Metabolic Oscillations and Glycolytic Phenotypes of Cancer Cells.

Cancer cells show several metabolic phenotypes depending on the cancer types and the microenvironments in tumor tissues. The glycolytic phenotype is one of the hallmarks of cancer cells and is considered to be one of the crucial features of malignant cancers. Here, we show glycolytic oscillations in the concentrations of metabolites in the glycolytic pathway in two types of cancer cells, HeLa cervical cancer cells and DU145 prostate cancer cells, and in two types of cellular morphologies, spheroids and monolayers. Autofluorescence from nicotinamide adenine dinucleotide (NADH) in cells was used for monitoring the glycolytic oscillations at the single-cell level. The frequencies of NADH oscillations were different among the cellular types and morphologies, indicating that more glycolytic cancer cells tended to exhibit oscillations with higher frequencies than less glycolytic cells. A mathematical model for glycolytic oscillations in cancer cells reproduced the experimental results quantitatively, confirming that the higher frequencies of oscillations were due to the higher activities of glycolytic enzymes. Thus, glycolytic oscillations are expected as a medical indicator to evaluate the malignancy of cancer cells with glycolytic phenotypes.

Coexistence of oscillatory and reduced states on a spherical field controlled by electrical potential.

The Belousov-Zhabotinsky (BZ) reaction was investigated to elucidate features of oscillations depending on the applied electrical potential, E. A cation-exchange resin bead loaded with the catalyst of the BZ reaction was placed on a platinum plate as a working electrode and then E was applied. We found that global oscillations (GO) and a reduced state coexisted on the bead at a negative value of E and that the source point of GO changed depending on E. The thickness of the reduced state was determined by a yellow colored region which corresponded to the distribution of Br2. The present studies suggest that the distribution of the inhibitor, Br-, which is produced from Br2, plays an important role in the existence of the reduced state and GO, and the source point of GO.

Chemical Wave Propagation in the Belousov-Zhabotinsky Reaction Controlled by Electrical Potential.

The Belousov-Zhabotinsky (BZ) reaction is an important experimental model for the study of chemical oscillations and waves far from the thermodynamic equilibrium. Earlier studies had observed that individual BZ microbeads can show both global oscillations and traveling waves, but failed to select these different dynamic states. Here, we report experiments, in which this control was achieved by an externally applied electrical potential. The spherical microbeads were first loaded with the catalyst, then immersed into a catalyst-free BZ solution, and finally placed onto a planar platinum electrode. For positive electrical potentials, we observed global oscillations, whereas negative potentials resulted in traveling waves. The spatio-temporal characteristics of these phenomena are discussed in relation to the activator, HBrO2, which is produced by an electrochemical reaction.

Modeling studies of heterogeneities in glycolytic oscillations in HeLa cervical cancer cells.

Previous experiments demonstrated that a population of HeLa cells starved of glucose or both glucose and serum exhibited a strong heterogeneity in the glycolytic oscillations in terms of the number of oscillatory cells, periods of oscillations, and duration of oscillations. Here, we report numerical simulations of this heterogeneous oscillatory behavior in HeLa cells by using a newly developed mathematical model. It is simple enough that we can apply a mathematical analysis, but capture the core of the glycolytic pathway and the activity of the glucose transporter (GLUT). Lognormal distributions of the values of the four rate constants in the model were obtained from the experimental distributions in the periods of oscillations. Thus, the heterogeneity in the periods of oscillations can be attributed to the difference in the rate constants of the enzymatic reactions. The activity of GLUT is found to determine whether the HeLa cells were oscillatory or non-oscillatory under the same experimental conditions. Simulation with the log-normal distribution of the maximum uptake velocity of glucose and the four randomized rate constants based on the log-normal distributions successfully reproduced the time-dependent number of oscillatory cells (oscillatory ratios) under the two starving conditions. The difference in the initial values of the metabolites has little effect on the simulated results.

Primordial oscillations in life: Direct observation of glycolytic oscillations in individual HeLa cervical cancer cells.

We report the first direct observation of glycolytic oscillations in HeLa cervical cancer cells, which we regard as primordial oscillations preserved in living cells. HeLa cells starved of glucose or both glucose and serum exhibited glycolytic oscillations in nicotinamide adenine dinucleotide (NADH), exhibiting asynchronous intercellular behaviors. Also found were spatially homogeneous and inhomogeneous intracellular NADH oscillations in the individual cells. Our results demonstrate that starved HeLa cells may be induced to exhibit glycolytic oscillations by either high-uptake of glucose or the enhancement of a glycolytic pathway (Crabtree effect or the Warburg effect), or both. Their asynchronous collective behaviors in the oscillations were probably due to a weak intercellular coupling. Elucidation of the relationship between the mechanism of glycolytic dynamics in cancer cells and their pathophysiological characteristics remains a challenge in future.

Periodic Oscillatory Motion of a Self-Propelled Motor Driven by Decomposition of H2 O2 by Catalase.

A self-propelled motor driven by the enzymatic reaction of catalase adsorbed onto a filter paper floating on an aqueous solution of H2 O2 was used to study nonlinear behavior in the motor's motion. An increase in the concentration of H2 O2 resulted in a change from no motion to irregular oscillatory motion, periodic oscillatory motion, and continuous motion. The mechanisms underlying oscillation and mode bifurcation are discussed based on experimental results on O2 bubble formation and growth on the underside of the motor.

Collective and individual glycolytic oscillations in yeast cells encapsulated in alginate microparticles.

Yeast cells were encapsulated into alginate microparticles of a few hundred micrometers diameter using a centrifuge-based droplet shooting device. We demonstrate the first experimental results of glycolytic oscillations in individual yeast cells immobilized in this way. We investigated both the individual and collective oscillatory behaviors at different cell densities. As the cell density increased, the amplitude of the individual oscillations increased while their period decreased, and the collective oscillations became more synchronized, with an order parameter close to 1 (indicating high synchrony). We also synthesized biphasic-Janus microparticles encapsulating yeast cells of different densities in each hemisphere. The cellular oscillations between the two hemispheres were entrained at both the individual and population levels. Such systems of cells encapsulated into microparticles are useful for investigating how cell-to-cell communication depends on the density and spatial distribution of cells.

Validation of sleep-staging accuracy for an in-home sleep electroencephalography device compared with simultaneous polysomnography in patients with obstructive sleep apnea.

Efforts to simplify standard polysomnography (PSG) in laboratories, especially for obstructive sleep apnea (OSA), and assess its agreement with portable electroencephalogram (EEG) devices are limited. We aimed to evaluate the agreement between a portable EEG device and type I PSG in patients with OSA and examine the EEG-based arousal index's ability to estimate apnea severity. We enrolled 77 Japanese patients with OSA who underwent simultaneous type I PSG and portable EEG monitoring. Combining pulse rate, oxygen saturation (SpO2), and EEG improved sleep staging accuracy. Bland-Altman plots, paired t-tests, and receiver operating characteristics curves were used to assess agreement and screening accuracy. Significant small biases were observed for total sleep time, sleep latency, awakening after falling asleep, sleep efficiency, N1, N2, and N3 rates, arousal index, and apnea indexes. All variables showed > 95% agreement in the Bland-Altman analysis, with interclass correlation coefficients of 0.761-0.982, indicating high inter-instrument validity. The EEG-based arousal index demonstrated sufficient power for screening AHI ≥ 15 and ≥ 30 and yielded promising results in predicting apnea severity. Portable EEG device showed strong agreement with type I PSG in patients with OSA. These suggest that patients with OSA may assess their condition at home.

The ecological roles of bacterial populations in the surface sediments of coastal lagoon environments in Japan as revealed by quantification and qualification of 16S rDNA.

Based on quantification and qualification of bacterial 16S rDNA, we verified the bacterial ecological characteristics of surface sediments of Lakes Shinji and Nakaumi, which are representative of coastal lagoons in Japan. Quantification and qualification of the 16S rDNA sequences was carried out using real time polymerase chain reaction and polymerase chain reaction denaturing gradient gel electrophoresis and non-metric multidimensional scaling, respectively. The results revealed that the copy number per gram of sediment ranged from 8.33 × 10(8) (Lake Nakaumi) to 1.69 × 10(11) (Honjo area), suggesting that bacterial carbon contributed only 0.05-9.64 % of the total carbon content in the samples. Compared with other aquatic environments, these results indicate that sedimentary bacteria are not likely to be important transporters of nutrients to higher trophic levels, or to act as carbon sinks in the lagoons. The bacterial compositions of Lake Shinji and Lake Nakaumi and the Honjo area were primarily influenced by sediment grain sizes and salinity, respectively. Statistical comparisons of the environmental properties suggested that the areas that were oxygen-abundant (Lake Shinji) and at a higher temperature (Honjo area) presented efficient organic matter degradation. The 16S rDNA copy number per gram of carbon and nitrogen showed the same tendency. Consequently, the primary roles of bacteria were degradation and preservation of organic materials, and this was affected by oxygen and temperature. These roles were supported by the bacterial diversity rather than the differences in the community compositions of the sedimentary bacteria in these coastal lagoons.

Toluene dioxygenase expression correlates with trichloroethylene degradation capacity in Pseudomonas putida F1 cultures.

Trichloroethylene (TCE) is extensively used in commercial applications, despite its risk to human health via soil and groundwater contamination. The stability of TCE, which is a useful characteristic for commercial application, makes it difficult to remove it from the environment. Numerous studies have demonstrated that TCE can be effectively removed from the environment using bioremediation. Pseudomonas putida F1 is capable of degrading TCE into less hazardous byproducts via the toluene dioxygenase pathway (TOD). Unfortunately, these bioremediation systems are not self-sustaining, as the degradation capacity declines over time. Fortunately, the replacement of metabolic co-factors is sufficient in many cases to maintain effective TCE degradation. Thus, monitoring systems must be developed to predict when TCE degradation rates are likely to decline. Herein, we show evidence that tod expression levels correlate with the ability of P. putida F1 to metabolize TCE in the presence of toluene. Furthermore, the presence of toluene improves the replication of P. putida F1, even when TCE is present at high concentration. These findings may be applied to real world applications to decide when the bioremediation system requires supplementation with aromatic substrates, in order to maintain maximum TCE removal capacity.

Oscillations and Dynamic Symbiosis in Cellular Metabolism in Cancer.

The grade of malignancy differs among cancer cell types, yet it remains the burden of genetic studies to understand the reasons behind this observation. Metabolic studies of cancer, based on the Warburg effect or aerobic glycolysis, have also not provided any clarity. Instead, the significance of oxidative phosphorylation (OXPHOS) has been found to play critical roles in aggressive cancer cells. In this perspective, metabolic symbiosis is addressed as one of the ultimate causes of the grade of cancer malignancy. Metabolic symbiosis gives rise to metabolic heterogeneities which enable cancer cells to acquire greater opportunities for proliferation and metastasis in tumor microenvironments. This study introduces a real-time new imaging technique to visualize metabolic symbiosis between cancer-associated fibroblasts (CAFs) and cancer cells based on the metabolic oscillations in these cells. The causality of cellular oscillations in cancer cells and CAFs, connected through lactate transport, is a key point for the development of this novel technique.

Glycolytic oscillations in HeLa cervical cancer cell spheroids.

Previous studies have unravelled glycolytic oscillations in cancer cells, such as HeLa cervical and DU145 prostate cancer cells, using a monolayer culture system. Here, we demonstrate glycolytic oscillations in HeLa cervical cancer cell spheroids. Experiments revealed that a small number of HeLa cells in spheroids exhibited heterogeneous oscillations with a higher frequency than those in monolayers. Model analyses and our previous experiments indicated that the higher frequencies of oscillations in spheroids were mostly due to the increase in glycolytic enzyme activity in the cells, and to the decrease in glucose concentration by diffusional transport of glucose from the surface to inside the spheroids, as well as the increase in cell density through spheroid formation. These results and our previous studies imply that more malignant cancer cells tend to exhibit glycolytic oscillations with higher frequencies than less malignant cells. Adjacent cells in spheroids oscillated within a 10% difference in frequency, but did not synchronize with each other. This suggests that weak cell-to-cell interactions might exist among HeLa cells connected with cadherins in the spheroid microenvironment; however, the interactions were not strong enough to induce synchronization of glycolytic oscillations.

Photoexcited chemical wave in the ruthenium-catalyzed Belousov-Zhabotinsky reaction.

The excitation of the photosensitive Belousov-Zhabotinsky (BZ) reaction induced by light stimulation was systematically investigated. A stepwise increase in the light intensity induced the excitation, whereas a stepwise decrease did not induce the excitation. The threshold values for the excitation were found to be a function of the initial and final light intensities, time variation in light intensity, and the concentration of NaBrO(3). The experimental results were qualitatively reproduced by a theoretical calculation based on a three-variable Oregonator model modified for the photosensitive BZ reaction. These results suggest that although the steady light irradiation is known to inhibit oscillation and chemical waves in the BZ system under almost all conditions, the stepwise increase in the light irradiation leads to the rapid production of an activator, resulting in the photoexcitation.

Real-time reverse transcription PCR analysis of trichloroethylene-regulated toluene dioxygenase expression in Pseudomonas putida F1.

Toluene dioxygenase (tod) is a multicomponent enzyme system in Pseudomonas putida F1. Tod can mediate the degradation of Trichloroethylene (TCE), a widespread pollutant. In this study, we try to explore the TCE-regulated tod expression by using real-time qRT-PCR. The minimal culture media were supplemented with glucose, toluene, or a mixture of glucose/toluene respectively as carbon and energy sources. The TCE was injected into each medium after a 12-hour incubation period. The TCE injection severely affected bacterial growth when cultured with toluene or toluene/glucose mixtures. The cell density dropped 61 % for bacteria growing in toluene and 36 % for bacteria in the glucose/toluene mixture after TCE injection, but the TCE treatment had little effect on bacteria supplied with glucose alone. The decrease in cell number was caused by the cytotoxicity of the TCE metabolized by tod. The results from the real-time qRT-PCR revealed that TCE was capable of inducing tod expression in a toluene-dependent manner and that the tod expression level increased 50 times in toluene and 3 times in the toluene/glucose mixture after 6 hours of TCE treatment. Furthermore, validation of the rpoD gene as a reference gene for P. putida F1 was performed in this study, providing a valuable foundation for future studies to use real-time qRT-PCR in the analysis of the P. putida F1 strain.

Bacterial community structure analysis of sediment in the Sagami River, Japan using a rapid approach based on two-dimensional DNA gel electrophoresis mapping with selective primer pairs.

A rapid approach based on two-dimensional DNA gel electrophroesis (2-DGE) mapping with selective primer pairs was employed to analyze bacterial community structure in sediments from upstream, midstream and downstream of Sagami River in Japan. The 2-DGE maps indicated that Alpha- and Delta-proteobacteria were major bacterial populations in the upstream and midstream sediments. Further bacterial community structure analysis showed that richness proportion of Alpha- and Delta-proteobacterial groups reflected a trend toward decreasing from the upstream to downstream sediments. The biomass proportion of bacterial populations in the midstream sediment showed a significantly difference from that in the other sediments, suggesting that there may be an environmental pressure on the midstream bacterial community. Lorenz curves, together with Gini coefficients were successfully applied to the 2-DGE mapping data for resolving evenness of bacterial populations, and showed that the plotted curve from high-resolution 2-DGE mapping became less linear and more an exponential function than that of the 1-DGE methods such as chain length analysis and denaturing gradient gel electrophoresis, suggesting that the 2-DGE mapping may achieve a more detailed evaluation of bacterial community. In conclusion, the 2-DGE mapping combined with the selective primer pairs enables bacterial community structure analysis in river sediment and thus it can also monitor sediment pollution based on the change of bacterial community structure.

Spontaneous Mode Switching of Self-Propelled Droplet Motion Induced by a Clock Reaction in the Belousov-Zhabotinsky Medium.

Interfacial chemical dynamics on a droplet generate various self-propelled motions. For example, ballistic and random motions arise depending on the physicochemical conditions inside the droplet and its environment. In this study, we focus on the relationship between oxidant concentrations in an aqueous droplet and its mode of self-propelled motion in an oil phase including surfactant. We demonstrated that the chemical conditions inside self-propelled aqueous droplets were changed systematically, indicating that random motion appeared at higher concentrations of oxidants, which were H2SO4 and BrO3-, and ballistic motion at lower concentrations. In addition, spontaneous mode switching from ballistic to random motion was successfully demonstrated by adding malonic acid, wherein the initially observed reduced state of the aqueous solution suddenly changed to the oxidized state. Although we only observed one-time transition and have not yet succeeded to realize alternation between ballistic (reduced state) and random motion (oxidized state), such spontaneous transitions are fundamental steps in realizing artificial cells and understanding the fundamental mechanisms of life-like behavior, such as bacterial chemotaxis originating from periodical run-and-tumble motion.

Switching between Two Oscillatory States Depending on the Electrical Potential.

Various spatiotemporal patterns were created on the surface or in the body of cation-exchange resin beads which were loaded with the catalyst of the Belousov-Zhabotinsky (BZ) reaction. Either global oscillations (GO) or traveling waves (TW) and the switching between them were observed in the previous papers, but it was not clear how chemicals contribute to the reaction inside/around the BZ bead. In this paper, we scanned the electrical potential, E, from +1 to -1 V (negative scan) and then turned from -1 to +1 V (positive scan) to control the switching between GO and TW. We found that the electrical switching potential from TW to GO, ETG, and from GO to TW, EGT, depended on the scanning direction of E and the diameter of the bead, d. The present study suggests that the electrode-induced increase of the inhibitor, Br-, and the activator, HBrO2, around the BZ bead plays an important role in determining ETG and EGT.

Feasible conditions for Japanese woody biomass utilization.

There is an abundance of woody biomass in Japan. However, its economic feasibility is limited. There have been several discussions on whether generation of heat or electrical power is more suitable for woody biomass. In this study, we clarified the feasibility of generating heat and electricity from the viewpoint of biomass unit price and biomass productivity based on actual operation data for the first time. It was determined that heat production is feasible for a small-scale biomass energy use of 2,660 t/year or more. Electrical power generation is feasible only for a large-scale biomass energy use of 13,100 t/year or more, but it allows use of woody biomass with a higher unit price.

A chemical oscillator based on the photoreduction of 2-methyl-1,4-benzoquinone.

This study investigated bromate-2-methyl-1,4-benzoquinone photoreaction in a batch reactor, in which the photoreduction of 2-methyl-1,4-benzoquinone plays a vital role in initiating and sustaining the reaction process. Transient oscillatory phenomena are observed over broad reaction conditions, but a phase diagram in the bromate and sulfuric acid concentration plane shows that the nonlinear behavior is more sensitive to the ratio of bromate and acid than their absolute concentrations. Light intensity and the photosensitive substance 2-methyl-1,4-benzoquinone exhibited opposite effects on the induction time of these oscillations, implying that illumination does more than just reduce 2-methyl-benzoquinone. The oscillatory behavior has been qualitatively reproduced with a generic model developed for bromate-aromatic compounds systems.