Wide-scope targeted analysis of bioactive lipids in human plasma by LC/MS/MS.

Quantitative information on blood metabolites can be used in developing advanced medical strategies such as early detection and prevention of disease. Monitoring bioactive lipids such as steroids, bile acids, and PUFA metabolites could be a valuable indicator of health status. However, a method for simultaneously measuring these bioactive lipids has not yet been developed. Here, we report a LC/MS/MS method that can simultaneously measure 144 bioactive lipids, including steroids, bile acids, and PUFA metabolites, from human plasma, and a sample preparation method for these targets. Protein removal by methanol precipitation and purification of bioactive lipids by solid-phase extraction improved the recovery of the targeted compounds in human plasma samples, demonstrating the importance of sample preparation methods for a wide range of bioactive lipid analyses. Using the developed method, we studied the plasma from healthy human volunteers and confirmed the presence of bioactive lipid molecules associated with sex differences and circadian rhythms. The developed method of bioactive lipid analysis can be applied to health monitoring and disease biomarker discovery in precision medicine.

rs12411980 single-nucleotide polymorphism related to PRTFDC1 expression is significantly associated with phantom tooth pain.

Phantom tooth pain (PTP) is one type of non-odontogenic neuropathic toothache, which rarely occurs after appropriate pulpectomy or tooth extraction. The cause of PTP is unknown. We investigated pain-related genetic factors that are associated with PTP. Four pain-associated genes, including G protein-coupled receptor 158 (GPR158) and phosphoribosyl transferase domain containing 1 (PRTFDC1), are adjacent to each other on the human genome. Some of these four genes or their genomic region may be related to PTP. We statistically analyzed associations between single-nucleotide polymorphisms (SNPs) in the genomic region and PTP in patients with PTP (PTP group), other orofacial pain (OFP group), and healthy control subjects. We then performed a database search of expression quantitative trait loci (eQTLs). For the seven SNPs that were significantly associated with PTP even after Bonferroni correction, we focused on the rs12411980 tag SNP (P = 9.42 × 10-4). Statistical analyses of the PTP group and healthy subject groups (group labels: NOC and TD) revealed that the rate of the GG genotype of the rs12411980 SNP was significantly higher in the PTP group than in the healthy subject groups (PTP group vs. NOC group: P = 2.92 × 10-4, PTP group vs. TD group: P = 5.46 × 10-4; percentage of GG: 30% in PTP group, 12% in NOC group, 11% in TD group). These results suggest that the GG genotype of the rs12411980 SNP is more susceptible to PTP. The rs2765697 SNP that is in strong linkage disequilibrium with the rs12411980 SNP is an eQTL that is associated with higher PRTFDC1 expression in the minor allele homozygotes in the healthy subject groups of the rs2765697 SNP. Thus, PRTFDC1 expression similarly increases in the minor allele homozygotes (GG genotype) in the healthy subject groups of the rs12411980 SNP, which would lead to greater susceptibility to PTP.

Loss of the sustained antidepressant-like effect of (2R,6R)-hydroxynorketamine in NMDA receptor GluN2D subunit knockout mice.

Ketamine, an N-methyl-d-aspartate (NMDA) receptor antagonist, has attracted attention for its acute and sustained antidepressant effects in patients with depression. Hydroxynorketamine (HNK), a metabolite of ketamine, exerts antidepressant effects without exerting ketamine's side effects and has attracted much attention in recent years. However, the detailed pharmacological mechanism of action of HNK remains unclear. We previously showed that the GluN2D NMDA receptor subunit is important for sustained antidepressant-like effects of (R)-ketamine. Therefore, we investigated whether the GluN2D subunit is involved in antidepressant-like effects of (2R,6R)-HNK and (2S,6S)-HNK. Treatment with (2R,6R)-HNK but not (2S,6S)-HNK exerted acute and sustained antidepressant-like effects in the tail-suspension test in wildtype mice. Interestingly, sustained antidepressant-like effects of (2R,6R)-HNK were abolished in GluN2D-knockout mice, whereas acute antidepressant-like effects were maintained in GluN2D-knockout mice. When expression levels of GluN2A and GluN2B subunits were evaluated, a decrease in GluN2B protein expression in the nucleus accumbens was found in stressed wildtype mice but not in stressed GluN2D-knockout mice. These results suggest that the GluN2D subunit and possibly the GluN2B subunit are involved in the sustained antidepressant-like effect of (2R,6R)-HNK.

Role of Exchange Cations and Layer Charge on the Dynamics of Confined Water.

Describing the dynamic behavior of water confined in clay minerals is a fascinating challenge and crucial in many research areas, ranging from materials science and geotechnical engineering to environmental sustainability. Water is the most abundant resource on Earth, and the high reactivity of naturally occurring hydrous clay minerals used since prehistoric times for a variety of applications means that water-clay interaction is a ubiquitous phenomenon in nature. We have attempted to experimentally distinguish the rotational dynamics and translational diffusion of two distinct populations of interlayer water, confined and ultraconfined, in the sodium (Na) forms of two smectite clay minerals, montmorillonite (Mt) and hectorite (Ht). Samples hydrated at a pseudo one-layer hydration (1LH) state under ambient conditions were studied with quasi-elastic neutron scattering (QENS) between 150 and 300 K. Using a simplified revised jump-diffusion and rotation-diffusion model (srJRM), we observed that while interlayer water near the ditrigonal cavity in Ht forms strong H-bonds to both adjacent surface O and structural OH, H-bonding of other more prevalent interlayer water with the surface O is weaker compared to Mt, inducing a higher temperature for dynamical changes of confined water. Given the lower layer charge and faster dynamics observed for Ht compared to Mt, we consider this strong evidence confirming the influence of the interlayer cation and surfaces on confined water dynamics.

Mouse Model of Parkinson's Disease with Bilateral Dorsal Striatum Lesion with 6-Hydroxydopamine Exhibits Cognitive Apathy-like Behavior.

Among the symptoms of Parkinson's disease (PD), apathy comprises a set of behavioral, affective, and cognitive features that can be classified into several subtypes. However, the pathophysiology and brain regions that are involved in these different apathy subtypes are still poorly characterized. We examined which subtype of apathy is elicited in a mouse model of PD with 6-hydroxydopamine (6-OHDA) lesions and the behavioral symptoms that are exhibited. Male C57/BL6J mice were allocated to sham (n = 8) and 6-OHDA (n = 13) groups and locally injected with saline or 4 µg 6-OHDA bilaterally in the dorsal striatum. We then conducted motor performance tests and apathy-related behavioral experiments. We then pathologically evaluated tyrosine hydroxylase (TH) immunostaining. The 6-OHDA group exhibited significant impairments in motor function. In the behavioral tests of apathy, significant differences were observed between the sham and 6-OHDA groups in the hole-board test and novelty-suppressed feeding test. The 6-OHDA group exhibited impairments in inanimate novel object preference, whereas social preference was maintained in the three-chamber test. The number of TH+ pixels in the caudate putamen and substantia nigra compacta was significantly reduced in the 6-OHDA group. The present mouse model of PD predominantly showed dorsal striatum dopaminergic neuronal loss and a decrease in novelty seeking as a symptom that is related to the cognitive apathy component.

Single-Nucleotide Polymorphisms of the PAR2 and IL-17A Genes Are Significantly Associated with Chronic Pain.

Patients with chronic pain are affected psychologically and socially. There are also individual differences in treatment efficacy. Insufficient research has been conducted on genetic polymorphisms that are related to individual differences in the susceptibility to chronic pain. Autoimmune disorders can lead to inflammation and chronic pain; therefore, we focused on the autoimmune-related protease-activated receptor 2 (PAR2/F2RL1) and interleukin 17A (IL-17A/IL17A) genes. PAR2 and IL-17A are associated with autoimmune diseases that lead to chronic pain, and PAR2 regulates T-helper (Th) cell activation and differentiation. We hypothesized that the PAR2 and IL-17A genes are associated with chronic pain. The present study used a case-control design to statistically examine associations between genetic polymorphisms and the vulnerability to chronic pain. The rs2243057 polymorphism of the PAR2 gene and rs3819025 polymorphism of the IL-17A gene were previously reported to be associated with pain- or autoimmune-related phenotypes. Thus, these polymorphisms were investigated in the present study. We found that both rs2243057 and rs3819025 were significantly associated with a susceptibility to chronic pain. The present findings revealed autoimmune-related genetic factors that are involved in individual differences in chronic pain, further aiding understanding of the pathomechanism that underlies chronic pain and possibly contributing to future personalized medicine.

Structure, Microheterogeneity, and Transport Properties of Ethaline Decoded by X-ray/Neutron Scattering and MD Simulation.

Ethaline, a deep eutectic solvent (DES) composed of choline chloride (ChCl)-ethylene glycol (EG) in a 1:2 molar ratio, is garnering significant interest for its wide potential applications. The nature of liquid formation and the structure of H-bonds within ethaline were investigated by X-ray scattering (XRS), neutron scattering (NS), and MD simulations. The sum of the dissociation energy barriers of Ch-EG (3.31 kJ·mol-1) and EG-Cl (4.28 kJ·mol-1) exceeds that of Ch-Cl (5.97 kJ·mol-1). This results in a more pronounced solvation of ChCl by EG compared to ChCl association, facilitating the solubilization of ChCl crystals by EG to form a DES. A partial radial distribution function (PDF) reveals that Cl- solvation is dominated by the hydroxyl group of EG, while the methylene group dominates Ch+ solvation. The spatial distribution function (SDF) shows that the distribution of EG and Cl- around Ch+ partially overlaps with that of the quaternary ammonium group. However, the center of mass distance of Ch-Cl (4.95 Å) is significantly lower than that of Ch-EG (5.65 Å), suggesting a favorable advantage for Cl- in this competition. Chain and ring structure distributions provide direct evidence of the microheterogeneity of ethaline. Hydroxyl groups on the EG promote the formation of a chain structure in ethaline, while methylene groups favor a ring structure. H-bond, carbon H-bond, and Cl- bridge bond restrict Cl- diffusion. This new understanding is crucial for a deeper comprehension of the microstructure of ethaline and for elucidating its mechanisms in applications.

Combinatorial characterization of metastable luminous silver cations.

Thermodynamically metastable glasses that can contain metastable species are important functional materials. X-ray absorption near-edge structure (XANES) spectroscopy is an effective technique for determining the valence states of cations, especially for the doping element in phosphors. Herein, we first confirm the valence change of silver cations from monovalent to trivalent in aluminophosphate glasses by X-ray irradiation using a combination of Ag L3-edge XANES, electron spin resonance, and simulated XANES spectra based on first-principles calculations. The absorption edge of the experimental and simulated XANES spectra demonstrate the spectral features of Ag(III), confirming that AgO exists as Ag(I)Ag(III)O2. A part of Ag(I) changes to Ag(III) by X-ray irradiation, and the generation of Ag(III) is saturated after high irradiation doses, in good agreement with conventional radiophotoluminescence (RPL) behaviour. The structural modelling based on a combination of quantum beam analysis suggests that the local coordination of Ag cations is similar to that of Ag(III), which is confirmed by density functional theory calculations. This demonstration of Ag(III) in glass overturns the conventional understanding of the RPL mechanism of silver cations, redefining the science of silver-related materials.

Effect of A118G (rs1799971) single-nucleotide polymorphism of the µ-opioid receptor OPRM1 gene on intraoperative remifentanil requirements in Japanese women undergoing laparoscopic gynecological surgery.

AIM: Abundant data are available on the effect of the A118G (rs1799971) single-nucleotide polymorphism (SNP) of the µ-opioid receptor OPRM1 gene on morphine and fentanyl requirements for pain control. However, data on the effect of this SNP on intraoperative remifentanil requirements remain limited. We investigated the effect of this SNP on intraoperative remifentanil requirements. METHODS: We investigated 333 Japanese women, aged 21-69 years, who underwent laparoscopic gynecological surgery for benign gynecological disease under total intravenous anesthesia at Juntendo University Hospital. Average infusion rates of propofol and remifentanil during anesthesia and the average bispectral index (BIS) during surgery were recorded. Associations among genotypes of the A118G and phenotypes were examined with the Mann-Whitney U test. RESULTS: The average propofol infusion rate was not different between patients with different genotypes. The average remifentanil infusion rate was significantly higher in patients with the AG or GG genotype than the AA genotype (p = 0.028). The average intraoperative BIS was significantly higher in patients with the GG genotype than the AA or AG genotype (p = 0.039). CONCLUSIONS: The G allele of the A118G SNP was associated with higher intraoperative remifentanil requirements and higher intraoperative BIS values but was not associated with propofol requirements. Given that remifentanil and propofol act synergistically on the BIS, these results suggest that the G allele of the A118G SNP is associated with lower effects of remifentanil in achieving adequate intraoperative analgesia and in potentiating the sedative effect of propofol on the BIS.

The integration of metabolic and proteomic data uncovers an augmentation of the sphingolipid biosynthesis pathway during T-cell differentiation.

Recent studies have highlighted the significance of cellular metabolism in the initiation of clonal expansion and effector differentiation of T cells. Upon exposure to antigens, naïve CD4+ T cells undergo metabolic reprogramming to meet their metabolic requirements. However, only few studies have simultaneously evaluated the changes in protein and metabolite levels during T cell differentiation. Our research seeks to fill the gap by conducting a comprehensive analysis of changes in levels of metabolites, including sugars, amino acids, intermediates of the TCA cycle, fatty acids, and lipids. By integrating metabolomics and proteomics data, we discovered that the quantity and composition of cellular lipids underwent significant changes in different effector Th cell subsets. Especially, we found that the sphingolipid biosynthesis pathway was commonly activated in Th1, Th2, Th17, and iTreg cells and that inhibition of this pathway led to the suppression of Th17 and iTreg cells differentiation. Additionally, we discovered that Th17 and iTreg cells enhance glycosphingolipid metabolism, and inhibition of this pathway also results in the suppression of Th17 and iTreg cell generation. These findings demonstrate that the utility of our combined metabolomics and proteomics analysis in furthering the understanding of metabolic transition during Th cell differentiation.

Comparative studies of hair shaft components between healthy and diseased donors.

Globally, the rapid aging of the population is predicted to become even more severe in the second half of the 21st century. Thus, it is expected to establish a growing expectation for innovative, non-invasive health indicators and diagnostic methods to support disease prevention, care, and health promotion efforts. In this study, we aimed to establish a new health index and disease diagnosis method by analyzing the minerals and free amino acid components contained in hair shaft. We first evaluated the range of these components in healthy humans and then conducted a comparative analysis of these components in subjects with diabetes, hypertension, androgenetic alopecia, major depressive disorder, Alzheimer's disease, and stroke. In the statistical analysis, we first used a student's t test to compare the hair components of healthy people and those of patients with various diseases. However, many minerals and free amino acids showed significant differences in all diseases, because the sample size of the healthy group was very large compared to the sample size of the disease group. Therefore, we attempted a comparative analysis based on effect size, which is not affected by differences in sample size. As a result, we were able to narrow down the minerals and free amino acids for all diseases compared to t test analysis. For diabetes, the t test narrowed down the minerals to 15, whereas the effect size measurement narrowed it down to 3 (Cr, Mn, and Hg). For free amino acids, the t test narrowed it down to 15 minerals. By measuring the effect size, we were able to narrow it down to 7 (Gly, His, Lys, Pro, Ser, Thr, and Val). It is also possible to narrow down the minerals and free amino acids in other diseases, and to identify potential health indicators and disease-related components by using effect size.

Genome-wide DNA methylation risk scores for schizophrenia derived from blood and brain tissues further explain the genetic risk in patients stratified by polygenic risk scores for schizophrenia and bipolar disorder.

BACKGROUND: Genetic and environmental factors contribute to the pathogenesis of schizophrenia (SZ) and bipolar disorder (BD). Among genetic risk groups stratified by combinations of Polygenic Risk Score (PRS) deciles for SZ, BD and SZ versus BD, genetic SZ risk groups had high SZ risk and prominent cognitive impairments. Furthermore, epigenetic alterations are implicated in these disorders. However, it was unclear whether DNA Methylation Risk Scores (MRSs) for SZ risk derived from blood and brain tissues were associated with SZ risk, particularly the PRS-stratified genetic SZ risk group. METHODS: Epigenome-wide association studies (EWASs) of SZ risk in whole blood were preliminarily conducted between 66 SZ patients and 30 healthy controls (HCs) and among genetic risk groups (individuals with low genetic risk for SZ and BD in HCs (n=30) and in SZ patients (n=11), genetic BD risk in SZ patients (n=25) and genetic SZ risk in SZ patients (n=30)) stratified by combinations of PRSs for SZ, BD and SZ versus BD. Next, differences in MRSs based on independent EWASs of SZ risk in whole blood, postmortem frontal cortex (FC) and superior temporal gyrus (STG) were investigated among our case‒control and PRS-stratified genetic risk status groups. RESULTS: Among case‒control and genetic risk status groups, 33 and 351 genome-wide significant differentially methylated positions (DMPs) associated with SZ were identified, respectively, many of which were hypermethylated. Compared with the low genetic risk in HCs group, the genetic SZ risk in SZ group had 39 genome-wide significant DMPs, while the genetic BD risk in SZ group had only six genome-wide significant DMPs. The MRSs for SZ risk derived from whole blood, FC and STG were higher in our SZ patients than in HCs in whole blood and were particularly higher in the genetic SZ risk in SZ group than in the low genetic risk in HCs and genetic BD risk in SZ groups. Conversely, the MRSs for SZ risk based on our whole-blood EWASs among genetic risk groups were also associated with SZ in the FC and STG. There were no correlations between the MRSs and PRSs. CONCLUSIONS: These findings suggest that the MRS is a potential genetic marker in understanding SZ, particularly in patients with a genetic SZ risk.

Sclerostin modulates mineralization degree and stiffness profile in the fibrocartilaginous enthesis for mechanical tissue integrity.

Fibrocartilaginous entheses consist of tendons, unmineralized and mineralized fibrocartilage, and subchondral bone, each exhibiting varying stiffness. Here we examined the functional role of sclerostin, expressed in mature mineralized fibrochondrocytes. Following rapid mineralization of unmineralized fibrocartilage and concurrent replacement of epiphyseal hyaline cartilage by bone, unmineralized fibrocartilage reexpanded after a decline in alkaline phosphatase activity at the mineralization front. Sclerostin was co-expressed with osteocalcin at the base of mineralized fibrocartilage adjacent to subchondral bone. In Scx-deficient mice with less mechanical loading due to defects of the Achilles tendon, sclerostin+ fibrochondrocyte count significantly decreased in the defective enthesis where chondrocyte maturation was markedly impaired in both fibrocartilage and hyaline cartilage. Loss of the Sost gene, encoding sclerostin, elevated mineral density in mineralized zones of fibrocartilaginous entheses. Atomic force microscopy analysis revealed increased fibrocartilage stiffness. These lines of evidence suggest that sclerostin in mature mineralized fibrochondrocytes acts as a modulator for mechanical tissue integrity of fibrocartilaginous entheses.