Biology subjects

Nishimura, M.

Publications and source records attributed to Nishimura, M..

10 recordsLinked to original sources

bioRxiv2025

Thalamus-cortex interactions drive cell type-specific cortical development in human pluripotent stem cell-derived assembloids

The thalamus plays a pivotal role in the development and function of neural circuits in the cerebral cortex. However, how thalamus-cortex interactions influence human cortical development remains unknown primarily because of the inaccessibility of the human embryonic brain. Here, we demonstrate thalamus-dependent gene expression, circuit organization, and neural activity during corticogenesis using human thalamocortical assembloids (hThCAs). Human cortical and thalamic organoids (hCOs and hThOs) derived from induced pluripotent stem cells exhibited region-specific gene expression and spontaneous neuronal activity. Upon the fusion of these organoids, hThCAs reconstructed reciprocal thalamus-cortex axonal projections and synaptic connections. Transcriptomic analysis revealed thalamus-dependent acceleration of cortical maturation, with upregulation of programs linked to axon development, subplate/cortical plate identity, and activity-regulated genes. Histological analysis showed expanded progenitor pools and increased deep-layer neurons within hThCAs. Wide-field Ca2+ imaging demonstrated that wave-like activity originated in the thalamic region and propagated to the cortical region. Furthermore, two-photon Ca2+ imaging of cortical neurons revealed that synchronous activity emerged exclusively in pyramidal tract (PT) and corticothalamic (CT) neurons, whereas intratelencephalic (IT) neurons remain asynchronous, highlighting cell type-specific circuit integration within hThCAs. These synchronized events were absent in isolated hCOs or in cortico-cortical assembloids, underscoring the specificity of thalamic input. Our findings suggest that diffusible thalamic cues broadly enhance progenitor expansion, while long-range thalamic input organizes cell type-specific synchronous activity. This study demonstrates the thalamus-dependent acquisition of mature cortical phenotypes in a cell type-specific manner in hThCAs, establishing developmental mechanisms linking regional interactions and cell type-specific circuit specification. Thus, hThCAs provide a tractable human platform for dissecting human-specific developmental processes and modeling neurodevelopmental disorders with disrupted thalamocortical communication at the molecular, cellular, and circuit levels. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/681772v1_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@95b599org.highwire.dtl.DTLVardef@c56085org.highwire.dtl.DTLVardef@43df08org.highwire.dtl.DTLVardef@1e7c4ed_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIhThCAs recapitulate the developmental sequence of reciprocal projections with synapses. C_LIO_LIhThCA cortical transcriptomes resemble the late fetal human cortex. C_LIO_LIThalamic paracrine signals promote progenitor expansion and cortical cytoarchitecture. C_LIO_LIWave-like activity originates in the thalamus and propagates across the cortex. C_LIO_LIThalamic input drives cell type-specific synchronization and circuit integration. C_LI

neuroscience↗

bioRxiv2025

A copper-dependent, redox-based hydrogen peroxide perception in plants

Redox-related molecules, such as quinones and reactive oxygen species (ROS), are important signaling molecules for all living organisms. A plant-specific leucine rich-repeat receptor-like kinase (LRR-RLK) CANNOT RESPOND TO DMBQ 1 (CARD1), also known as HYDROGEN-PEROXIDE-INDUCED Ca2+ INCREASES (HPCA1), perceives both quinones and ROS, but knowledge on key structural features is unknown. Here, we determine the structure of the CARD1 ectodomain and uncover its unique features. Structural studies, coupled with genetics and biochemical analysis, demonstrated that previously identified unique cysteine residues are not essential for signal perception in CARD1. Interestingly, CARD1 harbors a copper ion on the surface of the ectodomain via histidine-coordination that is crucial for hydrogen peroxide signaling. Our work reports the first copper-dependent redox receptor and highlights a unique perspective between a receptor and non-peptide stimuli during their perception.

plant biology↗

bioRxiv2025

Structural basis of RNA polymerase II transcription on the H3-H4 octasome

The H3-H4 octasome is a nucleosome-like particle in which two DNA gyres are wrapped around each H3-H4 tetramer disk, forming a clamshell-like configuration. In the present study, we performed in vitro RNAPII transcription assays with the H3-H4 octasome and found that RNAPII transcribed the H3-H4 octasome more efficiently than the nucleosome. RNAPII paused at only one position, superhelical location (SHL) -4 in the H3-H4 octasome, in contrast to pausing at the SHL(-5), SHL(-2), and SHL(-1) positions in the nucleosome. Cryo-electron microscopy analysis revealed that two H3-H4 tetramer disks are retained when the RNAPII paused at the SHL(-4) position of the H3-H4 octasome. However, when RNAPII reached the SHL(-0.5) position, five base pairs before the dyad position of the H3-H4 octasome, the proximal H3-H4 tetramer was disassembled but the distal H3-H4 tetramer still remained on the DNA. Therefore, RNAPII efficiently transcribes the H3-H4 octasome by stepwise H3-H4 tetramer disassembly.

molecular biology↗

bioRxiv2024

Conversion of silent synapses to AMPA receptor-mediated functional synapses in human cortical organoids

Despite the crucial role of synaptic connections and neural activity in the development and organization of cortical circuits, the mechanisms underlying the formation of functional synaptic connections in the developing human cerebral cortex remain unclear. We investigated the development of -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated synaptic transmission using human cortical organoids (hCOs) derived from induced pluripotent stem cells. Two-photon Ca2 imaging revealed an increase in the frequency and amplitude of spontaneous activity in hCOs on day 80 compared to day 50. Additionally, spontaneous neural activity in late-stage hCOs, but not in early-stage hCOs, was blocked by N-methyl-D-aspartate receptor (NMDAR) and AMPAR antagonists. However, transsynaptic circuit tracing with G-deleted rabies viral vectors indicated a similar number of synaptic connections in early- and late-stage hCOs. Notably, chemical labeling demonstrated a significant increase in AMPAR expression on the postsynaptic membrane and colocalization with NMDAR in late-stage hCOs. These results suggest that hCOs progressively organize excitatory synaptic transmission, concurrent with the transition from silent synapses lacking AMPARs to functional synapses containing NMDARs and AMPARs. This in vitro model of human cortical circuits derived from induced pluripotent stem cells reflects the developmental programs underlying physiological transitions, providing valuable insights into human corticogenesis and neurodevelopmental disorders. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=181 SRC="FIGDIR/small/629885v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1432b6dorg.highwire.dtl.DTLVardef@d8350borg.highwire.dtl.DTLVardef@8273f8org.highwire.dtl.DTLVardef@8bbabd_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2024

Spherical Echo-Planar Time-resolved Imaging (sEPTI) for rapid 3D quantitative T2* and Susceptibility imaging

PurposeTo develop a 3D spherical EPTI (sEPTI) acquisition and a comprehensive reconstruction pipeline for rapid high-quality whole-brain submillimeter T2* and QSM quantification. MethodsFor the sEPTI acquisition, spherical k-space coverage is utilized with variable echo-spacing and maximum kx ramp-sampling to improve efficiency and incoherency when compared to existing EPTI approaches. For reconstruction, an iterative rank-shrinking B0 estimation and odd-even high-order phase correction algorithms were incorporated into the reconstruction to better mitigate artifacts from field imperfections. A physics-informed unrolled network was utilized to boost the SNR, where 1-mm and 0.75-mm isotropic whole-brain imaging were performed in 45 and 90 seconds, respectively. These protocols were validated through simulations, phantom, and in vivo experiments. Ten healthy subjects were recruited to provide sufficient data for the unrolled network. The entire pipeline was validated on additional 5 healthy subjects where different EPTI sampling approaches were compared. Two additional pediatric patients with epilepsy were recruited to demonstrate the generalizability of the unrolled reconstruction. ResultssEPTI achieved 1.4 x faster imaging with improved image quality and quantitative map precision compared to existing EPTI approaches. The B0 update and the phase correction provide improved reconstruction performance with lower artifacts. The unrolled network boosted the SNR, achieving high-quality T2* and QSM quantification with single average data. High-quality reconstruction was also obtained in the pediatric patient using this network. ConclusionsEPTI achieved whole-brain distortion-free multi-echo imaging and T2* and QSM quantification at 0.75 mm in 90 seconds which has the potential to be useful for wide clinical applications.

bioengineering↗

bioRxiv2023

Conserved cysteine residues in Kaposis sarcoma herpesvirus ORF34 are necessary for viral production and viral pre-initiation complex formation.

Kaposis sarcoma herpesvirus (KSHV) ORF34 plays a significant role as a component of the viral pre-initiation complex (vPIC), which is indispensable for late gene expression across beta and gamma herpesviruses. Although the key role of ORF34 within the vPIC and its function as a hub protein have been recognized, further clarification regarding its specific contribution to vPIC functionality and interactions with other components is required. This study employed a deep-learning algorithm-assisted structural model of ORF34, revealing highly conserved amino acid residues across human beta- and gamma-herpesviruses localized in structured domains. Thus, we engineered ORF34 alanine-scanning mutants by substituting conserved residues with alanine. These mutants were evaluated for their ability to interact with other vPIC factors and restore viral production in cells harboring the ORF34-deficient KSHV-BAC. Our experimental results highlight the crucial role of the 4 cysteine residues conserved in ORF34: a tetrahedral arrangement consisting of a pair of C-Xn-C consensus motifs. This suggests the potential incorporation of metal cations in interacting with ORF24 and ORF66 vPIC components, facilitating late gene transcription, and promoting overall virus production by capturing metal cations. In summary, our findings underline the essential role of conserved cysteines in KSHV ORF34 for effective vPIC assembly and viral replication, thereby enhancing our understanding of the complex interplay between the vPIC components. IMPORTANCEThe initiation of late gene transcription is universally conserved across the gamma- and beta-herpesvirus families. This process employs a viral pre-initiation complex (vPIC), which is analogous to a cellular PIC. Although KSHV ORF34 is a critical factor for viral replication and is a component of the vPIC, the specifics of vPIC formation and the essential domains crucial for its function remain unclear. Structural predictions suggest that the 4 conserved cysteines (C170, C175, C256, and C259) form a tetrahedron that coordinates the metal cation. We further investigated the role of these conserved amino acids in interactions with other vPIC components, late gene expression, and virus production, to demonstrate for the first time that these cysteines are pivotal for these functions. This discovery not only deepens our comprehensive understanding of ORF34 and vPIC dynamics but also lays the groundwork for more detailed studies on herpesvirus replication mechanisms in future research.

microbiology↗

bioRxiv2022

Novel monoclonal antibodies showing broad neutralizing activity for SARS-CoV-2 variants including Omicrons BA.5 and BA.2.75

We identified novel neutralizing monoclonal antibodies against SARS-CoV-2 variants (including Omicron) from individuals received two doses of mRNA vaccination after they had been infected with wildtype. We named them MO1, MO2 and MO3. MO1 shows high neutralizing activity against authentic variants: D614G, Delta, BA.1, BA.1.1, BA.2, and BA.2.75 and BA.5. Our findings confirm that the wildtype-derived vaccination can induce neutralizing antibodies that recognize the epitopes conserved among the SARS-CoV-2 variants (including BA.5 and BA.2.75). The monoclonal antibodies obtained herein could serve as novel prophylaxis and therapeutics against not only current SARS-CoV-2 viruses but also future variants that may arise.

microbiology↗

bioRxiv2022

Extension of bacterial rDNA sequencing to concurrent epigenetic analysis and its application to 16S meta-epigenetics

Although polymerase chain reaction (PCR) amplification and sequencing of the 16S rDNA region has been used in a wide range of scientific fields, it does not provide DNA methylation information. We describe a simple add-on method to investigate 5-methylcytosine residues in the bacterial 16S rDNA region from clinical samples or flora. Single-stranded bacterial DNA after bisulfite conversion was preferentially amplified with multiple displacement amplification (MDA) at pH neutral, and the 16S rDNA region was analyzed using nested bisulfite PCR and sequencing. 16S rDNA bisulfite sequencing can provide clinically important bacterial DNA methylation status concurrently with intact 16S rDNA sequence information. We used this approach to identify novel methylation sites and a methyltransferase (M. MmnI) in Morganella morganii. Next, we analyzed bacterial flora from clinical specimens of small amount and identified different methylation motifs among Enterococcus faecalis strains. The method developed here, referred to as "add-on" to the conventional 16S rDNA analysis, is the most clinically used bacterial identification genetic test, which provides additional information that could not be obtained with the conventional method. Since the relationship between drug resistance in bacteria and DNA methylation status has been reported, bacterial epigenetic information would be useful in clinical testing as well. Our analysis suggests that M. MmnI has a promotive effect on erythromycin resistance. 16S rDNA bisulfite PCR and sequencing coupled with MDA at pH neutral is a useful add-on tool for analyzing 16S meta-epigenetics.

microbiology↗

bioRxiv2019

Kaposi’s sarcoma-associated herpesvirus ORF66 is essential for late gene expression and virus production via interaction with ORF34

Kaposis sarcoma-associated herpesvirus (KSHV) is closely associated with B-cell and endothelial cell malignancies. After the initial infection, KSHV retains its viral genome in the nucleus of the host cell and establishes a lifelong latency. During lytic infection, KSHV encoded lytic-related proteins are expressed in a sequential manner and are classified as immediate early, early, and late gene transcripts. The transcriptional initiation of KSHV late genes is thought to require the complex formation of the virus specific pre-initiation complex (vPIC), which may consist of at least 6 transcription factors (ORF18, 24, 30, 31, 34, and 66). However, the functional role of ORF66 in vPIC during KSHV replication remains largely unclear. Here, we generated ORF66-deficient KSHV using a BAC system to evaluate its role during viral replication. While ORF66-deficient KSHV demonstrated mainly attenuated late gene expression and decreased viral production, viral DNA replication was unaffected. CHIP analysis showed that ORF66 bound to the promoters of late gene (K8.1), but did not to those of latent gene (ORF72), immediate early gene (ORF16) and early gene (ORF46/47). Furthermore, we found that three highly conserved C-X-X-C sequences and a conserved leucine-repeat in the C-terminal region of ORF66 were essential for interaction with ORF34 and viral production. The interaction between ORF66 and ORF34 occurred in a zinc-dependent manner. Our data support a model, in which ORF66 serves as a critical vPIC component to promote late viral gene expression and viral production.\n\nIMPORTANCEKSHV ORF66, a late gene product, and vPIC are thought to contribute significantly to late gene expression during the lytic replication. However, the physiological importance of ORF66 in terms of viral replication and vPIC formation remains poorly understood. Therfore, we generated a ORF66-deficient BAC clone and evaluated its viral replication. Results showed that ORF66 played a critical role in virus production and the transcription of L genes. To our knowledge, this is the first report showing ORF66 function in virus replication using ORF66-deficient KSHV. We also clarified that ORF66 interacted with the transcription start site of K8.1 gene, a late gene. Furthermore, we identified the ORF34-binding motifs in the ORF66 C-terminus: three C-X-X-C sequences and a leucine-repeat sequence, which are highly conserved among {beta}- and {gamma}-herpesviruses. Our study provides insights into the regulatory mechanisms of not only the late gene expression of KSHV but also those of other herpesviruses.

microbiology↗

bioRxiv2018

CD36hi monocytes play immunoregulatory roles in human umbilical cord blood

The fetal and neonatal immune systems are uniquely poised to generate tolerance to self, maternal, and environmental antigens encountered in the womb and shortly after birth. The tolerogenic nature of fetal and neonatal immunity is a rising health concern with the spread of vertically transmitted viruses, such as the Zika virus. A variety of mechanisms contribute to fetal and neonatal tolerance, including a propensity to generate Foxp3+ regulatory T cells (Tregs). Here, we demonstrate that a subset of CD14+ monocytes expressing the scavenger molecule, CD36, is able to generate CD4+ and CD8+ T cells that express Foxp3 from umbilical cord blood (UCB). Monocyte-induced Foxp3+ T cells have potent suppressive functions on T cell proliferation and maintain Foxp3 expression over six weeks in vitro. Importantly, UCB-derived Foxp3+ T cells are distinguishable from adult peripheral blood (APB) CD4+CD25+ Tregs by surface antigen expression. While UCB-derived Foxp3+ T cells express prototypic Treg-associated surface antigens, such as CD25 and glucocorticoid-induced tumor necrosis factor-related receptor (GITR), only UCB-derived Foxp3+ T cells express CD26. In addition, most UCB-derived CD8+Foxp3+ T cells express CD31. Mechanistically, both APB and UCB-derived monocytes support the development of Foxp3+ T cells from naive T cells, but APB naive T cells are less efficient in expressing Foxp3 than UCB naive T cells. These data suggest that antigen presentation by CD36hi monocytes in the fetus leads to the development of a group of T cells that share some but not all phenotypes of adult thymus-derived Tregs.

immunology↗