Biology subjects

Ishida, M.

Publications and source records attributed to Ishida, M..

16 recordsLinked to original sources

bioRxiv2026

NECTIN4 defines a subtype-dependent tumor state coupling enfortumab vedotin targetability to antitumor immunity in bladder cancer

NECTIN4 is the target of enfortumab vedotin (EV), but its biological role in bladder cancer (BC) and its relationship to EV plus PD-1 blockade remain unclear. Integrating clinical multi-omics, spatial and single-cell immune profiling, and experimental models, we identify NECTIN4 as a subtype-dependent determinant of tumor state and antitumor immunity. In basal BC cells, NECTIN4 suppressed inflammatory and mesenchymal programs. Consistently, NECTIN4-high basal/squamous muscle-invasive BCs showed favorable survival, higher checkpoint inhibitor response rates, and a coordinated CD8+ T cell-inflamed microenvironment with no comparable associations in luminal papillary tumors. In orthotopic models, murine Nectin4 promoted CD8+ T cell-dependent tumor control, whereas human NECTIN4 conferred EV sensitivity. EV also attenuated NECTIN4-dependent TIGIT binding. Conversely, NECTIN4-low models were preferentially sensitive to HDAC inhibition. NECTIN4 therefore couples antibody-drug conjugate targetability to a subtype-specific tumor-immune state permissive for checkpoint blockade, whereas the NECTIN4-low state defines a distinct inflammatory-mesenchymal state with an alternative therapeutic vulnerability.

cancer biology↗

bioRxiv2026

Effects of periodic group housing opportunities on reproductive performance and welfare in sows

ObjectiveStall housing of pregnant sows raises welfare concerns, whereas conventional group housing systems often reduce space efficiency. This study evaluated the effects of periodic group housing (PG) on reproductive performance and welfare compared with continuous stall housing (CS). MethodsSows in the CS group (n = 15) were continuously housed in stalls. In the PG group (n = 15), sows were housed in groups of three and allocated 1 day of group housing and 6 days of stall housing per week over 10 weeks. During group housing sessions, the sows had access to a group housing area containing sawdust. Behavioral observations and salivary cortisol measurements were conducted on the first day of the stall housing session in weeks 1, 6, and 10. Behavioral indices were expressed as proportions based on 90 sampling points recorded at 1-min intervals. ResultsThe number of stillbirths was significantly lower in the PG group than in the CS group (0.63 vs. 1.49 piglets per litter). whereas other reproductive outcomes, including total litter size and average birth weight, did not differ. In older parity sows, the PG treatment markedly increased the proportion of time spent lying, suggesting reduced discomfort associated with restricted movement. Furthermore, the proportion of exploratory behavior decreased markedly, and drinking behavior showed a decreasing trend across parity levels in the PG group, suggesting partial satisfaction of motivations for environmental exploration and oral manipulation. The proportion of oral abnormal behavior showed a pronounced interaction between housing treatment and experimental week, increasing from week 1 to week 6 in the PG group. Salivary cortisol concentrations did not differ between the groups. ConclusionPG may improve reproductive performance and partially satisfy the behavioral motivations restricted under continuous stall housing. This system may represent a practical alternative for improving animal welfare while minimizing economic losses.

animal behavior and cognition↗

bioRxiv2025

Visualization of functional and effective connectivity underlying auditory descriptive naming

ObjectiveWe visualized functional and effective connectivity within specific white matter networks in response to auditory descriptive questions. MethodsWe investigated 40 Japanese-speaking patients with focal epilepsy and estimated connectivity measures using cortical high-gamma dynamics and MRI tractography. ResultsHearing a wh-interrogative at question onset enhanced inter-hemispheric functional connectivity, with left-to-right callosal facilitatory flows between the superior-temporal gyri, contrasted by functional connectivity diminution with right-to-left callosal suppressive flows between dorsolateral prefrontal regions. Processing verbs associated with concrete objects or adverbs increased left intra-hemispheric connectivity, with bidirectional facilitatory flows through extensive white matter pathways. Questions beginning with what, compared to where, induced greater neural engagement in the left posterior inferior-frontal gyrus at question offset, linked to enhanced functional connectivity and bidirectional facilitatory flows to the temporal lobe neocortex via the arcuate fasciculus. During overt responses, inter-hemispheric functional connectivity was enhanced, with bidirectional callosal flows between Rolandic areas, and individuals with higher IQ scores exhibited less prolonged neural engagement in the left posterior middle frontal gyrus. ConclusionsVisualization of directional neural interactions within white matter networks during overt naming is feasible. SignificancePhrase order may influence network dynamics in listeners, even when presented with auditory descriptive questions conveying similar meanings.

neuroscience↗

bioRxiv2024

A coral peptide has bactericidal activity against a global marine pathogen, Vibrio coralliilyticus

Scleractinian corals and their associated microorganisms, including endosymbiotic dinoflagellates and bacteria, constitute coral holobionts. Rising seawater temperatures weaken coral symbiotic relationships, thereby increasing susceptibility to infectious diseases and leading to disease outbreaks and subsequent population declines. The temperature-dependent coral pathogen, Vibrio coralliilyticus, poses one of the greatest threats to coral reefs because of global warming. However, coral immune defenses against this pathogen are poorly understood. We previously identified coral genes responding to V. coralliilyticus exposure by transcriptomic analysis of the reef-building coral, Acropora digitifera. Here, bioinformatic analysis identified digitiferin, a coral antimicrobial peptide (AMP), in the genome of A. digitifera. Recombinant digitiferin showed antibacterial activity against both Gram-positive and -negative bacteria. A 3D structural model and direct microscopic observations revealed that digitiferin damaged bacterial cell membranes by forming pores; however, initial growth inhibition tests in 1.5 % (w/v) NaCl showed no effect on pathogens. Because we found that digitiferin is secreted from epithelial cells into mucus and shows salt sensitivity, we hypothesized that it is active against pathogens in low-salt environments. We then investigated whether it showed antibacterial activity against pathogens in low-salt, moderate-osmolality conditions, using mannitol for osmoregulation instead of NaCl. The results showed that digitiferin exhibits bactericidal activity against V. coralliilyticus under salt-free conditions. To the best of our knowledge, this is the first study to characterize a scleractinian AMP that kills V. coralliilyticus. These findings contribute to a better understanding of coral immunity and may facilitate development of techniques to overcome coral Vibrio disease. SignificanceHealthy coral reefs are one of the most important marine ecosystems, supporting [~]25 % of all marine organisms. Coral reef ecosystems are threatened by coral infectious diseases, as rising seawater temperatures exacerbate pathogen infectivity. Thus, understanding coral immune systems is becoming increasingly important to protect coral reefs. This study reports the discovery of an antimicrobial peptide, digitiferin, from the reef-building coral, Acropora digitifera. Digitiferin is effective against Gram-positive and -negative bacteria, including the major coral pathogen, Vibrio coralliilyticus, and is secreted into mucus from ectodermal granular epithelial cells. As the first antimicrobial peptide known to have bactericidal activity against V. coralliilyticus, this discovery enhances our understanding of coral immunology and coral-pathogen interactions.

microbiology↗

bioRxiv2024

High-speed Intracellular Temperature Mapping Reveals the Existence of Non-Conductive Dissipation of Energy by Heating

Recently, intracellular thermometry has revealed temperature variations within cells. Although the biological significance of intracellular temperature change is recognized, the physical principles of intracellular temperature change remain a mystery. Here, we investigate intracellular heat transfer through intracellular temperature mapping using a fluorescent polymeric thermometer and high-speed fluorescence lifetime imaging microscopy. Through infrared laser irradiation-assisted heating, we track changes in temperature distribution to examine the mechanism of intracellular heat dissipation in comparison with heat conduction. Continuous heating induces the significantly slower relaxation of the average temperature of single cells compared with that of liposomes containing homogeneous aqueous solutions of comparable size; to the scale of seconds. We additionally elucidate that these phenomena are impacted by intracellular structures and molecules. Finally, we discover that this slow intracellular temperature relaxation originates from non-diffusive heat dissipation distinct from the conventional heat conduction model. Our results provide insights into the mechanisms of temperature variation in cells that are unresolved based on our current understanding, establishing a framework for understanding intracellular thermodynamics under non-equilibrium conditions.

biophysics↗

bioRxiv2024

Traveling wave chemotaxis of neutrophil-like HL-60 cells

The question of how changes in chemoattractant concentration translate into the chemotactic response of immune cells serves as a paradigm for the quantitative understanding of how cells perceive and process temporal and spatial information. Here, using a microfluidic approach, we analyzed the migration of neutrophil-like HL-60 cells to a traveling wave of the chemoattractants fMLP and leukotriene B4 (LTB4). We found that under a pulsatile wave that travels at a speed of 95 and 170 {micro}m/min, cells move forward in the front of the wave but slow down and randomly orient at the back due to temporal decrease in the attractant concentration. Under a slower wave, cells re-orient and migrate at the back of the wave; thus, cell displacement is canceled out or even becomes negative as cells chase the receding wave. FRET-based analysis indicated that these patterns of movement correlated well with spatiotemporal changes in Cdc42 activity. Furthermore, pharmacological perturbations suggested that migration in front of the wave depends on Cdc42, whereas that in the back of the wave depends more on PI3K/Rac and ROCK. These results suggest that pulsatile attractant waves may recruit or disperse neutrophils, depending on their speed and degree of cell polarization. Significance statementO_LIThe way neutrophil chemotaxis is directed by attractants is thought to depend on temporal and spatial concentration changes, however the response to a transient pulsatile stimulus has not been well explored. C_LIO_LIA moderately fast traveling wave of fMLP and leukotrieneB4 (LTB4) directs unidirectional cell migration. Under slow waves, cells respond to the back of the wave; thus, cell displacement is canceled out. Cdc42, PI3K, Rac, and ROCK contributed differently to responses at the front and back of the wave. C_LIO_LIThese findings suggest that traveling waves of attractant can guide immune cell recruitment and dispersal. C_LI

cell biology↗

bioRxiv2024

The transcriptomic landscape of monosomy X (45,X) during early human fetal and placental development

Monosomy X (45,X) is associated with Turner syndrome and pregnancy loss in humans, but the underlying mechanisms remain unclear. We therefore analyzed the transcriptomic landscape of clinically relevant human fetal 45,X tissues (including pancreas, liver, kidney, skin, placenta) with matched 46,XX and 46,XY control samples between 11-15 weeks post conception (n=78). Although most pseudoautosomal region 1 (PAR1) genes were lower in monosomy X tissues, we also found reduced expression of several key genes escaping X inactivation (e.g., KDM5C and KDM6A), and potentially clinically important transcripts such as genes implicated in ascending aortic aneurysm. In contrast, higher expression of an autosomal, long non-coding RNA (OVCH1-AS1) was seen in all 45,X tissues. In the placenta, lower expression of CSF2RA was demonstrated, likely contributing to immune dysregulation. Taken together, these findings provide novel insights into the biological consequences of a single X chromosome during early human development and potential insights in genetic mechanisms in Turner syndrome.

genetics↗

bioRxiv2023

Enhancer evolution as a driving force for lineage-specific paralog usage in the central nervous system.

Expression patterns of paralogous genes in the functionally homologous cells sometimes show differences across species. However, no reasonable explanation for the mechanism underlying such phenomena has been discovered. To understand this mechanism, the present study focused on the hypophysiotropic GnRH neurons in vertebrates as a model. These neurons express either gnrh1 or gnrh3 paralogs depending on species, and apparent switching of the expressed paralogs in them occurred at least four times in vertebrate evolution. First, we found redundant expressions of gnrh1 and gnrh3 in a single neuron in piranha and hypothesized that this situation may indicate an ancestral condition. We tested this hypothesis by examining the activity of piranha gnrh1/gnrh3 enhancers in zebrafish and medaka, in which the two gnrh paralogs are not co-expressed. Here, the gnrh1/gnrh3 enhancer of piranha induced reporter RFP/GFP co-expressions in a single hypophysiotropic GnRH neuron in both zebrafish and medaka. From these results, we propose that long-lasting ([~]550 My) redundancy after gnrh1/3 duplication in 1R/2R WGD may be the key to apparent switching of the paralog usage among the present-day species. Moreover, interspecies analyses of enhancers indicated that the loss of enhancers rather than changes in trans-regulatory elements drove the role-division of these paralogs.

evolutionary biology↗

bioRxiv2021

Tissue- and ethnicity-independent hypervariable DNA methylation states show evidence of establishment in the early human embryo

We analysed DNA methylation data from 30 datasets comprising 3,474 individuals, 19 tissues and 8 ethnicities at CpGs covered by the Illumina450K array. We identified 4,143 hypervariable CpGs ("hvCpGs") with methylation in the top 5% most variable sites across multiple tissues and ethnicities. hvCpG methylation was influenced but not determined by genetic variation, and was not linked to probe reliability, epigenetic drift, age, sex or cell heterogeneity effects. hvCpG methylation tended to covary across tissues derived from different germ-layers and hvCpGs were enriched for associations with periconceptional environment, proximity to ERV1 and ERVK retrovirus elements and parent-of-origin-specific methylation. They also showed distinctive methylation signatures in monozygotic twins. Together, these properties position hvCpGs as strong candidates for studying how stochastic and/or environmentally influenced DNA methylation states which are established in the early embryo and maintained stably thereafter can influence life-long health and disease.

genomics↗

bioRxiv2021

Optogenetic relaxation of actomyosin contractility uncovers mechanistic roles of cortical tension during cytokinesis

Actomyosin contractility generated cooperatively by nonmuscle myosin II and actin filaments plays essential roles in a wide range of biological processes, such as cell motility, cytokinesis, and tissue morphogenesis. However, it is still unknown how actomyosin contractility generates force and maintains cellular morphology. Here, we demonstrate an optogenetic method to induce relaxation of actomyosin contractility. The system, named OptoMYPT, combines a catalytic subunit of the type I phosphatase-binding domain of MYPT1 with an optogenetic dimerizer, so that it allows light-dependent recruitment of endogenous PP1c to the plasma membrane. Blue-light illumination was sufficient to induce dephosphorylation of myosin regulatory light chains and decrease in traction force at the subcellular level. The OptoMYPT system was further employed to understand the mechanics of actomyosin-based cortical tension and contractile ring tension during cytokinesis. We found that the relaxation of cortical tension at both poles by OptoMYPT accelerated the furrow ingression rate, revealing that the cortical tension substantially antagonizes constriction of the cleavage furrow. Based on these results, the OptoMYPT system will provide new opportunities to understand cellular and tissue mechanics.

cell biology↗

bioRxiv2020

DAJIN-assisted multiplex genotyping to validate the outcomes of CRISPR-Cas-based genome editing

Genome editing can introduce designed mutations into a target genomic site. Recent research has revealed that it can also induce various unintended events such as structural variations, small indels, and substitutions at, and in some cases, away from the target site. These rearrangements may result in confounding phenotypes in biomedical research samples and cause a concern in clinical or agricultural applications. However, current genotyping methods do not allow a comprehensive analysis of diverse mutations for phasing and mosaic variant detection. Here, we developed a genotyping method with an on-target site analysis software named Determine Allele mutations and Judge Intended genotype by Nanopore sequencer (DAJIN) that can automatically identify and classify both intended and unintended diverse mutations, including point mutations, deletions, inversions, and cis double knock-in at single-nucleotide resolution. Our approach with DAJIN can handle approximately 100 samples under different editing conditions in a single run. With its high versatility, scalability, and convenience, DAJIN-assisted multiplex genotyping may become a new standard for validating genome editing outcomes.

genomics↗

bioRxiv2020

Ethanolamine phosphate on the second mannose as bridge in GPI anchored proteins: Towards understanding inherited PIGG deficiency

Glycosylphosphatidylinositols (GPIs) are glycolipids that anchor many proteins (GPI-APs) on the cell surface. The core glycan of GPI precursor has three mannoses, which in mammals, are all modified by ethanolamine-phosphate (EthN-P). It is postulated that EthN-P on the third mannose (EthN-P-Man3) is the bridge between GPI to the protein and the second (EthN-P-Man2) is removed after GPI-protein attachment. However, EthN-P-Man2 may not be always transient, as mutations of PIGG, the enzyme that transfers EthN-P to Man2, result in inherited GPI deficiencies (IGDs), characterized by neuronal dysfunctions. Here, we show EthN-P on Man2 is the preferential bridge in some GPI-APs, among them, the ect-5-nucleotidase and netrin G2. We found that CD59, a GPI-AP, is attached via EthN-P-Man2 both in PIGB-knockout cells, in which GPI lacks Man3 and with a small fraction, in wild type cells. Our findings modify the current view of GPI anchoring and provide mechanistic bases of IGDs caused by PIGG mutations.

molecular biology↗

bioRxiv2020

Comparative mapping of crawling-cell morphodynamics in deep learning-based feature space

Navigation of fast migrating cells such as amoeba Dictyostelium and immune cells are tightly associated with their morphologies that range from steady polarized forms that support high directionality to those more complex and variable when making frequent turns. Model simulations are essential for quantitative understanding of these features and their origins, however systematic comparisons with real data are underdeveloped. Here, by employing deep-learning-based feature extraction combined with phase-field modeling framework, we show that a low dimensional feature space for 2D migrating cell morphologies obtained from the shape stereotype of keratocytes, Dictyostelium and neutrophils can be fully mapped by interlinked signaling network of cell-polarization and protrusion dynamics. Our analysis links the data-driven shape analysis to the underlying causalities by identifying key parameters critical for migratory morphologies both normal and aberrant under genetic and pharmacological perturbations. The results underscore the importance of deciphering self-organizing states and their interplay when characterizing morphological phenotypes.

cell biology↗

bioRxiv2019

Diverse Species-Specific Phenotypic Consequences of Loss of Function Sorting Nexin 14 Mutations

Mutations in the SNX14 gene cause spinocerebellar ataxia, autosomal recessive 20 (SCAR20) in both humans and dogs. SCAR20 is understood to involve subcellular disruption to autophagy and lipid metabolism. Previously reported studies on the phenotypic consequences of SNX14 mutations have been limited to in vitro investigation of patient-derived dermal fibroblasts, laboratory engineered cell lines and developmental analysis of zebrafish morphants. In addition, studies have investigated the biochemical roles of SNX14 homologues Snz (Drosophila) and Mdm1 (yeast) which have demonstrated an important role during lipid biogenesis. This study investigates the impact of constitutive Snx14 mutations in laboratory species: mice and zebrafish. Loss of SNX14 in mice was found to be embryonic lethal around mid-gestation. This is due to placental pathology that involves severe disruption to syncytiotrophoblast cell differentiation. Zebrafish carrying a homozygous, maternal zygotic snx14 genetic loss-of-function mutation contrasts with other vertebrates, being both viable and anatomically normal. Whilst no obvious behavioural effects were observed, elevated levels of neutral lipids and phospholipids resemble previously reported effects on lipid homeostasis in other species. The biochemical role of SNX14 therefore appears largely conserved through evolution while the overall consequences of loss of function varies considerably between species. New mouse and zebrafish models therefore provide valuable insights into the functional importance of SNX14 with distinct opportunities for investigating its cellular and metabolic function in vivo.

developmental biology↗

bioRxiv2019

ECM-body: A cell-free 3D biomimetic scaffold derived from intact planarian body

Extracellular matrix (ECM) plays key roles in shaping fates of stem cells, not only by providing suitable niche but also by mediating physical and biochemical cues. Despite intensive investigations on regeneration, the roles of ECM on fate determination of stem cells in animal with great regenerative potency, such as planarian, remained unclear. Here, we developed a method to decellularizing and isolating extracellular matrix from planarians. Although the isolated scaffold appears translucent, it contains all the internal features, resembling the structure of intact planarian, and which we thus called \"ECM-body\". Nuclear staining demonstrated that ECM-body contains very little or no cell remained. Histological sections displayed a well-preserved morphological integrity of the specimen. Scanning electron microscope showed porous surface on ECM-body, potentially suitable for housing cells. Furthermore, our preliminary experiment suggested that ECM-body can be utilized as biomimetic scaffold for cell culture as it may support survival of injected neoblasts.

developmental biology↗

bioRxiv2018

A Neurodevelopmental Disorder Caused by Mutations in the VPS51 Subunit of the GARP and EARP Complexes

GARP and EARP are related heterotetrameric protein complexes that associate with the cytosolic face of the trans-Golgi network and recycling endosomes, respectively. At these locations, GARP and EARP function to promote the fusion of endosome-derived transport carriers with their corresponding compartments. GARP and EARP share three subunits, VPS51, VPS52 and VPS53, and each has an additional complex-specific subunit, VPS54 or VPS50, respectively. The role of these complexes in human physiology, however, remains poorly understood. By exome sequencing, we have identified compound heterozygous mutations in the gene encoding the shared GARP/EARP subunit VPS51 in a six-year-old patient with severe global developmental delay, microcephaly, hypotonia, epilepsy, cortical vision impairment, pontocerebellar abnormalities, failure to thrive, liver dysfunction, lower extremity edema and dysmorphic features. The mutation in one allele causes a frameshift that produces a longer but highly unstable protein that is degraded by the proteasome. In contrast, the other mutant allele produces a protein with a single amino-acid substitution that is stable but assembles less efficiently with the other GARP/EARP subunits. Consequently, skin fibroblasts from the patient have reduced levels of fully-assembled GARP and EARP complexes. Likely because of this deficiency, the patients fibroblasts display altered distribution of the cation-independent mannose 6-phosphate receptor, which normally sorts acid hydrolases to lysosomes. Furthermore, a fraction of the patients fibroblasts exhibit swelling of lysosomes. These findings thus identify a novel genetic locus for a neurodevelopmental disorder and highlight the critical importance of GARP/EARP function in cellular and organismal physiology.

cell biology↗