Biology subjects

Nakao, R.

Publications and source records attributed to Nakao, R..

9 recordsLinked to original sources

bioRxiv2026

Reverse genetics system for emerging tick-borne orthonairovirus highlights dispensable N-terminal region of Gn associated with replication in tick vectors

A growing number of emerging and re-emerging tick-borne orthonairoviruses belonging to the Sulina and Tamdy genogroups have recently been identified in association with febrile diseases in East Asia. Yezo virus (YEZV) is one such viruses and is genetically distinct from well-characterized Crimean-Congo hemorrhagic fever virus. Orthonairovirus glycoproteins (Gn and Gc) expressed from a single glycoprotein precursor (GPC) gene mediate interaction with host cells, yet the organization and functions of the YEZV glycoproteins remain largely undefined. To characterize YEZV glycoproteins in the context of infection, we developed a reverse genetics system that enables recovery of recombinant YEZV entirely from cloned cDNAs. The recombinant wild-type virus exhibited growth properties comparable to those of the parental isolate in vitro and maintained pathogenicity in vivo. Proteomic analysis of purified virus particles produced in mammalian cells showed that peptide coverage of GPC-derived products began at residue 69. We therefore used the reverse genetics system to examine the functional importance of the GPC subregion upstream of residue 69. A mutant with a deletion of GPC residues 28 to 68 (rYEZV GPC{Delta}28-68), which retained the predicted signal peptide, was successfully recovered, suggesting that this subregion is dispensable for producing infectious virus. rYEZV GPC{Delta}28-68 propagated at levels comparable to those of the wild-type virus in mammalian cells and exhibited similar pathogenicity in a mouse model. Interestingly, the mutant reached lower viral titers in tick-derived ISE6 cells and in ticks in vivo, suggesting that this N-terminal subregion may contribute to virus replication in ticks. Together, these findings demonstrate the utility of the newly established reverse genetics system for dissecting the functions of the YEZV glycoproteins in mammalian and tick systems.

microbiology↗

bioRxiv2026

A universal plug-and-display vaccine platform for mucosal and systemic immunity using Bacillus subtilis membrane vesicles

Many bacterial species naturally secrete membrane vesicles (MVs) that mediate the intercellular transport of biomolecules, including nucleic acids, proteins, and metabolites. Beyond their native physiological roles, MVs hold considerable potential for biomedical applications. Here, we demonstrate that MVs from several Bacillus species exhibit potent intrinsic adjuvant activity, efficaciously eliciting immune responses and facilitating antigen-specific antibody production in mice. Exploiting this adjuvanticity, we engineered a highly adaptable universal vaccine platform that uses B. subtilis MVs as self-adjuvanting carriers. This system employs a modular "plug-and-display" architecture that covalently anchors recombinant antigens to the MV surface through a multi-step bioconjugation cascade. After validation of this methodology using a model antigen, we adapted the platform to target Yersinia pestis, the causative agent of plague. We formulated a Y. pestis vaccine by labeling the MV surface with a modified capsule antigen fraction 1 (mCaf1). Intranasal administration of the mCaf1-MV vaccine effectively elicited both systemic and mucosal immunity. Crucially, this vaccine conferred highly efficacious protection against a lethal Y. pestis infection in a murine model. These findings demonstrate the exceptional protective efficacy of the B. subtilis MV platform and highlight its broad potential for the rapid development of mucosal vaccines against diverse emerging pathogens.

bioengineering↗

bioRxiv2026

Periodontal pathogen-derived extracellular vesicles promote EGFR-dependent malignant traits in human pancreatic cancer cells

Extracellular vesicles (EVs) produced by periodontopathic bacteria have been implicated in systemic diseases, yet their mechanistic impact on pancreatic cancer (PAC) cell behavior remains poorly defined. Here, we show that EVs derived from Aggregatibacter actinomycetemcomitans (Aa), but not from Porphyromonas gingivalis or Fusobacterium nucleatum, robustly induce epithelial-mesenchymal transition (EMT), migration, and invasion in human PAC cells. These effects require the Aa genotoxin cytolethal distending toxin (Cdt), which is selectively packaged into Aa-derived EVs and efficiently delivered to host cells. EV-associated Cdt induces DNA damage, triggers a senescence-like transcriptional program, increases EGF sensitivity, and activates the EGFR-ERK/Akt signaling axis independently of TGF{beta} signaling, thereby promoting malignant traits in vitro. Notably, these responses occur in human but not murine PAC cells, revealing species-specific susceptibility to bacterial genotoxins. Together, these findings reveal a previously unrecognized cross-kingdom mechanism by which periodontopathic bacterial EVs promote metastatic traits in human pancreatic cancer cells through Cdt-mediated DNA damage and EGFR-ERK/Akt signaling.

microbiology↗

bioRxiv2025

Efficacy of natural marine sponges as a passive environmental DNA sampler for freshwater fish diversity monitoring

Environmental DNA (eDNA) analysis is a cost-effective and noninvasive tool for species and biodiversity monitoring in aquatic environments. Passive eDNA sampling is a novel alternative to conventional sampling methods such as water filtration. In this study, we examined the efficacy of the sponge skeleton as a passive eDNA sampler for monitoring freshwater fish. The performance of the passive sampling method was compared with that of standard water filtration in a river environment. Five DNA extraction methods were used in three experiments, and a suitable method for DNA extraction from sponge skeleton was identified. Quantitative fish metabarcoding using MiFish primers revealed no significant differences in species richness between the aqueous and passive sampling methods. Although both the sampling methods showed comparable trends in fish community structure, different clusters were identified for water sampling and passive samplers based on the differences in the DNA concentration of each fish species. The fish diversity in the passive samples was comparable among four DNA extraction methods (except for the filtration method) using the direct capture method. Our results demonstrate the efficacy of passive eDNA sampling for monitoring freshwater fish diversity and the potential use of sponge skeletons as absorption materials for passive eDNA samplers.

ecology↗

bioRxiv2022

Environmental DNA phylogeography: successful reconstruction of phylogeographic patterns of multiple fish species from a cup of water

Phylogeography is an integrative field of science linking micro- and macro-evolutionary processes, contributing to the inference of vicariance, dispersal, speciation, and other population-level processes. Phylogeographic surveys usually require considerable effort and time to obtain numerous samples from many geographical sites covering the distribution range of target species; this associated high cost limits their application. Recently, environmental DNA (eDNA) analysis has been useful not only for detecting species but also for assessing genetic diversity; hence, there has been growing interest in its application to phylogeography. As the first step of eDNA-based phylogeography, we examined (1) data screening procedures suitable for phylogeography and (2) whether the results obtained from eDNA analysis accurately reflect known phylogeographic patterns. For these purposes, we performed quantitative eDNA metabarcoding using group-specific primer sets in five freshwater fish species belonging to two taxonomic groups from a total of 94 water samples collected from western Japan. As a result, three-step data screening based on the DNA copy number of each haplotype detected successfully eliminated suspected false positive haplotypes. Furthermore, eDNA analysis could almost perfectly reconstruct the phylogenetic and phylogeographic patterns obtained for all target species with the conventional method. Despite existing limitations and future challenges, eDNA-based phylogeography can significantly reduce survey time and effort and is applicable for simultaneous analysis of multiple species in single water samples. eDNA-based phylogeography has the potential to revolutionise phylogeography.

molecular biology↗

bioRxiv2022

Quantitative environmental DNA metabarcoding reflects quantitative capture data of fish community obtained by electrical shocker

The simultaneous conservation of species richness and evenness is important to effectively reduce biodiversity loss and keep ecosystem health. Environmental DNA (eDNA) metabarcoding has been used as a powerful tool for identifying community composition, but it does not necessarily provide quantitative information due to several methodological limitations. Thus, the quantification of eDNA through metabarcoding is an important frontier of eDNA-based biomonitoring. Particularly, the qMiSeq approach has recently been developed as a quantitative metabarcoding method and has attracted much attention due to its usefulness. The aim here was to evaluate the performance of the qMiSeq approach as a quantitative monitoring tool for fish communities by comparing the quantified eDNA concentrations with the results of fish capture surveys. The eDNA water sampling and the capture surveys using the electrical shocker were conducted at a total of 21 sites in four rivers in Japan. As a result, we found significant positive relationships between eDNA concentrations of each species quantified by qMiSeq and both the abundance and biomass of each captured taxon at each site. Furthermore, for seven out of eleven taxa, a significant positive relationship was observed between quantified DNA concentrations by sample and the abundance and/or biomass. In total, our results demonstrated that eDNA metabarcoding with the qMiSeq approach is a suitable and useful tool for quantitative monitoring of fish communities. Due to the simplicity of the eDNA analysis, the eDNA metabarcoding with qMiSeq approach would promote further growth of quantitative monitoring of biodiversity. HighlightsO_LIeDNA analysis and capture surveys of fishes were conducted at a total of 21 sites. C_LIO_LIQuantitative eDNA metabarcoding (qMiSeq) successfully quantified the eDNA of fishes. C_LIO_LIFor each site, the estimated eDNA conc. reflected the abundance and biomass of fishes. C_LIO_LIFor each taxon, the estimated eDNA conc. were comparable among sites. C_LIO_LIqMiSeq is a suitable and useful tool for quantitative monitoring of fish community. C_LI

ecology↗

bioRxiv2020

BoLA-DRB3 gene haplotypes show divergence in native Sudanese cattle from Taurine and Zebu breeds

Autochthonous Sudanese cattle breeds, namely Baggara for beef and Butana and Kenana for dairy, are characterized by their adaptive characteristics and high performance in hot and dry agro-ecosystems, are used largely by nomadic and semi-nomadic pastoralists. Here we analyzed the diversity and genetic structure of the BoLA-DRB3 gene, a genetic locus linked to the immune response, for the indigenous cattle of Sudan and in the context of the global cattle repository. Blood samples (n=225) were taken from three indigenous breeds (Baggara; n=113, Butana; n= 60 and Kenana; n=52) distributed across six regions of Sudan. Nucleotide sequences were genotyped using the sequence-based typing method. Sequence electropherograms were analyzed using the Assign SBT software. We describe 53 alleles, including seven new, novel alleles. In the Baggara breed the number of alleles was 46 (40 previously reported and six new ones), 33 in the Kenana breed (28 previously reported and five new ones), and 33 in the Butana breed (28 previously reported and five new ones). Venn analysis of Sudanese breeds with Southeast Asian, European and American cattle showed 115 alleles of which 14 were unique to Sudanese breeds. Three of the alleles exhibited gene frequency of > 0.5%, representing 26% of the 53 alleles detected in the native Sudanese cattle. Observed versus expected heterozygosity was higher than 0.93 in all three breeds analyzed and equilibrium status revealed by Hardy-Weinberg Equilibrium suggests pure genetic drift. Gene frequency distributions of Baggara cattle showed an even distribution (P = 0.016), consistent with the theoretical proportion expected under balancing selection pressure as opposed to positive or neutral selection. In contrast, Butana and Kenana cattle (P = 0.225 and P = 0.138, respectively) were more congruent with neutral selection, similar to the results obtained for most of the cattle breeds analyzed so far. Sudanese cattle breeds were located within a narrow cloud in an intermediate position between the Zebu and Taurine breeds and close to other Southeast Asian breeds, in accordance with the composite origin of these native breeds, which is also reinforced by the presence of African and Zebu unique BoLA-DRB3 alleles within these breeds. The results of the Principal Component Analysis were in agreement with the overall clustering pattern observed on the NJ and/or UPGMA trees. These results contribute to our understanding of the genetic diversity and distribution pattern of BoLA-DRB3 gene alleles in Sudanese cattle breeds and provide insight into their uniqueness in their ability to survive arrays of tropical diseases and reproduce well in Sudans harsh environment. Author summaryAfrican cattle survive and adapt to a variety of diseases via acquired immunity capable of presenting antigens through the function of the major histocompatibility complex (MHC) or bovine leukocyte antigen (BoLA) in cattle. The aim of this study was to investigate how the immune system is structured and to what extent three economically important breeds in Sudan differ from exotic cattle. Here, we use the sequence-based typing approach to analyze BoLA-DRB3s genetic diversity linked to immunity against complex diseases that infect cattle. By examining 225 indigenous cattle belonging to three breeds in Sudan, we demonstrate that these cattle are unique from all known cattle by identifying seven new alleles; BoLA-DRB3*004:02Sp, BoLA-DRB3*011:02Sp, BoLA-DRB3*018:01Sp, BoLA-DRB3*021:01sp, BoLA-DRB3*024:18Sp, BoLA-DRB3*027:05sp, and BoLA-DRB3*032:01sp. When analyzing frequency of the protein pockets implicated in the antigen-binding function of the MHC complex by PCA we found that pockets 4 and 9 are the ones that best differentiate these native breeds from the rest. This may be attributed to high disease tolerance/susceptibility to tropical infections, such as those carried by ticks and intestinal parasites. Further studies are needed on these newly identified variants and their association with specific common disease(s). This finding is especially important for disease resistance/susceptibility association to help advise on candidate animals in selection schemes.

evolutionary biology↗

bioRxiv2020

Illumina iSeq 100 and MiSeq exhibit similar performance in freshwater fish environmental DNA metabarcoding

Environmental DNA (eDNA) analysis is a method of detecting DNA from environmental samples and is used as a biomonitoring tool. In recent studies, Illumina MiSeq has been the most extensively used tool for eDNA metabarcoding. The Illumina iSeq 100 (hereafter, iSeq), one of the high-throughput sequencers (HTS), has a relatively simple workflow and is potentially more affordable than other HTS. However, its utility in eDNA metabarcoding has still not been investigated. In the present study, we applied fish eDNA metabarcoding to 40 water samples from river and lake ecosystems to assess the difference in species detectability and composition between iSeq and MiSeq. To check differences in sequence quality and errors, we also assessed differences in read changes between the two HTS. There were similar sequence qualities between iSeq and MiSeq. Significant difference was observed in the number of species between two HTS, but no difference was observed in species composition between the two HTS. Additionally, the species compositions in common with the conventional method were the same between the two HTS. According to the results, using the same amplicon library for sequencing, two HTS would exhibit a similar performance of fish species detection using eDNA metabarcoding.

ecology↗

bioRxiv2020

Cystal structure of inhibitor-bound human MSPL/TMPRSS13 that can activate high pathogenic avian influenza

Infection of certain influenza viruses is triggered when its hemagglutinin (HA) is cleaved by host cell proteases such as proprotein convertases and type II transmembrane serine proteases (TTSP). HA with a monobasic motif is cleaved by trypsin-like proteases, including TMPRSS2 and HAT, while the multibasic motif found in high pathogenicity avian influenza HA is cleaved by furin, PC5/6, or MSPL. MSPL belongs to the TMPRSS family and preferentially cleaves [R/K]-K-K-R{downarrow} sequences. Here, we solved the crystal structure of the extracellular region of human MSPL in complex with an irreversible substrate-analog inhibitor. The structure revealed three domains clustered around the C-terminal -helix of the SPD. The inhibitor structure and its putative model show that the P1-Arg inserts into the S1 pocket, whereas the P2-Lys and P4-Arg interacts with the Asp/Glu-rich 99-loop that is unique to MSPL. Based on the structure of MSPL, we also constructed a homology model of TMPRSS2, which is essential for the activation of the SARS-CoV-2 spike protein and infection. The model may provide the structural insight for the drug development for COVID-19.

biophysics↗