Biology subjects

Okamoto, A.

Publications and source records attributed to Okamoto, A..

16 recordsLinked to original sources

bioRxiv2026

Initiating mammalian reproduction from a single male genome

Sex in mammals is unidirectionally determined by the configuration of sex chromosomes. In contrast, several species, including fishes, can switch sexes in response to environmental cues, illustrating the reproductive advantages of sexual plasticity. Thus, technologies that can bypass the unidirectional nature of mammalian sex determination could open new avenues in reproductive biology. Here, by establishing a method that enables robust Y-chromosome elimination in embryos, we provide a strategy to regulate sex in mice. We show that this approach efficiently induces male-to-female sex reversal by releasing the Y chromosome as micronuclei during early development. When integrated with optimized somatic cell nuclear transfer, it enabled the parallel production of both male and sex-reversed female mice from a single male genome source. Offspring were successfully obtained from crosses between these male and sex-reversed female clones, demonstrating that this "dual-sex cloning" strategy can initiate sexual reproduction solely from a male somatic genome. These findings highlight dual-sex cloning as a valuable platform for securing mammalian reproduction and biodiversity from limited genetic resources.

developmental biology↗

bioRxiv2026

N-Terminal Alkylation of Proteins with Triazole-4-carbaldehyde for Targeted Liposome Engineering

A platform method to associate native proteins and peptides onto a liposome membrane using site-specific N-terminal alkylation based on 1H-1,2,3-triazole-4-carbaldehyde (TA4C) is developed. The TA4C reagent reacts with the N-terminal -amino group of native proteins under mild aqueous conditions in a single step, without genetic engineering or protecting group strategies. Equipping TA4C with hexyl and nonyl chains provides a direct handle for tuning the association between the protein and membrane. N-terminal alkylation of green fluorescent protein (GFP) as a model proceeds in high yield (93% for the hexyl group and 70% for the nonyl group), and tethering the N-terminal alkyl group on GFP efficiently associates the protein with the liposomal membrane, as confirmed by confocal laser scanning microscopy and dynamic light scattering. We extended this strategy to an investigation of the GE11 peptide, a ligand for the epidermal growth factor receptor (EGFR). The liposome immobilized with GE11 peptide possessing an N-terminal alkyl group enables active targeting with EGFR-overexpressing A431 cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/742023v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@9c5ec3org.highwire.dtl.DTLVardef@117765org.highwire.dtl.DTLVardef@b5c471org.highwire.dtl.DTLVardef@1741293_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

Spatial transcriptomic analysis reveals coordinated gene expression in ovarian clear cell carcinoma and adjacent endometriosis in UK and Japanese patients

PurposeOvarian clear cell carcinoma (OCCC) is strongly associated with endometriosis and shows geographic variation in incidence. We investigated whether OCCC and adjacent endometriosis exhibit distinct transcriptional states and whether these patterns differ between United Kingdom (UK) and Japanese cohorts. Experimental DesignWe performed whole-transcriptome spatial profiling on specimens from 16 OCCC cases (8 UK, 8 Japan) in which tumor and endometriosis were both present. Gene expression was analyzed in tumor, endometriosis and stroma. ARID1A status was assessed by immunohistochemistry. ResultsMedian age was 59 years (range 26-82). 13/16 cases (81.3%) had early-stage disease. Tissue compartment rather than cohort of origin was the dominant source of variation across endometriosis and tumor regions. Endometriosis was enriched for inflammatory and immune-related pathways compared to tumor, whilst there was greater representation of chromatin and protein-DNA complex assembly pathways in tumor regions. These patterns were conserved across both cohorts and after stratification by ARID1A status. Mesenchymal-associated gene expression scores also significantly differed across stroma, endometriosis and tumor with clear compartmental separation. Cell type deconvolution analyses showed clear compositional differences between stromal and epithelial disease compartments. ConclusionsOCCC and coexisting endometriosis are transcriptionally distinct, with the dominant contrast being compartmental rather than geographic. ARID1A alone is unlikely to account for the principal spatial transcriptional states identified here. Further analyses will be required to ascertain whether these differences reflect genuine biological differences between OCCC and coexisting endometriosis or represent different stages of endometriosis-associated tumorigenesis. Translational RelevanceOvarian clear cell carcinoma often arises in association with endometriosis, yet the biological transition between these lesions remains poorly understood. Using spatial transcriptomics in matched tumor and adjacent endometriosis from Japanese and UK cohorts, we showed that endometriosis is characterized by inflammatory and antigen-presentation features, whereas tumor regions showed chromatin-organization and oncogenic transcriptional states. These patterns were largely maintained irrespective of ARID1A status and geographic background. In addition, spatial deconvolution suggested differences in local immune composition, with tumor regions showing relatively greater neutrophil- and T cell-associated signals. Together, our data suggest that OCCC and coexisting endometriosis share a spatially linked tissue context, but that tumor regions have distinct transcriptional profile and microenvironment that may be involved in the malignant transformation and inform interpretation of molecular classification in endometriosis-associated OCCC.

pathology↗

bioRxiv2025

Development of a sandwich ELISA for the detection of bovine A1 beta-casein

The genetic variant A2 beta-casein is associated with fewer digestive and absorption issues compared to A1 beta-casein, leading to increased global demand for A2 milk. However, contamination with the A1 variant during collection, transportation, or sterilization of A2 milk poses a risk, necessitating a verification test to ensure A2 milk does not contain A1 beta-casein. We developed an A1-specific monoclonal antibody (mAb) and a general mAb that reacts with both A1 and A2 variants using the iliac lymph node method. A sandwich ELISA was created using the general mAb as the capture antibody and the A1-specific mAb as the detection antibody to identify A1 beta-casein in milk. This ELISA successfully detected A1 beta-casein in raw and pasteurized A2 milk, including ultra-high temperature treated milk. The test identified A1 beta-casein when the A1 spike in A2 milk exceeded 1% in volume, indicating its capability to detect contamination from one A1A1 cow in a herd of one hundred A2A2 cows. The developed A1 beta-casein ELISA is suitable for high-throughput analysis and can be valuable for monitoring A1 beta-casein contamination in commercially produced A2 milk.

zoology↗

bioRxiv2025

Structural basis for the folding of PINK1 by the HSP90-CDC37 chaperone complex

PTEN-induced kinase 1 (PINK1) is a mitochondrial serine/threonine kinase that plays a central role in Parkin-dependent mitophagy. Mutations in PINK1 are associated with familial Parkinsons disease. PINK1 is a high-affinity client of the HSP90-CDC37 complex and is stabilized by this chaperone system. However, the molecular mechanism by which HSP90-CDC37 facilitates the folding of PINK1 remains unclear. Here, we present a cryogenic electron microscopy structure of the human PINK1-HSP90-CDC37 complex. The {beta}5 strand of the PINK1 N-lobe is accommodated in the central channel of the HSP90 dimer, which holds the PINK1 kinase domain in a partially unfolded state. The C-lobe and unique C-terminal extension (CTE) of PINK1 is folded. HSP90 covers the CTE of PINK1, which overlaps with interaction sites for TOM5, TOM20, and the PINK1 N-helix. The HPNI motif of CDC37 interacts with the C-lobe of PINK1, mimicking the HPNI motif in the N-lobe. The pathogenic mutation L347P is suggested to disrupt these interactions, while H271Q is located within the HPNI motif in the N-lobe of PINK1. These findings provide structural insights into the folding of PINK1 and its dysfunction in Parkinsons disease.

molecular biology↗

bioRxiv2025

Combined genomic and molecular analysis defines prognostic markers of relapse in stage IA-IC1 clear cell ovarian carcinoma

ObjectiveClear cell ovarian carcinoma (CCOC) is generally associated with a favourable prognosis, but up to 30% of low-stage cases relapse within five years. The benefit of adjuvant chemotherapy for low-stage CCOC (FIGO stage IA-IC1) remains uncertain. This study aimed to identify molecular and immune markers associated with relapse in a well-characterized CCOC cohort. MethodsUsing the Edinburgh Ovarian Cancer Database, we identified 85 CCOC cases. Targeted DNA sequencing assessed genomic alterations and tumour immune infiltration was evaluated using CD3 and CD8 immunohistochemistry on tissue microarrays. Tumours were stratified by stage (IA-IC1, IC2-II, III-IV), and both univariate and multivariate analysis applied for progression-free survival (PFS) included stage, age, genomic features, and immune markers. ResultsCommon genomic alterations included ARID1A (49%) and PIK3CA (42%) mutations, PIK3-AKT pathway perturbations (60%), and mismatch repair-related mutational signatures (25.9%). Genomic and molecular features were not significantly associated with tumour stage; however, low-stage tumours (IA-IC1) were enriched in CD3+ (40% cases) and CD8+ (29% cases) tumour-infiltrating lymphocytes (TILs) compared to higher-stage tumours (IC2-II: 13%/7%; III-IV: 9%/0% cases). In univariate analysis, low CD3+ TIL levels were significantly associated with reduced progression-free survival (HR = 4.4, P = 0.042, 95% CI 1.1-18). ARID1A wild-type status was linked to poorer PFS but only in low-stage tumours HR = 7.2, P = 0.088; 95% CI 0.74-72). Notably, the combination of ARID1A wild-type status and CD3+ depletion identified a high-risk subgroup among low-stage CCOC cases (HR = 11.7, P = 0.050; 95% CI 1-138), highlighting potential as a composite risk marker set. ConclusionsCombined ARID1A mutation and low CD3+ve TIL levels are suggestive of higher recurrence risk in low-stage CCOC patients. These findings support further investigation of targeted therapies or immune checkpoint inhibitors in larger CCOC cohorts. HighlightsO_LIWe report a well-characterized CCOC cohort with rich clinical, genomic and molecular data. C_LIO_LICommon mutations included ARID1A (49%) and PIK3CA (42%), with MSI in 9%, showing no significant enrichment across tumour stages. C_LIO_LILow-stage CCOC has higher levels of CD3+ & CD8+ tumour infiltrating leukocytes than in advanced-stage disease. C_LIO_LIARID1A wild-type tumours with CD3+ TIL depletion represent a high-risk subgroup of low-stage CCOC. C_LIO_LIFindings support further study and highlight potential markers for recurrence risk. C_LI

cancer biology↗

bioRxiv2024

Droplet Sequencing Reveals Virulence Gene Clusters in Oral Biofilm Extracellular Vesicles

Bacterial extracellular vesicles (BEVs), produced by a broad spectrum of bacteria, play a crucial role in enhancing intercellular communication through DNA transfer. A vital determinant of their gene transfer efficiency is the gene content diversity within BEVs, an aspect that conventional metagenomics fails to capture. Our study bridges this gap with a novel microdroplet-based sequencing technique that precisely details DNA content within hundreds of individual BEVs. This technique revealed a unique DNA profile in BEVs from the oral pathogen Porphyromonas gingivalis, pinpointing specific genomic regions related to DNA integration (e.g., DNA transposition and CRISPR-Cas systems). These enriched genes, overlooked by standard analyses that aggregate total read counts, indicate that our method offers a more focused view into the genetic contents of BEVs. Applying our technique to dental plaque-derived BEVs, we discovered a hundredfold higher prevalence of DNA encapsulation than previously estimated, with over 30% of BEVs containing DNA. Specifically, we identified a substantial presence of O-antigen biosynthesis genes, prominent hotspots of frequent horizontal gene transfer, from Alcaligenes faecalis. Given that O-antigens mediate host-bacterial interactions, this gene enrichment in the large fraction of BEVs suggests a potential novel pathway by which BEVs could influence pathogenicity within oral biofilms. Our research unveils critical insights into the potential functions of vesicular DNA in microbial communities, establishing a powerful platform for studying vesicular DNA in microbiomes. This technical breakthrough provides a foundational basis for future research in microbial communication and the development of potential therapeutic or diagnosis strategies. Significance StatementBEVs have been studied for decades, yet their roles in nature and disease are just beginning to be appreciated. Our study makes a significant leap in understanding the roles of BEVs as gene transfer vehicles. By developing a microdroplet-based sequencing technique, we have uncovered detailed DNA profiles within individual BEVs, a task beyond the capabilities of conventional metagenomic methods. This breakthrough highlights specific genomic regions enriched in BEVs from pure culture and human dental plaque. Furthermore, the high prevalence of biofilm BEVs enriched in O-antigen biosynthesis genes, suggests a potential impact on the pathogenicity of oral biofilms. This research establishes a new methodological platform for exploring the intricacies of BEV-mediated interactions in a complex microbial community.

microbiology↗

bioRxiv2023

Laser-induced auditory perception: transtympanic laser irradiation of the cochlea from the outer ear evokes an auditory-related neural and behavioral response

Brain-machine interfaces are emerging devices that act as a medium for communication between the neural system and external software and hardware. Over the last decade, there has been a discussion on applying infrared laser stimulation to the brain-machine interfaces, including cochlear implants. Infrared lasers can selectively activate neural populations without introducing exogenous agents to tissues. This study presents the first demonstration that laser irradiation of the cochlea, a peripheral sensory organ, can elicit a clear behavioral auditory response. Mongolian gerbils (Meriones unguiculatus) were divided into two groups, each subjected to classical conditioning where a reward water delivery was predicted by either cochlear laser stimulation or sound stimulus. Laser-conditioned animals successfully learned licking behavior, with conditioned responses and behavioral properties comparable to auditory-conditioned animals. The laser-evoked response was significantly inhibited by auditory masking, and auditory-conditioned animals demonstrated stimulus generalization to laser stimulation. In a subsequent experiment, simultaneous presentation of auditory and laser stimulation induced nonlinear amplification in both auditory cortical and behavioral responses, suggesting that combining auditory and laser stimulation can enhance auditory perception beyond the effects of each stimulus alone. These findings indicate that infrared laser irradiation of the cochlea can potentially evoke and enhance auditory perception, making it a promising candidate for implementation in auditory prostheses.

neuroscience↗

bioRxiv2022

Novel therapeutic strategies for injured endometrium: Autologous intrauterine transplantation of menstrual blood-derived cells from infertile patients

BackgroundMenstrual blood-derived cells show regenerative potential as a mesenchymal stem cell and may therefore be a novel stem cell source of treatment for refractory infertility with injured endometrium. However, there have been few pre-clinical studies using cells from infertile patients, which needs to be addressed before establishing an autologous transplantation. Herein, we aimed to investigate the therapeutic capacity of menstrual blood-derived cells from infertile patients on endometrial infertility. MethodsWe collected menstrual blood-derived cells from volunteers and infertile patients, and confirmed their mesenchymal stem cell phenotype by flowcytometry and induction of tri-lineage differentiation. We compared the proliferative and paracrine capacities of these cells. Furthermore, we also investigated the regenerative potential and safety concerns of the intrauterine transplantation of infertile patient-derived cells using a mouse model with mechanically injured endometrium. ResultsMenstrual blood-derived cells from both infertile patients and volunteers showed phenotypic characteristics of mesenchymal stem cells. In vitro proliferative and paracrine capacities for wound healing and angiogenesis were equal for both samples. Furthermore, the transplantation of infertile patient-derived cells into uterine horns of the mouse model ameliorated endometrial thickness, prevented fibrosis and improved fertility outcomes without any apparent complications. ConclusionsIn our preclinical study, intrauterine transplantation of menstrual blood-derived cells may be a novel and attractive stem cell source for the curative and prophylactic therapy for injured endometrium. Further studies will be warranted for future clinical application.

cell biology↗

bioRxiv2022

Safety margin evaluations for transtympanic laser stimulation of the cochlea from the outer ear in Mongolian gerbils (Meriones unguiculatus)

Infrared laser cochlear stimulation has been suggested as an alternative to traditional auditory prostheses. Previous research has mainly examined the short-term effects of laser stimulation, but to effectively use this technique, it is necessary to study whether prolonged laser exposure can safely and sustainably induce cochlear responses. This study assessed the effect of laser-induced cochlear damage using classically-conditioned Mongolian gerbils. The white noise was presented as a conditioned stimulus, and reward licking was recorded as a conditioned response. After training, the subjects cochlea was exposed to a continuous pulsed laser for 15 h. The licking rate drastically decreased after the exposure of 26.4 W/cm2, while it did not change after 6.6 W/cm2 or weaker. These findings showed, for the first time, that the safety margin of long-term, at least several hours, cochlear laser stimulation exists and will contribute to the safe and effective delimitation of laser parameters in future research.

bioengineering↗

bioRxiv2022

Chemoenzymatic fluorescence labeling of intercellularly contacting cells using lipidated sortase A

Methods to label intercellular contact attract particular attention due to their potential in cell biological and medical applications through analysis of intercellular communications. In this study, a simple and versatile method for chemoenzymatically labeling the intercellularly contacting cell was developed by using a cell-surface anchoring reagent of poly(ethylene glycol)(PEG)-lipid conjugate. The surfaces of each cell in cell pairs of interest were efficiently decorated with sortase A (SrtA) and triglycine peptide that were lipidated with PEG-lipid, respectively. In the mixture of the two cell populations, the triglycine-modified cells were enzymatically labeled with a fluorescent labeling reagent by contacting with the SrtA-modified cells both on the substrate and in cell suspensions. Such selective labeling of the contacting cells was confirmed by confocal microscopy and flow cytometry. The results show a proof of principle that the present method is a promising tool for selective visualization and quantification of the intercellularly contacting cells among cell mixtures in cell-cell communication analysis.

biochemistry↗

bioRxiv2021

Glutamine deficiency in solid tumors confers resistance to ribosomal RNA synthesis inhibitors

Ribosome biogenesis involves the processing of precursor ribosomal RNAs (pre-rRNAs) and sequential assembly with ribosomal proteins. Here we report that nutrient deprivation severely impairs pre-rRNA processing and leads to the accumulation of unprocessed rRNAs. Upon nutrient restoration, the accumulated pre-rRNAs are processed into mature rRNAs that are utilized for ribosome biogenesis. Failure to accumulate pre-rRNAs under nutrient deprivation leads to perturbed ribosome assembly during nutrient restoration and subsequent apoptosis via uL5/uL18-mediated activation of p53. Restoration of glutamine alone activates p53 by triggering uL5/uL18 translation. Induction of uL5/uL18 protein synthesis by glutamine was dependent on the translation factor eukaryotic elongation factor 2 (eEF2), which was in turn dependent on Raf/MEK/ERK signalling. Depriving cells of glutamine prevents the activation of p53 by rRNA synthesis inhibitors. Our data reveals a mechanism that cancer cells can exploit to suppress p53-mediated apoptosis during fluctuations in environmental nutrient availability.

cancer biology↗

bioRxiv2021

Deep learning generates custom-made logistic regression models for explaining how breast cancer subtypes are classified

Differentiating the intrinsic subtypes of breast cancer is crucial for deciding the best treatment strategy. Deep learning can predict the subtypes from genetic information more accurately than conventional statistical methods, but to date, deep learning has not been directly utilized to examine which genes are associated with which subtypes. To clarify the mechanisms embedded in the intrinsic subtypes, we developed an explainable deep learning model called a point-wise linear (PWL) model that generates a custom-made logistic regression for each patient. Logistic regression, which is familiar to both physicians and medical informatics researchers, allows us to analyze the importance of the feature variables, and the PWL model harnesses these practical abilities of logistic regression. In this study, we show that analyzing breast cancer subtypes is clinically beneficial for patients and one of the best ways to validate the capability of the PWL model. First, we trained the PWL model with RNA-seq data to predict PAM50 intrinsic subtypes and applied it to the 41/50 genes of PAM50 through the subtype prediction task. Second, we developed a deep enrichment analysis method to reveal the relationships between the PAM50 subtypes and the copy numbers of breast cancer. Our findings showed that the PWL model utilized genes relevant to the cell cycle-related pathways. These preliminary successes in breast cancer subtype analysis demonstrate the potential of our analysis strategy to clarify the mechanisms underlying breast cancer and improve overall clinical outcomes.

cancer biology↗

bioRxiv2019

FeGenie: a comprehensive tool for the identification of iron genes and iron gene neighborhoods in genomes and metagenome assemblies

Iron is a micronutrient for nearly all life on Earth. It can be used as an electron donor and electron acceptor by iron-oxidizing and iron-reducing microorganisms, and is used in a variety of biological processes, including photosynthesis and respiration. While it is the fourth most abundant metal in the Earths crust, iron is often limiting for growth in oxic environments because it is readily oxidized and precipitated. Much of our understanding of how microorganisms compete for and utilize iron is based on laboratory experiments. However, the advent of next-generation sequencing and the associated surge in publicly-available sequence data has now made it possible to probe the structure and function of microbial communities in the environment. To bridge the gap between our understanding of iron acquisition and utilization in model microorganisms and the plethora of sequence data available from environmental studies, we have created a comprehensive database of hidden Markov models (HMMs) that is based on genes related to iron acquisition, storage, and reduction/oxidation. Along with this database, we present FeGenie, a bioinformatics tool that accepts genome and metagenome assemblies as input and uses our comprehensive HMM database to annotate the provided datasets with respect to iron-related genes and gene clusters. An important contribution of this tool is the efficient identification of genes involved in iron oxidation and dissimilatory iron reduction, which have been largely overlooked by standard annotation pipelines. While this tool will not replace the reliability of culture-dependent analyses of microbial physiology, it provides reliable predictions derived from the most up-to-date genetic markers. FeGenies database will be maintained and continually-updated as new genetic markers are discovered. FeGenie is freely available: https://github.com/Arkadiy-Garber/FeGenie.

bioinformatics↗

bioRxiv2017

Propofol induces metabolic reprogramming and cell death in a mitochondrial electron transport chain-dependent manner

The intravenous anesthetic propofol (2,6-diisopropylphenol) has been used for the induction and maintenance of anesthesia in operating rooms and for sedation in intensive care units. Although there is no widely accepted definition of propofol infusion syndrome (PRIS), PRIS is defined as the development of metabolic acidosis, rhabdomyolysis, hyperkalemia, hepatomegaly, renal failure, arrhythmia, and progressive cardiac failure. In vitro evidence suggests that PRIS is related to the impaired mitochondrial function. There are indications that preexisting mitochondrial disorders predispose to PRIS. However, the precise molecular mechanisms, including mitochondrial defects and a metabolic conversion by propofol, are largely unknown as yet. To elucidate the underlying cellular and molecular mechanisms of PRIS, we investigated the effects of propofol on the cellular metabolic mode and cell death. We demonstrated that clinically relevant concentrations of propofol, used within a clinically relevant exposure time, suppressed the mitochondrial function, caused the generation of reactive oxygen species, and induced a metabolic switch, from oxidative phosphorylation to glycolysis, by targeting complexes I and III of mitochondria. The data also indicated that a predisposition to mitochondrial dysfunction, caused by a genetic mutation or pharmacological suppression of the electron transport chain by biguanides such as metformin and phenformin, promoted the cell death and caspase activation induced by propofol.

pharmacology and toxicology↗

bioRxiv2016

Fetal therapy model of myelomeningocele with three-dimensional skin using amniotic fluid cell-derived induced pluripotent stem cells

Myelomeningocele (MMC) is a congenital disease without genetic abnormalities. Neurological symptoms are irreversibly impaired after birth. No effective treatment has been reported to date. Only surgical repairs have reported so far. In this study, we performed antenatal treatment of MMC with an artificial skin using induced pluripotent stem cells (iPSCs) generated from a patient with Down syndrome (AF-T21-iPSCs) and twin-twin transfusion syndrome (AF-TTTS-iPSCs) to a rat model. We manufactured three-dimensional skin with epidermis generated from keratinocytes derived from AF-T21-iPSCs and AF-TTTS-iPSCs and dermis of human fibroblasts and collagen type I. For generation of epidermis, we developed a novel protocol using Y-27632 and epidermal growth factor. The artificial skin was successfully covered over MMC defect sites during pregnancy, implying a possible antenatal surgical treatment with iPSC technology.

cell biology↗