Semi-quantitative microfluidic paper-based analytical device for ionic silica detection.

Silicate ions (SiO32-), or ionic silica, are known to cause silica scaling in industrial water applications when excess amounts are present; hence, concentrations must be monitored and kept at a constant low level. Ionic silica is conventionally measured by spectrophotometry in the form of its silicomolybdic complex based on the molybdenum blue reaction, but the operation process is complicated and not suitable for on-site detection. To solve these issues, microfluidic paper-based analytical devices (µPADs) have been gaining attention as portable, low-cost analytical devices suitable for on-site detection. The foldable origami type device described in this work enabled silica detection based on the molybdenum blue reaction, in the range of 50-1000 mg/L, with a practically detectable lowest concentration of 50 mg/L. The device showed selectivity for silicate ions and stability over 21 days when stored at 4 °C. The semi-quantitative analytical performance makes the proposed paper-based device attractive for on-site industrial monitoring.

Clotrimazole inhibits the Wnt/ß-catenin pathway by activating two eIF2α kinases: The heme-regulated translational inhibitor and the double-stranded RNA-induced protein kinase.

The Wnt/ß-catenin signaling pathway controls cell proliferation and differentiation, and therefore, when this pathway is excessively activated, it causes tumorigenesis. Our chemical suppressor screening in zebrafish embryos identified antifungal azoles including clotrimazole, miconazole, and itraconazole, as Wnt/ß-catenin signaling inhibitors. Here we show the mechanism underlying the Wnt/ß-catenin pathway inhibition by antifungal azoles. Clotrimazole reduced ß-catenin revels in a proteasome-independent fashion. By gene knockdown of two translational regulators, heme-regulated translational inhibitor and double-stranded RNA-induced protein kinase, we show that they mediate the clotrimazole-induced inhibition of the Wnt/ß-catenin pathway. Thus, clotrimazole inhibits the Wnt/ß-catenin pathway by decreasing ß-catenin protein levels through translational regulation. Antifungal azoles represent genuine candidate compounds for anticancer drugs or chemopreventive agents that reduce adenomatous polyps.

An evaluation of the engraftment and the blood flow of porcine skin autografts inactivated by high hydrostatic pressure.

We previously reported that exposure to a high hydrostatic pressure (HHP) of 200 MPa could completely inactivate porcine skin without damaging the extracellular matrix. In this study, we used an autologous porcine skin graft model and explored whether the skin inactivated by HHP could be engrafted without inflammation to the residual cellular components. Twenty-one full-thickness skin grafts of 1.5 × 1.5 cm in size were prepared from a minipig (n = 2). Grafts were either nonpressurized or pressurized to 100, 150, 200, 300, 500, or 1000 MPa (n = 3) and randomly implanted on the fascia and removed at 1 and 4 weeks after grafting. All grafts showed complete engraftment at the macroscopic level and microcirculation was detected by a full-field laser speckle perfusion imager. The epidermis was removed and skin appendages were not observed in the grafts pressurized to more than 200 MPa. Azan and Elastica van Gieson staining showed no sign of dermal collagen fiber degeneration, while elastin fibers were observed. The fibroblasts and capillaries were observed to have infiltrated to dermis in all groups without severe inflammation. In conclusion, we showed that skin inactivated by HHP up to 1000 MPa could be engrafted successfully without removing cellular remnants. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1091-1101, 2017.

The superiority of the autografts inactivated by high hydrostatic pressure to decellularized allografts in a porcine model.

We are developing a novel skin regeneration therapy in which the inactivation of nevus tissue via high hydrostatic pressure (HHP) is used in the reconstruction of the dermis in combination with a cultured epidermal autograft. In this study, we used a porcine skin graft model to explore whether autologous skin including cellular debris inactivated by HHP or allogeneic skin decellularized by HHP is better for dermal reconstruction. Grafts (n = 6) were prepared for five groups each: autologous skin without pressurization group (control group), autologous skin inactivated by 200 MPa group, autologous skin inactivated by 1000 MPa group, allogeneic skin decellularized by 200 MPa group, and allogeneic skin decellularized by 1000 MPa group. All of the grafts at 1, 4, and 12 weeks showed complete engraftment macroscopically. The mean areas of the grafts of the control group (p < 0.01) and autologous 200 MPa group (p < 0.01) were larger than that of the allogeneic 1000 MPa group at four weeks after implantation. The thickness of the control group and autologous 200 MPa group was comparable, and that of the autologous 200 MPa group was significantly thicker than that of the allogeneic 200 MPa group (p < 0.01). This suggests that the autologous dermis was superior to the allogeneic decellularized dermis as a skin graft, and that HHP at 200 MPa provided a better outcome than HHP at 1000 MPa. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2653-2661, 2017.

Calcitonin gene-related peptide pre-administration acts as a novel antidepressant in stressed mice.

Calcitonin gene-related peptide (CGRP) is a neuropeptide that has potent vasodilator properties and is involved in various behavioral disorders. The relationship between CGRP and depression-like behavior is unclear. In this study, we used chronically stressed mice to investigate whether CGRP is involved in depression-like behavior. Each mouse was exposed to restraint and water immersion stress for 15 days. After stress exposure, mice were assessed using behavioral tests: open field test, forced swim test and sucrose preference test. Serum corticosterone levels, hippocampal proliferation and mRNA expression of neurotrophins were measured. After stress exposure, mice exhibited depression-like behavior and decreased CGRP mRNA levels in the hippocampus. Although intracerebroventricular CGRP administration (0.5 nmol) did not alter depression-like behavior after 15-day stress exposure, a single CGRP administration into the brain, before the beginning of the 15-day stress exposure, normalized the behavioral dysfunctions and increased nerve growth factor (Ngf) mRNA levels in stressed mice. Furthermore, in the mouse E14 hippocampal cell line, CGRP treatment induced increased expression of Ngf mRNA. The NGF receptor inhibitor K252a inhibited CGRP's antidepressant-like effects in stressed mice. These results suggest that CGRP expression in the mouse hippocampus is associated with depression-like behavior and changes in Ngf mRNA levels.

The rapid inactivation of porcine skin by applying high hydrostatic pressure without damaging the extracellular matrix.

We previously reported that high hydrostatic pressure (HHP) of 200 MPa for 10 minutes could induce cell killing. In this study, we explored whether HHP at 200 MPa or HHP at lower pressure, in combination with hyposmotic distilled water (DW), could inactivate the skin, as well as cultured cells. We investigated the inactivation of porcine skin samples 4 mm in diameter. They were immersed in either a normal saline solution (NSS) or DW, and then were pressurized at 100 and 200 MPa for 5, 10, 30, or 60 min. Next, we explored the inactivation of specimens punched out from the pressurized skin 10×2 cm in size. The viability was evaluated using a WST-8 assay and an outgrowth culture. The histology of specimens was analyzed histologically. The mitochondrial activity was inactivated after the pressurization at 200 MPa in both experiments, and no outgrowth was observed after the pressurization at 200 MPa. The arrangement and proportion of the dermal collagen fibers or the elastin fibers were not adversely affected after the pressurization at 200 MPa for up to 60 minutes. This study showed that a HHP at 200 MPa for 10 min could inactivate the skin without damaging the dermal matrix.

Time Course of Behavioral Alteration and mRNA Levels of Neurotrophic Factor Following Stress Exposure in Mouse.

Stress is known to affect neurotrophic factor expression, which induces depression-like behavior. However, whether there are time-dependent changes in neurotrophic factor mRNA expression following stress remains unclear. In the present study, we tested whether chronic stress exposure induces long-term changes in depression-related behavior, serum corticosterone, and hippocampal proliferation as well as neurotrophic factor family mRNA levels, such as brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3), and ciliary neurotrophic factor (CNTF), in the mouse hippocampus. The mRNA level of neurotrophic factors (BDNF, NGF, NT-3, and CNTF) was measured using the real-time PCR. The serum corticosterone level was evaluated by enzyme-linked immunosorbent assay, and, for each subject, the hippocampal proliferation was examined by 5-bromo-2-deoxyuridine immunostaining. Mice exhibited depression-like behavior in the forced-swim test (FST) and decreased BDNF mRNA and hippocampal proliferation in the middle of the stress exposure. After 15 days of stress exposure, we observed increased immobility in the FST, serum corticosterone levels, and BDNF mRNA levels and degenerated hippocampal proliferation, maintained for at least 2 weeks. Anhedonia-like behavior in the sucrose preference test and NGF mRNA levels were decreased following 15 days of stress. NGF mRNA levels were significantly higher 1 week after stress exposure. The current data demonstrate that chronic stress exposure induces prolonged BDNF and NGF mRNA changes and increases corticosterone levels and depression-like behavior in the FST, but does not alter other neurotrophic factors or performance in the sucrose preference test.

[Effects of 15-day chronic stress on behavior and neurological changes in the hippocampus of ICR mice].

  Numerous rodent models of depression have been reported, most requiring a long experimental period and significant effort. We explored a new potential mouse model for depression by investigating whether exposure to a 15-day chronic stress paradigm could induce depression-like behavior in ICR mice. Animals in the stress-exposed groups were subjected to 3 h of restraint while immersed in a 28°C water bath daily for 15 consecutive days. Immobility time in the forced swim test was increased in the chronic stress-exposed mice compared with the controls. Serum corticosterone levels were also much higher in the stressed mice than in the control mice. Hippocampal cell survival (BrdU-positive cells) and neurotrophic factor (NGF, TrkA) mRNA levels were significantly decreased in the chronic stress-exposed mice compared with controls. Administration of the anti-depressant drugs clomipramine (20 mg/kg/d) or imipramine (30 mg/kg/d) did not change the immobility time in the forced swim test, but treatment with lithium (100 mg/kg/d) did result in slight improvement. These results suggest that this 15-day chronic stress paradigm can induce depression-like behavior and neurological changes, in a short time and with minimal effort, facilitating the assessment of treatments for depression.

Study of low-temperature (4 degrees C) transport of mouse two-cell embryos enclosed in oviducts.

PURPOSE: We examined usefulness of a mouse embryo transportation system for low-temperature transport of oviducts containing mouse two-cell embryos. METHODS: Oviducts containing two-cell mouse embryos were stored at 4 degrees C for 36 h. After that, embryos were collected and cultured for 96 h in Potassium Simplex Optimized Medium (KSOM) medium and evaluated for their rate of development to hatched blastocysts. Embryos were transferred to recipients, and the rate of survival to live young was investigated. The oviducts were then transported from Yamagata to Kumamoto (distance of approx. 1,000 km). At the destination, embryos were implanted in recipient dams and were studied to evaluate their survival to live young. RESULTS: After preservation for 36 h at 4 degrees C, 68.3% of two-cell embryos developed to hatched blastocysts. As a result of transplanting 546 embryos into 25 recipients, 109 normal live young mice were obtained; the rate of development was 20.0%. Results of oviduct transport from Yamagata to Kumamoto indicated that 30.2% of transplanted embryos developed to live young. CONCLUSION: Low-temperature transport of two-cell embryos in oviducts is useful as a method of shipping mouse embryos between institutes.