Biology subjects

Sakaguchi, K.

Publications and source records attributed to Sakaguchi, K..

7 recordsLinked to original sources

bioRxiv2025

Autofluorescence-based Label-free Cell Counting Method in Suspension Culture with Microcarriers

To advance the industrialization of cultured meat and regenerative medicine, scalable and efficient cell culture techniques are essential. Among these, the suspension culture method using microcarriers has emerged as a promising approach for the large-scale cell culture technique. However, monitoring cell growth on the microcarriers remains challenging, particularly in developing cell counting techniques that can be seamlessly integrated into bioprocess workflows without cell detachment, fluorescence labeling and any parameter tuning in the analysis algorithm. In this study, we proposed a versatile image analysis-based cell counting method by using cellular autofluorescence without any parameter tuning. The proposed method estimates the number of cells by applying spatiotemporal averaging to the autofluorescence signals in the microscopic images. Using numerical and cell culture experiments, we demonstrated that the proposed method can estimates the number of cells accurately. This technique, which harnesses the ubiquitous autofluorescence inherent in living cells, offers a cost-effective and practical solution applicable to a broad range of fields requiring high-throughput cell quantification.

systems biology↗

bioRxiv2024

Identification of novel candidate genes associated symbiotic compatibility of soybean with rhizobia under natural conditions

A robust symbiotic relationship between soybean and rhizobia can enhance the yield and quality of soybeans by reducing nitrogen fertilizer inputs, thereby contributing to sustainable agriculture. The genetic interplay between soybean cultivars and the rhizobial species colonizing their roots under natural conditions remains underexplored. This study builds on the observation that the prevalence of rhizobial species associated with the soybean cultivars Peking and Tamahomare varies significantly. Herein, we performed a quantitative trait loci (QTL) analysis of the proportion of Rhizobium species present in the root nodules of these cultivars using recombinant inbred lines derived from a cross between Peking and Tamahomare. A major QTL was identified on chromosome 18, accounting for 42% of the phenotypic variation, and was subsequently localized to a 240 kb region. The RNA-seq analysis indicated that a single gene featuring nucleotide binding site-leucine-rich repeat domains exhibited markedly different expression levels in parent cultivars within the QTL region. As this locus is distinct from the chromosomal regions containing known nodule-related genes, such as Rj or rj, it likely represents a novel gene involved in symbiosis between rhizobia and soybeans. Further research of the function and role of this new gene has potential to improve soybean yield and contribute to sustainable agriculture under low nitrogen fertilizer conditions.

plant biology↗

bioRxiv2023

Direct reprogramming of oral epithelial progenitor cells to cancer stem cells at single cell resolution in vivo

Tumor initiation represents the first step in tumorigenesis during which normal progenitor cells undergo cell fate transition to cancer. Capturing this process as it occurs in vivo, however, remains elusive. Here we employ spatiotemporally controlled oncogene activation and tumor suppressor inhibition together with multiomics to unveil the processes underlying oral epithelial progenitor cell reprogramming into tumor initiating cells (TIC) at single cell resolution. TIC displayed a distinct stem-like state, defined by aberrant proliferative, hypoxic, squamous differentiation, and partial epithelial to mesenchymal (pEMT) invasive gene programs. YAP-mediated TIC programs included the activation of oncogenic transcriptional networks and mTOR signaling, and the recruitment of myeloid cells to the invasive front contributing to tumor infiltration. TIC transcriptional programs are conserved in human head and neck cancer and associated with poor patient survival. These findings illuminate processes underlying cancer initiation at single cell resolution, and identify candidate targets for early cancer detection and prevention.

cancer biology↗

bioRxiv2022

Insulin Effectiveness, a Measure Consisting of Insulin Sensitivity, Secretion, and Clearance, Captures Differences Between Diabetic and Nondiabetic States

ContextInsulin clearance is implicated in regulation of glucose homeostasis independently of insulin sensitivity and insulin secretion. ObjectiveTo understand the relation between blood glucose and insulin sensitivity, secretion, and clearance. MethodsWe performed a hyperglycemic clamp, a hyperinsulinemic-euglycemic clamp, and an oral glucose tolerance test (OGTT) in 47, 16, and 49 subjects with normal glucose tolerance (NGT), impaired glucose tolerance (IGT), and type 2 diabetes mellitus (T2DM), respectively. Mathematical analyses were retrospectively performed on this dataset. ResultsThe disposition index (DI), defined as the product of insulin sensitivity and secretion, showed a weak correlation with blood glucose levels, especially in IGT (r = 0.04; 95% CI, -0.63- 0.44). However, an equation relating DI, insulin clearance, and blood glucose levels was well conserved regardless of the extent of glucose intolerance. As a measure of the effect of insulin, we developed an index, designated "DI/cle," that is based on this equation and corresponds to DI divided by the square of insulin clearance. DI/cle was not impaired in IGT compared with NGT, possibly as a result of a decrease in insulin clearance in response to a reduction in DI, whereas it was impaired in T2DM relative to IGT. Moreover, DI/cle estimated from a hyperinsulinemic-euglycemic clamp, OGTT, or a fasting blood test were significantly correlated with that estimated from two clamp tests (r = 0.52; 95% CI, 0.37- 0.64, r = 0.43; 95% CI, 0.24- 0.58, and r = 0.54; 95% CI, 0.38- 0.68, respectively). ConclusionsDI/cle can serve as a new indicator for the trajectory of changes in glucose tolerance.

systems biology↗

bioRxiv2022

Development of a fluorescence reporter system to quantify transcriptional activity of endogenous p53 in living cells

The tumor suppressor p53 plays a central role in cellular stress responses by regulating transcription of multiple target genes. The temporal dynamics of p53 are thought to be important for its function: it encode input information and are decoded to induce distinct cellular phenotypes. However it remains unclear to what extent the temporal dynamics of p53 reflects the activity of p53-induced gene expression. In this study, we report a multiplexed reporter system that allows us to visualize the transcriptional activity of p53 at the single cell level. Our reporter system features simple and sensitive observation of the transcriptional activity of endogenous p53 to the response elements of various target genes. Using this system, we show that the transcriptional activation of p53 exhibits strong cell-to-cell heterogeneity. The transcriptional activation of p53 by etoposide is highly dependent on the cell cycle but not by UV-C. Finally, we show that our reporter system allows simultaneous visualization of the transcriptional activity of p53 and cell cycle. Our reporter system can thus be a useful tool for studying biological processes involving the p53 signaling pathway. Summary statementTumor suppressor protein p53 is one of the hub factor of the cellular stress responses. Our novel reporter system allows us to visualize the transcriptional activity of endogenous p53 easily and precisely.

cell biology↗

bioRxiv2020

PPM1D is a neuroblastoma oncogene and therapeutic target in childhood neural tumors.

Majority of cancers harbor alterations of the tumor suppressor TP53. However, childhood cancers, including unfavorable neuroblastoma, often lack TP53 mutations despite frequent loss of p53 function, suggesting alternative p53 inactivating mechanisms. Here we show that p53-regulating PPM1D at chromosome 17q22.3 is linked to aggressive tumors and poor prognosis in neuroblastoma. We identified that WIP1-phosphatase encoded by PPM1D, is activated by frequent segmental 17q-gain further accumulated during clonal evolution, gene-amplifications, gene-fusions or gain-of-function somatic and germline mutations. Pharmacological and genetic manipulation established WIP1 as a druggable target in neuroblastoma. Genome-scale CRISPR-Cas9 screening demonstrated PPM1D genetic dependency in TP53 wild-type neuroblastoma cell lines, and shRNA PPM1D knockdown significantly delayed in vivo tumor formation. Establishing a transgenic mouse model overexpressing PPM1D showed that these mice develop cancers phenotypically and genetically similar to tumors arising in mice with dysfunctional p53 when subjected to low-dose irradiation. Tumors include T-cell lymphomas harboring Notch1-mutations, Pten-deletions and p53-accumulation, adenocarcinomas and PHOX2B-expressing neuroblastomas establishing PPM1D as a bona fide oncogene in wtTP53 cancer and childhood neuroblastoma. Pharmacological inhibition of WIP1 suppressed the growth of neural tumors in nude mice proposing WIP1 as a therapeutic target in neural childhood tumors.

cancer biology↗

bioRxiv2017

Bidirectional Changes Of Hepatic And Peripheral Insulin Clearance Characterize Abnormal Temporal Patterns Of Serum Insulin Concentration In Diabetic Subjects

Insulin plays a central role in glucose homeostasis, and impairment of insulin action causes glucose intolerance and leads to type 2 diabetes mellitus (T2DM). A decrease in the transient peak and sustained increase of circulating insulin by glucose infusion accompany T2DM pathogenesis. However, the mechanism underlying this abnormal temporal pattern of circulating insulin concentration remains unknown. Here we show that bidirectional changes of hepatic and peripheral insulin clearance characterize this abnormal temporal pattern of circulating insulin concentration during the progression of T2DM. We developed a mathematical model using a hyperglycemic and hyperinsulinemic-euglycemic clamp in 111 subjects, including healthy normoglycemic and diabetic subjects. The hepatic and peripheral insulin clearance significantly increase and decrease, respectively, during the progression of glucose intolerance. The increased hepatic insulin clearance reduces the amplitude of circulating insulin concentration, whereas the decreased peripheral insulin clearance changes the temporal patterns of circulating insulin concentration from transient to sustained. These results provide insight that may be useful in treating T2DM associated with aberrant insulin clearance.

systems biology↗