Biology subjects

Nakamura, E.

Publications and source records attributed to Nakamura, E..

11 recordsLinked to original sources

bioRxiv2026

HNRNPC-mediated RNA processing promotes VHL-associated excessive vasculogenesis in von Hippel-Lindau syndrome revealed from patient-derived blood vessel models

Von Hippel-Lindau Syndrome (VHLS) is an inherited disorder characterized by excessive vascularization, which contributes to the development of hemangioblastomas and benign or malignant tumors, as well as renal cell carcinoma (RCC) and cysts in multiple organs. VHLS is caused by mutations in VHL tumor suppressor gene, which encodes a component of an E3 ubiquitin ligase complex. Although therapeutic agents targeting hypoxia-inducible factor 2 alpha (HIF2A), which is stabilized by VHL loss and promotes tumor angiogenesis, have been approved for clinical use, their therapeutic response remains limited in RCC patients associated with VHLS. Therefore, there is an unmet need for the development of alternative therapeutic approaches independent of HIF2A. Here, we establish a human iPSCs-based model of VHLS-associated excessive vasculogenesis using induced pluripotent stem cells (iPSCs) derived from VHLS patients and VHL-edited isogenic iPSC lines. Vascular cells (VCs) differentiated from these VHLS-specific iPSCs exhibited excessive blood vessel formation compared to healthy donor-derived VCs, leading to vascular lesion formation characterized by abundant blood vessels and abnormal cellular growth in vivo. Mechanistically, RNA-binding proteins (RBPs) were preferentially stabilized in the patient-VCs. Among these RBPs, the stabilization of heterogeneous nuclear ribonucleoprotein C1/C2 (HNRNPC), which is a substrate for pVHL-mediated protein degradation independent of HIF2A expression, was critical for the dysregulated vascularization caused by VHL inactivation in this model. These findings provide an iPSCs-derived vascular platform to elucidate VHLS mechanisms and identify HNRNPC as a potential therapeutic target for VHL-driven vascular lesion formations through an HIF2A-independent mechanism.

cell biology↗

bioRxiv2025

Turbulence orchestrates actin-mitochondria dynamics to preserve GPIbα and support iPSC-derived platelet biogenesis

The in vitro manufacturing of induced pluripotent stem cell-derived platelets (iPSC-platelets) remains limited by low yield and poor quality, thus impeding practical clinical application. To address this, we developed a turbulence-assisted system using defined physical stimulation to enhance platelet production from immortalized megakaryocyte progenitor cell lines (imMKCLs). This approach significantly increases the yield of glycoprotein Ib (GPIb) functional iPSC-platelets. However, the underlying mechanisms remained unclear. Here, we reveal that GPIb iPSC-platelets retain high mitochondrial membrane potential, low phosphatidylserine (PS) exposure, and active ATP-dependent flippases, preserving mitochondrial integrity and platelet functionality. In contrast, GPIb- platelets exhibit PS exposure, ADAM17 activation, and selective GPIb shedding. Notably, turbulent flow induces late-stage actin depolymerization, promoting even mitochondrial distribution within maturing imMKCLs and enhancing mitochondrial inheritance to iPSC-platelets. These findings highlight the key role of turbulence in regulating actin dynamics and mitochondrial allocation during platelet biogenesis, providing a mechanistic foundation for improving in vitro platelet production for therapeutic use.

cell biology↗

bioRxiv2025

Sequestration-Based Neural Networks That Operate Out of Equilibrium

Classification of high-dimensional information is a ubiquitous computing paradigm across diverse biological systems, including organs such as the brain, down to signaling between individual cells. Inspired by the success of artificial neural networks in machine learning, the idea of engineering genetic circuits that operate as neural networks emerges as a strategy to expand the classification capabilities of living systems. In this work, we design these biomolecular neural networks (BNNs) based on the molecular sequestration reaction, and experimentally characterize their behavior as linear classifiers for increasing levels of complexity. Initially, we demonstrate that a static, DNA-based system can effectively prototype a linear classifier, though we also identify its limitations to easily tune the slope of the decision boundary it generates. We then propose and experimentally validate a BNN at the protein level using a cell-free transcription-translation (TXTL) system, which overcomes the DNA-based systems limitation and behaves as a linear classifier even before it reaches its steady state (or, equivalently, out-of-equilibrium). Ultimately, we test a CRISPR-based design and its out-of-equilibrium behavior in a biological context by successfully constructing a linear classifier within mammalian cells. Overall, by leveraging mathematical modeling and experimental automation, we establish molecular sequestration as a universal scheme for implementing neural networks within living systems, paving the way for transformative advances in synthetic biology and programmable biocomputing systems.

synthetic biology↗

bioRxiv2025

Temporal dose inversion properties of adaptive biomolecular circuits

Cells have the capacity to encode and decode information in the temporal features of molecular signals. Many pathways, for example, generate either sustained or pulsatile responses depending on the context, and such diverse temporal behaviors have a profound impact on cell fate. Here we focus on how molecular pathways can convert the temporal features of dynamic signals, in particular how they can convert transient signals into persistent downstream events and vice versa. We refer to this type of behavior as temporal dose inversion, and we demonstrate that it can be achieved through adaptive molecular circuits. Using a cell-free synthetic gene circuit implementing an incoherent feedforward loop (IFFL), we experimentally demonstrate the temporal dose inversion. To understand the design principles of the temporal dose inversion, we analyze adaptive circuit motifs including incoherent feedforward loops (IFFLs) and negative feedback loops (NFLs). Through numerical simulations with expensive parameter exploration, we identify parametric regimes in which these circuits exhibit temporal dose inversion. We further examine more detailed biological models of the IFFL and NFL circuits, including enzymatic signaling models and gene regulatory network models, showing that the IFFL circuits is more likely to exhibit temporal dose inversion compared with the NFL circuits. Finally, we analyze a generalized IFFL topology, and we find that both the time delay in the inhibition pathway and the relative signal intensities of the activation and inhibition signals are key determinants for temporal dose inversion. Together, our results establish a design principle of temporal dose inversion on the adaptive biomolecular circuits and provide mechanistic insight into how molecular networks process temporal information in dynamic signals.

systems biology↗

bioRxiv2024

Neutrophil extracellular traps formation and deposition of fibrin and von Willebrand factor during human coronary atherogenesis

Background and aimsNeutrophil extracellular traps (NETs), fibrin, and the von Willebrand factor (VWF) are present in acute coronary thrombotic plaques. However, the presence and extent of NETs, fibrin, and VWF in early-to-unstable coronary atherosclerotic lesions remain unclear. This study aims to determine the presence and extent of neutrophils, NETs, fibrin, and VWF formation during human coronary atherogenesis. MethodsCoronary sections from autopsy patients with non-cardiac death (n=5, 37 sections) and ischemic heart diseases (n= 11, 65 sections) were classified by atherosclerosis classification: diffuse intimal thickening, pathological intimal thickening, fibrous cap atheroma, fibrocalcified plaque, thin-cap fibroatheroma, ruptured plaque, and intraplaque hemorrhage. We immunohistochemically assessed the expression of CD66b (neutrophils), citrullinated histone H3 (Cit-H3, a marker of NETs), fibrin, and VWF. ResultsNeutrophil and Cit-H3 expression were rarely observed, except in thin-cap fibroatheromas and ruptured plaques. Fibrin deposition was observed in pathological intimal thickening and necrotic cores of atheromas, and it was abundant in thin-cap fibroatheroma and ruptured plaques. VWF deposition was observed in the necrotic core of the atheromas and was abundant in ruptured plaques. In non-ruptured plaques, the immunopositive areas for Cit-H3 and fibrin were larger in hemorrhagic plaques than in non-hemorrhagic plaques. ConclusionsThese results suggest that NET formation is rare in stable coronary plaques, and fibrin formation begins in stable lesions and increases with plaque destabilization in the coronary artery. Intraplaque hemorrhage may promote NET formation and fibrin deposition in non-ruptured plaques. The abundance of neutrophilic infiltrates and VWF deposition may be a response to plaque rupture.

pathology↗

bioRxiv2024

Design of a sequestration-based network with tunable pulsing dynamics

Incoherent feedforward networks exhibit the ability to generate temporal pulse behavior. However, exerting control over specific dynamic properties, such as amplitude and rise time, poses a challenge and is intricately tied to the networks implementation. In this study, we focus on analyzing sequestration-based networks capable of exhibiting pulse behavior. By employing time-scale separation in the fast sequestration regime, we approximate the temporal dynamics of these networks. This approach allows us to establish a mapping that elucidates the impact of varying the kinetic rates and pulse specifications, including amplitude and rise time. Furthermore, we introduce a positive feedback mechanism to regulate the amplitude of the pulsing response.

synthetic biology↗

bioRxiv2024

Expression of fibroblast activation protein-α in human deep venous thrombus

BackgroundFibroblast activation protein- (FAP), a type-II transmembrane serine protease, is expressed during wound healing, in cancer-associated fibroblasts, and in chronic fibrosing diseases. However, its expression in deep vein thrombus (DVT) remains unclear. Therefore, in this study, we investigated FAP expression and localization in DVT. MethodsFirst, we pathologically accessed aspirated thrombi of patients with DVT (n=14), classifying thrombotic areas as follows; fresh, cellular lysis, endothelialization, fibroblastic reaction. Endothelialization and fibroblastic reaction were defined as organizing reactions. We immunohistochemically examined FAP-expressed areas and expressed cells. Second, we analyzed FAP expression in cultured dermal fibroblasts. ResultsAll the aspirated thrombi showed a mixture of at least three of the four thrombotic areas. Specifically, 83 % of aspirated thrombi showed fresh and organizing reactions within each thrombus. Immunohistochemical expression of FAP was restricted in organizing area. Further, FAP expression in the thrombi was mainly found in vimentin-positive or -smooth muscle actin-positive fibroblasts in double immunofluorescence. Some CD163-positive macrophages also showed FAP expression. FAP mRNA and protein levels significantly increased in cultured fibroblasts with low- proliferative activity under 0.1% fetal bovine serum (FBS) than that in fibroblasts under 10% FBS. Fibroblasts cultured in 10% FBS showed a significant decrease in FAP mRNA levels following supplementation with hemin, but not with thrombin. ConclusionsThe heterogeneous composition of the venous thrombi suggests the existence of a multistep thrombus formation process in human DVT. Further, fibroblasts or myofibroblasts may express FAP in organizing process in DVT. FAP expression may increase in fibroblasts with low-proliferative activity.

pathology↗

bioRxiv2023

Factor XI localization in human deep venous thrombus and function of activated factor XI on venous thrombus formation and hemostasis in rabbit

BackgroundNovel anticoagulants targeting coagulation factor XI (FXI)/activated FXI (FXIa) are under development. However, whether FXI is present in human deep vein thrombosis (DVT) and whether FXIa and activated factor X (FXa) play different roles in venous thrombus formation and hemostasis remain unclear. This study aimed to determine the presence of FXI in DVT and the effects of direct oral FXIa and FXa inhibitors on venous thrombus formation and hemostasis in rabbits and mural thrombus formation in flow chamber system. MethodsWe immunohistochemically assessed FXI localization in human aspirated DVT (n=15). Additionally, we compared thrombus formation induced by endothelial denudation and stenosis in jugular vein, and skin bleeding time and volume between rabbits treated with direct FXIa inhibitors (ONO-1600586) and FXa inhibitors (rivaroxaban). Ex vivo rabbit and human blood were perfused on a flow chamber under low shear rates (70/s). ResultFXI was localized in all DVT, predominantly in fibrin-rich areas. The FXI-immunopositive area in the non-organizing area was greater than that in the organizing area. Although FXIa and FXa inhibitors comparably inhibited venous thrombus formation, FXIa inhibitors did not affect bleeding time or volume in rabbits. FXIa or FXa inhibitors mildly or strongly inhibited fibrin formation at low shear rates respectively. Furthermore, the FXIa inhibitor suppressed human FXIa activity, thrombin generation, and fibrin formation during perfusion. ConclusionThe pathological findings of human DVT suggest FXIs role in human DVT. FXIa inhibitors may inhibit less fibrin formation than FXa inhibitors, and may explain the minor role of FXIa in hemostasis. EssentialO_LIPresence of factor XI (FXI) in venous thrombus and less bleeding in its inhibition are unclear. C_LIO_LIWe assessed FXI localization in deep vein thrombosis (DVT) and function of FXIa in rabbit. C_LIO_LIFXI localized in human DVT that provide a rationale for FXI inhibition in human DVT. C_LIO_LIFXIa inhibitor inhibited less fibrin formation than factor Xa inhibitor under low-shear rate. C_LI

pathology↗

bioRxiv2023

Deep neural generation of neuronal spikes.

In the brain, many regions work in a network-like association, yet it is not known how durable these associations are in terms of activity and could survive without structural connections. To assess the association or similarity between brain regions with a new "generating" approach, this study evaluated the similarity of activities of neurons at the cellular level within each region after disconnecting between regions. To this end, a multi-layer LSTM (Long-Short Term Memory) model was used. Surprisingly, the results revealed that generation of activity from one region to other regions that had been disconnected was possible with similar reproduction accuracy as generation between the same regions in many cases. Notably, not only firing rates but also synchronization of firing between neuron pairs, which is often used as neuronal representations, could be reproduced with considerable precision. Additionally, their accuracies were associated with the relative distance between brain regions and the strength of the structural connections that initially connected them. This outcome not only enables us to look into principles in neuroscience based on the potential to generate new informative data, but also creates neural activity that has not been measured in adequate amounts and could potentially lead to reduced animal experiments.

neuroscience↗

bioRxiv2020

Interleukin-11 is a Marker for Both Cancer- and Inflammation-Associated Fibroblasts that Contribute to Colorectal Cancer Progression

Interleukin (IL)-11 is a member of the IL-6 family of cytokines and involved in multiple cellular responses, including tumor development. However, the origin and functions of IL-11-producing (IL-11+) cells are not fully understood. To characterize IL-11+ cells in vivo, we generated Il11 reporter mice. IL-11+ cells appeared in the colon of three murine tumor models, and a murine acute colitis model. Il11ra1 or Il11 deletion attenuated the development of colitis-associated colorectal cancer. IL-11+ cells expressed fibroblast markers, and genes associated with cell proliferation and tissue repair. IL-11 induced STAT3 phosphorylation in colonic fibroblasts, suggesting the activation of IL-11+ fibroblasts. Analysis using the human cancer database revealed that genes enriched in IL-11+ fibroblasts were elevated in human colorectal cancer, and correlated with reduced disease-free survival. Together, our results suggested that tumor cells induced IL-11+ fibroblasts, and that a feed-forward loop between IL-11 and IL-11+ fibroblasts might contribute to tumor development.

cancer biology↗

bioRxiv2019

LaeA controls citric acid production through regulation of a citrate exporter encoding cexA in Aspergillus luchuensis mut. kawachii

The putative methyltransferase LaeA is a global regulator of metabolic and development processes in filamentous fungi. We characterized the homologous laeA genes of the white koji fungus Aspergillus luchuensis mut. kawachii (A. kawachii) to determine their role in the hyperproduction of citric acid. The {Delta}laeA strain exhibited a significant reduction in citric acid productivity. Cap-analysis gene expression (CAGE) revealed that laeA is required for the gene expression of a putative citrate exporter encoding cexA, which is critical for citric acid production. The deficit in the productivity of citric acid by the {Delta}laeA strain was rescued by the overexpression of cexA to a level comparable with that of the cexA-overexpressing {Delta}cexA strain. In addition, chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) analysis indicated that LaeA regulates the expression of cexA via methylation levels of the histones H3K4 and H3K9. These results indicate that LaeA is involved in citric acid production through epigenetic regulation of cexA in A. kawachii.\n\nIMPORTANCEA. kawachii has been traditionally used for production of the distilled spirit shochu in Japan. Citric acid produced by A. kawachii plays an important role in preventing microbial contamination during the shochu fermentation process. This study characterized homologous laeA genes; using CAGE, complementation test, and ChIP-qPCR, it was found that laeA is required for citric acid production though regulation of cexA in A. kawachii. The epigenetic regulation of citrate production elucidated in this study will be useful for controlling the fermentation processes of shochu.

microbiology↗