Hepatic glucuronidation of tetrabromobisphenol A and tetrachlorobisphenol A: interspecies differences in humans and laboratory animals and responsible UDP-glucuronosyltransferase isoforms in humans.

Tetrabromobisphenol A (TBBPA) and tetrachlorobisphenol A (TCBPA), bisphenol A (BPA) analogs, are endocrine-disrupting chemicals predominantly metabolized into glucuronides by UDP-glucuronosyltransferase (UGT) enzymes in humans and rats. In the present study, TBBPA and TCBPA glucuronidation by the liver microsomes of humans and laboratory animals (monkeys, dogs, minipigs, rats, mice, and hamsters) and recombinant human hepatic UGTs (10 isoforms) were examined. TBBPA glucuronidation by the liver microsomes followed the Michaelis-Menten model kinetics in humans, rats, and hamsters and the biphasic model in monkeys, dogs, minipigs, and mice. The CLint values based on the Eadie-Hofstee plots were mice (147) > monkeys (122) > minipigs (108) > humans (100) and rats (98) > dogs (81) > hamsters (47). TCBPA glucuronidation kinetics by the liver microsomes followed the biphasic model in all species except for minipigs, which followed the Michaelis-Menten model. The CLint values were monkeys (172) > rats (151) > mice (134) > minipigs (104), dogs (102), and humans (100) > hamsters (88). Among recombinant human UGTs examined, UGT1A1 and UGT1A9 showed higher TBBPA and TCBPA glucuronidation abilities. The kinetics of TBBPA and TCBPA glucuronidation followed the substrate inhibition model in UGT1A1 and the Michaelis-Menten model in UGT1A9. The CLint values were UGT1A1 (100) > UGT1A9 (42) for TBBPA glucuronidation and UGT1A1 (100) > UGT1A9 (53) for TCBPA glucuronidation, and the activities at high substrate concentration ranges were higher in UGT1A9 than in UGT1A1 for both TBBPA and TCBPA. These results suggest that the glucuronidation abilities toward TBBPA and TCBPA in the liver differ extensively across species, and that UGT1A1 and UGT1A9 expressed in the liver mainly contribute to the metabolism and detoxification of TBBPA and TCBPA in humans.

Development and psychometric validation of an educational support assessment scale for novice home healthcare nurses.

BACKGROUND: Educational support quality is an essential factor in determining the education of novice home healthcare nurses. This study developed a scale to assess the educational support provided by home healthcare agencies among novice home healthcare nurses. METHODS: Hypothetical components were derived from a literature review, including experiential learning theory. Expert panels evaluated the initial scale items, and the scale was tested with 3000 agencies from April to June 2022. A total of 627 valid responses were analyzed. RESULTS: Exploratory factor analysis produced a four-subscale structure consisting of 34 items that supported the hypothesized components. Cronbach's alphas ranged 0.889 to 0.961, and the intraclass correlation coefficients ranged 0.703 to 0.905 in the test-retest survey. CONCLUSIONS: The educational support assessment scale developed for novice home healthcare nurses is valid and reliable. Managers in home healthcare agencies should apply the results of assessments using the scale to improve their human resource development.

Simultaneous Screening of SARS-CoV-2 Omicron and Delta Variants Using High-Resolution Melting Analysis.

A novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strain, the Omicron variant (Pango lineage B.1.1.529), was identified in South Africa in late September 2021. This variant has multiple spike protein deletions and mutations, with 15 amino acid substitutions detected in the receptor-binding domain (RBD). These RBD substitutions are hypothesized to increase infectivity and reduce antibody affinity, which is supported by recent data showing that the Omicron variant spreads faster than the Delta variant (Pango lineage B.1.617.2). Thus, this increase in infectivity should lead to Omicron being the dominant variant and developing screening tests that discriminate between Omicron and Delta variants is urgently needed. In this study, we successfully developed a novel screening assay using high-resolution melting analysis, in which two genotypes at G446/L452 and S477/T478 RBD were determined (G446S/L452 and S477N/T478K for Omicron; G446/L452R and S477/T478K for Delta). Using synthetic DNA fragments, we confirmed both melting point and melting peak shape of the RBD Omicron variant was distinguishable from those of wild-type and the Delta variant. Although this study was conducted without clinical samples, these results suggest that our high-resolution melting (HRM)-based genotyping method can readily identify the Omicron and Delta variants. This simple method should contribute to the rapid identification of SARS-CoV-2 variants and thus prevent potential widespread infection and inflow of the Omicron variant.

Species-Specific Activation of Transient Receptor Potential Ankyrin 1 by Phthalic Acid Monoesters.

Phthalic acid (PA) diesters are widely used in consumer products, as plasticizers, and are ubiquitous environmental pollutants. There is a growing concern about their adjuvant effect on allergic diseases. Although its precise mechanism remains unknown, possible involvement of transient receptor potential ankyrin 1 (TRPA1) has been suggested. Hence, in this study, the activation of human and mouse TRPA1s by a series of PA di- and monoesters was investigated using a heterologous expression system in vitro. Consequently, it was found that monoesters activated human TRPA1, where EC50 values were in the order of mono-hexyl > mono-heptyl > mono-n-octyl > mono-2-ethylhexyl > mono-isononyl and mono-isodecyl esters. Significant species differences in TRPA1 activation by PA monoesters were also discovered; PA monoesters activated human TRPA1 but not mouse TRPA1 in a concentration-dependent manner up to 50 µM. These findings suggest that PA esters may exert TRPA1-dependent adverse effects on humans, which have never been demonstrated in experimental animals.

Regioselective Glucuronidation of Flavones at C5, C7, and C4' Positions in Human Liver and Intestinal Microsomes: Comparison among Apigenin, Acacetin, and Genkwanin.

Flavones, which are distributed in a variety of plants and foods in nature, possess significant biological activities, including antitumor and anti-inflammatory effects, and are metabolized into glucuronides by uridine 5'-diphosphate (UDP)-glucuronosyltransferase (UGT) enzymes in humans. In this study, apigenin, acacetin, and genkwanin, flavones having hydroxyl groups at C5, C7, and/or C4'positions were focused on, and the regioselective glucuronidation in human liver and intestinal microsomes was examined. Two glucuronides (namely, AP-7G and AP-4'G for apigenin, AC-5G and AC-7G for acacetin, and GE-5G and GE-4'G for genkwanin) were formed from each flavone by liver and intestinal microsomes, except for only GE-4'G formation from genkwanin by intestinal microsomes. The order of total glucuronidation activities was liver microsomes > intestinal microsomes for apigenin and acacetin, and liver microsomes < intestinal microsomes for genkwanin. The order of CLint values (x-intercept) based on v versus V/[S] plots for apigenin glucuronidation was AP-7G > AP-4'G in liver microsomes and AP-7G < AP-4'G in intestinal microsomes. The order of CLint values was AC-5G < AC-7G for acacetin and GE-5G < GE-4'G genkwanin glucuronidation in both liver and intestinal microsomes. This suggests that the abilities and roles of UGT enzymes in the glucuronidation of apigenin, acacetin, and genkwanin in humans differ depending on the chemical structure of flavones.

Isoform-Specific Quantification of Human Bitter Taste Receptor Transcripts Using Real-Time PCR Analysis.

Bitter taste receptors (TAS2Rs) are expressed by oral cavity cells in mammals and classically function as sensors for bitter compounds. There are 25 functional isoforms of human TAS2Rs, with individual bitter ligands. Each human TAS2R isoform is distributed in several tissues, such as the airway epithelia and gastrointestinal tract, and plays an important role in physiological functions. However, quantification of each isoform is difficult because of highly homologous sequences between some TAS2R isoforms. Therefore, differentiating the isoforms by their expression levels is suitable for clarifying the tissue-specific effects of bitter compounds. In this study, we developed a real-time quantitative PCR (qPCR) method to determine the expression of each TAS2R isoform. Using plasmid standards harboring each isoform, we confirmed that the current assay can quantify the gene expression of each isoform, with negligible interference from other isoforms. In addition, our methods can successfully discriminate between the mRNA expression of each isoform in human cell lines and tissues. Therefore, this qPCR method can successfully quantify the mRNA level of each TAS2R isoform. This method will contribute to a better understanding of the molecular mechanisms underlying the TAS2R ligand-activated signal transduction.

Development of a genotyping platform for SARS-CoV-2 variants using high-resolution melting analysis.

INTRODUCTION: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel coronavirus causing coronavirus disease 2019 (COVID-19), has been expanding globally since late 2019. SARS-CoV-2, an RNA virus, has a genome sequence that can easily undergo mutation. Several mutated SARS-CoV-2 strains, including those with higher infectivity than others, have been reported. To reduce SARS-CoV-2 transmission, it is crucial to trace its infection sources. Here, we developed a simple, easy-to-use genotyping method to identify SARS-CoV-2 variants using a high-resolution melting (HRM) analysis. METHODS: We investigated five mutation sites, A23403G, G25563T, G26144T, T28144C, and G28882A, which are known strain determinants according to GISAID clades (L, S, V, G, GH, and GR). RESULTS: We first employed synthetic DNA fragments containing the five characteristic sites for HRM analysis. All sequences clearly differentiated wild-type from mutant viruses. We then confirmed that RNA fragments were suitable for HRM analysis following reverse transcription. Human saliva did not negatively affect the HRM analysis, which supports the absence of a matrix effect. CONCLUSIONS: Our results indicate that this HRM-based genotyping method can identify SARS-CoV-2 variants. This novel assay platform potentially paves the way for accurate and rapid identification of SARS-CoV-2 infection sources.

[Analysis of Cyanide Compounds in Foods by the Purge Method Using a Midget Impinger].

Cyanogenic glycosides in loquat (Eriobotrya japonica) seeds, which are used in so-called health foods, pose a public concern in Japan due to their potential health risks. Several pretreatment methods, such as the steam distillation and Conway microdiffusion methods, have been established for the determination of cyanogenic glycoside concentrations in foods. However, these methods are time-consuming and have extremely low throughput. Therefore, we developed a simple and rapid method, called the purge method, to analyze cyanide compounds in seed-derived food products. Under this method, the aqueous extract of cyanogenic glycosides is treated with ß-glucosidase in a midget impinger, after which the liberated cyanide is purged into an absorbing solution. The concentration of cyanide in the adsorbent is then quantified using 4-pyridinecarboxylic acid-pyrazolone reagent. A single-laboratory method validation study was performed using amygdalin at a concentration of 10 ppm as cyanide ion. The validation parameter results (trueness, 83.9%; repeatability, 1.18%; intermediate precision, 4.67%) indicated that the developed method was suitable, precise and accurate. The purge method was used to analyze cyanide concentrations in commercially available food samples. Of the 10 samples tested (loquat seed powder, apricot kernel powder, and plum seed powder), three samples were found to contain cyanogenic glycosides at concentrations of >10 ppm as hydrogen cyanide, with the highest concentration detected being 861 ppm. These results clearly demonstrated the applicability of our method in determining cyanogenic glycosides in seed-derived food samples.

A Phosphonium Ylide as a Ligand for [3 + 2] Coupling Reactions of Epoxides with Heterocumulenes under Mild Conditions.

The potential of carbonyl-stabilized phosphonium ylides as ligands for novel catalysis was explored. We found that the combination of phosphonium ylides and metal halide salts efficiently catalyzed the reaction of epoxides with carbon dioxide under mild conditions. Five-membered cyclic carbonates, including disubstituted cyclic carbonates, were obtained in good yields with the use of 1 atm of carbon dioxide at 35 °C. Terminal epoxides could be converted to N-aryl oxazolidinones in the reaction with isocyanates under a similar catalytic system.

Development of demyelinating lesions in progressive multifocal leukoencephalopathy (PML): Comparison of magnetic resonance images and neuropathology of post-mortem brain.

Progressive multifocal leukoencephalopathy (PML) is a demyelinating disorder caused by opportunistic infection of JC polyomavirus (JCV). Today, increased attention has been focused on PML development in multiple sclerosis (MS) patients under disease-modifying therapies (DMT). Although in the acquired immunodeficiency syndrome (AIDS) era, PML was thought to be a rapidly progressive disease with poor prognosis, drug-associated PML is relatively slow in progress, and a favorable outcome may be expected with early diagnosis. However, early PML diagnosis on magnetic resonance imaging (MRI) is frequently difficult, and JCV DNA copy number in cerebrospinal fluid (CSF) is usually low. To facilitate early PML diagnosis on MRI, the pre-mortem images were compared with neuropathology of the post-mortem brain, and underlying pathology corresponding to the MRI findings was evaluated. As a result, PML lesions of the autopsied brain were divided into three parts, based on the disease extension patterns: (A) Progressive white matter lesion in the right frontoparietal lobe including the precentral gyrus. Huge demyelinated lesions were formed with fusions of numerous small lesions. (B) Central lesion including deep gray matters, such as the putamen and thalamus. The left thalamic lesion was contiguous with the pontine tegmentum. (C) Infratentorial lesion of brainstem and cerebellum. Demyelination in the pontine basilar region and in cerebellar white matter was contiguous via middle cerebellar peduncles (MCPs). In addition, (D) satellite lesions were scattered all over the brain. These observations indicate that PML lesions likely evolve with three steps in a tract-dependent manner: (1) initiation; (2) extension/expansion of demyelinating lesions; and (3) fusion. Understanding of the PML disease evolution patterns would enable confident early diagnosis on MRI, which is essential for favorable prognosis with good functional outcome.

Frontotemporal lobar degeneration due to P301L tau mutation showing apathy and severe frontal atrophy but lacking other behavioral changes: A case report and literature review.

The clinical features in cases that have mutations in the microtubule-associated protein tau gene but lack prominent behavioral changes remain unclear. Here, we describe detailed clinical and pathological features of a case carrying the P301L tau mutation that showed only apathy until the middle stage of the course. The mother of this case was suspected to have mild cognitive decline at age 46. However, before she was fully examined, she had a subarachnoid hemorrhage at age 49 and died at age 53. An autopsy was not done. The proband of this pedigree, a 60-year-old right-handed Japanese man at the time of death, began to make mistakes at work at the age of 51 years. Until age 54, he showed only mild apathy with bradykinesia. Insight was well spared. Parkinsonism and echolalia developed at age 55, and pyramidal signs and oral tendency at age 57. Personality change, disinhibition, stereotypy, or semantic memory impairment was not found throughout the course. The final neurological diagnosis was unspecified dementia. Pathological examination demonstrated numerous round four-repeat tau-positive three-repeat tau-negative or perinuclear ring-like neuronal cytoplasmic inclusions with many ballooned neurons in the frontal and temporal cortices and hippocampus. Genetic analysis using frozen brain tissue demonstrated a P301L tau mutation. Among 31 previously reported cases bearing the P301L tau mutation for which the data regarding initial symptoms are available, one clinical case showed only apathy with depression in the early stage. Given these findings, clinicians should be aware that a clinical course characterized only by apathy for several years, which can be misdiagnosed as a psychiatric disorder, is one of the clinical presentations associated with P301L tau mutation.

MEF2C mRNA expression and cognitive function in Japanese patients with Alzheimer's disease.

AIM: Despite continuing research into Alzheimer's disease (AD), its pathological mechanisms and modulating factors remain unknown. Several genes influence AD pathogenesis by affecting inflammatory pathways. Myocyte-enhancer factor 2C (MEF2C) is one such candidate gene for AD. METHODS: We examined MEF2C mRNA expression levels and methylation rates of CpG on its promoter region in peripheral leukocytes from Japanese AD patients compared with age- and sex-matched control subjects. RESULTS: In peripheral leukocytes, MEF2C mRNA expression levels in AD subjects were significantly lower than those in control subjects (0.86 ± 0.25 vs 0.99 ± 0.27, respectively, P = 0.007) and were correlated with the Alzheimer's Disease Assessment Scale (r = -0.345, P = 0.049) and the Mini Mental State Examination (r = 0.324, P = 0.02). No significant differences were found in methylation rates between AD and control subjects. CONCLUSION: MEF2C mRNA expression in leukocytes may be a biological marker for cognitive decline in AD.

Structural stability of flagellin subunit affects the rate of flagellin export in the absence of FliS chaperone.

FliS chaperone binds to flagellin FliC in the cytoplasm and transfers FliC to a sorting platform of the flagellar type III export apparatus through the interaction between FliS and FlhA for rapid and efficient protein export during flagellar filament assembly. FliS also suppresses the secretion of an anti-σ factor, FlgM. Loss of FliS results in a short filament phenotype although the expression levels of FliC are increased considerably due to an increase in the secretion level of FlgM. Here to clarify the rate limiting step of FliC export in the absence of FliS, we isolated bypass mutants from a Salmonella ΔfliS mutant. All the bypass mutations were identified in FliC. These bypass mutations increased the export rate of FliC by ca. twofold, allowing the bypass mutant cells to produce longer filaments than the parental ΔfliS cells. Both far-UV CD measurements and limited proteolysis revealed that the bypass mutations significantly destabilize the folded structure of FliC monomer. These results suggest that an unfolding step of FliC limits the export rate of FliC in the ΔfliS mutant, thereby producing short filaments. We propose that FliS promotes FliC docking at the FlhA platform to facilitate subsequent unfolding of FliC.

Pick's disease with neuronal four-repeat tau accumulation in the basal ganglia, brain stem nuclei and cerebellum.

It is very rare that cases of Pick's disease, a representative three-repeat (3R) tauopathy, also have significant four-repeat (4R) tau accumulation. Here, we report a Pick's disease case that clinically showed behavioral variant frontotemporal dementia without motor disturbance during the course, and pathologically had 3R tau-positive Pick bodies as well as numerous 4R tau-positive neuronal cytoplasmic inclusions (NCIs). Abundant 3R tau-positive 4R tau-negative spherical or horseshoe-shaped Pick bodies were found in the frontotemporal cortex, limbic region, striatum and pontine nucleus. On the other hand, many 4R tau-positive, 3R tau-negative, Gallyas-negative dot-, rod- or intertwined skein-like NCIs were found mainly in the subthalamic nucleus, pontine nucleus, inferior olivary nucleus and cerebellar dentate nucleus. Tufted astrocytes, astrocytic plaques, argyrophilic grains or globular glial inclusions were absent. Double-labeling immunofluorescence demonstrated that 3R tau was hardly accumulated in 4R tau-positive inclusions. On tau immunoblotting, while 60 and 64 kDa bands were demonstrated in the frontal cortex, 60, 64 and 68 kDa bands, as well as the 33 kDa tau fragments that are reported to be characteristic of progressive supranuclear palsy brains, were found in the basal ganglia and cerebellum. No mutation was identified in the tau gene. The present case suggests that, although probably rare, some Pick's disease cases have non-negligible 4R tau pathology in the subcortical nuclei, and that such 4R tau pathology can affect the evaluation of the distribution of AT8-positive tau pathology in Pick's disease cases.

DNA methylation changes at SNCA intron 1 in patients with dementia with Lewy bodies.

AIM: It is difficult to diagnose dementia with Lewy bodies (DLB) because it exhibits clinical and neuropathological overlap with both Alzheimer's disease and Parkinson's disease. The α-synuclein protein is a major component of Lewy bodies, and accumulation of α-synuclein aggregates causes synaptic dysfunction in DLB. Epigenetic changes at the synuclein alpha ( SNCA ) gene may be involved in DLB pathogenesis. METHODS: We examined DNA methylation rates at 10 CpG sites located in intron 1 of SNCA and SNCA mRNA expression in peripheral leukocytes to compare DLB patients (n = 20; nine men, 11 women; age = 78.8 ± 7.7 years) with healthy controls (n = 20; eight men, 12 women; age = 77.0 ± 6.9 years). RESULTS: The methylation rate at CpG 4 ( P = 0.002) and the overall mean methylation rate at these sites (P < 0.001) were significantly lower in DLB patients than in healthy controls after Bonferroni correction. Although SNCA126 , a partial form of SNCA mRNA expression, was significantly increased in DLB ( P = 0.017), there was no significant difference in total SNCA mRNA expression between DLB patients and healthy controls ( P = 0.165). No correlation was observed between SCNA mRNA expression levels and blood DNA methylation rates in either DLB or healthy controls. CONCLUSION: Our findings indicated that lower methylation rates may be a biomarker for DLB.

Paravertebral block catheter breakage by electrocautery during thoracic surgery.

Advantages of thoracic paravertebral analgesia (TPA) include placement of the catheter closer to the surgical field; however, the catheter can become damaged during the operation. We experienced a case of intraoperative TPA catheter breakage that prompted us to perform an experiment to investigate possible causes. A 50-year-old male underwent a thoracoscopic lower lobectomy under general anesthesia with TPA via an intercostal approach. Following surgery, it was discovered that the catheter had become occluded, as well as cut and fused, so we reopened the incision and removed the residual catheter. From that experience, we performed an experiment to examine electrocautery-induced damage in normal (Portex™, Smith's Medical), radiopaque (Perifix SoftTip™, BBraun), and reinforced (Perifix FX™, BBraun) epidural catheters (n = 8 each). Chicken meat was penetrated by each catheter and then cut by electrocautery. In the normal group, breakage occurred in 8 and occlusion in 6 of the catheters, and in the radiopaque group breakage occurred in 8 and occlusion in 7. In contrast, breakage occurred in only 3 and occlusion in none in the reinforced group, with the 5 without breakage remaining connected only by the spring coil. Furthermore, in 7 of the reinforced catheters, electric arc-induced thermal damage was observed at the tip of the catheter. A TPA catheter for thoracic surgery should be inserted via the median approach, or it should be inserted after surgery to avoid catheter damage during surgery.

Elevated TREM2 mRNA expression in leukocytes in schizophrenia but not major depressive disorder.

The pathological mechanisms of schizophrenia (SCZ) have not been clarified, but the microglia hypothesis has recently been discussed. We previously reported that the mRNA for a protein related to activation of microglia, triggering receptor expressed on myeloid cell 2 (TREM2), is expressed higher in peripheral leukocytes in SCZ than controls. In this study, we analyzed TREM2 mRNA expression in leukocytes from both SCZ and major depressive disorder (MDD) patients. We compared 50 SCZ patients and 42 MDD patients with age-matched controls. Levels of TREM2 mRNA in leukocytes were analyzed with quantitative real-time PCR method using TaqMan probe. TREM2 mRNA expression was significantly higher in leukocytes of SCZ subjects than controls, but the expression level was non-significantly different in MDD subjects. We observed a decrease in TREM2 mRNA expression in leukocytes from one SCZ patient after clozapine treatment. The expression did not change following ECT, but the expression level in this patient was still significantly higher than that in controls. We conclude that the high amount of TREM2 mRNA expression in leukocytes is specific to SCZ but not MDD and that changes in TREM2 mRNA expression may be a trait biomarker for SCZ.

Association Study and Meta-Analysis of Polymorphisms, Methylation Profiles, and Peripheral mRNA Expression of the Serotonin Transporter Gene in Patients with Alzheimer's Disease.

BACKGROUND/AIM: The aim of this study was to elucidate the relationship between Alzheimer's disease (AD) and the serotonin transporter gene (SLC6A4). METHODS: AD subjects (n = 43) and controls (n = 47) were recruited and evaluated. In leukocytes, we evaluated two polymorphisms in SLC6A4, the serotonin transporter length polymorphic region (5-HTT-LPR) and rs25531, as well as methylation rates of the SLC6A4 promoter region and the SLC6A4 mRNA expression level. We also performed a meta-analysis to examine the relationship between the frequency of the L allele and the risk of AD. RESULTS: The distributions of 5-HTT-LPR and rs25531 polymorphisms in AD subjects were not different from those of controls. Although the methylation rates in AD subjects were not significantly different from those of controls, the expression level in AD subjects was significantly higher than in controls. Additionally, the expression level in AD subjects was significantly correlated with apathy. Meta-analysis revealed that the L/L genotype significantly reduced the risk of AD, but only in the Caucasian population. CONCLUSION: Higher SLC6A4 mRNA expression in leukocytes in AD was associated with apathy regardless of SLC6A4 genotypes and methylation rates of the promoter region. The L/L genotype may reduce the risk of AD in the Caucasian population.

No abnormal hexanucleotide repeat expansion of C9ORF72 in Japanese schizophrenia patients.

Abnormal hexanucleotide repeat expansion of C9ORF72 is known to cause neurodegenerative disorders such as frontotemporal dementia. Additionally, patients with psychotic symptoms are more likely to have abnormal hexanucleotide repeat expansion than are patients without them. We investigated the hexanucleotide repeat sizes of C9ORF72 in 466 Japanese schizophrenia patients. We found no abnormal hexanucleotide repeat expansion. In conclusion, C9ORF72 may not be responsible for schizophrenia susceptibility in the Japanese population.