Impedance Measurement System for Assessing the Barrier Integrity of Three-Dimensional Human Intestinal Organoids.

Human pluripotent stem cell (hPSC)-derived intestinal organoids (HIOs) hold unprecedented promise for basic biology and translational applications. However, developing a quantitative method to evaluate the epithelial cell membrane integrity of HIOs as an in vitro intestinal barrier model is a major challenge because of their complex three-dimensional (3D) structure. In this study, we developed an impedance system to measure the change in electrical resistance of 3D HIOs depending on the integrity of the intestinal epithelial cell membrane, which can reflect functionality and maturity. The expression of intestinal maturation- and tight junction-related markers was significantly higher in HIOs matured in vitro by treatment with IL-2 than in control HIOs. Analysis of gap junction size indicated that mature HIOs have greater integrity, with approximately 30% more compact gaps than immature HIOs. We designed a multi-microchannel system controlled by the inhalation pressure where the HIO is loaded, which enhances the stability and sensitivity of the impedance signal. We demonstrated the applicability of the impedance system by showing the difference in resistance between control and mature HIOs, reflecting the expression of tight junction proteins and their maturation status. We also validated the impedance system by monitoring its resistance in real time during junctional damage to HIOs induced by a digestive agent. In summary, we suggest a quantitative method to directly quantify the physiological changes in complex 3D organoid structures based on impedance spectroscopy, which can be applied to noninvasively monitor live cells and therefore enable their use in subsequent experiments.

Separation of fine particles at different frequencies and HRTs using acoustic standing waves.

The objective of this study was to evaluate the separation of fine particles using several frequencies and hydraulic retention times (HRTs) in an acoustic standing wave reactor without any separate cooling devices. The acoustic standing wave reactor consisted of sufficient space (over 100 mm) between the transducer and reflector, resulting in a slight increase in temperature. However, the increase in temperature did not affect the formation of standing waves and particle aggregations in our experiments. The results indicated that the turbidity removal efficiencies of fine kaolin particles, when using frequencies of 580 kHz, 1, and 2 MHz, increased with longer standing wave operation time. Especially, the turbidity removal efficiencies for 1 and 2 MHz were higher than that for 580 kHz because the wavelength (λ) of the 580 kHz wave was longer than that of the 1 and 2 MHz waves. Furthermore, the turbidity removal efficiency of kaolin in a continuous reactor improved with increasing hydraulic retention times (HRTs), and the reactor was more effective with 1 and 2 MHz used in parallel instead of 1 and 2 MHz used individually under the same HRT conditions with the entrance length (EL) having no adverse effect.

Assessing Barrier Function in Psoriasis and Cornification Models of Artificial Skin Using Non-Invasive Impedance Spectroscopy.

Reconstructed epidermal equivalents (REEs) consist of two distinct cell layers - the stratum corneum (SC) and the keratinocyte layer (KL). The interplay of these layers is particularly crucial in pruritic inflammatory disorders, like psoriasis, where a defective SC barrier is associated with immune dysregulation. However, independent evaluation of the skin barrier function of the SC and KL in REEs is highly challenging because of the lack of quantitative methodologies that do not disrupt the counter layer. Here, a non-invasive impedance spectroscopy technique is introduced for dissecting the distinct contributions of the SC and KL to overall skin barrier function without disrupting the structure. These findings, inferred from the impedance spectra, highlight the individual barrier resistances and maturation levels of each layer. Using an equivalent circuit model, a correlation between impedance parameters and specific skin layers, offering insights beyond traditional impedance methods that address full-thickness skin only is established. This approach successfully detects subtle changes, such as increased paracellular permeability due to mild irritants and the characterization of an immature SC in psoriatic models. This research has significant implications, paving the way for detailed mechanistic investigations and fostering the development of therapies for skin irritation and inflammatory disorders.

Impedance spectroscopy of highly ordered nano-porous electrodes based on Au-AAO (anodic aluminum oxide) structure.

Electrochemical measurements using the microelectrodes are increasingly utilized for the label-free detection of the small amount of biological materials such as DNA, protein, and cells. However, the interfacial electrode impedance increases and may hinder the detection of weak signals as the size of electrode decreases. To enhance the measurement sensitivity while reducing the electrode size, in this study, microelectrodes employing a nanoporous structure were fabricated and characterized by using electrical impedance spectroscopy. We made the highly ordered honeycomb nanoporous structure of Anodic Aluminum Oxide (AAO) by electrochemical anodizing and formed Au layer on the surface of AAO (Au/AAO) by electroless Au plating method. The electrical characteristics of the fabricated Au/AAO electrodes were evaluated by using de Levie's model derived for the pore electrodes. As a result, the interfacial electrode impedance of the fabricated Au/AAO electrodes was 2-3 order lower than the value of the planar electrodes at frequencies below 1 kHz. It implies this nanoporous electrode could be directly applied to label free detection of biomaterials.

Long-Term Examination of Water Chemistry Changes Following Treatment of Cyanobacterial Bloom with Coagulants and Minerals.

The abundant growth in cyanobacterial blooms poses severe ecological threats with a high risk to aquatic organisms and global public health. Control of cyanobacterial blooms involves spraying cyanobacteria removal materials, including coagulants. However, little is known about the fate of the coagulated-cyanobacteria-laden water. Here, we examined long-term changes in water quality following treatment with various coagulants and minerals for cyanobacterial removal when the coagulated cyanobacterial cells were not removed from the water. An experiment in a controlled water system tested the effects of six different compounds, one conventional coagulant, two natural inorganic coagulants, and three minerals. All tested coagulants and minerals exhibited >75% of cyanobacterial removal efficiency. However, compared to the control, higher concentrations of nitrogen were observed from some samples treated during the experimental period. After 20 months, the final total phosphorus concentration of the raw water increased 20-fold compared to the initial concentration to 11.82 mg/L, indicating significant nutrient release over time. Moreover, we observed that the decomposition of sedimented cyanobacterial cells caused the release of intracellular contents into the supernatant, increasing phosphorous concentration over time. Therefore, cyanobacterial cells should be removed from water after treatment to prevent eutrophication and maintain water quality.

Quantification of non-alcoholic fatty liver disease progression in 3D liver microtissues using impedance spectroscopy.

Non-alcoholic fatty liver disease (NAFLD) has become a global pandemic. However, a pharmacological cure has not been approved for NAFLD treatment. The greatest barriers to the development of new treatments are the ambiguous criteria among the NAFLD stages and the lack of quantitative methodologies for its disease assessment in a translatable preclinical model. In this study, we developed impedance assessment systems to quantify NAFLD progression in three-dimensional (3D) liver microtissue (hMT). The hMT model undergoing NAFLD represents clinical-like characteristics for a range of stages, such as lipid accumulation, cell ballooning, and stiffening. Each stage can be quantitatively assessed by an impedance system with microchannels under constant or dynamic pressure, depending on the relevant mechanical and morphological changes used in the clinical assessment of NAFLD. We determined a correlation between the impedance parameters and pathophysiological characteristics, such as gap widening and cytoplasmic deformation associated with NAFLD progression using bioimpedance simulation, showing hMTs struggling to return to normal states. In addition, we identified the relative stiffness to assess fibrogenesis from the correlation of resistance change and elongation length into the smaller channel of hMTs. We hope this methodology will have a significant impact on drug development by facilitating improved NAFLD assessment.

Degradation of different pesticides in water by microplasma: the roles of individual radicals and degradation pathways.

Pesticides are emergent toxins often identified in aquatic environments. In the present study, microplasma was employed to reduce the pesticide content in water. The degradation efficacy, rate, and pathways of standard organophosphorus pesticides (namely, chlorpyrifos, chlorpyrifos oxone, and diazinone) and an organochlorine pesticide (namely, DDT solution) were evaluated using microplasma. High-performance liquid chromatography (HPLC) analysis was performed to elucidate the degradation efficiency of pesticides as a function of plasma-produced substances that originally contributed to the main reduction procedure. Microplasma produces several types of radicals or reactive substances, for instance dissolved ozone (O3), nitrogen oxides, hydroxyl radicals (OH radicals), and hydrogen peroxide (H2O2). The removal potential differs due to the existence or absence of varieties of plasma-produced substances. The functions of major plasma-produced species on pesticide removal were determined by a passive technique. Nitrogen oxides showed a key role in organophosphorus pesticide removal, whereas dissolved ozone and OH radicals played major roles in DDT degradation. HPLC data showed that plasma-induced pesticide removal showed first-order reaction kinetics. The pesticide removal pathways through microplasma were validated by investigating the achieved data from LC-MS and GC-MS.

TNFα enhances trovafloxacin-induced in vitro hepatotoxicity by inhibiting protective autophagy.

Trovafloxacin (TVX) is associated with idiosyncratic drug-induced liver injury (iDILI) and inflammation-mediated hepatotoxicity. However, the inflammatory stress-regulated mechanisms in iDILI remain unclear. Herein, we elucidated the novel role of tumor-necrosis factor alpha (TNFα), an inflammatory stress factor, in TVX-induced in vitro hepatotoxicity and synergistic toxicity. TVX specifically induced synergistic toxicity in HepG2 cells with TNFα, which inhibits autophagy. TVX-treated HepG2 cells induced protective autophagy by inhibiting the expression of mTOR signaling proteins, while ATG5 knockdown in HepG2 cells, responsible for the impairment of autophagy, enhanced TVX-induced toxicity due to the increase in cytochrome C release and JNK pathway activation. Interestingly, the expression of mTOR signal proteins, which were suppressed by TVX, disrupted the negative feedback of the PI3K/AKT pathway and TNFα rebounded p70S6K phosphorylation. Co-treatment with TVX and TNFα inhibited protective autophagy by maintaining p70S6K activity, which enhanced TVX-induced cytotoxicity. Phosphorylation of p70S6K was inhibited by siRNA knockdown and rapamycin to restore TNFα-inhibited autophagy, which prevented the synergistic effect on TVX-induced cytotoxicity. These results indicate that TVX activates protective autophagy in HepG2 cells exposed to toxicity and an imbalance in negative feedback regulation of autophagy by TNFα synergistically enhanced the toxicity. The finding from this study may contribute to a better understanding of the mechanisms underlying iDILI associated with inflammatory stress.

Metabolomic Analysis of Germinated Brown Rice at Different Germination Stages.

Brown rice (BR) is unpolished rice containing many bioactive compounds in addition to the basic nutrients of the rice grain. Herein, BR was germinated for up to 48 h to prepare germinated brown rice (GBR). The physiological and chemical changes in the GBR during germination were analyzed. GBR samples germinated for 48 h were in the radicle-emergence stage, but root formation was not observed. The change in the GBR metabolite profile during germination was analyzed to determine the effect of germination on the chemical profiles of the GBR samples. Twenty-five metabolites including acidic compounds, amino acids, sugars, lipid metabolites, and secondary metabolites were identified as the components that contributed to the variations in the GBR groups germinated for different time periods. Among the metabolites, the carbohydrates associated with energy production and lipid metabolites changed significantly. Based on the identified metabolites, a metabolomic pathway was proposed. Carbohydrate metabolism, citric acid cycle, and lipid metabolism were the main processes that were affected during germination. Although further studies on the relationship between the metabolite profile and nutritional quality of the GBR are needed, these results are useful for understanding the effect of germination on the physiological and chemical changes in BR.

Effects of operational conditions on sludge degradation and organic acids formation in low-critical wet air oxidation.

Wet air oxidation processes are to treat highly concentrated organic compounds including refractory materials, sludge, and night soil, and usually operated at supercritical water conditions of high temperature and pressure. In this study, the effects of operational conditions including temperature, pressure, and oxidant dose on sludge degradation and conversion into subsequent intermediates such as organic acids were investigated at low critical wet oxidation conditions. The reaction time and temperature in the wet air oxidation process was shown an important factor affecting the liquefaction of volatile solids, with more significant effect on the thermal hydrolysis reaction rather than the oxidation reaction. The degradation efficiency of sludge and the formation of organic acids were improved with longer reaction time and higher reaction temperature. For the sludge reduction and the organic acids formation under the wet air oxidation, the optimal conditions for reaction temperature, time, pressure, and oxidant dose were shown approximately 240 degrees C, 30min, 60atm, and 2.0L/min, respectively.

Human three-dimensional in vitro model of hepatic zonation to predict zonal hepatotoxicity.

BACKGROUND: Various hepatic models mimicking liver lobules have been investigated to evaluate the potential hepatotoxic effects of chemicals and drugs, but in vitro hepatic models of zonal hepatotoxicity have not yet been established. Herein, we developed a three-dimensional (3D) hepatic zonal channel to evaluate zone-specific hepatotoxicity. Based on the perivenous zone-3-like cytochrome P450 (CYP) expression patterns in metabolically active HepaRG cells treated with CHIR99021 (CHIR), which is an inducer of Wnt/ß-catenin signaling, this culture model represents a novel tool for exploring hepatic zonation. RESULTS: We generated and validated a 3D hepatic zonal channel model in which 3D HepaRG cells were well distributed in agarose hydrogel channels, and a linear gradient of CHIR was generated according to the zonal distance. According to the results from imaging analyses and bioanalytical experiments, acetaminophen (APAP) caused cytotoxicity in the zone-3 region of the 3D hepatic zonal channel, and the levels of nonphosphorylated ß-catenin, CYP2E, and apoptotic proteins were remarkably increased in the zone-3-like region. Finally, the applicability of the 3D hepatic zonal channel model for the high-throughput screening of zonal hepatotoxicity was successfully evaluated using hepatotoxic drugs, including tamoxifen, bromobenzene, and APAP. CONCLUSIONS: The results indicated that tamoxifen induced cytotoxic effects, regardless of the zonal distance, while the zone-3-specific hepatotoxic drugs bromobenzene and APAP induced greater cytotoxic effects on cells in the zone-3-like region. This finding highlights the potential of our 3D hepatic zonation model as a valuable tool for replicating and evaluating zonal hepatotoxicity by mimicking the spatial features of liver lobules.

The control of cell adhesion on a PMMA polymer surface consisting of nanopillar arrays.

We investigated the effect of arrayed nanostructures on the cell adhesion rate by forming nanopillars on a PMMA polymer surface, and demonstrated cell patterning tools for the polymer surface without biological or chemical reagents. Nanopillar arrayed structures with various heights (0, 50, 100, 150, and 200nm with 50nm of pitch size and 60nm of diameter) were formed on a PMMA surface by using nano-molding techniques with nanoporous AAO (anodic aluminum oxide) as a template. The nanopillar arrayed structures provide negative effects on the cell adhesion on the non-treated PMMA (moderate hydrophobic, ≥80° of contact angle), whereas slightly positive and no effects were shown by nanopillar structures on plasma (hydrophilic, ≤20°) and silane-treated PMMA (moderate, 40-70°), respectively. The microstructure on the polymer surface showed a 20% positive effect on the cell adhesion rate. As a result, nano or micro patterning structures could control the cell adhesion rate (15-120%) and it enabled the formation of closed cell patterns on the PMMA surface without chemical or biological surface treatments.