Biology subjects

Hirano, H.

Publications and source records attributed to Hirano, H..

8 recordsLinked to original sources

bioRxiv2025

Population-scale chemical response revealed by a barcoded yeast collection

Natural genetic variation shapes how microbial populations adapt to environmental and chemical challenges, but scalable approaches to map genotype-phenotype relationships across diverse genetic backgrounds remain limited. Here, we developed a systematically barcoded collection of 520 Saccharomyces cerevisiae natural isolates that captures the ecological, geographical and genetic diversity of the species. Using pooled barcode sequencing, we profiled fitness responses to over 600 bioactive and natural compounds, revealing broader and more polarized bioactivity than the standard yeast gene-deletion collection. Fitness-based clustering defined six major compound groups with reproducible, population-structured sensitivity patterns. Genome-wide association analysis identified significant genetic variants across 107 compounds, linking natural polymorphisms to chemical responses and involving genes in genome maintenance, ribosome biogenesis, vesicular trafficking and stress tolerance. Together, our barcoded natural population provides a scalable framework for chemical-genetic screening, enabling systematic dissection of how genetic diversity shapes microbial fitness and adaptation.

evolutionary biology↗

bioRxiv2025

Structural basis of lipid-mediated gating in a two-pore domain potassium channel

TOK1 is the first identified member of the K2P channel family and contains additional N- and C-terminal domains, displaying a configuration distinct from that of canonical K2P channels. Although recent advances in structural studies of K2P channels have elucidated their architectures, the structural basis of TOK1 has remained unknown, limiting our understanding of its unique configuration. Here, we present the cryo-electron microscopy (cryo-EM) structure of TOK1, unveiling its distinctive domain architecture. Furthermore, the structures of TOK1 in three distinct states provide mechanistic insights into its regulation through lipid binding and dissociation. Phosphorylation of TOK1 induces the formation of an additional lipid-binding site, leading to channel inactivation. Conversely, upon activation, the phospholipid dissociates, allowing ion permeation. Our comprehensive study, integrating cryo-EM structural analysis, molecular dynamics simulations, electrophysiological recordings, and mass spectrometry, elucidates the distinctive features of TOK1, an atypical K2P channel, and provides a framework for understanding lipid-mediated regulation within this family.

molecular biology↗

bioRxiv2025

Microstructural architecture of the argonauts' shell-like eggcase reveals the formation logic of a convergent extended phenotype

Argonautid octopods of the genus Argonauta possess a shell-like biomineralized external structure called an eggcase. A classical behavioral observation suggested that this structure is produced by the first dorsal arms rather than by the mantle tissue as seen in typical molluscan shells. In this study, we performed detailed microstructural analyses using scanning electron microscopy to investigate the characteristics of normal, undamaged eggcases and regions that had undergone post-damage repair. Our analysis revealed that the eggcase comprises five layers: an outermost organic membrane, an outer spherulitic-fibrous prismatic layer, a middle organic layer, an inner spherulitic-fibrous prismatic layer, and an innermost organic membrane. Both prismatic layers exhibit bidirectional growth from the middle organic layer, a unique feature not observed in typical molluscan shells. We propose a four-stage formation sequence to explain the observed microstructural architecture: nucleation on the organic scaffold, bidirectional crystal growth from the organic mid-layer, perpendicular growth relative to the eggcase surface, and final encapsulation by an organic membrane. Interestingly, the growth pattern and crystal shapes in the prismatic layers resemble of the internalized shells of cuttlefish, the calcified skeleton of stony corals, and avian eggshells, suggesting functional convergence. We also identified two repair mechanisms: reattachment of broken fragments and regeneration via new secretions. These findings call into question the previous assumptions about the role of the first dorsal arms in calcification. The eggcase also exemplifies the formation of a functionally and structurally complex extended phenotype through convergence.

zoology↗

bioRxiv2023

Quinoclamine inhibits Shiga toxin production in enterohemorrhagic Escherichia coli

ObjectivesEnterohemorrhagic Escherichia coli (EHEC) is responsible for the most severe symptoms of E. coli infections, including hemorrhagic colitis and hemorrhagic uremic syndrome. Shiga toxin 2 (Stx2) plays a significant role as a major virulence factor. The genes encoding Stx2 locate in lambda-like prophage on the EHEC genome. Consequently, Stx2 is expressed when production of the phage is induced by the SOS response. Antibiotic treatment is not recommended for curing the bacterial infection, because it is associated with severe hemorrhagic uremic syndrome. If Stx2 production is prevented, EHEC pathogenicity significantly decreases, and antibiotics may be available to treat the infection. MethodsWe conducted two independent screenings to identify Stx2 production inhibitors for libraries from the RIKEN Natural Product Depository (NPDepo); namely, screening of the Authentic Library, and two-round screening of the Pilot and Analog Libraries. ResultsThe screening of Authentic Library identified niclosamide as a Stx2 production inhibitor. Besides, two naphthoquinoids were identified after the two-round of screening of the Pilot and Analog Libraries. Niclosamide, and quinoclamine, which has structure shared in the two naphthoquinoids, prevented cell lysis via the phage production and ceased Stx2 production in EHEC. The SOS reporter assay indicated that quinoclamine prevented the SOS response in E. coli, whereas niclosamide did not. ConclusionsThese findings suggest that quinoclamine inhibited Stx2 production by preventing the SOS response, whereas niclosamide was involved in phage propagation following the SOS response. These compounds can be a potential therapeutic option to treat EHEC infections.

microbiology↗

bioRxiv2023

Time-deterministic cryo-optical microscopy

We developed a technique to rapidly freeze biological systems during observation under an optical microscope, which allows for detailed optical observation at a precise time in the biological dynamics at high spatial resolution and quantifiability under low-temperature conditions. Our study further found that ion distributions and molecular conformations can be fixed in space and time, demonstrated by visualization of calcium waves rapid-frozen at an instant using fluorescent ion indicators. These results show that time-deterministic cryo-optical microscopy can suspend cellular dynamics while maintaining molecular and ionic states, allowing us to view cellular dynamics in a way that has not been possible before. One-Sentence SummaryRapid freezing of dynamic biological systems during optical observation presents time information to the fixed samples

biophysics↗

bioRxiv2022

Identification of a novel family of benzimidazole species-selective Complex I inhibitors as potential anthelmintics

Soil-transmitted helminths (STHs) including Ascaris, hookworm, and whipworm are major human pathogens infecting over a billion people worldwide1,2. There are few existing classes of anthelmintics and resistance is increasing3-5 -- there is thus an urgent need for new classes of these drugs. Here we focus on identifying compounds that interfere with the unusual anaerobic metabolism that STHs use to survive the highly hypoxic conditions of the host gut6-9. This requires rhodoquinone (RQ), a quinone electron carrier that is not made or used by the STH hosts10. We previously showed that C. elegans also uses this rhodoquinone-dependent metabolism (RQDM)11 and established a high throughput assay for RQDM11. We screened a collection of 480 natural products for compounds that kill worms specifically when they rely on RQDM -- these 480 are representatives of a full library of ~25,000 natural products and derivatives12,13. We identify several classes of compound including a novel family of species selective inhibitors of Complex I. These Complex I inhibitors are based on a benzimidazole core but unlike commercial benzimidazole anthelmintics they do not target microtubules14-17. We screened over 1,200 benzimidazoles and identify the key structural requirements for species selective Complex I inhibition. We suggest that these novel benzimidazole species-selective Complex I inhibitors may be potential anthelmintics.

molecular biology↗

bioRxiv2022

Plant-based Production and Characterization of a Promising Fc-fusion Protein against Bone Mass Density Loss

Microgravity-induced bone loss is a main obstacle for long term space missions as it is difficult to maintain bone mass when loading stimuli is reduced. With a typical bone mineral density loss of 1.5% per month of microgravity exposure, the chances for osteoporosis and fractures may endanger astronauts health. Parathyroid Hormone or PTH (1-34) is an FDA approved treatment for osteoporosis, and may reverse microgravity-induced bone loss. However, PTH proteins requires refrigeration, daily subcutaneous injection, and have a short shelf-life, limiting its use in a resource-limited environment, like space. In this study, PTH was produced in an Fc-fusion form via transient expression in plants, to improve the circulatory half-life which reduces dosing frequency and to simplify purification if needed. Plant-based expression is well-suited for space medicine application given its low resource consumption and short expression timeline. The PTH-Fc accumulation profile in plant was established with a peak expression on day 5 post infiltration of 373 {+/-} 59 mg/kg leaf fresh weight. Once the PTH-Fc was purified, the amino acid sequence and the binding affinity to its target, PTH 1 receptor (PTH1R), was determined utilizing biolayer interferometry (BLI). The binding affinity between PTH-Fc and PTH1R was 2.30 x 10-6 M, similar to the affinity between PTH (1-34) and PTH1R (2.31 x 10-6 M). Its function was also confirmed in a cell-based receptor stimulation assay, where PTH-Fc was able to stimulate the PTH1R producing cyclic adenosine monophosphate (cAMP) with an EC50 of (8.54 {+/-} 0.12) x 10-9 M, comparable to the EC50 from the PTH (1-34) of 1.49 x 10-8 M. These results suggest that plant recombinant PTH-Fc exhibits a similar potency compared to PTH. Furthermore, it can be produced rapidly at high levels with minimal resources and reagents, making it ideal for production in low resource environments such as space.

synthetic biology↗

bioRxiv2017

Functional Annotation of Chemical Libraries across Diverse Biological Processes

Chemical-genetic approaches offer the potential for unbiased functional annotation of chemical libraries. Mutations can alter the response of cells to a compound, revealing chemical-genetic interactions that can elucidate a compounds mode of action. We developed a highly parallel and unbiased yeast chemical-genetic screening system involving three key components. First, in a drug-sensitive genetic background, we constructed an optimized, diagnostic mutant collection that is predictive all major yeast biological processes. Second, we implemented a multiplexed (768-plex) barcode sequencing protocol, enabling assembly of thousands of chemical-genetic profiles. Finally, based on comparison of the chemical-genetic profiles with a compendium of genome-wide genetic interaction profiles, we predicted compound functionality. Applying this high-throughput approach, we screened 7 different compound libraries and annotated their functional diversity. We further validated biological process predictions, prioritized a diverse set of compounds, and identified compounds that appear to have dual modes of action.

systems biology↗