Biology subjects

Uchiyama, J.

Publications and source records attributed to Uchiyama, J..

7 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

Novel Escherichia coli Phages Representing a Distinct Genus within Stephanstirmvirinae: Genome and Host Range Characteristics

Bacteriophages play crucial roles in microbial ecosystems and have potential biotechnological applications. However, our understanding of culturable phages remains limited. This study characterized six novel Escherichia coli phages isolated from pig farm wastewater and urban sewage using comprehensive genomic, morphological, and host-range analyses. Using multiple comparative approaches, including gene-sharing network analysis, average nucleotide identity (ANI), and nucleotide intergenomic similarity (NIS), we demonstrated that five of these phages form a distinct group within the subfamily Stephanstirmvirinae, potentially representing a novel genus provisionally named "Wecvirus". These phages were further classified into two distinct species within the proposed genus, each of which exhibits a unique host range pattern. This host specificity is reflected in the species-specific differences in the amino acid sequences of tail fibers, which are crucial for infection. The remaining phage, which was not classified as Wecvirus exhibited characteristics that challenged the current classification criteria, highlighting the need for more flexible taxonomic approaches. Our findings expand the understanding of phage diversity within Stephanstirmvirinae and contribute to the evolving phage taxonomy framework. ImportanceThe rapid emergence of antibiotic-resistant pathogens necessitates the development of alternative antimicrobial strategies, with phage therapy showing promising potential. However, the successful implementation of phage-based treatments requires a comprehensive understanding of phage diversity and host-pathogen interactions. Our study expands the known diversity within Stephanstirmvirinae by characterizing novel E. coli phages that constitute a distinct genus. The identification of species-specific host recognition mechanisms within this genus provides insights into phage-host adaptation. These findings contribute to our understanding of phage taxonomy and evolution, while also offering practical implications for the development of phage-based therapeutics against pathogenic E. coli strains. This research highlights the importance of continued exploration of novel phages for both fundamental understanding and therapeutic applications.

evolutionary biology↗

bioRxiv2022

Bacterial threat assessment of bacteriophage infection is mediated by intracellular polyamine accumulation and Gac/Rsm signaling

When eukaryotic cells are killed by pathogenic microorganisms, damage-associated and pathogen-associated signals are generated that alert other cells of nearby danger. Bacteria can detect the death of their kin; however, how bacteria make threat assessments of cellular injury is largely unexplored. Here we show that polyamines released by lysed bacteria serve as damage-associated molecules in Pseudomonas aeruginosa. In response to exogenous polyamines, Gac/Rsm and cyclic-di-GMP signaling is activated and intracellular polyamine levels increase. In the absence of a threat, polyamines are catabolized, and intracellular polyamines return to basal levels, but cells infected by bacteriophage increase and maintain intracellular polyamine levels, which inhibits phage replication. Phage species not inhibited by polyamines did not trigger polyamine accumulation by P. aeruginosa, suggesting polyamine accumulation and metabolism are targets in the phage-host arms-race. Our results suggest that like eukaryotic cells, bacteria can differentiate damage-associated and pathogen-associated signals to make threat assessments of cellular injury. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/486733v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1f69334org.highwire.dtl.DTLVardef@13a4422org.highwire.dtl.DTLVardef@4f3edeorg.highwire.dtl.DTLVardef@a86c43_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2021

pSNAP: Proteome-wide analysis of elongating nascent polypeptide chains

Cellular global translation is often measured using ribosome profiling or quantitative mass spectrometry, but these methods do not provide direct information at the level of elongating nascent polypeptide chains (NPCs) and associated co-translational events. Here we describe pSNAP, a method for proteome-wide profiling of NPCs by affinity enrichment of puromycin- and stable isotope-labeled polypeptides. pSNAP does not require ribosome purification and/or chemical labeling, and captures bona fide NPCs that characteristically exhibit protein N-terminus-biased positions. We applied pSNAP to evaluate the effect of silmitasertib, a potential molecular therapy for cancer, and revealed acute translational repression through casein kinase II and mTOR pathways. We also characterized modifications on NPCs and demonstrated that the combination of different types of modifications, such as acetylation and phosphorylation in the N-terminal region of histone H1.5, can modulate interactions with ribosome-associated factors. Thus, pSNAP provides a framework for dissecting co-translational regulations on a proteome-wide scale.

biochemistry↗

bioRxiv2021

Novel roles of small extracellular vesicles in regulating the quiescence and proliferation of neural stem cells

Neural stem cells (NSCs) quiescence plays pivotal roles in securing sustainable neurogenesis and avoiding stemness exhaustion in the adult brain. The maintenance of quiescence and transition between proliferation and quiescence are complex processes associated with multiple niche signals, and environmental stimuli. Though the mechanisms of the transitions between NSC states have been extensively investigated, they remain to be fully elucidated. Exosomes are small extracellular vesicles (sEVs) containing functional units such as proteins, microRNAs, and mRNAs. It has already been demonstrated that sEVs actively participate in cancer cell proliferation and metastasis. However, the role of sEVs in NSC quiescence has not been investigated. Here, we applied proteomics to analyze the protein cargos of sEVs derived from proliferating, quiescent, and reactivating NSCs. Our findings revealed expression level fluctuations of NSCs sEV protein cargo at different proliferative conditions. We also identified functional clusters of gene ontology annotations from differentially expressed proteins in three sources of exosomes. Moreover, the use of exosome inhibitors revealed the contribution of exosomes to NSC quiescence at the entrance into quiescence, as well as in quiescence maintenance. Exosome inhibition delayed the entrance into quiescence by proliferating NSCs and allowed quiescent NSCs to exit from the G0 phase of the cell cycle. Protein translation was significantly upregulated in both quiescent NSCs and quiescent-induced NSCs via the exosome inhibition. Our results demonstrated that NSC exosomes are involved in regulating the quiescence of NSCs and provide a functional prediction of NSCs exosome protein cargos in terms of cell-cycle regulation and protein synthesis.

developmental biology↗

bioRxiv2020

Quantitative nascent proteome profiling by dual pulse labeling with O-propargyl-puromycin and stable isotope labeled amino acids

Monitoring translational regulation in response to environmental signals is crucial for understanding cellular proteostasis. However, only limited approaches are currently available for quantifying acute changes in protein synthesis induced by stimuli. Recently, a clickable puromycin analog, O-propargyl-puromycin (OPP), was developed and applied to label the C-termini of nascent polypeptide chains (NPCs). Following affinity purification via a click reaction, OPP allows for a proteomic analysis of NPCs. Despite its advantage, the affinity purification of NPCs using magnetic beads or resins inherently suffers from significant non-specific protein binding, which hinders accurate quantification of the nascent proteins. To address this issue, we employed dual pulse labeling of NPCs with both OPP and stable isotope labeled amino acids to distinguish bona fide NPCs from non-specific proteins, thereby enabling the accurate quantitative profiling of NPCs. We applied this method to dissecting the transcription-coupled translation responses and quantified ~3,000 nascent proteins. We found that the translation of a subset of ribosomal proteins (e.g., RPSA, RPLP0) as well as signaling proteins (e.g., BCAR3, EFNA1, DUSP1) was significantly repressed by transcription inhibition. Together, the present method provides an accurate and broadly applicable nascent proteome profiling for many biological applications at the level of translation.

biochemistry↗

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↗