Microbial Water Quality Assessment of Private Wells Using 16S rRNA Gene Amplicon Sequencing with a Nanopore Sequencer.

Private wells are used daily worldwide as convenient household water sources. In Japan, where water supply coverage is high, well water is occasionally used for non-potable purposes, such as irrigation and watering. Currently, the main microbiological test of well water is designed to detect Escherichia coli, which is an indicator of fecal contamination, using culture methods. Water use such as watering generates bioaerosols, which may cause airborne infection. However, many causative bacteria of aerosol-derived infections, such as Legionella spp., are difficult to detect using culture methods. Thus, more comprehensive modern assessment is desirable for securing the microbiological quality of well water. Here, the bacterial community structure of five private wells located in different environments was examined using the rapid and portable MinION sequencer, which enabled us to identify bacteria to the species level based on full-length 16S ribosomal RNA (rRNA) gene sequences. The results revealed the differences in the bacterial community structures of water samples from the five wells and detected Legionella pneumophila and Aeromonas hydrophila as new candidate microbial indicators. The comprehensive analysis method used in this study successfully detected bacteria causing opportunistic infections, which are difficult to detect by conventional methods. This approach is expected to be routinely applied in the future as a highly accurate method for assessing the microbiological quality of private well water.

[Situations of Passive Smoking by Urine Cotinine Levels and Recognition of Passive Smoking in High School Students -Effectiveness of Smoking Prevention Education Using a Video Made by High School Students].

We investigated a situation of passive smoking and its damaging effects among high school students. Urine cotinine concentration was measured and quantified. Additionally, we evaluated the awareness of passive smoking and smoking regulations in high school students, and the educational effect on passive smoking using a questionnaire survey and educational videos produced by high school students. We conducted a self-reporting questionnaire survey with high school students before and after watching the video produced by the high school students. We gathered the scores of the Kano Social Nicotine Dependence Questionnaire (KTSND) and awareness of smoking restrictions. Consent was obtained through the questionnaire before watching the video and collecting urine samples. Urine cotinine concentrations from 54 samples were evaluated and indicated within the low value. The KTSND score significantly decreased for those who responded to both questionnaires, after watching the video. Furthermore, analysis of the KTSND questionnaire items showed a significant decrease in scores for lifestyle, stress, and smoking location. This suggests that the video produced in this study has a certain amount of educational effect on passive smoking and that the student-led educational method is effective. The survey using the KTSND revealed that there were some students who were not exposed to passive smoking, but instead had high smoking tolerance. Going forward, it will be necessary to promote education on passive smoking and smoking prevention by incorporating the video lecture and urine cotinine concentration was measured, as in this study, to encourage behavior that decreases passive smoking among high school students.

Homogeneous nucleation of corundum nanocrystallites by rapid heating of aluminum formate hydroxide-based precursor powder.

The high density nucleation of α-Al2O3 nanocrystallites was observed by rapid heating of the aluminum formate hydroxide-based precursor powder at 1200 °C for 50 s. The nucleation of α-Al2O3 nanocrystallites with less 10 nm in size from high purity aluminum oxide matrix has not been observed to our knowledge. Based on the results of XRD and TEM, α-Al2O3 nanocrystallites nucleated from the amorphous phase which formed after thermal decomposition of the precursor powder. Subsequently, α-Al2O3 with hollow rod-like morphology formed through coalescence and growth of nanocrystallites after heating at 1200 °C for 1 min. The results obtained in this paper indicates a possible beneficial effect of the rapid heating and cooling of the aluminum formate hydroxide-based precursor powder on the precipitation of α-Al2O3 nanocrystallites.

An in vitro ES cell-based clock recapitulation assay model identifies CK2α as an endogenous clock regulator.

We previously reported emergence and disappearance of circadian molecular oscillations during differentiation of mouse embryonic stem (ES) cells and reprogramming of differentiated cells, respectively. Here we present a robust and stringent in vitro circadian clock formation assay that recapitulates in vivo circadian phenotypes. This assay system first confirmed that a mutant ES cell line lacking Casein Kinase I delta (CKIδ) induced ∼3 hours longer period-length of circadian rhythm than the wild type, which was compatible with recently reported results using CKIδ null mice. In addition, this assay system also revealed that a Casein Kinase 2 alpha subunit (CK2α) homozygous mutant ES cell line developed significantly longer (about 2.5 hours) periods of circadian clock oscillations after in vitro or in vivo differentiation. Moreover, revertant ES cell lines in which mutagenic vector sequences were deleted showed nearly wild type periods after differentiation, indicating that the abnormal circadian period of the mutant ES cell line originated from the mutation in the CK2α gene. Since CK2α deficient mice are embryonic lethal, this in vitro assay system represents the genetic evidence showing an essential role of CK2α in the mammalian circadian clock. This assay was successfully applied for the phenotype analysis of homozygous mutant ES cells, demonstrating that an ES cell-based in vitro assay is available for circadian genetic screening.

Orally administered selective TRPV1 antagonist, JTS-653, attenuates chronic pain refractory to non-steroidal anti-inflammatory drugs in rats and mice including post-herpetic pain.

Chronic pain refractory to non-steroidal anti-inflammatory drugs (NSAIDs) is a major problem and drugs for such pain are needed. Many studies suggest that transient receptor potential vanilloid type 1 (TRPV1) is associated with NSAID-refractory chronic pain. Therefore, we investigated the involvement of TRPV1 in NSAID-refractory chronic pain using experimental models for NSAID-refractory chronic pain reflecting severe arthritic and postherpetic pain. The selective TRPV1 antagonist JTS-653 {(3S)-3-(hydroxymethyl)-4-(5-methylpyridin-2-yl)-N-[6-(2,2,2-trifluoroethoxy)pyridin-3-yl]-3,4-dihydro-2H-benzo[b][1,4]oxazine-8-carboxamide} reversed mechanical hyperalgesia on day 7 after injection of complete-Freund-adjuvant into the hindpaw in rats at 0.3 mg/kg, whereas indomethacin showed no effect. JTS-653 reduced chronic pain at 0.3 mg/kg in herpes simplex virus-1-inoculated mice that has been reported as NSAID-refractory pain. JTS-653 partially attenuated mechanical hyperalgesia in the L5 spinal nerve ligation model in rats at 0.3 mg/kg, whereas indomethacin showed no effect. Both JTS-653 and indomethacin reduced formalin-induced pain in the second phase, whereas they showed no effect in the first phase. JTS-653 did not affect the nociception of noxious thermal and mechanical stimuli and motor coordination in normal rats. These findings demonstrate the TRPV1 involvement in NSAID-refractory chronic pain reflecting severe arthritic and postherpetic pain. TRPV1 antagonists would be useful for the treatment of NSAID-refractory chronic pain.

JTS-653 blocks afferent nerve firing and attenuates bladder overactivity without affecting normal voiding function.

PURPOSE: We evaluated the role of TRPV1 in bladder overactivity based on afferent nerve firing and urodynamic parameters using the selective TRPV1 antagonist JTS-653. MATERIALS AND METHODS: We evaluated the effects of JTS-653 on the increased pelvic nerve discharge and intravesical pressure induced by intravesical infusion of 100 µM capsaicin in anesthetized rats. The effects of JTS-653 on the urodynamic parameters of bladder overactivity induced by intravesical infusion of 30 nM resiniferatoxin or 0.2% acetic acid, or on normal bladder activity were evaluated by cystometry in conscious rats. The effects of JTS-653 on carbachol induced contraction were investigated using bladder muscle strips. RESULTS: JTS-653 significantly suppressed the capsaicin induced increase in nerve discharge and intravesical pressure. Intravesical infusion of resiniferatoxin or acetic acid decreased the intercontraction interval and voided volume. JTS-653 significantly increased the intercontraction interval and voided volume in rats with resiniferatoxin or acetic acid induced bladder overactivity without affecting maximal voiding pressure. The antimuscarinic agent propiverine significantly decreased maximal voiding pressure but did not affect the intercontraction interval or voided volume in rats with acetic acid induced bladder overactivity. In normal rats JTS-653 showed no significant effects on the intercontraction interval, voided volume or maximal voiding pressure. JTS-653 did not affect carbachol induced contraction of the bladder muscle. CONCLUSIONS: Our findings suggest that TRPV1 is involved in bladder overactivity via afferent nerve activation but it is not associated with normal voiding function. A TRPV1 antagonist would be a useful drug for bladder overactivity with a different pharmacological profile than antimuscarinic agents.

Evoked potentials to gustatory stimulations in cases with unilateral facial nerve palsy.

Evoked potentials to taste stimulations were discussed. New our technique of objective measurement of gustatory function by an evoked potential technique will be introduced. As a result, introduction of taste solution on the tongue innervated by facial nerve in normal subjects resulted in positive responses. Absence of taste solution did not resulted in the positive response. In case with unilateral; facial nerve palsy, introduction of taste solution on the affected side did not resulted in the positive responses.

Pharmacological characterization of (3S)-3-(hydroxymethyl)-4-(5-methylpyridin-2-yl)-N-[6-(2,2,2-trifluoroethoxy)pyridin-3-yl]-3,4-dihydro-2H-benzo[b][1,4]oxazine-8-carboxamide (JTS-653), a novel transient receptor potential vanilloid 1 antagonist.

Transient receptor potential vanilloid 1 (TRPV1) activation in peripheral sensory nerve is known to be associated with various pain-related diseases, thus TRPV1 has been the focus as a target for drug discovery. In this study, we characterized the pharmacological profiles of (3S)-3-(hydroxymethyl)-4-(5-methylpyridin-2-yl)-N-[6-(2,2,2-trifluoroethoxy)pyridin-3-yl]-3,4-dihydro-2H-benzo[b][1,4]oxazine-8-carboxamide (JTS-653), a novel TRPV1 antagonist. JTS-653 displaced [(3)H]resiniferatoxin binding to human and rat TRPV1. JTS-653 competitively antagonized the capsaicin-induced activation of human TRPV1 with pA(2) values of 10.1. JTS-653 also inhibited proton-induced activation of human and rat TRPV1 with IC(50) values of 0.320 and 0.347 nM, respectively. Electrophysiological studies indicated that JTS-653 blocked heat-induced inward currents in rat TRPV1 with IC(50) values of 1.4 nM. JTS-653 showed weak or no inhibitory effects on other TRP channels, receptors, and enzymes. JTS-653 significantly prevented capsaicin-induced mechanical hyperalgesia at 1 mg/kg p.o. and attenuated carrageenan-induced mechanical hyperalgesia at 0.3 mg/kg p.o. JTS-653 significantly attenuated carrageenan-induced thermal hyperalgesia at 0.1 mg/kg p.o. and fully reversed at 0.3 mg/kg p.o. without affecting the volume of the carrageenan-treated paw. JTS-653 showed a transient increase of body temperature at 0.3 mg/kg p.o. These results indicated that JTS-653 is a highly potent and selective TRPV1 antagonist in vitro and in vivo and suggested that JTS-653 is one of the most potent TRPV1 antagonists. The profiles of JTS-653, high potency in vivo and transient hyperthermia, seem to be associated with polymodal inhibition of TRPV1 activation.

Formation of retinoylated proteins from retinoyl-CoA in rat tissues.

Retinoylation (acylation of proteins by retinoic acid) is considered as one mechanism of retinoic acid (RA) action occurring in cells in vitro and in vivo. Previously, our studies showed that in rat tissues the formation of retinoyl-CoA from RA, the first step of retinoylation, required ATP, CoA and MgCl(2). In the current study, we examined whether the transfer of retinoyl-CoA into proteins, the second step of retinoylation, occurs in rat tissues. [(3)H]-Labeled-retinoyl-CoA bound covalently to proteins in rat liver, kidney, testis, and brain. The levels of incorporation of retinoyl-CoA into proteins were higher in vitamin A-deficient rats than in normal ones. The formation of retinoylated proteins depended on the incubation time, and the concentrations of retinoyl-CoA and homogenate. The reaction was suppressed by fatty acyl-CoAs and palmitic acid, but not by arachidonic acid. The Vmax and Km values for retinoyl-CoA in the formation of retinoylated proteins using a crude liver extract were estimated to be 2,597.3 pmol/min/mg protein and 9.5 x 10(-5) M, respectively. Retinoylated proteins formed from retinoyl-CoA, including a 17 kDa protein exhibiting high radioactivity, disappeared in the presence of 2-mercaptoethanol, indicating that RA was linked to the proteins through a thioester bond. These results demonstrate that retinoylation in rat tissues occurs via retinoyl-CoA formed from RA. This process may play a significant physiological role in cells.

Evoked response to taste stimulations.

For the recording of gustatory evoked responses, the tip of a stimulator is pressed vertically on one side of the tongue until the trigger pulses are generated by a switch attached to the bottom of the stimulator. According to our results, no detectable response was observed in the absence of taste. The positive waves were distinguishable by using the technique of superimposition before averaging, and the positive wave was made clearer by averaging.

Evoked response to olfactory stimulations in anosmic patients.

In our study, we explored the influence of blast method introduction of odorant on evoked response. In normal patients, no detectable response was observed in the absence of an odor, and introduction of an odorant at the end of inspiration or during expiration did not result in any detectable positive response. In anosmic patients, glacial acetic acid, which is thought to be a strong trigeminal stimulating agent, evoked a negative response without detection of odor. Accordingly, the positively evoked response to odorant was thought to be elicited mainly by the odorant, not by the trigeminal stimulations or the auditory stimulations (or both).