Biology subjects

Yoshida, M.

Publications and source records attributed to Yoshida, M..

At least 73 records · Page 4Linked to original sources

bioRxiv2021

Mapping interindividual dynamics of innate immune response at single-cell resolution

Common genetic variants modulate the cellular response to viruses and are implicated in a range of immune pathologies, including infectious and autoimmune diseases. The transcriptional antiviral response is known to vary between infected cells from a single individual, yet how genetic variants across individuals modulate the antiviral response (and its cell-to-cell variability) is not well understood. Here, we triggered the antiviral response in human fibroblasts from 68 healthy donors, and profiled tens of thousands of cells using single-cell RNA-seq. We developed GASPACHO (GAuSsian Processes for Association mapping leveraging Cell HeterOgeneity), the first statistical approach designed to identify dynamic eQTLs across a transcriptional trajectory of cell populations, without aggregating single-cell data into pseudo-bulk. This allows us to uncover the underlying architecture and variability of antiviral response across responding cells, and to identify more than two thousands eQTLs modulating the dynamic changes during this response. Many of these eQTLs colocalise with risk loci identified in GWAS of infectious and autoimmune diseases. As a case study, we focus on a COVID-19 susceptibility locus, colocalised with the antiviral OAS1 splicing QTL. We validated it in blood cells from a patient cohort and in the infected nasal cells of a patient with the risk allele, demonstrating the utility of GASPACHO to fine-map and functionally characterise a genetic locus. In summary, our novel analytical approach provides a new framework for delineation of the genetic variants that shape a wide spectrum of transcriptional responses at single-cell resolution.

genetics↗

bioRxiv2021

NR4A3 is oxidative stress responsible transcription factor through HMOX1, and also controls cell cycle through CCNE1 and CDK2 in pancreatic islet derived 1.1B4 cells

The mechanism of antioxidant defense system is still controversial. As islet {beta}-cell is weak in oxidative condition, that causes diabetes mellitus, therefore, antioxidant defense system of human pancreatic islet derived 1.1B4 cell was analyzed. Cells were exposed to H2O2 and comprehensive gene expression was analyzed by Agilent human microarray. HMOX1 and NR4A3, member of orphan receptor, were up-regulated. Therefore, NR4A3 was knocked down with siRNA, then analyzed gene expression by microarray, and found that the knocked down cells were weak in oxidative stress. HMOX1 expression was strongly inhibited by siRNA of NR4A3, and NR4A3 responsible sequence of aaggtca was found near the HMOX1 gene, suggesting NR4A3 is oxidative stress responsible transcription factor through HMOX1 expression. The expression of CCNE1 and CDK2 was also inhibited by knocked down of NR4A3, it is suggested NR4A3 is also important transcription factor for cell growth regulation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/457070v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@18d0371org.highwire.dtl.DTLVardef@dd1eacorg.highwire.dtl.DTLVardef@108b1b9org.highwire.dtl.DTLVardef@1cbf899_HPS_FORMAT_FIGEXP M_FIG Hydrogen peroxide induces NR4A3 and binds to aaggtca sequence of HMOX1, and increased transcription of HMOX1. Resulting heme oxygenase produces biliverdin, antioxidants, from heme. NR4A3 also bind to aaggtca sequence of CDK2 and CCNE1, resulting CDK2 and Cyclin E. CDK2 bind to cyclin E and cell goes from G1 to S phase. C_FIG

genomics↗

bioRxiv2021

Does the rainbow trout ovarian fluid guide the spermatozoon on its way to the egg?

Fertilization of freshwater fish occurs in an environment that may negatively affect the gametes; therefore, the specific mechanisms triggering the encounters of gametes would be highly expedient. The egg and ovarian fluid (OF) are likely the primary sources of these triggers in fish that we confirmed here for rainbow trout Oncorhynchus mykiss. The ovarian fluid significantly affected the spermatozoa performance: it supported high velocity for a more extended period and changed the motility pattern from tumbling in water to straightforward moving in the ovarian fluid. Rainbow trout OF induced a trapping chemotaxis-like effect on activated male gametes, and this effect depended on the properties of the activating media. The interaction of the spermatozoa with the attracting agents was accompanied by their "turn-and-run" behavior involving asymmetric flagellar beating and Ca2+ concentration bursts in the bent flagella segment, characteristic of the chemotactic response. Collectively, the ovarian fluid creates the optimal environment for rainbow trout spermatozoa performance, being an effective promoter of fertilization. The individual peculiarities of the egg (ovarian fluid) - sperm interaction in rainbow trout reflect the specific features of the spawning process in this species.

ecology↗

bioRxiv2021

CatSper mediates the chemotactic behavior and motility of the ascidian sperm

Sperm motility, including chemotactic behavior, is regulated by changes in the intracellular Ca2+ concentration. The cation channel of sperm (CatSper), plays an important role in the regulation of intracellular Ca2+ concentration. In mammals, CatSper is the only Ca2+ channel that functions in the sperm, and the mice that lack the genes for the subunits of CatSper, which make up the pore region of the Ca2+ channel, are infertile due to the inhibition of hyperactivation of the sperm. CatSper is also thought to be involved in chemotaxis in sea urchins. In contrast, in the ascidian, Ciona intestinalis, the sperm-activating and -attracting factor (SAAF) interacts with Ca2+/ATPase, which is a Ca2+-pump. Although the existence of CatSper genes has been reported, it is not clear whether CatSper is the specific Ca2+ channel that functions in the ascidian sperm. Therefore, in this study, we generated Catsper3 knockout (KO) animals that found that they were significantly less motile, with few motile sperms not exhibiting any chemotactic behavior. These results suggest that CatSper plays important roles in the spermatogenesis and basic motility mechanisms of sperms in both ascidians and mammals.

developmental biology↗

bioRxiv2021

Structure-based Classification of Tauopathies

Ordered assembly of the tau protein into filaments characterizes multiple neurodegenerative diseases, which are called tauopathies. We previously reported that by electron cryo-microscopy (cryo-EM), tau filament structures from Alzheimers disease (1,2), chronic traumatic encephalopathy (CTE) (3), Picks disease (4) and corticobasal degeneration (CBD) (5) are distinct. Here we show that the structures of tau filaments from typical and atypical progressive supranuclear palsy (PSP), the most common tauopathy after Alzheimers disease, define a previously unknown, three-layered fold. Moreover, the tau filament structures from globular glial tauopathy (GGT, Types I and II) are similar to those from PSP. The tau filament fold of argyrophilic grain disease (AGD) differs from the above and resembles the four-layered CBD fold. The majority of tau filaments from aging-related tau astrogliopathy (ARTAG) also have the AGD fold. Surprisingly, tau protofilament structures from inherited cases with mutations +3/+16 in intron 10 of MAPT, the microtubule-associated protein tau gene, are identical to those from AGD, suggesting that a relative overproduction of four-repeat tau can give rise to the AGD fold. Finally, tau filament structures from cases of familial British dementia (FBD) and familial Danish dementia (FDD) are the same as those from Alzheimers disease and primary age-related tauopathy (PART). These structures provide the basis for a classification of tauopathies that also allows identification of new entities, as we show here for a case diagnosed as PSP, but with abundant spherical 4R tau inclusions in limbic and other brain areas. The structures of the tau fold of this new disease (Limbic-predominant Neuronal inclusion body 4R Tauopathy, LNT) were intermediate between those of GGT and PSP.

neuroscience↗

bioRxiv2020

White matter alterations in glaucoma and vision-deprived brains differ outside the visual system

The degree to which glaucoma has effects beyond the eye -in the brain- is unclear. We investigated white matter microstructure (WMM) alterations in 37 tracts of patients with glaucoma, monocular blindness and controls. We used reproducible methods and the advanced cloud computing platform brainlife.io. White matter tracts were subdivided into seven categories ranging from primarily involved in vision (the visual white matter) to primarily involved in cognition and motor control. WMM in both glaucoma and monocular blind subjects was lower than controls in the visual white matter, suggesting neurodegenerative mechanisms due to reduced sensory inputs. In glaucoma participants WMM differences from controls decreased outside the visual white matter. A test-retest validation approach was used to validate these results. The pattern of results was different in monocular blind participants, where WMM properties increased outside the visual white matter as compared to controls. The pattern of results suggests that whereas in the blind loss of visual input might promote white matter reorganization outside of the early visual system, such reorganization might be reduced or absent in glaucoma. The results provide indirect evidence that in glaucoma unknown factors might limit the brain plasticity effects that in other patient groups follow visual loss.

neuroscience↗

bioRxiv2020

Seeded assembly in vitro does not replicate the structures of α-synuclein filaments from multiple system atrophy

The propagation of conformational strains by templated seeding is central to the prion concept. Seeded assembly of -synuclein into filaments is believed to underlie the prion-like spreading of protein inclusions in a number of human neurodegenerative diseases, including Parkinson's disease, dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). We previously determined the atomic structures of -synuclein filaments from the putamen of five individuals with MSA. Here, we used filament preparations from three of these brains for the in vitro seeded assembly of recombinant human -synuclein. We find that the structures of the seeded assemblies differ from those of the seeds, suggesting that additional, as yet unknown, factors play a role in the propagation of pathology. Identification of these factors will be essential for understanding the prion-like spreading of -synuclein proteinopathies.

molecular biology↗

bioRxiv2020

Splicing modulators elicit global translational repression by condensate-prone proteins translated from introns

Chemical splicing modulators that bind to the spliceosome have provided an attractive venue for cancer treatment. Splicing modulators induce accumulation and subsequent translation of a subset of intron-retained mRNAs. Yet, the biological effect of proteins containing translated intron sequences remains unclear. Here we identified a number of truncated proteins generated upon treatment with the splicing modulator spliceostatin A (SSA) using genome-wide ribosome profiling and bio-orthogonal non-canonical amino-acid tagging (BONCAT) mass spectrometry. A subset of these truncated proteins has intrinsically disordered regions, forms insoluble cellular condensates, and triggers the proteotoxic stress response through JNK phosphorylation, thereby inhibiting the mTORC1 pathway. In turn, this reduces global translation. These findings indicate that creating an overburden of condensate-prone proteins derived from introns represses translation and prevents further production of harmful truncated proteins. This mechanism appears to contribute to the antiproliferative and proapoptotic activity of splicing modulators.

molecular biology↗

bioRxiv2020

A Novel DNA Chromatography Method to Distinguish M. abscessus Subspecies and Macrolide Susceptibility

RationaleThe clinical impact of infection with Mycobacterium abscessus complex (MABC), a group of emerging non-tuberculosis mycobacteria (NTM), is increasing. Mycobacterium abscessus subsp. abscessus/bolletii frequently shows natural resistance to macrolide antibiotics, whereas Mycobacterium abscessus subsp. massiliense is generally susceptible. Therefore, rapid and accurate discrimination of macrolide-susceptible MABC subgroups is required for effective clinical decisions about macrolide treatments for MABC infection. ObjectivesTo develop a simple and rapid diagnostic that can identify MABC isolates showing macrolide susceptibility. MethodsWhole genome sequencing (WGS) was performed for 148 clinical or environmental MABC isolates from Japan to identify genetic markers that can discriminate three MABC subspecies and the macrolide-susceptible erm(41) T28C sequevar. Using the identified genetic markers, we established PCR based- or DNA chromatography-based assays. Validation testing was performed using MABC isolates from Taiwan. Measurements and Main ResultsWe identified unique sequence regions that could be used to differentiate the three subspecies. Our WGS-based phylogenetic analysis indicated that M. abscessus carrying the macrolide-susceptible erm(41) T28C sequevar were tightly clustered, and identified 11 genes that were significantly associated with the lineage for use as genetic markers. To detect these genetic markers and the erm(41) locus, we developed a DNA chromatography method that identified three subspecies, the erm(41) T28C sequevar and intact erm(41) for MABC in a single assay within one hour. The agreement rate between the DNA chromatography-based and WGS-based identification was 99.7%. ConclusionsWe developed a novel, rapid and simple DNA chromatography method for identification of MABC macrolide susceptibility with high accuracy.

microbiology↗

bioRxiv2020

Reward-associated configural cues elicit theta oscillations of rat retrosplenial neurons phase-locked to LFP theta cycle

Previous behavioral studies implicated the retrosplenial cortex (RSC) in stimulus-stimulus associations, and also in retrieval of remote associative memory based on EEG theta oscillations. To investigate the neural mechanisms underlying these processes, RSC neurons and local field potentials (LFPs) were simultaneously recorded from well-trained rats performing a cue-reward association task. In the task, simultaneous presentation of two multimodal conditioned stimuli (configural CSs) predicted a reward outcome opposite to that associated with individual presentation of each elemental CS. Here, we show neurophysiological evidence that the RSC is involved in stimulus-stimulus association where configural CSs are discriminated from each elementary CS that is a constituent of the configural CSs, and that memory retrieval of rewarding CSs is associated with theta oscillation of RSC neurons during CS presentation, which is phase-locked to LFP theta cycles.

neuroscience↗

bioRxiv2020

Common properties of visually-guided saccadic behavior and bottom-up attention in marmoset, macaque, and human

The saccade is a stereotypic behavior whose investigation improves our understanding of how primate brains implement precise motor control. Furthermore, saccades offer an important window into the cognitive and attentional state of the brain. Historically, saccade studies have largely relied on macaque. However, the cortical network giving rise to the saccadic command is difficult to study in macaque because relevant cortical areas lie in sulci and are difficult to access. Recently, a New World monkey - the marmoset - has garnered attention as an attractive alternative to macaque because of its smooth cortical surface, its smaller body, and its amenability to transgenic technology. However, adoption of marmoset for oculomotor research has been limited due to a lack of in-depth descriptions of marmoset saccade kinematics and their ability to perform psychophysical and cognitive tasks. Here, we directly compare free-viewing and visually-guided behavior of marmoset, macaque, and human engaged in identical tasks under similar conditions. In video free-viewing task, all species exhibited qualitatively similar saccade kinematics including saccade main sequence up to 25{degrees} in amplitude. Furthermore, the conventional bottom-up saliency model predicted gaze targets at similar rates for all species. We further verified their visually-guided behavior by training them with step and gap saccade tasks. All species showed similar gap effect and express saccades in the gap paradigm. Our results suggest that the three species have similar natural and task-guided visuomotor behavior. The marmoset can be trained on saccadic tasks and thus can serve as a model for oculomotor, attention, and cognitive research. New & noteworthyWe directly compared the results of video free-viewing task and visually-guided saccade tasks (step and gap) among three different species: the marmoset, macaque and human. We found that all species exhibit qualitatively similar saccadic behavior and bottom-up saliency albeit with small differences. Our results suggest that the marmoset possesses similar neural mechanisms to macaque and human for saccadic control, and it is an appropriate model animal to study neural mechanisms for active vision and attention.

animal behavior and cognition↗

bioRxiv2020

Selective inhibitors of SIRT2 regulate perinuclear α-tubulin acetylation, migration, and invasion of breast cancer cells

Sirtuin 2 (SIRT2) is a protein deacylase enzyme that removes acetyl groups and longer chain acyl groups from post-translationally modified lysine residues. It affects diverse biological functions in the cell and has been considered a drug target in relation to both neurodegenerative diseases and cancer. Therefore, access to well-characterized and robust tool compounds is essential for the continued investigation of the complex functions of this enzyme. Here, we report a collection of probes that are potent, selective, stable in serum, water-soluble, amenable to cell culture experiments, and inhibit both SIRT2 deacetylation and demyristoylation. Compared to the current landscape of SIRT2 inhibitors, this is a unique ensemble of features built into a single compound. We expect the developed chemotypes to find broad application in the interrogation of SIRT2 functions in both healthy and diseased cells, and to provide a foundation for the development of future therapeutics.

biochemistry↗

bioRxiv2020

Distinct chemotactic behavior in the original Escherichia coli K-12 depending on forward-and-backward swimming, not on run-tumble movements

Most motile bacteria are propelled by rigid, helical, flagellar filaments and display distinct swimming patterns to explore their favorable environments. Escherichia coli cells have a reversible rotary motor at the base of each filament. They exhibit a run-tumble swimming pattern, driven by switching of rotatory direction which causes polymorphic flagellar transformation. Here we report a novel swimming mode in E. coli ATCC10798, which is one of the original K-12 clones. High-speed tracking of single ATCC10798 cells showed forward and backward swimming with an average turning angle of 150{degrees}. The flagellar helicity remained right-handed with a 1.3 m pitch and 0.14 m helix radius, which is assumed to be a curly type, regardless of motor switching; the flagella of ATCC10798 did not show polymorphic transformation. The torque and rotational switching of the motor was almost identical to the E. coli W3110 strain, which is a derivative of K-12 and a wild-type for chemotaxis. The single point mutation of N87K in FliC, one of the filament subunits, is critical to the change in flagellar morphology and swimming pattern, and lack of flagellar polymorphism. E. coli cells expressing FliC(N87K) sensed ascending a chemotactic gradient in liquid but did not form rings on a semi-solid surface. Based on these findings, we propose a flagellar polymorphism-dependent migration mechanism in structured environments.

microbiology↗

bioRxiv2020

Structures of α-synuclein filaments from multiple system atrophy

Synucleinopathies are human neurodegenerative diseases that include multiple system atrophy (MSA), Parkinsons disease, Parkinsons disease dementia (PDD) and dementia with Lewy bodies (DLB) (1). Existing treatments are at best symptomatic. These diseases are characterised by the presence in brain cells of filamentous inclusions of -synuclein, the formation of which is believed to cause disease (2, 3). However, the structures of -synuclein filaments from human brain are not known. Here we show, using electron cryo-microscopy, that -synuclein inclusions from MSA are made of two types of filaments, each of which consists of two different protofilaments. Non-proteinaceous molecules are present at the protofilament interfaces. By two-dimensional class averaging, we show that -synuclein filaments from the brains of patients with MSA and DLB are different, suggesting that distinct conformers (or strains) characterise synucleinopathies. As was the case of tau assemblies (4-9), the structures of -synuclein filaments extracted from the brains of individuals with MSA differ from those formed in vitro using recombinant proteins, with implications for understanding the mechanisms of aggregate propagation and neurodegeneration in human brain. These findings have diagnostic and potential therapeutic relevance, especially in view of the unmet clinical need to be able to image filamentous -synuclein inclusions in human brain.

neuroscience↗

bioRxiv2019

Evidence for a role for BK channels in the regulation of ADAM17 activity.

Large-conductance voltage and calcium activated channels, KCa1.1, have a large single conductance (~p250) and are highly selective for potassium ions. As a result they have been termed big potassium channels (BK channels). Because of the channels ability to integrate multiple physical and chemical signals they have received much attention in excitable cells. In comparison they have received relatively little attention in non-excitable cells in those of the immune system. Here we report evidence that the BK channel regulates ADAM17 activity. Upon macrophage activation, BK channels translocate to the cell membrane. Genetic or pharmacological inhibition of the cell membrane BK channels resulted in elevated TNF- release and increased metalloproteinase a disintegrin and metalloproteinase domain 17 (ADAM17) activity. Inhibitors of BK channels also increased IL-6R release, a second ADAM17 substrate. In comparison, a BK channel opener decreases TNF- release. Taken together, our results demonstrate a novel mechanism by which ion channel regulates ADAM17 activity. Given the broad range of ADAM17 substrates, this finding has implications in many fields of cell biology including immunology, neurology and cancer biology.

pharmacology and toxicology↗

bioRxiv2019

Long-read sequencing identifies GGC repeat expansion in human-specific NOTCH2NLC associated with neuronal intranuclear inclusion disease

Neuronal intranuclear inclusion disease (NIID) is a progressive neurodegenerative disease characterized by eosinophilic hyaline intranuclear inclusions in neuronal and somatic cells. The wide range of clinical manifestations in NIID makes ante-mortem diagnosis difficult 1-8, but skin biopsy realized its ante-mortem diagnosis 9,10 and many NIID cases have been diagnosed by skin biopsy11,12. Most cases of NIID are sporadic, but several familial cases are known. Using a large NIID family, we conducted linkage mapping, found a 58.1-Mb linked-region at 1p22.1-q21.3 with a maximum logarithm of odds (LOD) score of 4.21, and successfully identified a GGC repeat expansion in the 5 portion of NOTCH2NLC in all affected members by long-read sequencing, but not in unaffected members. We further found the similar expansions in additional 8 unrelated families with NIID as well as 39 sporadic NIID patients. Repeat-primed PCR consistently detected the GGC repeat expansion in all the familial and sporadic patients diagnosed by skin biopsy, but never in unaffected family members nor 200 controls. This shows that pathogenic changes in a human-specific gene evolutionarily generated by segmental duplication indeed causes a human disease.

genetics↗

bioRxiv2017

Pathways of DNA unlinking: A story of stepwise simplification

In Escherichia coli DNA replication yields interlinked chromosomes. Controlling topological changes associated with replication and returning the newly replicated chromosomes to an unlinked monomeric state is essential to cell survival. In the absence of the topoisomerase topoIV, the site-specific recombination complex XerCD-dif-FtsK can remove replication links by local reconnection. We previously showed mathematically that there is a unique minimal pathway of unlinking replication links by reconnection while stepwise reducing the topological complexity. However, the possibility that reconnection preserves or increases topological complexity is biologically plausible. In this case, are there other unlinking pathways? Which is the most probable? We consider these questions in an analytical and numerical study of minimal unlinking pathways. We use a Markov Chain Monte Carlo algorithm with Multiple Markov Chain sampling to model local reconnection on 491 different substrate topologies, 166 knots and 325 links, and distinguish between pathways connecting a total of 881 different topologies. We conclude that the minimal pathway of unlinking replication links that was found under more stringent assumptions is the most probable. We also present exact results on unlinking a 6-crossing replication link. These results point to a general process of topology simplification by local reconnection, with applications going beyond DNA.

bioinformatics↗

bioRxiv2017

Chaperonin facilitates protein folding by avoiding polypeptide collapse

Chaperonins assist folding of many cellular proteins, including essential proteins for cell viability. However, it remains unclear how chaperonin-assisted folding is different from spontaneous folding. Chaperonin GroEL/GroES facilitates folding of denatured protein encapsulated in its central cage but the denatured protein often escapes from the cage to the outside during reaction. Here, we show evidence that the in-cage-folding and the escape occur diverging from the same intermediate complex in which polypeptide is tethered loosely to the cage and partly protrudes out of the cage. Furthermore, denatured proteins in the chaperonin cage start their folding from extended conformations but not from compact conformations as usually observed in spontaneous folding. We propose that the formation of tethered intermediate of polypeptide is necessary to prevent polypeptide collapse at the expense of polypeptide escape. The tethering of polypeptide would allow freely mobile portions of tethered polypeptide to fold segmentally. The folding acceleration and deceleration by chaperonin for various substrate proteins can be explained by considering the tethering.

biochemistry↗