Biology subjects

Matsuoka, Y.

Publications and source records attributed to Matsuoka, Y..

At least 19 recordsLinked to original sources

bioRxiv2026

Oxidized phosphatidylinositol impairs lysosomal membrane repair to promote ferroptosis

Ferroptosis is a regulated form of cell death driven by iron-dependent and unrestrained lipid peroxidation, which generates phospholipid hydroperoxides that cause membrane rupture. Oxidized phospholipid species, including oxidized arachidonic acid-containing phosphatidylethanolamines (PE), are abundant during ferroptosis. However, previous studies have examined only a limited number of lipid species, and it remains unclear which oxidized phospholipids consistently arise across distinct cell types and ferroptosis-inducing conditions. Here, we comprehensively profiled oxidized phospholipids generated during ferroptosis across multiple cell lines and animal models. We identified PE 18:0_20:4;O3 and phosphatidylinositol (PI) 18:0_20:4;O3 as oxidized phospholipid species that are consistently detectable across all tested ferroptosis-inducing conditions. Furthermore, oxidized phosphatidylinositol impairs the phosphoinositide-initiated membrane tethering and lipid transport (PITT) pathway, a key mechanism for lysosomal membrane repair. These results indicate that the oxidized phospholipids identified here may serve as markers of ferroptosis while also acting as bioactive mediators that compromise lysosomal membrane homeostasis and repair.

cell biology↗

bioRxiv2026

An immune receptor pair consisting of NLR and MLKL confers stable resistance against Pyricularia oryzae pathotype Eluesine on wheat by recognition of three effectors

Kinase fusion proteins (KFPs) have emerged as an important group of immune receptors encoded by plant resistance genes. Here, we report a new type of gene pair that controls resistance of wheat to the blast fungus, Pyricularia oryzae. We cloned a fungal gene involved in avirulence of P. oryzae pathotype Eleusine on wheat and designated it PWT8. We also identified its corresponding resistance gene in wheat, and tentatively named it Rwt8. This resistance gene was located at the same locus as previously identified resistance genes Rwt3 and Rwt6. Molecular cloning revealed that Rwt3, Rwt6, and Rwt8 were the same gene consisting of an identical gene pair, one encoding an NLR and the other encoding a mixed lineage kinase-like (MLKL) protein. These two genes were closely linked in a head-to-head orientation and behaved as a single gene. This gene pair recognized three AVR genes, PWT3, PWT6, and PWT8, and was designated Rwt3.6.8. The distribution of Rwt3.6.8 in common wheat landraces suggested that the gene pair may have been a factor which the D genome provided to the genus Triticum to broaden its adaptability to various environments in the world, especially in Asia and Africa.

plant biology↗

bioRxiv2026

Off-target-free chemogenetic platform that decodes physiological roles of target GPCRs

G-protein-coupled receptors (GPCRs) are essential mediators of cellular processes, and their dysfunction is implicated in various diseases. Although pharmacological approaches are powerful for elucidating GPCR functions, careful consideration of potential off-target effects remains crucial. Here, we present an off-target-free chemogenetic platform to dissect the physiological roles of target GPCRs. As a proof-of-concept, we focused on the adenosine A2A receptor (A2AR), a prototypical GPCR involved in neurodegenerative diseases. Leveraging the ligand recognition mechanisms of A2AR, we designed a prominent chemogenetic pair comprising a clinical A2AR antagonist and an engineered A2AR mutant that is insensitive to the antagonist but retains responsiveness to adenosine. Using this pair, we uncovered the critical role of A2AR in neurite outgrowth under hypoxic conditions in neuron-like cells, while avoiding potential off-target effects. Furthermore, we demonstrate the generalizability of this chemogenetic approach to other class A GPCRs, offering a versatile platform for receptor-specific dissection of GPCR signaling.

biochemistry↗

bioRxiv2025

Evolution of HMA-integrated tandem kinases accompanied by expansion of target pathogens

Tandem kinase proteins (TKPs) are an emerging family of plant intracellular immune receptors that offer potential for developing novel disease resistance. We cloned Rmo2 and Rwt7, genes for resistance to the blast fungus, from barley and wheat, respectively, and discovered that they are orthologs encoding TKPs with an integrated N-terminal heavy metal-associated (HMA) domain. Rmo2 was collocated with Rpg1, a TKP gene for resistance to stem rust, while Rwt7 was collocated with WTK4, a TKP gene for resistance to powdery mildew. Domain swapping suggested that the HMA domain determines target effector specificity of these genes. We propose a model illustrating an evolutionary process in which a TKP gene has differentiated into paralogs and orthologs that recognize various effectors through diversification of their HMA domains.

plant biology↗

bioRxiv2025

Co-administration of intranasal parainfluenza virus vaccines expressing antigenically distinct SARS-CoV-2 S antigens elicits broad and durable immunity in hamsters

Intranasal COVID-19 vaccines with the ability to induce broad and durable mucosal and systemic immunity would be useful as stand-alone vaccines or in combination with injectable vaccines. Here, we evaluated in the hamster model the immunogenicity, breadth of immunity, and durability of protection elicited by co-administration of two live-attenuated bovine/human parainfluenza virus type 3 (B/HPIV3) vectors expressing antigenically distinct prefusion stabilized S proteins of the ancestral SARS-CoV-2 isolate (B/HPIV3/S-6P) or the Omicron/BA.5 variant (B/HPIV3/S-BA.5-2P). These vectors are being developed as bivalent pediatric vaccines against HPIV3 and SARS-CoV-2 and are based on bovine PIV3 with the fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins replaced by those of human PIV3. To broaden the S-specific antibody response, we evaluated co-administration of these B/HPIV3 S-expressing vectors. Each B/HPIV3 S-expressing vector induced robust serum anti-S IgG and IgA antibody levels to the antigen-matched S protein that were sustained for at least five months. Co-administration increased the breadth of the S-specific antibody response, spanning the antigenic breadths of the response elicited by each B/HPIV3 S-expressing vector individually. Animals that had received the mixture of vectors developed neutralizing antibodies to ancestral as well as recently circulating SARS-CoV-2 strains. Hamsters immunized intranasally were protected against Omicron/BA.5 challenge 5 months after immunization, with no weight loss, SARS-CoV-2 challenge virus replication, or increase in host inflammatory cytokines in the upper and lower airways detectable after the challenge, indicating durable protection. Thus, intranasal co-administration of live-attenuated B/HPIV3 expressing antigenically distinct S proteins induced broad and durable antibody responses and long-term protection against Omicron/BA.5 challenge. This approach warrants further development and may better protect against emerging SARS-CoV-2 variants. Author summaryCurrent SARS-CoV-2 vaccines protect against severe disease but are less efficient at blocking infection. Intranasal SARS-CoV-2 vaccines, however, have been shown to induce local immunity that better blocks infection at the nasal portal of entry and have been proposed as booster vaccines to induce better protection against SARS-CoV-2 variants. In the present study, we evaluated in hamsters an intranasal live-attenuated chimeric bovine/human parainfluenza virus type 3 (B/HPIV3) as a bivalent pediatric vaccine against PIV3 and SARS-CoV-2. Co-administration in a single intranasal dose of two B/HPIV3 vectors, one expressing the spike protein S from the antigenically distinct ancestral Wuhan-Hu-1 strain and one expressing S of Omicron/BA.5, induced broad and durable serum anti-S IgG and IgA antibody responses that remained strong for at least five months. Hamsters challenged with the Omicron/BA.5 strain five months after immunization were protected from weight loss, inflammatory responses, and challenge virus replication in the upper and lower airways after challenge. Thus, in the hamster model, intranasal immunization with live-attenuated B/HPIV3 expressing SARS-CoV-2 S can provide durable protection for several months, and combining two antigenically distinct B/HPIV3 S-expressing vectors substantially broadens the antibody response. This mucosal immunization approach may better protect against infection from emerging SARS-CoV-2 variants.

microbiology↗

bioRxiv2025

Searching for Acrolein-Scavenging Compounds in Plants: Isoalliin from Onion as an Excellent Scavenger

Reactive carbonyl species (RCS), such as acrolein (Acr), are generated through the degradation of lipid peroxides and exert cytotoxic effects. To identify natural RCS scavengers, we examined 80% ethanol extracts from 46 angiosperm species for Acr-trapping activity using an HPLC-based assay. Strong activities were observed in several taxa, including garlic, spinach, avocado, broccoli, and lotus. In garlic, the active metabolite was identified as S-allyl-L-cysteine sulfoxide (alliin), a characteristic Allium amino acid. Alliin and its S-(1E)-propenyl and S-methyl derivatives (isoalliin and methiin, respectively) trapped up to two Acr molecules at the amino group and exhibited higher activities than known scavengers such as carnosine and epigallocatechin gallate. These findings highlight S-alk(en)yl-L-cysteine sulfoxides as potent secondary antioxidants and suggest that structurally diverse RCS scavengers remain to be discovered in plants.

plant biology↗

bioRxiv2025

A highly efficient knock-in strategy using fast-annealed 3'-extended dsDNA templates for emerging model insects

CRISPR-Cas genome editing toolkits have expanded the scope of genetic studies in various emerging model organisms. However, their applications are limited mainly to knockout experiments due to technical difficulties in establishing knock-in strains, which enable in vivo molecular tagging-based experiments. Here, we investigated knock-in strategies in the harlequin ladybug Harmonia axyridis, a model insect for evolutionary developmental biology, which shows more than 200 color pattern variations within a species. We tested several knock-in strategies using synthetic DNA templates. We found that ssDNA templates generated founder knock-in strains efficiently (2.5-11%), whereas the 5 regions of ssDNA templates were frequently deleted when the insert length exceeded [~]40 bases. To overcome this limitation, we designed several 3 extended DNA templates. Fast-annealed 3-extended double-stranded DNA templates, which were designed for tagging endogenous proteins with epitope tags, showed high founder generation efficiency (9.9-20.9%) and accuracy (30.8-85.7%). This strategy is also applicable to the two-spotted cricket Gryllus bimaculatus, suggesting that the fast-annealed 3-extended dsDNA template is a versatile DNA template for generating knock-in strains in emerging model insects for developmental genetic studies. Summary statementFast-annealed 3-extended dsDNA templates facilitate efficient CRISPR-Cas9-mediated knock-in in emerging model insects.

bioengineering↗

bioRxiv2025

f4-statistics-based ancestry profiling and convolutional neural network phenotyping shed new light on the structure of genetic and spike shape diversity in Aegilops tauschii Coss.

Aegilops tauschii Coss., a progenitor of bread wheat, is an important wild genetic resource for breeding. The species comprises three genetically defined lineages (TauL1, TauL2, and TauL3), each displaying distinctive phenotypes in various agronomic traits, including spike shape. In the present work, we studied the relationship between population structure and spike shape variation patterns using a collection of 249 accessions. f4-statistics-based ancestry profiling confirmed the previously identified lineages and revealed a genetic component derived from TauL3 in the genomes of some southern Caspian and Transcaucasus TauL1 and TauL2 accessions. Spike shape variation patterns were analyzed using a convolutional neural network-based approach, trained on green and dry spike image datasets. This analysis showed that spike shape diversity is structured according to lineages and demonstrated that the lineages can be distinguished based on spike shape. The implications of these findings for the origins of common wheat and the intraspecific taxonomy of Ae. tauschii are discussed. Plain Language SummaryWild wheat, Aegilops tauschii, represents a vast reservoir of alleles that have not yet been utilized in breeding. These alleles may confer beneficial phenotypes, such as drought tolerance and disease resistance, when introduced into bread wheat. To fully leverage this reservoir, it is essential to quickly identify strains with potentially useful alleles. In Ae. tauschii, which consists of strain groups (lineages) with unique genetic makeups, this can be done by determining a strains lineage based on spike shape. In this work, we trained machine learning models for this purpose and found that spike shape diversity reflects lineage structure. These models demonstrated potential for practical use in assigning strains to their respective lineages based on spike shape. Our work opens new avenues for the application of machine learning in wheat improvement, as well as in the genetic and evolutionary studies of wheat morphology.

evolutionary biology↗

bioRxiv2024

Integrated hepatic ferroptosis gene signature dictates pathogenic features of ferroptosis

Background & AimsFerroptosis, a distinctive form of cell death induced by iron-dependent lipid peroxidation, is implicated in various biological processes, including liver diseases. Establishing an iron overload-induced ferroptosis model and identifying hepatic gene signatures associated with ferroptosis are crucial for understanding its role in liver pathogenesis. MethodsF-box and leucine-rich repeat protein 5 (FBXL5) is a substrate-recognition component of the SCF E3 ligase complex that restricts intracellular iron levels. In this study, we used liver-specific Fbxl5-null mice to establish an iron overload-induced ferroptosis model. Transcriptome analysis identified genes involved in hepatic ferroptosis. Integrating these gene signatures with another ferroptosis model enabled the assessment of ferroptosis-related pathology in murine liver injury models and in 174 patients undergoing liver resection surgery. ResultsIron overload induced severe liver damage in liver-specific Fbxl5-null mice, characterized by elevated liver enzymes, histopathological changes, and lipid peroxidation. Transcriptome analysis revealed a distinct set of genes associated with hepatic ferroptosis response. Generating a gene signature for evaluating ferroptosis enhanced the understanding of ferroptosis-related pathologies in liver diseases. Iron overload exacerbated liver damage in murine ischemia-reperfusion injury models via ferroptosis induction. In human patients, elevated serum iron levels correlated with sustained post-operative liver damage, indicating heightened susceptibility to ferroptosis. ConclusionHere, a murine model of iron overload-induced hepatic ferroptosis was established, and a gene signature indicative of hepatic ferroptosis response in both mice and humans was identified. These findings underscore the role of ferroptosis in liver injury progression and suggest potential therapeutic targets for liver disease intervention. HIGHLIGHTSO_LILiver-specific Fbxl5 knockout mice provide an iron-induced ferroptotic injury model C_LIO_LIIntegrated gene signature of iron- and acetaminophen-induced liver injury dictates ferroptosis C_LIO_LIIron overload aggravates hepatic ischemia-reperfusion injury in mice C_LIO_LIPatients with high iron levels show delayed post-operative liver damage recovery C_LI IMPACT AND IMPLICATIONSOur study elucidated the critical role of iron in liver disease pathogenesis and ischemia-reperfusion injury (IRI). By establishing a murine model of iron overload-induced ferroptosis, we confirmed that iron overload exacerbated hepatic IRI, underscoring the importance of ferroptosis in liver damage. Additionally, the development of an integrated gene signature for hepatic ferroptosis response provides a valuable tool for evaluating ferroptosis in liver diseases. Via analysis of patient data, we also highlighted the clinical relevance of ferroptosis in post-operative liver damage, offering insights into potential therapeutic strategies targeting iron and ferroptosis to improve outcomes in patients with liver diseases.

molecular biology↗

bioRxiv2024

Intranasal parainfluenza virus-vectored vaccine expressing SARS-CoV-2 spike protein of Delta or Omicron B.1.1.529 induces mucosal and systemic immunity and protects hamsters against homologous and heterologous challenge

The continuous emergence of new SARS-CoV-2 variants requires that COVID vaccines be updated to match circulating strains. We generated B/HPIV3-vectored vaccines expressing 6P-stabilized S protein of the ancestral, B.1.617.2/Delta, or B.1.1.529/Omicron variants as pediatric vaccines for intranasal immunization against HPIV3 and SARS-CoV-2 and characterized these in hamsters. Following intranasal immunization, these B/HPIV3 vectors replicated in the upper and lower respiratory tract and induced mucosal and serum anti-S IgA and IgG. B/HPIV3 expressing ancestral or B.1.617.2/Delta-derived S-6P induced serum antibodies that effectively neutralized SARS-CoV-2 of the ancestral and B.1.617.2/Delta lineages, while the cross-neutralizing potency of B.1.1.529/Omicron S-induced antibodies was lower. Despite the lower cross-neutralizing titers induced by B/HPIV3 expressing S-6P from B.1.1.529/Omicron, a single intranasal dose of all three versions of B/HPIV3 vectors was protective against matched or heterologous WA1/2020, B.1.617.2/Delta or BA.1 (B.1.1.529.1)/Omicron challenge; hamsters were protected from challenge virus replication in the lungs, while low levels of challenge virus were detectable in the upper respiratory tract of a small number of animals. Immunization also protected against lung inflammatory response after challenge, with mild inflammatory cytokine induction associated with the slightly lower level of cross-protection of WA1/2020 and B.1.617.2/Delta variants against the BA.1/Omicron variant. Serum antibodies elicited by all vaccine candidates were broadly reactive against 20 antigenic variants, but the antigenic breadth of antibodies elicited by B/HPIV3-expressed S-6P from the ancestral or B.1.617.2/Delta variant exceeded that of the S-6P B.1.1.529/Omicron expressing vector. These results will guide development of intranasal B/HPIV3 vectors with S antigens matching circulating SARS-CoV-2 variants. Author SummaryIntranasal COVID vaccines have the potential to stimulate respiratory mucosal immunity, effectively restricting replication of SARS-CoV-2 in the respiratory tract, thereby reducing virus shedding and transmission. To develop pediatric vaccines for intranasal immunization against HPIV3 and SARS-CoV-2, we use live-attenuated bovine-human parainfluenza virus vaccine, a pediatric intranasal parainfluenza virus vaccine candidate, designed to express the stabilized SARS-CoV-2 spike protein. We compared the immunogenicity and breadth of protection of the B/HPIV3-expressed ancestral, B.1.617.2/Delta, or B.1.1.529/Omicron variants following intranasal immunization in hamsters. All three B/HPIV3 vectors replicated in the respiratory tract, induced mucosal and serum anti-S IgA and IgG, and were protective in the hamster model against matched or heterologous WA1/2020, B.1.617.2/Delta or BA.1 (B.1.1.529.1)/Omicron SARS-CoV-2 challenge. Serum antibodies elicited by all intranasal vaccine candidates were broadly reactive against 20 antigenic variants of SARS-CoV-2, but the antigenic breadth of antibodies elicited by B/HPIV3-expressed stabilized S from the ancestral or B.1.617.2/Delta variant exceeded that of the S-6P B.1.1.529/Omicron expressing vector. Thus, these intranasal vectored SARS-CoV-2 vaccine candidates induce cross-protective SARS-CoV-2 immunity with antigenic breadth similar to that of injectable SARS-CoV-2 vaccines. These results will guide development of intranasal COVID vaccines based on B/HPIV3 vectors with S antigens matching current SARS-CoV-2 variants.

microbiology↗

bioRxiv2023

Identification of genes supporting cold resistance of mammalian cells: lessons from a hibernator

Susceptibility of human cells to cold stress restricts the use of therapeutic hypothermia and long-term preservation of organs at low temperatures. In contrast, cells of mammalian hibernators possess remarkable cold resistance, but little is known about the molecular mechanisms underlying this phenomenon. In this study, we conducted a gain-of-function screening of genes that confer cold resistance to cold-vulnerable human cells using a cDNA library constructed from the Syrian hamster, a mammalian hibernator, and identified Gpx4 as a potent suppressor of cold-induced cell death. Additionally, genetic or pharmacological inhibition of Gpx4 in a hamster cell line under prolonged cold culture led to cell death, which resembles ferroptosis characterized by accumulation of lipid peroxide and ferrous iron dependency. Genetic disruption of other ferroptosis-suppressing pathways, namely biopterin synthesis and mitochondrial or plasma membrane CoQ reduction pathways, accelerated cold-induced cell death under Gpx4 dysfunction. Collectively, ferroptosis-suppressing pathways protect the cells of a mammalian hibernator from cold-induced cell death and the augmentation of these pathways renders cold resistance to cells of non-hibernators, including humans.

cell biology↗

bioRxiv2023

Evolution of wheat blast resistance gene Rmg8 accompanied by differentiation of variants recognizing the powdery mildew fungus

Wheat blast, a devastating disease having spread recently from South America to Asia and Africa, is caused by Pyricularia oryzae pathotype Triticum which emerged in 1985. Rmg8 and Rmg7, genes for resistance to wheat blast found in common wheat and tetraploid wheat, respectively, recognize the same avirulence gene, AVR-Rmg8. Here, we show an evolutionary process in which resistance gene(s), which had obtained an ability to recognize AVR-Rmg8 before the differentiation of Triticum and Aegilops, has expanded its target pathogens. Molecular cloning revealed that Rmg7 was one of alleles of Pm4 (Pm4a), a gene for resistance to wheat powdery mildew on 2AL, whereas Rmg8 was its homoeolog on 2BL ineffective against wheat powdery mildew. Rmg8 variants with the ability to recognize AVR-Rmg8 were distributed not only in Triticum spp. but also in Aegilops speltoides, Ae. umbellulata, and Ae. comosa. This result suggests that the origin of resistance gene(s) recognizing AVR-Rmg8 dates back to the time before differentiation of A, B, S, U, and M genomes, that is, [~]5 million years before the emergence of its current target, the wheat blast fungus. Phylogenetic analyses suggested that, in the evolutionary process thereafter, some of their variants gained the ability to recognize the wheat powdery mildew fungus and evolved into genes for resistance to wheat powdery mildew.

plant biology↗

bioRxiv2022

Live-attenuated pediatric parainfluenza vaccine expressing 6P-stabilized SARS-CoV-2 spike protein is protective against SARS-CoV-2 variants in hamsters

The pediatric live-attenuated bovine/human parainfluenza virus type 3 (B/HPIV3)-vectored vaccine expressing the prefusion-stabilized SARS-CoV-2 spike (S) protein (B/HPIV3/S-2P) was previously evaluated in vitro and in hamsters. To improve its immunogenicity, we generated B/HPIV3/S-6P, expressing S further stabilized with 6 proline mutations (S-6P). Intranasal immunization of hamsters with B/HPIV3/S-6P reproducibly elicited significantly higher serum anti-S IgA/IgG titers than B/HPIV3/S-2P; hamster sera efficiently neutralized variants of concern (VoCs), including Omicron variants. B/HPIV3/S-2P and B/HPIV3/S-6P immunization protected hamsters against weight loss and lung inflammation following SARS-CoV-2 challenge with the vaccine-matched strain WA1/2020 or VoCs B.1.1.7/Alpha or B.1.351/Beta and induced near-sterilizing immunity. Three weeks post-challenge, B/HPIV3/S-2P- and B/HPIV3/S-6P-immunized hamsters exhibited a robust anamnestic serum antibody response with increased neutralizing potency to VoCs, including Omicron sublineages. B/HPIV3/S-6P primed for stronger anamnestic antibody responses after challenge with WA1/2020 than B/HPIV3/S-2P. B/HPIV3/S-6P will be evaluated as an intranasal vaccine to protect infants against both HPIV3 and SARS-CoV-2. AUTHOR SUMMARYSARS-CoV-2 infects and causes disease in all age groups. While injectable SARS-CoV-2 vaccines are effective against severe COVID-19, they do not fully prevent SARS-CoV-2 replication and transmission. This study describes the preclinical comparison in hamsters of B/HPIV3/S-2P and B/HPIV3/S-6P, live-attenuated pediatric vector vaccine candidates expressing the "2P" prefusion stabilized version of the SARS-CoV-2 spike protein, or the further-stabilized "6P" version. B/HPIV3/S-6P induced significantly stronger anti-S serum IgA and IgG responses than B/HPIV3/S-2P. A single intranasal immunization with B/HPIV3/S-6P elicited broad systemic antibody responses in hamsters that efficiently neutralized the vaccine-matched isolate as well as variants of concern, including Omicron. B/HPIV3/S-6P immunization induced near-complete airway protection against the vaccine-matched SARS-CoV-2 isolate as well as two variants. Furthermore, following SARS-CoV-2 challenge, immunized hamsters exhibited strong anamnestic serum antibody responses. Based on these data, B/HPIV3/S-6P will be further evaluated in a phase I study.

microbiology↗

bioRxiv2022

Lepidopteran prolegs are novel traits, not leg homologs

Lepidopteran larvae have both thoracic legs and abdominal prolegs, yet it is unclear whether these are serial homologs. We examined the role of three Hox genes in proleg development in Bicyclus anynana butterflies using CRISPR-Cas9 and discovered that under a partial segment abdominal-A (abd-A) knockout, both types of appendages can develop in the same segment, arguing for prolegs being a novel trait, not a leg serial homolog. We also discover that specificity protein (sp) genes do not co-localize with Distal-less (Dll) in prolegs, as they do in legs, and that the proleg gene-regulatory network (GRN) mostly resembles the head-horn GRN, another novel trait in the lepidoptera. We propose that larval prolegs evolved from the co-option of a partial limb GRN into novel embryonic coordinates in the abdomen. One-Sentence SummaryThe chubby legs of caterpillars are evolutionary innovations

evolutionary biology↗

bioRxiv2022

Intranasal pediatric parainfluenza virus-vectored SARS-CoV-2 vaccine candidate is protective in macaques

Pediatric SARS-CoV-2 vaccines are needed that elicit immunity directly in the airways, as well as systemically. Building on pediatric parainfluenza virus vaccines in clinical development, we generated a live-attenuated parainfluenza virus-vectored vaccine candidate expressing SARS-CoV-2 prefusion-stabilized spike (S) protein (B/HPIV3/S-6P) and evaluated its immunogenicity and protective efficacy in rhesus macaques. A single intranasal/intratracheal dose of B/HPIV3/S-6P induced strong S-specific airway mucosal IgA and IgG responses. High levels of S-specific antibodies were also induced in serum, which efficiently neutralized SARS-CoV-2 variants of concern. Furthermore, B/HPIV3/S-6P induced robust systemic and pulmonary S-specific CD4+ and CD8+ T-cell responses, including tissue-resident memory cells in lungs. Following challenge, SARS-CoV-2 replication was undetectable in airways and lung tissues of immunized macaques. B/HPIV3/S-6P will be evaluated clinically as pediatric intranasal SARS-CoV-2/parainfluenza virus type 3 vaccine. One-Sentence SummaryIntranasal parainfluenza virus-vectored COVID-19 vaccine induces anti-S antibodies, T-cell memory and protection in macaques.

immunology↗

bioRxiv2022

Ultrabithorax modifies a regulatory network of genes essential for butterfly eyespot development in a wing sector-specific manner

Nymphalid butterfly species often have a different number of eyespots in forewings and hindwings but how the hindwing identity gene, Ultrabithorax (Ubx), drives this asymmetry, is not fully understood. It is also unclear why eyespot serial homologs originated initially only in hindwings. To address these questions, we examined a three-gene regulatory network (GRN) for eyespot development in the hindwings of Bicyclus anynana butterflies, and compared it to the same network previously described for forewings. We also examined how Ubx interacted with each of these three eyespot-essential genes. We found similar genetic interactions between the three genes in fore and hindwings, but we discovered three regulatory differences: Antennapedia (Antp) merely enhances spalt (sal) expression in the eyespot foci in hindwings but is not essential for sal activation, as in forewings; Ubx up- regulates Antp in all hindwing eyespot foci but represses Antp outside these wing regions and; Ubx is regulating sal expression in a wing-sector specific manner, i.e., it does not affect sal expression in the wing sectors that contain eyespots in both fore and hindwings, but it positively regulates sal expression in the sectors that have hindwing-specific eyespots. These results suggest that Ubx, or its downstream targets, might have paid a role in the origin of eyespots, restricted initially to hindwings, via the positive regulation of sal, an essential gene for eyespot development. We propose that Antp got co-opted into the eyespot GRN at a later stage by coming under Ubx regulation. This Hox gene redundancy, together with a novel positive feedback loop between sal and Antp, might have allowed Antp to functionally replace Ubx in forewings and lead to the origin of forewing eyespots. Outside the eyespot focal regions, we discovered that Ubx is up-regulated by Distal-less along the wing margin. We propose a model for how the regulatory connections between these genes might have evolved to produce wing- and sector-specific variation in eyespot number.

evolutionary biology↗

bioRxiv2021

Developmental plasticity in male courtship in Bicyclus anynana butterflies is driven by hormone regulation of the yellow gene

The organizational role for hormones in the regulation of sexual behavior is currently poorly explored. Previous work showed that seasonal variation in levels of the steroid hormone 20-hydroxyecdysone (20E) during pupal development regulates plasticity in male courtship behavior in Bicyclus anynana butterflies. Wet season (WS) males, reared at high temperature, have high levels of 20-hydroxyecdysone (20E) during pupation and become active courters. Dry season (DS) males, reared at low temperatures, have lower levels of 20E and lower courtship rates. Rescue of WS courtship rates can be achieved via injection of 20E into DS male pupae, but it is still unknown whether 20E alters gene expression in the pupal brain, and if so, the identity of those targets. Using transcriptomics, qPCR, and behavioral assays with a transgenic knockout, we show that higher expression levels of the yellow gene in DS male pupal brains, relative to WS brains, represses courtship in DS males. Furthermore, injecting DS males with 20E downregulates yellow to WS levels 4 hours post-injection, revealing a hormone sensitive window that determines courtship behavior. These findings are in striking contrast to Drosophila, where yellow is required for active male courtship behavior. We conclude that 20E plays an organizational role during pupal brain development by regulating the expression of yellow, which is a repressor of the neural circuity for male courtship behavior in B. anynana. This work shows that similar to vertebrates, hormones can also play an organizational role in insect brains, leading to permanent changes in adult sexual behavior. Significance StatementBehavioral plasticity in adult insects is known to be regulated by hormones, which activate neural circuits in response to environmental cues. Here, we show that hormones can also regulate adult behavioral plasticity by altering gene expression during brain development, adjusting the insects behavior to predictable seasonal environmental variation. We show that seasonal changes in the hormone 20E alters expression of the yellow gene in the developing pupal brain of Bicyclus anynana butterflies, which leads to differences in male courtship behavior between the dry and wet seasonal forms. This work provides one of the first examples of the organizational role of hormones in altering gene expression and adult sexual behavior in the developing insect brain.

neuroscience↗

bioRxiv2021

Establishment of CRISPR/Cas9-based knock-in in a hemimetabolous insect: targeted gene tagging in the cricket Gryllus bimaculatus

Studies of traditional model organisms like the fruit fly Drosophila melanogaster have contributed immensely to our understanding of the genetic basis of developmental processes. However, the generalizability of these findings cannot be confirmed without functional genetic analyses in additional organisms. Direct genome editing using targeted nucleases has the potential to transform hitherto poorly-understood organisms into viable laboratory organisms for functional genetic study. To this end, here we present a method to induce targeted genome knock-out and knock-in of desired sequences in an insect that serves as an informative contrast to Drosophila, the cricket Gryllus bimaculatus. The efficiency of germ line transmission of induced mutations is comparable to that reported for other well-studied laboratory organisms, and knock-ins targeting introns yield viable, fertile animals in which knock-in events are directly detectable by visualization of a fluorescent marker in the expression pattern of the targeted gene. Combined with the recently assembled and annotated genome of this cricket, this knock-in/knock-out method increases the viability of G. bimaculatus as a tractable system for functional genetics in a basally branching insect.

developmental biology↗