Biology subjects

Sato, Y.

Publications and source records attributed to Sato, Y..

At least 91 records · Page 5Linked to original sources

bioRxiv2023

Unravelling ATP processing by the AAA+ protein p97 at the atomic level

The human enzyme p97 regulates various cellular pathways by unfolding hundreds of protein substrates in an ATP-dependent manner, making it an essential component of homeostasis and impactful pharmacological target. The hexameric complex undergoes substantial conformational changes in the course of its catalytic cycle. Here, we elucidate the molecular motions that occur at the active site in the temporal window immediately before and after ATP hydrolysis by merging cryo-EM, NMR spectroscopy and MD simulations. p97 populates a metastable reaction intermediate, the ADP.Pi state, which is poised between hydrolysis and product release. Detailed snapshots reveal that the active site is finely tuned to trap and eventually discharge the cleaved phosphate. Signalling pathways originating at the active site coordinate the action of the hexamer subunits and couple hydrolysis with allosteric conformational changes. Our multidisciplinary approach enables a glimpse into the sophisticated spatial and temporal orchestration of ATP handling by a prototype AAA+ protein.

biophysics↗

bioRxiv2023

Volumetric trans-scale imaging of massive quantity of heterogeneous cell populations in centimeter-wide tissue and embryo

We established a volumetric trans-scale imaging system with an ultra-large field-of-view (FOV) that enables simultaneous observation of millions of cellular dynamics in centimeter-wide three-dimensional (3D) tissues and embryos. Using a custom-made giant lens system with a magnification of 2x and a numerical aperture (NA) of 0.25, and a CMOS camera with more than 100 megapixels, we built a trans-scale scope AMATERAS-2, and realized fluorescence imaging with a transverse spatial resolution of approximately 1.1 {micro}m across an FOV of approximately 1.5 x 1.0 cm2. The 3D resolving capability was realized through a combination of optical and computational sectioning techniques tailored for our low-power imaging system. We applied the imaging technique to 1.2 cm-wide section of mouse brain, and successfully observed various regions of the brain with sub-cellular resolution in a single FOV. We also performed time-lapse imaging of a 1-cm-wide vascular network during quail embryo development for over 24 hours, visualizing the movement of over 4.0 x 105 vascular endothelial cells and quantitatively analyzing their dynamics. Our results demonstrate the potential of this technique in accelerating production of comprehensive reference maps of all cells in organisms and tissues, which contributes to understanding developmental processes, brain functions, and pathogenesis of disease, as well as high-throughput quality check of tissues used for transplantation medicine.

developmental 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

Monitoring single root hairs using a micro-chambered hydroponics system MiCHy reveals a new mode of action of the aminosteroid U73122

When using chemical probes and inhibitors for cell biological studies, the uniform and rapid access of the chemical to the sample is key for the accuracy of the analysis. Until now, however, this could only be accomplished by using expensive equipment and labor-intensive and often lengthy procedures. Here, we introduce MiCHy, a micro-chambered hydroponics system, as an accessible and versatile method for chemical treatments and demonstrate its use for imaging roots of Arabidopsis thaliana, the major model system for plant cell biological research. Using lipid biosensors in combination with established chemicals, such as the phosphoinositol 4-kinase (PI4K) inhibitor phenylarsine oxide and the phosphoinositide-specific phospholipase C (PI-PLC) inhibitor U73122, we demonstrate the suitability of MiCHy for studying the relationship between growth and lipid dynamics in root hairs. We further reveal novel effects of U73122 on the plasma membrane localization of phosphatidylinositol-4-phosphate 5-kinase 3 (PIP5K3), which is the first evidence demonstrating a role for U73122 besides its role as a PI-PLC inhibitor in plants.

plant biology↗

bioRxiv2023

Seed size control via phloem end by callose deposition/degradation of β-1,3-glucanase

Seed formation is crucial for lives of plants as well as humans; however, the mechanisms governing seed size require further investigation. Here, we present a new mechanism to modify the seed size by the newly identified phloem end that support nutrient transport, at the chalazal end of the ovule, however, blocked by callose deposition. Callose is removed after central cell fertilization (open state), allowing nutrients to be transported to the seed. However, if fertilization fails, callose deposition persists (closed state), preventing the phloem end from transporting nutrients. {beta}-1,3-glucanase genes, including putative plasmodesmata-associated proteins (AtBG_ppap), were identified as regulators of callose removal. The Atbg_ppap mutant had the phloem end in the closed state and produced smaller seeds due to incomplete callose degradation. In contrast, the AtBG_ppap overexpression line produced larger seeds than the wild type due to continuous callose degradation, indicating that the phloem end regulates substance flow via callose deposition/degradation.

plant biology↗

bioRxiv2023

Design of artificial molecular motor inheriting directionality and scalability.

Realizing artificial molecular motors with autonomous functionality and high performance is a major challenge in biophysics. Such motors not only provide new perspectives in biotechnology but also offer a novel approach for the bottom-up elucidation of biological molecular motors. Directionality and scalability are critical factors for practical applications. However, the simultaneous realization of both remains challenging. In this study, we propose a novel design for a rotary motor that can be fabricated using a currently available technology, DNA origami, and validate its functionality through simulations with practical parameters. We demonstrate that the motor rotates unidirectionally and processively in the direction defined by structural asymmetry, which induces kinetic asymmetry in motor motion. The motor also exhibits scalability, such that increasing the number of connections between the motor and stator allows for a larger speed, run length, and stall force. Statement of SignificanceBiological molecular motors are the most sophisticated nanomachines found in nature. Despite a long history of research, we have yet to understand how they work in detail. A simple and promising approach involves the building of artificial molecular motors. The process of achieving these goals helps us understand how the structures and mechanisms of the biological motors have been optimized through the evolution of their functions. Some artificial motors have already been achieved. However, each realization implements only a part of the characteristics of biological motors. In this work, we propose a novel motor mechanism that simultaneously implements two essential characteristics: scalability and directionality. These results will help us improve the biophysics of molecular motors and contribute to developing high-performance artificial molecular motors with possible industrial applications.

biophysics↗

bioRxiv2023

Polygenic Risk Score Associates with Atherosclerotic Plaque Characteristics at Autopsy

BackgroundPolygenic risk scores (PRS) for coronary artery disease (CAD) potentially improve cardiovascular risk prediction. However, their relationship with histopathologic features of CAD has never been examined systematically. MethodsFrom 4,327 subjects referred to CVPath by the State of Maryland Office Chief Medical Examiner (OCME) for sudden death between 1994 and 2015, 2,455 cases were randomly selected for genotyping. We generated PRS from 291 known CAD risk loci. Detailed histopathologic examination of the coronary arteries was performed in all subjects. The primary study outcome measurements were histopathologic plaque features determining severity of atherosclerosis, including %stenosis, calcification, thin-cap fibroatheromas (TCFA), and thrombotic CAD. ResultsAfter exclusion of cases with insufficient DNA sample quality or with missing data, 954 cases (mean age 48.8{+/-}14.7; 75.7% men) remained in the final study cohort. Subjects in the highest PRS quintile exhibited more severe atherosclerosis compared to subjects in the lowest quintile, with greater %stenosis (80.3%{+/-}27.0% vs. 50.4%{+/-}38.7%; adjusted p<0.001) and a higher frequency of calcification (69.6% vs. 35.8%; adjusted p=0.004) and TCFAs (26.7% vs. 9.5%; adjusted p=0.007). Even after adjustment for traditional CAD risk factors subjects within the highest PRS quintile had higher odds of severe atherosclerosis (i.e., [≥]75% stenosis; adjusted OR 3.77; 95%CI 2.10-6.78; p<0.001) and plaque rupture (adjusted OR 4.05; 95%CI 2.26-7.24; p<0.001). Moreover, subjects within the highest quintile had higher odds of CAD-associated cause of death, especially among those aged 50 years and younger (adjusted OR 4.08; 95%CI 2.01-8.30; p<0.001). No associations were observed with plaque erosion. ConclusionsThis is the first autopsy study investigating associations between PRS and atherosclerosis severity at the histopathologic level in subjects with sudden death. Our pathological analysis suggests PRS correlates with plaque burden and features of advanced atherosclerosis and may be useful as a method for CAD risk stratification, especially in younger subjects. HighlightsO_LIIn this autopsy study including 954 subjects within the CVPath Sudden Death Registry, high PRS correlated with plaque burden and atherosclerosis severity. C_LIO_LIThe PRS showed differential associations with plaque rupture and plaque erosion, suggesting different etiologies to these two causes of thrombotic CAD. C_LIO_LIPRS may be useful for risk stratification, particularly in the young. Further examination of individual risk loci and their association with plaque morphology may help understand molecular mechanisms of atherosclerosis, potentially revealing new therapy targets of CAD. C_LI Graphic AbstractA polygenic risk score, generated from 291 known CAD risk loci, was assessed in 954 subjects within the CVPath Sudden Death Registry. Histopathologic examination of the coronary arteries was performed in all subjects. Subjects in the highest PRS quintile exhibited more severe atherosclerosis as compared to subjects in the lowest quintile, with a greater plaque burden, more calcification, and a higher frequency of plaque rupture.

genetics↗

bioRxiv2023

Automated escape system: identifying prey's kinematic and behavioral features critical for predator evasion

O_LIIdentifying the kinematic and behavioral variables of prey that influence the evasion from predator attacks is essential not only for comprehending the determinants of successful predator evasion but also for shedding light on the evolution of specific traits and the dynamics of predator-prey relationships on a larger scale. However, quantifying the relationship between these variables and the success or failure of predator evasion is challenging, particularly for variables with small variations within prey species. One promising approach to address this challenge is the use of a simulated prey system, which allows us to manipulate the kinematic and behavioral features of prey and expose them to real predators. Nevertheless, creating a system that moves comparably to real prey animals remains difficult, especially for invertebrate and lower vertebrate species that respond quickly to predators and escape rapidly. C_LIO_LIIn this study, we have developed an automated escape system that is comparable to real prey species, responding to a predator within tens of milliseconds and escaping at over 1.0 m/s. The system automatically detects an approaching predator and pulls the prey away from the predator once the predator reaches a predetermined threshold distance. Reaction distance, response latency, as well as escaping speed, duration, and direction can be adjusted in the system. C_LIO_LIBy repeatedly measuring the response latency and escaping speed of the system, we demonstrated the systems ability to exhibit fast and rapid responses while maintaining consistency across successive trials. As a case study, we manipulated the escape speed and reaction distance of the prey to expose them to a predatory fish, Coreoperca kawamebari. The results show that both variables significantly affect the outcome of predator-prey interactions. C_LIO_LIThese findings indicate that the developed escape system is useful for identifying kinematic and behavioral features of prey that are critical for predator evasion. Moreover, due to its relatively low cost and customizability, we propose that this system can be applied to investigate various aspects of animal behaviors (e.g., eliciting escape responses by artificial stimuli) in different animal species. C_LI

animal behavior and cognition↗

bioRxiv2023

Type-2 diabetes alters hippocampal neural oscillations and disrupts synchrony between hippocampus and cortex

Type 2 diabetes mellitus (T2DM) increases the risk of neurological diseases, yet how brain oscillations change as age and T2DM interact is not well characterized. To delineate the age and diabetic effect on neurophysiology, we recorded local field potentials with multichannel electrodes spanning the somatosensory cortex and hippocampus (HPC) under urethane anesthesia in diabetic and normoglycemic control mice, at 200 and 400 days of age. We analyzed the signal power of brain oscillations, brain state, sharp wave associate ripples (SPW-Rs), and functional connectivity between the cortex and HPC. We found that while both age and T2DM were correlated with a breakdown in long-range functional connectivity and reduced neurogenesis in the dentate gyrus and subventricular zone, T2DM further slowed brain oscillations and reduced theta-gamma coupling. Age and T2DM also prolonged the duration of SPW-Rs and increased gamma power during SPW-R phase. Our results have identified potential electrophysiological substrates of hippocampal changes associated with T2DM and age. The perturbed brain oscillation features and diminished neurogenesis may underlie T2DM-accelerated cognitive impairment.

neuroscience↗

bioRxiv2023

Proteomic characterization of aging-driven changes in the mouse brain by co-expression network analysis

Brain aging causes a progressive decline in functional capacity and is a strong risk factor for dementias such as Alzheimers disease. To characterize age-related proteomic changes in the brain, we used quantitative proteomics to examine brain tissues, cortex and hippocampus, of mice at three age points (3, 15, and 24 months old), and quantified more than 7,000 proteins in total with high reproducibility. We found that many of the proteins upregulated with age were extracellular proteins, such as extracellular matrix proteins and secreted proteins, associated with glial cells. On the other hand, many of the significantly downregulated proteins were associated with synapses, particularly postsynaptic density, specifically in the cortex but not in the hippocampus. Our datasets will be helpful as resources for understanding the molecular basis of brain aging.

molecular biology↗

bioRxiv2023

Genome-wide neighbor effects predict genotype pairs that reduce herbivory in mixed planting

Genetically diverse populations can increase plant resistance to natural enemies. Yet, beneficial genotype pairs remain elusive due to the occurrence of both positive and negative effects of mixed planting on plant resistance, called associational resistance and susceptibility. We used genome-wide polymorphisms of the plant species Arabidopsis thaliana to identify genotype pairs that enhance associational resistance to herbivory. By quantifying neighbor interactions among 199 genotypes grown in a randomized block design, we predicted that 823 of the 19,701 candidate pairs could reduce herbivory through associational resistance. We planted such pairs with predicted associational resistance in mixtures and monocultures and found a significant reduction in herbivore damage in the mixtures. Our study highlights the potential application to assemble genotype mixtures with positive biodiversity effects.

ecology↗

bioRxiv2023

Characteristics of the rumen virome in Japanese cattle

The rumen microbiome is a highly complex ecosystem that includes bacteria, archaea, protozoa, fungi, and viruses. Viruses have a high potential to modify the rumen digestion of feeds via infection and cell lysis of prokaryotes in the rumen; however, understanding of the rumen virome is substantially less advanced due to limitations of the reference genome database. In this study, we conducted metagenomic sequencing of virus-like particles (VLPs) in the rumens of 22 Japanese cattle to construct a reference viral genome catalog of the rumen and uncover the rumen virome characteristics. We succeeded in construction of 8 232 nonredundant viral genomes ([≥]5 kb length and [≥]50% completeness). Among them, putative hosts of 1 223 virus genomes were predicted, and 1 053 virus genomes were taxonomically classified, mainly Siphoviridae, Myoviridae, and Podoviridae. Additionally, 2 764 putative auxiliary metabolic genes (AMGs) were identified in the viral genomes. Importantly, 22 viral genomes associated with archaea in the rumen were identified. Some archaeal viruses have AMGs related to DNA synthesis, suggesting that archaeal viruses control archaeal populations in the rumen and affect methane production from the rumen. Furthermore, we revealed that most rumen viruses were highly rumen-and individual-specific and related to rumen-specific prokaryotes. Overall, the rumen viral catalog and findings of this study will help future analyses to uncover the roles of rumen viruses in feed digestion, productivity, and methane production.

microbiology↗

bioRxiv2023

Integrative analysis of in vitro strategies to induce human vascularized cerebral organoids using single-cell RNA sequencing data

Cerebral organoids are three-dimensional in vitro cultured brains that mimic the function and structure of the human brain. One of the major challenges for cerebral organoids is the lack of functional vasculature. Without perfusable vessels, oxygen and nutrient supplies may be insufficient for long-term culture, hindering the investigation of the neurovascular interactions. Recently, several strategies for the vascularization of human cerebral organoids have been reported. However, the generalizable trends and variability among different strategies are unclear due to the lack of a comprehensive characterization and comparison of these vascularization strategies. In this study, we aimed to explore the effect of different vascularization strategies on the nervous system and vasculature in human cerebral organoids. We integrated single-cell RNA sequencing data of multiple vascularized and vascular organoids and fetal brains from publicly available datasets and assessed the protocol-dependent and culture-day-dependent effects on the cell composition and transcriptomic profiles in neuronal and vascular cells. We revealed the similarities and uniqueness of multiple vascularization strategies and demonstrated the transcriptomic effects of vascular induction on neuronal and mesodermal-like cell populations. Moreover, our data suggested that the interaction between neurons and mesodermal-like cell populations is important for the cerebrovascular-specific profile of endothelial-like cells. This study highlights the current challenges to vascularization strategies in human cerebral organoids and offers a benchmark for the future fabrication of vascularized organoids.

neuroscience↗

bioRxiv2022

Genome-wide association study highlights escape from aphids by delayed growth in Arabidopsis thaliana

Field studies have shown that plant phenological and architectural traits often explain substantial variation in herbivory. Although plant genes involved in physical and chemical defense are well studied, less is known about the genetic basis underlying effects of plant growth traits on herbivory. Here, we conducted a genome-wide association study (GWAS) of aphid abundance in a field population of Arabidopsis thaliana. This field GWAS detected a significant peak on the third chromosome of A. thaliana. Out of candidate genes near this significant genomic region, a mutant of a ribosomal gene (AT3G13882) exhibited slower growth and later flowering than a wild type under laboratory conditions. A no-choice assay with the turnip aphid, Lipaphis erysimi, found that aphids were unable to successfully establish on the mutant. These findings suggest the potential role of growth-related genes in altering herbivore abundance.

ecology↗

bioRxiv2022

Actin filaments accumulate in the nucleus during interphase and remain in the vicinity of condensing chromosomes during prophase to metaphase in zebrafish early embryos

In the cytoplasm, filamentous actin (F-actin) plays a critical role in cell regulation, including cell migration, stress fiber formation, and cytokinesis. Recent studies have shown that actin filaments that form in the nucleus are associated with diverse functions. Here, using live imaging of an F-actin-specific probe, superfolder GFP-tagged utrophin (UtrCH-sfGFP), we demonstrated the dynamics of nuclear actin in zebrafish (Danio rerio) embryos. In early zebrafish embryos up to around the high stage, UtrCH-sfGFP increasingly accumulated in nuclei during the interphase and reached a peak during the prophase. After nuclear envelope breakdown (NEBD), patches of UtrCH-sfGFP remained in the vicinity of condensing chromosomes during the prometaphase to metaphase. When zygotic transcription was inhibited by injecting -amanitin, the nuclear accumulation of UtrCH-sfGFP was still observed at the sphere and dome stages, suggesting that zygotic transcription may induce a decrease in nuclear F-actin. The accumulation of F-actin in nuclei may contribute to proper mitotic progression of large cells with rapid cell cycles in zebrafish early embryos, by assisting in NEBD, chromosome congression, and/or spindle assembly. Summary statementFilamentous actin accumulates in the nucleus of zebrafish early embryos and forms patches associating with condensing chromosomes during prophase.

cell biology↗

bioRxiv2022

Microbial mat compositions and localization patterns explain the virulence of black band disease in corals

Black band disease (BBD) in corals is characterized by a distinctive, band-like microbial mat, which spreads across the tissues and often kills infected colonies. The microbial mat is dominated by cyanobacteria but also commonly contains sulfide-oxidizing bacteria (SOB), sulfate-reducing bacteria (SRB), and other microbes. The migration rate in BBD varies across different environmental conditions including temperature, light, and pH. However, whether variations in the migration rates reflect differences in the microbial consortium within the BBD mat remains unknown. Here, we show that the micro-scale surface structure, bacterial composition, and spatial distribution differed across BBD lesions with different migration rates. The migration rate was positively correlated with the relative abundance of potential SOBs belonging to Arcobacteraceae localized in the middle layer within the mat and negatively correlated with the relative abundance of other potential SOBs belonging to Rhodobacteraceae. Our study highlights the microbial composition in BBD as an important determinant of virulence.

microbiology↗

bioRxiv2022

Aquaporin regulates cell rounding through vacuole formation during endothelial-to-hematopoietic transition

Endothelial-to-hematopoietic transition (EHT) is crucial for hematopoietic stem cell (HSC) generation. During EHT, the morphology of hemogenic endothelial cells (HECs) changes from flat and adherent to spherical HSCs, which detach from the dorsal aorta. HECs attain a rounded shape in a mitosis-independent manner before cell adhesion termination, suggesting an atypical cell-rounding mechanism. However, the direct mechanisms underlying this change in cell morphology during EHT remain unclear. Here, we show that large vacuoles were transiently formed in HECs and that aquaporin-1 (AQP1) was localized in the vacuole and plasma membranes. Overexpression of AQP1 in non-HECs induced ectopic vacuole expansion, cell rounding, and subsequent cell detachment from the endothelium into the bloodstream, mimicking EHT. Loss of redundant AQP functions by CRISPR/Cas9 gene editing in HECs impeded the morphological EHT. Our findings provide the first evidence indicating that morphological segregation of HSCs from endothelial cells is regulated by water influx into vacuoles. These findings provide important insights for further exploration of the mechanisms underlying cell/tissue morphogenesis through water-adoptive cellular responses. SUMMARY STATEMENTHemogenic endothelial cells transiently form large vacuoles during endothelial-to-hematopoietic transition. Aquaporin water channels regulate cell rounding and detachment of emerging hematopoietic stem cells through vacuole formation.

developmental biology↗

bioRxiv2022

Identification of lactic acid bacteria with reduced inflammatory and enhanced protective properties against influenza virus

Lactic acid bacteria (LAB) exert beneficial effects on health by regulating innate immunity in the intestinal tract. Many LAB that demonstrate anti-viral properties depend on the hosts inflammatory responses; however, a few LAB strains have low inflammatory and anti-viral properties. In this study, the LAB strain D279 (NITE_BP-03645, Latilactobacillus sakei) was isolated from among 741 LAB strains that were analyzed for their ability to induce interleukin 12 (IL-12), and was subsequently characterized. D279 induced the highest levels of IL-12 among the screened LAB. Further, D279 significantly activated anti-viral genes and preferentially induced IFN{lambda} in vitro, which plays a critical role in the epithelial tissue, conferring strong anti-influenza potency without inflammation, whereas IFN levels were relatively lower. Administration of pasteurized D279 to mice resulted in strong anti-influenza potency, including higher natural killer (NK) cell activity, and a reduced number of viruses in the lung compared to the control. Importantly, no D279-fed mice were sacrificed during the viral infection tests. Taken together, these results suggest that D279 administration confers beneficial effects with reduced inflammation, regulates innate immunity, and may be relevant for commercial use in the future.

microbiology↗