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Fukuda, S.

Publications and source records attributed to Fukuda, S..

At least 19 recordsLinked to original sources

Major microbiota transitions across liver disease progression are mediated by key taxa linking host environments and microbial communities

The gut microbiome plays a critical role in chronic liver disease, yet quantitative methods that integrate microbial community structure with clinical indices remain limited. Here, we applied an energy landscape analysis (ELA)-based framework to characterize transitions in gut microbiota along a clinical gradient of liver disease severity, as measured by the FibroScan-AST (FAST) score. The analysis revealed characteristic community states corresponding to low, intermediate, and high FAST scores, indicating that liver disease progression is associated with structured transitions in microbial community organization rather than simple changes in overall diversity. To further dissect community-level organization, we developed the community shaping index (CSI), which quantifies how strongly individual taxa are associated with specific community structures. Integrating CSI with species-environment association parameters identified key taxa with distinct ecological roles, including unclassified Subdoligranulum taxon, an unclassified Ruminococcus gnavus group taxon, and Streptococcus salivarius, that link host environmental conditions with community composition. Exploratory causal discovery suggested that Streptococcus salivarius and the unclassified Subdoligranulum may play distinct, potentially active roles in linking liver health with gut microbial organization, but with contrasting associations: S. salivarius was linked to disease-related liver deterioration and dysbiotic states, whereas the unclassified Subdoligranulum was associated with healthier liver function and coherent community organization. Overall, this ecologically grounded framework provides a unified and mechanistic foundation for analyzing microbiome structure and disease-associated transitions.

ecology↗

Polyvinylpyrrolidone enhances AAV transduction in vitro and shows preliminary utility for subretinal delivery

Adeno-associated virus (AAV) vectors are widely used for gene delivery, but inefficient transduction can require high vector doses. We tested whether unmodified linear polyvinylpyrrolidone (PVP), a pharmaceutical excipient, can improve AAV formulation without chemical modification of the vector or polymer. PVP10, PVP40, and PVP360 were evaluated in vitro across HEK293, HeLa, MEF, and CHO cells; selected formulations were tested after intravenous delivery, and 3% PVP40 was tested by subretinal delivery. In vitro, 1-3% PVP increased AAV8- and AAV9-mediated GFP expression across multiple cell lines. PVP360 showed broad activity in the initial cross-cell assay, whereas the HEK293 molecular-weight screen identified PVP40 and PVP360 as the most active formulations. The substantial fold increase observed in CHO cells largely reflected the low baseline transduction of the control group. MTT absorbance declined with concentrated PVP360, whereas PVP40 retained transduction-enhancing activity and was selected for local testing. In the HeLa AAV-DJ assay, the response pattern differed between MOI 1,000 and MOI 100; the largest observed increases in GFP-positive area occurred with 1.5% PVP10 and 1.5% PVP360 at MOI 100. Intravenous AAV9 delivery with PVP did not consistently increase ex vivo organ reporter signal. By contrast, subretinal delivery of AAV-PHP.eB with 3% PVP40 produced a 1.79- fold larger mean DsRed-positive area per retinal section, averaged within each eye (Welchs t-test p = 0.063; Bayesian Pr[{Delta} > 0] = 0.952). These findings support further evaluation of PVP40 for local subretinal AAV delivery.

bioengineering↗

Acquisition of Stickland-metabolizing bacteria during infancy prevents Clostridium botulinum infection

Infant botulism is caused by intestinal colonization with Clostridium botulinum, whereas healthy adults are resistant. Although the gut microbiota has long been implicated in protection against C. botulinum, its bacterial basis has remained unknown. Here, we show that specific amino acid-metabolizing bacteria confer resistance to C. botulinum colonization through nutrient competition. Longitudinally collected human infant microbiotas exhibited a transition from susceptibility to resistance after transplantation into germ-free mice. 5-Aminovalerate marked the resistant microbiota, implicating Stickland metabolism. Resistant microbiotas were enriched in Stickland-metabolizing Clostridia, including Clostridioides difficile. Metabolomics revealed overlapping amino acid utilization, and C. difficile suppressed C. botulinum expansion through amino acid competition. These findings demonstrate that acquisition of Stickland-metabolizing Clostridia during infancy prevents C. botulinum infection through competition for shared amino-acid-dependent nutritional niches.

microbiology↗

Epithelial Glutamate Retention Protects against Colitis

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract that encompasses ulcerative colitis and Crohns disease. Here we identify the cystine/glutamate antiporter xCT as being markedly upregulated in the inflamed intestinal epithelium of patients with IBD. To clarify its functional contribution to disease pathogenesis, we performed genetic loss-of-function study and found that inhibition of xCT confers robust protection against dextran sulfate sodium (DSS)-induced colitis in mice. Intestinal epithelial cell (IEC)-specific deletion of xCT markedly attenuated colitis severity, demonstrating that epithelial xCT upregulation acts as a disease-exacerbating factor in IBD. Mechanistically, xCT deficiency preserved intracellular glutamate levels and protein polyglutamylation, thereby maintaining epithelial barrier integrity and protecting IECs from inflammatory injury. Consistently, pharmacological inhibition of glutamine synthetase, which increases intracellular glutamate, exerted a potent anti-inflammatory effect on the DSS-induced colitis. These findings identify intracellular glutamate retention in IECs as a previously unrecognized mechanism of epithelial protection and highlight both inhibition of xCT-dependent glutamate efflux and suppression of glutamine synthetase as potential therapeutic strategies for IBD.

molecular biology↗

Co-targeting an AMPK--MAPK axis reprograms CAFs and suppresses PDAC

Pancreatic ductal adenocarcinoma (PDAC) is a fatal cancer characterized by limited therapeutic options and a highly treatment-resistant tumor microenvironment. Beyond tumor-intrinsic genetic alterations, growing evidence indicates that host-microbiome interactions influence cancer progression through microbial metabolites. However, how microbiome-derived metabolites influence oncogenic signaling in PDAC remains unclear. Here, integrated profiling revealed a consistent reduction of the microbial metabolite acetic acid in fecal samples from treatment-naive patients with PDAC and in a genetically defined Drosophila model recapitulating key PDAC driver alterations. Acetic acid activates AMP-activated protein kinase, and pharmacological activation of this pathway together with inhibition of mitogen-activated protein kinase signaling suppressed tumor growth in fly and mouse models. Combined pathway targeting restored AMPK activity and suppressed cancer-associated fibroblast activation. These findings identify a microbiome-associated metabolic vulnerability in PDAC and suggest that coordinated targeting of metabolic and oncogenic signaling may restrain tumor progression and improve therapeutic strategies.

cancer biology↗

Enhancing the imaging rate of high-speed atomic force microscopy using a combination of multiple techniques

Protein molecules, while functioning, undergo dynamic conformational changes and interactions with their partners, which can be directly observed by high-speed atomic force microscopy (HS-AFM). However, its imaging rate of approximately 10 frames per second (fps) has remained unchanged since its inception, despite the increasing demand for observing dynamic biomolecular processes that occur too fast to be captured at this time resolution. Here we report a combination of techniques that significantly enhances the imaging rate without disturbing the sample. HS-AFM imaging of the stator ring of F1-ATPase visualised two intermediate states, which could not be resolved with the previous HS-AFM system. The high-resolution molecular movies demonstrate how the catalytic subunits cooperate during the chemomechanical cycle.

biophysics↗

A human sensory neuron model for varicella-zoster virus latency and reactivation in vitro.

Varicella-zoster virus (VZV) is a ubiquitous human neurotropic alphaherpesvirus that establishes lifelong latency in sensory ganglionic neurons. Subsequent viral reactivation causes herpes zoster, a morbid disease often complicated by neuropathic pain. Mechanisms underlying VZV latency and reactivation are not understood, mostly due to the lack of permissive animal models and challenges of current in vitro latency modelling. Here, we evaluated HD10.6 cells, a simplified and easily expandable human sensory neuron line to model VZV latency and reactivation. Mature HD10.6 (mHD10.6) differentiated neurons supported productive VZV infection, viral DNA replication, production of infectious progeny, and viral spread in cultures. VZV infection was associated with limited cytopathic effects and ultrastructural changes. Infecting mHD10.6 neurons with cell-free VZV in the presence of antivirals resulted in a quiescent-persistent state, characterized by persistent VZV genomes with restricted VZV gene expression and absence of infectious virus. Importantly, VZV could be reactivated by treatment with capsaicin, as evidenced by increased lytic viral transcription and virus spread. In conclusion, this study establishes human HD10.6 neurons as a novel and scalable in vitro model for studying VZV latency and reactivation to identify virus and host factors governing latency that may serve as therapeutic targets to restrict VZV reactivation. ImportanceMost individuals carry latent varicella-zoster virus (VZV) in their dorsal root ganglia (DRG), which can reactivate to cause shingles and chronic pain. The mechanisms by which VZV establishes latency and triggers of reactivation are incompletely understood. Current platforms for the study of VZV latency do not easily support functional experiments (ganglia) or are difficult to expand and complicated by mixed neuronal populations (stem cell-derived neurons). Here, we demonstrate that matured HD10.6 (mHD10.6) cells derived from immortalized human DRG-derived neurons provide a clonal, scalable, and easy-to-expand platform for studying lytic, latent, and reactivated VZV infection in sensory neurons. We propose that the HD10.6 platform could provide the basis to conduct studies on the viral latent state that have hitherto not been possible.

microbiology↗

Hox - Meis-relayed topographical genetic switch underlies cardiopharyngeal neural crest diversification, revealed by multimodal analysis

Neural crest cells (NCCs) are multipotent migratory cells essential for cardiac development, yet the lineage trajectories and gene regulatory networks (GRNs) underlying their differentiation in the cardiopharyngeal region remain unclear. Here, we integrate single-cell RNA-seq, spatial transcriptomics, and multiomic analyses to construct a comprehensive map of NCC lineages in developing mouse cardiopharyngeal tissues. We identify a transition from Hox-positive pharyngeal NCCs to Hox-negative intracardiac populations associated with the outflow tract cushion, accompanied by a shift in Meis transcription factor binding and GRN architecture. By contrast, NCCs forming the aorticopulmonary septum and great vessel smooth muscle retain distinct Hox-codes. A Meis2-Sox9-Scx GRN defines a skeletogenic progenitor-like intermediate state that gives rise to coronary artery smooth muscle and semilunar valves. Our findings suggest that the loss of Hox-dependent regional identity enables pharyngeal NCCs to acquire new fates upon entering the cardiac cushion, providing insight into the developmental origins of coronary and valvular calcification.

developmental biology↗

Development of the genetic toolkit identifies capsular polysaccharides as a competitive colonization factor in Ruminococcus gnavus

Ruminococcus gnavus is a commensal bacterium in the human gut associated with various diseases, and its abundance often increases in the intestines of patients with active inflammatory bowel disease (IBD). However, the genetic factors that establish gut colonization and virulence remain elusive due to the lack of a system for genetic modification. This study develops several genetic tools, including a shuttle vector, an inducible promoter, fluorescent proteins, and gene disruption or deletion systems in R. gnavus to identify the genes involved in R. gnavus colonization. Using these genetic tools, we found the respective sortase enzymes necessary for both cell wall localization of the superantigens and capsular polysaccharide (CPS) production in R. gnavus ATCC 29149. We identified the putative new gene cluster responsible for CPS production in the adjacent region of the sortase gene and confirmed that CPS production is crucial for competitive fitness during colonization in germ-free mouse intestines and that CPS production is inversely correlated with the inflammatory activity of R. gnavus. Furthermore, CPS cluster-possessing strains tended to be more prevalent in healthy human isolates compared to those from Crohns disease patients. This implies that CPS production is involved in their inflammatory activities and pathogenesis of intestinal diseases such as IBD. We highlight the potential of precise gene modification systems to identify the genetic factors that facilitate intestinal colonization and pathogenesis in gut bacteria.

microbiology↗

Therapeutic ability of extracellular vesicles derived from angio-miRNA-modified mesenchymal stromal cells in ischemic animal models

BackgroundRegenerative therapy using extracellular vesicles (EVs) is expected to be an option for supportive therapy of chronic limb-threatening ischemia. In this study, we examined the angiogenic potential of EVs derived from genetically modified MSCs, focusing on the angio-miRNAs in these vesicles. MethodsBone marrow mesenchymal stromal cells (BM-MSCs) were transfected with a lentiviral vector containing specific angio-miRNAs (miRNA-126, -135b, or -210). EVs were separated from the culture medium of BM-MSCs via ultracentrifugation. The quality and quantity of the EVs were evaluated using nanoparticle tracking analysis, fluorometry, and an ExoScreen assay. Tube formation assays were conducted using human umbilical vein endothelial cells to assess the angiogenic potency of the EVs in vitro. The EVs were injected into the ischemic hind limb muscles of mice. The grades of limb ischemia, blood perfusion, and muscle tissues were analyzed to assess the angiogenic potency of the EVs in vivo. ResultsAngio-miRNAs were overexpressed in MSCs transfected with a lentiviral vector. Tube formation was significantly enhanced when endothelial cells were cultured with the EVs (P < 0.05). Compared to non-contact co-culture with BM-MSCs, EVs derived from modified BM-MSCs significantly promoted tube formation in endothelial cells (P < 0.05). The severity of limb ischemia on day 7 was alleviated when a combination of two or three EVs derived from modified BM-MSCs was injected compared with native EVs. Toe blood perfusion on day 14 significantly recovered when a combination of two or three EVs derived from modified BM-MSCs was injected (P < 0.05). Tissue analysis revealed that the number of newly formed endothelial cells in ischemic muscles increased when a combination of two or three EVs derived from modified BM-MSCs was injected. ConclusionsThe use of a combination of EVs derived from genetically modified MSCs stimulated angiogenesis both in vitro and in vivo.

cell biology↗

Submolecular-scale Hairpin DNA Folding Dynamics Studied by High-Speed AFM with Optical Tweezers

Optical tweezers have contributed to elucidate the folding mechanisms associated with biomolecules. By combining single-molecule or super-resolution techniques, imaging can also be performed while measuring or inducing force coupling with biochemical reactions; however, they cannot capture structural information beyond the fluorophore spatial resolution. To overcome this problem, here, we developed a technique that combines optical tweezers with high-speed atomic force microscopy (AFM). To solve the problem of incompatible instrumental configurations, we developed a unique optical tweezers measurement system that is specialized for high-speed AFM. Upon applying an external force to a synthesized DNA secondary structure, we successfully visualized the dissociation of the duplex structure. Furthermore, we succeeded in observing spontaneous reannealing of the duplex structure upon releasing the force, which demonstrates that the folding reaction can be reversibly controlled. We also reveal that along with duplex unfolding, a metastable secondary structure is generated and its topology changes transiently over time. The results indicate that this technique provides structural insights that cannot be obtained by conventional fluorescence techniques.

biophysics↗

Identification of new resistance QTL regions in loquat cultivar Champagne against Pseudomonas syringae pv. eriobotryae group C

Loquat canker, caused by Pseudomonas syringae pv. eriobotryae, is a bacterial disease that infects loquat (Eriobotrya japonica) and has been reported in several countries. Three pathotypes, A, B, and C, have been reported in Japan. The loquat cultivar Champagne is resistant to the loquat canker group C and possesses a qualitative trait governed by a recessive homozygous pse-c gene located on Linkage Group 3 (LG3), and quantitative traits located at unidentified loci. In this study, we identified novel quantitative trait loci (QTL) regions for resistance to group C in this cultivar. A seedling population with Tanaka (Pse-c/Pse-c) crossed with Champagne (pse-c/pse-c) was tested. The genetic map of Champagne includes a total of 1,016 SNP markers mapped across 17 LGs, covering a total distance of 1,301 cM and an average marker density of 1.4 cM/locus. In addition to minor potential QTLs, the major QTL for resistance to loquat canker group C was detected in the upper region of LG14, with the QTL contributing 6.9% to the disease index. The results of this study open new possibilities for resistance breeding against this disease. Highlights.A total of 1,016 SNP markers were mapped on a linkage map consisting of 17 linkage groups with a total distance of 1,301 cM. QTL analysis revealed a novel resistance QTL region in Champagne against loquat canker group C in the upper part of the LG14. The identified QTLs in this study provide new possibilities for resistance breeding in loquat.

plant biology↗

A stress sensor IRE1α is required for bacterial exotoxin-induced inflammasome activation in tissue-resident macrophages

Cholera toxin (CT), a bacterial exotoxin composed of one A subunit (CTA) and five B subunits (CTB), functions as an immune adjuvant. CTB can induce production of interleukin-1{beta} (IL-1{beta}), a proinflammatory cytokine, in synergy with a lipopolysaccharide (LPS), from resident peritoneal macrophages (RPMs) through the pyrin and NLRP3 inflammasomes. However, how CTB or CT activates these inflammasomes in the macrophages has been unclear. Here, we clarified the roles of IRE1, an endoplasmic reticulum (ER) stress sensor, in CT-induced IL-1{beta} production from RPMs. In RPMs, CTB is incorporated into ER and induced ER stress responses, depending on GM1, a cell membrane ganglioside. IRE1-deficient RPMs showed a significant impairment of CT- or CTB-induced IL-1{beta} production, indicating that IRE1 was required for CT- or CTB-induced IL-1{beta} production from RPMs. This study first demonstrates the critical roles of IRE1 in activation of both NLRP3 and pyrin inflammasomes in tissue-resident macrophages. One sentence summaryIRE1 is required for NLRP3 and pyrin-mediated IL-1{beta} production

immunology↗

Partially hydrolyzed guar gum attenuates the symptoms of SARS-CoV-2 infection through gut microbiota modulation in an animal model.

The coronavirus disease 2019 (COVID-19) pandemic has caused worldwide health issues. Although several vaccines have been developed, it is still difficult to prevent and reduce the inflammation caused by the infection. Studies have shown that there are correlations between the gut environment and severity of symptoms caused by SARS-CoV-2 infection. Several gut metabolites produced by the gut microbiota such as SCFAs and the secondary bile acid UDCA are reported to improve the survival rate of the host after viral infection in an animal model through modulation of the host immune system. Therefore, in this study, we attempted to use the prebiotic dietary fiber PHGG to modulate the gut microbiome and intestinal metabolites for improvement of host survival rate after SARS-CoV-2 infection in a Syrian hamster model. We were able to show that PHGG significantly improved the host survival rate and body weight reduction. Analysis of the gut microbiome, serum, and intestinal metabolites revealed that PHGG significantly increased the concentrations of several intestinal SCFAs, fecal secondary bile acids, and serum secondary bile acids. Furthermore, several microbial species and metabolites identified in this study are consistent with reports in humans. Taken together, our data suggest that PHGG is a candidate prebiotic food for reducing the morbidity of COVID-19.

microbiology↗

Renal tubular function and morphology revealed in kidney without labeling using three-dimensional dynamic optical coherence tomography

Renal tubule has distinct metabolic features and functional activity that may be altered during kidney disease. In this paper, we present label-free functional activity imaging of renal tubule in normal and obstructed mouse kidney models using three-dimensional (3D) dynamic optical coherence tomography (OCT) ex vivo. To create an obstructed kidney model, we ligated the ureter of the left kidney for either 7 or 14 days. Two different dynamic OCT (DOCT) methods were implemented to access the slow and fast activity of the renal tubules: a logarithmic intensity variance (LIV) method and a complex-correlation-based method. Three-dimensional DOCT data were acquired with a 1.3 m swept-source OCT system and repeating raster scan protocols. In the normal kidney, the renal tubule appeared as a convoluted pipe-like structure in the DOCT projection image. Such pipe-like structures were not observed in the kidneys subjected to obstruction of the ureter for several days. Instead of any anatomical structures, a superficial high dynamics appearance was observed in the perirenal cortex region of the obstructed kidneys. These findings suggest that volumetric DOCT can be used as a tool to investigate kidney function during kidney diseases.

bioengineering↗

Gut microbiome-based prediction of autoimmune neuroinflammation

Gut commensals are linked to neurodegenerative diseases, yet little is known about causal and functional roles of microbial risk factors in the gut-brain axis. Here, we employed a pre-clinical model of multiple sclerosis in mice harboring distinct complex microbiotas and six defined strain combinations of a functionally-characterized synthetic human microbiota. Discrete microbiota compositions resulted in different probabilities for development of severe autoimmune neuroinflammation. Nevertheless, assessing presence or the relative abundances of a suspected microbial risk factor failed to predict disease courses across different microbiota compositions. Importantly, we found considerable inter-individual disease course variations between mice harboring the same microbiota. Evaluation of multiple microbiome-associated functional characteristics and host immune responses demonstrated that the immunoglobulin A-coating index of Bacteroides ovatus before disease onset is a robust individual predictor for disease development. Our study highlights that the "microbial risk factor" concept needs to be seen in the context of a given microbial community network, and host-specific responses to that community must be considered when aiming for predicting disease risk based on microbiota characteristics.

microbiology↗

Facilitative interaction networks in experimental microbial community dynamics

Understanding potential roles of facilitative interactions between species is one of the major challenges in ecology and microbiology. However, we still have limited knowledge of entangled webs of facilitative interactions in ecosystems. By compiling whole-genome shotgun metagenomic data of an experimental microbial community, we tested the hypothesis that architecture of facilitative interaction networks could change through time. A metabolic modeling approach for estimating dependence between microbial genomes (species) allowed us to infer the network structure of potential facilitative interactions at 13 time points through the 110-day monitoring of experimental microbiomes. We then found that positive feedback loops, which were theoretically predicted to promote cascade breakdown of ecological communities, existed within the inferred networks of metabolic interactions prior to the drastic community-compositional shift observed in the microbiome time-series. We further applied "directed-graph" analyses to pinpoint potential keystone species located at the "upper stream" positions of such feedback loops. These analyses on facilitative interactions will help us understand key mechanisms causing catastrophic shifts in microbial community structure.

ecology↗

Metagenomic analysis of ecological niche overlap and community collapse in microbiome dynamics

Species utilizing the same resources often fail to coexist for extended periods of time. Such competitive exclusion mechanisms potentially underly microbiome dynamics, causing breakdowns of communities composed of species with similar genetic backgrounds of resource utilization. Although genes responsible for competitive exclusion among a small number of species have been investigated in pioneering studies, it remains a major challenge to integrate genomics and ecology for understanding stable coexistence in species-rich communities. Here, we show that community-scale analyses of functional gene redundancy can provide a useful platform for interpreting and predicting collapse of bacterial communities. Through 110-day time-series of experimental microbiome dynamics, we analyzed the metagenome-assembled genomes of co-occurring bacterial species. We then inferred ecological niche space based on the multivariate analysis of the genome compositions. The analysis allowed us to evaluate potential shifts in the level of niche overlap between species through time. We hypothesized that community-scale pressure of competitive exclusion could be evaluated by quantifying overlap of genetically determined resource-use profiles (metabolic pathway profiles) among coexisting species. We found that the degree of community compositional changes observed in the experimental microbiome was explained by the magnitude of gene-repertoire overlaps among bacterial species. The metagenome-based analysis of genetic potential for competitive exclusion will help us forecast major events in microbiome dynamics such as sudden community collapse (i.e., dysbiosis).

microbiology↗