, and cAMP response element binding protein (which are signaling molecules downstream of the Gs protein coupled with the A2aR), suppressed IL-17A production, suggesting that activation of A2aR induces IL-17A production by CD4+ T cells. Furthermore, immune subset studies revealed that adenosine induced hypersecretion of IL-17A by T-helper (Th)17 cells. These results indicate that adenosine is an endogenous modulator of neutrophilic inflammation. Administration of an A2aR antagonist to mice with experimental autoimmune encephalomyelitis led to marked amelioration of symptoms, suggesting that suppression of adenosine-mediated IL-17A production is an effective treatment for Th17-related autoimmune diseases.

immunology↗

bioRxiv2021

Tracheal motile cilia require CAMSAP3 for formation of central microtubule pair and coordinated beating

Motile cilia of multiciliated epithelial cells undergo synchronized beating to produce fluid flow along the luminal surface of various organs. Each motile cilium consists of an axoneme and a basal body, which are linked by a transition zone. The axoneme exhibits a characteristic 9+2 microtubule arrangement important for ciliary motion, but how this microtubule system is generated is not yet fully understood. Here we show that CAMSAP3, a protein that can stabilize the minus end of a microtubule, concentrates at multiple sites of the cilium-basal body complex, including the upper region of the transition zone or the axonemal basal plate where the central pair of microtubules (CP) initiates. CAMSAP3 dysfunction resulted in loss of the CP and partial distortion of the basal plate, as well as the failure of multicilia to undergo synchronized beating. These findings suggest that CAMSAP3 plays pivotal roles in the formation or stabilization of the CP by localizing at the basal region of the axoneme, and thereby supports the coordinated motion of multicilia in airway epithelial cells.

cell biology↗