Biology subjects

Kobayashi, D.

Publications and source records attributed to Kobayashi, D..

14 recordsLinked to original sources

bioRxiv2026

A bivalent Lassa virus mRNA vaccine protects guinea pigs from lethal infection with Lassa virus

Lassa virus (LASV), the causative agent of the life-threatening febrile disease, Lassa fever, remains a major public health concern in West Africa, where hundreds of thousands of infections occur annually. However, no licensed vaccine is currently available, highlighting the urgent need for effective preventive measures. We previously demonstrated that an mRNA-lipid nanoparticle (LNP) vaccine expressing LASV glycoprotein precursor (GPC; LASgpc-mRNA-LNP) conferred protection against lethal infection with a recombinant lymphocytic choriomeningitis virus expressing LASV GPC in mice. In this study, we extended our previous work with the aim of evaluating the efficacy of an mRNA-LNP vaccine expressing LASV nucleoprotein (NP; LASnp-mRNA-LNP) and a bivalent vaccine combining LASgpc-mRNA-LNP and LASnp-mRNA-LNP. Using a lethal CBA/N mouse model of infection with ML29, an LASV live-attenuated vaccine candidate encoding LASV GPC and NP, we found that LASgpc-mRNA-LNP and LASnp-mRNA-LNP each conferred protection in the absence of detectable neutralizing antibody responses. We identified LASV GPC- and NP-derived CD4 and CD8 T-cell epitope peptides in CBA/N mice and demonstrated that vaccination induced robust antigen-specific T-cell responses. Importantly, the bivalent vaccine provided complete protection against lethal challenge with doses at which monovalent vaccines failed to protect all mice, suggesting a potential dose-sparing benefit of the bivalent formulation. Furthermore, the bivalent vaccine fully protected guinea pigs against lethal challenge with authentic LASV. Together, these findings establish LASV NP, in addition to GPC, as a protective antigen in the mRNA vaccine platform and provide a rationale for developing bivalent mRNA-LNP vaccines to improve protection against Lassa fever.

microbiology↗

bioRxiv2026

Statins and genetic inhibition of the mevalonate pathway activate an ATF3-STMN2 regenerative program

Loss of neuronal regenerative capacity is a common feature of neurodegenerative disease and axonal injury, yet the transcriptional programs governing this state remain poorly defined. Stathmin-2 (STMN2), a tubulin-binding protein essential for axon maintenance and repair, is profoundly depleted following loss of nuclear TDP-43 in neurodegenerative disease. Here, we identify statins as potent inducers of STMN2 expression. Pharmacological and genetic suppression of the mevalonate pathway, and subsequent prevention of protein geranylgeranylation, restored STMN2 levels in TDP-43 deficient cells and promoted neurite growth. STMN2 induction was abrogated when using a statin analogue unable to interact with HMG-CoA reductase, and through co-administration of mevalonate or geranylgeranyl diphosphate substrates. RNA-seq revealed that statins induce a coordinated pro-regenerative transcriptional response, including activation of the AP-1 transcription factor complex gene, ATF3. Loss of ATF3 attenuated STMN2 induction in vitro, and diminished injury-induced Stmn2 upregulation in spinal motor neurons in vivo. These results demonstrate statins as modulators of ATF3 and STMN2 expression and highlight their therapeutic potential in neurodegenerative disease.

neuroscience↗

bioRxiv2026

A versatile, marker-free platform for life cycle-wide imaging in Plasmodium falciparum via integration of an exogenous gene cassette into a conserved intergenic locus

The development of a transgenic Plasmodium falciparum line that exhibits robust fluorescence throughout its life cycle in cultured cells and mosquito hosts is valuable for live imaging, tissue localization studies, and quantitative evaluations of parasite infectivity. In this study, we utilized Plasmodium-optimized genome editing to integrate an mCherry expression cassette into a specific intergenic locus without gene disruption; thereby successfully minimizing the physiological impact of exogenous gene introduction. The resulting transgenic parasite line, NF54-mCh, exhibited intense fluorescence across all developmental stages; including intraerythrocytic asexual and sexual blood stages; mosquito ookinete, oocyst, and sporozoite stages; and liver stages. Cultured NF54-mCh exhibited normal intraerythrocytic proliferation, gametocytogenesis, and gametocyte maturation, with efficient transmission to mosquito vectors. The marker brightness in salivary gland sporozoites allowed for visual, non-invasive identification of infected mosquitoes, thus facilitating dissection. Sporozoites dissected from salivary glands were highly infectious to livers in humanized mice; and allowed completion of the full life cycle, as evidenced by the appearance of ring-stage parasites in inoculated human erythrocytes. NF54-mCh serves as a parental line for performing additional genetic modifications, because the CRISPR/Cas9-based genome editing method is free of introduced drug resistance markers. The broader applicability of this strategy was validated by generating fluorescence reporter lines for use in the rodent malaria parasite model systems, Plasmodium berghei and Plasmodium yoelii. In summary, NF54-mCh represents a unique, versatile platform that will accelerate fundamental research and support the future development of innovative malaria control strategies, including studies on new vaccines and drug efficacy. IMPORTANCEA detailed understanding of the molecular and cellular biology of the human malaria parasite Plasmodium falciparum, particularly during infection processes, is essential for efforts in malaria control and elimination. In this study, we generated a marker-free transgenic P. falciparum line which exhibits constitutive, robust fluorescence throughout its developmental stages in erythrocytes, mosquitoes, and hepatocytes, without impairing parasite growth or fitness. The fluorescence marker gene was inserted into an intergenic locus and thus no genes were disrupted. The pfs47 gene is intact, which allows the parasites to evade the mosquito innate system; therefore, the parasite retains the ability for pfs47-dependent infection of refractory Anopheles strains. The transgenic parasites do not contain drug resistance markers, and thus there are no limitations for further genetic manipulation. This transgenic parasite line provides a powerful tool for the study of fundamental parasite infection mechanisms and for the molecular analysis of host-parasite interactions.

microbiology↗

bioRxiv2025

A cattle-derived human H5N1 isolate suppresses innate immunity despite efficient replication in human respiratory organoids

The H5N1 high pathogenicity avian influenza virus (HPAIV) of clade 2.3.4.4b, which spreads globally via wild birds, has become a major public health concern because it can infect a variety of mammals, including humans. In 2024, infection of dairy cattle with H5N1 HPAIV clade 2.3.4.4b was confirmed in the United States, and subsequent human cases were reported. Although these viruses are highly pathogenic in animal models, human infections have generally been mild, revealing a striking discrepancy. Here, we characterized the cattle-derived human H5N1 isolate A/Texas/37/2024 (TX37-H5N1) using three-dimensional human respiratory organoids derived from induced pluripotent stem (iPS) cells. Despite efficient replication, TX37-H5N1 induced minimal interferon and inflammatory cytokine responses. Bulk and single-cell RNA sequencing revealed reduced STAT1-mediated transcriptional activity in TX37-H5N1-infected organoids compared to the historic H5N1 human isolate A/Vietnam/1203/2004. These findings suggest that TX37-H5N1 fails to trigger the strong innate responses, including robust cytokine production, that are typically associated with severe H5N1 disease and are thought to contribute to cytokine storm-medicated pathogenesis. This attenuated response may help explain the discrepancy between the high pathogenicity of TX37-H5N1 in animal models and its mild clinical presentation in humans. While zoonotic influenza risk is often assessed using cell lines or animal models, our study highlights the value of using human respiratory organoids to evaluate human-specific virus-host interactions. This platform provides a complementary tool for assessing the risk of emerging avian influenza viruses. HighlightsO_LIHuman respiratory organoids were used to model zoonotic B3.13 H5N1 infections. C_LIO_LIA cattle-derived human isolate, TX37-H5N1, replicated more efficiently than historical VN1203-H5N1. C_LIO_LITX37-H5N1 suppressed STAT-IRF-mediated innate immune responses. C_LIO_LITX37-H5N1 was sensitive to baloxavir and oseltamivir but less sensitive to favipiravir. C_LIO_LIHuman respiratory organoids offer a complementary platform for zoonotic influenza risk assessment. C_LI In briefOur study used human iPSC-derived respiratory organoids to investigate the mild clinical presentation of zoonotic B3.13 H5N1 viruses. We found that TX37-H5N1 replicates efficiently but suppresses innate immune responses, providing mechanistic insight into species-specific pathogenesis and highlighting the utility of human organoids for zoonotic risk assessment.

microbiology↗

bioRxiv2025

Genetic evidence: zebrafish hoxba and hoxbb clusters are essential for the anterior-posterior positioning of pectoral fins

Vertebrate paired appendages, such as the pectoral fins in fish and the forelimbs in tetrapods, arise at specific regions along the anterior-posterior axis of the body. Hox genes have long been considered prime candidates for determining the anteroposterior positioning of these paired appendages during development. Evidence from various model organisms, including mouse and chick, supports a role for Hox genes in limb positioning. However, despite extensive phenotypic analyses of numerous single and compound Hox knockout mice, clear genetic evidence for substantial defects in limb positioning has been limited, leaving questions unresolved. In a previous study, we generated seven distinct hox cluster-deficient mutants in zebrafish. Here, we provide genetic evidence that zebrafish hoxba;hoxbb cluster-deleted mutants specifically exhibit a complete lack of pectoral fins, accompanied by the absence of tbx5a expression in pectoral fin buds. In these mutants, tbx5a expression in the pectoral fin field of the lateral plate mesoderm fails to be induced at an early stage, suggesting a loss of pectoral fin precursor cells. Furthermore, the competence to respond to retinoic acid is lost in hoxba;hoxbb cluster mutants, indicating that tbx5a expression cannot be induced in the pectoral fin buds. We further identify hoxb4a, hoxb5a, and hoxb5b as pivotal genes underlying this process. Although the frameshift mutations in these hox genes do not recapitulate the absence of pectoral fins, we demonstrate that deletion mutants at these genomic loci show the absence of pectoral fins with low penetrance. Our results suggest that, by establishing the expression domains along the anteroposterior axis, hoxb4a, hoxb5a, and hoxb5b within hoxba and hoxbb clusters cooperatively determine the positioning of zebrafish pectoral fins through the induction of tbx5a expression in the restricted pectoral fin field. Our findings also provide insights into the evolutionary origin of paired appendages in vertebrates.

developmental biology↗

bioRxiv2024

Data-independent acquisition (DIA)-based Label-free Redox Proteomics (DIALRP) Reveals Novel Oxidative Stress Responsive Translation Factors

Oxidative stress is a key factor in numerous physiological and pathological processes, including aging, cancer, and neurodegenerative diseases. Protein cysteine residues are particularly susceptible to oxidative stress-induced modifications that can alter their structure and function, thereby affecting intracellular signaling pathways. In this study, we developed a data-independent acquisition mass spectrometry (DIA-MS)-based label-free redox proteomics method, termed DIALRP, to comprehensively analyse cysteine oxidative modifications in the prostate cancer cell line DU145 under oxidative stress induced by menadione (MND). Through these analyses, we identified translation-related factors with significantly elevated cysteine oxidation upon MND treatment and evaluated their functional relevance. Notably, our data demonstrated that the inhibition of EIF2, EIF6, and EEF2 complex formation due to oxidative stress occurs during the cellular response to translational inhibition. These insights reveal a previously unrecognized mechanism of translation regulation under oxidative stress and provide a valuable framework for future studies on redox-mediated cellular processes.

molecular biology↗

bioRxiv2024

Segment-specific promoter activity for RNA synthesis in the genome of Oz virus, genus Thogotovirus

Oz virus (OZV), a tick-borne, six-segmented negative-strand RNA virus in the genus Thogotovirus, caused a fatal human infection in Japan in 2023. To investigate mechanisms of viral RNA synthesis, we developed an OZV minigenome assay used in mammalian cells. Comparisons of promoter activities across six segments revealed that Segment 5 exhibited markedly lower promoter activity. Unlike the other segments forming a "distal duplex", a double-strand RNA beginning at the 11th nucleotide on the 5 end and the 10th on the 3, Segment 5 partially lacks this feature. Introducing a mutation in Segment 5 distal duplex resulted in a substantial increase in promoter activity. Further, we examined the promoter structures of other viruses in the genus Thogotovirus using public database. Thogoto virus Segment 1 also partially lacks a base pair in the distal duplex. In six-segmented RNA viruses, promoter activities are varied, with notable differences in activities likely existing among segments. HighlightsO_LIA minigenome assay was developed to elucidate the RNA synthesis mechanism of Oz virus. C_LIO_LISegment 5 of the Oz virus exhibits significantly lower promoter activity compared to other segments. C_LIO_LIThe distal duplex region formed by double-strand RNA at the genomic ends is essential for Oz virus RNA synthesis. C_LIO_LIPromoter activities in six-segmented RNA viruses are inherently variable among segments. C_LI

microbiology↗

bioRxiv2023

Acto3D: user- and budget-friendly software for multichannel high-resolution three-dimensional imaging

Advances in fluorescence microscopy and tissue-clearing technology have revolutionized three-dimensional (3D) imaging of fluorescently labeled tissues, organs, and embryos. However, the complexity and high cost of existing software and computer solutions for such imaging limit its widespread adoption by researchers with limited resources. We here introduce Acto3D as a user- and budget-friendly, open-source computer software application designed to streamline the generation and observation of high-resolution 3D images of targets labeled with multiple fluorescent probes. Acto3D features an intuitive interface that simplifies the importation, visualization, and analysis of data sets, has an associated tool for annotation of vascular lumens, and incorporates multiple fluorescence channels for comprehensive imaging. Underpinned by an integrated graphics processing unit, Acto3D allows accurate image reconstruction and efficient data processing without the need for expensive high-performance computers. We validated the software by imaging mouse embryonic structures. Acto3D thus constitutes a cost-effective and efficient platform to support biological research.

developmental biology↗

bioRxiv2023

Ciliary protein CEP290 regulates focal adhesion via microtubule system in non-ciliated cells

Almost all differentiated mammalian cells have primary cilia on their surface. Ciliary dysfunction causes ciliopathy in humans. Centrosomal protein 290 (CEP290) is a ciliary protein that causes ciliopathies, localizes at the cilial base in ciliated cells, whereas it localizes to the centrosome in non-ciliated proliferating cells. The cilia-dependent function of CEP290 has been extensively studied; however, the cilia-independent function, which is likely responsible for the wider phenotypic spectra of CEP290-related ciliopathies, remains largely unknown. Here, we examined cilia-independent functions of CEP290 in non-ciliated cells. Our study showed that Cep290 function loss suppresses microtubule elongation due to microtubule organizing center malfunction. Surprisingly, CEP290 forms a complex with the adenomatous polyposis coli (APC) protein encoded by the adenomatous polyposis coli gene. The APC-CEP290 complex exists in the centrosome and on microtubule fibers. Notably, the reduced focal adhesion formation is likely responsible for the Cep290 mutant phenotypes, including impaired directed cell migration, shrunken cell shape, and reduced adhesive capacity to the extracellular matrix. The APC-CEP290 complex is consistently important for transporting a focal adhesion molecule, paxillin, to focal adhesions in non-ciliated cells. Thus, our findings provide a novel platform to better understand the ciliopathies.

cell biology↗

bioRxiv2022

Requirement of sequential hydrolysis by CD73 and ALP for uptake of vitamin B2 into cells

Extracellular hydrolysis of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) to riboflavin is thought to be important for cellular uptake of vitamin B2 because FAD and FMN are hydrophilic and do not pass the plasma membrane. However, it is not clear whether FAD and FMN are hydrolyzed by cell surface enzymes for vitamin B2 uptake. Here, we show that in human cells, FAD, a major form of vitamin B2 in plasma, is hydrolyzed by CD73 (also called ecto-5' nucleotidase) to FMN, then FMN is hydrolyzed by alkaline phosphatase to riboflavin, which is efficiently imported into cells. This process is impaired on the surface of glycosylphosphatidylinositol (GPI)-deficient cells due to lack of these GPI-anchored enzymes. During culture of GPI-deficient cells with FAD or FMN, hydrolysis of these forms of vitamin B2, intracellular levels of vitamin B2, vitamin B2-dependent pyridoxal 5'-phosphate formation, and mitochondrial functions were significantly decreased compared with those in GPI-restored cells. These results suggest that inefficient uptake of vitamin B2 might account for mitochondrial dysfunction seen in some cases of inherited GPI deficiency.

biochemistry↗

bioRxiv2022

AT-hook DNA-binding motif-containing protein one knockdown downregulates EWS-FLI1 transcriptional activity in Ewings sarcoma cells

Ewings sarcoma is the second most common bone malignancy in children or young adults and is caused by an oncogenic transcription factor by a chromosomal translocation between the EWSR1 gene and the ETS transcription factor family. However, the transcriptional mechanism of EWS-ETS fusion proteins is still unclear. To identify the transcriptional complexes of EWS-ETS fusion transcription factors, we applied a proximal labeling system called BioID in Ewings sarcoma cells. We identified AHDC1 as a proximal protein of EWS-ETS fusion proteins. AHDC1 knockdown showed a reduced cell growth and transcriptional activity of EWS-FLI1. AHDC1 knockdown also reduced BRD4 and BRG1 protein levels, both known as interacting proteins of EWS-FLI1. In addition, AHDC1 co-localized with BRD4. Our results suggest that AHDC1 supports cell growth through EWS-FLI1.

cancer biology↗

bioRxiv2022

Emergence of hyper insecticide-resistant dengue vectors in Indochina Peninsula: threats of concomitant knockdown resistance mutations

Aedes aegypti (Linnaeus, 1762) is the main mosquito vector for dengue and other arboviral infectious diseases. Control of this important vector highly relies on the use of insecticides, especially pyrethroids. Nevertheless, the development of pyrethroid resistance is a major obstacle to mosquito/disease control worldwide. Here, we focused on the mutations in the target site of pyrethroid insecticides, voltage-sensitive sodium channel (Vssc), and found that Ae. aegypti collected from Vietnam has the L982W allele in the Vssc at a high frequency (>79%). L982W mutation is located in the highly conserved region of Vssc that is associated with sodium-ion selectivity and permeation rate. Strains having the L982W allele showed similar or even higher levels of resistance to pyrethroids than those having V1016G, a typical knockdown resistance allele in Asia. Furthermore, concomitant mutations L982W+F1534C and V1016G+F1534C were confirmed, and strains having these multiple Vssc mutations exhibited incomparably higher levels of pyrethroid resistance than any other field population ever reported. Molecular modeling analysis confirmed that these concomitant mutant alleles could interfere with approaching pyrethroid to Vssc. Remarkably, >90% of Vssc of Ae. aegypti were occupied by these hyper insecticide-resistant haplotypes in Phnom Penh city, Cambodia. Analysis of whole Vssc coding genes suggested that Vsscs have evolved into stronger resistant forms efficiently through gene recombination events. At this point, L982W has never been detected in Vssc of Ae. aegypti from any other neighboring countries. We strongly emphasize the need to be vigilant about these strong resistance genes spreading to the world through Indochina Peninsula. Significance StatementThe high frequency (>78%) of the L982W allele was detected at the target site of the pyrethroid insecticide, the voltage-sensitive sodium channel (Vssc) of Aedes aegypti collected from Vietnam and Cambodia. Haplotypes having concomitant mutations L982W+F1534C and V1016G+F1534C were also confirmed in both countries, and their frequency was high (>90%) in Phnom Penh, Cambodia. Strains having these haplotypes exhibited substantially higher levels of pyrethroid resistance than any other field population ever reported. The L982W mutation has never been detected in any country of the Indochina Peninsula except Vietnam and Cambodia, but it may be spreading to other areas of Asia, which can cause an unprecedentedly serious threat to the control of dengue fever as well as other Aedes-borne infectious diseases.

neuroscience↗

bioRxiv2021

Water droplet-in-oil digestion method for single-cell proteomics

Recent advances in single-cell proteomics highlight the promise of sensitive analyses in limited cell populations. However, technical challenges remain for sample recovery, throughput, and versatility. Here, we first report a water droplet-in-oil digestion (WinO) method based on carboxyl-coated beads and phase transfer surfactants for proteomic analysis using limited sample amounts. This method was developed to minimize the contact area between the sample solution and the container to reduce the loss of proteins and peptides by adsorption. This method increased protein and peptide recovery 10-fold as well as the number of quantified transmembrane proteins compared to an in-solution digestion (ISD) method. The proteome profiles obtained from 100 cells using the WinO method highly correlated with those from 10000 cells using the ISD method. We successfully applied the WinO method to single-cell proteomics and quantified 462 proteins. Using the WinO method, samples can be easily prepared in a multi-well plate, making it a widely applicable and suitable method for single-cell proteomics.

systems biology↗

bioRxiv2021

Myosin phosphatase target subunit 1 governs integrity of the embryonic gut epithelium to circumvent atresia development in medaka, Oryzias latipes

Congenital intestinal atresia (IA) is a birth defect characterised by the absence or closure of part of the intestine. Although genetic factors are implicated, mechanistic understanding has been hindered by the lack of suitable animal models. Here, we describe a medaka (Oryzias latipes) mutant, generated by N-ethyl-N-nitrosourea (ENU) mutagenesis, that develops IA during embryogenesis. Positional cloning identified a nonsense mutation in mypt1, encoding myosin phosphatase target subunit 1. Mutant embryos exhibited ectopic accumulation of F-actin and phosphorylated myosin regulatory light chain (Mrlc) in the intestinal epithelium, consistent with disrupted actomyosin regulation. These cytoskeletal abnormalities were accompanied by epithelial disorganisation without notable alterations in cell proliferation, motility, or apoptosis. Inhibition of myh11a, encoding smooth muscle (SM) myosin heavy chain, ameliorated the IA phenotype but Blebbistatin treatment completely rescued the defect, suggesting a non-contractile role prior to SM maturation. Together, these findings demonstrate that mypt1 loss disrupts intestinal morphogenesis through actomyosin dysregulation. Given the recent clinical identification of IA associated with MYPT1 mutations, this medaka model offers a valuable platform to investigate the developmental and molecular basis of MYPT1-associated IA in human.

developmental biology↗