Biology subjects

Koiwai, K.

Publications and source records attributed to Koiwai, K..

8 recordsLinked to original sources

bioRxiv2026

Optical flow reveals motility signatures for inferring pathogenic bacterial mixture compositions via temporal convolutional networks

With the rapid expansion of global food demand, aquaculture has become a critical pillar for future food security. However, aquaculture systems remain highly vulnerable to pathogenic bacteria, and rapid identification of antagonistic microbes is essential for sustainable disease control. Conventional evaluation approaches rely on fluorescence labeling or post-culture assays, limiting the ability to quantify dynamic interactions in mixed microbial populations in a real-time and label-free manner. Here, we propose a computational framework for classifying the mixing ratio of Vibrio harveyi and environmental bacteria using time-series motion features extracted from microscopy videos. We defined 24 interpretable motility descriptors and employed a Temporal Convolutional Network (TCN) to learn their temporal structure. The proposed method achieved a classification accuracy of 93.3%, outperforming conventional static statistical approaches and alternative machine learning models. These findings indicate that mixture discrimination in microbial communities is governed not by absolute motility magnitude, but by collective alignment and its temporal stability. Our study establishes a time-resolved computational framework for quantifying dynamic collective order in mixed microbial populations and highlights its potential for label-free automated screening and robotic microbiological applications. Author summaryBacterial infections pose a major threat to aquaculture, and rapid identification of antagonistic microbes is essential for sustainable disease management. Existing screening approaches often require fluorescent labeling or post-culture analysis, making real-time evaluation of mixed bacterial populations difficult. In this study, we show that mixture ratios of Vibrio harveyi and environmental bacteria can be accurately classified from time-series motion features extracted from microscopy videos. By applying TCN to 24 interpretable motility descriptors, we achieved high classification accuracy without relying on fluorescent markers. Our analysis demonstrates that collective directional alignment and its temporal stability, rather than absolute swimming speed, are the key determinants of mixture discrimination. This work introduces a computational strategy for quantifying dynamic collective order in microbial communities and supports the development of label-free, automated screening platforms for microbiological applications.

microbiology↗

bioRxiv2025

Chromosome-scale genome assembly of Malcolmia littorea using long-read sequencing and single-pollen genotyping technologies

Malcolmia littorea, a member of the family Brassicaceae, is adapted to coastal and sandy environments and has become a model in studies of reproductive barriers. However, genomic resources for the species are limited. Here, with the aim of understanding the molecular mechanisms underlying key traits in M. littorea, including its survival under harsh conditions, we present a de novo genome assembly consisting of 10 chromosome-scale sequences. We employed a high-fidelity long-read sequencing technology for genome assembly. To anchor the sequences to chromosomes, we developed a single-pollen genotyping method to construct a genetic linkage map based on SNPs derived from transcriptomes of pollen grains, possessing recombinant haploid genomes. We built a genome assembly consisting of 10 chromosome-scale sequences (214 Mb in total) for M. littorea containing 30,861 predicted genes. A comparative genome analysis and gene prediction indicated that the genome of M. littorea is double the size of the Arabidopsis thaliana genome, consistent with a whole-genome duplication followed by gene subfunctionalization and/or neofunctionalization in M. littorea. This study provides a basis for research on M. littorea, an understudied species with ecological and evolutionary significance.

genomics↗

bioRxiv2023

Structural and functional insights into the complex formation of a trimeric autotransporter adhesin with a peptidoglycan-binding periplasmic protein

Trimeric autotransporter adhesins (TAAs) are outer membrane (OM) proteins that are widely distributed in gram-negative bacteria and are involved primarily in adhesion to biotic and abiotic surfaces, cell agglutination, and biofilm formation. TAAs consist of a passenger domain, which is secreted onto the cell surface, and a transmembrane domain, which forms a pore in the OM to secrete and anchor the passenger domain. Because the interactions between TAAs and chaperones or dedicated auxiliary proteins during secretion are short-lived, TAAs are thought to reside on the OM without forming complexes with other proteins after secretion. In this study, we aimed to clarify the interactions between an Acinetobacter TAA, AtaA, and a peptidoglycan (PG)-binding periplasmic protein, TpgA. Pull-down assays using recombinant proteins identified the interacting domains. X-ray crystallography at 2.6 [A] resolution revealed an A3B3 heterohexameric complex structure composed of the N-terminal domain of TpgA and the transmembrane domain of AtaA. TpgA-N consists of two short helices and three antiparallel {beta} strands, yielding an {beta}{beta}{beta} topology similar to BamE. However, the regions corresponding to BamE interfaces with BamA and BamD differ in TpgA-N. All-atom molecular dynamics simulations and mutational assays revealed that both electrostatic and hydrophobic interactions contribute to stable complex formation. Bioinformatic analyses indicate that the TAA-TpgA complex occurs in a wide range of species. These findings will contribute to a better understanding of TAAs and the cell envelope. ImportanceGram-negative bacteria have specialized secretion systems (SSs) that translocate molecules from the cytoplasm to the extracellular space. Type V SSs have a simpler structure consisting of a functional passenger domain and a transmembrane domain involved in the secretion and anchoring of the passenger domain. Here, we provide the first direct evidence that a trimeric autotransporter adhesin (TAA) exported by a type Vc system forms a stable complex with a peptidoglycan-binding periplasmic protein. The 2.6 [A] structure of the A3B3 heterohexamer, together with simulation and mutational data, reveals complementary electrostatic and hydrophobic contacts that stabilize flexible loops on the periplasmic face of the TAA transmembrane barrel. Conservation of the taa-tpgA gene cassette and of key interface residues across diverse genera suggests that this coupling is a common strategy for tuning TAA stability and indicates that the envelope architecture of many TAAs, including those related to pathogenicity, is more elaborate than previously appreciated.

microbiology↗

bioRxiv2023

scRNA-seq analysis of hemocytes of penaeid shrimp under virus infection

The classification of cells in non-model organisms has lagged behind that of model organisms using established cluster of differentiation marker sets. To reduce fish diseases, research is needed to better understand immune-related cells, or hemocytes, in non-model organisms like shrimp and other marine invertebrates. In this study, we used Drop-seq to examine how virus infection affected the populations of hemocytes in kuruma shrimp, Penaeus japonicus, which had been artificially infected with a virus. The findings demonstrated that virus infection reduced particular cell populations in circulating hemolymph and inhibited the expression of antimicrobial peptides. We also identified the gene sets that are likely to be responsible for this reduction. Additionally, we identified functionally unknown genes as novel antimicrobial peptides, and we supported this assumption by the fact that these genes were expressed in the population of hemocytes that expressed other antimicrobial peptides. In addition, we aimed to improve the operability of the experiment by conducting Drop-seq with fixed cells as a template and discussed the impact of methanol fixation on Drop-seq data in comparison to previous results obtained without fixation. These results not only deepen our understanding of the immune system of crustaceans but also demonstrate that single-cell analysis can accelerate research on non-model organisms.

cell biology↗

bioRxiv2021

Structural insight into host plasma membrane association and assembly of HIV-1 Matrix protein

HIV-1 continues to be a global health concern since AIDS was first recognized by the World Health Organization (WHO). It is estimated that there were 38 million people infected with HIV-1 and 1.5 million deaths in 2019 alone. A better understanding of the details of the HIV late-stage life cycle, involving Pr55Gag attachment to the membrane for the further oligomerization to release virion, will provide us new avenues for potential treatment. Inositol hexakisphosphate (IP6) is an abundant endogenous cyclitol molecule and its binding was linked to the oligomerization of Pr55Gag via the MA domain. However, the binding site of IP6 on MA was unknown and the structural details of this interaction were missing. Here, we present three high-resolution crystal structures of the MA domain in complex with IP6 molecules to reveal its binding mode. Additionally, extensive Differential Scanning Fluorimetry analysis combined with cryo- and ambient-temperature X-ray crystallography and computational biology identify the key residues that participate in IP6 binding. Our data provide novel insights about the multilayered HIV-1 virion assembly process that involves the interplay of IP6 with PIP2, a phosphoinositide essential for the membrane binding of Pr55Gag. IP6 and PIP2 have neighboring alternate binding sites within the same highly basic region (residues 18-33). This indicates that IP6 and PIP2 bindings are not mutually exclusive and may play a key role in coordinating virion particles membrane localization. Based on our three different IP6-MA complex crystal structures, we propose a new model that involves the IP6 coordination of the oligomerization of outer MA and inner CA domain 2D layers during assembly and budding.

microbiology↗

bioRxiv2021

Single-cell RNA-seq analysis reveals penaeid shrimp hemocyte subpopulations and cell differentiation process

Crustacean aquaculture is expected to be a major source of fishery commodities in the near future. Hemocytes are key players of the immune system in shrimps; however, their classification, maturation, and differentiation are still under debate. To date, only discrete and inconsistent information on the classification of shrimp hemocytes has been reported, showing that the morphological characteristics are not sufficient to resolve their actual roles. Our present study using single-cell RNA sequencing, revealed nine types of hemocytes of Marsupenaeus japonicus based on their transcriptional profiles. We identified markers of each subpopulation and the differentiation pathways involved in their maturation. We also discovered cell growth factors that might play crucial roles in hemocyte differentiation. Different immune roles among these subpopulations were suggested from the analysis of differentially expressed immune-related genes. These results provide a unified classification of shrimp hemocytes, which improves the understanding of its immune system.

cell biology↗

bioRxiv2020

Size matters: optimal mask diameter and box size for single-particle cryogenic electron microscopy

Recently it has been demonstrated that single-particle cryogenic electron microscopy (cryo-EM) at 200 keV is capable of determining protein structures, including those smaller than 100 kDa, at sub-3.0 [A] resolutions, without using significant defocus or a phase plate. However, the majority of near-atomic resolution cryo-EM structures has been determined using 300 keV. Consequently, many typical parameter settings for the cryo-EM computational image processing steps, especially those associated with the contrast transfer function, are based on the accumulated experience of 300 kV cryo-EM. We have therefore revised these parameters, established theoretical bases for criteria to find an optimal mask diameter and box size for a given dataset irrespective of acceleration voltage or protein size, and proposed a protocol. Considering the defocus distributions of the datasets, merely optimizing the mask diameters and box sizes yielded meaningful resolution improvements for the reconstruction of < 200 kDa proteins using 200 kV cryo-EM.

molecular biology↗

bioRxiv2019

An integrated approach unravels a crucial structural property for the function of the insect steroidogenic Halloween protein Noppera-bo

Ecdysteroids are the principal insect steroid hormones essential for insect development and physiology. In the last 18 years, several enzymes responsible for ecdysteroid biosynthesis, encoded by Halloween genes, have been identified and well characterized, both genetically and biochemically. However, none of these proteins have yet been characterized at the tertiary structure level. Here, we report an integrated in silico, in vitro, and in vivo analyses of the Halloween glutathione S-transferase (GST) protein, Noppera-bo (Nobo). We determine crystal structures of Drosophila melanogaster Nobo (DmNobo) complexed with glutathione and 17{beta}-estradiol, a DmNobo inhibitor. 17{beta}-estradiol almost fully occupied the putative ligand-binding pocket, and a prominent hydrogen bond formed between Asp113 of DmNobo and 17{beta}-estradiol. Asp113 is essential for inhibiting DmNobo enzymatic activity by 17{beta}-estradiol, as 17{beta}-estradiol does not inhibit and physically interacts less with the Asp113Ala DmNobo point mutant. Asp113 is highly conserved among Nobo proteins, but not among other GSTs, implying that Asp113 is important for endogenous Nobo function. Indeed, a homozygous nobo allele possessing the Asp113Ala point mutation exhibits embryonic lethality with undifferentiated cuticle structure, a phenocopy of complete loss-of-function nobo homozygotes. These results suggest that the nobo family of GST proteins has acquired a unique amino acid residue, which seems to be essential for binding an endogenous sterol substrate to regulate ecdysteroid biosynthesis. This is the first study to reveal the structural characteristics of insect steroidogenic Halloween proteins. This study also provides basic insight into applied entomology for developing a new type of insecticides that specifically inhibit ecdysteroid biosynthesis.\n\nSignificance StatementInsect molting and metamorphosis are drastic and dynamic biological processes and, therefore, have fascinated many scientists. Ecdysteroids represent one class of insect hormones that are indispensable for inducing molting and metamorphosis. It is well known that proteins responsible for catalyzing ecdysteroid biosynthesis reactions are encoded by \"Halloween\" genes, most of which have names of ghosts and phantoms. However, no studies have focused on the structural properties of these biosynthetic proteins. In this study, we addressed this unsolved issue and successfully unraveled a structural property that is crucial for the function of the fruit fly Halloween protein, Noppera-bo (a Japanese faceless ghost). This is the first study to reveal the structural characteristics of an insect steroidogenic Halloween protein.

biochemistry↗