Biology subjects

Tanaka, N.

Publications and source records attributed to Tanaka, N..

At least 19 recordsLinked to original sources

bioRxiv2026

Cooperative regulation of NF-E2 related factor 1 protein stability and transcriptional activation by endoplasmic reticulum-associated degradation system mediator, Selenoprotein S/K.

NRF1 is a key mediator of the proteasome recovery pathway, yet its regulation by ER-resident factors is not fully elucidated. Here, we demonstrate that selenoproteins SELS and SELK are critical regulators for NRF1 protein dynamics. SELS stabilizes NRF1, while SELK induces its insolubilization. Their deficiency leads to a hyper-accumulation and increased nuclear localization of NRF1 under proteasome inhibition condition. This results in an augmented transcriptional response of proteasome subunits. These results indicate that SELS and SELK cooperatively gate NRF1 activity by controlling its retrotranslocation and solubility, highlighting a novel layer of selenoprotein-mediated quality control in the proteostasis network.

biochemistry↗

bioRxiv2026

Regulation of sphingolipid synthesis by the C2H2 zinc finger transcription factor Com2 through ubiquitin-proteasome mediated degradation pathway

Membrane lipid synthesis is globally coordinated by a limited set of master transcription factors that regulate broad gene networks encoding lipid-metabolic enzymes and their regulators. Here, we identify the C2H2 zinc-finger transcription factor Com2 as a regulator of sphingolipid homeostasis in Saccharomyces cerevisiae that promotes transcription of downstream targets, including the protein kinase Ypk1, a key activator of sphingolipid synthesis. Com2 protein abundance increased upon treatment with myriocin, an inhibitor of sphingolipid synthesis, but rapidly decreased after addition of phytosphingosine (PHS), a precursor of complex sphingolipids; this decrease was blocked by proteasome inhibitors. These results suggest that Com2 is regulated in a sphingolipid-dependent manner through proteasome-mediated degradation. Moreover, a Com2 mutant in which lysine residues putatively involved in ubiquitination were replaced with arginine exhibited attenuated PHS-dependent degradation and elevated phosphorylation. Likewise, a mutant in which putative phosphorylation sites were replaced with alanine showed reduced PHS-dependent degradation. Together, these findings indicate that Com2 undergoes phosphorylation-dependent degradation via the ubiquitin-proteasome system in response to sphingolipid levels.

cell biology↗

bioRxiv2025

Multi-omics profiling with indoor-unmanned phenotyping reveals drought adaptation through constitutive ABF1 expression in wild rice

Improving drought resistance is crucial for stable crop production under climate change. Identifying the mechanisms of drought resistance using diverse genetic resources, including crop wild relatives, would be beneficial for molecular breeding. Here, we developed an indoor, unmanned phenotyping platform that can noninvasively and automatically collect temporal data on plant responses to drought stress. Using this system, we analyzed the phenotypic, transcriptomic, and environmental data of four cultivated rice varieties and five wild relatives. Multi-omics analysis revealed that one wild rice species exhibited drought adaptation through the constitutive expression of ABSCISIC ACID RESPONSIVE ELEMENT-BINDING FACTOR 1 (ABF1), which encodes a transcription factor that regulates drought resistance, before drought stress. Drought testing of introgression lines of cultivated rice with constitutive ABF1 expression revealed higher drought tolerance than in cultivars without a growth penalty. Our findings suggest that constitutive ABF1 expression contributes to drought adaptation in both cultivated and wild rice.

plant biology↗

bioRxiv2025

NOTCH1 Acts as a Tumor Suppressor That Induces Early Differentiation in Head and Neck Cancer

We identified frequent inactivating notch1 mutations in HNSCC over a decade ago, indicating its role as a tumor suppressor--unlike its oncogenic function in leukemias and salivary gland tumors. However, there has been much debate in the literature regarding a possible oncogenic role in HNSCC as well, based on reports that notch1 signaling drives tumor growth and a cancer stem cell phenotype in some HNSCC tumor lines and patient samples. Clarifying whether NOTCH1 occasionally functions as an oncogenic driver in HNSCC is crucial to the prognosis and personalized therapy of patients with either wild-type or mutated NOTCH1. Here we present a systematic and comprehensive investigation unequivocally demonstrating that notch1 signaling functions as a tumor suppressor in HNSCC regardless of mutation or activation status and leads to reduction in frequency of cancer stem cells. We develop a robust gene expression signature of notch1 activation based on experimental data that when applied to patient samples shows notch1 signaling is associated with very early differentiation, an altered tumor microenvironment, and better prognosis-- consistent with a tumor suppressive role. Our work unifies the field by reconciling conflicting data and providing critical insights into the biological and clinical significance of the NOTCH1 pathway in HNSCC.

cancer biology↗

bioRxiv2025

DNA Specimen Preservation using DESS and DNA Extraction in Museum Collections: A Case Study Report

Recent advances in DNA research have increased the necessity for museums to preserve not only morphological specimens but also their DNA, leading us to maintain tissue samples linked to specimens at -80{degrees}C. DNA analysis has become an essential tool for taxonomic research and biodiversity assessment; however, freezer storage for all samples is impractical due to space limitations and operational costs. This creates a pressing need to develop more widely applicable DNA preservation methods. We investigated the comparative effects of traditional preservation methods versus DESS (DMSO/EDTA/saturated NaCl solution) preservation on both morphology and DNA integrity using museum specimens from various taxonomic groups. Our results demonstrated that DESS preservation maintained high-quality DNA fragments exceeding >15 kb at room temperature across all examined species, with nematode samples maintaining DNA integrity even after 10 years of storage. When preserving whole organisms, the optimal preservation solution conditions for maintaining both morphological features and DNA integrity varied among species. Notably, DNA integrity was maintained even after complete evaporation of the DESS solution. These findings suggest that DESS utilization for specimen DNA preservation is effective across many species, not only for long-term storage in environments without freezer facilities but also for temporary preservation until freezing. Simple SummaryRecent advances in DNA research have increased the necessity for museums to preserve not only morphological specimens but also their DNA, leading us to maintain tissue samples linked to specimens at -80{degrees}C. However, as freezer storage for all samples is impractical, there is a pressing need to develop more widely applicable DNA preservation methods. In this study, we investigated the effectiveness of traditional preservation methods versus DESS (DMSO/EDTA/saturated NaCl solution) preservation with regards to both morphology and DNA integrity using our museum specimens. Our results demonstrated that DESS preservation maintained high molecular weight DNA at room temperature across all examined species. When preserving whole organisms, the optimal preservation solution conditions for maintaining both morphological features and DNA integrity varied among species. Notably, DNA integrity was maintained even after complete evaporation of the DESS solution. These findings suggest that DESS utilization for specimen DNA preservation is effective across many species, not only for long-term storage in environments without freezer facilities but also for temporary preservation until freezing is possible.

zoology↗

bioRxiv2024

Predicted constrained accessible regions mark regulatory elements and causal variants

Open chromatin regions (OCRs) define cell-type-specific regulatory elements across the genome, yet their functional significance varies, making it challenging to pinpoint biologically essential regions. Here, we introduce CAMBUS (Chromatin Accessibility Mutation Burden Score), a machine-learning framework that identifies active and evolutionarily constrained OCRs by leveraging surrounding DNA sequences. Applying CAMBUS to 29 immune cell types, we identified 66,043 constrained OCRs, which were substantially enriched in the known constraint genome (odds ratio=11.45 (95% confidence interval 9.33-14.05), P=4.7 x 10-68), while 90% of these OCRs were not prioritized by existing constraint metrics. These OCRs were highly enriched for known enhancers and super-enhancers, independent of known epigenetic markers and annotated regions, and overlapped with regulatory elements implicated in immune-mediated diseases and experimentally validated functional variants, including rare variants, particularly in leukocyte-related traits. Furthermore, CAMBUS revealed cell-type-specific transcriptional regulatory landscapes, linking genetic constraint with gene regulation in immune cells and identifying plausible connections between 1,533 causal variants and 70 complex traits. By defining biologically constrained regulatory elements at high resolution, CAMBUS provides a framework for understanding the selective pressures shaping the non-coding genome and its role in human health and disease.

genetics↗

bioRxiv2024

Giant extrachromosomal element "Inocle" potentially expands the adaptive capacity of the human oral microbiome

Survival strategy of bacteria is expanded by extrachromosomal elements (ECEs). However, their genetic diversity and functional roles for adaptability are largely unknown. Here, we discovered a novel family of intracellular ECEs using 56 saliva samples by developing an efficient microbial DNA extraction method coupled with long-read metagenomics assembly. Even though this ECE family was not hitherto unidentified, our global prevalence analysis using 476 salivary metagenomic datasets elucidated that these ECEs reside in 74% of the population. These ECEs, which we named, "Inocles", are giant plasmid-like circular genomic elements of 395 kb in length, having Streptococcus as a host bacterium. Inocles encode a series of genes that contribute to intracellular stress tolerance, such as oxidative stress and DNA damage, and cell wall biosynthesis and modification involved in the interactions with oral epithelial cells. Moreover, Inocles exhibited significant positive correlations with immune cells and proteins responding to microbial infection in peripheral blood. Intriguingly, we examined and found their marked reductions among 68 patients of head and neck cancers and colorectal cancers, suggesting its potential usage for a novel biomarker of gastrointestinal cancers. Our results suggest that Inocles potentially boost the adaptive capacity of host bacteria against various stressors in the oral environment.

genomics↗

bioRxiv2024

A beta-Galactosidase acting on unique galactosides: the structure and function of a beta-1,2-galactosidase from Bacteroides xylanisolvens, an intestinal bacterium

Galactosides are major carbohydrates that are found in plant cell walls and various prebiotic oligosaccharides. Studying the detailed biochemical functions of {beta}-galactosidases in degrading these carbohydrates is important. In particular, identifying {beta}-galactosidases with new substrate specificities could help in the production of potentially beneficial oligosaccharides. In this study, we identified a {beta}-galactosidase with novel substrate specificity from Bacteroides xylanisolvens, an intestinal bacterium. The enzyme did not show hydrolytic activity toward natural {beta}-galactosides during the first screening. However, when -D-galactosyl fluoride (-GalF) as a donor substrate and galactose or D-fucose as an acceptor substrate were incubated with a nucleophile mutant, reaction products were detected. The galactobiose produced from the -GalF and galactose was identified as {beta}-1,2-galactobiose using NMR. Kinetic analysis revealed that this enzyme effectively hydrolyzed {beta}-1,2-galactobiose and {beta}-1,2-galactotriose. In the complex structure with methyl {beta}-galactopyranose as a ligand, the ligand is only located at subsite +1. The 2-hydroxy group and the anomeric methyl group of methyl {beta}-galactopyranose faces in the direction of subsite -1 and the solvent, respectively. This observation is consistent with the substrate specificity of the enzyme regarding linkage position and chain length. Overall, we concluded that the enzyme is a {beta}-galactosidase acting on {beta}-1,2-galactooligosaccharides. SynopsisThe structural and functional analysis of {beta}-galactosidase from an intestinal bacterium led to the discovery of a new {beta}-galactosidase hydrolyzing unique {beta}-1,2-galactooligosaccharides.

biochemistry↗

bioRxiv2024

Phosphodiesterase-5 inhibition inhibits epithelial ATP release and restores detrusor contractility in rats with type 2 diabetes via an increase in bladder blood flow

PurposeThe bladder dysfunction associated with type 2 diabetes mellitus (T2DM) involves urine storage and voiding disorders. We evaluated the pathologic conditions of bladder wall in a rat model of T2DM and evaluated the effects of the phosphodiesterase-5 (PDE-5) inhibitor tadalafil (TA). Materials and MethodsMale Otsuka Long-Evans Tokushima Fatty (OLETF) rats and Long-Evans Tokushima Otsuka (LETO) rats comprised T2DM and control groups. TA was orally administered for 12 weeks. The bladder blood flow and ATP released from the bladder epithelium were measured using laser speckle imaging and an organ bath bladder distention test. The expression levels of markers of hypoxia, pro-inflammatory cytokines, and growth factors in the bladder wall were measured by real-time PCR and ELISA. The contractions of bladder strips in response to KCl and carbachol were monitored in OLETF rats. ResultsThe bladder blood flow was impaired and there was greater ATP release and vesicular nucleotide transporter (VNUT) expression in the OLETF rats than in the LETO rats, but these effects were suppressed by TA administration. Furthermore, the high expression of HIF-1, 8-OHdG, IL-6, TNF-, IGF-1, and bFGF in the OLETF rats was reduced by TA administration. In the OLETF rats, the contractile responses of bladder strips to KCl and carbachol were impaired, but were restored by TA administration. ConclusionsThe impairment of bladder blood flow in rats with T2DM is associated with greater ATP release and the upregulation of VNUT, markers of hypoxia, proinflammatory cytokines, and growth factors in the bladder epithelium. PDE5 inhibition has the potential to prevent the storage and voiding dysfunction associated with T2DM.

pharmacology and toxicology↗

bioRxiv2024

Effective Gene Therapy for Metachromatic Leukodystrophy Achieved with Minimal Lentiviral Genomic Integrations

Metachromatic leukodystrophy (MLD) is a fatal lysosomal storage disease (LSD) characterized by the deficient enzymatic activity of arylsulfatase A (ARSA). Combined autologous hematopoietic stem cell transplant (HSCT) with lentiviral (LV) based gene therapy has great potential to treat MLD. However, if enzyme production is inadequate, this could result in continued loss of motor function, implying a high vector copy number (VCN) requirement for optimal enzymatic output. This may place children at increased risk for genomic toxicity due to higher VCN. We increased the expression of ARSA cDNA at single integration by generating novel LVs, optimizing ARSA expression, and enhancing safety. In addition, our vectors achieved optimal transduction in mouse and human HSC with minimal multiplicity of infection (MOI). Our top-performing vector (EA1) showed at least 4X more ARSA activity than the currently EU-approved vector and a superior ability to secrete vesicle-associated ARSA, a critical modality to transfer functional enzymes from microglia to oligodendrocytes. Three-month-old Arsa-KO MLD mice transplanted with Arsa-KO BM cells transduced with 0.6 VCN of EA1 demonstrated behavior and CNS histology matching WT mice. Our novel vector boosts efficacy while improving safety as a robust approach for treating early symptomatic MLD patients.

molecular biology↗

bioRxiv2024

Beta-Glucanase superfamily identified by sequential, functional, and structural analyses

{beta}-1,2-Glucans are natural glucose polymers that play important physiological roles, including as symbiotic or pathogenic factors and in osmoregulation. Phylogenetically new glycoside hydrolase (GH) families have recently been identified from {beta}-1,2-glucanase (SGL) sequences from bacteria (GH144) and a fungus (GH162). In this study, we identified four phylogenetically new groups (Groups 1-4), and determined that these families, together with GH144, GH162, and GH189, a family of transglycosylase domains in cyclic {beta}-1,2-glucan synthases, form a superfamily. Biochemical analysis of six proteins in these groups revealed that the proteins in Groups 1-3 showed hydrolytic activity specific to {beta}-1,2-glucan. The kinetic parameters of the enzymes of Groups 1-3 were similar to GH144 and GH162 SGLs, indicating that these enzymes were SGLs. Optical rotation analysis revealed that the SGLs followed an anomer-inverting mechanism. Structural analysis and prediction of the proteins in Groups 1-4, GH144, GH162, and GH189 suggested that Groups 1-3 and GH144 had the same reaction mechanism. Nevertheless, Groups 1-3 were dispersed irregularly in the superfamily. Overall, we determined that Groups 1-3 were new GH families, GHxxx, GHyyy, and GHzzz, respectively, and proposed that this superfamily be called an SGL superfamily because of the phylogenetical, functional, and structural relationships within the superfamily. HighlightsWide variety of glycoside hydrolases is far beyond our understanding. Functional and structural analysis identified three new glycoside hydrolase families. Molecular evolution with irregular changes in reaction mechanism was revealed.

biochemistry↗

bioRxiv2023

Functional and structural analysis of a cyclization domain in a cyclic beta-1,2-glucan synthase

Cyclic {beta}-1,2-glucan synthase (CGS) is a key enzyme in production of cyclic {beta}-1,2-glucans (C{beta}Gs) which are involved in bacterial infection or symbiosis to host organisms. Nevertheless, a mechanism of cyclization, the final step in the CGS reaction, has not been fully understood. Here we performed functional and structural analyses of the cyclization domain of CGS alone from Thermoanaerobacter italicus (TiCGSCy). We first found that {beta}-glucosidase-resistant compounds are produced by TiCGSCy with linear {beta}-1,2-glucans as substrates. The 1H-NMR analysis revealed that these products are C{beta}Gs. Next, action pattern analyses using {beta}-1,2-glucooligosaccharides revealed a unique reaction pattern: exclusive transglycosylation without hydrolysis and a hexasaccharide as the minimum length of the substrate. Nevertheless, longer substrate {beta}-1,2-glucooligosaccharides are preferred, being consistent with the fact that CGSs generally produce C{beta}Gs with degrees of polymerization of around 20. Finally, the overall structure of the cyclization domain of TiCGSCy was found to be similar to those of {beta}-1,2-glucanases in phylogenetically different groups. Meanwhile, the identified catalytic residues indicated clear difference in the reaction pathways between these enzymes. Therefore, we propose a novel reaction mechanism of TiCGSCy. Thus, we propose that the present group of CGSs should be defined as a new glycoside hydrolase family, GHxxx. Statements and DeclarationsCompeting Interests: The authors declare no competing interests. Key PointsIt was clearly evidenced that cyclization domain alone produces cyclic {beta}-1,2-glucans. The domain exclusively catalyzes transglycosylation without hydrolysis. The present catalytic domain should be defined as a new glycoside hydrolase family.

biochemistry↗

bioRxiv2023

Essential role of an acidic endosomal environment in JAK3-mediated signal transduction

The endosome, an acidic organelle, is a platform for endocytic trafficking and signal transduction. Chloroquine, an intercellular acidic vesicle neutralizer (IAVN), has been used to treat autoimmune diseases. Its immunosuppressive mechanism remains unclear, although several modes of action attributable to lysosomal dysfunction have been postulated. This study found that IAVNs, including chloroquine, can selectively interfere with common cytokine receptor {gamma} chain ({gamma}c) cytokine-induced signal transduction. Our data show that JAK3 targets endosomal membranes in an acidic pH-dependent manner, which is essential for {gamma}c-cytokine-induced functional signal complex formation and signal activation. Among IAVNs, Monensin and similar ionophores were particularly effective in suppressing {gamma}c-cytokine-induced cell proliferation in vitro. Monensin exerted therapeutic effects in a collagen-induced arthritis mouse model by interfering with T helper 17 cells. Our findings help further understand the immunosuppressive mechanism of IAVNs and unveil the spatial regulation of JAK3-mediated signal transduction via endosomes.

immunology↗

bioRxiv2023

Thrombospondin 2 is a key determinant of fibrogenesis in NAFLD

Hepatic overexpression of the thrombospondin 2 gene (THBS2) and elevated levels of circulating thrombospondin 2 (TSP2) have been observed in patients with chronic liver disease. The current study aimed to identify the specific cells expressing THBS2/TSP2 in non-alcoholic fatty liver disease (NAFLD) and investigate the underlying mechanism behind THBS2/TSP2 up-regulation. Comprehensive NAFLD liver gene datasets, including single-cell RNA sequencing (scRNA-seq), in-house NAFLD liver tissue, and LX-2 cells derived from human hepatic stellate cells (HSCs), were analyzed using a combination of computational biology, genetic, immunological, and pharmacological approaches. Analysis of the genetic dataset revealed the presence of 1433 variable genes in patients with advanced fibrosis NAFLD, with THBS2 ranked among the top 2 genes. Quantitative polymerase chain reaction examination of NAFLD livers showed a significant correlation between THBS2 expression and fibrosis stage (r=0.349, p<0.001). In support of this, scRNA-seq data and in situ hybridization demonstrated that the THBS2 gene was highly expressed in HSCs of NAFLD patients with advanced fibrosis. Pathway analysis of the gene dataset revealed THBS2 expression to be associated with the transforming growth factor beta (TGF{beta}) pathway and collagen gene activation. Moreover, the activation of LX-2 cells with TGF{beta} increased THBS2/TSP2 and collagen expression independently of the TGF{beta}-SMAD2/3 pathway. THBS2 gene knockdown significantly decreased collagen expression in LX-2 cells. In conclusion, THBS2/TSP2 is highly expressed in HSCs and plays a role in regulating fibrogenesis in NAFLD patients. THBS2/TSP2 may therefore represent a potential target for anti-fibrotic therapy in NAFLD. (241 words) One-sentence summariesThrombospondin 2 represent a potential target for anti-fibrotic therapy in NAFLD.

molecular biology↗

bioRxiv2023

The stability of NPM1 oligomers regulated by acidic disordered regions controls the quality of liquid droplets

A nucleolus is a typical membrane-less nuclear body that is formed by liquid-liquid phase separation (LLPS) of its components. A major component that drives LLPS in the nucleolus is nucleophosmin (NPM1). The oligomer formation and inter-oligomer interactions of NPM1 are suggested to cooperatively contribute to the induction of LLPS. However, the molecular mechanism of how the quality of the liquid droplets formed by NPM1 is regulated is currently not well understood. In this manuscript, we revealed the regulatory mechanism of NPM1 oligomer formation and its relationship with the ability to form liquid droplets. Molecular dynamics simulations and mutant protein analyses suggest that the acidic amino acids in the N-terminal and central disordered regions of NPM1 disturb the key interactions between monomers. We also demonstrate that mutants with attenuated oligomer stability form liquid droplets as do the wild-type; the fluidity of the formed liquid droplets was greater than that of the wild-type. These results suggest that the stability of NPM1 oligomers is a critical determinant of liquid droplet quality. Furthermore, we observed that when the net negative charges in the acidic disordered regions were increased by phosphomimetic mutations at Ser125, the NPM1 oligomer stability decreased, which increased the fluidity of the liquid droplets. Our results provide a novel mechanistic insight into how nucleolar dynamics are regulated during the cell cycle.

biochemistry↗

bioRxiv2021

Transcriptional regulation of sphingolipid metabolism in budding yeast

Global control for the synthesis of lipids constituting a bilayer of cell membranes is known to be with a small number of transcription factors called master transcriptional regulators, which target a wide range of genes encoding lipid metabolism enzymes and/or their regulators. Although master transcriptional regulators of glycerophospholipids and sterols have been identified in both yeast and mammals, this aspect of sphingolipid metabolism is not yet understood. In the present study, we identified the C2H2-type zinc finger transcription factor, Com2, as a master transcriptional regulator of sphingolipid metabolism in the budding yeast, Saccharomyces cerevisiae. The target of rapamycin complex 2 (TORC2)-activated protein kinase Ypk1 is known to regulate sphingolipid metabolism. Activated Ypk1 stimulates the activity of serine palmitoyl transferase (SPT), the first-step enzyme in sphingolipid biosynthesis, by phosphorylating and inhibiting Orm1/2, a negative regulator of SPT. This regulation of SPT activity is thought to be a major pathway in the regulation of sphingolipid metabolism. In the present study, we found that inhibition of sphingolipid synthesis upregulates the expression of Com2, which in turn leads to the concomitant expression of Ypk1. The upregulation of Ypk1 expression was found to be dependent on a putative Com2-binding site in the YPK1 promoter. Our results also suggested that Com2 senses intracellular sphingolipid levels through a pathway independent of TORC2-Ypk1-mediated sensing of sphingolipids. Our results revealed an additional layer of mechanistic regulation that allows cells to maintain appropriate levels of sphingolipid biosynthesis and to rapidly induce this process in response to environmental stresses. Significance StatementOne of the major regulatory mechanisms involved in the control of lipid metabolism in bilayers of biological membranes is regulation at the transcriptional level by master transcriptional regulators that control the transcription of genes encoding lipid metabolism enzymes and/or their regulators. In the present study, we identified the C2H2-type zinc finger transcription factor Com2 as a master transcriptional regulator in sphingolipid metabolism. We found that Com2 regulates sphingolipid metabolism by transcriptionally controlling the expression of Ypk1, which regulates Orm1/2, a negative regulator of serine palmitoyl transferase, the first-step enzyme in sphingolipid biosynthesis, through phosphorylation. Our study revealed a new layer of regulation that allows the maintenance of an appropriate level of sphingolipid biosynthesis for a rapid response to environmental stresses.

cell biology↗

bioRxiv2021

Developing a Biomimetic Evaluation Method for Antiviral Coatings Using Artificial Saliva Droplets.

Respiratory infections pose a serious threat worldwide, and many new antiviral agents and coatings have been developed to reduce the overall risk of viral infection. Here, we evaluate the methodology used to test these antiviral coatings and developed a novel system that is more similar to "real-world" conditions. Contact infection is largely mediated via contact with saliva containing the active virus released as droplets by coughing or sneezing, with these droplets adhering to objects and surfaces and subsequently entering the human body via indirect contact with the mucous membranes. Here, we evaluated the antiviral effect of a known antiviral coating agent using an artificial saliva based system, where artificial saliva containing phages were sprayed onto the antiviral coating under various conditions associated with viral replication and infectious spread. We used a commercially available antiviral coating in this evaluation, and M13 bacteriophages as model viruses. This method enables simple biomimetic evaluations of any products antiviral effects.

bioengineering↗

bioRxiv2021

CZON-cutter: a CRISPR-Cas9 system with multiplexed organelle imaging in a simple unicellular alga

The simple cellular structure of the unicellular alga Cyanidioschyzon merolae consists of one nucleus, one mitochondrion, one chloroplast, and one peroxisome per cell and offers unique advantages to investigate mechanisms of organellar proliferation and the cell cycle. Here, we describe an engineered clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system, CZON-cutter, for simultaneous genome editing and organellar visualization. We engineered a C. merolae strain expressing a nuclear-localized Cas9-Venus nuclease to target editing at a locus defined by a single-guide RNA (sgRNA). We then successfully edited the algal genome and visualized the mitochondrion and peroxisome in transformants by fluorescent protein reporters with different excitation wavelengths. Fluorescent protein labeling of organelles in living transformants allows validation of phenotypes associated with organellar proliferation and the cell cycle, even when the edited gene is essential. Combined with the exceptional biological features of C. merolae, CZON-cutter will be instrumental for investigating cellular and organellar division in a high-throughput manner. SummaryAn engineered CRISPR-Cas9 system, named CZON-cutter, for simultaneous genome editing and fluorescent protein labeling of organelles in Cyanidioschyzon merolae can be used to validate intracellular function of a particular gene, even if it is essential.

molecular biology↗