Biology subjects

Tsuchiya, K.

Publications and source records attributed to Tsuchiya, K..

14 recordsLinked to original sources

bioRxiv2026

Three-dimensional vascular microenvironments uncover endothelial plasticity during TGF-β2-driven vascular remodeling

The tumor microenvironment plays a pivotal role in tumor development, harboring elements such as endothelial cells, immune cells, fibroblasts, and soluble factors such as transforming growth factor-{beta} (TGF-{beta}) family. TGF-{beta} family regulates cell development and promotes tumor invasion, metastasis, angiogenesis, and endothelial-to-mesenchymal transition (EndoMT). Here, we investigate the effects of TGF-{beta} signaling on vascular remodeling using a three-dimensional (3D) vascular network in a microfluidic device. Using both a co-culture (3D-Co) and simplified endothelial monoculture (3D-CM), we demonstrate that TGF-{beta} signaling reduces the quality and functionality of the vasculature by regressing them. In addition, we observed the upregulation of EndoMT-related markers in mRNA and protein expressions, suggesting the induction of EndoMT in 3D vascular networks. The increased vascular permeability stimulated by TGF-{beta}2 also supports the loss of endothelial identity in the 3D-Co. Transcriptomic analysis revealed the coordinated activation of pathways associated with cell migration and EndoMT, along with the suppression of cell cycle progression. A comparative analysis of two-dimensional (2D) and 3D cultures revealed a fundamentally distinct endothelial response to TGF-{beta}2 in the 3D context, including metabolic reprogramming. These findings demonstrate that the 3D microenvironment critically modulates endothelial responses to TGF-{beta} and enables the emergence of vascular phenotypes not captured in 2D systems. This study provides a more physiologically relevant platform to investigate endothelial dysfunction and vascular remodeling.

bioengineering↗

bioRxiv2025

Highly selective SGLT2 inhibitors suppress glucose uptake in alpha-TC1 cells, while glucagon secretion is not affected.

The existence of sodium-glucose cotransporter 2 (SGLT2) in pancreatic alpha cells and its potential roles in glucagon secretion remain controversial, despite its well-established function in renal glucose reabsorption. While some studies suggest SGLT2 presence and its involvement in glucagon regulation, others deny its expression in alpha cells. To clarify this dispute, we investigated the functional effects of highly selective SGLT2 inhibitors, dapagliflozin and empagliflozin, on glucose uptake, intracellular ATP levels, and glucagon secretion in alpha-TC1 cells, a widely used model for glucagon-secreting cells in culture. The SGLT2 inhibitors significantly suppressed basal glucose uptake in alpha-TC1 cells, indicating functional SGLT2 presence. However, the inhibitors did not affect glucagon secretion. Neither the SGLT2 inhibitors nor the more potent glucose transport inhibitor, cytochalasin B, altered intracellular ATP levels or glucagon secretion. In contrast, pharmacological inhibition of K/ATP channels increased glucagon secretion without affecting glucose uptake or ATP levels. These results suggest that while SGLT2 is functionally present at low levels and mediates basal glucose uptake in alpha-TC1 cells, its inhibition has insufficient influence on intracellular ATP levels, and therefore glucagon secretion remains stable. Furthermore, our observations support predominant involvement of K/ATP channels in regulating glucagon secretion. Further studies in human purified pancreatic alpha cells in addition to islets are warranted to fully elucidate SGLT2s role in alpha-cell physiology.

pharmacology and toxicology↗

bioRxiv2025

Xenopus laevis as an infection model for human pathogenic bacteria

Animal infection models are essential for understanding bacterial pathogenicity and corresponding host immune responses. In this study, we investigated whether juvenile Xenopus laevis could be used as an infection model for human pathogenic bacteria. Xenopus frogs succumbed to intraperitoneal injection containing the human pathogenic bacteria Staphylococcus aureus, Pseudomonas aeruginosa, and Listeria monocytogenes. In contrast, non-pathogenic bacteria Bacillus subtilis and Escherichia coli did not induce mortality in Xenopus frogs. The administration of appropriate antibiotics suppressed mortality caused by S. aureus and P. aeruginosa. Strains lacking the agr locus, cvfA (rny) gene, or hemolysin genes in S. aureus, LIPI-1-deleted mutant of L. monocytogenes, which attenuate virulence within mammals, exhibited reduced virulence in Xenopus frogs compared to their respective wild-type counterparts. Bacterial distribution analysis revealed that S. aureus persisted the blood, liver, heart, and muscles of Xenopus frogs until death. These results suggested that intraperitoneal injection of human pathogenic bacteria induces sepsis-like symptoms in Xenopus frogs supporting their use as a valuable animal model for evaluating antimicrobial efficacy and identifying virulence genes in various human pathogenic bacteria.

microbiology↗

bioRxiv2024

Automated Xeno-Free Chondrogenic Differentiation from Human Embryonic Stem Cells: Enhancing Efficiency and Ensuring High-Quality Mass Production

IntroductionRepairing damaged cartilage poses significant challenges, particularly in cases of congenital cartilage defects such as microtia or congenital tracheal stenosis, or as a consequence of traumatic injury, as the regenerative potential of cartilage is inherently limited. Stem cell therapy and tissue engineering offer promising approaches to overcome these limitations in cartilage healing. However, the challenge lies in the size of cartilage-containing organs, which necessitates a large quantity of cells to fill the damaged areas. Therefore, pluripotent stem cells that can proliferate indefinitely are highly desirable as a cell source. This study aims to delineate the differentiation conditions for cartilage derived from human embryonic stem cells (ESCs) and to develop an automated cell culture system to facilitate mass production for therapeutic applications. MethodsCartilage cell sheets were derived from human ESCs (SEES2, clinical trial-compatible line) by forming embryoid bodies (EBs) with either conventional manual culture or a benchtop multi-pipetter and an automated medium exchange integrated cell incubator, using xeno-free media. Cell sheets were implanted into the subcutaneous tissue of immunodeficient NOG mice to obtain cartilage tissue. The properties of cartilage tissues were examined by histological staining and quantitative PCR analysis. ResultsWe have optimized an efficient xeno-free system for cartilage production with the conventional culture method and successfully transitioned to an automated system. Differentiated cartilage was histologically uniform with cartilage-specific elasticity and strength. The cartilage tissues were stained by alcian blue, safranin O, and toluidine blue, and quantitative PCR showed an increase in differentiation markers such as ACAN, COL2A1, and Vimentin. Automation significantly enhanced the efficiency of human ESC-derived chondrocyte differentiation. The number of constituent cells within EBs and the seeding density of EBs were identified as key factors influencing chondrogenic differentiation efficiency. By automating the process of chondrogenic differentiation, we achieved scalable production of chondrocytes. ConclusionsBy integrating the differentiation protocol with an automated cell culture system, there is potential to produce cartilage of sufficient size for clinical applications in humans. The resulting cartilage tissue holds promise for clinical use in repairing organs such as the trachea, joints, ears, and nose.

cell biology↗

bioRxiv2023

Identification of key virus--prokaryote infection pairs that contribute to viral shunt in a freshwater lake

Viruses infect and kill productive prokaryotes in a density-or frequency-dependent manner and affect carbon cycling. However, the effects of the stratification transition, including the stratified and destratified periods, on the changes in prokaryotic/viral communities and the interactions among them remain unclear. We conducted a monthly survey of the surface and deep layers of a large and deep freshwater lake (Lake Biwa, Japan) for a year and analyzed the prokaryotic production and prokaryotic/viral metagenome. Our analysis (including 1 608 prokaryotes and 13 761 viruses) revealed that 19 prokaryotic species, accounting for [~]40% of total abundance, might be suppressed by viruses when prokaryotic production is higher. This suggests that a small proportion of prokaryotes contribute to a large amount of prokaryotic abundance, and these prokaryotes are infected and lysed by viruses, driving the viral shunt in the freshwater lake. Furthermore, we found that annual vertical mixing might yield a similar rate of community change between the surface and deep layers. This finding might be valuable in understanding how the communities change when the stratification of freshwater lakes is affected by global warming in the future.

microbiology↗

bioRxiv2023

Interleg coordination is not strictly controlled during walking

In human walking, the left and right legs move alternately, half a stride out of phase with each other. Although various parameters, such as stride frequency, stride length, and duty factor, vary with walking speed, the antiphase relationship of the leg motion remains unchanged. This is the case even during running. However, during walking in left-right asymmetric situations, such as walking with unilateral leg loading, walking along a curved path, and walking on a split-belt treadmill, the relative phase between left and right leg motion shifts from the antiphase condition to compensate for the asymmetry. In addition, the phase relationship fluctuates significantly during walking of elderly people and patients with neurological disabilities, such as those caused by stroke or Parkinsons disease. These observations suggest that appropriate interleg coordination is important for adaptive walking and that interleg coordination is strictly controlled during walking of healthy young people. However, the control mechanism of interleg coordination remains unclear. In the present study, we derive a quantity that models the control of interleg coordination during walking of healthy young people by taking advantage of a state-of-the-art method that combines big data science with nonlinear dynamics. This is done by modeling this control as the interaction between two coupled oscillators through the phase reduction theory and Bayesian inference method. However, the results were not what we expected. Specifically, we found that the relative phase between the motion of the legs is not actively controlled until the deviation from the antiphase condition exceeds a certain threshold. In other words, the control of interleg coordination has a dead zone like that in the case of the steering wheel of an automobile. Such forgoing of control presumably enhances energy efficiency and maneuverability during walking. Furthermore, the forgoing of control in specific situations, where we expect strict control, also appears in quiet standing. This suggests that interleg coordination in walking and quiet standing have a common characteristic strategy. Our discovery of the dead zone in the control of interleg coordination not only provides useful insight for understanding gait control in humans, but also should lead to the elucidation of the mechanisms involved in gait adaptation and disorders through further investigation of the dead zone.

bioengineering↗

bioRxiv2021

Microtubule-associated proteins promote microtubule generation in the absence of γ-tubulin in human colon cancer cells

{gamma}-Tubulin complex acts as the predominant microtubule (MT) nucleator that initiates MT formation and is therefore an essential factor for cell proliferation. Nonetheless, cellular MTs are formed after experimental depletion of the {gamma}-tubulin complex, suggesting that cells possess other factors that drive MT nucleation. Here, by combining gene knockout, auxin-inducible degron, RNA interference, MT depolymerisation/regrowth assay, and live microscopy, we identified four microtubule-associated proteins (MAPs), ch-TOG, CLASP1, CAMSAPs, and TPX2, which are involved in {gamma}-tubulin-independent MT generation in human colon cancer cells. In the mitotic MT regrowth assay, nucleated MTs organised non-centriolar MT organising centres (ncMTOCs) in the absence of {gamma}-tubulin. Depletion of CLASP1 or TPX2 substantially delayed ncMTOC formation, suggesting that they promote MT nucleation in the absence of {gamma}-tubulin. In contrast, depletion of CAMSAPs or ch-TOG did not affect the timing of ncMTOC appearance. CLASP1 also accelerates {gamma}-tubulin-independent MT regrowth during interphase. Thus, MT generation can be promoted by MAPs without the {gamma}-tubulin template.

cell biology↗

bioRxiv2021

m6A demethylase ALKBH5 promotes tumor cell proliferation by destabilizing IGF2BPs target genes and worsens the prognosis of patients with non-small cell lung cancer

The modification of N6-methyladenosine (m6A) in RNA and its eraser ALKBH5, an m6A demethylase, play important roles across various steps of human carcinogenesis. However, the involvement of ALKBH5 in non-small cell lung cancer (NSCLC) development remains to be completely elucidated. The current study revealed that the expression of ALKBH5 were increased in NSCLC and increased expression of ALKBH5 worsened the prognosis of patients with NSCLC. In vitro study revealed that ALKBH5 knockdown suppressed cell proliferation ability of PC9 and A549 cells as well as promoted G1 arrest and increased the number of apoptotic cells. Furthermore, ALKBH5 overexpression increased the cell proliferation ability of the immortalized cell lines. Microarray analysis and western blotting revealed that the expression of CDKN1A or TIMP3 were increased by ALKBH5 knockdown. These alterations were offset by a double knockdown of both ALKBH5 and one of the IGF2BPs. The decline of mRNAs was, at least partly, owing to the destabilization of these mRNAs by one of the IGF2BPs. In conclusions, the ALKBH5-IGF2BPs axis promotes cell proliferation and tumorigenicity, which in turn causes the unfavorable prognosis of NSCLC.

cancer biology↗

bioRxiv2020

Dynamical determinants of different spine movements and gait speeds in rotary and transverse gallops

Quadruped gallop is categorized into two types: rotary and transverse. While the rotary gallop involves two types of flight with different spine movements, the transverse gallop involves only one type of flight. The rotary gallop can achieve faster locomotion than the transverse gallop. To clarify these mechanisms from a dynamic viewpoint, we developed a simple model and derived periodic solutions by focusing on cheetahs and horses. The solutions gave a criterion to determine the flight type: while the ground reaction force does not change the direction of the spine movement for the rotary gallop, it changes for the transverse gallop, which was verified with the help of animal data. Furthermore, the criterion provided the mechanism by which the rotary gallop achieves higher-speed than the transverse gallop based on the flight duration. These findings improve our understanding of the mechanisms underlying different gaits that animals use.

bioinformatics↗

bioRxiv2019

The WIPE assay for selection and elimination of HIV-1 provirus in vitro using latency-reversing agents

Persistence of HIV-1 latent reservoir cells during antiretroviral therapy is a major obstacle for curing HIV-1. Latency-reversing agents (LRAs) are under development to reactivate and eradicate latently infected cells; however, there are few useful models for evaluating LRA activity in vitro. Here, we established a long-term cell culture system harboring thousands of different HIV-1-infected cell clones with a wide distribution of HIV-1 provirus similar to that observed in vivo. A combination of an LRA and antiretroviral therapy (ART) significantly reduced viral rebound upon treatment interruption. Experimental investigation and mathematical modeling demonstrated that addition of LRA to ART induced latency-reversing effect and contributed to the eradication of replication competent HIV-1. The widely distributed intact provirus elimination (WIPE) assay will be useful for optimizing therapeutics against HIV-1 latency and investigating mechanistic insights into the clonal selection of heterogeneous HIV-1-infected cells.

microbiology↗

bioRxiv2019

Synthetic peptide-induced internalization of biomolecules into various plant and algal cells via micropinocytosis

Efficient intracellular delivery of biomolecules is important for many different biological and biotechnological applications in living organisms, and is a prerequisite for certain types of fundamental and applied research. One major challenge is the delivery of unmodified, functional cargoes in a simple, time-efficient, and high-throughput manner. Herein, we present an efficient strategy that uses fusion peptides containing cell penetrating peptide, endosomal escape domain, and a sarcosine linker to introduce biomolecules, namely fluorescent protein and dextran, via macropinocytosis into the cells of various land plants and microalgae. Our peptide-mediated delivery system allows for high-throughput delivery of functional biomolecules within a few minutes to a few hours as well as open new possibilities for biology and biotechnology using difficult-to-transfect cell types.

plant biology↗

bioRxiv2018

Importin-β targets HURP to kinetochore-fibers in coordination with Ran-GTP in human mitotic cells

During mitosis, a bipolar spindle is assembled around chromosomes to efficiently capture chromosomes. Previous work proposed that a chromosome-derived Ran-GTP gradient promotes spindle assembly around chromosomes by liberating spindle assembly factors (SAFs) from inhibitory importins. However, Rans dual functions in interphase nucleocytoplasmic transport and mitotic spindle assembly have made it difficult to assess its mitotic roles in somatic cells. Here, using auxin-inducible degron technology in human cells, we developed acute mitotic degradation assays to dissect Rans mitotic roles systematically and separately from its interphase function. In contrast to the prevailing model, we found that the Ran pathway is not essential for spindle assembly activities that occur at sites spatially separated from chromosomes, including activating NuMA for spindle pole focusing or for targeting TPX2. In contrast, Ran-GTP is required to localize HURP and HSET specifically at chromosome-proximal regions. We demonstrated that Ran-GTP and importin-{beta} coordinately promote HURPs dynamic microtubule binding-dissociation cycle near chromosomes, which results in stable kinetochore-fiber formation. Intriguingly, this pathway acts to establish proper spindle length preferentially during prometaphase, rather than metaphase. Together, we propose that the Ran pathway is required to activate SAFs specifically near chromosomes, but not generally during human mitotic spindle assembly. Ran-dependent spindle assembly is likely coupled with parallel pathways to activate SAFs, including NuMA, for spindle pole focusing away from chromosomes. HighlightsO_LIUsing auxin-inducible degron technology, we developed mitotic degradation assays for the Ran pathway in human cells. C_LIO_LIThe Ran pathway is non-essential to activate NuMA for spindle pole focusing. C_LIO_LIThe Ran pathway dynamically polarizes HURP and defines mitotic spindle length preferentially during prometaphase. C_LIO_LIRan-GTP is required to activate SAFs specifically near chromosomes, but not generally, in human mitotic cells. C_LI

cell biology↗

bioRxiv2018

A new highly sensitive real-time quantitative-PCR method for detection of BCR-ABL1 to monitor minimal residual disease in chronic myeloid leukemia after discontinuation of imatinib

Tyrosine kinase inhibitors (TKIs) targeting the BCR-ABL1 fusion protein, encoded by the Philadelphia chromosome, have drastically improved the outcomes for patients with chronic myeloid leukemia (CML). Although several real-time quantitative polymerase chain reaction (RQ-PCR) kits for the detection of BCR-ABL1 transcripts are commercially available, their accuracy and efficiency in laboratory practice require reevaluation. We have developed a new in-house RQ-PCR method to detect minimal residual disease (MRD) in CML cases. MRD was analyzed in 102 patients with CML from the DOMEST study, a clinical trial to study the rationale for imatinib mesylate discontinuation in Japan. The BCR-ABL1/ABL1 ratio was evaluated using the international standard (IS) ratio, where IS < 0.01% was defined as a major molecular response. At enrollment, BCR-ABL1 transcripts were undetectable in all samples using a widely-applied RQ-PCR method performed in the commercial laboratory, BML (BML Inc., Tokyo, Japan); however, the in-house method detected the BCR-ABL1 transcripts in five samples (5%) (mean IS ratio: 0.0062 {+/-} 0.0010%). After discontinuation of imatinib, BCR-ABL1 transcripts were detected using the in-house RQ-PCR in 21 patients (21%) that were not positive using the BML method. Nineteen samples were also tested using a commercially available RQ-PCR assay kit with a detection limit of IS ratio, 0.0007% (ODK-1201, Otsuka Pharmaceutical Co., Tokyo, Japan). This method detected low levels of BCR-ABL1 transcripts in 14 samples (74%), but scored negative for five samples (26%) that were positive using the in-house method. These data suggest that our new in-house RQ-PCR method is effective for monitoring MRD in CML.

cancer biology↗

bioRxiv2018

A decay effect of bacterial growth rate associated with genome reduction

Bacterial growth is an important topic in microbiology and of crucial importance to better understand living cells. Bacterial growth dynamics are quantitatively examined using various methods to determine the physical, chemical or biological features of growing populations. Due to methodological differences, the exponential growth rate, which is a parameter that is representative of growth dynamics, should be differentiated. This study experimentally verified the differentiation in growth rates attributed to different methodologies, and demonstrated that the most popular method, optical turbidity, led to the determination of a lower growth rate in comparison to the methods based on colony formation and ATP abundance, due to a decay effect of reading OD600 during a population increase. Accordingly, the logistic model, which is often applied to growth data reading the OD600, was revised by introducing a new parameter: the decay rate, to compensate for the lowered estimation in growth rates. The modified logistic model not only presented an improved goodness of fit in comparison to the original model but also led to an intriguing finding of a correlation between genome reduction and the decay rate. The decay effect seemed to be partially attributed to the decrease in cell size accompanied by a population increase and was medium dependent. In summary, the present study provides not only a better theoretical tool for the high-throughput studies on bacterial growth dynamics linking with experimental data using optical turbidity to the theoretical analysis with biological importance, but also a valuable insight for understanding the genome evolution and fitness increase in microbial life.

microbiology↗