Development of selective blockers for Ca2+-activated Cl- channel using Xenopus laevis oocytes with an improved drug screening strategy

<p>Abstract</p> <p>Background</p> <p>Ca²⁺-activated Cl^-channels (CaCCs) participate in many important physiological processes. However, the lack of effective and selective blockers has hindered the study of these channels, mostly due to the lack of good assay system. Here, we have developed a reliable drug screening method for better blockers of CaCCs, using the endogeneous CaCCs in <it>Xenopus laevis </it>oocytes and two-electrode voltage-clamp (TEVC) technique.</p> <p>Results</p> <p>Oocytes were prepared with a treatment of Ca²⁺ionophore, which was followed by a treatment of thapsigargin which depletes Ca²⁺stores to eliminate any contribution of Ca²⁺release. TEVC was performed with micropipette containing chelerythrine to prevent PKC dependent run-up or run-down. Under these conditions, Ca²⁺-activated Cl^-currents induced by bath application of Ca²⁺to oocytes showed stable peak amplitude when repetitively activated, allowing us to test several concentrations of a test compound from one oocyte. Inhibitory activities of commercially available blockers and synthesized anthranilic acid derivatives were tested using this method. As a result, newly synthesized <it>N</it>-(4-trifluoromethylphenyl)anthranilic acid with trifluoromethyl group (-CF₃) at <it>para </it>position on the benzene ring showed the lowest IC₅₀.</p> <p>Conclusion</p> <p>Our results provide an optimal drug screening strategy suitable for high throughput screening, and propose <it>N</it>-(4-trifluoromethylphenyl)anthranilic acid as an improved CaCC blocker.</p

C-terminally mutated tubby protein accumulates in aggresomes

The tubby protein (Tub), a putative transcription factor, plays important roles in the maintenance and function of neuronal cells. A splicing defect-causing mutation in the 3&apos;-end of the tubby gene, which is predicted to disrupt the carboxy-terminal region of the Tub protein, causes maturity-onset obesity, blindness, and deafness in mice. Although this pathological Tub mutation leads to a loss of function, the precise mechanism has not yet been investigated. Here, we found that the mutant Tub proteins were mostly localized to puncta found in the perinuclear region and that the C-terminus was important for its solubility. Immunocytochemical analysis revealed that puncta of mutant Tub co-localized with the aggresome. Moreover, whereas wild-type Tub was translocated to the nucleus by extracellular signaling, the mutant forms failed to undergo such translocation. Taken together, our results suggest that the malfunctions of the Tub mutant are caused by its misfolding and subsequent localization to aggresomes.ope

Accuracy of analytical approaches to thermal response test interpretation

Thermal conductivity obtained via thermal response test (TRT) is one of the essential parameters for the design of shallow geothermal systems, especially a borehole heat exchanger (BHE). During TRT analysis, several factors (e.g., start time of analysis and test duration) could influence the estimated thermal conductivity. In addition, the influence of the factors may be different depending on the characteristics of the borehole and ground. This study investigated the effects of the start time and test duration through numerically generated TRT data under a diversity of environment and BHE information such as the thermal conductivity of grouting and ground. The generated numerical data were analyzed with three analytical approaches having different assumptions about the heat source to interpret the sensitivity of factors in the TRT analysis. By conducting the analytical sensitivity analysis, the importance of determining the appropriate start time and test duration could be emphasized when designing the test

Effect Of Nitric-Oxide Post-Oxidation Annealing On High-Temperature Oxidized 4H SiC.

Metal-Oxide-Semiconductor FETs using 4H-SiC have been investigated intensively because 4H-SiC semiconductor has excellent physical properties for power-device applications

Characteristics of Sawdust and Cocopeat Beddings, and Their Usefulness According to the Fan and Pen Location for Rearing Hanwoo Cattle

This study was designed to examine the characteristics of sawdust and cocopeat bedding materials, including physicochemical properties (Exp. I) and on-farm trial (Exp. II). In Exp. I, the proportion of particle size was in the order of sawdust> cocopeat India>cocopeat Vietnam (pcocopeat Vietnam>sawdust (p<0.05), thus cocopeat treatments showed 4.4 times higher bedding cost than sawdust. The water absorption rates were 702.0% in cocopeat India, 678.3% in cocopeat Vietnam, and 444.0% in sawdust, showing cocopeat had approximately 1.5 times higher water absorption rate than sawdust. Moisture evaporation rates after 12 h of air blowing (2.00 m/s) were higher (p<0.05) in cocopeat Vietnam (80.4%) than sawdust (71.2%) and cocopeat India (72.8%). In vitro ammonia emissions were higher (p<0.05) in sawdust (2.71 mg/m2/h) than cocopeat India (1.59 mg/m2/h) and Vietnam (1.22 mg/m2/h), and total ammonia emissions were higher (p<0.05) in sawdust (37.02 mg/m2) than cocopeat India (22.51 mg/m2) and Vietnam (13.60 mg/m2). In Exp. II, an on-farm trial was conducted with 48 Hanwoo cattle in 16 pens using the same bedding materials as in Exp. I, with fan (blowing 2.00 m/s) and no fan treatments, and feed bunk side (FB) and water supply side (WS) within a pen (4.5 m, width×9.0 m, length). Beddings were replaced with fresh bedding materials when moisture concentrations were over 65%. No interactions among treatments were detected for moisture concentration and increment rates, and ammonia concentrations, but a significant effect was observed (p<0.01) for each of the treatments. Both concentrations and increment rate of moisture were higher (p<0.01) in the beddings without fan than with fan. Moisture concentrations and increment rate within a pen were also higher (p<0.01) in FB than WS. Thus, the whole no-fan-FB and sawdust-fan-FB were replaced with fresh bedding material between 4 to 5 experimental weeks. The ammonia concentrations and pH of beddings were not significantly different among treatments. Therefore, using cocopeat bedding with a blowing fan can extend twice the bedding utilization period, and WS within a pen showed twice the bedding-life compared to FB. Despite the outstanding characteristics of cocopeat compared with sawdust, using cocopeat as an alternative for sawdust bedding is not recommended for cattle management, considering it has 4.4 times higher bedding cost and a dust production problem

Humanized Anti-hepatocyte Growth Factor Monoclonal Antibody (YYB-101) Inhibits Ovarian Cancer Progression

Current chemotherapy regimens have certain limitations in improving the survival rates of patients with advanced ovarian cancer. Hepatocyte growth factor (HGF) is important in ovarian cancer cell migration and invasion. This study assessed the effects of YYB-101, a humanized monoclonal anti-HGF antibody, on the growth and metastasis of ovarian cancer cells. YYB-101 suppressed the phosphorylation of the HGF receptor c-MET and inhibited the migration and invasion of SKOV3 and A2780 ovarian cancer cells. Moreover, the combination of YYB-101 and paclitaxel synergistically inhibited tumor growth in an in vivo ovarian cancer mouse xenograft model and significantly increased the overall survival (OS) rate compared with either paclitaxel or YYB-101 alone. Taken together, these findings suggest that YYB-101 has therapeutic potential in ovarian cancer when combined with conventional chemotherapy agents.1

Design and Characterization of a High Resolution Microfluidic Heat Flux Sensor with Thermal Modulation

A complementary metal-oxide semiconductor-compatible process was used in the design and fabrication of a suspended membrane microfluidic heat flux sensor with a thermopile for the purpose of measuring the heat flow rate. The combination of a thirty-junction gold and nickel thermoelectric sensor with an ultralow noise preamplifier, a low pass filter, and a lock-in amplifier can yield a resolution 20 nW with a sensitivity of 461 V/W. The thermal modulation method is used to eliminate low-frequency noise from the sensor output, and various amounts of fluidic heat were applied to the sensor to investigate its suitability for microfluidic applications. For sensor design and analysis of signal output, a method of modeling and simulating electro-thermal behavior in a microfluidic heat flux sensor with an integrated electronic circuit is presented and validated. The electro-thermal domain model was constructed by using system dynamics, particularly the bond graph. The electro-thermal domain system model in which the thermal and the electrical domains are coupled expresses the heat generation of samples and converts thermal input to electrical output. The proposed electro-thermal domain system model is in good agreement with the measured output voltage response in both the transient and the steady state