Phenotypic screening using waveform analysis of synchronized calcium oscillations in primary cortical cultures.

At present, in vitro phenotypic screening methods are widely used for drug discovery. In the field of epilepsy research, measurements of neuronal activities have been utilized for predicting efficacy of anti-epileptic drugs. Fluorescence measurements of calcium oscillations in neurons are commonly used for measurement of neuronal activities, and some anti-epileptic drugs have been evaluated using this assay technique. However, changes in waveforms were not quantified in previous reports. Here, we have developed a high-throughput screening system containing a new analysis method for quantifying waveforms, and our method has successfully enabled simultaneous measurement of calcium oscillations in a 96-well plate. Features of waveforms were extracted automatically and allowed the characterization of some anti-epileptic drugs using principal component analysis. Moreover, we have shown that trajectories in accordance with the concentrations of compounds in principal component analysis plots were unique to the mechanism of anti-epileptic drugs. We believe that an approach that focuses on the features of calcium oscillations will lead to better understanding of the characteristics of existing anti-epileptic drugs and allow to predict the mechanism of action of novel drug candidates.

A novel method for predicting the efficacy of topical drugs on onychomycosis: A comparison of efinaconazole and luliconazole.

To be effective against onychomycosis, topically applied drugs have to reach the infection site at an effective concentration to exert antifungal activity against the parasitic form of dermatophytes. We established a novel in vitro method for predicting drug efficacy at the infection site and verified the method by comparing the efficacy of two azole class topical anti-onychomycosis drugs. To predict drug efficacy in the nail plate, a human nail permeability test was conducted and the activities of the free-drugs in the upper, middle, and lowest layers of the nail plate were determined by measuring the growth inhibitory zone. Efinaconazole permeated the nail more efficiently than luliconazole, and the amount of efinaconazole in the middle and lowest layers was higher compared with that of luliconazole. Efinaconazole demonstrated antifungal activities at the concentrations in all of the nail layers, whereas luliconazole was only active at the concentrations in the upper and middle layers. The results could be explained by differences in their affinity for keratin and nail permeability. The established method enables the evaluation of nail permeability and anti-arthrospore activity of free-drugs in the nail plate to predict drug efficacy. This method will be useful for new topical drug development.

The Potential of Digital Polymerase Chain Reaction for Improving Diagnostic Yield of Nontuberculous Mycobacteria Pulmonary Disease.

INTRODUCTION: Many patients with nontuberculous mycobacteria pulmonary disease are asymptomatic. The disease diagnosis is confirmed in only a small proportion of patients with radiological findings suspicious for nontuberculous mycobacteria pulmonary disease. Thus, many patients remained undiagnosed. Here, we evaluated the diagnostic value of digital polymerase chain reaction (PCR) in nontuberculous mycobacteria pulmonary disease. METHODS: We prospectively evaluated 123 patients with radiological findings suspicious for nontuberculous mycobacteria pulmonary disease. Digital PCR was performed using bronchial lavage fluid, sputum, saliva, blood, and urine. RESULTS: The culture of bronchial washing fluid was positive for nontuberculous mycobacteria in 53 patients and negative in 70. The positive detection rate of nontuberculous mycobacteria by digital PCR in patients with positive culture (n = 53) was as follows: bronchial lavage fluid 100%, sputum 62.9%, saliva 41.5%, blood 7.5%, and urine 3.8%. All patients with two or more positive partitions for nontuberculous mycobacteria in the digital PCR of bronchial lavage fluid showed nontuberculous mycobacteria growth in the bronchial lavage fluid culture. The digital PCR analysis of the bronchial lavage fluid showed a high sensitivity (100%), specificity (85.7%), positive predictive value (84.1%), negative predictive value (100%), and a high concordance rate (91.9%) with the bronchial lavage fluid culture results. In addition, the culture of bronchial lavage fluid was positive for nontuberculous mycobacteria in patients with two or more positive partitions in the digital PCR of sputum and saliva with a combined positive predictive value of 81.1%. CONCLUSION: Digital PCR analysis of nontuberculous mycobacteria in bronchial lavage fluid shows a high concordance rate with the bronchial lavage fluid culture results and a high positive predictive value using both sputum and saliva, suggesting the potential usefulness of dPCR for diagnosis of nontuberculous mycobacteria pulmonary disease in clinical practice.

Couple Reports on Intimate Partner Violence and their Health Impact: Evidence From a Population-Based Survey in Japan.

Recent studies have shown that couples' reports of intimate partner violence (IPV) are not necessarily consistent. This study investigated the associations between patterns of partner victimization and perpetration reports and health and perceived wellbeing. Using household survey data from a probabilistic urban sample of adults aged 25 to 50 years and their partners, we identified 1,467 heterosexual pairs who completed the modified Japanese version of the Conflict Tactics Scale 2 Short Form. We classified responses into four categories: congruent report of no IPV, incongruent respondent victimization report, incongruent partner perpetration report, and congruent report of IPV. We used analysis of covariance to compare physical and mental health (measured by the SF-8 Health Survey Questionnaire) and life satisfaction among the four groups. The results showed that 22.4% of respondents were involved in IPV, and approximately 75% of them had incongruent reports. Compared with congruent reports of no IPV, respondents with an incongruent victimization report and an incongruent partner perpetration report scored significantly lower on physical health (-0.96, p = .03 and -1.16, p = .04, respectively). Those with an incongruent victimization report and congruent report of IPV had lower mental health scores (-2.32, p = .00 and -2.21, p = .00, respectively) and lower life satisfaction (-0.32, p = .00 and -0.21, p = .01, respectively). The results indicated that the respondent's victimization report was associated with worse mental health regardless of their partner's perpetration report, and physical health was worse only in cases with incongruent reports. Cases with a partner perpetration report but no respondent victimization report showed worse physical health but no negative effects on mental health and perceived wellbeing. These results indicate that research and practice regarding IPV should consider both partners' experiences of violence when assessing the impact of health and wellbeing on victims.

Scalable Total Syntheses and Structure-Activity Relationships of Haouamines†A, B, and Their Derivatives as Stable Formate Salts.

Haouamines A, B, and their derivatives were synthesized via Suzuki-Miyaura coupling and three key cyclization reactions as follows: the newly developed palladium(0)-catalyzed arylative cyclization of phenylalanine-derived alkyne-aldehydes with 2-bromoarylboronic acid (an "anti-Wacker"-type cyclization); BF3 ⋅OEt2 -promoted Friedel-Crafts-type cyclization of symmetrical electron-rich aromatic rings adjacent to a tertiary allylic alcohol leading to the indeno-tetrahydropyridine skeleton; and (cyanomethyl)trimethylphosphonium iodide-mediated macrocyclization of amino alcohols to afford aza-paracyclophane precursors. The palladium-catalyzed reduction of mono- and di-triflate intermediates in the later stages enabled the alteration of both the position and number of hydroxyl groups on the C-ring. The instability of haouamine B was dramatically improved by salt formation with formic acid. An unambiguous evaluation of the cytotoxicity of the prepared haouamine derivative formates with and without hydroxyl groups at different positions on the C-ring indicated that the catechol structure in haouamine B produced weak cytotoxicity.

Inhibition of the glutamine transporter SNAT1 confers neuroprotection in mice by modulating the mTOR-autophagy system.

The pathophysiological role of mammalian target of rapamycin complex 1 (mTORC1) in neurodegenerative diseases is established, but possible therapeutic targets responsible for its activation in neurons must be explored. Here we identified solute carrier family 38a member 1 (SNAT1, Slc38a1) as a positive regulator of mTORC1 in neurons. Slc38a1flox/flox and Synapsin I-Cre mice were crossed to generate mutant mice in which Slc38a1 was selectively deleted in neurons. Measurement of 2,3,5-triphenyltetrazolium chloride (TTC) or the MAP2-negative area in a mouse model of middle cerebral artery occlusion (MCAO) revealed that Slc38a1 deficiency decreased infarct size. We found a transient increase in the phosphorylation of p70S6k1 (pp70S6k1) and a suppressive effect of rapamycin on infarct size in MCAO mice. Autophagy inhibitors completely mitigated the suppressive effect of SNAT1 deficiency on neuronal cell death under in vitro stroke culture conditions. These results demonstrate that SNAT1 promoted ischemic brain damage via mTOR-autophagy system.

A new strategy for taste masking on bitter drug by other combined drug in fixed-dose combination: bitterness of Amlodipine besylate could be masked efficiently by Valsartan.

OBJECTIVES: The aim of this study was to evaluate the bitterness of amlodipine besylate (AML) combined with other five antihypertensive drugs: alacepril, benazepril, hydrochlorothiazide, telmisartan (TEL) and valsartan (VAL), which have possibility of usage as a fixed-dose combination (FDC) drugs. METHODS: The bitterness of individual six drugs and AML combined with each of the five drugs was evaluated using taste sensor SA402B (Intelligent Sensor Technology Inc.). AML combined with TEL or VAL was evaluated by taste sensor and human gustatory sensation tests. The interaction between AML with TEL or VAL was evaluated by 1 H-NMR. KEY FINDINGS: The bitterness of AML was significantly decreased by addition of VAL, whereas it remained unchanged by the addition of TEL in taste sensor and human gustatory sensation test. In the 1 H-NMR spectrum of AML with VAL, signal shifts of protons in AML were observed compared to that in AML alone. On the other hand, in the 1 H-NMR spectrum of AML with TEL, signal shifts of protons in AML were not observed. CONCLUSIONS: It was suggested that when VAL was mixed with AML, the electrostatic interactions between positive charged amino group of AML and negative charged tetrazole group of VAL were caused, and thereby led the suppression the bitterness of AML.

Evaluation of the effects of ontogenetic or maturation functions and chronic heart failure on the model analysis for the dose-response relationship of warfarin in Japanese children.

PURPOSE: We previously demonstrated that the rational pediatric dosage of warfarin can be well-described by a SIZE parameter that includes an allometry exponent of weight. On the other hand, allometry alone is considered to be insufficient to predict drug clearance in neonates and infants. The primary purpose of the present study was to evaluate the effects of incorporation of the maturation process into the analysis model for the dose-response relationship of warfarin in Japanese children. In addition, we evaluated the effect of chronic heart failure (CHF) on the response to warfarin as an independent risk factor for increased anticoagulant effects. METHODS: Thirty-eight patients with stable anticoagulation by warfarin were enrolled. During a mean follow-up period of 4.74 ± 3.51 years, 1092 data points including prothrombin time-international normalized ratio (PT-INR) were obtained. The data were subjected to multiple regression analysis to identify covariates related to the anticoagulant effects. RESULTS: Two different models describing the maturation process did not improve the predictive performance for the dose-response relationship in pediatric patients. In addition to the SIZE-normalized daily dose, the vitamin K epoxide reductase complex 1 (VKORC1) genotype, and concomitant use of bosentan, CHF was identified as a covariate increasing the anticoagulant effects of warfarin to 118%. CONCLUSION: The SIZE parameter was useful even without incorporation of maturation models to describe the response to warfarin in pediatric patients, and our longitudinal follow-up study design with multiple observations was beneficial to detect changes within individual subjects.

The possible clinical impact of risperidone on P-glycoprotein-mediated transport of tacrolimus: A case report and in vitro study.

The authors encountered the case of an 8-fold increase in the concentration/dose (C/D) ratio of tacrolimus (TAC) following the coadministration of voriconazole (VRCZ) in a hematopoietic stem cell transplantation (HSCT) recipient. The interaction observed was much greater than expected and the patient had also been treated with oral risperidone (RSP). It was hypothesized that cytochrome P450 (CYP)3A inhibition of the small intestine by voriconazole and P-glycoprotein (P-gp) inhibition of the small intestine by risperidone exerted a synergistic effect on the bioavailability of tacrolimus. The aim of the present study was to evaluate the effect of risperidone on the P-gp-mediated transport of tacrolimus. The transcellular transport of P-gp substrates was examined in Caco-2 and P-gp-expressing renal epithelial LLC-GA5-COL150 cells. In Caco-2 cells, the apical-basal (A-B) transport of rhodamine123 (Rh123) after a 120 min incubation was increased by 47.1%, whereas that in the B-A direction was decreased by 61.7% in the presence of risperidone (100 µm). These results indicate that risperidone showed an inhibitory effect on the P-gp-mediated transport of Rh123. In LLC-GA5-COL150 cells, the A-B transport of tacrolimus after 120 min incubation was increased by 21.7% in the presence of risperidone (100 µm), whereas that in the B-A direction was decreased by 10.7%. These results suggest that risperidone was at least partly involved in the mechanism of the marked increase in the C/D ratio of tacrolimus. This case report provides new insights into the diversity of drug interactions of tacrolimus triggered by the combination of two concomitant drugs.

Distinct Mechanism of Cysteine Oxidation-Dependent Activation and Cold Sensitization of Human Transient Receptor Potential Ankyrin 1 Channel by High and Low Oxaliplatin.

Oxaliplatin, a third-generation platinum-based chemotherapeutic agent, displays unique acute peripheral neuropathy triggered or enhanced by cold, and accumulating evidence suggests that transient receptor potential ankyrin 1 (TRPA1) is responsible. TRPA1 is activated by oxaliplatin via a glutathione-sensitive mechanism. However, oxaliplatin interrupts hydroxylation of a proline residue located in the N-terminal region of TRPA1 via inhibition of prolyl hydroxylase (PHD), which causes sensitization of TRPA1 to reactive oxygen species (ROS). Furthermore, PHD inhibition endows cold-insensitive human TRPA1 (hTRPA1) with ROS-dependent cold sensitivity. Since cysteine oxidation and proline hydroxylation regulate its activity, their association with oxaliplatin-induced TRPA1 activation and acquirement of cold sensitivity were investigated in the present study. A high concentration of oxaliplatin (1 mM) induced outward-rectifier whole-cell currents and increased the intracellular Ca2+ concentration in hTRPA1-expressing HEK293 cells, but did not increase the probability of hTRPA1 channel opening in the inside-out configuration. Oxaliplatin also induced the rapid generation of hydrogen peroxide, and the resultant Ca2+ influx was prevented in the presence of glutathione and in cysteine-mutated hTRPA1 (Cys641Ser)-expressing cells, whereas proline-mutated hTRPA1 (Pro394Ala)-expressing cells showed similar whole-cell currents and Ca2+ influx. By contrast, a lower concentration of oxaliplatin (100 µM) did not increase the intracellular Ca2+ concentration but did confer cold sensitivity on hTRPA1-expressing cells, and this was inhibited by PHD2 co-overexpression. Cold sensitivity was abolished by the mitochondria-targeting ROS scavenger mitoTEMPO and was minimal in cysteine-mutated hTRPA1 (Cys641Ser or Cys665Ser)-expressing cells. Thus, high oxaliplatin evokes ROS-mediated cysteine oxidation-dependent hTRPA1 activation independent of PHD activity, while a lower concentration induces cold-induced cysteine oxidation-dependent opening of hTRPA1 via PHD inhibition.

Reduction in Vegetable Intake Disparities With a Web-Based Nutrition Education Intervention Among Lower-Income Adults in Japan: Randomized Controlled Trial.

BACKGROUND: No existing Web-based nutrition education interventions have been evaluated in light of socioeconomic status just in Japan. OBJECTIVE: The aim was to investigate the effect of a Web-based intervention program on reducing vegetable intake disparities between low- and middle-income Japanese adults. METHODS: In this randomized controlled trial, participants were assessed at three time points-baseline, postintervention (5 weeks later), and a follow-up after 3 months-from October 2015 to March 2016. We collected data via a Japanese online research service company from 8564 adults aged 30 to 59 years. Participants were stratified according to national population statistics for gender and age, and randomly selected. They were then randomly allocated into intervention (n=900) and control (n=600) groups such that both groups contained an equal number of individuals with low and middle income. The intervention program encouraged behavior change using behavioral theories and techniques tailored to their assumed stage of change. The outcome was vegetable intake servings per day (1 serving being approximately 70 g). RESULTS: Out of 900 participants who started, 450 were from the middle income group (of which 386 or 85.7% completed the intervention), and 450 were from the low income group (of which 371 or 82.4% completed). In the intervention group, vegetable intake increased in the low-income participants from baseline to postintervention (0.42 servings, 95% CI 0.11-0.72). A two-way analysis of variance showed that low-income participants had significant main effects of group (η2=0.04, P=.01) and time (η2=0.01, P<.001), and a significant interaction (η2=0.01, P=.009). Middle-income participants also had a significant main effect of time (η2=0.01, P=.006) and a significant interaction (η2=0.01, P=.046). CONCLUSIONS: This Web-based nutritional education intervention could fill the vegetable intake gap between low- and middle-income adults in Japan, and is expected to prevent noncommunicable and lifestyle-related diseases. Further intervention program improvements are necessary to maintain and increase vegetable intake for other groups. TRIAL REGISTRATION: Current Controlled Trials (UMIN-ICDR): UMIN000019376; https://upload.umin.ac.jp/cgi-open-bin/ icdr_e/ctr_view.cgi?recptno=R000022404 (Archived by WebCite at http://www.webcitation.org/6u9wihBZU).

Cooling the lower abdomen to reduce postpartum blood loss: A randomized controlled trial.

BACKGROUND: Cooling the lower abdomen is one of the Japanese traditional non-pharmacological prophylactic managements for postpartum hemorrhage. This study aimed to evaluate the effectiveness of cooling the lower abdomen in reducing postpartum blood loss compared with no intervention. In both cases, women delivered vaginally without prophylactic oxytocin in the third stage of labor. METHODS: In this randomized controlled trial, the lower abdomen was cooled by placement of an 8.6°C icepack during the first 2 h after placental delivery. The primary outcome was measured as the total blood loss within 2 h after delivery. This study had 80% power at the two tails of 5% significance level to detect the mean difference (MD, 70 g) in total blood loss within 2 h after delivery between the two groups. The sample size was calculated as 144 women (72 women per group). RESULTS: Between January and May 2016, 160 women were randomly assigned to the intervention group (cooling the lower abdomen, n = 81) or the control group (n = 79). Baseline characteristics were similar between groups, with the exception of mean blood loss during the third stage of labor. The primary outcome was not reduced by cooling, compared with no intervention (mean blood loss, 513.3 vs. 478.1 g, respectively; MD = 35.2 g; 95% confidence interval = -65.3-135.7). No adverse events occurred; however, seven (8.7%) women in the intervention group declined to continue cooling the lower abdomen because of discomfort. CONCLUSION: Compared with the control group, cooling the lower abdomen did not reduce the total amount of blood loss up to 2 h after delivery. TRIAL REGISTRATION: UMIN-CTR UMIN000019834.

Taste-Masking Effect of Chlorogenic Acid (CGA) on Bitter Drugs Evaluated by Taste Sensor and Surface Plasmon Resonance on the Basis of CGA-Drug Interactions.

The purpose of this study was to evaluate the taste-masking effects of chlorogenic acid (CGA) on bitter drugs using taste sensor measurements and surface plasmon resonance (SPR) analysis of CGA-drug interactions. Six different bitter drugs were used: amlodipine besylate (AMD), diphenhydramine hydrochloride (DPH), donepezil hydrochloride (DNP), rebamipide (RBM), diclofenac sodium (DCF) and etodolac (ETD). Taste sensor outputs were significantly inhibited by the addition of CGA to all drugs. The inhibition ratio of the taste sensor output decreased in the following order DPH>DNP>AMD≈DCF≈RBM≈ETD. The association rate constant (ka) for the interaction between drugs and CGA as evaluated by SPR measurement also decreased in the following order DPH>DNP>AMD>DCF≈ETD≈RBM. It was suggested that basic drugs (AMD, DNP, DPH) associate more easily with CGA than acidic drugs (DCF, RBM, ETD). The inhibition ratios (%) of the taste sensor output of bitter drugs caused by CGA and the association rate constants (ka) between the drugs and CGA were significantly correlated (rs=0.886, p<0.05, Spearman's correlation test). Our findings suggest that the taste-masking effects of CGA are due to its direct association with the drugs. CGA may therefore be a useful taste-masking agent for basic drugs.

Suppression in Bitterness Intensity of Bitter Basic Drug by Chlorogenic Acid.

The purpose of the study was to evaluate suppression of the bitterness intensity of bitter basic drugs by chlorogenic acid (CGA) using the artificial taste sensor and human gustatory sensation testing and to investigate the mechanism underlying bitterness suppression using 1H-NMR. Diphenhydramine hydrocholoride (DPH) was the bitter basic drug used in the study. Quinic acid (QNA) and caffeic acid (CFA) together form CGA. Although all three acids suppressed the bitterness intensity of DPH in a dose-dependent manner as determined by the taste sensor and in gustatory sensation tests, CFA was less effective than either CGA or QNA. Data from 1H-NMR spectroscopic analysis of mixtures of the three acids with DPH suggest that the carboxyl group, which is present in both QNA and CGA but not CFA, interact with the amine group of DPH. This study showed that the bitterness intensity of DPH was suppressed by QNA and CGA through a direct electrostatic interaction with DPH as confirmed in 1H-NMR spectroscopic analysis. CGA and QNA may therefore be useful bitterness-masking agents for the basic drug DPH.

A randomized-controlled trial focusing on socio-economic status for promoting vegetable intake among adults using a web-based nutrition intervention programme: study protocol.

BACKGROUND: Web-based nutritional education programmes appear to be comparable to those delivered face-to-face. However, no existing web-based nutrition education or similar programme has yet been evaluated with consideration of socio-economic status. The objective of a nutritional education programme of promoting vegetable intake designed a randomized controlled trial (RCT) is to evaluate the results of intervention and to determine how socio-economic status influences the programme effects. METHODS/DESIGN: Participants will be randomly sampled individuals (aged 30-59) stratified according national population statistics for sex, age, and household income. Participants were consented to survey participation (n = 1500), and will be randomly divided into intervention and control groups. The intervention period is 5 weeks with one step of diet-related education per week. The main outcome of the programme is dietary behaviour as eating vegetable (350 g per day, five small bowl). To encourage behavioural changes, the programme contents are prepared using behavioural theories and techniques tailored to the assumed group stages of behavioural change. In the first step, we employ the health belief model to encourage a shift from the pre-contemplative to the contemplative phase; in the second and third steps, social cognitive theory is used to encourage transition to the preparatory phase; in the fourth step, social cognitive theory and strengthening social support are used to promote progression to the execution phase; finally, in the fifth step, strengthening social capital and social support are used to promote the shift to the maintenance phase. The baseline, post intervention and follow-up survey was assessed using a self-administered questionnaire. For process evaluation, we use five items relating to programme participation and satisfaction. A follow-up survey of participants will be carried out 3 months after intervention completion. DISCUSSION: The fact that this study is an RCT with an established control group is a strong advantage. Information and communications technology is not limited by time or place. If we could show this web-based nutrition education programmes has a positive effect, it may be an appropriate tool for reaching individuals in lower socio-economic state. TRIAL REGISTRATION: Current Controlled Trials UMIN-ICDR UMIN 000019376 (Registered October 16, 2015).

Effects of Brief Communication Skills Training for Workers Based on the Principles of Cognitive Behavioral Therapy: A Randomized Controlled Trial.

OBJECTIVE: Stimulating communication is an important workplace issue. We investigated the effects of a brief communication skills training (CST) program based on the principles of cognitive behavioral therapy (CBT). METHODS: A randomized controlled trial was conducted on 206 white-collar workers. The intervention group underwent a 2-hour CST group training conducted by an occupational physician. RESULT: The results of the intention-to-treat analysis using a mixed-effects model showed that there was a significant interaction between group and time observed for the item "thinking together to solve problems and issues" (P = 0.02). The effect size (Cohen d) was 0.35 (95% confidence interval, 0.07 to 0.62). CONCLUSIONS: The present study suggests that a brief CST based on the principles of CBT could improve the communication behavior of workers.

Bone Resorption Is Regulated by Circadian Clock in Osteoblasts.

We have previously shown that endochondral ossification is finely regulated by the Clock system expressed in chondrocytes during postnatal skeletogenesis. Here we show a sophisticated modulation of bone resorption and bone mass by the Clock system through its expression in bone-forming osteoblasts. Brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (Bmal1) and Period1 (Per1) were expressed with oscillatory rhythmicity in the bone in vivo, and circadian rhythm was also observed in cultured osteoblasts of Per1::luciferase transgenic mice. Global deletion of murine Bmal1, a core component of the Clock system, led to a low bone mass, associated with increased bone resorption. This phenotype was recapitulated by the deletion of Bmal1 in osteoblasts alone. Co-culture experiments revealed that Bmal1-deficient osteoblasts have a higher ability to support osteoclastogenesis. Moreover, 1α,25-dihydroxyvitamin D3 [1,25(OH)2 D3 ]-induced receptor activator of nuclear factor κB ligand (Rankl) expression was more strongly enhanced in both Bmal1-deficient bone and cultured osteoblasts, whereas overexpression of Bmal1/Clock conversely inhibited it in osteoblasts. These results suggest that bone resorption and bone mass are regulated at a sophisticated level by osteoblastic Clock system through a mechanism relevant to the modulation of 1,25(OH)2 D3 -induced Rankl expression in osteoblasts. © 2017 American Society for Bone and Mineral Research.

The intrinsic microglial clock system regulates interleukin-6 expression.

Similar to neurons, microglia have an intrinsic molecular clock. The master clock oscillator Bmal1 modulates interleukin-6 upregulation in microglial cells exposed to lipopolysaccharide. Bmal1 can play a role in microglial inflammatory responses. We previously demonstrated that gliotransmitter ATP induces transient expression of the clock gene Period1 via P2X7 purinergic receptors in cultured microglia. In this study, we further investigated mechanisms underlying the regulation of pro-inflammatory cytokine production by clock molecules in microglial cells. Several clock gene transcripts exhibited oscillatory diurnal rhythmicity in microglial BV-2 cells. Real-time luciferase monitoring also showed diurnal oscillatory luciferase activity in cultured microglia from Per1::Luciferase transgenic mice. Lipopolysaccharide (LPS) strongly induced the expression of pro-inflammatory cytokines in BV-2 cells, whereas an siRNA targeting Brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (Bmal1), a core positive component of the microglial molecular clock, selectively inhibited LPS-induced interleukin-6 (IL-6) expression. In addition, LPS-induced IL-6 expression was attenuated in microglia from Bmal1-deficient mice. This phenotype was recapitulated by pharmacological disruption of oscillatory diurnal rhythmicity using the synthetic Rev-Erb agonist SR9011. Promoter analysis of the Il6 gene revealed that Bmal1 is required for LPS-induced IL-6 expression in microglia. Mice conditionally Bmal1 deficient in cells expressing CD11b, including microglia, exhibited less potent upregulation of Il6 expression following middle cerebral artery occlusion compared with that in control mice, with a significant attenuation of neuronal damage. These results suggest that the intrinsic microglial clock modulates the inflammatory response, including the positive regulation of IL-6 expression in a particular pathological situation in the brain, GLIA 2016. GLIA 2017;65:198-208.

An Alteration in the Cecal Microbiota Composition by Feeding of 1-Kestose Results in a Marked Increase in the Cecal Butyrate Content in Rats.

Functional food ingredients, including prebiotics, have been ardently developed for improving the intestinal environment. Fructooligosaccarides (FOS), including fructans, are the well researched and commercialized prebiotics. However, to our knowledge, few studies have been conducted on the physiological effects of each component of FOS as prebiotics. 1-Kestose, a component of FOS, is composed of one glucose and two fructose molecules, and is considered as a key prebiotic component in short-chain FOS. In the present study, we examined the effects of dietary 1-kestose using 0.5-5% 1-kestose diets on cecal microbiota composition and cecal contents of short-chain fatty acids and lactate in rats. The findings indicate that dietary 1-kestose induced cecal hypertrophy and alterations in the cecal microbiota composition, including a marked increase in the cell number of Bifidobacterium spp. These alterations were associated with significant increases in acetate and lactate, and a marked increase in butyrate in cecal contents. Furthermore, dietary 1-kestose induced a significant decrease in serum insulin concentration in rats fed 2.5-5% 1-kestose diet. These findings suggest a potential of 1-kestose to be a prebiotic for improving the metabolism of the host.