Biology subjects

Miyazaki, Y.

Publications and source records attributed to Miyazaki, Y..

13 recordsLinked to original sources

bioRxiv2026

Novel cell wall-associated genes that enable Cryptococcus neoformans to evade dectin-1-mediated innate immune recognition.

The fungal pathogen Cryptococcus neoformans contains about 200 {micro}g of {beta}-1,3-glucan (1,3BG) per 1 mg of dry cell weight when grown under standard culture conditions (YPD medium at 30 {degrees}C under aerobic conditions). However, 1,3BG exposure is tightly suppressed, even in capsule-deficient strains, allowing the fungus to evade recognition by the immune receptor dectin-1 and anti-1,3BG antibodies. Although other pathogenic fungi mask 1,3BG with -1,3-glucan (1,3AG) to evade dectin-1 recognition, the factors responsible for 1,3BG masking and dectin-1 evasion in C. neoformans remain incompletely understood. To identify capsule-independent 1,3BG masking and dectin-1-evasion factors, we generated a series of cell wall-related gene deletion strains in the capsule-deficient strain cap59{Delta} by using CRISPR-Cas9 and screened for mutants that failed to evade dectin-1 binding. We found eight deletants (cap59{Delta}/mpk1{Delta}, cap59{Delta}/chs3{Delta}, cap59{Delta}/kre5{Delta}, cap59{Delta}/crz1{Delta}, cap59{Delta}/kre6{Delta}/skn1{Delta}, cap59{Delta}/hxl1{Delta}, cap59{Delta}/uge1{Delta}, and cap59{Delta}/ugt1{Delta}) that exhibited increased binding to dectin-1 or anti-1,3BG antibody, or both. As a similar phenotype was not observed in cap59{Delta}/ags1{Delta}, 1,3AG-mediated masking of 1,3BG appears to play a limited role in C. neoformans. These eight deletants induced significantly greater secretion of IL-6 and IL-1{beta} from dendritic cells (DCs) than did cap59{Delta}. This enhanced inflammatory response was markedly attenuated in dectin-1-deficient DCs, indicating that the increased immunogenicity was driven by 1,3BG exposure and subsequent dectin-1 recognition. Collectively, these findings demonstrate that multiple genes involved in maintaining cell wall integrity--including those involved in {beta}-1,6-glucan and chitosan biosynthesis--are essential for regulating 1,3BG exposure and enabling C. neoformans to evade dectin-1-mediated immune recognition. HighlightsO_LIWe identified new capsule-independent {beta}-1,3-glucan-masking genes in C. neoformans C_LIO_LIGene deletants had higher dectin-1 deposition than parental acapsular cap59{Delta} C_LIO_LIDeletion of these genes enhanced IL-6 and IL-1{beta} secretion by dendritic cells C_LIO_LIThe enhanced cytokine response was suppressed in dendritic cells lacking dectin-1. C_LIO_LIDeletant strains may serve as new whole-cell antigens for cryptococcal vaccines C_LI

microbiology↗

bioRxiv2026

HAC1 contributes to stress adaptation and virulence in the emerging fungal pathogen Candida auris

Candida auris (Candidozyma auris) is an emerging multidrug-resistant fungal pathogen that poses a significant global health threat. However, the molecular mechanisms underlying its virulence remain incompletely understood. In this study, we performed in vivo transcriptome analysis using an immunosuppressed mouse gastrointestinal infection model to identify genes associated with host-adaptation and virulence during infection. By comparing fungal transcriptomes obtained from colonization and dissemination sites with those from in vitro cultures, we identified genes that were consistently upregulated during infection. Among these genes, the unfolded protein response regulator HAC1 was selected as a candidate virulence-associated gene for further analysis. RT-PCR and sequencing analyses revealed that HAC1 mRNA in C. auris undergoes an unconventional splicing event of 287 bp that is enhanced under ER stress conditions. The excised region spans the annotated open reading frame boundary, suggesting that the translated region of HAC1 may require re-evaluation. Notably, a proportion of HAC1 transcripts appeared to be spliced even under non-stress conditions, indicating a detectable basal level of UPR activation. Differences in splicing dynamics were also observed among clade strains. Functional analyses demonstrated that deletion of HAC1 increased sensitivity to ER stress and heat stress. The HAC1 deletion mutant also exhibited reduced virulence in both Galleria mellonella and immunosuppressed mouse infection models, as evidenced by delayed host mortality and decreased fungal burdens, respectively. These findings indicate that HAC1 contributes to ER stress adaptation, thermotolerance, and survival in the host environment, and identify HAC1 as a virulence-associated gene in C. auris.

microbiology↗

bioRxiv2025

pH-Sensitive Amino Lipid-Driven Pore Formation Enables Endosomal Escape of Lipid Nanoparticles

Lipid nanoparticles (LNPs) have transformed nucleic acid delivery for vaccines and gene therapies, yet their efficiency remains limited by incomplete endosomal escape. While experimental studies have revealed endosomal membrane disruption facilitating cytosolic release, the molecular basis of this process remains poorly understood. Here, we employ molecular simulations to elucidate how LNPs fuse with the endosomal membrane and release their payloads. We identify multiple fusion pathways, with a dominant stalk-pore mechanism. Our simulations reproduce and rationalize key experimental observations, including the transfer of ionizable lipids from LNPs to the membrane, which promotes nucleic acid reorientation and facilitates stalk formation and expansion. Furthermore, lipid shape, pH sensitivity, membrane tension, and nucleic acid encapsulation emerge as critical molecular determinants of endosomal escape efficiency. These findings advance our mechanistic understanding of intracellular delivery and provide a framework for the rational design of LNPs with improved performance for gene therapies and RNA vaccines.

biophysics↗

bioRxiv2024

Stratification of viral shedding patterns in saliva of COVID-19 patients

Living with COVID-19 requires continued vigilance against the spread and emergence of variants of concern (VOCs). Rapid and accurate saliva diagnostic testing, alongside basic public health responses, is a viable option contributing to effective transmission control. Nevertheless, our knowledge regarding the dynamics of SARS-CoV-2 infection in saliva is not as advanced as our understanding of the respiratory tract. Here we analyzed longitudinal viral load data of SARS-CoV-2 in saliva samples from 144 patients with mild COVID-19 (a combination of our collected data and published data). Using a mathematical model, we quantified individual-level viral dynamics and stratified them into three groups using a clustering approach. Notably, the three groups exhibited distinct differences viral RNA detection durations: 11.5 days (95% CI: 10.6 to 12.4), 17.4 days (16.6 to 18.2), and 30.0 days (28.1 to 31.8), respectively. Surprisingly, this stratified grouping remained unexplained despite our analysis of 47 types of clinical data, including basic demographic information, clinical symptoms, results of blood tests, and vital signs. Additionally, we quantified the expression levels of 92 micro-RNAs in a subset of saliva samples, but these also failed to explain the observed stratification, although the mir-1846 level may have been weakly correlated with peak viral load. Our study provides insights into SARS-CoV-2 infection dynamics in saliva, highlighting the challenges in predicting the duration of viral RNA detection without indicators that directly reflect an individuals immune response, such as antibody induction. Given the significant individual heterogeneity in the kinetics of saliva viral shedding, identifying biomarker(s) for viral shedding patterns will be crucial for improving public health interventions in the era of living with COVID-19.

microbiology↗

bioRxiv2023

Improved Protein Model in SPICA Force Field

The previous version of the SPICA coarse-grained (CG) force field (FF) protein model focused primarily on membrane proteins and successfully reproduced the dimerization free energies of several transmembrane helices and stable structures of various membrane protein assemblies. However, that model had limited accuracy when applied to other proteins, such as intrinsically disordered proteins (IDPs) and peripheral proteins, because the dimensions of the IDPs in an aqueous solution were too compact, and protein binding on the lipid membrane surface was over-stabilized. To improve the accuracy of the SPICA FF model for the simulation of such systems, in this study we introduce protein secondary structure-dependent nonbonded interaction parameters to the backbone segments and re-optimize almost all nonbonded parameters for amino acids. The improved FF proposed here successfully reproduces the radius of gyration of various IDPs, the binding sensitivity of several peripheral membrane proteins, and the dimerization free energies of several transmembrane helices. The new model also shows improved agreement with experiments on the free energy of peptide association in water. In addition, an extensive library of nonbonded interactions between proteins and lipids, including various glycerophospholipids, sphingolipids, and cholesterol, allows the study of specific interactions between lipids and peripheral and transmembrane proteins. Hence, the new SPICA FF (version 2) proposed herein is applicable with high accuracy for simulating a wide range of protein systems. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/557852v2_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@8b026dorg.highwire.dtl.DTLVardef@4c622forg.highwire.dtl.DTLVardef@c2081forg.highwire.dtl.DTLVardef@a34b69_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

Atypical cell death in the growth plate chondrocytes of Tric-b-knockout mice

TRIC-A and TRIC-B proteins form homotrimeric cation-permeable channels in the sarco/endoplasmic reticulum (SR/ER) and nuclear membranes and are thought to contribute to counterionic flux coupled with store Ca2+ release in various cell types. Serious mutations in the TRIC-B locus cause autosomal recessive osteogenesis imperfecta (OI), which is characterized by insufficient bone mineralization. We have reported that Tric-b-knockout mice can be used as an OI model. Here we report irregular cell death in proliferating growth plate chondrocytes in developing Tric-b-knockout bones. In the knockout chondrocytes, excess pro-collagen fibers were occasionally accumulated in severely dilated ER elements. Of the major ER stress pathways, the PERK pathway was preferentially hyperactivated in the knockout chondrocytes, and most likely altered gene expression to induce apoptosis-related proteins including CHOP and caspase 12. In Ca2+ imaging experiments, the knockout chondrocytes exhibited aberrant Ca2+ handling; ER Ca2+ release was impaired, and intracellular Ca2+ concentration was elevated. Our data suggest that Tric-b deficiency directs growth plate chondrocytes to pro-apoptotic stages by compromising cellular Ca2+-handling and exacerbating ER stress, leading to atypical apoptotic cell death.

developmental biology↗

bioRxiv2022

CiDRE+ M2c macrophages hijacked by SARS-CoV-2 cause COVID-19 severity

Infection of the lungs with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) via the angiotensin I converting enzyme 2 (ACE2) receptor induces a type of systemic inflammation known as a cytokine storm. However, the precise mechanisms involved in severe coronavirus disease 2019 (COVID-19) pneumonia are unknown. Here, we show that interleukin-10 (IL-10) changed normal alveolar macrophages into ACE2-expressing M2c-type macrophages that functioned as spreading vectors for SARS-CoV-2 infection. The depletion of alveolar macrophages and blockade of IL-10 attenuated SARS-CoV-2 pathogenicity. Furthermore, genome-wide association and quantitative trait locus analyses identified novel mRNA transcripts in human patients, COVID-19 infectivity enhancing dual receptor (CiDRE), which has unique synergistic effects within the IL-10-ACE2 system in M2c-type macrophages. Our results demonstrate that alveolar macrophages stimulated by IL-10 are key players in severe COVID-19. Collectively, CiDRE expression levels are potential risk factors that predict COVID-19 severity, and CiDRE inhibitors might be useful as COVID-19 therapies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=195 HEIGHT=200 SRC="FIGDIR/small/510331v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1e96c82org.highwire.dtl.DTLVardef@1d2dc32org.highwire.dtl.DTLVardef@7689d1org.highwire.dtl.DTLVardef@520d17_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2022

ROR1 plays a critical role in pancreatic tumor-initiating cells with a partial EMT signature.

Tumor-initiating cells are the major drivers of chemoresistance and relapse, making them attractive targets for cancer therapy. However, the identity of tumor- initiating cells in human pancreatic ductal adenocarcinoma (PDAC) and the key molecules underlying their traits remain poorly understood. Here, we show that a partial epithelial-mesenchymal transition (EMT)-like subpopulation marked by high expression of receptor tyrosine kinase-like orphan receptor 1 (ROR1) is the origin of heterogeneous tumor cells in PDAC. We demonstrate that ROR1 depletion suppresses tumor growth, recurrence after chemotherapy, and metastasis. Mechanistically, ROR1 induces the expression of AURKB by activating E2F to enhance PDAC proliferation. Furthermore, epigenomic analyses reveal that ROR1 is transcriptionally dependent on YAP/BRD4 binding at the enhancer region, and targeting this pathway reduces ROR1 expression and prevents PDAC growth. Collectively, our findings reveal a critical role of ROR1high cells as tumor-initiating cells and the functional importance of ROR1 in PDAC progression, thereby highlighting its therapeutic targetability. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=192 HEIGHT=200 SRC="FIGDIR/small/499868v2_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@167bf72org.highwire.dtl.DTLVardef@49df52org.highwire.dtl.DTLVardef@1e480a3org.highwire.dtl.DTLVardef@b987bf_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2022

Hypoxic conditions promote Candida glabrata colonization in the intestinal tract and EPA6 plays a significant role in hypoxic adhesion to intestinal cells

Candida glabrata, a fungal pathogen colonizing mucocutaneous membranes and indwelling medical devices, is associated with invasive infections, especially in immunocompromised individuals. Candidiasis could be of endogenous and exogenous origins. Endogenous infections are considered to derive from the invasion of Candida species colonizing the digestive mucosa. Investigations of the gut-to-bloodstream translocation mechanisms of Candida species remain limited, although environmental oxygen levels have been recently suggested to alter the human fungal pathogen phenotypes. Moreover, human fungal pathogens, including Candida, colonizing or invading less oxygenated tissues encounter altered oxygen circumstances. Therefore, phenotype investigation under hypoxic conditions could provide valuable novel insights into the host-pathogen interaction mechanisms. This study aimed to elucidate the adhesion capabilities and mechanisms of C. glabrata depending on various oxygen levels. We performed C. glabrata adhesion assays to Caco-2 cells under aerobic, microaerobic (5 vol% oxygen), and anaerobic conditions, conducted RNA-seq to identify candidate genes functioning on hypoxic adhesion. We then generated deletants of these genes and evaluated both their adhesion to Caco-2 cells under anaerobic conditions and their colonization ability in the hypoxic intestinal tract in a mouse model. We observed significant differences in Caco-2 cell adhesion in response to different oxygen levels. Under hypoxic conditions, the C. glabrata adhesion capability increased and the expression levels of seven adhesion-related genes were up-regulated. Among these mutants, the adhesion capability of epa6{Delta} decreased the most. The epa6{Delta} mutant exhibited significantly lower intestinal colonization in mice than the wild-type. To the best of our knowledge, this study first describes the hypoxic adjustment of C. glabrata to intestinal cell adhesion, in which EPA6 plays the most significant role. If Epa6p function could be inhibited, it may contribute to reducing endogenous infection. Phenotype investigation under hypoxic conditions could provide valuable novel insights into the host-pathogen interaction mechanisms. Author SummaryCandida glabrata is the second most common pathogen of Candida infections (i.e., candidiasis), colonizing mucocutaneous membranes, indwelling medical devices, thereby causing bloodstream- and medical device-related infections and often leading to high morbidity and mortality. Candidiasis could be of endogenous and exogenous origins. Endogenous infections are considered to derive from the invasion of Candida species colonizing the digestive mucosa. Investigations of the gut-to-bloodstream colonizing and translocation mechanisms of Candida species remain limited. Interestingly, recent studies suggest that environmental oxygen levels could alter the human fungal pathogen phenotypes. This study thus focused on the relationship between the colonization and adhesion capability of C. glabrata in the gastrointestinal tract depending on the environmental oxygen level to address the underlying mechanisms. Our results suggest that anaerobic conditions promote C. glabrata adhesion and EPA6 plays a significant role in hypoxic adhesion, opening new perspectives in various affiliated fields and related research domains. If Epa6p function could be inhibited, it may contribute to control the colonization in the gut and following translocation. C. glabrata is known to be low-susceptible to azole antifungals. A novel antifungal agent type, such as one targeting these adhesive molecules, should thus be considered and further related studies would be necessary.

microbiology↗

bioRxiv2021

The SPICA Coarse-Grained Force Field for Proteins and Peptides

A coarse-grained (CG) model for peptides and proteins was developed as an extension of the SPICA (Surface Property fItting Coarse grAined) force field (FF). The model was designed to examine membrane proteins that are fully compatible with the lipid membranes of the SPICA FF. A preliminary version of this protein model was created using thermodynamic properties, including the surface tension and density in the SPICA (formerly called SDK) FF. In this study, we improved the CG protein model to facilitate molecular dynamics (MD) simulation with a reproduction of multiple properties from both experiments and all-atom (AA) simulations. The side chain analogs reproduced the transfer free energy profiles across the lipid membrane and demonstrated reasonable dimerization free energies in water compared to those from AA-MD. A series of peptides/proteins adsorbed or penetrated into the membrane simulated by the CG-MD correctly predicted the penetration depths and tilt angles of peripheral and transmembrane peptides/proteins comparable to those in the orientation of protein in membrane (OPM) database. In addition, the dimerization free energies of several transmembrane helices within a lipid bilayer were comparable to those from experimental estimation. Application studies on a series of membrane protein assemblies, scramblases, and poliovirus capsids demonstrated a good performance of the SPICA FF.

biophysics↗

bioRxiv2021

C-type natriuretic peptide facilitates autonomic Ca2+ entry in growth plate chondrocytes for stimulating bone growth

The growth plates are cartilage tissues found at both ends of developing bones, and vital proliferation and differentiation of growth plate chondrocytes are primarily responsible for bone growth. C-type natriuretic peptide (CNP) stimulates bone growth by activating natriuretic peptide receptor 2 (NPR2) which is equipped with guanylate cyclase on the cytoplasmic side, but its signaling pathway is unclear in growth plate chondrocytes. We previously reported that transient receptor potential melastatin-like 7 (TRPM7) channels mediate intermissive Ca2+ influx in growth plate chondrocytes, leading to activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) for promoting bone growth. In this report, we provide experimental evidence indicating a functional link between CNP and TRPM7 channels. Our pharmacological data suggest that CNP-evoked NPR2 activation elevates cellular cGMP content and stimulates big-conductance Ca2+-dependent K+ (BK) channels as a substrate for cGMP-dependent protein kinase (PKG). BK channel-induced hyperpolarization likely enhances the driving force of TRPM7-mediated Ca2+ entry and seems to accordingly activate CaMKII. Indeed, ex vivo organ culture analysis indicates that CNP-facilitated bone growth is abolished by chondrocyte-specific Trpm7 gene ablation. The defined CNP signaling pathway, the NPR2-PKG-BK channel-TRPM7 channel-CaMKII axis, likely pinpoints promising target proteins for developing new therapeutic treatments for divergent growth disorders.

cell biology↗

bioRxiv2021

Ecosystem size and complexity dictate riverine biodiversity

Larger ecosystems support more species; this ubiquitous pattern is the foundation of current conservation schemes. However, many ecosystems possess a complex spatial structure that cannot be represented by area, and the role of such complexity in scaling biodiversity is largely unknown. Here, we use theory and extensive fish community data from two distinct geographic regions (Japan and United States) to show that ecosystem size and complexity dictate riverine biodiversity. We found that larger and more branched complex river networks harbored greater species richness due to increased space and environmental heterogeneity. The complexity effect was comparable to the size effect, and this pattern has emerged regardless of ecological contexts. The dual control of biodiversity may be a pervasive feature that has far-reaching implications for biodiversity conservation. One sentence summaryThis study provides the first evidence that ecosystem size and complexity play comparable roles in regulating biodiversity.

ecology↗

bioRxiv2018

Cas9/CRISPR genome editing to demonstrate the contribution of Cyp51A Gly138Ser to azole resistance in Aspergillus fumigatus

Azole resistance in Aspergillus fumigatus is predominantly associated with increased expression of Cyp51A (lanosterol 14-demethylase), the target enzyme of azole antifungal agents, or with single-nucleotide polymorphisms (SNPs) in cyp51A. Although several SNPs that may be linked to low susceptibility in azole-resistant isolates have previously been reported, few studies have been conducted to conclusively demonstrate the contribution of SNPs to decreased azole susceptibility. An A. fumigatus strain was isolated from the sputum of a 74-year-old male receiving long-term voriconazole treatment for chronic progressive pulmonary aspergillosis. Etest antifungal susceptibility testing showed low susceptibility to voriconazole, itraconazole, and posaconazole. Nucleotide sequencing of cyp51A from this isolate revealed the mutations Gly138Ser (GGC[->]AGC) and Asn248Lys (AAT[->]AAA) compared with the cyp51A of azole-susceptible isolates. PCR-amplified DNA fragments containing cyp51A with or without the mutations of interest and a hygromycin marker were simultaneously introduced along with the Cas9 protein and in vitro-synthesized single-guide RNA into protoplasts of the azole-resistant/susceptible strains. Etest azole susceptibility testing of recombinant strains showed an increased susceptibility via the replacement of Ser138 by glycine. In contrast, azole susceptibility was slightly decreased when a Ser138 mutation was introduced into the azole-susceptible strain AfS35, indicating that the serine at position 138 of Cyp51A contributes to low susceptibility in the azole-resistant isolate. Genetic recombination, which has been hampered thus far in clinical isolates, can now be achieved using Cas9/CRISPR genome editing. This technique could be useful to investigate the contribution of other SNPs of cyp51A to azole resistance.

microbiology↗