Biology subjects

Sawada, K.

Publications and source records attributed to Sawada, K..

9 recordsLinked to original sources

bioRxiv2026

Catalog of gut microbiota alterations associated with anticancer therapies across multiple cancer types

BackgroundAnticancer therapies can alter the gut microbiota and may affect gut bacteria associated with treatment response. However, most longitudinal studies have focused on specific cancer types or individual treatment regimens, and systematic analyses across diverse cancer therapies remain limited. We analyzed longitudinal fecal microbiota profiles using 16S ribosomal RNA gene amplicon sequencing in the pan-cancer SCRUM-Japan MONSTAR-SCREEN cohort. We included 528 paired pre- and post-treatment fecal samples from 264 patients with advanced solid tumors across 18 cancer types and characterized the gut microbiota alterations associated with 22 anticancer drugs and related clinical factors. ResultsAcross the cohort, Shannon diversity did not significantly change after treatment (mean, 3.81 vs. 3.78; P = 0.58), and pre- and post-treatment samples exhibited no clear separation in ordination space. However, within-patient analysis detected a subtle but significant longitudinal microbiota shift (paired PERMANOVA, P = 0.0001), highlighting the importance of accounting for paired sampling. Clustering of genus-level compositional alterations revealed patient groups with distinct degrees of microbiota alteration, with the largest shifts associated with antibiotic exposure, transition from normal stool to diarrhea, and specific treatment regimens. Multivariable regression analysis of 22 anticancer drugs identified drug-bacteria associations and demonstrated that drugs with similar mechanisms of action, including epidermal growth factor receptor (EGFR) inhibitors and immune checkpoint inhibitors, exhibited similar microbiota change profiles. Targeted analyses highlighted concordant reductions in the Christensenellaceae R-7 group among EGFR inhibitor-exposed patients and depletion of Faecalibacterium among immune checkpoint inhibitor-exposed patients. ConclusionsThis study provides a cross-cancer catalog of microbiota alterations associated with anticancer therapies and highlights therapy-related shifts in the gut ecosystem, including patterns shared by drugs with similar mechanisms of action.

microbiology↗

bioRxiv2026

Molecular basis of promiscuous chemokine-engagement by the Duffy antigen receptor

The Duffy blood group antigen, encoded by a seven transmembrane protein known as Duffy Antigen Receptor for Chemokines (DARC), or Atypical Chemokine Receptor 1 (ACKR1), serves as a key receptor for the malarial parasite, Plasmodium vivax, on erythrocytes. DARC exhibits remarkable functional divergence compared to prototypical chemokine receptors and other G protein-coupled receptors (GPCRs) as it does not engage canonical signal-transducers such as G-proteins, GPCR regulatory kinases (GRKs), and {beta}-arrestins. DARC is a highly promiscuous receptor interacting with several homeostatic and inflammatory C-C and C-X-C subtype chemokines, and similar to other chemokine receptors, these interactions are modulated by post-translational tyrosine sulfation in the N-terminus. A single nucleotide polymorphism in DARC leading to Gly42Asp substitution forms the basis for Fya vs. Fyb Duffy blood group antigen classification with differential impact on Plasmodium vivax infection and cancer progression. However, the molecular basis of promiscuous chemokine-binding by DARC and the modulation by receptor sulfation and Fya/Fyb allelic variation remains unclear. Here, we design a sortase-mediated chemical-ligation strategy to generate purified DARC with naturally-occurring tyrosine sulfation at the N-terminus, and determine high-resolution cryo-EM structures in complex with a C-C type chemokine, CCL7, and a C-X-C type chemokine, CXCL8. We observe that similar to CCL7, CXCL8 engages with DARC primarily through the N-terminus of the receptor, which is in stark contrast with the two-site binding mechanism displayed by other chemokine receptors. Interestingly, the E-L-R motif in CXCL8 engages a pseudo-R-D motif in DARC leading to superficial engagement unlike CXCR2, a prototypical chemokine receptor sharing the same agonist. Moreover, tyrosine sulfation and Fyb allelic variation leads to a repositioning of the receptor N-terminus on the core domain of CXCL8, resulting in distinct interaction network imparting greater binding affinity. Taken together, our study presents novel insights into promiscuous chemokine-binding to DARC, tyrosine sulfation-mediated fine-tuning of chemokine engagement, and a generalizable sortase-mediated chemical-ligation platform applicable to other chemokine receptors.

biochemistry↗

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

Molecular mechanism of naturally-encoded signaling-bias at the complement anaphylatoxin receptors

The conceptual framework of biased signaling has revolutionized our understanding of GPCR signaling and regulatory paradigms, and greatly impacted the efforts focused on the discovery of GPCR-targeted therapeutics. However, the mechanistic basis of biased signaling remains primarily defined based on synthetic ligands and receptor mutants with relatively limited progress in understanding naturally-encoded signaling-bias. Here, we present fundamental molecular and structural insights into naturally-encoded signaling-bias at the complement anaphylatoxin C5a receptors namely, C5aR1 and C5aR2. We first discover that C5a-d-Arg, the naturally-occurring version of C5a lacking the terminal arginine, exhibits robust G-protein signaling-bias at C5aR1, characterised by attenuated {beta}arr recruitment. This signaling-bias manifests in both cytokine release from primary human immune cells, and in vivo, during neutrophil mobilization. We combine the cryo-EM structures of C5a/C5a-d-Arg-C5aR1 complexes with MD simulation, site-directed mutagenesis, and cellular experiments to elucidate that the G-protein-bias exhibited by C5a-d-Arg results from a distinct orientation of TM7 and helix 8 in C5aR1 leading to inefficient GRK recruitment and receptor phosphorylation. Next, we determine the first cryo-EM structures of C5aR2, a naturally-encoded {beta}-arrestin-biased receptor, in an apo state, complexed with the natural agonists C5a and C5a-d-Arg, and three peptide agonists including a first-in-class, newly discovered C5aR2-selective agonist, R8Y. These structural snapshots reveal key differences between the binding of C5a and C5a- d-Arg to C5aR1 and C5aR2, and provide a molecular basis of functional specialization at these two receptors. Moreover, the structural insights also allow us to decipher the molecular basis of naturally-encoded signaling-bias at C5aR2 originating from a shallower cytoplasmic interface with hydrophobic interior pocket that is not permissive to efficient G-protein-coupling and activation. Finally, we also engineer and characterize loss-of-function and gain-of-function variants of C5aR1 and C5aR2, which in turn corroborate and validate the structural observations presented here. Collectively, our findings offer crucial insights into previously lacking molecular mechanisms of the naturally-encoded signaling-bias at GPCRs, which have broad implications not only for the general framework of biased-signaling, but also for novel therapeutic design.

biochemistry↗

bioRxiv2025

Genetic diversity in horseshoe bat ACE2 and sarbecovirus spike proteins mutually shape one another

Angiotensin-converting enzyme 2 (ACE2) serves as the entry receptor for a wide diversity of sarbecoviruses naturally harboured by horseshoe bats (genus Rhinolophus). Despite the extensive circulation of these viruses in many horseshoe bat species, the potential interactions between virus and receptor evolution remain poorly understood. We sampled individuals of the intermediate horseshoe bat (Rhinolophus affinis) across Vietnam and identified 15 genotypes of ACE2 proteins, 10 of which are previously unreported. Phylogenetic analysis and infectivity assays with a panel of 36 sarbecovirus spike proteins indicated that the R. affinis ACE2 phylogeny has geographic structuring and genotypes originating from different geographic regions exhibit distinct infectivity phenotypes. We detected site-specific positive selection on ACE2 site 24 with the associated substitutions largely affecting the receptors sarbecovirus infectivity profile. Together, our findings suggest that the R. affinis within-species ACE2 diversity has likely been shaped through selection by past sarbecovirus infection. Similarly on the virus end, we use mutagenesis assays and structural analysis through cryo-EM, to delineate the proximal evolution of the Ra22QT77 spike defined by specialization to the ACE2 genotypes of horseshoe bats found in and near southern Vietnam, where the virus was sampled. Our findings contribute to a better understanding of host-sarbecovirus co-evolution dynamics and provide valuable insights into the receptor usage determinants of these viruses. HighlightsO_LIWe identify a total of 15 ACE2 genotypes in R. affinis bats from Vietnam. C_LIO_LIACE2 intraspecific polymorphism is geographically separated and associated with distinct sarbecovirus infectivity. C_LIO_LISite 24 of R. affinis ACE2 experiences positive selection and controls susceptibility to sarbecoviruses. C_LIO_LIBat ACE2s and sarbecovirus spikes are bidirectionally shaped by each others evolution. C_LI

microbiology↗

bioRxiv2025

Molecular fingerprints of a convergent mechanism orchestrating diverse ligand recognition and species-specific pharmacology at the complement anaphylatoxin receptors

Complement anaphylatoxin receptors (C3aR and C5aR1) are prototypical G protein-coupled receptors (GPCRs) playing crucial physiological roles in innate immunity by combating pathogenic infections and orchestrating inflammatory responses. They continue to be important therapeutic targets for multiple disorders including autoimmune diseases, acute and chronic inflammation, and allergy-related conditions. Recent structural coverage has provided important insights into their activation and signaling, however, confounding observations in the literature related to ligand efficacy and functional responses, especially in different model systems, present a major challenge for drug discovery efforts. Here, we systematically and comprehensively profile a broad set of natural and synthetic ligands at C3aR and C5aR1 and discover a previously unanticipated level of functional specialization in terms of species-specific pharmacology and receptor activation. Taking a lead from this, we determine seventeen cryo-EM structures of different ligand-receptor-G-protein complexes and uncover distinct orientation of agonists between the human and mouse receptors despite an overlapping positioning in the orthosteric binding pocket. Combined with extensive mutagenesis and functional assays, these structural snapshots allow us to decode and validate a convergent molecular mechanism involving a "Five-Point-Switch" in these receptors that orchestrates the recognition and efficacy of diverse agonists. We also identify species-specific differences at the level of phosphorylation patterns encoded in the carboxyl-terminus of these receptors and directly visualize their impact on {beta}arr binding and activation using cryo-EM structures. Interestingly, we observe that {beta}arrs engage with the mouse C5aR1 using a variation of previously discovered P-X-P-P phosphorylation motif via a "Sliding-Mechanism" and also exhibit distinct oligomeric state for the human vs. mouse receptors. Taken together, this study elucidates functional specialization at the complement anaphylatoxin receptors and underlying molecular mechanisms, offering a previously lacking framework with direct and immediate implications for the development of novel therapeutics.

biochemistry↗

bioRxiv2022

Human blastoid from primed human embryonic stem cells

Artificial human blastoids are used to investigate early embryo development, pregnancy failures, and birth deficiencies, previously impossible without human embryos. Recent methods generating blastoids used human naive pluripotent stem cells, which are prone to genomic instability during in vitro culturing. Here, we introduce a simple, robust, and scalable method for generating human blastoids from more stable, primed human pluripotent stem cells (hPSC). Using a non-cell-adhesive hydrogel, hPSC aggregates formed an asymmetric blastoid structure with a cellular distribution similar to that of a human blastocyst, with in vitro implantation capability. Single-cell RNA-seq followed by cellular trajectory analysis revealed that hydrogel promoted differentiation to tri-lineage cells associated with a blastocyst. This model will allow studies on the underlying mechanisms of human pre- and postimplantation processes, consider elaborating on the potential implications of the model on assistive reproductive technology. One Sentence SummaryNo more than 125 characters and spaces.

bioengineering↗

bioRxiv2022

In vitro generation of human embryonic stem cell-derived heart organoids possessing physiological ion currents

Recently, methods for in vitro organogenesis have been broadly developed due to their strong potential for human applications in medicine. In the present study, we optimized the culturing method of human heart organoids (hHOs) from embryonic stem cells (ESCs) in the presence of the highly concentrated laminin-entactin and fibroblast growth factor 4. The resulting hHOs showed distinctive cardiac morphology with atrium- and ventricle-like chambers composed of cardiac cells as well as expressed the integral proteins of gap junctions and ion channels. In fact, isolated cardiomyocytes from these hHOs exhibited Na and Ca currents by patch clamp analysis. These results indicated that the present method will provide a powerful tool for cardiac safety assessment of newly developed drugs as an in vitro human ESC-derived test system. One-Sentence SummaryFGF4 and ECM contribute to the generation of human HOs with heart compartments and electrophysiological properties.

bioengineering↗

bioRxiv2020

Patterns of functional and structural brain information are preserved over generations

There is clear evidence of intergenerational transmission of life values, cognitive traits, psychiatric disorders, and even aspects of daily decision making. To investigate biological substrates of this phenomenon, brain has received increasing attention as a measurable biomarker and potential target for intervention. However, no previous study has quantitatively and comprehensively investigated the effects of intergenerational transmission on functional and structural brain networks from parents to their children. Here, by employing an unusually large cohort dataset, we show that patterns of functional and structural brain networks are preserved over a generation. We also demonstrate that several demographic and behavioural phenotypes have effects on brain similarity. Collectively, our results provide a comprehensive picture of neurobiological substrates of parent-child similarity, and demonstrate the usability of our dataset for investigating the neurobiological substrates of intergenerational transmission.

neuroscience↗