Biology subjects

Okubo, Y.

Publications and source records attributed to Okubo, Y..

7 recordsLinked to original sources

bioRxiv2026

A versatile, marker-free platform for life cycle-wide imaging in Plasmodium falciparum via integration of an exogenous gene cassette into a conserved intergenic locus

The development of a transgenic Plasmodium falciparum line that exhibits robust fluorescence throughout its life cycle in cultured cells and mosquito hosts is valuable for live imaging, tissue localization studies, and quantitative evaluations of parasite infectivity. In this study, we utilized Plasmodium-optimized genome editing to integrate an mCherry expression cassette into a specific intergenic locus without gene disruption; thereby successfully minimizing the physiological impact of exogenous gene introduction. The resulting transgenic parasite line, NF54-mCh, exhibited intense fluorescence across all developmental stages; including intraerythrocytic asexual and sexual blood stages; mosquito ookinete, oocyst, and sporozoite stages; and liver stages. Cultured NF54-mCh exhibited normal intraerythrocytic proliferation, gametocytogenesis, and gametocyte maturation, with efficient transmission to mosquito vectors. The marker brightness in salivary gland sporozoites allowed for visual, non-invasive identification of infected mosquitoes, thus facilitating dissection. Sporozoites dissected from salivary glands were highly infectious to livers in humanized mice; and allowed completion of the full life cycle, as evidenced by the appearance of ring-stage parasites in inoculated human erythrocytes. NF54-mCh serves as a parental line for performing additional genetic modifications, because the CRISPR/Cas9-based genome editing method is free of introduced drug resistance markers. The broader applicability of this strategy was validated by generating fluorescence reporter lines for use in the rodent malaria parasite model systems, Plasmodium berghei and Plasmodium yoelii. In summary, NF54-mCh represents a unique, versatile platform that will accelerate fundamental research and support the future development of innovative malaria control strategies, including studies on new vaccines and drug efficacy. IMPORTANCEA detailed understanding of the molecular and cellular biology of the human malaria parasite Plasmodium falciparum, particularly during infection processes, is essential for efforts in malaria control and elimination. In this study, we generated a marker-free transgenic P. falciparum line which exhibits constitutive, robust fluorescence throughout its developmental stages in erythrocytes, mosquitoes, and hepatocytes, without impairing parasite growth or fitness. The fluorescence marker gene was inserted into an intergenic locus and thus no genes were disrupted. The pfs47 gene is intact, which allows the parasites to evade the mosquito innate system; therefore, the parasite retains the ability for pfs47-dependent infection of refractory Anopheles strains. The transgenic parasites do not contain drug resistance markers, and thus there are no limitations for further genetic manipulation. This transgenic parasite line provides a powerful tool for the study of fundamental parasite infection mechanisms and for the molecular analysis of host-parasite interactions.

microbiology↗

bioRxiv2025

Foot placement coordination is impaired in people with Parkinson's Disease

BackgroundGait instability is a common and disabling symptom of Parkinsons disease (PD), contributing to frequent falls and reduced quality of life. While clinical balance tests and spatiotemporal gait measures can predict fall risk, they do not fully explain the underlying control mechanisms. In healthy individuals, foot placement is actively adjusted based on an internal estimate of the center of mass state to maintain gait stability, known as foot placement control. This estimation relies on the integration of multisensory information, which has been shown to be impaired in PD, potentially disrupting the control of gait stability through foot placement. ObjectiveTo investigate whether foot placement coordination during overground walking is impaired in people with PD. MethodsFifty people with PD and 51 healthy older adults walked overground for ten minutes at self-selected, comfortable walking speed. Foot placement errors were quantified as the deviation between the actual foot placement and the predicted placement derived from the center of mass kinematic state during the preceding swing phase. ResultsFoot placement errors were significantly higher in people with PD than in healthy older adults in both mediolateral (p < .05) and anteroposterior directions (p < .0001), at both mid-swing and terminal swing. Relative explained variance in mediolateral direction was significantly higher in people with PD compared to healthy older adults (p < .005). ConclusionWe provide first evidence of impaired coordination between the center of mass and foot placement in PD. Future work should investigate a causal relationship between impaired foot placement control, sensorimotor integration and gait instability.

pathology↗

bioRxiv2024

A Reference Tissue Implementation of Simultaneous Multifactor Bayesian Analysis (SiMBA) of PET Time Activity Curve Data

PET analysis is conventionally performed as a two-stage process of quantification followed by analysis. We recently introduced SiMBA (Simultaneous Multifactor Bayesian Analysis), a hierarchical model that performs quantification and analysis for all brain regions of all individuals at once, and in so doing improves both the accuracy of parameter estimation as well as inferential efficiency. However until now, SiMBA has only been implemented for the two-tissue compartment model. We have now extended this general approach to also allow a non-invasive reference tissue implementation that includes both the full reference tissue model and the simplified reference tissue model. In simulated data, SiMBA improves quantitative parameter estimation accuracy, reducing error by, on average, 57% for binding potential (BPND). In considerations of statistical power, our simulation studies indicate that the efficiency of SiMBA modeling approximately corresponds to improvements that would require doubling the sample size if using conventional methods, with no increase in the false positive rate. We applied the model to PET data measured with [11C]AZ10419369, which binds selectively to the serotonin 1B receptor, in datasets collected at three different PET centres (n=139, n=44 and n=39). We show that SiMBA yields replicable inferences by comparing associations between PET parameters and age in the different datasets. Moreover, we show that time activity curve data from different centres can be combined in a single SiMBA model using covariates to control between-centre parameter differences, in order to harmonise data between centres. In summary, we present a novel approach for noninvasive quantification and analysis of PET time activity curve data which improves quantification and inferences, enables effective between-centre data harmonisation, and also yields replicable outcomes. This method has the potential to significantly expand the range of research questions which can be meaningfully tested using conventional sample sizes with PET imaging.

neuroscience↗

bioRxiv2022

Reprogramming of three-dimensional microenvironments for in vitro hair follicle induction

During embryonic development, reciprocal interactions between epidermal and mesenchymal layers trigger hair follicle morphogenesis. This study revealed that microenvironmental reprogramming via control over these interactions enabled hair follicle induction in vitro. A key approach is to modulate spatial distributions of epithelial and mesenchymal cells in their spontaneous organization. The de novo hair follicles with typical morphological features emerged in aggregates of the two cell types, termed hair follicloids, and hair shafts sprouted with near 100% efficiency in vitro. The hair shaft length reached [~]3 mm in culture. Typical trichogenic signaling pathways were upregulated in hair follicloids. Owing to replication of hair follicle morphogenesis in vitro, production and transportation of melanosomes were also monitored in the hair bulb region. This in vitro hair follicle model might be valuable for better understanding hair follicle induction, for evaluating hair growth as well as the inhibition of hair growth by drugs, and modeling gray hairs in a well-defined environment. TeaserIn tissue morphogenesis, different types of cells harmonize in a pre-programmed manner using messenger systems such as epithelial-mesenchymal interactions. Organoids are a promising tool to elucidate such mechanisms on a molecular level. This work describes a strategy for reprograming three-dimensional microenvironments to trigger the initiation of in vitro regeneration of hair follicle organoids. Hair follicle organoids generated fully matured hair follicles, enabling the monitoring of hair follicle morphogenesis in vitro and determination of signaling pathways involved in early hair follicle morphogenesis. The principles uncovered herein may be relevant to other organ systems and will contribute to our understanding of developmental phenomena in physiological and pathological processes, eventually opening up new research avenues for the development of new treatment strategies.

bioengineering↗

bioRxiv2021

Multi-omics analysis reveals cross-organism interactions in coral holobiont

Corals create an ecosystem, called a holobiont, with intracellular algae (zooxanthellae) and resident bacteria. Zooxanthellae and some bacteria play major roles in the physiological properties of the coral host. However, because of the difficulties in experimental verification of cross-organism interactions, the mechanisms underpinning these interactions are largely unknown. To address this, we here generated and then analyzed multi-omics datasets for corals, zooxanthellae, and bacteria collected at Okinawa, Japan, from November 2014 to September 2016. Using cross-organism co-expression analysis, we successfully characterized the host-alga relationship in the coral holobiont. Specifically, we observed that the coral host dominates the zooxanthellae. The multi-omics analysis also suggested that infection with coral-associated bacteria Endozoicomonas likely involves coral-like ephrin ligands, triggering an immune response of the coral host. This study highlights the potential of the multi-omics approach to elucidate coral-microbe interactions.

systems biology↗

bioRxiv2018

Exposure to unpredictable trips and slips while walking can improve balance recovery responses with minimum predictive gait alterations

INTRODUCTIONThis study aimed to determine if repeated exposure to unpredictable trips and slips while walking can improve balance recovery responses when predictive gait alterations (e.g. slowing down) are minimised.\n\nMETHODSTen young adults walked on a 10-m walkway that induced slips and trips in fixed and random locations. Participants were exposed to a total of 12 slips, 12 trips and 6 non-perturbed walks in three conditions: 1) right leg fixed location, 2) left leg fixed location and 3) random leg and location. Kinematics during non-perturbed walks and previous and recovery steps were analysed.\n\nRESULTSThroughout the three conditions, participants walked with similar gait speed, step length and cadence(p>0.05). Participants extrapolated centre of mass (XCoM) was anteriorly shifted immediately before slips at the fixed location (p<0.01), but this predictive gait alteration did not transfer to random perturbation locations. Improved balance recovery from trips in the random location was indicated by increased margin of stability and step length during recovery steps (p<0.05). Changes in balance recovery from slips in the random location was shown by reduced backward XCoM displacement and reduced slip speed during recovery steps (p<0.05).\n\nCONCLUSIONSEven in the absence of most predictive gait alterations, balance recovery responses to trips and slips were improved through exposure to repeated unpredictable perturbations. A common predictive gait alteration to lean forward immediately before a slip was not useful when the perturbation location was unpredictable. Training balance recovery with unpredictable perturbations may be beneficial to fall avoidance in everyday life.

physiology↗

bioRxiv2018

IP3R2-independent Ca2+ release from the endoplasmic reticulum in astrocytes

Accumulating evidence indicates that astrocytes are actively involved in the physiological and pathophysiological functions of the brain. Intracellular Ca2+ signaling, especially Ca2+ release from the endoplasmic reticulum (ER), is considered to be crucial for the regulation of astrocytic functions. Mice with genetic deletion of inositol 1,4,5-trisphosphate receptor type 2 (IP3R2) are reportedly devoid of astrocytic Ca2+ signaling, and thus widely used to explore the roles of Ca2+ signaling in astrocytic functions. While functional deficits in IP3R2-knockout (KO) mice have been found in some reports, no functional deficit was observed in others. Thus, there remains a controversy regarding the functional significance of astrocytic Ca2+ signaling. To address this controversy, we re-evaluated the assumption that Ca2+ release from the ER is abolished in IP3R2-KO astrocytes using a highly sensitive imaging technique. We expressed the ER luminal Ca2+ indicator G-CEPIA1er in cortical and hippocampal astrocytes to directly visualize spontaneous and stimulus-induced Ca2+ release from the ER. We found attenuated but significant Ca2+ release in response to application of norepinephrine to IP3R2-KO astrocytes. This IP3R2-independent Ca2+ release induced only minimal cytosolic Ca2+ transients but induced robust Ca2+ increases in mitochondria that are frequently in close contact with the ER. These results indicate that ER Ca2+ release is retained and is sufficient to increase the Ca2+ concentration in close proximity to the ER in IP3R2-KO astrocytes.

neuroscience↗