Biology subjects

Endo, Y.

Publications and source records attributed to Endo, Y..

At least 19 recordsLinked to original sources

bioRxiv2026

A High-Sensitivity and High-Throughput LC-MS Workflow for Low-Input and Single-Cell Proteomics

Single-cell proteomics (SCP) requires sample preparation that minimizes sample loss and highly sensitive LC-MS analysis because the amount of protein available from a single cell is extremely limited. Here, we developed an integrated workflow for low-input and single-cell proteomics based on single-cell proteomics by LMNG-assisted sample preparation (scpLASP), which enables microliter-scale sample preparation using standard laboratory consumables. Systematic optimization of peptide reconstitution conditions identified 0.01% n-dodecyl-{beta}-D-maltoside (DDM) combined with 0.01% lauryl maltose neopentyl glycol (LMNG) as the condition yielding the highest number of identified peptides among those tested for low-input samples. Optimization of the LC mobile-phase composition identified 0.5% acetic acid with 1% dimethyl sulfoxide (DMSO) as the optimal condition, increasing the mean number of identified protein groups from 6,767 with 0.1% formic acid to 7,409 for 250 pg HeLa digests analyzed using an Orbitrap Astral Zoom. Shortening the LC gradient from 11 to 5.5 min enabled higher analytical throughput while maintaining mean identification counts of 6,558 and 7,088 protein groups from 100 and 250 pg HeLa digests, respectively, with median protein-level coefficients of variation of 9.20% and 6.99%. When the optimized workflow was applied to HEK293-derived Expi293F single cells using the same 5.5-min LC gradient, an effective analytical throughput of 135 samples per day was achieved with an injection volume of 4.5 {micro}L, accounting for sample-loading time. This workflow identified a median of 5,655 protein groups per cell using an empirical spectral library generated from 20-cell samples. Overall, this study establishes a sensitive and high-throughput workflow for low-input and single-cell proteomics that combines low-loss microliter-scale sample preparation with optimized LC-MS analysis, achieving high proteome depth and analytical throughput without requiring specialized nanoliter-scale sample-preparation devices.

biochemistry↗

bioRxiv2026

Structural determination of human nucleosomes reconstituted by the ExACT platform

The structure and function of eukaryotic chromatin have been extensively studied using conventional salt dialysis-based nucleosome assembly, which has provided fundamental structural insights into nucleosomes as well as a mechanistic understanding of their roles in DNA replication, repair and gene expression. Recently, we developed a labor-saving and time-efficient nucleosome assembly method using a wheat germ cell-free Expression and Assembly Coupled Technology (ExACT), which is emerging as a powerful tool for chromatin research. Here we report cryo-electron microscopy (cryo-EM) structures of human H3.1- and H3.3-containing nucleosomes assembled using this approach and validated by deep-learning-based amino acid-wise model quality (DAQ) scoring. The structures of H3.1- and H3.3-nucleosomes are nearly identical to previously reported models, confirming the structural fidelity of the method. In addition, we determined the previously unreported structure of the primate-specific H3.X-containing nucleosome. Together, these findings validate the cell-free co-expression nucleosome assembly platform and establish this method as a robust framework for biochemically investigating chromatin dynamics.

molecular biology↗

bioRxiv2026

Development of an Ethylenediaminetetraacetic Acid-Enhanced Deep Proteomic Profiling Method for Dried Blood Spots and Its Application in Mouse Disease Models

Dried blood spots (DBS) are well-established microsamples used in clinical testing and newborn screening. However, their use in deep proteomics is hindered by highly abundant blood proteins and inefficient protein recovery from filter paper matrices. The non-targeted analysis of non-specifically DBS-absorbed proteins (NANDA) workflow partially overcomes the impact of abundant blood proteins and has enabled the identification of over 5,000 proteins from DBS samples. Nonetheless, residual abundant proteins, including hemoglobin and fibrinogen, constrain deep proteomic analysis. Therefore, this study aimed to evaluate the effects of the metal chelator ethylenediaminetetraacetic acid (EDTA) on the depth of DBS proteomic analysis. An optimized EDTA-enhanced NANDA protocol that incorporated a 100 mM EDTA wash step was compatible with standard DBS collection procedures and required no modification of current clinical workflows, markedly enhancing the depletion of abundant proteins and facilitating its potential use in clinical and translational settings. When combined with Orbitrap Astral data-independent acquisition mass spectrometry, this approach enabled the single-shot identification of more than 7,000 proteins from DBS samples; to the best of our knowledge, this represents the deepest proteome coverage reported to date, and the workflow further supported high-throughput and highly reproducible analyses. Additionally, its application to mouse disease models revealed disease-specific systemic immune signatures from minimal blood volumes. Collectively, these results establish EDTA-enhanced NANDA as a practical and scalable workflow that overcomes longstanding limitations of DBS proteomics, thereby enabling deep, high-throughput, minimally invasive proteomic profiling across diverse biological and experimental contexts.

molecular biology↗

bioRxiv2026

11β-HSD2 buffers fetal glucocorticoid exposure inducing Per1 expression under maternal stress

Glucocorticoids (GCs) have been proposed as maternal-fetal communication signals. However, fetal circadian rhythms are initially shielded from maternal entrainment, in addition to delayed circadian clock emergence due to CLOCK suppression. Premature CLOCK/BMAL1 activation disrupts Hes7-driven somite-like structure in gastruloids. Given the genomic proximity of Per1 to Hes7 and their transcriptional ripple effect, the physiological significance of delayed cell-autonomous circadian clock development and the temporal program of maternal-fetal communication during the developmental process have remained unclear. Here, based on a marked decline in Hsd11b2, encoding a GC-inactivating 11{beta}-HSD2 enzyme, during organogenesis, we performed split-litter embryo-transfer experiments in which Hsd11b2 knockout (KO) and wild-type (WT) embryos shared the same maternal environment. Amniotic fluid (AF) GCs remained low and arrhythmic under basal conditions. In contrast, maternal stress caused a pronounced GC surge and Per1 induction in KO, suggesting that 11{beta}-HSD2 buffers acute maternal GC surges. Despite the genomic proximity of Per1 to Hes7 and their transcriptional ripple effect, stress-associated and pharmacological GC exposure recapitulated no overt segmentation defects in vivo. Embryonic stem cell-derived gastruloid assays confirmed that neither GC exposure nor Per1 induction arrested Hes7 oscillations, whereas premature CLOCK/BMAL1 activation impaired these processes even in Hes7 KO gastruloid with ectopic rescue, suggesting that interference with the segmentation clock is mediated by premature CLOCK/BMAL1 activation, not by GC-induced Per1 expression. These findings clearly show that maternal GC signals are selectively buffered during early development. In addition, suppression of CLOCK/BMAL1 activity preserves segmentation clock function, indicating delayed circadian clock emergence is actively regulated during embryogenesis. Significance StatementGCs have been proposed as maternal-fetal communication signals. However, initially, circadian clock is not only suppressed but also shielded from maternal entrainment. Premature CLOCK/BMAL1 activation can disrupt Hes7-driven somitogenesis. In a split-litter Hsd11b2-knockout model, AF GCs remained low and arrhythmic basally but surged after maternal stress in KO embryos, inducing Per1. Despite a genomic position effect of Per1-Hes7 and their putative transcriptional coupling, stress-associated or pharmacological GC exposure did not cause segmentation defects in vivo or disrupt Hes7 oscillations in vitro, whereas CLOCK/BMAL1-driven arrest of Hes7 oscillations persisted in gastruloids despite ectopic Hes7 rescue. These findings identify 11{beta}-HSD2 as a developmental buffer and support the physiological importance of the temporal architecture controlling the timing of circadian clock development.

developmental biology↗

bioRxiv2025

Conserved gene- and network-level thermal memory intervals in two divergent perennial crucifers in nature

Some biological responses persist long after the initial stimulus has disappeared--a phenomenon known as cellular memory. In its long-term form, cellular memory often reflects interactions between cis-acting chromatin states and diffusible trans-acting regulators, which are experimentally difficult to separate in vivo. A key challenge is to develop a quantitative framework that captures cellular memory length even without prior mechanistic knowledge. The FLOWERING LOCUS C (FLC) gene illustrates this problem and opportunity: a Polycomb/Trithorax cis-acting chromatin switch at FLC produces bistable ON/OFF transcriptional states, while trans-acting factors such as VIN3 and FT modulate transitions between those states. In laboratory studies, FLC memory is classically viewed as persistence of repression or activation after the inducing signal disappears; field studies additionally show that FLC integrates fluctuating temperatures over past intervals, revealing time-integrative mode of memory. To quantify such long-term effects systematically, we formalized the thermal memory interval (TMI), the time window of past environmental cues that best predicts current gene expression--as a transferable metric. We applied TMI to the VIN3-FLC-FT module in two perennial Brassicaceae with divergent life histories: Arabidopsis halleri subsp. gemmifera, an established ecological model, and Eutrema japonicum, introduced here to identify features generalizable beyond a single species. Using long-term expression and meteorological data, TMIs distinguished spring versus autumn FLC states and revealed distributed memory across the VIN3-FLC-FT network, with intervals from 1-150 days, extending previously reported FLC timescales. While TMIs require dense time-series data and do not by themselves reveal molecular mechanism, they offer a robust, quantitative, and generalizable framework: TMIs can be extended to other genes and to alternative environmental or physiological variables, enabling direct, comparative quantification of cellular memory across genes, species, and contexts.

molecular biology↗

bioRxiv2025

Kidney-specific lipid metabolism underlies stable chemical individuality in domestic cats and other felids

Domestic cats uniquely accumulate lipid droplets (LDs) in renal proximal tubular epithelial cells, but their chemical composition and biological functions remain unknown. Here, we identify a distinctive renal specialization in felids: lipid droplets enriched in branched-chain fatty acids (BFAs). These BFAs accumulate as triglyceride components and are excreted in urine as free fatty acids with stable and individually distinctive profiles. Behavioral assays show that cats discriminate urine samples based on BFA composition, even against complex volatile backgrounds, suggesting BFAs as robust, metabolically encoded olfactory signatures for individual recognition. Comparative analyses of non-domesticated felids reveal that BFA production is broadly conserved but exhibits both species-specific and individual variation. These findings demonstrate how a core metabolic pathway has been evolutionarily co-opted into a chemical signature system. This discovery highlights an unusual integration of renal physiology with social communication and expand our understanding of lipid metabolism, organ-level adaptation, and the evolution of chemical individuality in mammals. Significance statementUnique lipid droplets in felid kidneys reveal how organ-specific metabolism can be adapted for social communication.

zoology↗

bioRxiv2025

Whole-genome sequencing revealed local genetic differences and multi-layered genetic structure in brown bear (Ursus arctos) populations

A multi-layered genetic structure shaped by multiple demographic events across different time periods has been well documented in some species. However, evaluating this pattern remains challenging in many wide-ranging wildlife because genome-wide datasets with broad geographic coverage are still scarce. Brown bears are genetically differentiated among regional populations, but the genomic data are still limited and the demographic events remain incompletely understood. Here, we generated whole-genome resequencing data from brown bears in Asian local populations previously reported to show significant variation and combined these data with ancient samples for population genomic analyses. Our results support previously reported demographic events, such as the early divergence of Central Asian populations. Furthermore, we found genetic differences among Asian brown bear populations, suggesting that population-specific demographic histories occurring at different times have contributed to their unique genetic characteristics. In the Central and West Asian populations, divergence times estimated from autosomal genomes differed by hundreds of thousands of years from those inferred from mitochondrial DNA and Y-chromosomal DNA haplotypes, suggesting that gene flow continued after regional dispersal. In addition, the Y-chromosomal phylogeny and f4 statistics indicated genetic differentiation between the Kazakhstan and Tibetan populations, likely resulting from male-biased introgression into the Tibetan population. Finaly, the Hokkaido and Etorofu (Iturup) populations retained alleles derived from hybridization with polar bears, likely due to the limited impact of post-LGM population expansion. These findings indicate that the genetic variation among brown bear populations is shaped by multiple demographic events occurring at different times, resulting in a multi-layered genetic structure. Significance StatementMulti-layered genetic structure is well known in some species, but remains poorly understood in wild species due to limited genomic data. In this study, we newly sequenced nine brown bears in Asia which included bears that genome data in their area is unreported. By combining these with modern and ancient genomes, we conducted population genomic analyses to characterize local genetic variation and detect hybridization with related species such as the polar bear. We identified (1) substantial divergence time gaps in Central and West Asian populations between autosomal genomes and mitochondrial and Y-chromosomal haplotypes, (2) male-biased introgression into the Tibetan population, and (3) limited impact of recent expansion between island and Eurasian continental populations. These findings suggest that the genetic structure of the populations has been shaped by multiple demographic events at different times, resulting in a multi-layered genetic structure and providing new insights into how wildlife genetic diversity is shaped.

evolutionary biology↗

bioRxiv2024

A novel RyR2-selective stabilizer prevents and ameliorates severe arrhythmias in stress-induced ventricular tachycardia

Background and PurposeAberrant activation of the type 2 ryanodine receptor (RyR2) causes lethal arrhythmias, such as catecholaminergic polymorphic ventricular tachycardia (CPVT). Developing drugs that suppress RyR2 hyperactivation may be key to novel arrhythmia treatments. This study evaluated the antiarrhythmic potential of Ryanozole, a recently developed novel RyR2 modulator with high affinity and selectivity, using CPVT mouse models harboring mutant RyR2s. Experimental approachIn vitro effects of Ryanozole were evaluated by ER Ca2+-based assay and [3H]Ryanodine binding assay using RyR2-expressing HEK293 cells. Two lines of mice with different arrhythmia severity, RyR2-R420W and -K4750Q, were employed for in vivo assessments. Intracellular Ca2+ signals were analyzed in isolated cardiomyocytes using Cal520. Antiarrhythmic effects were evaluated by ECG under catecholaminergic challenge in anesthetized mice and during spontaneous arrhythmias in conscious mice. ECG and echocardiographic parameters were evaluated before and after drug administration. Key resultsRyanozole inhibited both wild type and mutant RyR2s with similar IC50 of 15-40 nM. The inhibition was more potent at lower cytosolic Ca2+ concentrations. It suppressed Ca2+ waves and Ca2+ sparks without affecting action potential-evoked Ca2+ transients. Ryanozole effectively prevented adrenaline-induced arrhythmias and rapidly terminated spontaneous arrhythmias during daily activity. Importantly, Ryanozole did not impair cardiac conduction or contractility, unlike conventional antiarrhythmic drugs. Conclusions and implicationsRyanozole strongly suppresses RyR2 under diastolic Ca2+ conditions, thereby preventing the arrhythmogenic trigger of aberrant Ca2+ release. This mechanism likely provides potent antiarrhythmic effects while preserving cardiac function. Ryanozole is a promising therapeutic candidate treating RyR2-mediated arrhythmias, such as CPVT.

pharmacology and toxicology↗

bioRxiv2024

Secreted phospholipase PLA2G12A-driven lysophospholipid signaling via lipolytic modification of extracellular vesicles facilitates pathogenic Th17 differentiation

Lipogenesis-driven metabolic flux is crucial for differentiation of pathogenic Th17 cells. Although our previous CRISPR-based screening identified PLA2G12A as a key player in this process, it has remained obscure how this secreted phospholipase A2 isoform controls Th17 differentiation. Here we show that global, T cell-specific, or fibroblast-specific deletion of PLA2G12A prevents Th17 differentiation and associated diseases including psoriasis and arthritis. PLA2G12A acts on Th17-derived extracellular vesicles (EVs) to produce lysophospholipids including the ROR{gamma}t activator 1-oleoyl-lysophosphatidylethanolamine. These lysophospholipids are further converted by autotaxin to lysophosphatidic acid (LPA), which assists Th17 differentiation mainly via LPA2 receptor. Moreover, PLA2G12A promotes the secretion and uptake of EVs by Th17 cells and alters their cargo contents. Defective Th17 differentiation by PLA2G12A deficiency is rescued by supplementation with PLA2G12A-modified EVs. Importantly, a PLA2G12A-blocking antibody prevents Th17 differentiation and ameliorates psoriasis and arthritis models. Thus, targeting the PLA2G12A-EV-lysophospholipid axis may be useful for treatment of Th17-related diseases.

immunology↗

bioRxiv2023

Universal pretreatment development for low-input proteomics using Lauryl Maltose Neopentyl Glycol

In recent years, the demand for low-input proteomics, most notably single-cell proteomics (SCP), has increased. In this study, we developed a Lauryl Maltose Neopentyl Glycol (LMNG)-assisted sample preparation (LASP) method that suppresses the loss of proteins and peptides in samples by adding LMNG, a surfactant, to the digested solution and removing the LMNG simply via reversed phase solid-phase extraction. The advantage of removing LMNG during sample preparation for general proteomic analysis is that it prevents mass spectrometry (MS) contamination. When the LASP method was applied to the low-input SP3 method and on-bead digestion in immunoprecipitation-MS, the recovery of the digested peptides was greatly improved. Furthermore, we established a simple and operationally easy sample preparation method for SCP based on the LASP method (scpLASP) and identified a median of 1,175 proteins from a single HEK239F cell using liquid chromatography (LC)-MS/MS with a throughput of 80 samples per day.

biochemistry↗

bioRxiv2023

Liver-specific LXR inhibition represses reverse cholesterol transport in cholesterol-fed mice

ObjectiveHigh density lipoprotein (HDL) exerts an anti-atherosclerotic effect via reverse cholesterol transport (RCT). Several phases of RCT are transcriptionally controlled by Liver X receptors (LXRs). Although macrophage LXRs reportedly promote RCT, it is still uncertain whether hepatic LXRs affect RCT in vivo. Approach and ResultsTo address this question, we induced hepatic overexpression of sulfotransferase family cytosolic 2B member 1 (Sult2b1) in mice. Sult2b1 facilitates generation of sulfated cholesterol, resulting in reduced production of LXR ligands (oxysterols), which impairs LXR signaling. Adenoviral vectors expressing Sult2b1 (Ad-Sult2b1) or luciferase were intravenously injected into mice under a normal or high-cholesterol diet. Hepatic Sult2b1 overexpression resulted in reduced expression of LXR-target genes - ATP-binding cassette transporter G5/G8, cholesterol 7 hydroxylase and LXR itself - respectively reducing or increasing cholesterol levels in HDL and apolipoprotein B-containing lipoproteins (apoB-L). A macrophage RCT assay revealed that Sult2b1 overexpression inhibited fecal excretion of macrophage-derived 3H-cholesterol only under a high-cholesterol diet. In a HDL kinetic study, Ad-Sult2b1 promoted catabolism/hepatic uptake of HDL-derived cholesterol, thereby reducing fecal excretion. We next performed an in vitro lipoprotein production assay which revealed a Sult2b1-mediated reduction/increase in HDL or apoB-L secretion from hepatocytes, respectively. Finally, in LXR/{beta} double knockout mice, hepatic Sult2b1 overexpression increased apoB-L levels, but there were no differences in HDL levels or RCT compared to the control, indicating that Sult2b1-mediated effects on HDL/RCT and apoB-L were distinct: the former was LXR-dependent, but not the latter. ConclusionsHepatic LXR inhibition negatively regulates circulating HDL levels and RCT by reducing LXR-target gene expression. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/527401v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@18ff927org.highwire.dtl.DTLVardef@12e776corg.highwire.dtl.DTLVardef@1a26ebaorg.highwire.dtl.DTLVardef@113b5c6_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2021

Neural mechanism of experience-dependent sensory gain control in C. elegans

Animals sensory systems adjust their responsiveness to environmental stimuli that vary greatly in their intensity. Here we report the neural mechanism of experience-dependent sensory adjustment, especially gain control, in the ASH nociceptive neurons in Caenorhabditis elegans. Using calcium imaging under gradual changes in stimulus intensity, we find that the ASH neurons of naive animals respond to concentration increases in a repulsive odor 2-nonanone regardless of the magnitude of the concentration increase. However, after preexposure to the odor, the ASH neurons exhibit significantly weak responses to a small gradual increase in odor concentration while their responses to a large gradual increase remain strong. Thus, preexposure changes the slope of stimulus-response relationships (i.e., gain control). Behavioral analysis suggests that this gain control contributes to the preexposure-dependent enhancement of odor avoidance behavior. Mathematical analysis reveals that the ASH response consists of fast and slow components, and that the fast component is specifically suppressed by preexposure. In addition, genetic analysis suggests that G protein signaling is required for the fast component. Thus, our integrative study demonstrates how prior experience dynamically modulates stimulus-response relationships in sensory neurons, eventually leading to adaptive modulation of behavior.

neuroscience↗

bioRxiv2021

Electric shock causes fear-like persistent behavioral response in the nematode Caenorhabditis elegans

Behavioral persistency reflects internal brain states, which are the foundations of multiple brain functions. However, experimental paradigms that enable genetic analyses of behavioral persistency and its associated brain functions have been limited. Here we report novel persistent behavioral responses caused by electric stimuli in the nematode Caenorhabditis elegans. When the animals on bacterial food are stimulated by alternating current, their movement speed suddenly increases more than 2-fold, which persists for minutes even after the electric stimulation is terminated. Genetic analyses reveal that multiple types of voltage-gated channels are required for the response, possibly as the sensors, and neuropeptide signaling regulates the duration of the persistent response. Additional behavioral analyses indicate that the animals response to electric shock is scalable and has a negative valence. These properties, along with persistence, have been recently regarded as essential features of emotion, suggesting that the animals response to electric shock may express a form of emotion, such as fear.

neuroscience↗

bioRxiv2021

Generation of iPSCs from endangered Grevy's zebra and comparative transcriptomic analysis of mammalian PSCs

Induced pluripotent stem cells (iPSCs) can provide a biological resource for functional and conservation research in various species. This expectation has led to generation of iPSCs from various species, including those identified as endangered species. However, the understanding of species variation in mammalian iPSCs is largely unknown. Here, to gain insight into the species variation in iPSCs, we the first generated iPSCs from the endangered species Grevys zebra (Equus grevyi; gz-iPSCs) for the first time in the world. We isolated primary fibroblasts cell from an individual that had died of natural causes at a zoo and reprogrammed the fibroblasts into iPSCs. We confirmed their pluripotency and differentiation potential and performed RNA sequencing analysis. The gz-iPSC transcriptome showed that the generated gz-iPSCs robustly expressed genes associated with pluripotency and reprogramming processes, including epithelial-to-mesenchymal and mesenchymal-to-epithelial transitions. Comparative transcriptomics with other species revealed patterns of gene expression among mammalian PSCs and detected evolutionary conservation of pluripotency-associated genes and the plausible importance of the translation process. This study provides new insights into the evolution of mammalian PSCs, and the species conservation and variation of PSCs will advance our understanding of the early development of mammals.

cell biology↗

bioRxiv2021

Quantitative modeling of regular retinal microglia distribution

1Microglia are resident immune cells in the central nervous system (CNS), showing a regular distribution. Advancing microscopy and image processing techniques have contributed to elucidating microglias morphology, dynamics, and distribution. However, the mechanism underlying the regular distribution of microglia remains to be elucidated. First, we quantitatively confirmed the regularity of the distribution pattern of microglial soma. Second, we formulated a mathematical model that includes factors that may influence regular distribution. Next, we experimentally quantified the model parameters (cell movement, process formation, and ATP dynamics). The resulting model simulation from the measured parameters showed that direct cell-cell contact is most important in generating regular cell spacing. Finally, we tried to specify the molecular pathway responsible for the repulsion between neighboring microglia. Author summaryMicroglia are resident immune cells in the central nervous system. It is known that the microglia cells show a regular distribution, but the mechanism underlying the regular distribution remains to be elucidated. In the present study, we quantitatively assayed the regularity of the microglia soma distribution using the image processing technique. Next, we formulated a mathematical model of cell distribution that includes factors that may influence regular distribution. Next, we experimentally quantified the model parameters using organ culture. As a result, we obtained parameters for cell migration, cell process dynamics, and extracellular ATP dynamics. Then we undertook numerical simulation of the mathematical model using the parameters obtained by the experiments. The resulting model simulations showed that direct cell-cell contact is the most important factor in generating regular spacing. Finally, we screened possible molecular pathways involved in the regular spacing of microglia, which confirmed the validity of the model.

cell biology↗

bioRxiv2021

Stem cell therapy for skin regeneration using mesenchymal stem cells derived from the progeroid Werner syndrome-specific iPS cells

Adult progeria, Werner syndrome (WS), is an autosomal recessive disorder that develops accelerated aging-associated symptoms after puberty. Refractory skin ulcer of limbs, which is one of the symptoms specific to WS, is seriously painful and sometimes results in amputation. In recent years, cell therapy using mesenchymal stem cells (MSCs) has been attracting attention; however, the effect of WS-derived MSCs on skin ulcers is still unclear. In this study, we generated iPS cells from a patient with WS and a normal subject, differentiated them into MSCs (WS- and NM-iMSC, respectively), and performed cell therapy to a refractory skin ulcer mouse model. As a result, WS-iMSC recapitulated premature senescence phenotypes in vitro. Upon subcutaneous injection around the wounds of mice, WS-iMSC was significantly inferior in wound healing effect compared to NM-iMSC. Proteome and transcriptome analysis revealed altered expression of genes related to angiogenesis, inflammation, and proliferation in WS-iMSC with remarkable downregulation of VEGF, a potent angiogenic factor. In addition, simultaneous administration of recombinant human VEGF and WS-iMSC improved the wound healing effect in vivo. These results indicate that the expression of angiogenic factors is reduced in WS-iMSC, and its supplementation restores the wound healing ability. This finding may pave the way to develop the treatment of intractable skin ulcers of WS.

cell biology↗

bioRxiv2020

Noise-induced scaling in skull suture interdigitation

Sutures, the thin, soft tissue between skull bones, serve as the major craniofacial growth centers during postnatal development. In a newborn skull, the sutures are straight; however, as the skull develops, the sutures wind dynamically to form an interdigitation pattern. Moreover, the final winding pattern had been shown to have fractal characteristics. Although various molecules involved in suture development have been identified, the mechanism underlying the pattern formation remains unknown. In a previous study, we reproduced the formation of the interdigitation pattern in a mathematical model combining an interface equation and a convolution kernel. However, the generated pattern had a specific characteristic length, and the model was unable to produce a fractal structure with the model.In the present study, we focused on the anterior part of the sagittal suture and formulated a new mathematical model with time–space-dependent noise that was able to generate the fractal structure. We reduced our previous model to represent the linear dynamics of the centerline of the suture tissue and included a time–space-dependent noise term. We showed theoretically that the final pattern from the model follows a scaling law due to the scaling of the dispersion relation in the full model, which we confirmed numerically. Furthermore, we observed experimentally that stochastic fluctuation of the osteogenic signal exists in the developing skull, and found that actual suture patterns followed a scaling law similar to that of the theoretical prediction.Author summary Skull sutures (thin, undifferentiated tissue between bones) act as the growth centers for the skull. Sutures are straight at birth but later develop an interdigitated pattern that ultimately becomes a fractal structure. While our previous mathematical model of sutures generated a periodic pattern, the mechanism underlying the fractal structure formation remained to be elucidated. Here, we focused only on the anterior part of the sagittal suture and formulated a reduced model representing the initial linear phase of pattern formation with the addition of a time–space-dependent noise term. We showed analytically that the model generates patterns with a scaling law. This result was confirmed numerically and experimentally.View Full Text

developmental biology↗

bioRxiv2020

Aberrant claudin-6-adhesion signal promotes endometrial cancer progression via estrogen receptor α

Cell adhesion proteins not only maintain tissue integrity but also possess signaling abilities to organize diverse cellular events in physiological and pathological processes; however, the underlying mechanism remains obscure. Among cell adhesion molecules, the claudin (CLDN) family often possesses aberrant expression in various cancers, but the biological relevance and molecular basis have not yet been established. Here, we show that high CLDN6 expression promotes endometrial cancer progression and represents the poor prognostic marker. The second extracellular domain and Y196/200 of CLDN6 were required to recruit and activate Src-family kinases (SFKs) and to stimulate malignant phenotypes. Importantly, we demonstrate that the CLDN6/SFK/PI3K-dependent AKT and SGK (serum- and glucocorticoid-regulated kinase) signalings target Ser518 in the human estrogen receptor and ligand-independently activate target genes in endometrial cancer cells, resulting in cancer development. The identification of this machinery highlights regulation of the transcription factors by cell adhesion to advance tumor progression.

cancer biology↗