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Fujita, K.

Publications and source records attributed to Fujita, K..

At least 19 recordsLinked to original sources

Thalamocortical credit routing from the basal ganglia and cerebellum: anatomical constraints and circuit principles

Animals learn to select actions that maximize reward in complex environments. A central challenge in this process is credit routing: behavioral outcomes must be routed back to the neural populations that contributed to generating the relevant actions. One possible route is through thalamocortical pathways conveying learning signals from the basal ganglia and cerebellum, where plasticity is shaped by reward-prediction errors. However, how these pathways route such signals back to specific cortical circuits remains unclear. To address this question, we combined anatomical circuit mapping in mice with closed-loop circuit modeling. Anterograde transsynaptic tracing revealed that thalamic neurons receiving input from the substantia nigra pars reticulata or the lateral cerebellar nucleus were largely segregated, with partial overlap in the ventromedial thalamus. Nevertheless, their cortical axons shared a pronounced enrichment in layer 1a, most prominently in secondary motor cortex. In a brain- wide synergistic loop-circuit model, learning was enhanced when both pathways delivered additive and gain-modulating feedback aligned with the cortical action readout. Local activity- based calibration established this alignment, whereas dendritic-spike- and reward-prediction- error-dependent thalamocortical plasticity further refined behavioral output. Module-specific feedback was essential for a brain-machine interface task requiring the simultaneous conditioning of multiple target neurons. Together, these findings define a credit-routing architecture in which laminar convergence, plastic alignment, and modular feedback link parallel subcortical learning systems to cortical action representations.

neuroscience↗

Topical Activatable Fluorescence Probes for Rapid Intraoperative Detection of Peritoneal Dissemination in High-Grade Serous Ovarian Carcinoma

BackgroundEpithelial ovarian cancer (EOC) is one of the most lethal gynecologic malignancies, largely because most patients are diagnosed at an advanced stage with peritoneal dissemination. High-grade serous carcinoma (HGSC), the most common and aggressive EOC subtype, requires complete cytoreductive surgery to improve the prognosis; however, minute disseminated lesions are often missed by conventional intraoperative inspection. Here, we aimed to develop fluorescence probes for rapid and sensitive intraoperative detection of HGSC peritoneal dissemination. MethodsWe screened a hydroxymethyl rhodamine green (HMRG)-based fluorescence probe library consisting of 381 protease- and aminopeptidase-reactive fluorescence probes using tumor and non-tumor specimens from patients with HGSC. The target enzyme of the hit probes was identified by means of enzyme assays, immunohistochemistry, and LC/MS analysis. Diagnostic utility was evaluated ex vivo using clinical specimens and in vivo using a peritoneal dissemination mouse model. ResultsThree probes--EK-HMRG, NA-HMRG and DA-HMRG--were selected as promising candidates for the detection of peritoneal dissemination in HGSC. Puromycin-sensitive aminopeptidase (PSA) was identified as a novel target enzyme of these probes. EK-HMRG, NA-HMRG and DA-HMRG rapidly detected peritoneal dissemination just a few millimeters in size with high sensitivity and specificity in clinical HGSC specimens and in a peritoneal dissemination mouse model after topical application. ConclusionsThe PSA-targeting topical fluorescence probes EK-HMRG, NA-HMRG and DA-HMRG are promising tools for real-time, highly sensitive intraoperative visualization of peritoneal dissemination in HGSC, and are promising candidates to improve complete resection rates.

cancer biology↗

Early detection of pancreatic ductal adenocarcinoma by single-molecule profiling of pancreatic enzyme activities

Pancreatic ductal adenocarcinoma (PDAC) remains a leading cause of cancer-related mortality, largely due to diagnosis at advanced stages. Early detection through minimally invasive liquid biopsy holds promise for improving patient outcomes. Here, we report a blood-based liquid biopsy platform based on single-molecule enzyme activity profiling (SEAP), which detects proteoform-level alterations in circulating pancreatic enzymes at single-molecule resolution. Using a tissue-centric biomarker discovery strategy, we identified activity signatures of pancreas-specific digestive enzymes associated with PDAC. A combinatorial classifier detected PDAC (stage I-IV) with 95.5% specificity and 75.0% sensitivity (74.2% for stage I-II) across 690 blood samples collected from multiple hospitals and biobanks. Performance was further validated in an independent cohort enriched for early-stage disease, where 54.2% (13/24) of stage IA, 64.3% (9/14) of stage IB, and 21.4% (3/14) of stage 0 lesions were classified as positive. These findings support the clinical potential of SEAP for early detection of PDAC.

biochemistry↗

Single-Molecule Activity Profiling of Glycosidase Proteoforms Using Water-Soluble Fluorogenic Probes

In this study, we report a series of highly water-soluble fluorogenic probes for glycosidases useful for microdevice-based single-molecule enzyme activity profiling (SEAP). The assay was able to detect trace glycosidase activities in blood with high sensitivity and with proteoform resolution. This platform revealed the potential of -mannosidase as sensitive blood-based liquid-biopsy biomarkers for liver injury, highlighting the potential of this approach for activity-based diagnostics.

biochemistry↗

Intranuclear Quantum Sensing with Fluorescent Nanodiamond Enabled by Electron-Irradiation and Surface-Chemical Optimization for Microinjection

Fluorescent nanodiamonds (FNDs) containing nitrogen-vacancy (NV) centers are promising quantum sensors for intracellular measurements, yet nuclear applications remained out of reach because optically detected magnetic resonance (ODMR) signals are weak and capillary delivery is inefficient. This study addresses both constraints by optimizing the electron irradiation dose to balance NV creation and charge-state stability, and by grafting hyperbranched polyglycerol with terminal carboxyl groups (HPGCOOH) to suppress aggregation and prevent needle clogging. The optimized dose yields strong ODMR contrast while preserving fluorescence suitable for microscopy. HPGCOOH surfaces enable smooth and reproducible microinjection through fine capillaries. Using this strategy, the microinjection of ODMR-active FNDs into the nuclei of living COS7 cells is achieved, and clear intranuclear spectra comparable to cytoplasmic readouts are obtained. Furthermore, field-of-view temperature sensing across multiple cell nuclei is demonstrated, enabling quantitative and spatially resolved thermal mapping within the genomic environment. This methodology provides a practical route to nuclear quantum sensing and opens opportunities for nanoscale physicochemical measurements within the genomic environment.

biophysics↗

Platinum Cross-linked Collagen Matrices with Tunable Stiffness as a Platform to Investigate Cellular Mechanosensing

The mechanical properties of the cellular microenvironment are key regulators of cellular physiology. Although the field of cancer mechanobiology has attracted attention, the availability of matrix systems with independently and precisely tunable mechanical properties remains limited. Our group previously developed a collagen gel with enhanced mechanical strength by cross-linking collagen molecules using a platinum complex. In this study, we investigated the tunability of the mechanical properties of the platinum cross-linked collagen gel (PCG) and demonstrated that mechanical parameters can be controlled by varying the amount of the platinum complex. In addition, we examined how matrix mechanical properties modulate the phenotypes of lung adenocarcinoma A549 cells using the collagen matrix. Although A549 cells exhibited significant morphological alterations on stiffer matrices, these changes were not accompanied by classical epithelial-to-mesenchymal transition (EMT). Instead, they were associated with the upregulation of diverse gene expression related to cancer malignancy. We focused on maternal embryonic leucine zipper kinase (MELK) whose gene expression increased on stiffer matrices. Consistently, A549 cells cultured on stiffer matrices displayed enhanced sensitivity to a MELK-targeting anticancer drug. These findings highlight the potential of the matrices with tunable mechanical parameters not only to provide variety of physiological microenvironment but also to advance anticancer drug screening when combined with gene expression analysis. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/720034v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@419074org.highwire.dtl.DTLVardef@72ef67org.highwire.dtl.DTLVardef@1c36e17org.highwire.dtl.DTLVardef@170dc33_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIPlatinum cross-linked collagen gel enables independent tuning of compressive and shear elasticities. C_LIO_LICellular functions may be regulated by matrix mechanical parameters through distinct mechanisms. C_LIO_LICorrelation analysis between matrix mechanical parameters and cancer cell gene expression provides a rational strategy for therapeutic drug screening. C_LI

bioengineering↗

Enzyme activity as an actionable axis for small-molecule precision oncology

Current precision oncology--molecular targeted therapies and immunotherapies--relies on genomic or expressed biomarkers, yet most cancer patients remain ineligible for these treatments. Here, we establish enzyme activity as an actionable and orthogonal axis for precision cancer medicine. Strategic activity-based screening of mouse organs and human clinical specimens with a panel of enzyme-reactive fluorescence probes identified {beta}-galactosidase 1 (GLB1) and {beta}-hexosaminidases (HEX) as broadly elevated tumor-selective biomarkers. Leveraging these activities, we developed 7-ethyl-10-hydroxycamptothecin (SN38)-based GLB1-and HEX-reactive prodrugs. These prodrugs exhibited dramatically reduced systemic toxicities and improved therapeutic windows, compared to a clinically used SN38-based prodrug, irinotecan (CPT-11). Both prodrugs demonstrated activity-dependent therapeutic efficacy, affording a dramatic reduction of tumor volumes across multiple in vivo models, including a subcutaneous patient-derived xenograft (PDX) of lung squamous cell carcinoma that lacked genetic alterations targeted by current precision medicine. Furthermore, this strategy is broadly applicable across various cytotoxic payloads, establishing a generalizable platform for small-molecule precision medicines. Our results define an enzyme-targeting paradigm for precision oncology, in which fluorescence probes serve as companion diagnostic tools to guide development and selection of appropriately targeted prodrugs, which are expected to provide safer and more efficacious treatment options for cancer patients with elevated enzyme activities.

cancer biology↗

Lipoxins Regulate Intercalated Disk-Associated Signaling and Immune Remodeling in Dilated Cardiomyopathy

We investigated whether pro-resolving lipid mediators of the lipoxin family can attenuate fibrosis and inflammation in muscle LIM protein knockout (MLPko) mice, a model of dilated cardiomyopathy (DCM). Male and female MLPko mice received either vehicle or a mix of lipoxin-A4 and lipoxin-B4 three times per week for six weeks. Cardiac function was assessed using echocardiography, and fibrosis and DCM-associated cardiac signaling was evaluated through histology, immunofluorescence and immunoblot analyses. Flow cytometry and RNA sequencing (RNAseq) was performed to identify changes in cardiac gene expression and characterize macrophage subpopulations, respectively. Flow cytometry showed increased inflammatory CD11c+ M1-like macrophages and reduction of CD206+ M2-like macrophages in MLPko hearts compared to wild-type controls. Lipoxin treatment partially reversed the macrophage imbalance and showed mild improvements in cardiac physiology in MLPko males. RNAseq analyses revealed sex-dependent alterations in the expression of pro-fibrotic and inflammation-related genes, suggesting changes in extracellular matrix (ECM) integrity and composition, and to the adaptive immune response. Intriguingly, several ECM proteins showed unexpected localizations at cardiac intercalated disks, which are known to be involved in DCM etiology. Further analysis identified lipoxin-dependent reduction in the DCM-associated expression of intercalated disk components only in lipoxin-treated MLPko males. Lipoxins also modulated key cardiac signaling pathways in a sex-specific manner, including Erk1/2 and PKC-linked Ankrd1/Carp1, which is associated with DCM development in MLPko mice. While lipoxins do not directly reverse cardiac dysfunction or fibrosis in MLPko mice, they may provide sex-specific protective effects by modulating DCM-related cardiac signaling pathways and by influencing immune-cell populations.

cell biology↗

Quantitative MRI Reveals Bone Marrow Regeneration Following Targeted Marrow Irradiation and Transplantation in a Sickle Cell Disease Model

Sickle cell disease (SCD) is associated with chronic bone marrow stress, altered hematopoiesis, and reduced adiposity. Whether marrow-selective conditioning followed by transplantation normalizes these abnormalities remains unclear. We investigated bone marrow remodeling in Townes mice by comparing SCD control (SCD-Con) with mice that received total marrow irradiation (TMI) followed by bone marrow transplantation (SCD-TMI-BMT). Multiparametric micro-MRI at 7 T quantified proton density water fraction (PDWF), proton density fat fraction (PDFF), and R2*(1/T2*), and micro-CT assessed trabecular structure in the femur. SCD-Con marrow showed higher water content (elevated PDWF), reduced adiposity (lower PDFF), and imaging features consistent with erythroid hyperplasia and elevated iron burden (shorter T2* with reciprocal increase in R2*). In contrast, SCD-TMI-BMT mice demonstrated smaller R2*, reduced PDWF, and partial restoration of adiposity, accompanied by reciprocal shifts in R2*, consistent with decreased cellular iron and marrow remodeling. Micro-CT revealed an improved trabecular architecture after BMT compared to SCD control. MRI imaging biomarkers aligned with histologic evidence of reduced cellularity and larger adipocyte voids. In conclusion, a TMI-BMT SCD model promotes partial normalization of the marrow microenvironment. Multiparametric MRI with micro-CT provides a practical, non-invasive framework for monitoring marrow remodeling and skeletal health after curative therapy.

bioengineering↗

From Single Molecular Detail to Subcellular Dynamics: Real-Time Kinetics Study of dPA Uptake with Raman-based Spectroscopy

Real-time observation of cellular transformations at the subcellular level provides insight into the dynamic biochemical processes underlying cell function and disease. However, many challenges remain in developing methods to analyze metabolic transformations in cells quantitatively. Our study applies Raman spectroscopy (RS) and stimulated Raman scattering (SRS) to link single-molecule resolution with cellular-scale lipid turnover tracking. Using HL-60 cells as a model, we monitor the uptake kinetics of deuterated palmitic acid (dPA), a marker of de novo lipogenesis, from the first minutes to hours. SRS enables microscopic, high-temporal-resolution visualization of dPA incorporation across entire cell volumes, capturing both its distribution and uptake rate. Complementary RS measurements identify distinct metabolic phases: rapid incorporation into pre-existing lipid droplets (LDs) followed by the emergence of new, partially unsaturated LDs associated with cytochrome-linked metabolism. Statistical and spectral analyses reveal cell-to-cell heterogeneity, emphasizing the complexity of lipid metabolism at both the cellular and subcellular levels. By integrating SRS and RS data into time-dependent kinetic profiles, we establish a unified submicron-to-cellular model of fatty acid metabolism. This multi-resolution spectroscopic approach demonstrates how real-time, label-based tracking can uncover dynamic metabolic heterogeneity, advancing understanding of the fatty acid uptake and metabolism in the microscale.

cell biology↗

Phase-targeting rapid cryofixation of the beating heart and histological analysis unveil contractile state-dependent sarcomere dynamics

The heart is a functional syncytium consisting of numerous cardiomyocytes that repetitively exhibit coordinated contractions/relaxations. However, the extent to which myocyte sarcomere arrangements in the heart differ across beats is unknown. To examine this, we conducted cardiac phase-targeting rapid cryofixation of Langendorff-perfused rat hearts. We adjusted the timepoint of cryogen exposure to the electrically paced heart and observed phase-dependent differences in the sarcomere length (SL) of subepicardial myocytes by -actinin immunohistochemistry, namely a significantly shorter SL during systole than during diastole. We detected spatially inhomogeneous SL distributions by generating a heatmap of the myocardium. For peak systole the SL heatmap exhibited nearly uniform SL shortening within and among the individual myocytes with some myocardia exhibiting nonuniform SLs. During diastole, the heart showed predominant SL elongation, which was also accompanied by patchy distributions of locally short-SL regions, reflecting inhomogeneous SLs. This SL inhomogeneity was attenuated by pharmacological relaxation by 2,3-butanedione monoxime. The heatmap of the rapidly-frozen heart during ventricular fibrillation also revealed inhomogeneous SLs within and among individual myocytes. Overall, cardiac phase-targeting cryofixation unveiled in-depth behaviors on SL in the heart. Our cryofixation strategy will open a new horizon to clarify precise spatiotemporal changes in sarcomere structures and understand cardiac functions.

biophysics↗

Aβ-HMGB1 complex is a pathogenic molecule at the advanced stage of Alzheimer's disease

Multiple molecules including A{beta}, tau and other inflammatory molecules mediate Alzheimers disease (AD) pathology. High mobility group box 1 (HMGB1), which is released from necrotic cells and binds to Toll-like receptors (TLRs) of surrounding neurons and microglia, also mediates AD pathology from the early stage. Paradoxically, HMGB1 concentration in cerebrospinal fluid (CSF) at the advanced stage of AD is not higher than that at the early stage. Here we show that A{beta}-HMGB1 complexes are generated in neurons undergoing secondary necrosis around A{beta} plaques in the AD brain at the advanced stage. A{beta}-HMGB1 complex triggers neurite degeneration and necrosis of human normal iPSC-derived neurons via binding to TLR4. Further, two anti-HMGB1 antibodies that inhibit its interaction with TLR4 successfully suppress the toxicity of A{beta}-HMGB1 complex to human iPSC-derived neurons, and recover cognitive impairment and A{beta}-HMGB1 complex-related brain pathology in AD model mice, while such therapeutic effects were not obvious with an anti-A{beta} antibody (lecanemab) approved for human AD patients. Enzyme-Linked Immuno Sorbent Assay (ELISA) revealed plasma level of A{beta}-HMGB1 complex was increased in a part of AD patients at the advanced stage. These findings for the molecular basis of toxicity to neurons suggest the significance of A{beta}-HMGB1 complex at the advanced stage of AD pathology, and might explain the discrepancy between A{beta} burdens and clinical symptoms of human AD patients treated with anti-A{beta} antibody.

neuroscience↗

PQBP1-dependent alternative RNA splicing underlies high calorie diet-induced cognitive impairment

High calorie-high fat diet (HFD) has been implicated as a pathological modifier of brain diseases including neurodegenerative dementias, but the detailed molecular mechanisms remain largely unknown. Here we report that HFD suppresses PPAR{gamma}-mediated transcriptional expression of PQBP1, a RNA splicing factor implicated in human intellectual disability and Alzheimers disease. RNAseq-based comprehensive analyses of alternative RNA splicing (AS) in HFD-fed mice for 1 or 6 weeks and in PQBP1-cKO mice reveal their common changes, which weigh on synapse-related genes. Betweenness-based extraction of core molecules from the common changes reveals CASK, Cacnb1 and Cyfip2 as key molecules of the network. Both CASK and Cacnb1 regulate STXBP1, a causative gene for infantile epilepsy syndrome and an essential factor for synapse vesicle release, via their direct interaction. In addition, our analysis suggests that Syt1 plays a role specifically in HFD for 1 week. HFD-induced AS isoforms of CASK, Cacnb1, Cyfip2 and Syt1 impair pre-synapse vesicle release in primary neurons. AAV-PQBP1, AAV-CASK, AAV-Cacnb1, AAV-Cyfip2 or AAV-Syt1 rescues synapse and/or cognitive dysfunctions in HFD mice, genetically supporting the pathological PQBP1-presynase axis in HFD. Moreover, immunohistochemistry experiments suggest that the pathological axis plays roles not only in excitatory neurons, but also in inhibitory neurons of the brain. Collectively, our results unravel a novel molecular mechanism for brain dysfunction when mice are exposed to a HFD.

neuroscience↗

Low-Background Cancer Imaging With a Bioorthogonal Fluorescence Probe and Engineered Reporter Enzyme Bearing a Targeting Moiety

Combinatorial use of an antibody-reporter enzyme conjugate and a fluorescence probe activated by the enzyme is a powerful strategy for fluorescence-guided cancer surgery. However, conventional probes for typical reporter enzymes are insufficiently bioorthogonal, resulting in high background signals in non-target tissues. We screened a library of HMRef (rhodol derivative)-based fluorescence probes bearing various sugar moieties, and discovered that HMRef-{beta}-D-fucose is bioorthogonal in mammalian systems, but is activated by a metagenomic glycosidase, Td2F2. Directed evolution generated a mutant with a kcat/Km value for HMRef-{beta}-D-fucose of 3.3 x 105/M/sec, 7.3 times higher than that of wild-type Td2F2 and comparable to that of {beta}-galactosidase (LacZ) with a corresponding probe. Theoretical calculation suggested that E296G mutation in Td2F2 causes structural changes that facilitate the probes access to the enzymes active site. In a proof-of-concept study, cancer cells were visualized with a minimal background in the mesentery of a mouse model of peritoneally disseminated human-ovarian-cancer-derived SKOV-3 cells, which endogenously express HER2, by using HMRef-{beta}-D-fucose together with engineered Td2F2 conjugated/fused to a HER2-binding antibody/nanobody.

bioengineering↗

Photodynamic Therapy for Deep Organ Cancer by Implantable Wireless OLEDs

Metronomic photodynamic therapy (mPDT) is a method of continuously delivering low-intensity light to a cancer lesion. Since high light intensity is not required, the light device can be miniaturized and implanted in the body. Nevertheless, for clinical implementation, it is essential to develop a light source apparatus that can effectively illuminate the entire tumor and operate stably without damaging internal organs. To address this issue, an organic light-emitting diode (OLED) optical device that has an area large enough to cover the entire lesion and is powered by a wireless power supply has been developed. The device is thin and lightweight and it functioned stably in the abdominal cavity of rats for more than two weeks without causing foreign body reactions. By covering the tumor site of rat liver cancer with this device and continuously illuminating the tumor with appropriate administration of a photosensitizer, the entire tumor under the emitting surface was eliminated through apoptosis without leaving any residual tumor margins. This is the first report of a robust antitumor effect on parenchymal organ cancers by mPDT using a biocompatible, wirelessly powered OLED device, which represents a potential new deep organ cancer therapy.

bioengineering↗

Raman flow cytometry using time delay integration

Raman flow cytometry offers chemically sensitive, label-free measurement of cells and particles; however, the technique suffers from low cell throughput due to the weak Raman signal. Here, we demonstrate the use of time-delay integration to achieve Raman flow cytometry combined with dual-sided line illumination. The use of line illumination from both sides of the cell flow capillary kept the cell stream in the detection area by balancing optical force from the illumination lines. The time-delay integration allowed accumulation of Raman signals from flowing cells without sacrificing the spectrum readout rate. With the developed system, we achieved Raman flow cytometry at throughputs of 32 and 75 events per second for cell and particle detection, respectively. We applied the technique for analyzing lipid uptake in HepG2 cells and degranulation in bone marrow-derived murine mast cells.

biophysics↗

Raman microscopy of cryofixed biological specimens for high-resolution and high-sensitivity chemical imaging

Raman microscopy is an emerging molecular imaging technology, yet its signal-to-noise-ratio (SNR) in measurements of biological specimens is severely limited due to the small cross-section of Raman scattering. Here, we present Raman imaging techniques of cryofixed specimens to overcome SNR limitations by enabling long exposure of specimens under highly stabilized low-temperature conditions. The observation of frozen specimens in a cryostat at a constant low temperature immediately after rapid freezing enabled the improvement of SNR and significantly enhanced spatial and spectral resolution. We also confirmed that the cryofixation can preserve physicochemical states of specimens by observing alkyne-labeled coenzyme Q in cytosol and hemeproteins in acute ischemic myocardium, which cannot be done by fixation using chemical reagents. Finally, we applied the technique for multiplex Raman imaging of label-free endogenous molecules and alkyne-tagged molecules in cryofixed HeLa cells, demonstrating its capability of high-content imaging of complex biological phenomena while maintaining physiological conditions.

biophysics↗

Dynamic coordination of the lever-arm swing of human myosin II in thick filaments on actin

Muscle myosins work in motor ensembles and must adapt their power stroke in response to mechanical actions by surrounding motors. Understanding the coordination of power strokes is essential for bridging microscopic molecular functions and macroscopic muscle contractions, but the details of this phenomenon remain elusive. Here we used high-speed atomic force microscopy to visualize the individual dynamics (lever-arm swing) of the myosin head bound to actin in DNA origami-based synthetic thick filaments. We observed spatially local lever-arm coordination, and our three-dimensional numerical model explained how mechanical communication between myosins achieved coordination. In a sarcomere model, the local coordination was spatially periodic and propagated toward the contraction direction. We confirmed that a structural mismatch between myosin head spacing (42.8 nm) and the actin helical pitch (37 nm) caused the coordination while improving contraction speed and energy efficiency. Our findings reveal a key physical basis of efficient muscle contraction.

biophysics↗