Biology subjects

Takahashi, C.

Publications and source records attributed to Takahashi, C..

11 recordsLinked to original sources

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

Reliable Detection of SGLT2 Protein through Knockout-based Antibody Characterization and Optimized Procedures

IntroductionSodium-glucose cotransporter 2 (SGLT2) is a key mediator of renal glucose reabsorption. Its pharmacological inhibition exerts cardio- and reno-protective benefits. Despite widespread clinical interest, reliable detection of SGLT2 protein remains challenging due to concerns regarding the specificity of available antibodies. MethodsThis study assessed the specificity of eight commercially available anti-SGLT2 antibodies by immunohistochemistry and Western blotting. Genetically engineered Sglt2-deficient mice and rats, generated via clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) technology, were employed as definitive negative controls. Additionally, human kidney tissues, including renal cell carcinoma samples, were analyzed. ResultsAmong the antibodies tested, few exhibited robust specificity, characterized by substantial immunostaining with minimal background in wild-type kidney tissues and complete absence of staining in Sglt2-deficient samples. In renal cell carcinoma samples, a validated antibody detected SGLT2 immunostaining in proximal tubules of non-tumor regions but not in tumor areas. Subcellular localization studies revealed that SGLT2 was enriched within proximal tubular microvilli, partially overlapping with its co-factor PDZK1IP1 (MAP17). LRP2 (megalin) and NHE3 were placed at the microvillar base and did not colocalize with SGLT2. Western blotting identified a specific SGLT2 band at approximately 55 kDa in kidney lysates using several antibodies under optimized procedures. This band was shifted to approximately 45 kDa after enzymatic removal of N-linked glycans. One antibody detected a weak band at the same molecular mass even in kidney lysates from Sglt2-deficient rodents. ConclusionsConsiderable variability exists in the specificity of commercially available anti-SGLT2 antibodies. Only a limited number of antibodies are suitable for reliable detection of SGLT2 in rodent and human samples. Rigorous antibody characterization, including the use of knockout controls and optimized experimental conditions, is essential to ensure reproducibility and prevent misinterpretation in studies investigating the biological and pathophysiological roles of SGLT2.

molecular biology↗

bioRxiv2025

Ubiquitination-activated TAB-TAK1-IKK-NF-kB axis modulates gene expression for cell survival in the lysosomal damage response

The lysosomal damage response is important for the maintenance of cellular homeostasis. Although the mechanisms underlying the repair and autophagic elimination of damaged lysosomes have been elucidated, the early signal transduction pathways and genes induced in response to lysosomal damage remain elusive. We performed transcriptome and proteome analyses and found that the TAB-TAK1- IKK-NF-{kappa}B axis is activated by K63-linked ubiquitin chains that accumulate on damaged lysosomes. This activates the expression of various transcription factors and cytokines that promote anti-apoptosis and intercellular signaling. The findings highlight the crucial role of ubiquitin-regulated signal transduction and gene expression in cell survival and cell-cell communication in response to lysosomal damage. The results suggest that the ubiquitin system is not only involved in the removal of damaged lysosomes by lysophagy, but also functions in the activation of cellular signaling for cell survival.

cell biology↗

bioRxiv2025

Genome-wide mapping of the Galleria mellonella larvae transcription start sites during fungal infection and treatment

Using Low Quantity single strand CAGE (LQ-ssCAGE), we mapped the transcription start sites (TSS). We annotated the 5 end of the invertebrate Galleria mellonella, an upcoming and booming experimental model in infectious disease and immunology research. However, the current genome annotation of this model organism lacks annotation of the 5 end and TSS information. G. mellonella larva was infected with the fungal pathogen Madurella mycetomatis to map TSS under healthy and infection conditions. Larvae were first treated with itraconazole or ravuconazole, and then RNA-seq and LQ-ssCAGE libraries were prepared and sequenced 4, 30, and 52 hours following infection. The LQ-ssCAGE data was processed to identify CAGE transcription start site (CTSS), uni-, and bi-directional clusters. LQ-ssCAGE enabled us to precisely identify 39,410 TSS and 249 active enhancers; we assigned genomic features to the resulting TSSs and enhancers. The majority of the TSS peaks are annotated as promoter regions, while the enhancers were annotated as intergenic and genic. Furthermore, we confirmed the quality of TSS calling by promoter shapes and GC bias. Furthermore, we identified a set of super-enhancers and predicted de-novo motifs. The raw and processed data was deposited to NCBI GEO GSE282923. CTSS, TSS peaks, and enhancers coordinated are available through the ZENBU Genome browser. In this study, we reported the first atlas of TSS and active enhancers of G. mellonella.

genomics↗

bioRxiv2024

Molecular characterization of response to etrolizumab and anti-TNF reveals treatment resistance in ulcerative colitis is associated with the abundance of residual neutrophil subsets and inflammatory fibroblast populations

Ulcerative colitis (UC) is a chronic inflammatory large bowel disease characterized by immune cell infiltration and continuous erosion of intestinal crypts, causing severe ulceration and abdominal pain. In the etrolizumab Phase 3 studies, transcriptional analyses of colonic biopsies revealed reductions in genes associated with aEb7+ intraepithelial lymphocytes with etrolizumab but not adalimumab. Both treatments significantly reduced stromal and myeloid cell-associated genes, with changes associated with MCS remission status. Generation of a single-cell atlas from inflamed and uninflamed colonic biopsies from UC patients led to the identification of thirty-six discrete cell populations, including cells of the myeloid compartment. The UC atlas was used to generate cell-specific signatures, allowing for cellular deconvolution of the Phase 3 datasets. It revealed significant reductions in neutrophil subsets, monocyte-derived macrophages, and inflammatory fibroblasts, as well as increases in colonic epithelial cells common to both etrolizumab and adalimumab. Pseudo-time trajectory analyses identified four unique neutrophil subsets with unique cell phenotypes reflecting changes in cell state or differentiation from PADI4hi, OSMhi, MX1hi, and ultimately to CXCR4hi populations. PADI4hi and OSMhi neutrophils exhibited high levels of proteases (MMP9, LYZ), inflammatory cytokines (CXCL1, IL1B, OSM), and abundant cytokine or chemokine receptors (CXCR1, CXCR2). MX1 populations expressed markers indicating prior IFN exposure (MX1, IFIT1). In contrast, more differentiated or mature neutrophils exhibited high levels of CXCL2, TNF-a, and CXCR4, as well as angiogenic factors like VEGFA. PADI4hi and OSMhi neutrophils, we predict, have abundant cytokine and chemokine interactions with inflammatory fibroblasts within the inflamed colon, such as OSM: OSMR and IL1B: IL1R1 interactions. Changes in PADI4hi and OSMhi neutrophils were closely associated with MCS remission in both etrolizumab and adalimumab-treated patients. In contrast, only minor changes in CXCR4hi neutrophils were observed and not associated with clinical outcomes. Our results suggest that neutrophils are not only heterogeneous in phenotype but have abundant cell-cell interactions in inflamed colonic tissue that are likely implicated in maintaining chronic disease activity. We hypothesize that limiting the interactions between neutrophils and other myeloid cells with resident cells such as inflammatory fibroblasts may reduce the production of inflammatory mediators and limit activation and infiltration of neutrophils, which may be necessary for achieving greater rates of clinical remission in response to interventional agents.

immunology↗

bioRxiv2022

Iron regulatory pathways differentially expressed during Madurella mycetomatis grain development in Galleria mellonella

BackgroundMycetoma is a neglected, chronic granulomatous infection of the subcutaneous tissue, most often caused by the fungal pathogen Madurella mycetomatis. Characteristic of the infection is the formation of grains. However, knowledge of the function and formation of the grain is limited. To map the processes leading to M. mycetomatis grain formation, we used a Galleria mellonella larvae infection model and time-course transcriptomic profiling. MethodsGalleria mellonella larvae were infected with M. mycetomatis genome strain mm55. At 4h, 24h, 72h and 168h post-inoculation, RNA was extracted from larval content. Two types of sequencing libraries were prepared for time-course transcriptomic profiling and analysis. FindingsIn the infected G. mellonella, 88.0% of the RNA sequence reads mapped to G. mellonella, while only 0.01% mapped to M. mycetomatis. Differential Gene Expression analysis revealed that 3,498 G. mellonella and 136 M. mycetomatis genes were differentially expressed during infection. Most of the enriched GO terms of both host and pathogen are linked to energy pathways, nucleobase metabolic process as well as cation and iron transport. Genes related to iron transport were highly expressed by both G. mellonella (transferrin and ferritin) and M. mycetomatis (SidA, SidD and SidI). A protein-protein interaction network analysis of D. melanogaster homologous genes in M. mycetomatis revealed the expression of the entire siderophore biosynthesis pathway throughout infection. InterpretationThe identification of the importance of iron acquisition during grain formation can be exploited as a potential novel diagnostic and therapeutic strategy for mycetoma. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSMycetoma is a chronic, neglected tropical infectious disease, characterised by a large subcutaneous mass and the formation of black grains in the affected tissue. Treatment for mycetoma is disappointing as in 25-50% of the patients recurrences are noted and up to 15% of patients will have to undergo amputation. The main reason behind this poor treatment response is the formation of protective structures by the pathogen upon entering the human body. These structures are called grains and provide a strong barrier for antifungal agents. Although grains are the hallmark of mycetoma, it is currently not known how these grains are formed. To improve the current therapy, it is important to gain insights in grain formation. Added value of this studyWe unravel the processes leading to grain formation and development in an invertebrate model of Madurella mycetomatis grain. We were able to build a model of grain formation and demonstrated that iron sequestering plays an important role in this process. Our findings were an important milestone in understanding the pathogenesis of mycetoma which has been a mystery for decades. Implications of all the available evidenceThe findings, will provide leads for future drug development of mycetoma treatment and therefore, improve patients live and end the need for amputations.

microbiology↗

bioRxiv2022

NFYA promotes the malignant behavior of triple-negative breast cancer through the regulation of lipid metabolism

Two splicing variants exist in NFYA, which exhibits high expression in many human tumor types, and their expression balance is known to correlate with prognosis in breast cancer, but functional differences are still unclear. Here, we demonstrate that NFYAv1, the long-form variant, upregulates the transcription of ACACA and FASN, essential lipogenic enzymes, to enhance the malignant behavior of triple-negative breast cancer (TNBC). Loss of the NFYAv1-lipogenesis axis strongly suppresses the malignant behavior in vitro and in vivo, indicating that the NFYAv1-lipogenesis axis is essential for TNBC malignant behavior and that the axis might be a potential therapeutic target for TNBC. Furthermore, mice deficient in lipogenic enzymes such as Acly, Acaca, and Fasn exhibit embryonic lethality, but our Nfyav1-deficient mice have no apparent developmental abnormalities. Taken together, our results elucidate NFYAv1-lipogenesis axis has significant tumor-promoting effects and the potential for NFYAv1 to be a safer therapeutic target for TNBC.

cancer biology↗

bioRxiv2022

The germline factor DDX4 contributes to the chemoresistance of small cell lung cancer cells

Human cancers often re-express germline factors, yet their mechanistic role in oncogenesis and cancer progression remains unknown. Here we demonstrate that DDX4, a germline factor and RNA helicase conserved in all multicellular organisms, contributes to epithelial mesenchyme transition (EMT)-like features and cisplatin resistance in small cell lung cancer (SCLC) cells. DDX4 depletion in H69AR and SHP77 cell lines decreased motility and resistance to cisplatin, whereas its overexpression increased these features. Proteomic analysis suggests that DDX4 upregulates metabolic protein expression related to DNA repair and immune/inflammatory response, suggesting its fundamental function may be in regulating cellular metabolism. Consistent with these trends in cell lines, DDX4 depletion compromised in vivo tumor development while its overexpression enhanced tumor growth even after cisplatin treatment in nude mice. Although the DDX4 expression level in somatic tumors is generally low compared to that in the germline, the relatively higher DDX4 expression in SCLC patients correlates with decreased survival and shows increased expression of EMT and cisplatin resistance markers. Taken together, we conclude that DDX4 influences the survival of SCLC patients by altering cellular metabolism in response to environmental cues such as drug treatments. This fundamental function of DDX4 as a germline factor might be applicable in other cancer types that express DDX4 and may serve as a key to combat specific tumors that are highly resistant to treatments. HighlightsO_LIDDX4 contributes to cellular motility and drug resistance in SCLC cells. C_LIO_LIDDX4-overexpression globally alters the proteome and suppresses cytokine production. C_LIO_LIDDX4 promotes tumorigenesis and drug resistance in vitro and in vivo. C_LIO_LIDDX4 expression correlates with survival in SCLC patients and with immune/inflammatory response both in cell lines and patient samples. C_LI

cancer biology↗

bioRxiv2021

Aerobic glycolysis in cancer cells supplies ATP while preventing excess metabolic thermogenesis

Adenosine triphosphate (ATP) regeneration by substrate-level phosphorylation is a general feature of cancer metabolism, even under normoxic conditions (aerobic glycolysis). However, it is unclear why cancer cells prefer inefficient aerobic glycolysis over the highly efficient process of oxidative phosphorylation for ATP regeneration. To investigate the metabolic principles underlying aerobic glycolysis, we performed 13C-metabolic flux analysis of 12 cultured cancer cell lines and explored the metabolic constraints required to reproduce the results using in silico metabolic simulations. We found that the measured flux distribution can be reproduced by maximizing the ATP consumption in the flux balance analysis considering a limitation of metabolic heat dissipation (enthalpy change). It suggests that aerobic glycolysis may be preferable because metabolic heat production during one mol of ATP regeneration by aerobic glycolysis was less than that produced by oxidative phosphorylation (OXPHOS). Consistent with the simulation, OXPHOS inhibition induced metabolic redirection to aerobic glycolysis while maintaining the intracellular temperature. Furthermore, the dependency on aerobic glycolysis was partly alleviated upon culturing at low temperatures. Our data suggest that metabolic thermogenesis is an important factor in understanding aerobic glycolysis in cancer cells and that an advantage of aerobic glycolysis is the reduction in metabolic heat generation during ATP regeneration.

cancer biology↗

bioRxiv2020

Involvement of I-BAR protein IRSp53 in tumor cell growth via extracellular microvesicle secretion

Cellular protrusions mediated by the membrane-deforming I-BAR domain protein IRSp53 are involved in cell migration, including metastasis. However, the role of IRSp53 in cell proliferation remains unclear. Here, we examined the role of IRSp53 in cell proliferation and found that it acts through secretion. Coculture of gingiva squamous carcinoma Ca9-22 cells and their IRSp53-knockout cells restored proliferation to parental Ca9-22 cell levels, suggesting possible secretion dependent on IRSp53. Notably, the amounts of microvesicle fraction proteins that were secreted into the culture medium were reduced in the IRSp53-knockout cells. The IRSp53-knockout cells exhibited decreased phosphorylation of mitogen-activated protein kinase, suggesting the decrease in the proliferation signals. The phosphorylation was restored by the addition of the microvesicles. In mice xenograft Ca9-22 cells, IRSp53-containing particles were secreted around the xenograft, indicating that IRSp53-dependent secretion occurs in vivo. In a tumor mice model, IRSp53 deficiency elongated lifespan. In some human cancers, the higher levels of IRSp53 mRNA expression was found to be correlated with shorter survival years. Therefore, IRSp53 is involved in tumor progression and secretion for cellular proliferation.

cell biology↗

bioRxiv2017

Scaling up DNA data storage and randomaccess retrieval

Current storage technologies can no longer keep pace with exponentially growing amounts of data. 1 Synthetic DNA offers an attractive alternative due to its potential information density of ~ 1018 B/mm3, 107 times denser than magnetic tape, and potential durability of thousands of years.2 Recent advances in DNA data storage have highlighted technical challenges, in particular, coding and random access, but have stored only modest amounts of data in synthetic DNA. 3,4,5 This paper demonstrates an end-to-end approach toward the viability of DNA data storage with large-scale random access. We encoded and stored 35 distinct files, totaling 200MB of data, in more than 13 million DNA oligonucleotides (about 2 billion nucleotides in total) and fully recovered the data with no bit errors, representing an advance of almost an order of magnitude compared to prior work. 6 Our data curation focused on technologically advanced data types and historical relevance, including the Universal Declaration of Human Rights in over 100 languages,7 a high-definition music video of the band OK Go,8 and a CropTrust database of the seeds stored in the Svalbard Global Seed Vault.9 We developed a random access methodology based on selective amplification, for which we designed and validated a large library of primers, and successfully retrieved arbitrarily chosen items from a subset of our pool containing 10.3 million DNA sequences. Moreover, we developed a novel coding scheme that dramatically reduces the physical redundancy (sequencing read coverage) required for error-free decoding to a median of 5x, while maintaining levels of logical redundancy comparable to the best prior codes. We further stress-tested our coding approach by successfully decoding a file using the more error-prone nanopore-based sequencing. We provide a detailed analysis of errors in the process of writing, storing, and reading data from synthetic DNA at a large scale, which helps characterize DNA as a storage medium and justify our coding approach. Thus, we have demonstrated a significant improvement in data volume, random access, and encoding/decoding schemes that contribute to a whole-system vision for DNA data storage.

bioengineering↗