Biology subjects

Taguchi, A.

Publications and source records attributed to Taguchi, A..

13 recordsLinked to original sources

bioRxiv2026

Structural insights into fosfomycin efflux by a streptococcal ABC transporter

Gram-positive bacteria encode a broad array of ABC transporters that mediate substrate translocation across the cell membrane, with some contributing to their survival under environmental stresses such as antimicrobial exposure. While several of these transporters have been shown to exhibit multidrug efflux activity, the functional roles of many others remain unknown. Here, using an efflux pump screen in the opportunistic human pathogen Streptococcus pneumoniae, we identified a previously uncharacterized type IV ABC transporter (FoeAB) that confers resistance to the antibiotic fosfomycin. We show that purified FoeAB mediates fosfomycin transport in a liposome-reconstituted system and provide evidence that it functions as a multidrug efflux pump with substrate preferences distinct from those of known efflux pumps. Furthermore, we present cryogenic electron microscopy (cryo-EM) structures of FoeAB in inward- and outward-facing states, which reveal conformational changes associated with nucleotide binding and identify residues important for substrate transport. Collectively, these findings expand the known repertoire of antibiotic-exporting ABC transporters in Gram-positive bacteria and provide structural insight into its transport mechanism. SIGNIFICANCEEfflux pumps often play major roles in antibiotic resistance in clinically important pathogens, yet many of these transporters remain poorly defined. In this study, we identify and mechanistically characterize FoeAB, a previously unrecognized type IV ABC transporter in streptococcal species that mediates efflux of the antibiotic fosfomycin. By combining functional assays with structural studies of FoeAB in multiple conformational states, we propose a model for how this transporter recognizes its substrate and harnesses nucleotide-driven conformational changes to mediate substrate export. Our findings point to a previously underappreciated efflux capabilities in Gram-positive bacteria, highlighting the need for better characterization of membrane transporters to clarify their roles in bacterial physiology and drug resistance.

microbiology↗

bioRxiv2026

Integrating spatial and single-cell multi-omics analysis of induced pluripotent stem cell-derived cervical adenocarcinoma model

Human papillomavirus 18 (HPV18) preferentially infects cervical stem cell-like cells and is strongly associated with adenocarcinoma. However, the mechanisms underlying differentiation into cervical adenocarcinoma remain unclear due to the lack of appropriate experimental models. We aimed to establish a model of HPV18-associated cervical adenocarcinoma and elucidate its molecular and cellular differentiation mechanisms. HPV18 E6/E7 were introduced into induced pluripotent stem cell-derived reserve cell-like cells (iRCs) to generate tumor models. Spatial transcriptomics and single-cell multi-omics analyses were performed to integrate histological and molecular data. A distinct component (Gland_A) exhibited morphological and immunohistochemical features of cervical adenocarcinoma and was efficiently induced in iRC-18 tumors. Gland_A showed increased chromatin accessibility and elevated expression of FOXA1, FOXA2, and ALDH1A1. Analysis of clinical samples confirmed enrichment of ALDH1A1 in HPV-associated adenocarcinomas. This model recapitulates key features of HPV18-associated cervical adenocarcinoma and provides insights into its differentiation mechanisms.

cancer biology↗

bioRxiv2025

Hox - Meis-relayed topographical genetic switch underlies cardiopharyngeal neural crest diversification, revealed by multimodal analysis

Neural crest cells (NCCs) are multipotent migratory cells essential for cardiac development, yet the lineage trajectories and gene regulatory networks (GRNs) underlying their differentiation in the cardiopharyngeal region remain unclear. Here, we integrate single-cell RNA-seq, spatial transcriptomics, and multiomic analyses to construct a comprehensive map of NCC lineages in developing mouse cardiopharyngeal tissues. We identify a transition from Hox-positive pharyngeal NCCs to Hox-negative intracardiac populations associated with the outflow tract cushion, accompanied by a shift in Meis transcription factor binding and GRN architecture. By contrast, NCCs forming the aorticopulmonary septum and great vessel smooth muscle retain distinct Hox-codes. A Meis2-Sox9-Scx GRN defines a skeletogenic progenitor-like intermediate state that gives rise to coronary artery smooth muscle and semilunar valves. Our findings suggest that the loss of Hox-dependent regional identity enables pharyngeal NCCs to acquire new fates upon entering the cardiac cushion, providing insight into the developmental origins of coronary and valvular calcification.

developmental biology↗

bioRxiv2025

Novel therapy for gastric cancer peritoneal dissemination using genetically modified dental pulp cells and astatine-211

Peritoneal dissemination of gastric cancer is a terminal stage with limited treatment options and a five-year survival rate below 10%. To develop a more effective treatment, we created a new approach that uses genetically engineered human dental pulp cells (DPCs) that express the sodium/iodide symporter (NIS). These cells (NIS-DPCs) deliver astatine-211 (At-211) directly to tumor sites. When injected intraperitoneally into a mouse model of gastric cancer dissemination, DPCs, which were isolated and expanded, showed strong tumor-homing ability. Chemotaxis mediated by the CXCR4/SDF-1 axis has been reported to play a role in this accumulation. In addition to histological analysis, fluorescent imaging confirmed the selective accumulation of NIS-DPCs within tumor lesions. Introducing the NIS gene markedly increased SLC5A5 expression, enabling efficient At-211 uptake. To avoid nonspecific binding, sodium At-211 was utilized. High-resolution alpha-particle imaging visualized alpha-ray emission specifically from NIS-DPCs, confirming the radionuclides intracellular retention. In vivo, the sequential administration of NIS-DPCs, followed by Na[At-211], resulted in the pronounced regression of peritoneal tumors and a significant extension of survival compared to controls. The therapeutic mechanism involves three coordinated steps: tumor-directed migration of NIS-DPCs, At-211 uptake via NIS transporters, and localized alpha-particle-mediated cytotoxicity. This study introduces the novel concept of cell-based alpha-radiotherapy, which integrates regenerative and nuclear medicine. Due to the well-documented safety profile of DPCs in human clinical trials (J-REPAIR, jRCT1080224505), the NIS-DPC platform emerges as a promising approach for the precise, localized irradiation of disseminated gastric cancer and other challenging malignancies.

cancer biology↗

bioRxiv2024

Structural basis of nucleotide selectivity in pyruvate kinase

Nucleoside triphosphates are indispensable in numerous biological processes, with enzymes involved in their biogenesis playing pivotal roles in cell proliferation. Pyruvate kinase (PYK), commonly regarded as the terminal glycolytic enzyme that generates ATP in tandem with pyruvate, is also capable of synthesizing a wide range of nucleoside triphosphates from their diphosphate precursors. Despite their substrate promiscuity, some PYKs show preference towards specific nucleotides, suggesting an underlying mechanism for differentiating nucleotide bases. However, the thorough characterization of this mechanism has been hindered by the paucity of nucleotide-bound PYK structures. Here, we present crystal structures of Streptococcus pneumoniae PYK in complex with four different nucleotides. These structures facilitate direct comparison of the protein-nucleotide interactions and offer structural insights into its pronounced selectivity for GTP synthesis. Notably, this selectivity is dependent on a sequence motif in the nucleotide recognition site that is widely present among prokaryotic PYKs, particularly in Firmicutes species. We show that pneumococcal cell growth is significantly impaired when expressing a PYK variant with compromised GTP and UTP synthesis activity, underscoring the importance of PYK in maintaining nucleotide homeostasis. Our findings collectively advance our understanding of PYK biochemistry and prokaryotic metabolism.

biochemistry↗

bioRxiv2022

Cardiac neural crest lineage diversity and underlying gene regulatory networks revealed by multimodal analysis

Neural crest cells (NCCs), a multipotent stem cell population, contribute to cardiac development as a source of the outflow septum, vascular smooth muscle and semilunar valves. However, genetic programs underlying lineage diversification of cardiac NCCs remain largely unknown. Using single-cell (sc) and spatial transcriptomics, we demonstrate multiple NCC subpopulations with distinct gene expression signatures; smooth muscle(-like), non-muscle mesenchymal, and Schwann cell progenitor/melanoblast-like cells. Integrative scRNA-seq and scATAC-seq analyses predict lineage trajectories starting from immature NCCs, which bifurcate into smooth muscle(-like) and non-muscle mesenchymal lineages in association with hierarchical transcription factor networks. Combinatory analyses with Cre-mediated genetic lineage tracing characterize intermediate NCCs at the bifurcation as Sox9+/Scx+ tendon and cartilage progenitor-like cells with genetic programs, some of which are common to skeletal tissues whereas others are unique to cardiac NCCs. These findings provide a basis for understanding the roles of NCCs in cardiac development and pathogenesis particularly associated with calcification.

developmental biology↗

bioRxiv2021

Young APPKI NL-G-F/NL-G-F mice display high-fat diet-induced metabolic disturbances and specific disorders associated with brain energy homeostasis.

AimType 2 diabetes mellitus (T2DM) is an increased risk factor for Alzheimers disease (AD); however, the relationship between the two conditions is controversial. High-fat diet (HFD) causes cognitive impairment with/without A{beta} accumulation in middle-aged or aged transgenic (Tg) and knock-in (KI) AD mouse models, except for metabolic disorders, which commonly occur in all mice types. Alternatively, whether HFD in early life impacts energy metabolism and neurological phenotypes in young AD mouse models remains unknown. In the present study, we examined the effects of HFD on young APPKI NL-G-F/NL-G-F mice, one of the novel knock-in (KI)-AD mouse models. MethodsThe mice were categorized by diet into two experimental groups, normal diet (ND) and HFD. Four-week-old WT and APPKI NL-G-F/NL-G-F mice were fed ND or HFD for nine weeks. Both types of mice on ND and HFD were examined during young adulthood. ResultsHFD causes T2DM-related metabolic disturbances in young WT and APPKI NL-G-F/NL-G-F mice and specific impairment of brain energy homeostasis only in young APPKI NL-G-F/NL-G-F mice. However, HFD-induced metabolic dysfunctions had no impact on behaviors, A{beta} levels, and specific IRS1 modifications in both young APPKI NL-G-F/NL- G-F mice and young WT mice. ConclusionHFD in early life is effective in causing metabolic disturbances in young WT and APPKI NL-G-F/NL-G-F mice but is ineffective in inducing neurological disorders in young mice, which suggests that the aging effects along with long-term HFD cause neurological alterations.

neuroscience↗

bioRxiv2021

Pharmacologic rescue of circadian β-cell failure through P2Y1 purinergic receptor identified by small-molecule screen

The mammalian circadian clock drives daily oscillations in physiology and behavior through an autoregulatory transcription feedback loop present in central and peripheral cells. Ablation of the core clock within the endocrine pancreas of adult animals impairs the transcription and splicing of genes involved in hormone exocytosis and causes hypoinsulinemic diabetes. However, identification of druggable proteins and pathways to ameliorate the burden of circadian metabolic disease remains a challenge. Here, we generated {beta} cells expressing a nano-luciferase reporter within the proinsulin polypeptide to screen 2,640 pharmacologically-active compounds and identify insulinotropic molecules that bypass the secretory defect in clock mutant {beta} cells. We validated lead compounds in primary mouse islets and identified known modulators of ligandgated ion channels and G-protein coupled receptors, including the antihelmintic ivermectin. Single-cell electrophysiology in circadian mutant mouse and human cadaveric islets validated ivermectin as a glucose-dependent secretagogue. Genetic, genomic, and pharmacologic analyses established that the molecular clock controls the expression of the purinergic P2Y1 receptor to mediate the insulinotropic activity of ivermectin. These findings identify the P2Y1 purinergic receptor as a target to rescue circadian {beta}-cell failure and establish a chemical genetic screen for endocrine therapeutics.

cell biology↗

bioRxiv2021

Biochemical reconstitution defines new functions for membrane-bound glycosidases in assembly of the bacterial cell wall

The peptidoglycan cell wall is a macromolecular structure that encases bacteria and is essential for their survival. Proper assembly of the cell wall requires peptidoglycan synthases as well as membrane-bound cleavage enzymes that control where new peptidoglycan is made and inserted. We are only beginning to understand the roles of peptidoglycan cleavage enzymes in cell wall assembly. Previous studies have shown that two membrane-bound proteins in Streptococcus pneumoniae, here named MpgA and MpgB, are important in maintaining cell wall integrity. MpgA was predicted to be a lytic transglycosylase based on its homology to Escherichia coli MltG while the enzymatic activity of MpgB was unclear. Using nascent peptidoglycan substrates synthesized in vitro from the peptidoglycan precursor Lipid II, we report that both MpgA and MpgB are muramidases. We show that replacing a single amino acid in E. coli MltG with the corresponding amino acid from MpgA results in muramidase activity, allowing us to predict from the presence of this amino acid that other putative lytic transglycosylases actually function as muramidases. Strikingly, we report that MpgA and MpgB cut nascent peptidoglycan at different positions along the sugar backbone relative to the reducing end. MpgA produces much longer peptidoglycan oligomers and we show that its cleavage site selectivity is controlled by the LysM-like subdomain, which is also present in MltG. We propose that MltGs ability to complement loss of MpgA in S. pneumoniae despite performing different cleavage chemistry is because it can cleave nascent peptidoglycan at the same distance from the lipid anchor.

microbiology↗

bioRxiv2020

Organization of Peptidoglycan Synthesis in Nodes and Separate Rings at Different Stages of Cell Division of Streptococcus pneumoniae

Bacterial peptidoglycan (PG) synthesis requires strict spatial and temporal organization to reproduce specific cell shapes. In the ovoid-shaped, pathogenic bacterium Streptococcus pneumoniae (Spn), septal and peripheral (sidewall-like) PG synthesis occur simultaneously at midcell. To uncover the organization of proteins and activities that carry out these two modes of PG synthesis, we examined Spn cells vertically oriented onto their poles to image the division plane at the high lateral resolution of 3D-SIM (structured-illumination microscopy). Using fluorescent D-amino acid (FDAA) probes, we show that areas of new transpeptidase (TP) activity catalyzed by penicillin-binding proteins (PBPs) separate into a pair of concentric rings early in division, representing peripheral PG (pPG) synthesis (outer ring) and the leading-edge (inner ring) of septal PG (sPG) synthesis. Fluorescently tagged PBP2x or FtsZ locate primarily to the inner FDAA-marked ring, whereas PBP2b and FtsX remain in the outer ring, suggesting roles in sPG or pPG synthesis, respectively. Short pulses of FDAA labeling revealed an arrangement of separate regularly spaced "nodes" of TP activity around the division site of predivisional cells. Control experiments in wild-type and mutant strains support the interpretation of nodal spacing of TP activity, and statistical analysis confirmed that the number of nodes correlates with different ring diameters. This nodal pattern of FDAA labeling is conserved in other ovoid-shaped species. Tagged PBP2x, PBP2b, and FtsX proteins also exhibited nodal patterns with spacing comparable to that of FDAA labeling. Together, these results reveal a highly ordered PG synthesis apparatus in ovococcal bacteria at different stages of division. SIGNIFICANCEThe spatial organization of PBPs and their TP activity at division septa is not well understood. In some bacteria, TP activity and PBP localization seem to be nodal (also called punctate), whereas in other bacteria, discrete foci of PBP activity are infrequently or not observed. Here we report two basic properties of the organization of PBPs and TP activity in the ovoid-shaped bacterium Spn. First, there is distinct spatial separation of the sPG machine, including FtsZ, from the pPG synthesis machine at the midcell of dividing Spn cells. Second, in predivisional cells, PBPs and TP activity are organized heterogeneously into regularly spaced nodes, whose number and dynamic distribution are likely driven by the PG synthesis of PBP:SEDS complexes.

microbiology↗

bioRxiv2019

Lysine demethylase 7a regulates the anterior-posterior development in mouse by modulating the transcription of Hox gene cluster

Temporal and spatial colinear expression of the Hox genes determines the specification of positional identities during vertebrate development. Post-translational modifications of histones contribute to transcriptional regulation. Lysine demethylase 7A (Kdm7a) demethylates lysine 9 di-methylation of histone H3 (H3K9me2) and participates in the transcriptional activation of developmental genes. However, the role of Kdm7a during mouse embryonic development remains to be elucidated. Here, we show that Kdm7a-/- mouse exhibits an anterior homeotic transformation of the axial skeleton, including an increased number of presacral elements. Importantly, posterior Hox genes (caudally from Hox9) are specifically downregulated in the Kdm7a-/- embryo, which correlates with increased levels of H3K9me2. These observations suggest that Kdm7a controls the transcription of posterior Hox genes, likely via its demethylating activity, and thereby regulating the murine anterior-posterior development. Such epigenetic regulatory mechanisms may be harnessed for the proper control of coordinate body patterning in vertebrates.

molecular biology↗

bioRxiv2019

Association between Japanese community health workers willingness to continue service and two categories of motives: altruistic and self-oriented

BackgroundAs population aging progresses, volunteers in health field are expected to play a key role in health promotion and disease prevention, which may improve community residents health and well-being and at the same time help curve healthcare cost. The objective of this study is to examine the effects of self-oriented motives and altruistic motives as explanatory factors for Japanese Community Health Workers (CHWs) desire to continue their service. Unraveling the relative effects of these two types of motivation on CHW retention may lead to policy and practical implications for recruiting, training, and supporting CHWs in Japan. Haddad (2007) observed that citizens in Japan generally have a sense of governmental and individual responsibility for dealing with social problems. Applying these insights to CHWs, we hypothesize that altruistic motives have more potent influence on volunteers willingness to continue to serve than self-oriented motives.\n\nMethodsThree cities in Shiga prefecture, Japan agreed to participate in the study. Anonymous, self-administered questionnaire was mailed to all CHWs who work in the three communities. The survey data were collected in March and April, 2013. A total of 417 questionnaires were mailed to CHWs, of which 346 were completed and returned (response rate 83.0%). Nine questionnaires missing response to the question concerning willingness to continue serving were removed from the analysis. The final analysis used 337 questionnaires (effective response rate 80.8%).\n\nResultsOne hundred ninety-nine (59.1%) of the respondents answered the question about willingness to continue CHW affirmatively, and 138 (40.9%) negatively. Controlling for other relevant factors, those with self-oriented motives in serving as CHWs were more likely to state they are willing to continue to serve (OR:1.54, confidence interval 1.00-2.37) than those without such motives. Contrary to our hypothesis, the effect of altruistic motives on respondents willingness to continue serving as CHWs was slightly larger than that of self-oriented motives; the odds ratio was 1.56(confidence interval 1.08-2.27).\n\nConclusionOne practical implication of the research is that learning more about the twin motives, self-oriented and altruistic, of volunteers and tailoring the content of CHW training to address those motives may be beneficial.

scientific communication and education↗

bioRxiv2018

FtsW is a peptidoglycan polymerase that is activated by its cognate penicillin-binding protein

The peptidoglycan cell wall is essential for the survival and shape maintenance ofbacteria.1 For decades it was thought that only penicillin-binding proteins (PBPs) effected peptidoglycan synthesis. Recently, it was shown that RodA, a member of the Rod complex involved in side wall peptidoglycan synthesis, acts as a peptidoglycan polymerase.2-4 RodA is absent or dispensable in many bacteria that contain a cell wall; however, all of these bacteria have a RodA homologue, FtsW, which is a core member of the divisome complex that is essential for septal cell wall assembly.5,6 FtsW was previously proposed flip the peptidoglycan precursor Lipid II to the peripasm,7,8 but we report here that FtsW polymerizes Lipid II. We show that FtsW polymerase activity depends on the presence of the class B PBP (bPBP) that it recruits to the septum. We also demonstrate that the polymerase activity of FtsW is required for its function in vivo. Our findings establish FtsW as a peptidoglycan polymerase that works with its cognate bPBP to produce septal peptidoglycan during cell division.

microbiology↗