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Ogawa, T.

Publications and source records attributed to Ogawa, T..

32 records · Page 2Linked to original sources

Relationship Between Domain and Function of the Yeast RNase T2, Rny1p, Which Mediates rRNA Degradation upon Starvation

RNase T2 is ubiquitous across diverse organisms, playing essential roles despite its simple enzymatic activity. In Saccharomyces cerevisiae, RNase T2, known as Rny1p, localizes in vacuoles and mediates rRNA degradation during autophagy of ribosomes. In this study, we elucidated novel aspects of ribosome degradation mechanisms and the function of Rny1p. First, we discovered that most ribosomes are degraded by selective autophagy, where Rsa1p is the specific receptor of ribosomes to be degraded. Complex structure prediction suggested that Rsa1p also interacts with Atg8p. Furthermore, we observed that the accumulation of rRNA in vacuoles, due to the lack of Rny1p, leads to a decrease in bulk autophagic activity. This decrease in autophagic activity may explain the inability of Rny1p-deficient strains to adapt to starvation conditions. Second, our structural prediction and biochemical analyses indicate that a C-terminal extension, characteristic in fungal RNase T2 including Rny1p, is not necessary for rRNA degradation but for anchoring to the cell wall. Together with molecular phylogenetic analysis, a species-specific role of RNase T2 conferred by the C-terminal extension is suggested.

molecular biology↗

Capsular polysaccharide-mediated protein loading onto extracellular membrane vesicles of a fish intestinal bacterium, Shewanella vesiculosa HM13

Bacterial extracellular membrane vesicles (EMVs) play various physiologically important roles mediated by cargo proteins. However, our understanding of the molecular mechanism underlying cargo loading onto EMVs is limited. In this study, we analyzed the mechanism of cargo protein loading onto EMVs from a fish intestinal Gram-negative bacterium, Shewanella vesiculosa HM13. This strain secretes EMVs carrying a major cargo protein, P49. Near the P49 gene, we found genes having homology to genes involved in protein secretion and surface polysaccharide-chain synthesis. Among them, the deletion of genes encoding homologs of a flippase involved in bacterial extracellular polysaccharide synthesis (HM3343), phosphoethanolamine transferase (HM3344), and glycerophosphodiester phosphodiesterase (HM3345) resulted in the loss of capsular polysaccharide (CPS) of EMVs. We conducted an in vitro P49 loading assay onto P49-free EMVs to examine whether P49 was loaded onto the EMVs via its interaction with the CPS of the EMVs. We found that purified P49 was loaded onto EMVs harboring CPS in vitro, whereas it was not loaded onto EMVs from the mutants lacking CPS production due to the loss of HM3343, HM3344, and HM3345. Transmission electron microscopy of EMVs loaded with P49 in vitro and in vivo showed spherical nanoparticles around the EMVs, whereas such particles were not observed for EMVs without loaded P49, implying that P49 constitutes those particles on the surface of EMVs. These results indicate that P49 is loaded onto EMVs via its interaction with the CPS of EMVs. IMPORTANCEElucidating the mechanisms of cargo loading onto bacterial extracellular membrane vesicles (EMVs) is important to understand their biogenesis and to develop their applications. Here, we show that the major cargo protein of EMVs from a fish intestinal Gram-negative bacterium, Shewanella vesiculosa HM13, is loaded onto EMVs through its interaction with capsular polysaccharide (CPS) of EMVs. Genes involved in CPS synthesis were also identified. To our knowledge, there have been no reports describing the cargo protein-loading mechanism in which CPS serves as the protein-binding scaffold for EMVs. Thus, this study represents a new mode of protein loading onto EMVs. The results deepen our understanding of cargo loading onto EMVs and would contribute to development of their applications.

microbiology↗

Two cyclic electron flows around photosystem I differentially participate in C4 photosynthesis

C4 plants assimilate CO2 more efficiently than C3 plants because of their C4 cycle that concentrates CO2. However, the C4 cycle requires additional ATP molecules, which may be supplied by the cyclic electron flow around photosystem I. One cyclic electron flow route, which depends on a chloroplast NADH dehydrogenase-like (NDH) complex, is suggested to be crucial for C4 plants despite the low activity in C3 plants. The other route depends on proton gradient regulation 5 (PGR5) and PGR5-like photosynthetic phenotype 1 (PGRL1), which is considered a major cyclic electron flow route to generate the proton gradient across the thylakoid membrane in C3 plants. However, its contribution to C4 photosynthesis is still unclear. In this study, we investigated the contribution of the two cyclic electron flow routes to the NADP-malic enzyme subtype of C4 photosynthesis in Flaveria bidentis. We observed that the suppression of the NDH-dependent route drastically delayed growth and decreased the CO2 assimilation rate to approximately 30% of the wild-type rate. On the other hand, the suppression of the PGR5/PGRL1-dependent route did not affect plant growth and resulted in a CO2 assimilation rate that was approximately 80% of the wild-type rate. Our data indicate that the NDH-dependent cyclic electron flow substantially contributes to the NADP-malic enzyme subtype of C4 photosynthesis and that the PGR5/PGRL1-dependent route cannot complement the NDH-dependent route in F. bidentis. These findings support the fact that during the C4 evolution, the photosynthetic electron flow may be optimized to provide the energy required for C4 photosynthesis.

plant biology↗

Global inflammatory response in in vitro organ cultured testes using single-cell RNA-sequencing

In vitro functional sperm production is important for understanding spermatogenesis and for the treatment of male infertility. Here, we describe similarities and differences between testis tissues in vivo and in vitro and clarify abnormalities in the early stage of in vitro spermatogenesis at single-cell resolution. While in vitro spermatogenesis progressed similarly to in vivo spermatogenesis until the early pachytene spermatocyte stage, a noticeable acute inflammatory response occurred in immune cells and non-immune testicular somatic cells immediately after cultivation. Inhibitor treatment revealed that NLRP3 inflammasome signaling is key to the inflammation. We observed damaged/dead germ cell accumulation in cultured testis, which may be due to dysfunctional phagocytosis by Sertoli cells. Our data revealed abnormal testicular milieu of in vitro cultured testes caused by tissue-wide sterile inflammation, in which the danger-associated molecular pattern-NLRP3 inflammasome axis may be a key element.

molecular biology↗

Hidden pathway for cytokine receptor activation: Structural insights into a marine sponge-derived lectin that activates the thrombopoietin receptor via recognition of the fucose moiety

N-glycan-mediated activation of the thrombopoietin receptor (MPL) under pathological conditions has been implicated in myeloproliferative neoplasms induced by mutant calreticulin, which forms an endogenous receptor-agonist complex that constitutively activates the receptor. However, the molecular basis for this mechanism has not been studied because no external agonists existed. We describe the structure and function of a marine sponge-derived MPL agonist, thrombocorticin (ThC), a homodimerized lectin with calcium-dependent fucose-binding properties. ThC-induced activation persists due to limited receptor internalization. The strong synergy between ThC and thrombopoietin suggests that ThC catalyzes the formation of receptor dimers on the cell surface. MPL is subject to sugar-mediated activation, where the kinetics differ from those of cytokines. This result suggests the presence of diverse receptor activation pathways in human thrombopoiesis. One-sentence summaryA marine sponge lectin catalyzes thrombopoietin receptor dimerization and activation, exhibiting strong synergy with thrombopoietin, and modulates internalization of the receptor.

cell biology↗

TAS-Seq: a robust and sensitive amplification method for beads-based scRNA-seq

Single-cell RNA-sequencing (scRNA-seq) is valuable for analyzing cellular heterogeneity. Cell composition accuracy is critical for analyzing cell-cell interaction networks from scRNA-seq data. We developed terminator-assisted solid-phase cDNA amplification and sequencing (TAS-Seq) for scRNA-seq based on a terminator, terminal transferase, and nanowell/bead-based scRNA-seq platform; TAS-Seq showed high tolerance to variations in the terminal transferase reaction, which complicate the handling of existing terminal transferase-based scRNA-seq methods. In murine and human lung samples, TAS-Seq yielded scRNA-seq data that were highly correlated with flow-cytometric data, showing higher gene-detection sensitivity and more robust detection of important cell-cell interactions and expression of growth factors/interleukins in cell subsets than 10X Chromium v2 and Smart-seq2. Expanding TAS-Seq application will improve understanding and atlas construction of lung biology at the single-cell level.

genomics↗

EEG-based neurofeedback with network components extraction: data-driven approach by multilayer ICA extension and simultaneous EEG-fMRI measurements

Several studies have reported advanced treatments for depressive symptoms, such as real-time neurofeedback (NF) with functional MRI (fMRI) and/or electroencephalogram (EEG). NF focusing on a regularization of brain activity associated with the amygdala or functional connectivity (FC) between the executive control network (ECN) and default mode network (DMN) has been applied to reduce depressive symptoms. However, it is practically difficult to install the fMRI-NF system and to consistently provide treatment, because of high cost. Additionally, no practical signal processing techniques have been developed extracting FC-related features from EEG signals, particularly when no physical forward models are available. In this regard, stacked pooling and linear components estimation (SPLICE), recently proposed as a multilayer extension of independent component analysis (ICA) and related independent subspace analysis (ISA), can be a promising alternative. The resting-state EEG network features can be correlated with fMRI network activity corresponding to the DMN or ECN. This may enable the modulation of the target FC-related features in EEG-based NF. In this study, we developed a real-time EEG NF system for improving depressive symptoms by using the SPLICE. Utilizing information from the fMRI biomarkers, we evaluated our paradigm for effectiveness with regard to upregulation of the dorsolateral prefrontal cortex /middle frontal gyrus or downregulation of the precuneus/posterior cingulate cortex. We conducted an NF experiment in participants with subclinical depression; the participants were divided into the NF group (n=8) and the sham group (n=9). We found a significant reduction and a large effect size in the rumination response scale (RRS) score (reflection) in the NF group, compared to the sham group. However, we did not find a significant relationship between the training score and difference in symptoms. This suggests that increased controllability of the EEG signals did not directly reduce the RRS reflection score. This could be due to various reasons such as improper feature extraction, individual differences, and the targeted brain regions. In this paper, we also discuss the possible ways to modify our NF protocol including the design of the experiment, sample size, and online processing. We then discuss way to improve the NF training, based on our results.

neuroscience↗

Localization of planteose hydrolysis during seed germination of Orobanche minor

Root parasitic weeds of the Orobanchaceae, such as witchweeds (Striga spp.) and broomrapes (Orobanche and Phelipanche spp.), cause serious losses in agriculture worldwide. No practical method to control these parasitic weeds has been developed to date. Understanding the characteristic physiological processes in the life cycles of root parasitic weeds is particularly important to identify specific targets for growth modulators. In our previous study, planteose metabolism was revealed to be activated soon after the perception of strigolactones in germinating seeds of O. minor. Nojirimycin inhibited planteose metabolism and impeded seed germination of O. minor, indicating that planteose metabolism is a possible target for root parasitic weed control. In the present study, we investigated the distribution of planteose in dry seeds of O. minor by matrix-assisted laser desorption/ionization-mass spectrometry imaging. Planteose was detected in tissues surrounding--but not within--the embryo, supporting its suggested role as a storage carbohydrate. Biochemical assays and molecular characterization of an -galactosidase family member, OmAGAL2, indicated the enzyme is involved in planteose hydrolysis in the apoplast around the embryo after the perception of strigolactones to provide the embryo with essential hexoses for germination. These results indicated that OmAGAL2 is a potential molecular target for root parasitic weed control. HighlightPlanteose accumulated in tissues surrounding the embryo in Orobanche minor dry seeds and was indicated to be hydrolyzed in the apoplast around the embryo by -galactosidase during germination.

plant biology↗

Combining an alarmin HMGN1 peptide with PD-L1 blockade facilitates stem-like CD8+ T cell expansion and results in robust antitumor effects

BackgroundThe expansion of intratumoral stem-like CD8+ T (Tstem) cells provides a potential approach to improving the therapeutic efficacy of immune checkpoint blockade (ICB). Thus, here we demonstrate a strategy to facilitate Tstem cell expansion by combining an alarmin high-mobility group nucleosome binding domain 1 (HMGN1) peptide with programmed death-ligand 1 (PD-L1) blockade. MethodsThe antitumor effects of HMGN1, anti-PD-L1 antibody, and their combined treatment were monitored in the B16F10, LLC, Colon26, or the EO771 tumor-bearing mice. The comprehensive immunologic analyses, such as high-dimensional flow cytometry, transcriptome analysis, and single-cell RNA sequencing, were used to investigate the cellular and molecular mechanisms of antitumor immune responses after treatments. ResultsThe HMGN1 peptide synergizes with PD-L1 blockade in augmenting the number of mature DCs enriched in immunoregulatory molecules (mregDCs) in tumors, and enhancing their MHC class I antigen-presenting program, which is correlated with the expansion of intratumoral Tstem cells, specifically promoting the Tstem cells but restricting terminally exhausted CD8+ T (Tex) cells, owing to the regulatory molecules expressed on mregDCs. ConclusionOur results indicate that HMGN1 peptide serves as an immunoadjuvant to promote effective anti-PD-L1 immunotherapy and implicate that mregDCs play a role beyond facilitating Tstem cell expansion.

immunology↗

Asymmetric effective connectivity within frontoparietal motor network underlying motor imagery and motor execution

Both imagery and execution of motor controls consist of interactions within a neuronal network, including frontal motor-related regions and posterior parietal regions. To reveal neural representation in the frontoparietal motor network, two approaches have been proposed thus far: one is decoding of actions/modes related to motor control from the spatial pattern of brain activity; another is to estimate directed functional connectivity, which means a directed association between two brain regions within motor regions. However, directed connectivity among multiple regions of the motor network during motor imagery (MI) or motor execution (ME) has not been investigated. Here, we attempted to characterize the directed functional connectivity within the frontoparietal motor-related networks between the MI and ME conditions. We developed a delayed sequential movement and imagery task to evoke brain activity associated with data under ME and MI via functional magnetic resonance imaging scanning. We applied a causal discovery approach, linear non-Gaussian acyclic causal model, to identify directed functional connectivity among the frontoparietal motor-related brain regions for each condition. We demonstrated higher directed functional connectivity from the contralateral dorsal premotor cortex (dPMC) to the primary motor cortex (M1) in ME than in MI. We mainly identified significant direct effects of the dPMC and ventral premotor cortex (vPMC) to the parietal regions. In particular, connectivity from the dPMC to the superior parietal lobule in the same hemisphere showed significant positive effects across all conditions. Contrastingly, interlateral connectivities from the vPMC to the superior parietal lobule showed significantly negative effects across all conditions. Finally, we found positive effects from A1 to M1 in the same hemisphere, such as the audio-motor pathway. These results indicated that the sources of motor command originated in d/vPMC influenced M1 and parietal regions as achieving ME and MI. Additionally, sequential sounds may functionally facilitate temporal motor processes.

neuroscience↗

Multi-sample Full-length Transcriptome Analysis of 22 Breast Cancer Clinical Specimens with Long-Read Sequencing

Although transcriptome alteration is considered as one of the essential drivers of carcinogenesis, conventional short-read RNAseq technology has limited researchers from directly exploring full-length transcripts, only focusing on individual splice sites. We developed a pipeline for Multi-Sample long-read Transcriptome Assembly, MuSTA, and showed through simulations that it enables construction of transcriptome from the transcripts expressed in target samples and more accurate evaluation of transcript usage. We applied it to 22 breast cancer clinical specimens to successfully acquire cohort-wide full-length transcriptome from long-read RNAseq data. By comparing isoform existence and expression between estrogen receptor positive and triple-negative subtypes, we obtained a comprehensive set of subtype-specific isoforms and differentially used isoforms which consisted of both known and unannotated isoforms. We have also found that exon-intron structure of fusion transcripts tends to depend on their genomic regions, and have found three-piece fusion transcripts that were transcribed from complex structural rearrangements. For example, a three-piece fusion transcript resulted in aberrant expression of an endogenous retroviral gene, ERVFRD-1, which is normally expressed exclusively in placenta and supposed to protect fetus from maternal rejection, and expression of which were increased in several TCGA samples with ERVFRD-1 fusions. Our analyses of real clinical specimens and simulated data provide direct evidence that full-length transcript sequencing in multiple samples can add to our understanding of cancer biology and genomics in general.

genomics↗

Recovery of cryo-injured rabbit urethras by biofabricated C-shaped adipose-derived mesenchymal cell structures

Urethral tissue damage can cause stress urinary incontinence or urinary retention, and there are few long-term, effective treatments. For structural and functional recovery of urethral tissues, we designed and constructed C-shaped structures composed of adipose-derived mesenchymal cells (AMCs) by using a three-dimensional (3D)-bioprinter system, which could be transplanted in damaged urethras without obstructing the lumen. We determined if transplantation of the biofabricated structures could reconstruct the urethral tissues. AMCs were harvested from rabbits, cultured and labeled with PKH26 to form spheroids. The spheroids were assembled on a custom-designed C-shaped support by a 3D bioprinter. Urethras of rabbits were cryo-injured by spraying with liquid nitrogen for 20 seconds, incised and biofabricated structure was autologously transplanted. Control rabbits were treated similarly but without transplantation structure. Two and four weeks after surgery, the control urethras were partially constricted; however, the structure-transplanted urethras were patent. The AMCs within the structures differentiated into skeletal muscle, smooth muscle, nerve, or endothelial cells. Some cells contained growth factors and cytokines. Therefore, biofabricated C-shaped AMC structures have potential to be an effective treatment for urethral recovery. Summary StatementThree-dimensional bioprinter was used to biofabricate novel C-shaped structures composed of adipose-derived mesenchymal cells (AMCs) and have the potential to be an effective treatment for urethral recovery.

bioengineering↗

Unleashing floret fertility by a mutated homeobox gene improved grain yield during wheat evolution under domestication

Floret fertility is a key trait to determine the number of grains per inflorescence in cereals. During wheat (Triticum sp.) evolution, floret fertility has been increased and current bread wheat (T. aestivum L.) produces three to five grains per spikelet; however, little is known about the genetic basis controlling floret fertility. Here we identify the quantitative trait locus Grain Number Increase 1 (GNI1), encoding a homeodomain leucine zipper class I (HD-Zip I) transcription factor. GNI1 evolved in the Triticeae through gene duplication and functionalization. GNI1 was predominantly expressed in the most apical floret primordia and parts of the rachilla, suggesting that GNI1 inhibits rachilla growth and development. GNI1 expression decreased during wheat evolution, and as a consequence, more fertile florets and grains per spikelet are being produced. Genetic analysis revealed that the reduced-function allele of GNI1-A contributes to increase the number of fertile florets per spikelet. The knockdown of GNI1 in transgenic hexaploid wheat improved fertile floret and grain number. Furthermore, wheat plants carrying the impaired allele increased grain yield under field conditions. Our findings illuminate that gene duplication and functionalization generated evolutionary novelty for floret fertility (i.e. reducing floral numbers) while the mutations towards increased grain production were under selection during wheat evolution under domestication.\n\nSignificance StatementGrain number is a fundamental trait for cereal grain yield; but its underlying genetic basis is mainly unknown in wheat. Here we show for the first time a direct link between increased floret fertility, higher grain number per spike and higher plot-yields of wheat in the field. We have identified GNI1 gene encoding an HD-Zip I transcription factor responsible for increased floret fertility. The wild type allele imposes an inhibitory role specifically during rachilla development, indicating that expression of this protein actively shuts-down grain yield potential; whereas, the reduced-function allele enables more florets and grains to be produced. GNI1 evolved through gene duplication in Triticeae and its mutations were under parallel human selection during wheat and barley evolution under domestication.

plant biology↗

Cortical control of locomotor muscle activity through muscle synergies in humans: a neural decoding study

Walking movements are orchestrated by the activation of a large number of muscles. The control of numerous muscles during walking is believed to be simplified by flexible activation of groups of muscles called muscle synergies. Although significant corticomuscular connectivity during walking has been reported, the level at which the cortex controls locomotor muscle activity (i.e., muscle synergy or individual muscle level) remains unclear. Here, we examined cortical involvement in muscle control during walking by brain decoding of the activation of muscle synergies and individual muscles from electroencephalographic (EEG) signals using linear decoder models. First, we demonstrated that activation of locomotor muscle synergies was decoded from slow cortical waves with significant accuracy. In addition, we found that decoding accuracy for muscle synergy activation was greater than that for individual muscle activation and that decoding of individual muscle activation was based on muscle synergy-related cortical information. Taken together, these results provide indirect evidence that the cerebral cortex hierarchically controls multiple muscles through a few muscle synergies during walking. Our findings extend the current understanding of the role of the cortex in muscular control during walking and could accelerate the development of effective brain-machine interfaces for people with locomotor disabilities.

neuroscience↗