Biology subjects

Fujita, H.

Publications and source records attributed to Fujita, H..

At least 37 records · Page 2Linked to original sources

bioRxiv2022

Estimation of crossbridge-state during cardiomyocyte beating using second harmonic generation

Estimation of dynamic change of crossbridge formation in living cardiomyocytes is expected to provide crucial information for elucidating cardiomyopathy mechanisms, efficacy of an intervention, and other parameters. Here, we developed an assay system to dynamically measure second harmonic generation (SHG) polarization in pulsating cardiomyocyte and proved that the SHG anisotropy derived from myosin filaments in disease-model cardiomyocytes depended on their crossbridge status, providing an evaluation method for myosin force generation. Experiments utilizing an inheritable mutation that induces excessive myosin-actin interactions revealed that the correlation between sarcomere length and SHG anisotropy represents crossbridge formation ratio during pulsation. Furthermore, the present method found that ultraviolet irradiation induced an increased population of attached crossbridges that lost force-generating ability upon myocardial differentiation, causing acquired dysfunction. Taking an advantage of infrared two-photon excitation in SHG microscopy, myocardial dysfunction could be intravitally evaluated in a Drosophila disease model. Thus, along with the establishment of the methodology, we successfully demonstrated the applicability and effectiveness of the present method to evaluate the actomyosin activity of a drug or genetic defect on living cardiomyocytes.

biophysics↗

bioRxiv2022

Core species and interactions prominent in fish-associated microbiome dynamics

In aquatic ecosystems, the health of fish depends greatly on the dynamics of microbial community structure in the background environment. Nonetheless, finding microbes with profound impacts on fishs performance out of thousands of candidate species remains a major challenge. We here show that time-series analyses of microbial population dynamics illuminate core components and structure of fish-associated microbiomes. By targeting eel aquaculture microbiomes as model systems, we reconstructed the population dynamics of 9,605 bacterial and 303 archaeal species/strains across 128 days. Due to the remarkable increase/decrease of constituent microbial populations, the taxonomic compositions of microbiomes changed drastically through time. We then found that some specific microbial taxa showed positive relationship with eels activity level even after excluding cofounding effects of environmental parameters (pH and dissolved oxygen level) on population dynamics. In particular, a vitamin B12-producing bacteria, Cetobacterium somerae, consistently showed strong positive associations with eels activity level across the replicate time-series of the five aquaculture tanks. Network theoretical and metabolic modeling analyses further suggested that the highlighted bacterium formed compartments of close microbe-to-microbe interactions with some other bacterial taxa, forming potential core microbiomes with positive impacts on eels. Overall, these results suggest that integration of microbiology, ecological theory, and network science allows us to explore core species and interactions embedded within complex dynamics of fish-associated microbiomes.

ecology↗

bioRxiv2022

Silicon as a microfluidic material for imaging and incubation of droplets

Droplet microfluidics has become a powerful tool in life sciences, underlying digital assays, single-cell sequencing or directed evolution, and it is making foray in physical sciences as well. Imaging and incubation of droplets are crucial, yet they are encumbered by the poor optical, thermal and mechanical properties of PDMS - the de facto material for microfluidics. Here we show that silicon is an ideal material for droplet chambers. Si chambers pack droplets in a crystalline and immobile monolayer, are immune to evaporation or sagging, boost the number of collected photons, and tightly control the temperature field sensed by droplets. We use the mechanical and optical benefits of Si chambers to image [~]1 million of droplets from a multiplexed digital assay - with an acquisition rate similar to the best in-line methods. Lastly, we demonstrate their applicability with a demanding assay that maps the thermal dependence of Michaelis-Menten constants with an array of [~]150,000. The design of the Si chambers is streamlined to avoid complicated fabrication and improve reproducibility, which makes Silicon a complementary material to PDMS in the toolbox of droplet microfluidics. Significance StatementAs the technological engine behind single-cell sequencing and digital assays, droplets microfluidics has revolutionized life science and molecular diagnosis, and is making foray into physical sciences as well. Observing droplets in a controlled manner is becoming crucial, but PDMS - the de facto material of microfluidics - hampers imaging and incubation. Here we revisit silicon as a microfluidic material and show that its superior mechanical, optical and thermal performances improve the throughput and operation of droplets assay.

biochemistry↗

bioRxiv2022

Alternative stable states, nonlinear behavior, and predictability of microbiome dynamics

Microbiome dynamics are both crucial indicators and drivers of human health, agricultural output, and industrial bio-applications. However, predicting microbiome dynamics is notoriously difficult because communities often show abrupt structural changes, such as "dysbiosis" in human microbiomes. We here integrate theoretical and empirical bases for anticipating drastic shifts of microbial communities. We monitored 48 experimental microbiomes for 110 days and observed that various community-level events, including collapse and gradual compositional changes, occurred according to a defined set of environmental conditions. We then confirmed that the abrupt community changes observed through the time-series could be described as shifts between "alternative stable states" or dynamics around complex attractors. Furthermore, collapses of microbiome structure were successfully anticipated by means of the diagnostic threshold defined with the energy landscape analysis of statistical physics or that of a stability index of nonlinear mechanics. These results indicate that abrupt microbiome events in complex microbial communities can be forecasted by extending classic ecological concepts to the scale of species-rich microbial systems.

ecology↗

bioRxiv2022

Stable states and keystone species in soil microbiomes of agroecosystems

Theory predicts that biological communities can have multiple basins of attraction in terms of their species/taxonomic compositions. The presence of such basins of community structure has been examined in classic empirical studies on forest-savanna transitions and those on eutrophication in freshwater lakes. Nonetheless, it remains a major challenge to extend the investigations of multistability to species-rich microbial communities. By targeting soil microbiomes, we infer the stability landscapes of community structure based on the concepts of statistical physics. Our analysis on the compiled dataset involving 11 archaeal, 332 bacterial, and 240 fungal families detected from > 1,500 agroecosystem soil samples suggested that both prokaryotic and fungal community compositions could be classified into several basins of attraction. We also found that the basins differed greatly in their associations with crop disease prevalence in agroecosystems. A further analysis highlighted microbial taxa potentially playing key roles in transitions between basins with different ecosystem-scale functions. The statistical framework commonly applicable to diverse microbial and non-microbial communities will reorganize our understanding of relationship among community structure, stability, and functions.

microbiology↗

bioRxiv2022

Single-molecule tracking of Nanog and Oct4 in mouse embryonic stem cells

Nanog and Oct4 are core transcription factors in a gene regulatory network that regulate hundreds of target genes for pluripotency maintenance in mouse embryonic stem cells. To understand their function as a gatekeeper or a pioneer factor at the molecular scale, we quantified the residence time on target loci, fluctuation at the loci, and interaction clustering by visualizing single molecules of Nanog and Oct4 in a living nucleus during the pluripotency loss. Interestingly, Nanog interacted longer with its target loci in the lower Nanog expression state or at the onset of differentiation, indicating the possibility of a new feedback mechanism to maintain Nanog expression. The interaction time of Nanog and Oct4 corresponded to their fluctuation and interaction clustering, which depended on their expression or differentiation state, respectively. The DNA viscoelasticity near the Oct4 target locus remained flexible during the differentiation, reflecting its role as a pioneer factor. Based on these results, we propose a new feedback mechanism for pluripotency maintenance in which Nanog function is prolonged, corresponding to chromatin condensation, and Oct4 reopens the condensation.

biophysics↗

bioRxiv2022

Mice generated with induced pluripotent stem cells derived from mucosal-associated invariant T cells

The function of mucosal-associated invariant T (MAIT) cells, a burgeoning member of innate-like T cells abundant in humans and implicated in many diseases, remains obscure. To explore this, mice with a rearranged T cell receptor (TCR) or {beta} locus, specific for MAIT cells, were generated via induced pluripotent stem cells derived from MAIT cells and designated V19 and V{beta}8 mice, respectively. Both mice expressed large amounts of MAIT cells. The MAIT cells from these mice were activated by cytokines and an agonist to produce IFN-{gamma} and IL-17. While V{beta}8 mice showed resistance in a cancer metastasis model, V19 mice did not. Adoptive transfer of MAIT cells from the latter into the control mice, however, recapitulated the resistance. This has implications for understanding the role of MAIT cells in health and disease and in developing treatments for the plethora of diseases in which MAIT cells are implicated.

immunology↗

bioRxiv2022

Cryo-EM structures of human zinc transporter ZnT7 reveal the mechanism of Zn2+ uptake into the Golgi apparatus

Zinc ions (Zn2+) are vital to most cells, with the intracellular concentrations of Zn2+ being tightly regulated by multiple zinc transporters located at the plasma and organelle membranes. We herein present the 2.8-2.9 [A]-resolution cryo-EM structures of a Golgi-localized human Zn2+/H+ antiporter ZnT7 (hZnT7) in its outward- and inward-facing forms. Cryo-EM analyses showed that hZnT7 exists as a homodimer via tight interactions in both the cytosolic and transmembrane (TM) regions of two protomers, each of which contains a single Zn2+-binding site in its TM domain. hZnT7 undergoes a TM-helix rearrangement to create a negatively charged cytosolic cavity for Zn2+ entry in the inward-facing form and a widened luminal cavity for Zn2+ release in the outward-facing form. An exceptionally long cytosolic histidine-rich loop characteristic of hZnT7 can bind at least two Zn2+ ions, likely facilitating Zn2+ recruitment from the cytosol. Unique mechanisms of hZnT7-mediated Zn2+ uptake into the Golgi are proposed.

molecular biology↗

bioRxiv2022

Targeted deletion of uterine glandular Foxa2 induces embryonic diapause in mice

Embryonic diapause is a reproductive strategy in which embryo development and growth is temporarily arrested within the uterus to ensure the survival of neonates and mothers during unfavorable conditions. Pregnancy is reinitiated when conditions become favorable for neonatal survival. The mechanism of how the uterus enters diapause remains unclear. Mice with uterine deletion of Foxa2, a transcription factor, are infertile. In this study, we show that dormant blastocysts are recovered from these mice on day 8 of pregnancy with persistent uterine Msx1 expression, a gene critical to maintaining the uterine quiescent state, suggesting that these mice enter embryonic diapause. Leukemia inhibitory factor (LIF) can resume implantation in these mice. Although estrogen is critical for implantation in progesterone (P4)-primed uterus, our current model reveals that FOXA2-independent estrogenic effects are detrimental to sustaining uterine quiescence. Interestingly, P4 and anti-estrogen can prolong uterine quiescence in the absence of FOXA2. While we find that Msx1 expression persists in the uterus deficient in Foxa2, the complex relationship of FOXA2 with Msx genes and estrogen receptors remains to be explored.

developmental biology↗

bioRxiv2021

Reprogramming and redifferentiation of mucosal-associated invariant T cells reveals tumor inhibitory activity

Mucosal-associated invariant T (MAIT) cells belong to a family of innate-like T cells that bridge innate and adaptive immunities. Although MAIT cells have been implicated in tumor immunity, it currently remains unclear whether they function as tumor promoting or inhibitory cells. Therefore, we herein used induced pluripotent cell (iPSC) technology to investigate this issue. Murine MAIT cells were reprogrammed into iPSCs and redifferentiated towards MAIT-like cells (m-reMAIT cells). m-reMAIT cells were activated by an agonist and MR1-tetramer, a reagent to detect MAIT cells, in the presence and absence of antigen-presenting cells. This activation accompanied protein tyrosine phosphorylation and the production of T helper (Th)1-, Th2-, and Th17-cytokines and inflammatory chemokines. Upon adoptive transfer, m-reMAIT cells migrated to different organs with maturation in mice. Furthermore, m-reMAIT cells prolonged mouse survival upon tumor inoculation through the NK cell-mediated reinforcement of cytolytic activity. Collectively, the present results demonstrated the utility and role of m-reMAIT cells in tumor immunity, and will contribute to insights into the function of MAIT cells in immunity.

cancer biology↗

bioRxiv2021

Synergistic and offset effects of fungal species combinations on plant performance

In natural and agricultural ecosystems, survival and growth of plants depend substantially on microbes in the endosphere and rhizosphere. Although numerous studies have reported the presence of plant-growth promoting bacteria and fungi in below-ground biomes, it remains a major challenge to understand how sets of microbial species positively or negatively affect plants performance. By conducting a series of single- and dual-inoculation experiments of 13 endophytic and soil fungi targeting a Brassicaceae plant species, we here evaluated how microbial effects on plants depend on presence/absence of co-occurring microbes. The comparison of single- and dual-inoculation experiments showed that combinations of the fungal isolates with the highest plant-growth promoting effects in single inoculations did not yield highly positive impacts on plant performance traits (e.g., shoot dry weight). In contrast, pairs of fungi including small/moderate contributions to plants in single-inoculation contexts showed the greatest effects on plants among the 78 fungal pairs examined. These results on the offset and synergistic effects of pairs of microbes suggest that inoculation experiments of single microbial species/isolates can result in the overestimation or underestimation of microbial functions in multi-species contexts. Because keeping single-microbe systems in outdoor conditions is impractical, designing sets of microbes that can maximize performance of crop plants is an important step for the use of microbial functions in sustainable agriculture.

microbiology↗

bioRxiv2021

MARCH8 targets cytoplasmic lysine residues of various viral envelope glycoproteins

The host transmembrane protein MARCH8 is a RING finger E3 ubiquitin ligase that downregulates various host transmembrane proteins, such as MHC-II. We have recently reported that MARCH8 expression in virus-producing cells impairs viral infectivity by reducing virion incorporation of not only HIV-1 envelope glycoproteins but also vesicular stomatitis virus G-glycoprotein through two different pathways. However, the MARCH8 inhibition spectrum remains largely unknown. Here, we investigate the antiviral spectrum of MARCH8 using HIV-1 pseudotyped with a variety of viral envelope glycoproteins. Pseudotyping experiments revealed that viral envelopes derived from the rhabdovirus, arenavirus, coronavirus, and togavirus (alphavirus) families were sensitive to MARCH8-mediated inhibition. Lysine mutations at the cytoplasmic tails of rabies virus-G, lymphocytic choriomeningitis virus glycoproteins, SARS-CoV and SARS-CoV-2 spike proteins, and Chikungunya virus and Ross River virus E2 proteins conferred resistance to MARCH8. Immunofluorescence showed impaired downregulation of the mutants of these viral envelopes by MARCH8, followed by lysosomal degradation, suggesting that MARCH8-mediated ubiquitination leads to intracellular degradation of these envelopes. Indeed, rabies virus-G and Chikungunya virus E2 proteins proved to be clearly ubiquitinated. We conclude that MARCH8 has inhibitory activity on a variety of viral envelope glycoproteins whose cytoplasmic lysine residues are targeted by this antiviral factor.

microbiology↗

bioRxiv2020

Direct uptake mechanism in lysosome required for neuromuscular homeostasis

The degradation of cellular components plays an essential role in homeostasis. However, the known degradation pathways cannot account for the levels of proteolysis in cells. Here, we demonstrate that cytosolic proteins are imported into lysosomes in an ATP-dependent manner for degradation through a direct uptake mechanism distinct from any known pathway. SIDT2, a lysosomal membrane protein previously reported as an RNA transporter, translocates substrate proteins across the lysosomal membrane. Furthermore, we identify a dominant-negative mutation in SIDT2 that causes neuropathy and distal myopathy with rimmed vacuoles, a protein aggregation disease in humans. We generate Sidt2 knockout mice, recapitulating the characteristic features of this disease. Our results reveal a novel degradation pathway and illustrate its crucial role in cellular proteostasis, physiology, and pathophysiology. One Sentence SummaryDiscovery of a novel proteolytic pathway in cells, the dysfunction of which leads to protein aggregation disease in humans.

cell biology↗

bioRxiv2020

Epitope Prediction of Antigen Protein using Attention-Based LSTM Network

B-cells inducing antigen-specific immune responses in vivo produce large amounts of antigen-specific antibodies by recognizing the subregions (epitope regions) of antigen proteins. They can inhibit their functioning by binding antibodies to antigen proteins. Predicting of epitope regions is beneficial for the design and development of vaccines aimed to induce antigen-specific antibody production. However, prediction accuracy requires improvement. The conventional epitope region prediction methods have focused only on the target sequence in the amino acid sequences of an entire antigen protein and have not thoroughly considered its sequence and features as a whole. In the present paper, we propose a deep learning method based on short-term memory with an attention mechanism to consider the characteristics of a whole antigen protein in addition to the target sequence. The proposed method achieves better accuracy compared with the conventional method in the experimental prediction of epitope regions using the data from the immune epitope database.

bioinformatics↗

bioRxiv2020

Are mice born normally with an egg-only diet?

Chicken eggs (egg) contain high amounts of important proteins and fat with a very low amount of carbohydrates, and also contain all vitamins and minerals needed for the chick to develop. We mated 8-week-old mice consuming an egg-only diet, and examined whether mice got pregnant and mice gave birth. Then we continued to raise newborn mice only consuming an egg-only diet compared to normal chew diet. We found that the rate of pregnancy of the mice consuming an egg-only diet was not different from that of the mice consuming normal chew diet. Further, we found that the number of pupus born from the mother consuming only eggs was not different from that of the mother consuming normal chew diet. However, the newborn mice from the mother fed only eggs did not grow healthy when they consume egg-only diet. Only few mice were alive at one month after birth. Then we change the diet just before or just after birth from egg only diet to normal chew diet and we found that both group grew healthy more than one month. These results indicate that mice are born healthy only with egg only diet.

developmental biology↗

bioRxiv2020

Modular output circuits of the fastigial nucleus mediate diverse motor and nonmotor functions of the cerebellar vermis

The cerebellar vermis, long associated with axial motor control, has been implicated in a surprising range of neuropsychiatric disorders and cognitive and affective functions. Remarkably little is known, however, about the specific cell types and neural circuits responsible for these diverse functions. Here, using single-cell gene expression profiling and anatomical circuit analyses of vermis output neurons in the mouse fastigial (medial cerebellar) nucleus, we identify five major classes of glutamatergic projection neurons distinguished by gene expression, morphology, distribution, and input-output connectivity. Each fastigial cell type is connected with a specific set of Purkinje cells and inferior olive neurons and in turn innervates a distinct collection of downstream targets. Transsynaptic tracing indicates extensive disynaptic links with cognitive, affective, and motor forebrain circuits. These results indicate that diverse cerebellar vermis functions are mediated by modular synaptic connections of distinct fastigial cell types which differentially coordinate posturomotor, oromotor, positional-autonomic, orienting, and vigilance circuits.

neuroscience↗

bioRxiv2020

MARCH8 inhibits viral infection by two different mechanisms

Membrane-associated RING-CH 8 (MARCH8) inhibits infection with both HIV-1 and vesicular stomatitis virus G-glycoprotein (VSV-G)-pseudotyped viruses by reducing virion incorporation of envelope glycoproteins. The molecular mechanisms by which MARCH8 targets envelope glycoproteins remain unknown. Here, we show two different mechanisms by which MARCH8 inhibits viral infection. Viruses pseudotyped with the VSV-G mutant, in which cytoplasmic lysine residues were mutated, were insensitive to the inhibitory effect of MARCH8, whereas those with a similar lysine mutant of HIV-1 Env remained sensitive to it. Indeed, the wild-type VSV-G, but not its lysine mutant, was ubiquitinated by MARCH8. Furthermore, the MARCH8 mutant, which had a disrupted cytoplasmic tyrosine motif that is critical for intracellular protein sorting, did not inhibit HIV-1 Env-mediated infection, while it still impaired infection by VSV-G-pseudotyped viruses. Overall, we conclude that MARCH8 reduces viral infectivity by downregulating envelope glycoproteins through two different mechanisms mediated by a ubiquitination-dependent or tyrosine motif-dependent pathway.

microbiology↗

bioRxiv2019

Serpentine mountain uplift in northern Japan triggered the divergence of a narrow endemic from a widespread (sub)arctic Asia-Alaska species complex of Lagotis (Plantaginaceae)

In the circumboreal region, plants often have extremely-wide species ranges. Lagotis minor-glauca species complex widespread from (sub)arctic Asia to Alaska, however, have two allied narrow endemics in northern Japan: a serpentine plant L. takedana endemic to the Yubari Mountains (Mt. Yubari) and a non-serpentine plant L. yesoensis endemic to the Taisetsu Mountains (Mt. Taisetsu). Elucidating their origins sheds light on drivers for secondary-speciation of widespread circumboreal plants. To infer phylogenetic distinctiveness of two narrow endemics with those related taxa, which contained 25 out of all the 29 species of the genus, chloroplast DNA (cpDNA), nuclear ribosomal (nrITS), two low copy nuclear gene (LCN) markers and genome-wide single-nucleotide polymorphism genotyping (MIG-seq) were used. In the result of cpDNA analyses, the Lagotis minor-glauca species complex formed a clade. Within the clade, L. yesoensis and a portion of L. glauca samples formed a subclade. However, monophyly of each of the four species was not supported. In the results of nrITS and two LCN analyses, L. takedana was monophyletic, while monophyly was not recovered for each L. yesoensis, L. glauca, and L. minor. Based on a Bayesian dating analysis using nrITS data, the age of the most recent common ancestor of L. takedana was Ma (95% confidence interval: 0.05-1.75 Ma). Possible scenario is that an ancestral linage being adapted to serpentine soils migrated into the alpine habitat of Mt. Yubari, that was formed with mountain uplift by the early Pleistocene, and subsequently reproductively isolated from non-serpentine populations and speciated. The contrasting result of L. yesoensis, that was phylogenetically indistinct, is possibly explained by incorrect taxonomy, or alternatively, shallow history and incomplete lineage sorting. In Mt. Taisetsu, massive volcanic eruptions had occurred the Early Pleistocene and even after the last glacial period, suggesting that alpine plants have not migrated into and established populations in Mt. Taisetsu until very recently. To fully resolve the phylogeny of the three species L. yesoensis, L. glauca, and L. minor, further analyses using high resolution molecular markers are needed. The present study illustrated that two narrow endemics in northern Japan diverged from the widespread species include phylogenetically distinctive and indistinctive species, owing to historical orogeny and ecological factors.

ecology↗