Biology subjects

Saito, H.

Publications and source records attributed to Saito, H..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Platelet extracellular vesicles and their mitochondrial content significantly improve survival and cytokine levels when transfused into mice with sepsis or H1N1 infections

BACKGROUNDMitochondrial transplantation has recently gained prominence as a novel technique primarily focused on addressing ischemiareperfusion injuries and rare mitochondrial mutation diseases. Platelets, abundant in the bloodstream, play a crucial role in immune function. Upon activation, platelets release mitochondria encapsulated within extracellular vesicles, here referred to as "mitlets". These mitlets exhibit a preference for being internalized by immune cells circulating in the bloodstream, enhancing their cellular energetics. Herein, we hypothesized that the transplantation of mitlets between young animals and aged animals may exert a significant influence on the progression of infectious diseases. STUDY DESIGN AND METHODSIn this study, murine models of Influenza H1N1 infection and sepsis were employed to investigate disease dynamics. Specifically, mitlets isolated from young and healthy mice were transplanted into cohorts of mice of the same age afflicted by H1N1 infection, or into aged mice subjected to polymicrobial infection and sepsis. Survival outcomes and the quantification of cytokine levels were assessed across experimental groups to elucidate the potential therapeutic effects of mitlet transplantation. RESULTSIn the matched-age H1N1 infection model, as predicted, mitlet transplantation did not yield a statistically significant improvement in survival, although it did show a trend towards a reduction in the circulating inflammatory cytokine burden. In the young-to-old sepsis model, the transplantation of mitlets was associated with a significant enhancement in survival rates and a substantial reduction in bacterial loads and circulating cytokine levels. DISCUSSIONOur findings suggest that mitochondrial transplantation may constitute a safe and promising avenue for enhancing the immune systems capacity to counter infectious threats. This pilot investigation sets the stage for further exploration. It is plausible that in the future, immune senescence resulting from diminished mitochondrial energy production could be ameliorated through such transplantation interventions. As a consequence, this approach holds substantial potential as a novel immunotherapeutic strategy for the management of infectious diseases.

molecular biology↗

bioRxiv2023

Structural flexibility of apolipoprotein E-derived arginine-rich peptides improves their cell penetration capability

Amphipathic arginine-rich peptide, A2-17, exhibits moderate perturbation of lipid membranes and the highest cell penetration among its structural isomers. We investigated the direct cell-membrane penetration mechanism of the A2-17 peptide. We designed structurally constrained versions of A2-17, stapled (StpA2-17) and stitched (StchA2-17), whose -helical conformations were stabilized by chemical crosslinking. Circular dichroism confirmed that StpA2-17 and StchA2-17 had higher -helix content than A2-17 in aqueous solution. Upon liposome binding, only A2-17 exhibited a coil-to-helix transition. Confocal microscopy revealed that A2-17 had higher cell penetration efficiency than StpA2-17 in HeLa cells. Partitioning into lipid membranes was more prominent for StchA2-17 than for A2-17 or StpA2-17; StchA2-17 remained on the cell membrane without cell penetration. Tryptophan fluorescence analysis suggested that A2-17 and its analogs had similar membrane-insertion positions between the interface and hydrophobic core. Atomic force microscopy demonstrated that A2-17 reduced the mechanical rigidity of liposomes to a greater extent than StpA2-17 and StchA2-17. Finally, electrophysiological analysis showed that A2-17 induced a higher charge influx through transient pores in a planer lipid bilayer than StpA2-17 and StchA2-17. These findings indicate that structural flexibility, which enables diverse conformations of A2-17, leads to a membrane perturbation mode that contributes to cell membrane penetration.

biophysics↗

bioRxiv2023

Neuromechanical Justification of Lower-Limb Functional Tests for a Return to Running: A Muscle Coordination Analysis

IntroductionThis study aimed to explore the shared muscle synergies between running and functional tests that are commonly used when considering the return to running (RTR) after sports-related injuries. We hypothesized that shared muscle synergies would differ among tasks, providing insights into prioritizing functional tests in the context of RTR. MethodsTen healthy male participants were recruited to perform running and 9 functional tasks and their 16 lower limb and trunk muscle activities were recorded using electromyography (EMG). Non-negative matrix factorization (NMF) was applied to the collected EMG data to explore shared muscle synergies between running and the functional tasks. We compared the percentages of shared synergies and temporal patterns between running and each functional test. ResultsAlthough all functional tests exhibited shared muscle synergies with running, the walk (75% [40%-100%]), single leg hops with 30% of maximum distance (SLH30) (60% [20%-100%]), and stepup (63% [0%-100%]) tasks displayed significantly higher percentages of shared synergies compared to other tests. However, significant differences in temporal patternss were observed between running and all functional tasks, indicating varying activation profiles of shared muscle synergies. ConclusionAlthough all functional tests shared muscle synergies with running, variations in the degree of shared synergies and temporal patterns imply that walking, SLH30, and step-up tests may be the most beneficial in predicting running behavior post-ACL injuries. However, functional tests cannot fully replicate running dynamics, suggesting the need for a careful interpretation when assessing readiness for RTR.

neuroscience↗

bioRxiv2023

Monoterpene glucosides accumulated in Eustoma grandiflorum roots promote hyphal branching in arbuscular mycorrhizal fungi

Host plant-derived strigolactones trigger hyphal branching in arbuscular mycorrhizal (AM) fungi, initiating a symbiotic interaction between land plants and AM fungi. However, our previous studies revealed that gibberellin-treated Eustoma grandiflorum (Gentianaceae) activates rhizospheric hyphal branching in AM fungi using unidentified molecules other than strigolactones. In this study, we analyzed independent transcriptomic data of E. grandiflorum and found that the gentiopicroside (GPS) and swertiamarin (SWM), which are characteristic monoterpene glucosides in Gentianaceae, were highly biosynthesized in gibberellin-treated E. grandiflorum roots. Moreover, these metabolites considerably promoted hyphal branching in the Glomeraceae AM fungi Rhizophagus irregularis and R. clarus. GPS treatment also enhanced R. irregularis colonization of the monocotyledonous crop Allium schoenoprasum. Interestingly, these metabolites did not provoke the germination of the root parasitic plant Orobanche minor. Altogether, our study unveiled the crucial role of GPS and SWM in activating the symbiotic relationship between AM fungi and E. grandiflorum.

plant biology↗

bioRxiv2023

Background environment modulates motor contagions in humans

Motor contagions refer to implicit effects in ones actions induced by the observation of actions made by others. A plethora of studies over the last two decades have exhibited that observed, as well as predicted, actions can induce various kinds of motor contagions in a human observer. However, motor contagion has always been investigated in regard to different features of an observed action and it remains unclear whether the environment, in which an observed action takes place, modulates motor contagions as well. Here we investigated the effect of the observed environment on motor contagions using an empirical hand steering task in which the participants were required to move a cursor through visual channels of different shapes. We observed the movement time of observers to be influenced by both the movement of the cursor they observed, as well as the background (channel shape) in which the cursor movement was observed. Observers consistently made faster movements after observing steering movements in a narrowing channel compared to a widening channel. These results show a distinct effect of the environment, in which an observed action occurs, on ones own movement.

neuroscience↗

bioRxiv2023

Detecting and validating influential organisms for rice growth: An ecological network approach

How to achieve sustainable food production while reducing environmental impacts is a major concern in agricultural science, and advanced breeding techniques are promising for achieving such goals. However, rice is usually grown under field conditions and influenced by surrounding ecological community members. How ecological communities influence the rice performance in the field has been underexplored despite the potential of ecological communities to establish an environment-friendly agricultural system. In the present study, we demonstrate an ecological-network-based approach to detect potentially influential, previously overlooked organisms for rice (Oryza sativa). First, we established small experimental rice plots, and measured rice growth and monitored ecological community dynamics intensively and extensively using quantitative environmental DNA metabarcoding in 2017 in Japan. We detected more than 1000 species (including microbes and macrobes such as insects) in the rice plots, and nonlinear time series analysis detected 52 potentially influential organisms with lower-level taxonomic information. The results of the time series analysis were validated under field conditions in 2019 by field manipulation experiments. In 2019, we focused on two species, Globisporangium nunn and Chironomus kiiensis, whose abundance was manipulated in artificial rice plots. The responses of rice, namely, the growth rate and gene expression patterns, were measured before and after the manipulation. We confirmed that, especially in the G. nunn-added treatment, rice growth rate and gene expression pattern were changed. In the present study, we demonstrated that intensive monitoring of an agricultural system and the application of nonlinear time series analysis were helpful to identify influential organisms under field conditions. Although the effects of the manipulations were relatively small, the research framework presented here has future potential to harness the ecological complexity and utilize it in agriculture. Our proof-of-concept study would be an important basis for the further development of field-basis system management.

ecology↗

bioRxiv2023

Gravitational and mechanical forces drive mitochondrial translation through the cell adhesion-FAK axis

Life on Earth has evolved in a form suitable for the gravitational force of 1 x g. Although the pivotal role of gravity in gene expression has been revealed by multiomics approaches in space-flown samples and astronauts, the molecular details of how mammalian cells harness gravity have remained unclear. Here, we showed that mitochondria utilize gravity to activate protein synthesis within the organelle. Genome-wide ribosome profiling revealed reduced mitochondrial translation in mammalian cells and Caenorhabditis elegans under both microgravity at the International Space Station and simulated microgravity in a 3D-clinostat on the ground. We found that attenuation of cell adhesion through laminin-integrin interactions causes the phenotype. The downstream signaling pathway including FAK, RAC1, PAK1, BAD, and Bcl-2 family proteins in the cytosol, and mitochondrial fatty acid synthesis (mtFAS) pathway in the matrix maintain mitochondrial translation at high level. Mechanistically, a decreased level of mitochondrial malonyl-CoA, which is consumed by activated mtFAS, leads to a reduction in the malonylation of the translational machinery and an increase in the initiation and elongation of in organello translation. Consistent with the role of integrin as a mechanosensor, we observed a decrease in mitochondrial translation via the minimization of mechanical stress in mouse skeletal muscle. Our work provides mechanistic insights into how cells convert gravitational and mechanical forces into translation in an energy-producing organelle.

molecular biology↗

bioRxiv2022

Degenerate oligonucleotide primer MIG-seq: an effective PCR-based method for high-throughput genotyping

Multiplexed inter-simple sequence repeats genotyping by sequencing (MIG-seq) is an next-generation sequencing library construction method developed for the analysis of DNA in ecology. Although MIG-seq can generate libraries from low-quality DNA, few polymorphisms can be obtained in species with small genomes. In this study, we developed degenerate oligonucleotide primer MIG-seq (dpMIG-seq) as an effective polymorphism discovery method that allows for variation in the number of polymorphisms while retaining the advantages of MIG-seq, including independence from DNA quality. In dpMIG-seq, a proportion of the simple sequence repeats in the primer sequence of the first PCR in MIG-seq was changed to degenerate oligonucleotides to enable annealing to a wider range of sequences. In tests of several crop species other than wheat, the number of loci that could be sequenced using dpMIG-seq with a data volume of 0.3 gigabases (Gb) was increased compared with that sequenced using MIG-seq. In wheat, the number of polymorphisms obtained via dpMIG-seq was higher than that obtained via MIG-seq when a data volume of about [≥]2 Gb was obtained. In dpMIG-seq, different loci could be sequenced by changing the positions of the degenerate oligonucleotides. By applying dpMIG-seq, we constructed a linkage map consisting of 5,142 markers for the rice inter-subspecies F2 population, and we detected quantitative trait loci for heading date in the regions where known heading-related genes were located. Overall, our results show that dpMIG-seq is a useful tool for the genetic analysis of crop species.

genetics↗

bioRxiv2022

Antagonistic interactions between odorants alter human odor perception.

The olfactory system detects a vast number of odorants using hundreds of olfactory receptors (ORs), the largest group of the G protein-coupled receptor (GPCR) superfamily. Each OR is activated by specific odorous ligands. Like other GPCRs, activation of ORs may be blocked through antagonism. Recent reports highlight widespread antagonisms in odor mixtures influencing olfactory neuron activities. However, it is unclear if and how these antagonisms influence perception of odor mixtures. Here we show that odorant antagonisms at the receptor level alter odor perception. Using a large-scale heterologous expression, we first identified a set of human ORs that are activated by methanethiol and hydrogen sulfide, two extremely potent volatile sulfur malodors. We then screened odorants that block activation of these ORs and identified a set of antagonists, including {beta}-ionone. Finally, human sensory evaluation revealed that odor intensity and unpleasantness of methanethiol were decreased by {beta}-ionone. Odor intensity of {beta}-ionone itself is not correlated with the degree of suppression of malodor sensation. Suppression was also not observed when methanethiol and {beta}-ionone were simultaneously introduced to different nostrils. Together, our data supports the model that odor sensation is altered through antagonistic interactions at the level of the ORs.

neuroscience↗

bioRxiv2022

Hydroxylation site-specific and production-dependent roles of endogenous oxysterols in cellular cholesterol homeostasis

The cholesterol metabolites oxysterols play central roles in cholesterol feedback control. They modulate the activity of two master transcription factors that control cholesterol homeostatic responses, sterol regulatory element-binding protein-2 (SREBP-2) and liver X receptor (LXR). Although the role of exogenous oxysterols has been well-established, whether endogenously synthesized oxysterols similarly control both SREBP-2 and LXR to those added exogenously remains poorly explored. Here, we carefully validate the role of oxysterols enzymatically synthesized within cells in cholesterol homeostatic responses. We first show that SREBP-2 responds more sensitively to exogenous oxysterols than LXR. We then show that endogenous 25-hydroxycholesterol (25-HC), 27-HC, and 24S-HC synthesized by CH25H, CYP27A1, and CYP46A1, respectively, suppress SREBP-2 activity at different degrees by stabilizing Insig proteins whereas 7a-HC has little impact on SREBP-2. The results explain the hydroxylation site-specific role of endogenous oxysterols. On the other hand, the expression of CH25H, CYP46A1, CYP27A1, or CYP7A1 fails to induce LXR target gene expression. We also show the 25-HC production-dependent suppression of SREBP-2 using a tetracycline-inducible CH25H expression system. Moreover, we quantitatively determine the specificity of the four cholesterol hydroxylases in living cells. Based on these results, we propose that endogenous side-chain oxysterols primarily regulate the activity of SREBP-2, not LXR.

biochemistry↗

bioRxiv2022

Embodiment of Supernumerary Robotic Limbs in Virtual Reality

The supernumerary robotic limb system expands the motor function of human users by adding extra artificially designed limbs. It is important for us to embody the system as if it is a part of ones own body and to maintain cognitive transparency in which the cognitive load is suppressed. Embodiment studies have been conducted with an expansion of bodily functions through a "substitution" and "extension." However, there have been few studies on the "addition" of supernumerary body parts. In this study, we developed a supernumerary robotic limb system that operates in a virtual environment, and then evaluated whether the extra limb can be regarded as a part of ones own body using a questionnaire and whether the perception of peripersonal space changes with a visuotactile crossmodal congruency task. We found that the participants can embody the extra-limbs after using the supernumerary robotic limb system. We also found a positive correlation between the perceptual change in the crossmodal congruency task and the subjective feeling that the number of ones arms had increased (supernumerary limb sensation). These results suggest that the addition of an extra body part may cause the participants to feel that they had acquired a new body part that differs from their original body part through a functional expansion.

neuroscience↗

bioRxiv2022

Xeno-free induced pluripotent stem cell-derived neural progenitor cells for in vivo applications

Cell-based therapies are a promising treatment paradigm for neurodegenerative diseases and other brain injuries. Despite recent advances in stem cell technology, major concerns have been raised regarding the feasibility and safety of cell therapies for clinical applications. Here, we generate good manufacturing practice (GMP)-compatible neural progenitor cells (NPCs) from transgene- and xeno-free induced pluripotent stem cells (iPSCs) that can be smoothly adapted for clinical applications. The produced NPCs have a stable gene-expression over at least 15 passages and can be scaled for up to 1018 cells per initially seeded 106 cells. To ensure a pure NPC population for in vivo applications, we reduce risks of iPSC contamination using micro RNA-switch technology as a safety checkpoint. Using lentiviral transduction with a fluorescent and bioluminescent dual-reporter construct, combined with non-invasive in vivo bioluminescent imaging, we longitudinally tracked the grafted cells in healthy wild-type and genetically immunosuppressed mice as well as in a mouse model of ischemic stroke. Long term in-depth characterization revealed that transplanted cells have the capability to survive and spontaneously differentiate into functional and mature neurons throughout a time course of a month.

neuroscience↗

bioRxiv2022

Mito-FUNCAT-FACS reveals cellular heterogeneity in mitochondrial translation

Mitochondria possess their own genome that encodes components of oxidative phosphorylation (OXPHOS) complexes, and mitochondrial ribosomes within the organelle translate the mRNAs expressed from mitochondrial genome. Given the differential OXPHOS activity observed in diverse cell types, cell growth conditions, and other circumstances, cellular heterogeneity in mitochondrial translation can be expected. Although individual protein products translated in mitochondria have been monitored, the lack of techniques that address the variation in overall mitochondrial protein synthesis in cell populations poses analytic challenges. Here, we adapted mitochondrial-specific fluorescent noncanonical amino acid tagging (FUNCAT) for use with fluorescence-activated cell sorting (FACS) and developed mito-FUNCAT-FACS. The click chemistry-compatible methionine analog L-homopropargylglycine (HPG) enabled the metabolic labeling of newly synthesized proteins. In the presence of cytosolic translation inhibitors, HPG was selectively incorporated into mitochondrial nascent proteins and conjugated to fluorophores via the click reaction (mito-FUNCAT). The application of in situ mito-FUNCAT to flow cytometry allowed us to disentangle changes in net mitochondrial translation activity from those of the organelle mass and detect variations in mitochondrial translation in cancer cells. Our approach provides a useful methodology for examining mitochondrial protein synthesis in individual cells.

molecular biology↗

bioRxiv2021

Post-transcriptionally regulated genes are essential for pluripotent stem cell survival

The effects of transcription factors on the maintenance and differentiation of pluripotent stem cells (PSCs) have been well studied. However, the importance of post-transcriptional regulatory mechanisms, which cause the quantitative dissociation of mRNA and protein expression, has not been explored in detail. Here, by combining transcriptome and proteome profiling, we identified 228 post-transcriptionally regulated genes with strict upregulation of the protein level in PSCs. Among them, we found that 84 genes were vital for the survival of PSCs and HDFs, including 20 genes that were specifically necessary for the survival of PSCs. These 20 proteins were upregulated only in PSCs and not in differentiated cells derived from the three germ layers. Subcellular fractionation of the mRNA showed that the expression of most of the 20 proteins was regulated at the mRNA localization stage from the nucleus to the cytoplasm, but their translation efficiency was constant. Together, these results revealed that post-transcriptionally regulated genes have a crucial role in PSC survival.

molecular biology↗

bioRxiv2021

Programmable mammalian translational modulators by CRISPR-associated proteins

The complexity of synthetic genetic circuits relies on repertories of biological circuitry with high orthogonality. Although post-transcriptional circuitry relying on RNA-binding proteins (RBPs) qualifies as a repertory, the limited pool of regulatory devices hinders network modularity and scalability. Here we propose CaRTRIDGE (Cas-Responsive Translational Regulation Integratable into Diverse Genomic Engineering) to repurpose CRISPR-associated (Cas) proteins as translational modulators. We demonstrate that a set of Cas proteins are able to repress (OFF) or activate (ON) the translation of mRNAs that contain a Cas-binding RNA motif in the 5-UTR. We designed 81 different types of translation OFF and ON switches and verified their functional characteristics. Many of them functioned as efficient translational regulators and showed orthogonality in mammalian cells. By interconnecting these switches, we designed and built artificial circuits, including 60 translational AND gates. Moreover, we show that various CRISPR-related technologies, including anti-CRISPR and split-Cas9 platforms, can be repurposed to control translation. Our Cas-mediated translational regulation is compatible with transcriptional regulation by Cas proteins and increases the complexity of synthetic circuits with fewer elements. CaRTRIDGE builds protein-responsive mRNA switches more than ever and leads to the development of both Cas-mediated genome editing and translational regulation technologies.

synthetic biology↗

bioRxiv2021

Flexible recruitments of fundamental muscle synergies in the trunk and lower limbs for highly variable movements and postures

The extent to which muscle synergies represent the neural control of human behavior remains unknown. Here, we tested whether certain sets of muscle synergies that are fundamentally necessary across behaviors exist. We measured the electromyographic activities of 26 muscles including bilateral trunk and lower limb muscles during 24 locomotion, dynamic and static stability tasks, and extracted the muscle synergies using non-negative matrix factorization. Our results showed that 13 muscle synergies that may have unique functional roles accounted for almost all 24 tasks by combinations of single and/or merging of synergies. Therefore, our results may support the notion of the low dimensionality in motor outputs, in which the central nervous system flexibly recruits fundamental muscle synergies to execute diverse human behaviors. Further studies using manipulations of the central nervous system and/or neural recording are required the neural representation with such fundamental components of muscle synergies.

neuroscience↗

bioRxiv2021

Tracheal motile cilia require CAMSAP3 for formation of central microtubule pair and coordinated beating

Motile cilia of multiciliated epithelial cells undergo synchronized beating to produce fluid flow along the luminal surface of various organs. Each motile cilium consists of an axoneme and a basal body, which are linked by a transition zone. The axoneme exhibits a characteristic 9+2 microtubule arrangement important for ciliary motion, but how this microtubule system is generated is not yet fully understood. Here we show that CAMSAP3, a protein that can stabilize the minus end of a microtubule, concentrates at multiple sites of the cilium-basal body complex, including the upper region of the transition zone or the axonemal basal plate where the central pair of microtubules (CP) initiates. CAMSAP3 dysfunction resulted in loss of the CP and partial distortion of the basal plate, as well as the failure of multicilia to undergo synchronized beating. These findings suggest that CAMSAP3 plays pivotal roles in the formation or stabilization of the CP by localizing at the basal region of the axoneme, and thereby supports the coordinated motion of multicilia in airway epithelial cells.

cell biology↗

bioRxiv2020

Suppression of amyloid-β secretion from neurons by cis-9, trans-11-octadecadienoic acid, an isomer of conjugated linoleic acid

Conjugated linoleic acid (CLA) comprises several geometric and positional isomers of the parental linoleic acid (LA). Two of the isomers, cis-9, trans-11 CLA (c9,t11 CLA) and trans-10, cis-12 CLA (t10,c12 CLA) exert various biological activities. However, the effect of CLA on generation of neurotoxic amyloid-{beta} (A{beta}) protein remains unclear. We found that c9,t11CLA significantly suppressed generation of A{beta} in primary cultures of mouse neurons. CLA treatment did not affect the levels of {beta}-site APP-cleaving enzyme 1 (BACE1), a component of active {gamma}-secretase complex presenilin 1 amino-terminal fragment (PS1 NTF), or A{beta} protein precursor (APP) in cultured neurons. BACE1 activity in lysate of neurons treated with c9,t11 CLA, but not t10,c12 CLA, decreased slightly, although c9,t11 CLA did not directly affect the activity of recombinant BACE1. Interestingly, localization of BACE1 and APP in early endosomes increased in neurons treated with c9,t11 CLA; concomitantly, the localization of both proteins was reduced in late endosomes, where APP is predominantly cleaved by BACE1. c9,t11 CLA and t10,c12 CLA appeared to be incorporated into membrane phospholipids, as the level of CLA-containing lysophosphatidylcholine (CLA-LPC) increased dramatically in neurons incubated with CLA. Taken together, our findings indicate that accumulation of c9,t11 CLA-LPC, but not t10,c12 CLA-LPC, in neuronal membranes suppresses amyloidogenic cleavage of APP, thereby contributing to preservation of brain neurons by suppressing neurotoxic A{beta} production in aged subjects.

biochemistry↗