Biology subjects

Sakamoto, J.

Publications and source records attributed to Sakamoto, J..

8 recordsLinked to original sources

bioRxiv2026

Fully Two-Photon-Driven RESOLFT Microscopy for Super-Resolution Imaging inside Tissue

Two-photon excitation (2PE) microscopy reaches deep into tissue but remains diffraction-limited, and existing 2PE-STED and 2PE-RESOLFT schemes still rely on single-photon depletion or photoswitching. Here we present 2P-RESOLFT microscopy, in which excitation and reversible photoswitching are both two-photon-driven. Using the positive photoswitchable protein Padron2, we achieved 205-nm lateral resolution and resolved previously unreported membrane protrusions of {beta} cells inside pancreatic pseudoislets.

biophysics↗

bioRxiv2025

Wntless interacts with Notch signaling to balance the generation of neurons and gliocytes in vertebrate dorsal diencephalon

Wnt chaperon Wntless (Wls) mediates the intracellular transport of Wnts and plays important roles in early vertebrate brain development. Spatially restricted induction of Wls denotes the earliest differentiation of non-telencephalic cells in human brain organoids. In zebrafish developing diencephalon, loss-of-Wls reduces the formation of habenula (HA) neurons but how Wls influences HA neurogenesis is unclear and whether Wls regulates gliocyte development is unknown. Here we report that the formations of cholinergic, substance P-ergic or glutamatergic neurons in HA are reduced differentially but the generation of gliocyte-derived choroid plexus (ChP) epithelia is increased in wls null mutants. At earlier stage, three-dimensional gene expression analyses revealed that while neurog1 expressions in HA progenitor zones are reduced, the expressions of Notch downstream effector her6 are increased and expanded into HA progenitor zones in wls mutants. Over-expressing Her6 in neurog1-positive cells reduced neurog1 expressions in HA progenitor zones. These results indicate that Wls restricts the expressions of Notch effector her6 to promote the specification of neurog1 proneurons and demotes the generation of ChP epithelia in zebrafish embryonic dorsal diencephalon.

developmental biology↗

bioRxiv2025

Excessive chondrogenesis in the blastema initiates during hypomorphic limb regeneration in Xenopus froglet, but not during patterned limb regeneration in Xenopus tadpoles and newts

Xenopus laevis tadpoles and newts regenerate a limb almost completely after amputation, with recapitulation of the patten formation of the limb. Metamorphosed Xenopus froglets form a cone-shaped regenerating blastema, similar to tadpoles and newts, but ultimately regenerate only a hypomorphic cartilaginous spike. Previous study suggested that excessive chondrogenesis, distinct from Xenopus tadpoles, may occur in the regenerating limb of a froglet and may prevent pattern formation during regeneration. However, it remains unclear whether excessive chondrogenesis actually occurs in froglet blastemas. If it does, when does it initiate and how does it progress in the blastemas? To answer these questions, we examined the extent of chondrogenesis in regenerating blastemas which have the common morphological shapes observed in newts (Pleurodeles waltl), Xenopus laevis tadpoles, and froglets. To evaluate excessive chondrogenesis, we developed a simplified procedure using immunofluorescence for cartilage markers (Sox9 or Col2a1) and quantitative image analysis. Our analysis revealed that signs of excessive chondrogenesis were detected not in newts and tadpoles but in froglets blastemas. During limb regeneration in froglets, the first sign of excessive chondrogenesis was detected in the cone-shaped blastema at the medium bud (MB) stage, and excessive chondrogenesis progressed to a more severe state as the blastema grew. These results indicate that excessive chondrogenesis initiates specifically in froglet blastemas at the MB stage at the latest and progresses in a definite spatio-temporal manner. Further elucidation of the mechanisms underlying froglet-specific excessive chondrogenesis in the blastema may lead to the recovery of patterned limb regeneration in froglets with adequate inhibition of chondrogenesis.

developmental biology↗

bioRxiv2024

Implication of S-1 biomarkers on aging and prognosis in elderly patients with lung cancer treated by adjuvant S-1 chemotherapy: Accompanying results of the Setouchi Lung Cancer Group Study 1201

BACKGROUNDSThe management of elderly patients presents several challenges due to age-related declines; however recent recommendations advocate for age not being the sole determinant for adjuvant treatment decisions in patients with non-small cell lung cancer (NSCLC). Aging may alter expression levels of 5-fluorouracil (5-FU) biomarkers. METHODSExpression changes with aging were explored using The Cancer Genome Atlas (TCGA) database. 5-FU-related biomarker expressions, including thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), orotate phosphoribosyltransferase, epidermal growth factor receptor (EGFR), and excision repair cross-complementation group-1 (ERCC1), were assessed by the quantitative reverse-transcription PCR assays in 89 NSCLCs elderly patients ([≥] 75 years old) receiving adjuvant S-1, an oral fluoropyrimidine agent, therapy in a SLCG1201 trial. RESULTS: TCGA database analysis (n=955) indicated decreased TS expression with aging, particularly in those 75 or older. In the SCLG1201 trial, univariate analysis revealed that high EGFR and low TS expressions correlated with favorable recurrence-free survival (RFS)(p=0.0264) and overall survival (OS)(p=0.0365), respectively. Multivariable analysis confirmed pathological stage as an independent prognostic factor for both RFS and OS (p=0.0052 and 0.0352, respectively). EGFR mutant tumors (n=27) showed significantly higher DPD (p=0.0066) and EGFR (p<0.0001) expressions, and lower TS(p=0.0125) and ERCC1(p=0.0015) expressions. CONCLUSIONDespite pathological stage being an independent prognostic factor, high EGFR and low TS expressions may predict better clinical outcomes in elderly NSCLC patients using adjuvant S-1. The age-related decrease in TS expression supports the potential benefit of 5-FU-based therapy in them compared to younger patients. Further research is warranted to validate these clinical implications.

pharmacology and toxicology↗

bioRxiv2024

Amyloid-β-induced Alteration of Fast and Localized Calcium Elevations in Cultured Astrocytes

Alzheimers disease (AD) is a progressive neurodegenerative disorder that causes cognitive decline. Uncovering the mechanisms of neurodegeneration in the early stages is essential to establish a treatment for AD. Recent research has proposed the hypothesis that amyloid-{beta} (A{beta}) oligomers elicit an excessive glutamate release from astrocytes toward synapses through intracellular free Ca2+ ([Ca2+]i) elevations in astrocytes, finally resulting in neuronal dendritic spine loss. Under physiological conditions, astrocytic [Ca2+]i elevations range spatially from microdomains to network-wide propagation and temporally from milliseconds to tens of seconds. Astrocytic localized and fast [Ca2+]i elevations might correlate with glutamate release; however, the A{beta}-induced alteration of localized, fast astrocytic [Ca2+]i elevations remains unexplored. In this study, we quantitatively investigated the A{beta} dimers-induced changes in the spatial and temporal patterns of [Ca2+]i in a primary culture of astrocytes by two-photon excitation spinning-disk confocal microscopy. The frequency of fast [Ca2+]i elevations occurring locally in astrocytes ([≤]0.5 s, [≤]35 {micro}m2) and [Ca2+]i event occupancy relative to cell area significantly increased after exposure to A{beta} dimers. The effect of A{beta} dimers appeared dose-dependently above 500 nM, and these A{beta} dimers-induced [Ca2+]i elevations were primarily mediated by a metabotropic purinergic receptor (P2Y1 receptor) and Ca2+ release from the endoplasmic reticulum. Our findings suggest that the A{beta} dimers-induced alterations and hyperactivation of astrocytic [Ca2+]i is a candidate cellular mechanism in the early stages of AD.

neuroscience↗

bioRxiv2022

The shh limb enhancer is activated in patterned limb regeneration but not in hypomorphic limb regeneration in Xenopus laevis

Xenopus young tadpoles regenerate a limb with the anteroposterior (AP) pattern, but metamorphosed froglets regenerate a hypomorphic limb after amputation. The key gene for AP patterning, shh, is expressed in a regenerating limb of the tadpole but not in that of the froglet. Genomic DNA in the shh limb-specific enhancer, MFCS1 (ZRS), is hypermethylated in froglets but hypomethylated in tadpoles: shh expression may be controlled by epigenetic regulation of MFCS1. Is MFCS1 specifically activated for regenerating the AP-patterned limb? We generated transgenic Xenopus laevis lines that visualize the MFCS1 enhancer activity with a GFP reporter. The transgenic tadpoles showed GFP expression in hoxd13- and shh-expressing domains of developing and regenerating limbs, whereas the froglets showed no GFP expression in the regenerating limbs despite having hoxd13 expression. Genome sequence analysis and co-transfection assays using cultured cells revealed that Hoxd13 can activate Xenopus MFCS1. These results suggest that MFCS1 activation correlates with regeneration of AP-patterned limbs and that re-activation of epigenetically inactivated MFCS1 would be crucial to confer the ability to non-regenerative animals for regenerating a properly patterned limb.

developmental biology↗

bioRxiv2020

Effect of belt electrode-skeletal muscle electrical stimulation on immobilization-induced muscle fibrosis

PurposeMacrophage accumulation in response to decreasing myonuclei may be the major mechanism underlying immobilization-induced muscle fibrosis in muscle contracture, an intervention strategy suppressing these lesions is necessary. Therefore, this research investigated the effect of belt electrode-skeletal muscle electrical stimulation (B-SES), a new electrical stimulation device, to the macrophage accumulation via myonuclei decrease in immobilization-induced muscle fibrosis. Materials and Methods18 Wistar male rats were divided into the control group, immobilization group (with plaster cast fixation to immobilize the soleus muscles in a shortened position for 2 weeks), and B-SES group (with muscle contractile exercise through B-SES during the immobilization period). B-SES stimulation was performed at a frequency of 50 Hz and an intensity of 4.7 mA, muscle contractile exercise by B-SES was applied to the lower limb muscles for 20 minutes/session (twice a day) for 2 weeks (6 times/week). The bilateral soleus muscles were used for histological, immunohistochemical, biochemical, and molecular biological analyses. ResultsThe number of myonuclei was significantly higher in the B-SES group than in the immobilization group, and there was no significant difference between the B-SES and control groups. The cross-sectional area of type I and II myofibers in the immobilization and B-SES groups was significantly lower than that in the control group, and the cross-sectional area of type I myofibers in the B-SES group was higher than that in the immobilization group. However, Atrogin-1 and MuRF-1 mRNA expression in the immobilization and B-SES groups was significantly higher than those in the control group. Additionally, the number of macrophages, IL-1{beta}, TGF-{beta}1, and -SMA mRNA expression, and hydroxyproline expression was significantly lower in the control and B-SES groups than those in the immobilization group. ConclusionThis research surmised that muscle contractile exercise through B-SES prevented immobilization-induced muscle fibrosis, and this alteration suppressed the development of muscle contracture.

pathology↗

bioRxiv2019

Heart Rate n-Variability (HRnV) and Its Application to Risk Stratification of Chest Pain Patients in the Emergency Department

BackgroundChest pain is one of the most common complaints among patients presenting to the emergency department (ED). Causes of chest pain can be benign or life threatening, making accurate risk stratification a critical issue in the ED. In addition to the use of established clinical scores, prior studies have attempted to create predictive models with heart rate variability (HRV). In this study, we proposed heart rate n-variability (HRnV), an alternative representation of beat-to-beat variation in electrocardiogram (ECG) and investigated its association with major adverse cardiac events (MACE) for ED patients with chest pain. MethodsWe conducted a retrospective analysis of data collected from the ED of a tertiary hospital in Singapore between September 2010 and July 2015. Patients >20 years old who presented to the ED with chief complaint of chest pain were conveniently recruited. Five to six-minute single-lead ECGs, demographics, medical history, troponin, and other required variables were collected. We developed the HRnV-Calc software to calculate HRnV parameters. The primary outcome was 30-day MACE, which included all-cause death, acute myocardial infarction, and revascularization. Univariable and multivariable logistic regression analyses were conducted to investigate the association between individual risk factors and the outcome. Receiver operating characteristic (ROC) analysis was performed to compare the HRnV model (based on leave-one-out cross-validation) against other clinical scores in predicting 30-day MACE. ResultsA total of 795 patients were included in the analysis, of which 247 (31%) had MACE within 30 days. The MACE group was older and had a higher proportion of male patients. Twenty-one conventional HRV and 115 HRnV parameters were calculated. In univariable analysis, eleven HRV parameters and 48 HRnV parameters were significantly associated with 30-day MACE. The multivariable stepwise logistic regression identified 16 predictors that were strongly associated with the MACE outcome; these predictors consisted of one HRV, seven HRnV parameters, troponin, ST segment changes, and several other factors. The HRnV model outperformed several clinical scores in the ROC analysis. ConclusionsThe novel HRnV representation demonstrated its value of augmenting HRV and traditional risk factors in designing a robust risk stratification tool for patients with chest pain at the ED.

bioengineering↗