Biology subjects

Ozaki, Y.

Publications and source records attributed to Ozaki, Y..

8 recordsLinked to original sources

bioRxiv2025

The chicken retrovirus-like gene ENS-1/ERNI and its LTR Soprano are involved in primordial germ cell development.

Cultured chicken primordial germ cells (PGCs) are attractive tools for reproductive biology and for preserving and modifying genetic resources. However, it remains difficult to establish cell lines from some breeds or females. In the present study, we performed transcriptome analyses of cultured PGCs from different breeds and found that the expression of the Galliformes-specific endogenous retrovirus-like gene ENS-1/ERNI correlated with the establishment efficiency of PGC lines. CRISPRi targeting the ENS-1/ERNI long terminal repeat (LTR) sequence (Soprano) broadly reduced homeostasis-related genes including ribosome, endoplasmic reticulum, and oxidative phosphorylation. These PGCs exhibited abnormal cell cycle arrest under DNA-damaged conditions, suggesting involvement in the quality control mechanisms of chicken PGCs. Furthermore, when transplanted into blastoderms, these PGCs showed a reduction in colonization of the genital ridges. These results indicate the involvement of Soprano LTR and ENS-1/ERNI in the development of chicken PGCs and their adaptation to the in vitro environment.

cell biology↗

bioRxiv2023

Fibroblast Nrf2 inhibits profibrotic transcription with Ddx54 and mitigates pathological fibrosis in the mouse heart and kidney

BackgroundTissue fibrosis is a common feature of many organ dysfunctions, such as heart failure and chronic kidney disease. However, no fundamental treatment has been developed. This study aims to identify novel molecular mechanisms for antifibrotic intervention, focusing on fibroblast activation. MethodsWe performed a forward genetic screen using a genome-wide CRISPR library in the context of transforming growth factor {beta} (TGF-{beta})-mediated connective tissue growth factor (CTGF) expression, and used unbiased techniques such as Cleavage Under Targets and Tagmentation (CUT&Tag) and proximity-dependent biotin labeling by TurboID to reveal the detailed molecular mechanisms. ResultsCRISPR library screening identified a number of players in both the canonical Smad pathway and the non-canonical pathway. In addition to the known factors, the Keap1-Nrf2 pathway was identified as a predominant regulator of TGF-{beta}-mediated CTGF expression. Keap1 deletion and consequent Nrf2 activation broadly suppressed profibrotic gene expression, independently of conventional antioxidant effects. CUT&Tag revealed that Nrf2 bound to the proximity of fibrosis-related genes including Ctgf and Fn1. Subsequent individual analysis revealed Smad3 and RNA polymerase II binding to the Nrf2 peak site, which was attenuated by Keap1 deletion. TurboID experiments further discovered that Nrf2 interacts with Ddx54, which acts as a corepressor. Consistently, Keap1 deletion-mediated repression of profibrotic gene expression was reversed by additional Ddx54 deletion. The impact of the Keap1-Nrf2 pathway on pathological fibrosis was examined using tamoxifen-inducible fibroblast-specific Keap1 knockout mice. Pressure overload for 4 weeks robustly induced cardiac hypertrophy, fibrosis and contractile dysfunction. However, deletion of Keap1 in the Postn lineage attenuated these cardiac pathologies. The anti-fibrotic effects of Keap1 deletion were also confirmed in renal fibrosis in the unilateral ureteral obstruction (UUO) model. ConclusionsFibroblast Nrf2 transcriptionally represses fibrosis-related genes in cooperation with the corepressor Ddx54. Fibroblast-specific deletion of Keap1 attenuated pathological fibrosis in pressure overload heart failure and renal fibrosis.

cell biology↗

bioRxiv2023

Tomato Response Evaluation through Fertilization and PGRs application Under Temperature Differentiation in late Winter

This study evaluated the exogenous application of PGRs substitute chemical fertilization without compromising the growth and yield of tomato in fluctuated day-night temperature and humidity stressed late winter. Two-factor experiment comprising chemical fertilizers at 100, 110, 90 and 80 % of recommended doses besides control and PGRs of GA3; NAA, 4-CPA and SA @ 50 ppm including control was conducted where treatments were assigned in triplicates. Results revealed no significant variation among the fertilizer doses (80% to 110% of recommendation) regarding growth and yield contributing traits while among the PGRs, GA3 @ 50 ppm produced maximum number of flower clusters plant-1 (16.85), flowers (8.80) and fruits (5.79) cluster-1, single fruit weight (67.83 g) and fruit yield (6.61 kg plant-1) of tomato that was statistically identical with the findings of SA. But significant reduction in yield was noted in NAA and 4-CPA (1.20 kg and 1.21 kg plant-1, respectively). Interestingly, GA3 and SA in combination with any doses of the studied fertilizers maximize the tomato morphological and reproductive traits while fertilizer plus NAA and 4-CPA interaction gave the inferior results. Further, correlation matrix and PCA findings revealed that five fertilizer doses have no distinctiveness whereas GA3 and SA has distinct position than other PGRs with the maximum dependent variables those were contributed positively in the total variations. The study findings suggested that 20% fertilizer requirement could be reduced with the substitution of GA3 and SA @ 50 ppm for successful cultivation of tomato in late winter having the extreme environmental issues.

physiology↗

bioRxiv2023

Deep mutational scanning of whole SARS-CoV-2 spike in an inverted infection system

In order to investigate SARS-CoV-2 mutations and their impact on immune evasion and infectivity, we developed a Deep Mutational Scanning (DMS) platform utilizing an inverted infection assay to measure spike expression, ACE2 affinity, and viral infectivity in human cells. Surprisingly, our analysis reveals that spike protein expression, rather than ACE2 affinity, is the primary factor affecting viral infectivity and correlated with SARS-CoV-2 evolution. Notably, within the N-terminal domain (NTD), spike expression and infectivity-enhancing mutations are concentrated in flexible loops. We also observed that Omicron variants BA.1 and BA.2 exhibit immune evasion through receptor binding domain (RBD) mutations, although these mutations reduce structural stability. Interestingly, the NTD has evolved to increase stability, compensating for the RBD instability and resulting in heightened overall infectivity. Our findings, available in SpikeScanDB, emphasize the importance of spike expression levels and compensatory mutations in both the NTD and RBD domains for shaping Omicron variant infectivity.

microbiology↗

bioRxiv2021

Engineered ACE2 counteracts vaccine-evading SARS-CoV-2 Omicron variant

The novel SARS-CoV-2 variant, Omicron (B.1.1.529) contains an unusually high number of mutations (>30) in the spike protein, raising concerns of escape from vaccines, convalescent sera and therapeutic drugs. Here we analyze the alteration of neutralizing titer with Omicron pseudovirus. Sera obtained 3 months after double BNT162b2 vaccination exhibit approximately 18-fold lower neutralization titers against Omicron than parental virus. Convalescent sera from Alpha and Delta patients allow similar levels of breakthrough by Omicron. Domain-wise analysis using chimeric spike revealed that this efficient evasion was primarily achieved by mutations clustered in the receptor-binding domain, but that multiple mutations in the N-terminal domain contributed as well. Omicron escapes a therapeutic cocktail of imdevimab and casirivimab, whereas sotrovimab, which targets a conserved region to avoid viral mutation, remains effective. The ACE2 decoy is another virus-neutralizing drug modality that is free, at least in theory, from complete escape. Deep mutational analysis demonstrated that, indeed, engineered ACE2 prevented escape for each single-residue mutation in the receptor-binding domain, similar to immunized sera. Engineered ACE2 neutralized Omicron comparable to Wuhan and also showed a therapeutic effect against Omicron infection in hamsters and human ACE2 transgenic mice. Like previous SARS-CoV-2 variants, some sarbecoviruses showed high sensitivity against engineered ACE2, confirming the therapeutic value against diverse variants, including those that are yet to emerge. One Sentence SummaryOmicron, carrying [~]30 mutations in the spike, exhibits effective immune evasion but remains highly susceptible to blockade by engineered ACE2.

microbiology↗

bioRxiv2021

Identification, crystallization and epitope determination of public TCR shared and expanded in COVID-19 patients

T cells play pivotal roles in protective immunity against SARS-CoV-2 infection. Follicular helper T (Tfh) cells mediate the production of antigen-specific antibodies; however, T cell receptor (TCR) clonotypes used by SARS-CoV-2-specific Tfh cells have not been well characterized. Here, we first identified and crystallized public TCR of Tfh clonotypes that are shared and expanded in unhospitalized COVID-19-recovered patients. These clonotypes preferentially recognized SARS-CoV-2 spike (S) protein epitopes which are conserved among emerging SARS-CoV-2 variants. These clonotypes did not react with S proteins derived from common cold human coronaviruses, but cross-reacted with symbiotic bacteria, which might confer the publicity. Among SARS-CoV-2 S epitopes, S864-882, presented by frequent HLA-DR alleles, could activate multiple public Tfh clonotypes in COVID-19-recovered patients. Furthermore, S864-882-loaded HLA tetramer preferentially bound to CD4+ T cells expressing CXCR5. In this study, we identified and crystallized public TCR for SARS-CoV-2 that may contribute to the prevention of COVID-19 aggravation.

immunology↗

bioRxiv2020

Iodine staining as a useful probe for distinguishing amyloid fibril polymorphs

ABSTRACTIt is recently suggested that amyloid polymorphism, i.e., structural diversity of amyloid fibrils, has a deep relationship with pathology. However, its prompt recognition is almost halted due to insufficiency of analytical methods for detecting polymorphism of amyloid fibrils sensitively and quickly. Here, we propose that iodine staining, a historically known reaction that was firstly found by Virchow, can be used as a method for distinguishing amyloid polymorphs. When insulin fibrils were prepared and iodine-stained, they exhibited different colors depending on polymorphs. Each of the colors was inherited to daughter fibrils by seeding reactions. The colors were fundamentally represented as a sum of three absorption bands in visible region between 400-750 nm, and the bands showed different titration curves against iodine, suggesting that there are three specific iodine binding sites. The analysis of resonance Raman spectra and polarization microscope suggested that several polyiodide ions composed of I3− and/or I5− were formed on the grooves or the edges of β-sheets. It was concluded that the polyiodide species and conformations formed are sensitive to surface structure of amyloid fibrils, and the resultant differences in color will be useful for detecting polymorphism in a wide range of diagnostic samples.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biochemistry↗

bioRxiv2019

Exploration of insulin amyloid polymorphism using Raman spectroscopy and imaging

We aimed to investigate insulin amyloid fibril polymorphism caused by salt effects and heating temperature, and to visualize the structural differences of the polymorphisms in situ using Raman imaging without labeling. The time course monitoring for amyloid formation was carried out in an acidic condition without any salts and with two species of salts (NaCl and Na2SO4) by heating at 60, 70, 80, and 90 . The intensity ratio of two Raman bands at 1672 and 1657 cm-1 due to {beta}-sheet and -helix structures was revealed to be an indicator of amyloid fibril formation, and the relative proportion of the {beta}-sheet structure was higher in the case with salts, especially at a higher temperature and with Na2SO4. In conjunction with the secondary structural changes of proteins, the S-S stretching vibrational mode of a disulfide bond ([~]514 cm-1) and the ratio of the tyrosine doublet R(I850/I826) were also found to be markers distinguishing polymorphisms of insulin amyloid fibrils by principal component analysis (PCA). Especially, amyloid fibrils with Na2SO4 media formed the g-g-g conformation of disulfide bond at a higher rate and without any salts; on the contrary, the g-g-g conformation was partially transformed into the g-g-t conformation at higher temperatures. The different environments of the hydroxyl groups of the tyrosine residue were assumed to be caused by fibril polymorphism. Raman imaging using these marker bands also successfully visualized the two- and three-dimensional structural differences of amyloid polymorphisms. The present results indicate the potential of Raman imaging as a diagnostic tool for polymorphisms in tissues of amyloid-related diseases.\n\nStatement of SignificanceOur results revealed three Raman markers distinguishing amyloid fibril polymorphisms caused by salt and temperature effects; the relative proportion of protein secondary structures (-helix and {beta}-sheet), the ratio of tyrosine doublet, and the conformational differences of disulfide bonds. The lower values of tyrosine doublet in the case with salts were interpreted as the anions rob the hydration water from proteins which induced protein misfolding. Using these parameters, Raman images captured their higher order structural differences in situ without labeling. The images of hydrogen bonds strength variations due to tyrosine doublet is believed to include significant novelty. The present results imply the potential of Raman imaging for use as a diagnostic imaging tool for tissues with amyloid-induced diseases.

biophysics↗