Biology subjects

Yoshida, A.

Publications and source records attributed to Yoshida, A..

35 records · Page 2Linked to original sources

bioRxiv2024

Involvement of neurons in the non-human primate anterior striatum in proactive inhibition

Behaving as desired requires selecting the appropriate behavior and inhibiting the selection of inappropriate behavior. This inhibitory function involves multiple processes, such as reactive and proactive inhibition, instead of a single process. In this study, macaque monkeys were required to perform a task in which they had to sequentially select (accept) or refuse (reject) a choice. Neural activity was recorded from the anterior striatum, which is considered to be involved in behavioral inhibition, focusing on the distinction between proactive and reactive inhibitions. We identified neurons with significant activity changes during the rejection of bad objects. Cluster analysis revealed three distinct groups, of which one showed obviously increased activity during object rejection, suggesting its involvement in proactive inhibition. This activity pattern was consistent irrespective of the rejection method, indicating a role beyond mere saccadic suppression. Furthermore, minimal activity changes during the fixation task indicated that these neurons were not primarily involved in reactive inhibition. In conclusion, these findings suggest that the anterior striatum plays a crucial role in cognitive control and orchestrates goal-directed behavior through proactive inhibition, which may be critical in understanding the mechanisms of behavioral inhibition dysfunction that occur in patients with basal ganglia disease.

neuroscience↗

bioRxiv2024

Immunogenicity and safety of a live-attenuated SARS-CoV-2 vaccine candidate based on multiple attenuation mechanisms

mRNA vaccines against SARS-CoV-2 were rapidly developed and effective during the pandemic. However, some limitations remain to be resolved, such as the short-lived induced immune response and certain adverse effects. Therefore, there is an urgent need to develop new vaccines that address these issues. While live-attenuated vaccines are a highly effective modality, they pose a risk of adverse effects, including virulence reversion. In the current study, we constructed a live-attenuated vaccine candidate, BK2102, combining naturally occurring virulence-attenuating mutations in the NSP14, NSP1, spike and ORF7-8 coding regions. Intranasal inoculation with BK2102 induced humoral and cellular immune responses in Syrian hamsters without apparent tissue damage in the lungs, leading to protection against a SARS-CoV-2 D614G and an Omicron BA.5 strains. The neutralizing antibodies induced by BK2102 persisted for up to 364 days, which indicated that they confer long-term protection against infection. Furthermore, we confirmed the safety of BK2102 using transgenic (Tg) mice expressing human ACE2 (hACE2), that are highly susceptible to SARS-CoV-2. BK2102 did not kill the Tg mice, even when virus was administered at a dose of 106 plaque-forming units (PFU), while 102 PFU of the D614G strain or an attenuated strain lacking the furin cleavage site (FCS) of the spike was sufficient to kill mice. These results suggest that BK2102 is a promising live-vaccine candidate strain that confers long-term protection without significant virulence.

microbiology↗

bioRxiv2023

A Deep Learning Approach to Detecting Temporal Characteristics of Cortical Regions

One view of the neocortical architecture is that every region functions based on a universal computational principle. Contrary to this, we postulated that each cortical region has its own specific algorithm and functional properties. This idea led us to hypothesize that unique temporal patterns should be associated with each region, with the functional commonalities and variances among regions reflecting in the temporal structure of their neural signals. To investigate these hypotheses, we employed deep learning to predict electrodes locations in the macaque brain using single-channel ECoG signals. To do this, we first divided the brain into seven regions based on anatomical landmarks, and trained a deep learning model to predict the electrode location from the ECoG signals. Remarkably, the model achieved an average accuracy of 33.6%, significantly above the chance level of 14.3%. All seven regions exhibited above-chance prediction accuracy. The models feature vectors identified two main clusters: one including higher visual areas and temporal cortex, and another encompassing the remaining other regions.These results bolster the argument for unique regional dynamics within the cortex, highlighting the diverse functional specializations present across cortical areas.

neuroscience↗

bioRxiv2023

Opposing functions of glutamatergic inputs between the globus pallidus external segment and substantia nigra pars reticulata

The indirect pathway of the basal ganglia, including the subthalamic nucleus (STN) and globus pallidus external segment (GPe), is believed to play a crucial role in suppressing involuntary movements. However, recent evidence suggests the STN and GPe also facilitate voluntary movements. This study hypothesized that excitatory inputs from the STN to the GPe contribute to this facilitation, and that excitatory projections to the substantia nigra pars reticulata (SNr) are involved in the inhibition. To disrupt the STN-GPe or STN-SNr projections in monkeys during choice and fixation tasks, glutamate receptor inhibitors were injected into the GPe or SNr, which induced delayed saccade latencies toward good choices in the choice task (GPe) and caused frequent reflexive saccades to objects in the fixation task (SNr). Our findings suggest excitatory inputs to the GPe and SNr work in opposing manners, providing new insights that redefine our understanding of the functions of basal ganglia pathways. HighlightsO_LISTN and GPe neuronal activity increased when good objects were chosen C_LIO_LISNr activity increased when rejecting bad objects and decreased when accepting good objects C_LIO_LIExcitatory inputs inactivation in the GPe caused delayed saccade to good objects C_LIO_LIExcitatory projection inhibition to the SNr suppressed involuntary saccade control C_LI

neuroscience↗

bioRxiv2023

Migratory pattern of zoonotic Toxocara cati and T. canis in experimentally infected pigs

Over a billion people are infected with Toxocara canis or T. cati, the roundworms of dogs and cats. Historically, T. canis has been considered the main responsible of human toxocarosis but as serodiagnosis cannot discriminate the two species, this remains unresolved. We assessed the migratory pattern of T. cati and T. canis in a pig model and found them to be equally infective. Overall, they had a similar migration pattern reaching multiple organs and tissues, including mesenteric lymph nodes, liver, lungs and diaphragm. We recovered larvae of both species in the brain, suggesting that T. cati also can cause neurological toxocarosis in humans. Both species induced systemic eosinophilia and histopathological changes in lungs, livers and mesenteric lymph nodes. This study emphasizes the importance of T. cati as a zoonotic agent and the need to develop diagnostic methods that can differentiate between sources of infection in humans.

microbiology↗

bioRxiv2022

piRNA-like small RNAs target transposable elements in a Clade IV parasitic nematode

The small RNA (sRNA) pathways identified in the model organism Caenorhabditis elegans are not widely conserved across nematodes. For example, the PIWI pathway and PIWI-interacting RNAs (piRNAs) are involved in regulating and silencing transposable elements (TE) in most animals but have been lost in nematodes outside of the Caenorhabditis elegans group (Clade V), and little is known about how nematodes regulate TEs in the absence of the PIWI pathway. Here, we investigated the role of sRNAs in the Clade IV parasitic nematode Strongyloides ratti by comparing two genetically identical adult stages (the parasitic female and free-living female). We identified putative small-interfering RNAs, microRNAs and tRNA-derived sRNA fragments that are differentially expressed between the two adult stages. Two classes of sRNAs were predicted to regulate TE activity including (i) a parasite-associated class of 21-22 nt long sRNAs with a 5 uracil (21-22Us) and monophosphate modification, and (ii) 27 nt long sRNAs with a 5 guanine/adenine (27GAs) and polyphosphate modification. The 21-22Us show striking resemblance to the 21U PIWI-interacting RNAs found in C. elegans, including an AT rich upstream sequence, overlapping loci and physical clustering in the genome.

genomics↗

bioRxiv2021

A method for the generation of pseudotyped virus particles bearing SARS coronavirus spike protein in high yields

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has threatened human health and the global economy. Development of additional vaccines and therapeutics is urgently required, but such development with live virus must be conducted with biosafety level 3 confinement. Pseudotyped viruses have been widely adopted for studies of virus entry and pharmaceutical development to overcome this restriction. Here we describe a modified protocol to generate vesicular stomatitis virus (VSV) pseudotyped with SARS-CoV or SARS-CoV-2 Spike protein in high yield. We found that pseudovirions produced with the conventional transient expression system lacked coronavirus Spike protein at their surface as a result of inhibition of parental VSV infection by overexpression of this protein. Establishment of stable cell lines with an optimal expression level of coronavirus Spike protein allowed the efficient production of progeny pseudoviruses decorated with Spike protein. This improved VSV pseudovirus production method should facilitate studies of coronavirus entry and development of antiviral agents.

microbiology↗

bioRxiv2021

Visualization of subcortical structures in non-human primates in vivo by Quantitative Susceptibility Mapping at 3T MRI

Magnetic resonance imaging (MRI) is now an essential tool in the field of neuroscience involving non-human primates (NHP). Structural MRI scanning using T1-weighted (T1w) or T2-weighted (T2w) images provides anatomical information, particularly for experiments involving deep structures such as the basal ganglia and cerebellum. However, for certain subcortical structures, T1w and T2w images fail to reveal important anatomical details. To better visualize such structures in the macaque brain, we applied a relatively new method called quantitative susceptibility mapping (QSM), which enhances tissue contrast based on the local tissue magnetic susceptibility. To evaluate the visualization of important structures, we quantified the the contrast-to-noise ratios (CNRs) of the ventral pallidum (VP), globus pallidus external and internal segments (GPe and GPi), substantia nigra (SN), subthalamic nucleus (STN) in the basal ganglia and the dentate nucleus (DN) in the cerebellum. For these structures, the QSM method significantly increased the CNR, and thus the visibility, beyond that in either the T1w or T2w images. In addition, QSM values of some structures were correlated to the age of the macaque subjects. These results indicate that the QSM method can enable the clear identification of certain subcortical structures that are invisible in more traditional scanning sequences. HighlightsO_LINHP subcortical structures are challenging to see in conventional T1w and T2w images C_LIO_LIWe applied quantitative susceptibility mapping (QSM) to identify them easily C_LIO_LIQSM clearly visualized basal ganglia and cerebellar nucleus of high brain iron content C_LIO_LICNRs of some subcortical nucleus were significantly higher in QSM C_LIO_LIQSM values of several subcortical nucleus increased with age C_LI

neuroscience↗

bioRxiv2021

Multi-Scale LM/EM Neuronal Imaging from Brain to Synapse with a Tissue Clearing Method, ScaleSF

The mammalian brain is organized over sizes that span several orders of magnitude, from synapses to the entire brain. Thus, a technique to visualize neural circuits across multiple spatial scales (multi-scale neuronal imaging) is vital for deciphering brain-wide connectivity. Here, we developed this technique by coupling successive light microscope/electron microscope (LM/EM) imaging with an ultrastructurally-preserved tissue clearing method, ScaleSF. Our multi-scale neuronal imaging incorporates 1) brain-wide macroscopic observation, 2) mesoscopic circuit mapping, 3) microscopic subcellular imaging, and 4) EM imaging of nanoscopic structures, allowing seamless integration of structural information from the brain to synapses. We applied the technique to three neural circuits of two different species, mouse striatofugal, mouse callosal, and marmoset corticostriatal projection systems, and succeeded in the simultaneous interrogation of their circuit structure and synaptic connectivity in a targeted way. Our multi-scale neuronal imaging will significantly advance the understanding of brain-wide connectivity by expanding the scales of objects.

neuroscience↗

bioRxiv2021

Interaction Between Phosphoinositide 3-Kinase and the VDAC2 Channel Tethers Endosomes to Mitochondria and Promotes Endosome Maturation

Intracellular organelles of mammalian cells communicate with each other during various cellular processes. The functions and molecular mechanisms of such interorganelle association remain largely unclear, however. We here identified voltage-dependent anion channel 2 (VDAC2), a mitochondrial outer membrane protein, as a binding partner of phosphoinositide 3-kinase (PI3K), a regulator of clathrin-independent endocytosis downstream of the small GTPase Ras. VDAC2 was found to tether endosomes positive for the Ras-PI3K complex to mitochondria in response to cell stimulation with epidermal growth factor and to promote clathrin-independent endocytosis as well as endosome maturation at membrane contact sites. With a newly developed optogenetics system to induce mitochondrion-endosome association, we found that, in addition to its structural role in such association, the pore function of VDAC2 is also required for the promotion of endosome maturation. Our findings thus uncover a previously unappreciated role of mitochondrion-endosome association in the regulation of endocytosis and endosome maturation. HighlightsO_LIThe mitochondrial protein VDAC2 binds PI3K and tethers endosomes to mitochondria C_LIO_LIVDAC2 promotes clathrin-independent endocytosis C_LIO_LIVDAC2-PI3K interaction induces acidification of endosomes associated with mitochondria C_LIO_LIThe pore function of VDAC2 also contributes to endosome maturation at contact sites C_LI

cell biology↗

bioRxiv2020

Cortico-subcortical functional connectivity profiles of resting-state networks in marmosets and humans

Understanding the similarity of cortico-subcortical networks topologies between humans and nonhuman primate species is critical to study the origin of network alternations underlying human neurological and neuropsychiatric diseases. The New World common marmoset (Callithrix jacchus) has become popular as a non-human primate model for human brain function. Most marmoset connectomic research, however, has exclusively focused on cortical areas, with connectivity to subcortical networks less extensively explored. In this study, we aimed to first isolate patterns of subcortical connectivity with cortical resting-state networks (RSNs) in awake marmosets using resting-state functional magnetic resonance imaging (RS-fMRI), then to compare these networks to those in humans using connectivity fingerprinting. While we could match several marmoset and human RSNs based on their functional fingerprints, we also found a few striking differences, for example strong functional connectivity of the default mode network with the superior colliculus in marmosets that was much weaker in humans. Together, these findings demonstrate that many of the core cortico-subcortical networks in humans are also present in marmosets, but that small, potentially functionally relevant differences exist.

neuroscience↗

bioRxiv2019

Localization of BCR-ABL to stress granules contributes to its oncogenic function

The oncogenic tyrosine kinase BCR-ABL activates a variety of signaling pathways and plays a causative role in the pathogenesis of chronic myelogenous leukemia (CML); however, the subcellular distribution of this chimeric protein remains controversial. Here, we report that BCR-ABL is localized to stress granules and that its granular localization contributes to BCR-ABL-dependent leukemogenesis. BCR-ABL-positive granules were not colocalized with any markers for membrane-bound organelles but were colocalized with HSP90a, a component of RNA granules. The number of such granules increased with thapsigargin treatment, confirming that the granules were stress granules. Given that treatment with the ABL kinase inhibitor imatinib and elimination of the N-terminal region of BCR-ABL abolished granule formation, kinase activity and the coiled-coil domain are required for granule formation. Whereas wild-type BCR-ABL rescued the growth defect in IL-3-depleted Ba/F3 cells, mutant BCR-ABL lacking the N-terminal region failed to do so. Moreover, forced tetramerization of the N-terminus-deleted mutant could not restore the growth defect, indicating that granule formation, but not tetramerization, through its N-terminus is critical for BCR-ABL-dependent oncogenicity. Our findings together provide new insights into the pathogenesis of CML by BCR-ABL and open a window for developing novel therapeutic strategies for this disease.

cell biology↗

bioRxiv2019

Changes in extracellular matrix gene and protein expressions in human trabecular meshwork cells in response to mechanical fluid flow stimulation

PurposeTo investigate the changes in extracellular matrix (ECM) gene and protein expressions in human trabecular meshwork (HTM) cells in response to mechanical fluid flow stimulation.\n\nMethodsHTM cells were cultured on a glass plate and exposed to shear stress (0, 0.2, and 1.0 dyne/cm2) for 12 hours. Changes in gene expressions were evaluated using microarray analysis. The representative genes related to ECM metabolism underwent real-time reverse-transcriptase polymerase chain reaction. Fibronectin (FN) and collagen (COL) IV levels in the supernatant were evaluated using immunoassays. Rho-associated coiled-coil-containing protein kinase (ROCK) activity also was investigated.\n\nResultsAfter stimulation, transforming growth factor {beta}2 mRNA levels were significantly (p < 0.01) lower than that of the static control (0 dyne/cm2 for 12 hours). Matrix metalloproteinase 2 mRNA levels were significantly (p < 0.05) higher than the static control. COL type 1 alpha 2 mRNA, COL type 4 alpha 2 mRNA, and COL type 6 alpha 1 mRNA levels were significantly (p < 0.05, < 0.01, and < 0.05, respectively) higher than the static control. The mean {+/-} standard deviation FN levels (ng/mL) in the supernatant after stimulation (0, 0.2, 1.0 dyne/cm2) were 193.7 {+/-} 7.6, 51.5 {+/-} 21.8, and 34.9 {+/-} 23.6, respectively (p < 0.01). The FN and COL IV levels and ROCK activity were significantly (p < 0.01 and < 0.05, respectively) lower than the static control.\n\nConclusionsChanges in gene and protein expressions related to ECM metabolism occurred in HTM cells after stimulation. Specifically, the suppression of FN and COL IV production might explain the importance of mechanical fluid flow stimulation on maintenance of the normal aqueous humor outflow.

molecular biology↗

bioRxiv2019

Folding latency of fluorescent proteins affects the mitochondrial localization of fusion protein

The discovery of fluorescent proteins (FPs) has revolutionized cell biology. The fusion of targeting sequences to FPs enables the investigation of cellular organelles and their dynamics; however, occasionally, such fluorescent fusion proteins (FFPs) exhibit behavior different from that of the native proteins. Here, we constructed a color pallet comprising different organelle markers and found that FFPs targeted to the mitochondria were mislocalized when fused to certain types of FPs. Such FPs included several variants of Aequorea victoria green FP (aqGFP) and a monomeric variant of the red FP. Because the FFPs that are mislocalized include FPs with faster maturing or folding mutations, the increase in the maturation rate is likely to prevent their expected localization. Indeed, when we reintroduced amino acid substitutions so that the FP sequences were equivalent to that of wild-type aqGFP, FFP localization to the mitochondria was significantly enhanced. Moreover, similar amino acid substitutions improved the localization of mitochondria-targeted pHluorin, which is a pH-sensitive variant of GFP, and its capability to monitor pH changes in the mitochondrial matrix. Our findings demonstrate the importance of selecting FPs that maximize FFP function.

cell biology↗

bioRxiv2019

Towards HCP-Style Macaque Connectomes: 24-Channel 3T Multi-Array Coil, MRI Sequences and Preprocessing

Macaque monkeys are an important model species for understanding cortical organization of primates, yet tools and methods for noninvasive image acquisition (e.g. MRI RF coils and pulse sequence protocols) and image data preprocessing have lagged behind those developed for humans. To resolve the structural and functional characteristics of the relatively thin macaque cortex, high spatial, temporal, and angular resolutions are required while maintaining high signal-to-noise ratio to ensure good image quality. To address these challenges, we developed a macaque 24-channel receive coil for 3-T MRI with parallel imaging capabilities. This coil enabled adaptation of the Human Connectome Project (HCP) image acquisition protocols to the macaque brain. We also adapted HCP preprocessing methods optimized for the macaque brain, including spatial minimal preprocessing of structural, functional MRI (fMRI), and diffusion MRI (dMRI). The coil provided high signal-to-noise ratio and high efficiency in data acquisition, allowing four- and five-fold acceleration for dMRI and fMRI, respectively. Automated parcellation of cortex, reconstruction of cortical surface, removal of artefacts and nuisance signals in fMRI, and distortion correction of dMRI performed well, and the overall quality of basic neurobiological measures was comparable with those for the HCP. The resulting HCP-style in vivo macaque MRI data show considerable promise for analyzing cortical architecture and functional and structural connectivity using advanced methods that have previously only been available for humans.\n\nHighlights[tpltrtarr] 24-channel 3T MR receive coil designed for the smaller macaque brain.\n[tpltrtarr]In vivo macaque imaging protocols adapted according to guidelines from the HCP.\n[tpltrtarr]Parallel imaging yields five- and four-fold acceleration in fMRI and dMRI sampling.\n[tpltrtarr]HCPs minimal preprocessing and denoising pipelines adapted for macaques.\n[tpltrtarr]The multi-modal MRI data show considerable promise for HCP-style analyses.

neuroscience↗

bioRxiv2018

Characterization of the gastric motility response to human motilin and erythromycin in human motilin receptor-expressing transgenic mice

Motilin is a gastrointestinal peptide hormone that stimulates gastrointestinal motility. Motilin is produced primarily in the duodenum and jejunum. Motilin receptors (MTLRs) are G protein-coupled receptors that may represent a clinically useful pharmacological target as they can be activated by erythromycin. The functions of motilin are highly species-dependent and remain poorly understood. As a functional motilin system is absent in rodents such as rats and mice, these species are not commonly used for basic studies. In this study, we examine the usefulness of human MTLR-overexpressing transgenic (hMTLR-Tg) mice by identifying the mechanisms of the gastric motor response to human motilin and erythromycin.\n\nThe distribution of hMTLR was examined immunohistochemically in male wild-type (WT) and hMTLR-Tg mice. The contractile response of gastric strips was measured isometrically in an organ bath, while gastric emptying was determined using phenol red.\n\nhMTLR expression was abundant in the gastric smooth muscle layer but more potently expressed in the myenteric plexus of hMTLR-Tg mice but not WT mice. hMTLR was not co-localized with vesicular acetylcholine transporter, a marker of cholinergic neurons in the myenteric plexus. Treatment with human motilin and erythromycin caused concentration-dependent contraction of gastric strips obtained from hMTLR-Tg mice but not from WT mice.\n\nThe contractile response to human motilin and erythromycin in hMTLR-Tg mice was affected by neither atropine nor tetrodotoxin and was totally absent in Ca2+-free conditions. Furthermore, intraperitoneal injection of erythromycin significantly promoted gastric emptying in hMTLR-Tg mice but not in WT mice.\n\nHuman motilin and erythromycin stimulate the gastric motor response in hMTLR-Tg mice. This action is mediated by direct contraction of smooth muscle via the influx of extracellular Ca2+. Thus, hMTLR-Tg mice may be useful for the evaluation of MTLR agonists as gastric prokinetic agents.

pharmacology and toxicology↗

bioRxiv2017

Morphological changes of plasma membrane and protein assembly during clathrin-mediated endocytosis

Clathrin-mediated endocytosis (CME) proceeds through a series of morphological changes of the plasma membrane induced by a number of protein components. Although the spatiotemporal assembly of these proteins has been elucidated by fluorescence-based techniques, the protein-induced morphological changes of the plasma membrane have not been fully clarified in living cells. Here we visualize membrane morphology together with protein localizations during CME by utilizing high-speed atomic force microscopy combined with a confocal laser scanning unit. The plasma membrane starts to invaginate ~30 seconds after clathrin starts to assemble, and the aperture diameter increases as clathrin accumulates. Actin rapidly accumulates around the pit and induces a small membrane swelling, which within 30 seconds rapidly covers the pit irreversibly. Inhibition of actin turnover abolishes the swelling and induces a reversible open-close motion of the pit, indicating that actin dynamics are necessary for efficient and irreversible pit closure at the end of the CME.

cell biology↗