Biology subjects

Sakaguchi, M.

Publications and source records attributed to Sakaguchi, M..

11 recordsLinked to original sources

bioRxiv2025

Genotype-first approach revealed unrecognized breed-specific genetic diseases in dogs

Numerous dogs are affected by genetic diseases because of their unique breeding situation. The identification of pathogenic variants in dogs not only has veterinary benefits but also leads to the development of new human disease models. However, fewer pathogenic variants have been identified in dogs than in humans, and standard genetic approaches, such as large-scale case/control studies and family analyses, often fail to identify novel causative variants in dogs. To address this, we applied a genotype-first approach by developing a multiplex polymerase chain reaction (PCR)-based targeted sequencing method for dogs, analyzing 203 genes--most of which are related to human disease--from 6,333 dogs collected from a veterinary hospital, and characterizing pathogenic variant carriers. In total, 120 pathogenic variants in 83 genes were identified. Frequent pathogenic variants were enriched in specific dog breeds. Dogs homozygous for a pathogenic CHEK2 variant, frequently identified in French Bulldogs (5.74%, homozygous), showed a five-fold increased risk of cancer (P = 3.45 x 10-2). A pathogenic NOD2 variant was detected in half of the Italian Greyhounds. However, their medical records did not document the typical clinical symptoms observed in humans with NOD2 deficiency. Some pathogenic variants were present in three or fewer dogs, and 11.0% of these dogs exhibited symptoms potentially associated with gene defects. These rare variants could become common within a short period due to the canine breeding system to induce breed-specific genetic diseases. These findings suggest that the genotype-first approach successfully identified unrecognized breed-specific genetic diseases. Further characterization of dogs with pathogenic variants enable to define clinical diagnosis, providing an important spontaneous model for human genetic diseases.

genomics↗

bioRxiv2025

Independent component analysis disentangles fluorescence signals from diffusing single molecules

Multiparameter measurements offer new possibilities of single-molecule fluorescence experiments for detecting and quantifying heterogeneity in freely diffusing molecules. However, data analysis remains challenging due to limited photon rates from individual molecules. Here, we present independent fluorescence component analysis (IFCA), a universal analytical framework that applies independent component analysis (ICA) to unmix multiparameter fluorescence signals. In this work, we developed an efficient algorithm of ICA for multiparameter photon data based on third-order cumulant tensor decomposition. To validate its performance, we applied IFCA to a static mixture of fluorescent dyes and found that IFCA enables robust separation of signals from five species in a nanomolar mixture solution with a 5 {micro}s binning time. We further analyzed a photon dataset from a FRET-labeled DNA construct using IFCA, demonstrating its capability to quantitatively characterize signals from subpopulations exchanging on a sub-millisecond time scale in a model-independent manner. These findings highlight the high ability of IFCA for quantitative analysis of complex molecular systems exploiting these advantages.

biophysics↗

bioRxiv2024

T-scope V4: miniaturized microscope for optogenetic tagging in freely behaving animals

A miniaturized microscope (i.e., miniscope) enables the imaging of neuronal activity using calcium sensors while simultaneously manipulating that activity using opsins in freely moving animals. However, many miniscopes use light-emitting diodes with broadband emission, leading to unintentional opsin stimulation by light intended solely for calcium sensor activation (a phenomenon referred to as "biological crosstalk"). To address this issue, we previously developed a miniscope including a port for chosen light sources, such as lasers, by restructuring the open-source UCLA Miniscope v3. However, targeting the same neuronal soma for both excitable opsin stimulation and calcium sensor imaging remained a challenge. Here, we integrated features from the UCLA Miniscope v4 into our new T-scope V4 miniscope. In optogenetic tagging experiments, we demonstrated that a 445-nm blue laser can be used to image neuronal activity with the calcium sensor GCaMP6s without inadvertently stimulating the ChrimsonR opsin, allowing for simultaneous neuronal activity imaging and manipulation in freely moving mice. Thus, the T-scope V4 can serve as a powerful tool for probing causal relationships between neuronal activity and its function in living animals. HighlightsO_LIWe developed the T-scope V4 that integrates features of the UCLA Miniscope v4 C_LIO_LIThis miniscope prevents "biological crosstalk" between sensors and opsins C_LIO_LIThis tool can help probe causal brain-behavior relationships in living animals C_LI

neuroscience↗

bioRxiv2024

ves1α genes expression is the major determinant of Babesia bovis-infected erythrocytes cytoadhesion to endothelial cells

Babesia bovis causes the most pathogenic form of babesiosis in cattle, resulting in high mortality in naive adults. This parasite invades red blood cells (RBCs) within the bovine hosts where they multiply and produce clinical disease. Babesia bovis exports numerous proteins into invaded RBCs changing its properties. Thus, the infected RBCs (iRBCs) are capable to cytoadhere in the microvasculature of internal organs and brain, leading to respiratory distress, neurologic signs, and mortality. Variant Erythrocyte Surface Antigen 1 (VESA1) is one of those exported proteins by B. bovis which represents a major virulence factor due to its central role in immune evasion by antigenic variation and intravascular parasite sequestration. VESA1 is a heterodimer protein encoded by ves1 and ves1{beta} multigene family and localized on the ridges, the focal point for cytoadhesion. To gain further insights into the molecular mechanisms of cytoadhesion of B. bovis, we panned the parasites with bovine brain microvasculature endothelial cells, which resulted in obtaining several clones with different cytoadherence abilities. The transcriptome analysis of 2 high and 2 low cytoadherent clones revealed that ves1 sequences were diversified, likely resulting from genomic recombination. On the other hand, ves1{beta} sequences were almost identical among these 4 clones. Insertion and expression of ves1 of a clone with high binding into ef-1 locus of a low binging clone increased cytoadherence confirming the role of ves1 suggested by our transcriptome data. Whole genome sequencing of cytoadherent clones revealed active locus of ves1 on chromosome 2. These results suggest that VESA1a proteins encoded by ves1 genes determine the cytoadherence specificity and/or cytoadherence strength of B. bovis and they are in the active site for recombination. Author summaryBabesia bovis is an apicomplexan intraerythrocytic protozoan parasite which causes the most pathogenic form of babesiosis in cattle. This pathogenicity is the result of parasite multiplication and cytoadherence of infected red blood cells (iRBCs) in the microvasculature of brain and internal organs and is mediated by B. bovis surface exposed ligand, Variant Erythrocyte Surface Antigen 1 (VESA1). Here using parasite panning assay, transcriptomics, and genetic tools, we showed that VESA1a is the main determinant of B. bovis cytoadhesion. VESA1 are large hypervariable proteins (>100kDa) consisting of VESA1a and VESA1b subunits encoded by ves1 and ves1{beta} multigene family. Panning B. bovis with bovine brain endothelial cells resulted in obtaining cytoadherent parasite clones with different binding abilities. Comparative transcriptome analysis revealed diversification of ves1 sequences. Insertion and expression of ves1 of a clone with high-binding ability in the genome of a low-binding clone increased cytoadherence confirming the role of ves1. Mapping RNA-seq on the genome of cytoadherent clones revealed the locus of active transcription and this locus was suggested to be the active site for recombination which promoted the production of variants of ves1 with different binding abilities. Altogether, our results provide new insights into B. bovis cytoadhesion and VESA1 biology.

microbiology↗

bioRxiv2024

Transcriptome Analysis Identified SPP1+ Monocytes as a Key in Extracellular Matrix Formation in Thrombi

Thrombi follow various natural courses. They are known to become harder over time and may persist long-term; some of them can also undergo early spontaneous dissolution and disappearance. Hindering thrombus stability may contribute to the treatment of thrombosis and the prevention of embolisms. However, the detailed mechanisms underlying thrombus maturation remain unknown. Using RNA sequencing, we revealed the transcriptional landscape of thrombi retrieved from the cerebral vessels and identified SPP1 as a hub gene related to extracellular matrix formation. Immunohistochemistry confirmed the expression of osteopontin in monocytes/macrophages in the thrombi, particularly in older thrombi. Single-cell RNA sequencing of thrombi from the pulmonary artery revealed increased communication between SPP1-high monocytes/macrophages and fibroblasts. These data suggest that SPP1-high monocytes/macrophages play a crucial role in extracellular matrix formation in thrombi and provide a basis for new antithrombotic therapies targeting thrombus maturation. TeaserSPP1+ monocytes play a key role in thrombus maturation, which can be a potential target for novel antithrombotic therapies.

molecular biology↗

bioRxiv2024

Critical role of Babesia bovis spherical body protein 3 in ridge formation on infected red blood cells

Babesia bovis, an apicomplexan intraerythrocytic protozoan parasite, causes serious economic loss to cattle industries around the world. Infection with this parasite leads to accumulation of infected red blood cells (iRBCs) in the brain microvasculature that results in severe clinical complications known as cerebral babesiosis. Throughout its growth within iRBCs, the parasite exports various proteins to the iRBCs that lead to the formation of protrusions known as "ridges" on the surface of iRBCs, which serve as sites for cytoadhesion to endothelial cells. Spherical body proteins (SBPs; proteins secreted from spherical bodies, which are organelles specific to Piroplasmida) are exported into iRBCs, and four proteins (SBP1-4) have been reported to date. In this study, we elucidated the function of SBP3 using an inducible gene knockdown (KD) system. Localization of SBP3 was assessed by immunofluorescence assay, and only partial colocalization was detected between SBP3 and SBP4 inside the iRBCs. In contrast, colocalization was observed with VESA-1, which is a major parasite ligand responsible for the cytoadhesion. Immunoelectron microscopy confirmed localization of SBP3 at the ridges. SBP3 KD was performed using the glmS system, and effective KD was confirmed by Western blotting, immunofluorescence assay, and RNA-seq analysis. The SBP3 KD parasites showed severe growth defect suggesting its importance for parasite survival in the iRBCs. VESA-1 on the surface of iRBCs was scarcely detected in SBP3 KD parasites, whereas SBP4 was still detected in the iRBCs. Moreover, abolition of ridges on the iRBCs and reduction of iRBCs cytoadhesion to the bovine brain endothelial cells were observed in SBP3 KD parasites. Immunoprecipitation followed by mass spectrometry analysis detected the host Band 3 multiprotein complex, suggesting an association of SBP3 with iRBC cytoskeleton proteins. Taken together, this study revealed the vital role of SBP3 in ridge formation and its significance in the pathogenesis of cerebral babesiosis. Author summaryBabesia bovis causes a high-mortality complication called cerebral babesiosis in cattle, similar to cerebral malaria in humans. Both complications are caused by the cytoadhesion of infected red blood cells (iRBCs) to the host brain endothelial cells. These parasites export numerous proteins to the host iRBCs and produce protrusions on the iRBCs that are called ridges for B. bovis and knobs for Plasmodium falciparum. Ridges and knobs play an important role in cytoadhesion as they are the sites of adherence; however, the molecules responsible for ridge formation remain unknown. In this study, we showed that SBP3 is a crucial protein for ridge formation. The SBP3 knockdown parasites showed severe growth defects and abolition of ridges on the iRBCs, and cytoadhesion of iRBCs to the bovine brain endothelial cells was significantly reduced. An immunoprecipitation experiment suggested an association of SBP3 with the host Band 3 multiprotein complex. Although there is no similarity in amino acid sequences, we suggest SBP3 to be a functional ortholog of KAHRP in P. falciparum. In summary, our results shed light on the molecular mechanism of ridge formation and the pathogenesis of B. bovis.

microbiology↗

bioRxiv2023

The CaliAli tool for long-term tracking of neuronal population dynamics in calcium imaging

Neuron-tracking algorithms exhibit suboptimal performance in calcium imaging when the same neurons are not consistently detected, as unmatched features hinder intersession alignment. CaliAli addresses this issue by employing an alignment-before-extraction strategy that incorporates vasculature information to improve the detectability of weak signals and maximize the number of trackable neurons. By excelling in neural remapping and high spatial overlap scenarios, CaliAli paves the way toward further understanding long-term neural network dynamics.

neuroscience↗

bioRxiv2023

Structure and its transformation of elliptical nege-like virus Tanay virus

Negeviruses that infect insects are recently identified virus species that are phylogenetically related to several plant viruses. They exhibit a unique virion structure, an elliptical core with a short projection. Negeviruses encode two structural proteins, a glycoprotein that forms a short projection, and an envelope protein that forms an elliptical core. The glycoprotein has been reported only in the negeviruses genes, and not in phylogenetically related plant viruses genes. In this report, we first describe the three-dimensional electron cryo-microscopy (cryo-EM) structure of Tanay virus (TANAV), one of the nege-like viruses. TANAV particle demonstrates a periodical envelope structure consisting of three layers surrounding the centered viral RNA. The elliptical core dynamically changes its shape under acidic and even low detergent conditions to form bullet-like or tubular shapes. The further cryo-EM studies on these transformed TANAV particles reveal their overall structural rearrangement. These findings suggest putative geometries of TANAV and its transformation in the life cycle, and the potential importance of the short projection for enabling cell entry to the insect hosts. Impact statementNegeviridae has recently been declared as a virus family that includes virus species exhibiting a unique particle structure that differs from other known viruses. They are known to be common mosquito viruses isolated around the world, but also phylogenetically related to several plant viruses that impair crop production. Therefore, the negeviruses may also play a role in plant ecosystems that threaten agriculture. However, the mechanism of infection and assembly of the negeviruses as well as their structure were unknown. In this study, intact and dissociated structures of the TANAV were first examined using cryo-EM single particle analysis (SPA) and electron cryo-tomography (cryo-ET). These results reveal new structural geometries of the TANAV particle and its dynamic transformation under acidic and even low-detergent conditions, providing new insights into the infection and assembly mechanism in negeviruses.

biophysics↗

bioRxiv2020

Preclinical study of DNA vaccines targeting SARS-CoV-2

To fight against the worldwide COVID-19 pandemic, the development of an effective and safe vaccine against SARS-CoV-2 is required. As potential pandemic vaccines, DNA/RNA vaccines, viral vector vaccines and protein-based vaccines have been rapidly developed to prevent pandemic spread worldwide. In this study, we designed plasmid DNA vaccine targeting the SARS-CoV-2 Spike glycoprotein (S protein) as pandemic vaccine, and the humoral, cellular, and functional immune responses were characterized to support proceeding to initial human clinical trials. After intramuscular injection of DNA vaccine encoding S protein with alum adjuvant (three times at 2-week intervals), the humoral immunoreaction, as assessed by anti-S protein or anti-receptor-binding domain (RBD) antibody titers, and the cellular immunoreaction, as assessed by antigen-induced IFN{gamma} expression, were up-regulated. In IgG subclass analysis, IgG2b was induced as the main subclass. Based on these analyses, DNA vaccine with alum adjuvant preferentially induced Th1-type T cell polarization. We confirmed the neutralizing action of DNA vaccine-induced antibodies via two different methods, a binding assay of RBD recombinant protein with angiotensin-converting enzyme 2 (ACE2), a receptor of SARS-CoV-2, and pseudovirus assay. Further B cell epitope mapping analysis using a peptide array showed that most vaccine-induced antibodies recognized the S2 and RBD subunits, but not the S1 subunit. In conclusion, DNA vaccine targeting the spike glycoprotein of SARS-CoV-2 might be an effective and safe approach to combat the COVID-19 pandemic.

immunology↗

bioRxiv2020

Epitope Prediction of Antigen Protein using Attention-Based LSTM Network

B-cells inducing antigen-specific immune responses in vivo produce large amounts of antigen-specific antibodies by recognizing the subregions (epitope regions) of antigen proteins. They can inhibit their functioning by binding antibodies to antigen proteins. Predicting of epitope regions is beneficial for the design and development of vaccines aimed to induce antigen-specific antibody production. However, prediction accuracy requires improvement. The conventional epitope region prediction methods have focused only on the target sequence in the amino acid sequences of an entire antigen protein and have not thoroughly considered its sequence and features as a whole. In the present paper, we propose a deep learning method based on short-term memory with an attention mechanism to consider the characteristics of a whole antigen protein in addition to the target sequence. The proposed method achieves better accuracy compared with the conventional method in the experimental prediction of epitope regions using the data from the immune epitope database.

bioinformatics↗

bioRxiv2018

Potential Application of Bacteriophages in Enrichment Culture for Improved Prenatal Streptococcus agalactiae Screening

Vertical transmission of Streptococcus agalactiae can cause neonatal infections. A culture test in the late stage of pregnancy is used to screen for the presence of maternal S. agalactiae for intrapartum antibiotic prophylaxis. For the test, vaginal-rectal swab sampling is immediately followed by enrichment culture and bacterial identification. In some cases, Enterococcus faecalis competes with and overgrowths S. agalactiae in the enrichment culture. Consequently, the identification test occasionally yields false-negative results. Bacterial viruses, bacteriophages (phages), infect and kill specific host bacteria. In the current study, we explored the feasibility of using phages to minimize the undesirable E. faecalis outgrowth and facilitate S. agalactiae detection in an experimental setting. Phage mixture was prepared using three phages that specifically infect E. faecalis: phiEF24C, phiEF17H, and phiM1EF22. The mixture inhibited the growth of 86.7% (26/30) of E. faecalis strains tested in the enrichment broth. When single strains of E. faecalis and S. agalactiae were inoculated in the enrichment broth containing the phage mixture, bacterial growth was inhibited or facilitated, respectively. Further, several sets of S. agalactiae and E. faecalis strains were co-cultured, and bacteria were detected on chromogenic agar after the enrichment culture. S. agalactiae was dominant after plating a phage mixture-treated co-culture, while it was barely detected after plating the untreated co-culture. Considering these observations, the phage mixture can be employed in the S. agalactiae culture test to increase test accuracy.

microbiology↗