Biology subjects

Kuroda, S.

Publications and source records attributed to Kuroda, S..

32 records · Page 2Linked to original sources

bioRxiv2022

Verification of extracellular vesicle-mediated functional mRNA delivery via RNA editing

The secretion and delivery of mRNA by extracellular vesicles (EVs) may contribute to intercellular communications. Several reporter assays have been developed to quantify EV-mediated functional delivery of mRNA into recipient cells. However, mRNA delivery efficiency can often be overestimated by experimental artifacts, resulting in "pseudo-delivery" of reporter proteins rather than mRNA. In this study, we revealed that substantial amounts of reporter proteins expressed in donor cells are secreted into the medium and interfere with the reporter assay. To eliminate this pseudo-delivery, we established a functional RNA delivery assay that employs an RNA editing tool, enabling the verification of bona fide delivery of mRNA into recipient cells. The donor cells expressed a reporter gene containing a stop codon in a non-functional open reading frame. After EV-mediated delivery of reporter mRNAs to the recipient cells, guide RNAs and RNA editing enzymes (dCas13b-hADAR2 fusion proteins) correct the RNA sequence and induce the expression of functional reporter proteins in the recipient cells. Using this system, we showed that EVs containing alphavirus-derived replicon successfully delivered functional RNA and expressed the reporter proteins. The RNA delivery assay using RNA editing enables the precise analysis of EV-mediated mRNA delivery.

cell biology↗

bioRxiv2021

Engineering of extracellular vesicles for small molecule-regulated cargo loading and cytoplasmic delivery of bioactive proteins

Cytoplasmic delivery of functional proteins into target cells remains challenging for many biological agents to exert their therapeutic effects. Extracellular vesicles (EVs) are expected to be a promising platform for protein delivery; however, efficient loading of proteins of interest (POIs) into EVs remains elusive. In this study, we utilized small compound-induced heterodimerization between FK506 binding protein (FKBP) and FKBP12-rapamycin-binding (FRB) domain, to sort bioactive proteins into EVs using the FRB-FKBP system. When CD81, a typical EV marker protein, and POI were fused with FKBP and FRB, respectively, rapamycin induced the binding of these proteins through FKBP-FRB interaction and recruited the POIs into EVs. The released EVs, displaying virus-derived membrane fusion protein, delivered the POI cargo into recipient cells and their functionality in the recipient cells was confirmed. Furthermore, we demonstrated that CD81 could be replaced with other EV-enriched proteins, such as CD63 or HIV Gag. Thus, the FRB-FKBP system enables the delivery of functional proteins and paves the way for EV-based protein delivery platforms.

bioengineering↗

bioRxiv2021

Omics-based label-free metabolic flux inference reveals dysregulation of glucose metabolism in liver associated with obesity

Glucose homeostasis is maintained by modulation of metabolic flux. Enzymes and metabolites regulate the involved metabolic pathways. Dysregulation of glucose homeostasis is a pathological event in obesity. Analyzing metabolic pathways and the mechanisms contributing to obesity-associated dysregulation in vivo is challenging. Here, we introduce OMELET: Omics-Based Metabolic Flux Estimation without Labeling for Extended Trans-omic Analysis. OMELET uses metabolomic, proteomic, and transcriptomic data to identify changes in metabolic flux, and to quantify contributions of metabolites, enzymes, and transcripts to the changes in metabolic flux. By evaluating the livers of fasting ob/ob mice, we found that increased metabolic flux through gluconeogenesis resulted primarily from increased transcripts, whereas that through the pyruvate cycle resulted from both increased transcripts and changes in substrates of metabolic enzymes. With OMELET, we identified mechanisms underlying the obesity-associated dysregulation of metabolic flux in liver. HighlightsO_LIWe created OMELET to infer metabolic flux and its regulation from multi-omic data. C_LIO_LIGluconeogenic and pyruvate cycle fluxes increased in fasting ob/ob mice. C_LIO_LITranscripts increases mediated the increase in gluconeogenic fluxes in ob/ob mice. C_LIO_LIIncreases in transcripts and substrates enhanced pyruvate cycle flux in ob/ob mice. C_LI

systems biology↗

bioRxiv2021

Reporter gene assay for membrane fusion of extracellular vesicles

AbstractExtracellular vesicles (EVs) secreted by living cells are expected to deliver biological cargo molecules, including RNA and proteins, to the cytoplasm of recipient cells. There is an increasing need to understand the mechanism of intercellular cargo delivery by EVs. However, the lack of a feasible bioassay has hampered our understanding of the biological processes of EV uptake, membrane fusion, and cargo delivery to recipient cells. Here, we describe a reporter gene assay that can measure the membrane fusion efficiency of EVs during cargo delivery to recipient cells. When EVs containing tetracycline transactivator (tTA)-fused tetraspanins are internalized by recipient cells and fuse with cell membranes, the tTA domain is exposed to the cytoplasm and cleaved by protease to induce tetracycline responsive element (TRE)-mediated reporter gene expression in recipient cells. This assay (designated as EV-mediated tetraspanin-tTA delivery assay, ETTD assay), enabled us to assess the cytoplasmic cargo delivery efficiency of EVs in recipient cells. With the help of a vesicular stomatitis virus-derived membrane fusion protein, the ETTD assay could detect significant enhancement of cargo delivery efficiency of EVs. Furthermore, the ETTD assay could evaluate the effect of potential cargo delivery enhancers/inhibitors. Thus, the ETTD assay may contribute to a better understanding of the underlying mechanism of the cytoplasmic cargo delivery by EVs.

cell biology↗

bioRxiv2020

Real-time luminescence assay for cytoplasmic cargo delivery of extracellular vesicles

Extracellular vesicles (EVs) have been considered to deliver biological cargos between cells and mediate intercellular communication. However, the mechanisms that underlie the biological process of EV uptake and cytoplasmic cargo release in recipient cells are largely unknown. Quantitative and real-time assays for assessment of the cargo delivery efficiency inside recipient cells have not been feasible. In this study, we developed an EV cargo delivery (EVCD) assay using a split luciferase called the NanoBiT system. Recipient cells expressing LgBiT, a large subunit of luciferase, emit luminescence when the EV cargo proteins fused with a small luminescence tag (HiBiT tag) that can complement LgBiT are delivered to the cytoplasm of recipient cells. Using the EVCD assay, the cargo delivery efficiency of EVs could be quantitatively measured in real time. This assay was highly sensitive in detecting a single event of cargo delivery per cell. We found that modification of EVs with a virus-derived fusogenic protein significantly enhanced the cytoplasmic cargo delivery; however, in the absence of a fusogenic protein, the cargo delivery efficiency of EVs was below the threshold of the assay. The EVCD assay could assess the effect of entry inhibitors on EV cargo delivery. Furthermore, using a luminescence microscope, the cytoplasmic cargo delivery of EVs was directly visualized in living cells. This assay could reveal the biological mechanism of the cargo delivery processes of EVs.

cell biology↗

bioRxiv2019

Trans-omic analysis reveals allosteric and gene regulation-axes for altered glucose-responsive liver metabolism associated with obesity

Impaired glucose tolerance associated with obesity causes postprandial hyperglycemia and can lead to type 2 diabetes. To study the differences in liver metabolism in the healthy and obese states, we constructed and analyzed trans-omic glucose-responsive metabolic networks with layers for metabolites, expression data for metabolic enzyme genes, transcription factors, and insulin signaling proteins from the livers of healthy and obese mice. We integrated multi-omic time-course data from wild-type (WT) and leptin-deficient obese (ob/ob) mice after orally administered glucose. In WT mice, metabolic reactions were rapidly regulated (within 10 minutes of oral glucose administration) primarily by glucose-responsive metabolites, especially ATP and NADP+, which functioned as allosteric regulators and substrates of metabolic enzymes, and by Akt-dependent glucose-responsive genes encoding metabolic enzymes. In ob/ob mice, most rapid regulation by glucose-responsive metabolites was absent; instead, glucose administration produced slow changes in the expression of metabolic enzyme-encoding genes to alter metabolic reactions in a time scale of hours. Few common regulatory events occurred in both the healthy and obese mice. Thus, our trans-omic network analysis revealed regulation of liver metabolism in response to glucose is mediated through different mechanisms in the healthy and obese states: Rapid changes in allosteric regulators and substrates and in gene expression dominate the healthy state, and slow transcriptional regulation dominates the obese state.\n\nOne Sentence SummaryRapid changes in regulatory metabolites and gene expression dominate the healthy state, and slow transcriptional regulation dominates the obese state.

systems biology↗

bioRxiv2019

Single-cell information analysis reveals small intra- and large intercellular variations increase cellular information capacity

Cells transmit information about extracellular stimulation through signaling pathways to control cellular function. A signaling pathway can be regarded as a communication channel. In the analysis of channels of cell populations, intercellular variation is considered noise. However, intercellular variation enables individual cells to encode different information. Therefore, at the single-cell level, each cell can be regarded as an independent channel. Thus, we propose that responses of cells of the same type in tissues, such as the fibers in a skeletal muscle, should be regarded as a multiple-cell channel composed of single-cell channels, in which intercellular variation contains information. Here, we applied electrical pulses to individual myotubes from cultured C2C12 cells or dissociated skeletal muscle fibers and measured Ca2+ responses or contraction, respectively, to estimate information capacity in a biological system. For each muscle cell system, we found that a single-cell channel transmitted more information than did a cell-population channel, indicating that the cellular response is consistent with each cell (low intracellular variation) but different among individual cells (high intercellular variation). As cell number and thus the number of single-cell channels increased, a multiple-cell channel transmitted more information by incorporating the differences among individual cells. Thus, a tissue with small intracellular and large intercellular variations has the capacity to distinguish differences in stimulation intensity to precisely control physiological function.\n\nOne Sentence SummarySmall intracellular and large intercellular variations increase information transmission for precise control of tissue function.

systems biology↗

bioRxiv2018

Development of a microfluidic cell culture and monitoring system for intracellular signaling studies

We describe a microfluidic cell culture and monitoring system that temporally controls molecule concentrations around cells cultured in a small space. The simple system consists of three syringe pumps and a microfluidic device with two inlet ports and two outlet ports. Each syringe pump discharges or draws culture medium, solutions containing signal molecules, or cell suspensions through a port in a programmed flow rate sequence. Signal molecule solutions of differing concentration are merged in a microchannel, mixed immediately, and transported into the cell culture chamber. Regulating the flow rate ratio of syringe pumps over time enables dynamic control of the concentration of signal molecules in the cell culture chamber. The system provides various time-dependent waveforms of concentration over cultured cells, including pulse, rectangular, and triangular. The practical performance of the system for concentration control was evaluated using fluorescent dye imaging. The system was also used with CHO-K1 cells to measure intracellular Ca2+ concentrations, which vary with extracellular ATP levels. When a rectangular pulse of ATP was applied to the cells, Ca2+ levels increased quickly. By contrast, several Ca2+ peaks were observed in response to stepwise increases in ATP concentration. Single-cell Ca2+ responses to ATP pulse stimulation were analyzed by quantitative fluorescence imaging. Hierarchical clustering and quantitative analysis of single-cell data revealed the diversity of Ca2+ responses to ATP pulse stimulation. These results demonstrate that the microfluidic cell culture system is useful for studying a variety of cellular responses, including cell signaling.

bioengineering↗

bioRxiv2018

Logical design of oral glucose ingestion pattern minimizing blood glucose in humans

Excessive increase in blood glucose level after eating increases the risk of macroangiopathy, and a method for not increasing the postprandial blood glucose level is desired. However, a logical design method of the dietary ingestion pattern controlling the postprandial blood glucose 2 level has not yet been established. We constructed a mathematical model of blood glucose control by oral glucose ingestion in 3 healthy human subjects, used the model to predict an optimal glucose ingestion pattern, and showed that the optimal ingestion pattern minimized the peak value of blood glucose level. Subjects orally ingested 3 doses of glucose by bolus or over 2 hours, and blood glucose, insulin, C-peptide and incretins were measured for 4 hours. We constructed an ordinary differential equation model that reproduced the time course data of the blood glucose and blood hormone levels. Using the model, we predicted that intermittent ingestion 30 minutes apart was the optimal glucose ingestion patterns that minimized the peak value of blood glucose level. We confirmed with subjects that this intermittent pattern decreased the peak value of blood glucose level. This approach could be applied to design optimal dietary ingestion patterns.\n\nIn BriefAs a forward problem, we measured blood glucose and hormones in three human subjects after oral glucose ingestion and constructed a mathematical model of blood glucose control. As an inverse problem, we used the model to predict the optimal oral glucose ingestion pattern that minimized the peak value of blood glucose level, and validated the pattern with the subjects.\n\nHighlightsO_LIModeling blood glucose concentrations predicts an intermittent ingestion pattern is optimal\nC_LIO_LIHuman validation shows ingestion at 30-minute intervals limits peak blood glucose\nC_LIO_LIWe provide a strategy to design optimal dietary ingestion patterns\nC_LI

systems biology↗

bioRxiv2018

\"Small and many\" is the strategy for robust and efficient information transfer in dendritic spines

A dendritic spine is a small structure on the dendrites of a neuron that processes input timing information from other neurons. Tens of thousands of spines are present on a neuron. Why are spines so small and many? Because of the small number of molecules in the spine volume, biochemical reactions become stochastic. Therefore, we used the stochastic simulation model of N-methyl-D-aspartate receptor (NMDAR)-mediated Ca2+ increase to address this issue. NMDAR-mediated Ca2+ increase codes the input timing information between prespiking and postspiking. We examined how much the input timing information is encoded by Ca2+ increase against prespiking fluctuation. We found that the input timing information encoded in the spine volume (10-1 m3) is more robust against prespiking fluctuation than that in the cell volume (103 m3). We further examined the mechanism of the robust information transfer in the spine volume. We demonstrated that the necessary and sufficient condition for robustness is that the stochastic NMDAR-mediated Ca2+ increase (intrinsic noise) becomes much larger than the prespiking fluctuation (extrinsic noise). The condition is satisfied in the spine volume, but not in the cell volume. Moreover, we compared the information transfer in many small \"spine-volume\" spines with that in a single large \"cell-volume\" spine. We found that many small \"spine-volume\" spines is much more efficient for information transfer than a single large \"cell-volume\" spine when prespiking fluctuation is large. Thus, robustness and efficiency are two functional reasons why dendritic spines are so small and many.\n\nSignificanceA dendritic spine is a small platform for information processing in a neuron, and tens of thousands of spines are present on a neuron. Why are spines so small and many? Here we addressed this issue using stochastic simulation of NMDAR-mediated Ca2+ increase in a spine. We demonstrated that smallness of a spine enables the robust information transfer against input fluctuation, and that many small spines are much efficient for information transfer than a single large cell. This is the first demonstration that shows the advantage of the \"small and many\" of spines in information processing. The \"small and many\" strategy may be used not only in spines of a neuron, but also in other small and many intracellular organelles.

biophysics↗

bioRxiv2018

Dynamic gait transition in the Scolopendromorpha scolopocryptops rubiginosus L. Koch centipede

Crawling using locomotory waves is a common method of locomotion for limbless and many-legged invertebrates. It is generally believed that the direction of locomotory waves is fixed for a given species. However, by recording and performing detailed analyses of the gait patterns of a Scolopendromorpha scolopocryptops rubiginosus L. Koch centipede in various conditions, we found that it dynamically generated its gait to allow for locomotory waves that varied in direction. By introducing the wave-index order parameter to characterise locomotory waves, we showed that gait patterns were associated with control of stride rather than rotation frequency.

animal behavior and cognition↗

bioRxiv2017

Trans-omic analysis reveals fed and fasting insulin signal across phosphoproteome, transcriptome, and metabolome

The concentration and temporal pattern of insulin selectively regulate multiple cellular functions. To understand how insulin dynamics are interpreted by cells, we constructed a trans-omic network of insulin action in FAO hepatoma cells from three networks--a phosphorylation-dependent cellular functions regulatory network using phosphoproteomic data, a transcriptional regulatory network using phosphoproteomic and transcriptomic data, and a metabolism regulatory network using phosphoproteomic and metabolomic data. With the trans-omic regulatory network, we identified selective regulatory networks that mediate differential responses to insulin. Akt and Erk, hub molecules of insulin signaling, encode information of a wide dynamic range of dose and time of insulin. Down-regulated genes and metabolites in glycolysis had high sensitivity to insulin (fasting insulin signal); up-regulated genes and dicarboxylic acids in the TCA cycle had low sensitivity (fed insulin signal). This integrated analysis enables molecular insight into how cells interpret physiologically fed and fasting insulin signals.\n\nHighlightsO_LIWe constructed a trans-omic network of insulin action using multi-omic data.\nC_LIO_LIThe trans-omic network integrates phosphorylation, transcription, and metabolism.\nC_LIO_LIWe classified signaling, transcriptome, and metabolome by sensitivity to insulin.\nC_LIO_LIWe identified fed and fasting insulin signal flow across the trans-omic network.\nC_LI

systems biology↗

bioRxiv2017

System Identification Using Compressed Sensing Reveals Signaling-Decoding System By Gene Expression

Cells decode information of signaling activation at a scale of tens of minutes by downstream gene expression with a scale of hours to days, leading to cell fate decisions such as cell differentiation. However, no system identification method with such different time scales exists. Here we used compressed sensing technology and developed a system identification method using data of different time scales by recovering signals of missing time points. We measured phosphorylation of ERK and CREB, immediate early gene expression products, and mRNAs of decoder genes for neurite elongation in PC12 cell differentiation and performed system identification, revealing the input-output relationships between signaling and gene expression with sensitivity such as graded or switch-like response and with time delay and gain, representing signal transfer efficiency. We predicted and validated the identified system using pharmacological perturbation. Thus, we provide a versatile method for system identification using data with different time scales.\n\nHighlightsO_LIWe developed a system identification method using compressed sensing.\nC_LIO_LIThis method allowed us to find a pathway using data of different time scales.\nC_LIO_LIWe identified a selective signaling-decoding system by gene expression.\nC_LIO_LIWe validated the identified system by pharmacological perturbation.\nC_LI\n\neTOC BlurbWe describe a system identification method of molecular networks with different time-scale data using a signal recovery technique in compressed sensing.

systems 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↗