Biology subjects

Ohnishi, Y.

Publications and source records attributed to Ohnishi, Y..

10 recordsLinked to original sources

bioRxiv2026

Body-wrapping anterior flagella drive ultrafast swimming in bacterial zoospores

Most bacteria swim at [~]10 body lengths per second (bl s-1), yet some microorganisms move far faster, and the physical design principles enabling such extreme motility remain poorly understood. Here we uncover an ultrafast swimming strategy in Actinoplanes missouriensis zoospores, which reach up to 560 m s-1 ([~]500 bl s-1), the fastest relative swimmer reported for any microorganism. High-speed imaging shows that propulsion is driven by an anterior, body-wrapping bundle of short flagella that rotate synchronously as a right-handed helix at [~]150 Hz. This architecture generates high thrust without extreme motor speeds and contrasts with the canonical rear-bundle paradigm of bacterial swimming. Microfluidic assays further demonstrate that this propulsion mode enhances dispersal across the flow interface, providing a mechanism for rapid colonization during the transient motile phase of the life cycle. Furthermore, chemotaxis-dependent reorientation is observed, suggesting that zoospore swimming can be directionally regulated. These results identify a new locomotion principle in which supramolecular organization of multiple flagella, rather than motor speed alone, sets the upper limits of bacterial swimming and offers inspiration for the design of high-performance microswimmers.

microbiology↗

bioRxiv2025

Promotion of seed germination in Echinochloa crus-galli with atmospheric air plasma

Plasma technology has attracted increasing attention for potential agricultural applications, and it has been investigated for decades particularly for its ability to promote seed germination. However, most studies have focused on crop species, and little is known about its potential use for weed management. In this study, we examined whether air plasma influences the germination of Echinochloa crus-galli, a major paddy field weed. When seeds were cultivated without stratification, direct irradiation with air plasma did not enhance germination. In contrast, once dormancy was partially released by cold stratification, plasma treatment significantly promoted germination. Interestingly, the effect disappeared after prolonged chilling, when dormancy was completely released. These results suggest that plasma promotes germination by acting on the dormancy-breaking process. In addition, plasma-activated water (PAW) also stimulated germination, although its effect was weaker than that of gaseous plasma. Overall, this study demonstrates that air plasma can promote the germination of grass weed seeds, providing a potential strategy for weed control by inducing germination prior to crop seeding.

plant biology↗

bioRxiv2025

Oxidation of ΔFOSB at Cys172 Controls Hippocampal Gene Targets and Learning

Imbalance of reduction/oxidation (redox) in the brain is associated with numerous diseases including Alzheimers disease (AD), substance abuse disorders, and stroke. Moreover, cognitive decline can be caused by neuronal dysfunction that precedes cell death, and this dysfunction is in part produced by altered gene expression. However, the mechanisms by which redox state controls gene expression in neurons are not well understood. {Delta}FOSB is a neuronally enriched transcription factor critical for orchestrating gene expression underlying memory, mood, and motivated behaviors. It is dysregulated in many conditions including AD. We showed recently that {Delta}FOSB forms a redox-sensitive disulfide bond between cysteine 172 (C172) of {Delta}FOSB and C279 of its preferred binding partner JUND. This bond works as a redox switch to control DNA-binding, based on studies of recombinant proteins in vitro. Here, we show that this redox control of {Delta}FOSB function in vitro is conserved in vivo. We show that {Delta}FOSB C172 forms a redox-sensitive disulfide bond with JUND that regulates the stability of this AP1-transcription factor complex and its binding to DNA in cells. We also validate the formation of {Delta}FOSB-containing complexes held together via disulfide bonds in mouse brain in vivo. We show that exogenous oxidative stress reduces {Delta}FOSB binding to gene targets in mouse brain and that Fosb C172S knock-in mice, which lack a functional {Delta}FOSB redox switch, are insensitive to this oxidation-dependent reduction in target gene binding, demonstrating that {Delta}FOSB is regulated by a redox switch that modulates binding to target genes in the hippocampus. Finally, we demonstrate that FosB C172S knock-in mice are less sensitive to cognitive dysfunction induced by oxidative stress. This evidence supports {Delta}FOSB as an important mediator of oxidative stress-driven changes in gene expression and cognition and implicates {Delta}FOSB as a possible therapeutic target for diseases associated with oxidative stress in the brain, including AD.

neuroscience↗

bioRxiv2024

Systemic identification of functionally conserved lncRNA metabolic regulators in human and mouse livers

BACKGROUND & AIMSUnlike protein-coding genes, the majority of human long non-coding RNAs (lncRNAs) lack conservation based on their sequences, posing a challenge for investigating their role in a pathophysiological context for clinical translation. This study explores the hypothesis that non-conserved lncRNAs in human and mouse livers may share similar metabolic functions, giving rise to functionally conserved lncRNA metabolic regulators (fcLMRs). METHODSWe developed a sequence-independent strategy to select putative fcLMRs, and performed extensive analysis to determine the functional similarities of putative human and mouse LMR pairs (h/mLMRs). RESULTSWe found that several pairs of putative fcLMRs share similar functions in regulating gene expression. We further demonstrated that a pair of fcLMRs, h/mLMR1, robustly regulated triglyceride levels by modulating the expression of a similar set of lipogenic genes. Mechanistically, h/mLMR1 binds to PABPC1, a regulator of protein translation, via short motifs on either lncRNA with divergent sequences but similar structures. This interaction inhibits protein translation, activating an amino acid-mTOR-SREBP1 axis to regulate lipogenic gene expression. Intriguingly, PABPC1-binding motifs on each lncRNA fully rescued the functions of their corresponding LMRs in the opposite species. Given the elevated expression of h/mLMR1 in humans and mice with hepatic steatosis, the PABPC1-binding motif on hLMR1 emerges as a potential non-conserved human drug target whose functions can be fully validated in a physiologically relevant setting before clinical studies. CONCLUSIONSOur study supports that fcLMRs represent a novel and prevalent biological phenomenon, and deep phenotyping of genetic mLMR mouse models constitutes a powerful approach to understand the pathophysiological role of lncRNAs in the human liver.

physiology↗

bioRxiv2023

HERC3 E3 ligase provides an ERAD branch eliminating select membrane proteins

Aberrant proteins located in the endoplasmic reticulum (ER) undergo rapid ubiquitination by multiple ubiquitin (Ub) E3 ligases and are retrotranslocated to the cytosol as part of the ER-associated degradation (ERAD). Despite several ERAD branches involving different Ub E3 ligases, each with distinct substrate specificity, the molecular machinery responsible for these ERAD branches in mammalian cells remains not fully understood. In this study, we have discovered a cytosolic Ub ligase called HERC3, which fulfills a distinct role in facilitating the ERAD of select polytopic membrane proteins. Using a series of multiplex knockdown/knockout experiments, we have demonstrated that HERC3 functions independently of the ER-embedded ubiquitin ligases RNF5 and RNF185 (RNF5/185) to facilitate the ubiquitination, retrotranslocation, and ERAD of misfolded CFTR. Furthermore, HERC3 collaborates with RNF5/185 to enhance the association of UBQLN proteins, thereby augmenting the retrotranslocation and ERAD of misfolded CFTR. While RNF5/185 participates in the ERAD process of both misfolded ABCB1 and CFTR, HERC3 specifically promotes the ERAD of CFTR, likely due to its ability to interact with the less hydrophobic membrane-spanning domains of CFTR. HERC3 may detect exposed transmembrane domains on the cytoplasmic surface of the ER, thereby facilitating the recruitment of UBQLN and subsequently accelerating the ERAD of select polytopic membrane proteins.

cell biology↗

bioRxiv2023

Single transcript-level metabolic responsive landscape of human liver transcriptome

Direct knowledge of gene regulation in human liver by metabolic stimuli could fundamentally advance our understanding of metabolic physiology. This information, however, is largely unknown, and this void is deeply rooted in a paradox that is caused by the inaccessibility of human liver to treatments and the insufficient annotation of liver transcriptome. Recent advances have uncovered immense complexity of transcriptome, i.e., multiple transcripts produced from one gene and extensive RNA modifications, which are all highly regulated and often condition-dependent. Establishing an inclusive annotation to study liver transcriptome dynamics thus requires human liver samples of diverse conditions, which are paradoxically unavailable due to the inaccessibility of human liver to treatments. In this work, we addressed these challenges by coupling an isogenic humanized mouse model with Nanopore single-molecule direct RNA sequencing (DRS). We first generated mice that carried humanized livers of identical genetic background, which were equivalent to clones of a single human liver, and then subjected the mice to representative metabolic treatments. We then analyzed the humanized livers with Nanopore DRS, which directly reads full-length native RNAs to determine the expression level, m6A modification and poly(A) tail length of all RNA transcript isoforms. Thus, our system allows for constructing a de novo annotation of human liver transcriptomes reflecting metabolic responses and studying transcriptome dynamics in conjunction. Our analysis uncovered a vast number of novel genes and transcripts that have not been previously reported. Our transcript-level analysis of human liver transcriptomes also identified a multitude of regulated metabolic pathways that were otherwise invisible using conventional short read RNA-seq. We also revealed for the first time the dynamic changes in m6A and poly(A) tail length of human liver transcripts many of which are transcribed from key metabolic genes. Furthermore, we performed comparative analyses of gene regulation between human and mouse and between two individuals using the liver-specific humanized mice. This revealed that transcriptome dynamics are highly species- and genetic background-dependent, which may only be faithfully studied in a humanized system that entails clones of the same human liver. Hence our work revealed a complex metabolic responsive landscape of human liver transcriptome and also provided a framework to understand transcriptome dynamics of human liver in response to physiologically relevant metabolic stimuli.

systems biology↗

bioRxiv2022

Engineering the substrate specificity of toluene degrading enzyme XylM using biosensor XylS and machine learning

Enzyme engineering using machine learning has been developed in recent years. However, to obtain a large amount of data on enzyme activities for training data, it is necessary to develop a high-throughput and accurate method for evaluating enzyme activities. Here, we examined whether a biosensor-based enzyme engineering method can be applied to machine learning. As a model experiment, we aimed to modify the substrate specificity of XylM, a rate-determining enzyme in a multistep oxidation reaction catalyzed by XylMABC in Pseudomonas putida. XylMABC naturally converts toluene and xylene to benzoic acid and toluic acid, respectively. We aimed to engineer XylM to improve its conversion efficiency to a non-native substrate, 2,6-xylenol. Wild-type XylMABC slightly converted 2,6-xylenol to 3-methylsalicylic acid, which is the ligand of the transcriptional regulator XylS in P. putida. By locating a fluorescent protein gene under the control of the Pm promoter to which XylS binds, a XylS-producing Escherichia coli strain showed higher fluorescence intensity in a 3-methylsalicylic acid concentration-dependent manner. We evaluated the 3-methylsalicylic acid productivity of XylM variants using the fluorescence intensity of the sensor strain as an indicator. The obtained data provided the training data for machine learning for the directed evolution of XylM. Two cycles of machine learning-assisted directed evolution resulted in the acquisition of XylM-D140E-V144K-F243L-N244S with 15 times higher productivity than wild-type XylM. These results demonstrate that an indirect enzyme activity evaluation method using biosensors is sufficiently quantitative and high-throughput to be used as training data for machine learning. The findings expand the versatility of machine learning in enzyme engineering.

bioengineering↗

bioRxiv2022

Mechanism-based crosslinking probes capture E. coli ketosynthase FabB in conformationally-distinct catalytic states.

Ketosynthases (KS) catalyse essential carbon-carbon bond forming reactions in fatty acid biosynthesis using a two-step, ping-pong reaction mechanism. In E. coli, there are two homodimeric elongating KSs, FabB and FabF, both of which possess overlapping substrate selectivity. However, FabB is essential for the biosynthesis of unsaturated fatty acids (UFAs) required for cell survival in the absence of exogenous UFAs. Additionally, FabB has reduced activity towards substrates longer than 14 carbons, whereas FabF efficiently catalyses the elongation of saturated C14 and unsaturated C16:1 acyl-acyl carrier protein (ACP) complexes. In this study, we solved two crosslinked crystal structures of FabB in complex with ACPs functionalized with long-chain fatty acid crosslinking probes that approximate catalytic steps. Both homodimeric structures possess asymmetric substrate binding pockets, suggestive of cooperative relationships between the two FabB monomers when engaged with C14 and C16 acyl chains. In addition, these structures capture an unusual rotamer of the active site gating residue, F392, potentially representative of the catalytic state prior to substrate release. These structures demonstrate the utility of mechanism-based crosslinking methods to capture and elucidate at near atomic resolution conformational transitions accompanying KS-mediated catalysis. SynopsisCrystal structures of KS-ACP crosslinked complex elucidate chain length preference and substrate processing mechanism of E. coli FabB.

biochemistry↗

bioRxiv2020

Overlapping and non-overlapping roles of the class-I histone deacetylase-1 corepressors, LET-418, SIN-3, and SPR-1 in Caenorhabditis elegans embryonic development

Histone deacetylases (HDACs) are divided into four classes. Class-I HDAC, HDAC-1 forms three types of complexes, namely the Nucleosome Remodeling Deacetylase complex, the Sin3 complex, and the CoREST complex, with specific corepressor component Mi2/CHD-3, Sin3, and RCOR1 in human, respectively. The functions of these HDAC-1 complexes are regulated by their corepressors, however, their exact mechanistic roles in several biological processes remain unexplored, such as in embryonic development. Here, we report that each of the corepressors, LET-418, SIN-3, and SPR-1, the homologous of Mi2, Sin3, and RCOR1, respectively, were expressed throughout Caenorhabditis elegans embryonic development and served essential roles in the process. Moreover, genetic analysis suggested that three pathways (i.e., LET-418- SIN-3-SPR-1, SIN-3-SPR-1, and LET-418) participated in embryonic development. Our terminal-phenotype observations of single mutants of each corepressor implied that LET-418, SIN-3, and SPR-1 played similar roles in promoting advancement to the middle and late embryonic stages. Genome-wide comparative-transcriptome analysis indicated that 47.5% and 42.3% of genes were commonly increased and decreased in sin-3 and spr-1 mutants, respectively. These results suggest that among the three pathways studied, the SIN-3-SPR-1 pathway mainly serves to regulate embryonic development. Comparative-Gene Ontology analysis indicated that these three pathways played overlapping and distinct roles in regulating C. elegans embryonic development.

developmental biology↗

bioRxiv2020

Multilevel integrative transcriptome analyses in humans and humanized mice define in vivo human lncRNA metabolic regulators

A growing number of long non-coding RNAs (lncRNAs) have emerged as vital metabolic regulators in research animals suggesting that lncRNAs could also play an important role in human metabolism. However, most human lncRNAs are non-conserved, vastly limiting our ability to identify human lncRNA metabolic regulators (hLMRs). As the sequence-function relation of lncRNAs has yet to be established, the identification of lncRNA metabolic regulators in animals often relies on their regulations by experimental metabolic conditions. But it is very challenging to apply this strategy to human lncRNAs because well-controlled human data are much limited in scope and often confounded by genetic heterogeneity. In this study, we establish an efficient pipeline to identify putative hLMRs that are metabolically sensitive, disease-relevant, and population applicable. We first progressively processed human transcriptome data to select human liver lncRNAs that exhibit highly dynamic expression in the general population, show differential expression in a metabolic disease population, and response to dietary intervention in a small disease cohort. We then experimentally demonstrated the responsiveness of selected hepatic lncRNAs to defined metabolic milieus in a liver-specific humanized mouse model. Furthermore, by extracting a concise list of protein-coding genes that are persistently correlated with lncRNAs in general and metabolic disease populations, we predicted the specific function for each hLMR. Using gain- and loss-of-function approaches in humanized mice as well as ectopic expression in conventional mice, we were able to validate the regulatory role of one non-conserved hLMR in cholesterol metabolism. Mechanistically, this hLMR binds to an RNA-binding protein, PTBP1, to modulate the transcription of cholesterol synthesis genes. In summary, our study provides a pipeline to overcome the variabilities intrinsic to human data to enable the efficient identification and functional definition of hLMRs. The combination of this bioinformatic framework and humanized murine model will enable broader systematic investigation of the physiological role of disease-relevant human lncRNAs in metabolic homeostasis.

physiology↗