Analysis of vitamin D metabolites in biological samples using a nanoluc-based vitamin D receptor ligand sensing system: NLucVDR.

Many assays are currently being developed to measure the levels of vitamin D metabolites in various samples (such as blood, urine, and saliva). This study focused on the measurement of vitamin D metabolites in serum and urine using the NLucVDR assay system, which consists of a split-type nanoluciferase and ligand-binding domain (LBD) of the human vitamin D receptor. Blood and urine samples were collected from 23 participants to validate the NLucVDR assay. The 25(OH)D3 levels in the serum and urine determined by the NLucVDR assay showed good correlations with those determined by standard analytical methods (ECLIA for serum and LC-MS/MS for urine), with correlation coefficients of 0.923 and 0.844 for serum and urine samples, respectively. In the case of serum samples, 25(OH)D3 levels determined by the NLucVDR assay were in good agreement with those determined by ECLIA. Therefore, the NLucVDR assay is a useful tool for measuring serum 25(OH)D3 levels. The contribution of each vitamin D metabolite to the luminescence intensity obtained during the NLucVDR assay depends on its concentration and affinity for NLucVDR. Thus, the contribution of 25(OH)D3 in serum appears to be much higher than that of the other metabolites. In contrast, the 25(OH)D3 levels in the urine determined by the NLucVDR assay were more than 20-fold higher than those determined by a standard analytical method (LC-MS/MS), suggesting that some vitamin D metabolite(s) in the urine remarkably increased the luminescence intensity of the NLucVDR assay. Notably, the 25(OH)D3 concentration in the urine determined by the NLucVDR assay and the serum 25(OH)D3 concentration determined by standard analytical methods showed a significant positive correlation (r = 0.568). These results suggest that the analysis of a small amount of urine using the NLucVDR assay may be useful for predicting the serum 25(OH)D3 levels.

Development of nanoluciferase-based sensing system that can specifically detect 1α,25-dihydroxyvitamin D in living cells.

Previously, we reported a FLucN-LXXLL+LBD-FLucC system that detects VDR ligands using split firefly luciferase techniques, ligand binding domain (LBD) of VDR, and LXXLL sequences that interact with LBD after VDR ligand binding. In vivo, 25-hydroxyvitamin D3 (25(OH)D3) and 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) act as VDR ligands that bind to VDR, and regulate bone-related gene expression. Therefore, the amount of 25(OH)D3 and 1α,25(OH)2D3 are indicators of bone-related diseases such as rickets and osteoporosis. In this study, we have developed a novel LgBiT-LXXLL+LBD-SmBiT system using NanoLuc Binary Technology (NanoBiT), which has an emission intensity several times higher than that of the split-type firefly luciferase. Furthermore, by using genetic engineering techniques, we attempted to construct a novel system that can specifically detect 1α,25(OH)2D3. Because histidine residues at positions 305 and 397 play important roles in forming a hydrogen bond with a hydroxyl group at position C25 of 25(OH)D3 and 1α,25(OH)2D3, His305 and His397 were each substituted by other amino acids. Consequently, the three mutant VDRs, H305D, H397N, and H397E were equally useful to detect 1α,25(OH)2D3 specifically. In addition, among the 58 variants of the LXXLL sequences, LPYEGSLLLKLLRAPVEE showed the greatest increase in luminescence upon the addition of 25(OH)D3 or 1α,25(OH)2D3. Thus, our novel system using NanoBiT appear to be useful for detecting native vitamin D or its derivatives.

Intake of L-serine before bedtime prevents the delay of the circadian phase in real life.

BACKGROUND: It has been shown in laboratory experiments using human subjects that ingestion of the non-essential amino acid L-serine before bedtime enhances the advance of circadian phase induced by light exposure the next morning. In the present study, we tested the effect of ingestion of L-serine before bedtime on circadian phase in real life and whether its effect depends on the initial circadian phase. METHODS: The subjects were 33 healthy male and female university students and they were divided into an L-serine group (n = 16) and a placebo group (n = 17). This study was conducted in a double-blind manner in autumn and winter. After a baseline period for 1 week, the subjects took 3.0 g of L-serine or a placebo 30 min before bedtime for 2 weeks. Saliva was collected twice a week at home every hour under a dim light condition from 20:00 to 1 h after habitual bedtime. Dim light melatonin onset (DLMO) was used as an index of phase of the circadian rhythm. RESULTS: DLMO after intervention was significantly delayed compared to the baseline DLMO in the placebo group (p = 0.02) but not in the L-serine group. There was a significant difference in the amount of changes in DLMO between the two groups (p = 0.04). There were no significant changes in sleeping habits after intervention in the two groups. There were significant positive correlations between advance of DLMO and DLMO before intervention in the L-serine group (r = 0.53, p < 0.05) and the placebo group (r = 0.69, p < 0.01). There was no significant difference in the slopes of regression lines between the two groups (p = 0.71), but the intercept in the L-serine group was significantly higher than that in the placebo group (p < 0.01). The levels of light exposure were not significantly different between the two groups. CONCLUSIONS: Our findings suggest that intake of L-serine before bedtime for multiple days might attenuate the circadian phase delay in the real world and that this effect does not depend on the initial circadian phase. TRIAL REGISTRATION: This study is registered with University Hospital Medical Information Network in Japan (UMIN000024435. Registered on October 17, 2016).

l-Serine Enhances Light-Induced Circadian Phase Resetting in Mice and Humans.

Background: The circadian clock is modulated by the timing of ingestion or food composition, but the effects of specific nutrients are poorly understood.Objective: We aimed to identify the amino acids that modulate the circadian clock and reset the light-induced circadian phase in mice and humans.Methods: Male CBA/N mice were orally administered 1 of 20 l-amino acids, and the circadian and light-induced phase shifts of wheel-running activity were analyzed. Antagonists of several neurotransmitter pathways were injected before l-serine administration, and light-induced phase shifts were analyzed. In addition, the effect of l-serine on the light-induced phase advance was investigated in healthy male students (mean ± SD age 22.2 ± 1.8 y) by using dim-light melatonin onset (DLMO) determined by saliva samples as an index of the circadian phase.Results: l-Serine administration enhanced light-induced phase shifts in mice (1.86-fold; P < 0.05). Both l-serine and its metabolite d-serine, a coagonist of N-methyl-d-aspartic acid (NMDA) receptors, exerted this effect, but d-serine concentrations in the hypothalamus did not increase after l-serine administration. The effect of l-serine was blocked by picrotoxin, an antagonist of γ-aminobutyric acid A receptors, but not by MK801, an antagonist of NMDA receptors. l-Serine administration altered the long-term expression patterns of clock genes in the suprachiasmatic nuclei. After advancing the light-dark cycle by 6 h, l-serine administration slightly accelerated re-entrainment to the shifted cycle. In humans, l-serine ingestion before bedtime induced significantly larger phase advances of DLMO after bright-light exposure during the morning (means ± SEMs-l-serine: 25.9 ± 6.6 min; placebo: 12.1 ± 7.0 min; P < 0.05).Conclusion: These results suggest that l-serine enhances light-induced phase resetting in mice and humans, and it may be useful for treating circadian disturbances.

Rice bran extract containing acylated steryl glucoside fraction decreases elevated blood LDL cholesterol level in obese Japanese men.

People who frequently consume whole grains show a lower incidence of arteriosclerotic disease than people who consume primarily refined grains. We examined whether or not rice bran extract containing the acylated steryl glucosides (ASG) fraction decreases blood LDL cholesterol levels in obese Japanese men with high blood levels of LDL cholesterol. The study utilized a randomized, double-blind design. A total of 51 subjects were randomly allocated to either a rice bran extract containing ASG fraction (RB-ASG) group or a placebo group. Subjects in the RB-ASG group received 30-50 mg/day of RB-ASG, and the placebo group took 9 capsules/day for 12 weeks. Before and after intake, height, weight, body fat percentage, systolic and diastolic blood pressure were measured, blood was collected, and visceral fat area, subcutaneous fat area, and abdominal circumference were determined based on umbilical computed tomography. Percentage decreases in blood LDL cholesterol, non-HDL cholesterol, LDL/HDL ratio, abdominal circumference and subcutaneous fat area were significantly better in the RB-ASG group than in the placebo group. These findings suggest that RB-ASG fraction may reduce blood LDL cholesterol levels and the risk of arteriosclerosis in obese Japanese men with high LDL cholesterol levels.

Effect of brazilian propolis on exacerbation of respiratory syncytial virus infection in mice exposed to tetrabromobisphenol a, a brominated flame retardant.

Tetrabromobisphenol A (TBBPA), a brominated flame retardant, has been found to exacerbate pneumonia in respiratory syncytial virus- (RSV-) infected mice. We examined the effect of Brazilian propolis (AF-08) on the exacerbation of RSV infection by TBBPA exposure in mice. Mice were fed a powdered diet mixed with 1% TBBPA alone, 0.02% AF-08 alone, or 1% TBBPA and 0.02% AF-08 for four weeks and then intranasally infected with RSV. TBBPA exposure increased the pulmonary virus titer and level of IFN- γ , a representative marker of pneumonia due to RSV infection, in the lungs of infected mice without toxicity. AF-08 was significantly effective in reducing the virus titers and IFN- γ level increased by TBBPA exposure. Also, AF-08 significantly reduced proinflammatory cytokine (TNF- α and IL-6) levels in the lungs of RSV-infected mice with TBBPA exposure, but Th2 cytokine (IL-4 and IL-10) levels were not evidently increased. Neither TBBPA exposure nor AF-08 treatment affected the anti-RSV antibody production in RSV-infected mice. In flow cytometry analysis, AF-08 seemed to be effective in reducing the ratio of pulmonary CD8a(+) cells in RSV-infected mice with TBBPA exposure. TBBPA and AF-08 did not exhibit anti-RSV activity in vitro. Thus, AF-08 probably ameliorated pneumonia exacerbated by TBBPA exposure in RSV-infected mice by limiting excess cellular immune responses.