5-Hydroxymethylcytosine (5hmC) is beginning to be expected to play a role as a diagnostic and prognostic marker of diseases. On the other hand, we developed the methylated-site display-amplified fragment length polymorphism (MSD-AFLP), an affordable, large-scale (approximately 40,000-50,000 CpG sites), highly sensitive methylation profiling method, and applied it to environmental health and disease biomarker discovery research. Herein, we attempted to modify the MSD-AFLP method to detect 5hmC. To validate this method, we compared hydroxymethylation levels among tissues using mouse samples to determine whether this method could detect tissue-specific differential 5hmC. We also considered combining this method with next generation sequencing (NGS). Comparisons using AFLP revealed that in some sites, the variation in hydroxymethylated DNA was greater than that in methylated DNA between tissues. Therefore, we determined the hydroxymethylation levels at these sites using Glucosylation-mediated restriction enzyme sensitive qPCR (gRES-qPCR) to confirm the accuracy of the AFLP analysis. The differences in hydroxymethylation levels between tissues were similar between the two methods. The protocol for combination with NGS was evaluated by comparing the AFLP data with 11 DNA fragments. The differences in hydroxymethylation levels between tissues were similar between the two methods. Cluster analysis demonstrated that NGS data were comparable to AFLP data in detecting differential and contrasting hydroxymethylation patterns across tissues. This method, based on the MSD-AFLP technique, will contribute to various epigenetics-based research, including the discovery of biomarkers and therapeutic drug targets.

Patients undergoing hemodialysis face a significantly elevated risk of cardiovascular disease (CVD) mortality. Uremic toxins, particularly indoxyl sulfate (IS) and p-cresyl sulfate (PCS), accumulate in patients undergoing dialysis and contribute to cardiovascular complications. Although IS and PCS concentrations are often assessed individually, this pilot study developed and validated an integrated scoring system combining these values to improve CVD mortality prediction. This prospective study included 66 patients undergoing hemodialysis at the Hitoyoshi Medical Center between 2008–2018. The IS + PCS score was calculated using Cox proportional hazards analysis. The primary endpoint was CVD death. The IS + PCS score was strongly associated with CVD mortality. IS alone exhibited high sensitivity (91.67%) but low specificity (44.44%), whereas PCS alone showed high specificity (92.59%) but low sensitivity (25.00%). The IS + PCS score achieved balanced sensitivity (83.33%) and specificity (57.41%), with a higher F1 score (0.44) than that of IS alone (0.41) and PCS alone (0.32). Leave-one-out cross-validation confirmed internal validity (mean C-index: 0.645). The IS + PCS score demonstrated potential as a prognostic predictor of CVD mortality in patients undergoing hemodialysis. While the score achieved balanced sensitivity and specificity, with an F1 score exceeding that of the individual markers, time-dependent receiver operating characteristic analysis showed no superiority over IS alone at any time point. These findings suggest that the integrated score combines the complementary strengths of both toxins, but requires validation in larger cohorts to establish clinical superiority.

Objective: Attention-deficit/hyperactivity disorder (ADHD) is a common neurodevelopmental disorder with early onset. Sleep disturbances in children with ADHD may impair attention and learning and exacerbate symptoms. Maintaining good sleep is crucial in ADHD treatment. This study investigated the effects of ADHD medications on subjective and objective sleep in children under 18, employing actigraphy and a questionnaire. Methods: This single-centre prospective observational study enrolled patients under 18 receiving methylphenidate (MPH), lisdexamfetamine (LDX), atomoxetine (ATX), or guanfacine (GXR) between June 1 and August 31, 2022. Patients using other drugs were excluded. Subjective sleep was assessed using the Athens Insomnia Scale (AIS), and objective sleep variables were analysed using an actigraph worn on the waist. Results: A total of 21 patients were enrolled. AIS total scores of 4 or more were significantly more common in GXR-treated patients (P = 0.009) than in ATX-treated patients. However, other variables were not statistically significant. Among patients receiving non-psychostimulant monotherapy, sleep time was longer (P = 0.045), and postural changes were more frequent (P < 0.001) than in those receiving combined psychostimulant and non-psychostimulant therapy. Conclusions: Findings suggest ATX may improve subjective sleep more effectively than GXR. A trend toward shorter sleep period time was observed in the combined group, likely due to the arousal effects of psychostimulants. Reduced postural changes in the combined group may indicate altered sleep structure.

Hinoki cypress (Chamaecyparis obtusa) is an evergreen conifer endemic to Japan. The benefits of hinoki fragrance for the central nervous system have garnered increasing attention, and its inhalation is gaining popularity. While hinoki essential oils are available from various manufacturers, the growing regions of the trees and plant parts used for extraction differ among products, and the characteristic benefits of each product are not clearly defined. In this study, we performed gas chromatography with flame ionization detection (GC-FID) and GC-MS analysis on ten commercial hinoki essential oils, one from leaf and nine derived from wood, to examine differences in volatile components between plant parts and among wood-derived samples. The results showed that sabinene, bornyl acetate and α-terpinyl acetate were the major components in leaf-derived oils, while α-pinene and δ-cadinene predominated in wood-derived oils. Among the wood-derived samples, the ratio of α-pinene to δ-cadinene was categorized into three distinct patterns: samples with higher α-pinene content, samples with higher δ-cadinene content, and samples in which both components were present in nearly equal amounts. Differences in chemical composition and the relative proportion of components may influence the physiological effects of hinoki essential oil. Evaluating how these variations affect health benefits could contribute to product quality assessment and help establish tailored applications for each specific chemical profile.

The developing brain establishes mature neural circuits through processes such as neuronal differentiation and synapse formation. However, these processes are susceptible to disruption by external factors, including exposure to environmental chemicals. These disruptions have been associated with neurodevelopmental disorders, such as autism spectrum disorder (ASD) and attention-deficit/hyperactivity disorder (ADHD). This underscores the increasing significance of developmental neurotoxicity (DNT) assessment. However, conventional DNT testing methods are extremely costly and time-consuming, making broad in vivo coverage of the large number of chemicals in use impractical. Therefore, there is an increasing international demand for more efficient, quantitative screening approaches. Bioluminescence imaging (BLI) using reporter transgenic (Tg) mice enables the noninvasive visualization of gene expression and signal transduction in living animals and has been widely applied in fields such as oncology and regenerative medicine. This review highlights recent advances in the application of reporter Tg mice to assess DNT and explores their potential applications in DNT screening, chemical risk assessment, and future research on developmental neurotoxicity.