Biology subjects

Hirota, K.

Publications and source records attributed to Hirota, K..

At least 19 recordsLinked to original sources

bioRxiv2026

Nautilus shell morphomics reveals microstructural heterogeneity alongside structural continuity across component boundaries

The nautilus (Nautilus sp.) is an early-branching cephalopods. It retains several conchiferan synapomorphies, including an external planispiral biomineralized shell. The shells complex structure allows it to withstand hydrostatic pressure, control buoyancy, and protect against external hazards. In this study, we comprehensively examined shell microstructures across different shell components and regions representing various ontogenetic stages in two adult museum shell specimens. We found that the nautilus shell is composed of five microstructural types (spherulitic, prismatic, nacreous, semi-prismatic, and irregularly oriented prismatic structures) organized into layered architectures within individual shell components and coordinated across the shell as an integrated system. Our observations highlight transitions between distinct microstructures within and across shell components and local variation within individual components such as the dorsal and ventral shell walls, suggesting that these patterns may contribute to shell strength and overall mechanical performance. Variation in caecum morphology suggests that this structure may be developmentally plastic and subject to relatively relaxed structural constraints. These findings show that the Nautilus shell is an integrated biomineral system in which diverse microstructures are organized across shell components to meet functional demands and provide the mechanical strength needed for survival.

evolutionary biology↗

bioRxiv2026

BRCA2 loss triggers a downward spiral of genomic instability via ROS-dependent metabolic collapse

BRCA2 plays a central role in maintaining genome integrity through homologous recombination and replication-fork protection, yet the compensatory networks sustaining BRCA2-deficient cells remain unclear. Here we show BRCA2 enforces a homeostatic mechanism aligning mitochondrial respiration with DNA repair capacity in both cancer and non-cancer contexts. Genome-wide CRISPR screening identified glutathione metabolism and base-excision repair as the key compensatory networks sustaining BRCA2-deficient cells by detoxifying mitochondria-derived reactive oxygen species. BRCA2 loss provokes an acute mitochondrial ROS surge, causing 8-oxoguanine accumulation and a systemic metabolic crisis marked by NAD+ and glutathione depletion. PARP inhibitor targets DNA replication vulnerabilities, increasing the cellular requirement for BRCA2. The resulting oxidative burden primes cells for TP53-dependent apoptosis in G1 during olaparib treatment, which extends cytotoxicity beyond canonical S-phase stress. These findings indicate BRCA2 prevents metabolic flux from outpacing repair capacity, providing a rationale for combining PARP inhibition with redox modulation to enhance efficacy and overcome resistance. HighlightsO_LIAcute BRCA2 loss induces ROS and mitochondrial dysfunction creating a metabolic scar C_LIO_LIOxidative lesions drive PARP hyperactivation and precipitate a cellular NAD crisis C_LIO_LIPARP inhibitors provoke TP53-dependent apoptosis in G1 beyond replication stress in S phase C_LIO_LIGlutathione deficiency exacerbates bone marrow failure under BRCA2 depletion C_LIO_LIBRCA2 tightly couples mitochondrial redox homeostasis to genomic maintenance C_LI

cell biology↗

bioRxiv2026

Benchmarking long-context genome language models on biosynthetic gene clusters

Recent advances in language models for natural language processing have spread to the field of genomics, driving the development of genome language models (gLMs) to decipher genomic information. Cutting-edge long-context gLMs are promising approaches for understanding and designing biological complexity, but their evaluation remains underdeveloped. In this study, we introduce BGCs-Bench, a unified benchmark focused on biosynthetic gene clusters for assessing long-range genomic modeling on three downstream tasks: biosynthetic class prediction, taxonomic classification and coding sequence annotation. Using BGCs-Bench, we perform systematic and layer-wise evaluations of the embedding representations of long-context gLMs, demonstrating that layer selection is crucial for downstream task performance. In addition to the evaluation results, the logit lens analysis of autoregressive gLMs suggests that StripedHyena-based models consist of earlier layers to encode biologically meaningful information from input DNA sequences and deeper layers to optimize embeddings for sequence generation. These findings provide insights for more effective development and application of long-context gLMs.

bioinformatics↗

bioRxiv2025

PDS5 proteins control genome architecture by limiting the lifetime of cohesin-NIPBL complexes

Cohesin-NIPBL complexes extrude genomic DNA into loops that are constrained by CTCF boundaries. This process has important regulatory functions and weakens the separation between euchromatic and heterochromatic compartments. Cohesin can also bind PDS5A or PDS5B, which do not support loop extrusion but are required for the formation of CTCF boundaries. How PDS5 proteins perform this function is unknown. Here we show by in vitro single-molecule imaging that PDS5 proteins stop loop extrusion by facilitating the dissociation of NIPBL from cohesin. Hi-C experiments suggest that this function is required for the establishment of CTCF boundaries in cells. In silico modelling indicates that PDS5 proteins enable the separation between compartments by limiting cohesins velocity and chromatin-residence time. The degree of this compartmentalization depends on the frequency with which chromatin is extruded relative to the time it takes for compartments to form. These results identify PDS5 proteins as key regulators of genome organization. HighlightsO_LIPDS5 proteins stop loop extrusion by facilitating dissociation of NIPBL from cohesin. C_LIO_LIPDS5 proteins strengthen CTCF boundaries by limiting the lifetime of cohesin-NIPBL. C_LIO_LIPDS5 proteins regulate compartmentalization. C_LIO_LICompartmentalization is governed by polymer relaxation and loop extrusion dynamics. C_LI

molecular biology↗

bioRxiv2025

DeepRES: Deep learning enables reaction-based comprehensive enzyme screening

BackgroundEnzymes accelerate biochemical reactions in living organisms, thus playing an important role in metabolism. Although metabolic pathway databases are growing, many metabolic reactions, termed orphan enzymes, have not been annotated to gene sequences, which hinders functional annotation in genomic analysis. Moreover, protein databases contain many proteins of unknown function. Owing to this gap between known proteins and enzymatic reactions, various proteins of unknown function may be orphan enzymes; however, available tools cannot adequately predict these links. ResultsIn this study, we developed DeepRES, an AI-based framework for comprehensive enzyme screening, to explore novel enzyme candidates from proteins of unknown function for reactions of interest. DeepRES implements enzyme screening via two steps: classification of enzymes and non-enzymes and prediction of catalytic capabilities for enzyme-reaction pairs. The two deep learning models comprising DeepRES showed comparable or superior performance to that of existing software. We performed screening of 1,255 orphan enzymes involved in the microbiome using DeepRES and successfully identified candidate proteins for 897 orphan enzymes. We then used those candidates as references for genomic analysis and explored novel biosynthetic gene clusters from microbial genomes to obtain promising candidate gene clusters, including those related to anthocyanin degradation. ConclusionsComprehensive enzyme screening via DeepRES, which is the first computational tool designed to associate orphan enzymes with proteins of unknown function, is expected to facilitate high-throughput identification of orphan enzyme-encoding genes. Furthermore, DeepRES can be easily integrated into the current genomic analysis pipeline to extend the functional annotation.

bioinformatics↗

bioRxiv2025

Microstructural architecture of the argonauts' shell-like eggcase reveals the formation logic of a convergent extended phenotype

Argonautid octopods of the genus Argonauta possess a shell-like biomineralized external structure called an eggcase. A classical behavioral observation suggested that this structure is produced by the first dorsal arms rather than by the mantle tissue as seen in typical molluscan shells. In this study, we performed detailed microstructural analyses using scanning electron microscopy to investigate the characteristics of normal, undamaged eggcases and regions that had undergone post-damage repair. Our analysis revealed that the eggcase comprises five layers: an outermost organic membrane, an outer spherulitic-fibrous prismatic layer, a middle organic layer, an inner spherulitic-fibrous prismatic layer, and an innermost organic membrane. Both prismatic layers exhibit bidirectional growth from the middle organic layer, a unique feature not observed in typical molluscan shells. We propose a four-stage formation sequence to explain the observed microstructural architecture: nucleation on the organic scaffold, bidirectional crystal growth from the organic mid-layer, perpendicular growth relative to the eggcase surface, and final encapsulation by an organic membrane. Interestingly, the growth pattern and crystal shapes in the prismatic layers resemble of the internalized shells of cuttlefish, the calcified skeleton of stony corals, and avian eggshells, suggesting functional convergence. We also identified two repair mechanisms: reattachment of broken fragments and regeneration via new secretions. These findings call into question the previous assumptions about the role of the first dorsal arms in calcification. The eggcase also exemplifies the formation of a functionally and structurally complex extended phenotype through convergence.

zoology↗

bioRxiv2025

Chromosomal fusions and subsequent rearrangements shaped octopus genomes

Why some animal groups retain ancestral chromosomal complements while others change significantly is a fundamental question in evolutionary genomics. Few systems exist where accumulations of chromosomal changes can be studied in the context of morphological innovation. In coleoid cephalopods (octopus, squid, cuttlefish), an ancient coleoid chromosomal rearrangement event (ACCRE) has led to a substantial increase in the chromosome number and a new set of chromosomal homologies. Compared to typical molluscan or bilaterian genomes, ACCRE has enabled the origin of many novel regulatory regions in coleoid cephalopods. However, the discrepancies between extant octopodiform (octopus, [~]30 chromosomes) and decapodiform (squid and cuttlefish, [~]46 chromosomes) karyotypes and the direction of these evolutionary changes remain unexplained. Here we provide a draft genome assembly of the vampire squid Vampyroteuthis sp., the largest cephalopod genome sequenced to-date (over 10 gigabasepairs). Through syntenic comparisons, we infer that this basally branching octopodiform species shows partial retention of the chromosomal complement of Decapodiformes, indicating its more ancestral state and the derived nature of the octopod karyotype. Together with the analysis of a new chromosome-level assembly of the pelagic octopod Argonauta hians, we identified irreversible chromosomal fusion-with-mixing events followed by inter-chromosomal translocations in octopods. We show that this secondary reduction and mixing within octopod chromosomes has enabled the origin of a more entangled genomic configuration, shedding light onto the early evolutionary transitions within the clade. Our results offer broader insights into general patterns of chromosomal evolution following large-scale rearrangement events in animal genomes. Significance statementHow changes to the ancient animal synteny result in novel chromosomal homologies is difficult to dissect due to the lack of intermediate states. Here we report that the genome of Vampyroteuthis, one of the largest animal genomes sequenced to-date (over 11 gigabasepairs), despite its phylogenetic position within the octopodiform cephalopods, partially retains squid and cuttlefish chromosomal complement, reflecting an ancestral karyotypic state that existed at the time of divergence between these cephalopod lineages. Our findings reveal karyotype reductions through chromosomal fusions were followed by inter-chromosomal translocations in octopods, leading to a more specialized genomic and gene regulatory architecture. These data show how chromosomal fusions can act as drivers of further inter-chromosomal rearrangements in animal genomes.

genomics↗

bioRxiv2025

Quantitative analysis of the frequency of chromosome loss following DSB induction

Numerical abnormalities in chromosomal states, referred to as aneuploidy, is commonly observed in many cancer cells. Although numerous internal and external factors induce aneuploidy, the primary cause of aneuploidy in humans remains unclear. DNA damage is identified as a potential cause of aneuploidy by inducing chromosome segregation errors. However, a direct relationship between DNA damage and aneuploidy remains poorly understood. A major reason for this is the extremely low frequency of aneuploidy in cultured cells, making quantitative analyses challenging. In this study, we investigated the relationship between DNA damage and aneuploidy in cell lines containing minichromosomes. These chromosomes are more prone to loss than normal chromosomes, with the rate of loss substantially increased following exposure to various DNA-damaging agents. To determine whether damaged chromosomes were subjected to direct loss or whether chromosome loss occurred as an indirect consequence of a prolonged G2 phase or other factors, we used the CRISPR-Cas9 system to introduce a single DNA double-strand break (DSB) on a minichromosome. The rate of minichromosome loss increased by approximately seven-fold compared with that of the control. Furthermore, the loss rate was significantly elevated in the absence of KU70, a key factor in non-homologous end joining, and upon inhibition of ataxia telangiectasia mutated (ATM), a DNA damage checkpoint protein. Finally, two closely spaced nicks, believed to generate a 5-overhang, were also shown to induce minichromosome loss. These findings indicated that a single DSB or two closely spaced nicks can cause aneuploidy if improperly repaired in vertebrates.

molecular biology↗

bioRxiv2024

Ca2+-evoked sperm cessation determines embryo number in mammals.

In mammals, fertilization takes place followed by fetus development in the maternal body, so the number of fetuses that exceeds the mothers capacity increases the frequency of stunted growth and stillbirths, while an excessive number of pups increases postnatal stunting and maternal child-rearing stress, which is detrimental to offspring procreation and species maintenance. Therefore, various control mechanisms are thought to be at work to ensure that fertilization occurs in appropriate numbers. Sperm are not capable of fertilization immediately after they are produced in the testis, and must undergo a stepwise activation process including capacitation, hyper motility activation or acrosome reaction before they meet the egg in the oviduct, where fertilization takes place, contributing positively on fertilization, which in turn ensures the number of fetuses for the maintenance of species. In contrast, in this study, we found that a subpopulation of sperm is derived in a Ca2+-dependent manner during the process for sperm to acquire fertility, which may regulate the number of fetuses. When sperm were harvested from the epididymis of mice and activated in vitro, a subpopulation of sperm emerged from the sperm population in which Lypd4 was expressed on the sperm surface at a ratio up to 30%. The sperm in this subpopulation were rather small in size, more permeable to dyes, and had already ceased motility. We further characterized this sperm population by surface antigen screening using various monoclonal antibodies and found the expression of several proteins, such as CD55, ICOS or Ccr3, specific to this population. The emergence of this sperm population was induced at a concentration of about 4% of Ca2+ in body fluids and was independent of capacitation or acrosome reaction, and also apoptotic process. This subpopulation also appeared over time in sperm ejaculated into the female body, accounting for about 50% of the sperm that reached the oviduct in an hour. When this sperm subpopulation was then removed from the entire population using anti-Lypd4 antibody followed by in utero insemination, the fertilization rate of the oocytes collected from the oviducts doubled. Such a sperm subpopulation was also observed in macaque monkeys, and removal of this subpopulation increased the egg penetration rate of sperm, suggesting that this sperm subpopulation exists commonly in mammals and that the mothers acceptable fetal number is adjusted by systematically sterilizing a certain number of sperm.

developmental biology↗

bioRxiv2024

DeepES: Deep learning-based enzyme screening to identify orphan enzyme genes

MotivationProgress in sequencing technology has led to determination of large numbers of protein sequences, and large enzyme databases are now available. Although many computational tools for enzyme annotation were developed, sequence information is unavailable for many enzymes, known as orphan enzymes. These orphan enzymes hinder sequence similarity-based functional annotation, leading gaps in understanding the association between sequences and enzymatic reactions. ResultsTherefore, we developed DeepES, a deep learning-based tool for enzyme screening to identify orphan enzyme genes, focusing on biosynthetic gene clusters and reaction class. DeepES uses protein sequences as inputs and evaluates whether the input genes contain biosynthetic gene clusters of interest by integrating the outputs of the binary classifier for each reaction class. The validation results suggested that DeepES can capture functional similarity between protein sequences, and it can be implemented to explore orphan enzyme genes. By applying DeepES to 4744 metagenome-assembled genomes, we identified candidate genes for 236 orphan enzymes, including those involved in short-chain fatty acid production as a characteristic pathway in human gut bacteria. Availability and implementationDeepES is available at https://github.com/yamada-lab/DeepES. Model weights and the candidate genes are available at Zenodo (https://doi.org/10.5281/zenodo.11123900).

bioinformatics↗

bioRxiv2023

Endogenous oncogenic KRAS expression increases cell proliferation and motility in near-diploid hTERT RPE-1 cells

About 18% of all human cancers carry a mutation in the KRAS gene making it among the most sought-after anti-cancer targets. However, mutant KRas protein has proved remarkably undruggable. The recent approval of the first generation of RAS inhibitors therefore marks a seminal milestone in the history of cancer research. Inevitably though, it also raises the predictable challenges of limited drug efficacies and acquired resistance. Hence, new approaches that improve our understanding of the tumorigenic mechanisms of oncogenic RAS within more physiological settings continue to be essential. Here, we have employed the near-diploid human hTERT RPE-1 cells to generate isogenic cell lines in which one of the endogenous KRAS alleles carries an oncogenic KRAS mutation at glycine 12. Cells with a KRASG12V/+, KRASG12C/+, or KRASG12D/+ genotype, together with wild-type KRASG12G(WT)/+ cells, reveal that oncogenic KRAS.G12X mutations increase cell proliferation rate, while further analyses showed that KRASG12V/+ cells had increased cell motility and reduced focal adhesions. EGF-induced ERK phosphorylation was marginally increased in KRASG12V/+cells, while EGF-induced AKT phosphorylation was comparable between KRASG12V/+and KRASG12G(WT)/+ cells. Interestingly, the KRASG12V/+cells were more sensitive to hydroxyurea and a MEK inhibitor, U0126, but more resistant to a PI3K inhibitor, PIK-90, than the KRASG12G(WT)/+cells. A combination of low doses of hydroxyurea and U0126 showed an additive inhibition on growth rate that was greater in KRASG12V/+than wild-type cells. Collectively, these cell lines will be a valuable resource for studying oncogenic RAS signalling and developing effective anti-KRAS reagents with minimum cytotoxicity on wild-type cells.

cell biology↗

bioRxiv2023

DNA metabarcoding workflow utilizing nanopore long-read sequencing and consensus generation for rapid identification of fungal taxa with high phylogenetic resolution

The study of microbiota has been revolutionized by the development of DNA metabarcoding. This sequence-based approach enables the direct detection of microorganisms without the need for culture and isolation, which significantly reduces analysis time and offers more comprehensive taxonomic profiles across broad phylogenetic lineages. While there has been an accumulating number of researches on bacteria, molecular phylogenetic analysis of fungi still remains challenging due to the lack of standardized tools and the incompleteness of reference databases limiting the accurate and precise identification of fungal taxa. Here, we present a DNA metabarcoding workflow for characterizing fungal microbiota with high taxonomic resolution. This method involves amplifying longer stretches of ribosomal RNA operons and sequencing them using nanopore long-read sequencing technology. The resulting reads were error-polished to generate consensus sequences with 99.5-100% accuracy, which were then aligned against reference genome assemblies. The efficacy of this method was explored using a polymicrobial mock community and patient-derived specimens, demonstrating the marked potential of long-read sequencing combined with consensus calling for accurate taxonomic classification. Our approach offers a powerful tool for the rapid identification of pathogenic fungi and has the promise to significantly improve our understanding of the role of fungi in health and disease.

microbiology↗

bioRxiv2022

PCNA recruits cohesin loader Scc2/NIPBL to ensure sister chromatid cohesion

Sister chromatid cohesion is essential for faithful chromosome segregation and genome duplication during cell division. Failure to establish cohesion during S phase by the ring-shaped multiprotein complex cohesin leads to genomic instability1-4. Replisome-associated proteins are required to generate cohesion by two independent pathways5. One mediates conversion of cohesins bound to unreplicated DNA ahead of replication forks into cohesive entities behind them, while the second promotes cohesin de novo loading onto newly-replicated DNAs6. The latter process depends on the cohesin loader Scc2/NIPBL and the alternative PCNA loader CTF18-RFC. However, the precise mechanism of de novo cohesin loading during replication is unknown. Here we show that PCNA physically recruits yeast cohesin loader Scc2 via its C-terminal PCNA-interacting protein motif. Binding to PCNA is crucial, as scc2-pip mutant deficient in Scc2-PCNA interaction is defective in cohesion when combined with replisome mutants of the cohesin conversion pathway. Moreover, scc2-pip mutant becomes inviable without its partner Scc4/MAU2 that localizes cohesin loader to centromeres. Importantly, the role of NIPBL recruitment to PCNA for cohesion generation is conserved in vertebrate cells. Our results demonstrate that PCNA, the maestro of replication-linked functions, is also crucially involved in the cohesion establishment through de novo cohesin loading onto replicated DNA.

cell biology↗

bioRxiv2022

A high-throughput 384-well CometChip platform reveals a role for 3-methyladenine in the cellular response to etoposide-induced DNA damage

The Comet or single-cell gel electrophoresis assay is the gold standard for analysis of cellular, nuclear genome damage. However, low throughput limits its application for large-scale studies. To overcome these limitations, a 96-well CometChip platform was recently developed that increases throughput and reduces variation due to simultaneous processing and automated analysis of 96 samples. To advance throughput further, we developed a 384-well CometChip platform that allows analysis of [~]100 cells per well. The 384-well CometChip extends the capacity by 4-fold as compared to the 96-well system, enhancing application for larger DNA damage analysis studies. The overall sensitivity of the 384-well CometChip is consistent with that of the 96-well system, sensitive to genotoxin exposure and to loss of DNA repair capacity. We then applied the 384-well platform to screen a library of protein kinase inhibitors to probe each as enhancers of etoposide induced DNA damage and found that 3-methyladenine significantly increased levels of etoposide-induced DNA damage. Our results suggest that a 384-well CometChip is useful for large-scale DNA damage analyses, which may have increased potential in the evaluation of chemotherapy efficacy, compound library screens, population-based analyses of genome damage and evaluating the impact of environmental genotoxins on genome integrity.

cancer biology↗

bioRxiv2021

Gene recruitments and dismissals in argonaut octopus genome provide insights to pelagic lifestyle adaptation and shell-like eggcase reacquisition

The paper nautilus, Argonauta argo, also known as the greater argonaut, is a species of octopods distinctly characterized by its pelagic lifestyle and by the presence of a spiral-shaped shell-like eggcase in females. The eggcase functions by protecting the eggs laid inside it, and by building and keeping air intakes for buoyancy. To reveal the genomic background of the species adaptation to pelagic lifestyle and the acquisition of its shell-like eggcase, we sequenced the draft genome sequence of the species. The genome size was 1.1 Gb, which is the smallest among the cephalopods known to date, with the top 215 scaffolds (average length 5,064,479 bp) covering 81% (1.09 Gb) of the total assembly. A total of 26,433 protein-coding genes were predicted from 16,802 assembled scaffolds. From these, we identified nearly intact HOX, Parahox, Wnt clusters and some gene clusters probably related to the pelagic lifestyle, such as reflectin, tyrosinase, and opsin. For example, opsin might have undergone an extensive duplication in order to adapt to the pelagic lifestyle, as opposed to other octopuses, which are mostly the benthic. Our gene models also discovered several genes homologous to those related to calcified shell formation in Conchiferan Mollusks, such as Pif-like, SOD, and TRX. Interestingly, comparative genomics analysis revealed that the homologous genes for such genes were also found in the genome of the octopus, which does not have a shell, as well as the basal cephalopods Nautilus. Therefore, the draft genome sequence of A. argo we presented here had not only helped us to gain further insights into the genetic background of the dynamic recruitment and dismissal of genes for the formation of an important, converging extended phenotypic structure such as the shell and the shell-like eggcase, but also the evolution of lifestyles in Cephalopods and the octopods, from benthic to pelagic.

genomics↗

bioRxiv2021

Independent adoptions of a set of proteins found in the matrix of the mineralized shell-like eggcase of Argonaut octopuses

The Argonaut octopus, commonly called the paper nautilus, has a spiral-coiled shell-like eggcase. As the main characteristics, the eggcase has no internal septum, is composed entirely of calcite with chitosan being the main polycarbonate and is reportedly formed by organic materials secreted from the membranes of the arms. Meanwhile, the biomineralized external "true" shells of the Mollusks, which includes the Cephalopods, are secreted from the mantle tissue. Therefore, the histological origin of the two shells is completely different. The question of how the Argonauts, which phylogenetically diverged from the completely shell-less octopuses, could form a converging shell-like external structure has thus intrigued biologists for a long time. To answer this question, we performed a multi-omics analysis of the transcriptome and proteome of the two congeneric Argonaut species, Argonauta argo and A. hians. Our result indicates that the shell-like eggcase is not a homolog of the shell, even at the protein level, because the Argonauts apparently recruited a different set of protein repertoires to as eggcase matrix proteins (EcMPs). However, we also found the homologs of three shell matrix proteins (SMPs) of the Conchiferan Mollusks, Pif-like, SOD, and TRX, in the eggcase matrix. The proteins were also found in the only surviving shelled Cephalopods, the nautiloid Nautilus pompilius. Phylogenetic analysis revealed that homologous genes of the Conchiferan SMPs and EcMPs were found in the draft genome of shell-less octopuses. Our result reported here thus suggests that the SMP-coding genes are conserved in both shelled and shell-less Cephalopods. Meanwhile, the Argonauts adopted some of the SMP-coding genes and other non-SMP-coding genes, to form a convergent, non-homologous biomineralized external structure, the eggcase, which is autapomorphic to the group.

evolutionary biology↗

bioRxiv2021

Inhibiting SARS-CoV-2 infection in vitro by suppressing its receptor, angiotensin-converting enzyme 2, via aryl-hydrocarbon receptor signal

Since understanding molecular mechanisms of SARS-CoV-2 infection is extremely important for developing effective therapies against COVID-19, we focused on the internalization mechanism of SARS-CoV-2 via ACE2. Although cigarette smoke is generally believed to be harmful to the pathogenesis of COVID-19, cigarette smoke extract (CSE) treatments were surprisingly found to suppress the expression of ACE2 in HepG2 cells. We thus tried to clarify the mechanism of CSE effects on expression of ACE2 in mammalian cells. Because RNA-seq analysis suggested that suppressive effects on ACE2 might be inversely correlated with induction of the genes regulated by aryl hydrocarbon receptor (AHR), the AHR agonists 6-formylindolo(3,2-b)carbazole (FICZ) and omeprazole (OMP) were tested to assess whether those treatments affected ACE2 expression. Both FICZ and OMP clearly suppressed ACE2 expression in a dose-dependent manner along with inducing CYP1A1. Knock-down experiments indicated a reduction of ACE2 by FICZ treatment in an AHR-dependent manner. Finally, treatments of AHR agonists inhibited SARS-CoV-2 infection into Vero E6 cells as determined with immunoblotting analyses detecting SARS-CoV-2 specific nucleocapsid protein. We here demonstrate that treatment with AHR agonists, including CSE, FICZ, and OMP, decreases expression of ACE2 via AHR activation, resulting in suppression of SARS-CoV-2 infection in mammalian cells.

molecular biology↗

bioRxiv2021

Novel Thyroid-Specific Autoantibodies in Patients with Immune-Related Adverse Events Involving the Thyroid Gland

AimsProgrammed cell death-1 (PD-1) blockade therapy frequently results in immune-related adverse events involving the thyroid gland (thyroid irAEs). Although clinical features of thyroid irAEs are known, the mechanisms remain unclear. Here, we conducted a pilot study to investigate mechanisms of thyroid irAE development from the perspective of autoantibodies. MethodsWe performed immunoprecipitation-based assays using sera of 3 patients who developed thyroid irAEs with PD-1 blockade therapy by nivolumab and HEK293T cell lysates, including overexpressed proteins of interest (NKX2-1, PAX8, FOXE1, and HHEX; thyroid-specific transcriptional factors). The pellets were analyzed by western blot to detect the HiBit tag attached to the C-terminus of the proteins. ResultsRelevant changes to NKX2-1 bands were not seen in all 3 patients, but PAX8 bands were augmented in patient 2 with lung cancer and patient 3 with renal cell carcinoma. In addition, FOXE1 bands were augmented in patient 1 with malignant melanoma and patient 3, and a HHEX band was augmented in patient 3. Thus, we revealed novel thyroid-specific autoantibodies, PAX8Ab, FOXE1Ab, and HHEXAb. Expression patterns of the antigens recognized by these antibodies were not identical to the primary sites, so autoimmune responses in thyroid irAE may originate from the thyroid gland, and not the malignancy. Considering that TPOAb rather than TgAb is often negative in patients with thyroid irAEs, other mechanisms such as cytotoxic T cell and antigenicity of thyroglobulin may be involved. ConclusionsAlthough the significance of these novel autoantibodies needs further examination, the present study provides new insights for thyroid autoimmunity.

immunology↗