Biology subjects

Ohashi, A.

Publications and source records attributed to Ohashi, A..

7 recordsLinked to original sources

bioRxiv2025

High-Density Microdroplet Cultivation Reveals the Essential Role of Microbial Interactions in the Growth of Environmental Microbes

The significance of microbial interactions in nature is not well understood because of the limitations of techniques to assess microbial growth at the single-cell level under conditions of high cell density (more than 107 cells/mL). To address this limitation for further solution of microbial uncultivability, we developed a Gel Microdroplet Aggregate in Oil (GMD-agg) cultivation method. This method provides a high inoculum cell density (>108 cells/mL) but maintains pure cultures of each growth unit. Millions of hydrogel particles (10-30 {micro}m in diameter) entrapping single cells with medium softly aggregated in oil (GMD-oil cultivation) is the key structure of this method. In this study, to assess the effect of microbial interactions on the cultivation of environmental microorganisms, soil and activated sludge samples were cultured, and colony formation and diversity during cultivation in GMDs were investigated. Results showed that the cultivation efficiency of GMD-agg was approximately 10 times higher than that of the method in which microbial interactions were canceled. In addition, the growth of taxonomies containing many uncultured microorganisms, such as Verrucomicrobia, Planctomycetia, Acidobacteria, and Vicinimibacteria, was observed. Furthermore, when isolated strains were co-cultured at high cell densities with either inter-or intra-species, we observed a higher cultivation efficiency than when microbial interactions were canceled. Rebuilding growth-promoting microbial interactions using isolated strains demonstrated that microbial interactions positively influence microbial growth. These findings indicate the effectiveness of this cultivation method, reveal the crucial role of microbial interactions in the growth of environmental microorganisms, and contribute to solving the issue of microbial uncultivability. IMPORTANCEThe overwhelming majority of environmental microbes remain uncultured, limiting our understanding of their physiology and ecological roles. Although microbial interactions have been predicted as one of the key factors for the growth of uncultivable microbial types, the effect of these interactions on cultivability remains poorly understood. In this study, we developed a new droplet-based co-cultivation approach that promotes microbial interactions while maintaining pure cultures and enables growth tracking at the single-cell level. This method significantly improved cultivability (approximately 10 times), including the growth of taxa that are difficult to cultivate. Direct observation of microbial growth in the community using this method clearly demonstrated that microbial interactions are essential for the growth of diverse microbial types. These findings underscore the importance of microbial interactions in cultivation and offer a basis for radically expanding microbial bioresources, manipulating microbial communities, and exploring previously unrecognized microbial interactions.

microbiology↗

bioRxiv2025

The integration mechanism of the four positional cues--anterior, posterior, dorsal, and ventral--in axolotl limb regeneration

Axolotls (Ambystoma mexicanum) exhibit a remarkable ability to regenerate limbs after amputation. Classical experiments have suggested that contact between cells derived from distinct orientations--dorsal, ventral, anterior, and posterior--within the regenerating blastema is necessary for accurate limb pattern formation. However, the molecular basis for this requirement has remained largely unknown. Here, we demonstrate that both dorsal and ventral tissues are required for limb formation via induction of Shh expression, which plays a crucial role in limb patterning. Using the accessory limb model (ALM), we induced position-specific blastemas lacking cells derived from a single orientation (anterior, posterior, dorsal, or ventral). Patterned limb formation occurred only in blastemas containing both dorsal- and ventral-derived cells. We further observed that Shh expression requires dorsoventral contact within a blastema, highlighting the necessity of dorsoventral contact for inducing Shh expression. In addition, we identified WNT10B and FGF2 as dorsal- and ventral-mediated signals, respectively, that create the inductive environment for Shh expression. Our findings clarify the role of dorsal and ventral cells in inducing Shh, a mechanism that has rarely been studied in the context of limb regeneration and pattern formation. This model provides new insights into how cells with different positional identities drive the regeneration process.

developmental biology↗

bioRxiv2024

Common marmosets use body posture as multi-functional signal to solicit, maintain, and modify social play

Social play is a highly active social interaction, characterized by rapid exchanges of various behaviors with multiple partners. Many primates use bodily expressions during social play, yet the potential signaling function of these expressions remains unclear. This study investigated whether common marmosets (Callithrix jacchus) use body posture as signal to regulate play. We recorded play within three captive common marmoset family groups using multiple cameras simultaneously to capture the fast-paced and high frequency behaviors. Three distinct signals (i.e. supine, hide, stalk) and six distinct play types (i.e. wrestle, chase, pounce, touch, catch, pull) were identified. We used a multi-state time-to-event model to analyze the sequences of play, including short-and long-term transitions between different states (i.e. signal, play, or rest/nothing). Our data-driven approach accounted for uncertainty in the duration of play bouts, using probabilistic classification rather than arbitrary bout thresholds. The resulting classifications allowed us to assess the social function of signals by comparing play behavior to a resting state baseline. We found that the presence of a signal: (1) increases the probability to play; (2) extends the duration of play; (3) leads to more diverse play; and (4) increases the probability of play fighting. Marmosets also show turn-taking of signaling and initiating subsequential play. These results show that marmosets use postures as communicative signals to initiate and change play dynamics, and thereby establish a mutual understanding of the joint action. The two-fold contribution of this study concerns novel analytical methods and a deeper conceptual understanding of primate communication. Play and its signals are important elements in the evolution of language, and our research contributes to its further understanding.

animal behavior and cognition↗

bioRxiv2023

Unique episymbiotic relationship between Gracilibacteria and Zoogloea in activated sludge flocs in a municipal wastewater treatment plant

Among the various bacteria present in activated sludge, uncultivated Patescibacteria (also known as the Candidate Phyla Radiation/CPR superphylum) are ubiquitous at the class or phylum level. Patescibacteria have a highly restricted metabolic capacity and are thought to be episymbiotic/endosymbiotic or predatory. However, only a limited number of Patescibacteria and their hosts have been identified. Therefore, many Patescibacteria have not been (co-)cultured and identified by fluorescence in situ hybridization (FISH) or electron microscopy. Little is known about the morphology, metabolic potential, and hosts of Gracilibacteria (formerly GN02 or BD1-5) which belong to Patescibacteria. In our previous study, we confirmed the presence of Gracilibacteria in activated sludge and successfully recovered its high-quality genome. In this study, we designed new probes to visualize members of Gracilibacteria in activated sludge and identified its host using FISH. The FISH observations revealed that Gracilibacteria, which formed loosely associated clusters, were located within dense clusters of Zoogloea, which were dominant in the activated sludge. The metagenome-assembled genomes (MAGs) of Zoogloea possessed genes related to extracellular polymeric substance (EPS) biosynthesis, floc formation, and nutrient removal, including a polyhydroxyalkanoate (PHA) accumulation pathway. The MAGs of Gracilibacteria possessed genes associated with type IV pili, competence protein EC (ComEC), and PHA degradation, which suggests that they have a Zoogloea-dependent lifestyle in activated sludge flocs. These findings clearly indicate a new symbiotic relationship between Gracilibacteria and Zoogloea, and to the best of our knowledge, this is the first study to show this interaction.

microbiology↗

bioRxiv2021

Fluctuation of cellular differentiation in limb regeneration is regulated by Pde4b in urodele amphibians

Urodele amphibians, Pleurodeles waltl and Ambystoma mexicanum, have organ-level regeneration capability, such as limb regeneration. Multipotent cells are induced by an endogenous mechanism in amphibian limb regeneration. It is well known that dermal fibroblasts receive regenerative signals and turn into multipotent cells, called blastema cells. However, the induction mechanism of the blastema cells from matured dermal cells was unknown. We previously found that BMP2, FGF2, and FGF8 (B2FF) could play sufficient roles in blastema induction in urodele amphibians. Here, we show that B2FF treatment can induce dermis-derived cells that can participate in multiple cell lineage in limb regeneration. We first established a newt dermis-derived cell line and confirmed that B2FF treatment on the newt cells provided plasticity in cellular differentiation in limb regeneration. Interspecies comparative analysis clarified that Pde4b upregulation by B2FF specifically took place in the newt cells. Blocking PDE4B signaling by Rolipram suppressed dermis-to-cartilage transformation and the mosaic knockout animals showed consistent results. Our results are a valuable insight into how dermal fibroblasts acquire multipotency during the early phase of limb regeneration via an endogenous program in amphibian limb regeneration.

developmental biology↗

bioRxiv2019

Long read sequencing reveals a novel class of structural aberrations in cancers: identification and characterization of cancerous local amplifications

Here we report identification of a new class of local structural aberrations in lung cancers. The whole-genome sequencing of cell lines using a long read sequencer, PromethION, demonstrated that typical cancerous mutations, such as point mutations, large deletions and gene fusions can be detected also on this platform. Unexpectedly, we revealed unique structural aberrations consisting of complex combinations of local duplications, inversions and micro deletions. We further analyzed and found that these mutations also occur in vivo, even in key cancer-related genes. These mutations may elucidate the molecular etiology of patients for whom causative cancerous events and therapeutic strategies remain elusive.

genomics↗

bioRxiv2019

Cultivating in situ triggers growth of previously uncultivated microorganisms via a growth initiation factor in nature

Most microorganisms resist cultivation under standard laboratory conditions. On the other hand, to cultivate microbes in a membrane-bound device incubated in nature (in situ cultivation) is an effective approach. In the present study, we applied in situ cultivation to isolate diverse previously uncultivated marine sponge-associated microbes and comparatively analyzed this methods efficiencies with those of the conventional method. Then, we attempted to clarify the key and unknown mechanism of in situ cultivation by focusing on growth triggering via growth initiation factor. We hypothesized that majority of environmental microorganisms are in nongrowing state and requiring \"growth initiation factor\" for the recovery and that can be provided from environments. Consequently, significantly more novel and diverse microbial types were isolated via in situ cultivation than by standard direct plating (SDP). Next, the effect of the sponge extract on starvation recovery was compared between strains derived from in situ and SDP cultivation. Adding small amounts of the sponge extracts to the medium elevated the colony-formation efficiencies of the in situ strains at the starvation recovery step, while it showed no positive effect on that of SDP strains. Conversely, specific growth rates or carrying capacities of all tested strains were not positively affected. These results indicate that, 1) the sponge extract contains chemical compounds that facilitate starvation recovery, these substances selectively worked on the in situ strains, and 2) growth initiation factor in the sponge extract did not continuously promote growth activity but worked as triggers for regrowth (resuscitation from dormancy).\n\nImportanceMost microbial species resist cultivation under laboratory condition. This is critical impediment for both academic and applied microbiology, and thus clarification of the mechanism of microbial uncultivability is highly demanded. Several evidences have been reported that to cultivate microbes in a membrane-bound device incubated in nature (in situ cultivation) is an effective approach. However, the mechanism behind this approach has not been clarified. The present study shows the evidence that 1) initiating growth is a key for cultivating previously uncultivated microbes rather than simple growth promotion, and 2) growth initiation factor (signaling-like compounds) in natural environments stimulate microbial resuscitation from a nongrowing state. Since no study has focused on growth initiation for cultivation of previously uncultivated microorganisms, the discovery shown in the present study provides a new insight into microorganisms previously considered uncultivable and a microbial growth controlling system in nature.

microbiology↗