Biology subjects

Nemoto, T.

Publications and source records attributed to Nemoto, T..

26 records · Page 2Linked to original sources

bioRxiv2023

Signal Improved ultra-Fast Light-sheet Microscope (SIFT) for large tissue imaging

Light-sheet fluorescence microscopy (LSFM) in conjunction with tissue clearing techniques enables morphological investigation of large tissues faster and with excellent optical sectioning. Recently, cleared tissue axially swept light-sheet microscope (ctASLM) demonstrated three-dimensional isotropic resolution in millimeter-scaled tissues. But ASLM based microscopes suffer from low detection signal and slow imaging speed. Here we report a simple and efficient imaging platform that employs precise control of two fixed distant light-sheet foci to carry out ASLM. This allowed us to carry out full field of view (FOV) imaging at 40 frames per second (fps) which is a four-fold improvement compared to the current state-of-the-art. In addition, in a particular frame rate, our method doubles the signal compared to the current ASLM technique. To augment the overall imaging performance, we also developed a deep learning based tissue information classifier that enables faster determination of tissue boundary. We demonstrated the performance of our imaging platform on various cleared tissue samples and demonstrated its robustness over a wide range of clearing protocols.

biophysics↗

bioRxiv2023

In-silico monitoring of directed evolution convergence to unveil best performing variants with credibility score

Directed evolution (DE) is a versatile protein-engineering strategy, successfully applied to a range of proteins, including enzymes, antibodies, and viral vectors. However, DE can be time-consuming and costly, as it typically requires many rounds of selection to identify desired mutants. Next-generation sequencing allows monitoring of millions of variants during DE and can be leveraged to reduce the number of selection rounds. Unfortunately the noisy nature of the sequencing data impedes the estimation of the performance of individual variants. Here, we propose ACIDES that combines statistical inference and in-silico simulations to improve performance estimation in DE by providing accurate statistical scores. We tested ACIDES first on a novel random-peptide-insertion experiment and then on several public datasets from DE of viral vectors and phage-display. ACIDES allows experimentalists to reliably estimate variant performance on the fly and can aid protein engineering pipelines in a range of applications, including gene therapy.

bioinformatics↗

bioRxiv2022

Complete loss of RelA and SpoT homologs in Arabidopsis reveals the importance of the plastidial stringent response in the interplay between chloroplast metabolism and plant defense response

The highly phosphorylated nucleotide, guanosine tetraphosphate (ppGpp), functions as a secondary messenger in bacteria and chloroplasts. The accumulation of ppGpp alters plastidial gene expression and metabolism, which are required for proper photosynthetic regulation and robust plant growth. However, because four plastid-localized ppGpp synthases/hydrolases function redundantly, the impact of the loss of ppGpp-dependent stringent response on plant physiology remains unclear. We used the CRISPR/Cas9 technology to generate an Arabidopsis thaliana mutant lacking all four ppGpp synthases/hydrolases, and characterized its phenotype. The mutant showed 20-fold less ppGpp levels than the wild type (WT) under normal growth conditions, and exhibited leaf chlorosis and increased expression of defense-related genes as well as salicylic acid and jasmonate levels upon transition to nitrogen-starvation conditions. These results demonstrate that proper levels of ppGpp in plastids are required for controlling not only plastid metabolism but also phytohormone signaling, which is essential for plant defense.

plant biology↗

bioRxiv2022

Cold-induced Suspension and Resetting of Ca2+ and Transcriptional Rhythms in the Suprachiasmatic Nucleus Neurons

Mammalian circadian rhythms are coordinated by the master clock located in the hypothalamic suprachiasmatic nucleus (SCN). Under severe environmental conditions, such as during the harsh winter season for food, certain mammalian species reduce their basal metabolism and thermogenesis, thereby undergoing torpor, a controlled state of hypothermia, which naturally returns to the normothermic state. A long-lasting debate focused on whether the SCN with a temperature-compensated clock remains functional during hypothermia. However, so far, no direct and quantitative evidence has been reported of temperature sensitivity in living SCN neurons. In this study, we performed dual-color fluorescence imaging of clock gene transcriptions and intracellular Ca2+ in mouse SCN neurons, using slices at various temperatures. We demonstrated that the Bmal1 transcription and Ca2+ circadian rhythms persisted at 22{degrees}C-28{degrees}C, although the two rhythms underwent temporal dissociation at 22 {degrees}C. Notably, Ca2+, Bmal1, and Per2 rhythms were suspended at 15{degrees}C, coupled with a significant Ca2+ increase, and all rhythms were reset by rewarming to 35{degrees}C. Upon rewarming, the Ca2+ rhythm showed stable oscillations immediately, whereas the Bmal1 and Per2 rhythms took several days to reach stable oscillations and recover their phase relationship with the Ca2+ rhythm. Taken together, we concluded that SCN neurons remain functional under moderate hypothermic conditions at approximately 22{degrees}C-28{degrees}C but stop ticking time in deep hypothermia at 15{degrees}C and that the rhythms reset after deep hypothermia. These data also indicate that the stable Ca2+ oscillation precedes clock gene transcriptional rhythms in the SCN neurons.

neuroscience↗

bioRxiv2022

Engineering of an electrically charged hydrogel implanted into a traumatic brain injury model for stepwise neuronal tissue reconstruction

Neural regeneration is extremely difficult to achieve. In traumatic brain injuries, the loss of brain parenchyma volume hinders neural regeneration. In this study, neuronal tissue engineering was performed by using electrically charged hydrogels composed of cationic and anionic monomers in a 1:1 ratio (C1A1 hydrogel), which served as an effective scaffold for the attachment of neural stem cells (NSCs). In the 3D environment of porous C1A1 hydrogels engineered by the cryogelation technique, NSCs differentiated into neuroglial cells. The C1A1 porous hydrogel was implanted into brain defects in a mouse traumatic damage model. The VEGF-immersed C1A1 porous hydrogel promoted host-derived vascular network formation together with the infiltration of macrophages/microglia and astrocytes into the gel. Furthermore, the stepwise transplantation of GFP-labeled NSCs supported differentiation to glial and neuronal cells. Therefore, this two-step method for neural regeneration may become a new approach for therapeutic brain tissue reconstruction after brain damage in the future. One Sentence SummaryBrain tissue reconstruction using charged hydrogel and stepwise NCS injection

bioengineering↗

bioRxiv2020

Elevated blood pressure in high fat diet-exposed low birth weight rat offspring is most likely caused by elevated glucocorticoid levels

Being delivered as a low birthweight (LBW) infant is a risk factor for elevated blood pressure and future problems with cardiovascular and cerebellar diseases. Premature babies have been reported to possess a lower number of nephrons, but the mechanisms by which blood pressure is elevated in full-term LBW infants remain unclear. We generated a fetal low-carbohydrate and calorie-restricted model rat, and some individuals showed postnatal growth failure caused by increased miR-322 expression in the liver and decreased growth hormone receptor expression. Using this model, we examined how a high-fat diet-induced mismatch between prenatal and postnatal environments could elevate blood pressure after growth. Although LBW rats fed standard chow had slightly higher blood pressure than control rats, their blood pressure was significantly higher than controls when exposed to a high-fat diet. Observation of glomeruli subjected to PAM staining showed no difference in number or size. Aortic and cardiac angiotensin II receptor expression was altered with compensatory responses. Blood aldosterone levels were not different between control and LBW rats, but blood corticosterone levels were significantly higher in the latter with high-fat diet exposure. Administration of metyrapone, a steroid synthesis inhibitor, reduced blood pressure to levels comparable to controls. We showed that high-fat diet exposure causes impairment of the pituitary glucocorticoid feedback via miR-449a. These results clarify that LBW rats have increased blood pressure due to high glucocorticoid levels when they are exposed to a high-fat diet. These findings suggest a new therapeutic target for hypertension of LBW individuals.

physiology↗

bioRxiv2019

SCAP promotes CARTS biogenesis at endoplasmic reticulum-Golgi contact sites

In response to cholesterol deprivation, SCAP escorts SREBP transcription factors from the endoplasmic reticulum (ER) to the Golgi complex for their proteolytic activation, leading to gene expression for cholesterol synthesis and uptake. Here we show that in cholesterol-fed cells ER-localized SCAP interacts through Sac1 phosphoinositide 4-phosphate (PI4P) phosphatase with a VAP/OSBP complex, which mediates counter-transport of ER cholesterol and Golgi PI4P at ER-Golgi contact sites. SCAP knockdown inhibited the turnover of PI4P perhaps due to a cholesterol transport defect and altered the subcellular distribution of the VAP/OSBP complex. As in the case of perturbation of lipid transfer complexes at ER-Golgi contact sites, SCAP knockdown inhibited the biogenesis of the trans-Golgi network-derived transport carriers CARTS, which was reversed by expression of wild-type SCAP but not cholesterol sensing-defective mutants. Altogether, our findings reveal a new role of SCAP under cholesterol-fed conditions in the facilitation of CARTS biogenesis at ER-Golgi contact sites, depending on the ER cholesterol. SummarySCAP is the key regulatory protein in cholesterol metabolism. Wakana et al. describe a new role of SCAP in controlling Golgi PI4P turnover and the biogenesis of the Golgi-derived transport carries CARTS via cholesterol/PI4P exchange machinery at ER-Golgi contact sites.

cell biology↗

bioRxiv2019

Optical clearing of living brains with MAGICAL to extend in vivo imaging

To understand brain functions, it is important to observe directly how multiple neural circuits are performing in living brains. However, due to tissue opaqueness, observable depth and spatiotemporal resolution are severely degraded in vivo. Here, we propose an optical brain clearing method for in vivo fluorescence microscopy, termed MAGICAL (Magical Additive Glycerol Improves Clear Alive Luminance). MAGICAL enabled two-photon microscopy to capture vivid images with fast speed, at cortical layer V and hippocampal CA1 in vivo. Moreover, MAGICAL promoted conventional confocal microscopy to visualize finer neuronal structures including synaptic boutons and spines in unprecedented deep regions, without intensive illumination leading to phototoxic effects. Fluorescence Emission Spectrum Transmissive Analysis (FESTA) showed that MAGICAL improved in vivo transmittance of shorter wavelength light, which is vulnerable to optical scattering thus unsuited for in vivo microscopy. These results suggest that MAGICAL would transparentize living brains via scattering reduction.

neuroscience↗