Unveiling the vitamin E profile in rice bran extracellular vesicles: evaluation of extraction and preparation methods.

Extracellular vesicles (EVs) are nanoscale entities secreted by various cells, encapsulating various nucleic acids and proteins that play important roles in cellular activities. Although rice bran is known for its richness in phytochemicals such as tocopherol and tocotrienol, the distribution of these compounds within EVs has not been extensively studied. The objective of this study was to detect and analyze the presence of vitamin E in EVs extracted from rice bran. We investigated several EV extraction methods, including rotation, vortex mixing, and ultrasonication, followed by post-extraction techniques such as ultracentrifugation, ultrafiltration, and lyophilization. Vitamin E in the EVs from rice bran was analyzed using LC-FLD. This study is the first to identify tocopherol and tocotrienol in rice bran-derived EVs. Our results indicate that ultracentrifugation followed by rotation is the most effective method for the preparation of rice bran-derived EVs. Notably, the vitamin E profile in EVs varies depending on the preparation method and differs from that in rice bran extracts. The pronounced presence of vitamin E in EVs suggests unique pharmacokinetics and underscores the potential of EVs as carriers for drug delivery systems. This study not only confirms the presence of vitamin E in EVs, but also underscores the potential of EVs and their phytochemical content for therapeutic applications.

Enzymatic and selective production of alkyl α-d-glucopyranosides by the α-glucosyl transfer enzyme derived from Xanthomonas campestris WU-9701.

Several alkyl glucosides exhibit various bioactivities. 1-Octyl ß-d-glucopyranoside produced by organic synthesis is used as a nonionic surfactant. However, no convenient method has been developed for the selective production of alkyl α-glucosides (α-AGs), such as 1-octyl α-d-glucopyranoside (α-OG). Therefore, we developed a simple method for selective production of α-AGs using the glucosyl transfer enzyme XgtA, (E.C. 3.2.1.20), derived from Xanthomonas campestris WU-9701. When 0.80 M alkyl alcohol and 2.5 units XgtA were incubated in 2.0 mL of 30 mM HEPES-NaOH buffer (pH 8.0) containing 1.2 M maltose at 45 °C, a specific α-AG corresponding to each alkyl alcohol (C2-C10) was detected. Under the standard conditions, we examined the selective production of α-OG from 1-octanol and maltose using XgtA. The reaction product was isolated and identified as α-OG via 1H nuclear magnetic resonance and nuclear overhauser effect spectroscopy analyses. No other glucosylated products, such as maltotriose, were detected in the reaction mixture. Under the standard conditions at 45 °C for 96 h, 243 mM α-OG (71 g/L) was produced in one batch production. Moreover, the addition of glucose isomerase to the reaction mixture decreased the concentration of glucose released via the reaction and increased the amount of α-OG produced; 359 mM α-OG (105 g/L) was maximally produced at 96 h. In conclusion, this study demonstrates the selective production of α-AGs using a simple enzymatic reaction, and XgtA has the potential to selectively produce various α-AGs.

Identification of epigenetically active L1 promoters in the human brain and their relationship with psychiatric disorders.

Long interspersed nuclear element-1 (LINE-1, L1) affects the transcriptome landscape in multiple ways. Promoter activity within its 5'UTR plays a critical role in regulating diverse L1 activities. However, the epigenetic status of L1 promoters in adult brain cells and their relationship with psychiatric disorders remain poorly understood. Here, we examined DNA methylation and hydroxymethylation of the full-length L1s in neurons and nonneurons and identified "epigenetically active" L1s. Notably, some of epigenetically active L1s were retrotransposition competent, which even had chimeric transcripts from the antisense promoters at their 5'UTRs. We also identified differentially methylated L1s in the prefrontal cortices of patients with psychiatric disorders. In nonneurons of bipolar disorder patients, one L1 was significantly hypomethylated and showed an inverse correlation with the expression level of the overlapping gene NREP. Finally, we observed that altered DNA methylation levels of L1 in patients with psychiatric disorders were not affected by the surrounding genomic regions but originated from the L1 sequences. These results suggested that altered epigenetic regulation of the L1 5'UTR in the brain was involved in the pathophysiology of psychiatric disorders.

Diagnostic and prognostic biomarkers for progressive fibrosing interstitial lung disease.

No biomarkers have been identified in bronchoalveolar lavage fluid (BALF) for predicting fibrosis progression or prognosis in progressive fibrosing interstitial lung disease (PF-ILD). We investigated BALF biomarkers for PF-ILD diagnosis and prognosis assessment. Overall, 120 patients with interstitial pneumonia who could be diagnosed with PF-ILD or non PF-ILD were enrolled in this retrospective study. PF-ILD was diagnosed according to Cottin's definition. All patients underwent bronchoscopy and BALF collection. We evaluated blood and BALF parameters, high-resolution computed tomography (HRCT) patterns, and spirometry data to identify factors influencing PF-ILD diagnosis and prognosis. On univariate logistic analysis, age, sex, the BALF white blood cell fraction (neutrophil, lymphocyte, eosinophil, and neutrophil-to-lymphocyte ratio), BALF flow cytometric analysis (CD8), and an idiopathic pulmonary fibrosis/usual interstitial pneumonia pattern on HRCT were correlated with PF-ILD diagnosis. Multivariate logistic regression analysis revealed that sex (male), age (cut-off 62 years, area under the curve [AUC] 0.67; sensitivity 0.80; specificity 0.47), white blood cell fraction in BALF (NLR, neutrophil, and lymphocyte), and CD8 in BALF (cut-off 34.2; AUC 0.66; sensitivity, 0.74; specificity, 0.62) were independent diagnostic predictors for PF-ILD. In BALF, the NLR (cut-off 8.70, AUC 0.62; sensitivity 0.62; specificity 0.70), neutrophil count (cut-off 3.0, AUC 0.59; sensitivity 0.57; specificity 0.63), and lymphocyte count (cut-off 42.0, AUC 0.63; sensitivity 0.77; specificity 0.53) were independent diagnostic predictors. In PF-ILD patients (n = 77), lactate dehydrogenase (cut-off 275, AUC 0.69; sensitivity 0.57; specificity 0.78), Krebs von den Lungen-6 (cut-off 1,140, AUC 0.74; sensitivity 0.71; specificity 0.76), baseline forced vital capacity (FVC) (cut-off 1.75 L, AUC 0.71; sensitivity, 0.93; specificity, 0.46), and BALF neutrophil ratio (cut-off 6.0, AUC 0.72; sensitivity 0.79; specificity 0.80) correlated with death within 3 years. The BALF cellular ratio, particularly the neutrophil ratio, correlated with the diagnosis and prognosis of PF-ILD. These findings may be useful in the management of patients with interstitial pneumonia.

Upregulation of IL-4 receptor signaling pathway in circulating ILC2s from asthma patients.

Background: Group 2 innate lymphoid cells (ILC2s) produce type 2 cytokines by stimulation with epithelial cell-derived cytokines and are implicated in the pathogenesis of various allergic diseases, including asthma. However, differences in the molecular characteristics of ILC2s between patients with asthma and healthy subjects remain unclear. Objective: We sought to evaluate differences in cytokine production capacity and gene expression profile of ILC2s in the peripheral blood of patients with asthma and healthy subjects. Methods: We evaluated ILC2s derived from 15 patients with asthma and 7 healthy subjects using flow cytometry, live-cell imaging of secretion activity analysis, and RNA-sequencing. Results: ILC2s were sorted as CD45+Lineage-CRTH2+CD127+CD161+ cells from the peripheral blood of patients with asthma and healthy subjects, and the number of ILC2s was decreased in patients with asthma (851 ± 1134 vs 2679 ± 3009 cells/20 mL blood; P = .0066). However, patient-derived ILC2s were activated to produce more IL-5 and IL-13 in response to stimulation with IL-2, IL-33, and thymic stromal lymphopoietin compared with healthy subject-derived ILC2s (P = .0032 and P = .0085, respectively). Furthermore, RNA-sequencing analysis revealed that patient-derived ILC2s had different gene expression profiles, such as increased expression in cell growth-related genes (CDKN1b, CCNG2, CCND2, CCN1), prostaglandin E receptor (PTGER2), and IL-4 receptor. In addition, a gene set of the IL-4 receptor signaling pathway was significantly upregulated in ILC2s in patients with asthma (P = .042). Conclusions: Our results suggest that circulating ILC2s in patients with asthma are preactivated via the IL-4 receptor signaling pathway and produce IL-5 and IL-13 vigorously by stimulation.

The Positivity of Phosphorylated STAT3 Is a Novel Marker for Favorable Prognosis in Germinal Center B-Cell Type of Diffuse Large B-Cell Lymphoma.

On the basis of immunohistochemistry, diffuse large B-cell lymphoma (DLBCL) is categorized as a germinal center B-cell (GCB) or non-GCB subtype. Recent integrated genomic analyses have highlighted the importance of the JAK-STAT3 pathway in the molecular pathogenesis of DLBCL. However, its relevance to clinical outcomes remains controversial. Therefore, we evaluated the extent of the nuclear expression of phosphorylated STAT3 (pSTAT3), a surrogate marker of signal transducer and activator of transcription 3 (STAT3) activation, by immunohistochemistry. We also analyzed the potential relationship between pSTAT3 positivity (defined as ≥40% positive neoplastic cells) and clinicopathologic characteristics in 294 patients with DLBCL. pSTAT3 was detected in 122 patients (42%), with a higher rate in the non-GCB subtype than in the GCB subtype (57% vs. 28%, P<0.001). Factors potentially activating STAT3, MYD88L265P, and Epstein-Barr virus-encoded small RNA were identified in the pSTAT3-positive non-GCB subtype, whereas the pSTAT3-positive GCB subtype often showed STAT3 mutations and lacked EZH2 mutations and the rearrangements of BCL2 and MYC. Multivariate analyses revealed that the pSTAT3-positive GCB subtype showed a favorable prognosis (HR: 0.17; 95% confidence interval, 0.04-0.7; P=0.014). These findings suggest that pSTAT3 positivity may have a unique impact on the clinicopathologic characteristics of DLBCL, making it a promising novel marker for the favorable prognosis of patients with the GCB subtype.

Identification and functional characterization of the extremely long allele of the serotonin transporter-linked polymorphic region.

SLC6A4, which encodes the serotonin transporter, has a functional polymorphism called the serotonin transporter-linked polymorphic region (5-HTTLPR). The 5-HTTLPR consists of short (S) and long (L) alleles, each of which has 14 or 16 tandem repeats. In addition, the extralong (XL) and other rare alleles have been reported in 5-HTTLPR. Although they are more frequent in Asian and African than in other populations, the extent of variations and allele frequencies (AFs) were not addressed in a large population. Here, we report the AFs of the rare alleles in a large number of Japanese subjects (N = 2894) consisting of two cohorts. The first cohort (case-control study set, CCSS) consisted of 1366 subjects, including 485 controls and 881 patients with psychosis (bipolar disorder or schizophrenia). The second cohort (the Arao cohort study set, ACSS) consisted of 1528 elderly subjects. During genotyping, we identified 11 novel 5-HTTLPR alleles, including 3 XL alleles. One novel allele had the longest subunit ever reported, consisting of 28 tandem repeats. We named this XL28-A. An in vitro luciferase assay revealed that XL28-A has no transcriptional activity. XL28-A was found in two unrelated patients with bipolar disorder in the CCSS and one healthy subject in the ACSS who did not show depressive symptoms or a decline in cognitive function. Therefore, it is unlikely that XL28-A is associated with psychiatric disorders, despite its apparent functional deficit. Our results suggest that unraveling the complex genetic variations of 5-HTTLPR will be important for further understanding its role in psychiatric disorders.

A Phase 1 Study of KHK4083: A Single-Blind, Randomized, Placebo-Controlled Single-Ascending-Dose Study in Healthy Adults and an Open-Label Multiple-Dose Study in Patients With Ulcerative Colitis.

OX40 plays an essential role in maintaining late T-cell proliferation and survival by suppressing apoptosis and by inducing T-cell memory formation. Here, we report the results of the phase 1 study of KHK4083, a fully human antimonoclonal antibody specific for OX40. In this study, we aimed to assess the safety and tolerability of a single intravenous or subcutaneous administration of KHK4083 compared with placebo in healthy Japanese and Caucasian subjects and determined the pharmacokinetics (PK) and immunogenicity. Also, we assessed the preliminary efficacy and pharmacodynamics of multiple intravenous doses in Japanese patients with moderate to severe ulcerative colitis (UC). Drug-related treatment emergent adverse events occurred in 21 healthy subjects (58.3%) and 5 patients with UC (62.5%) after administration of KHK4083. There were no serious adverse events. The PK profile of a single intravenous dose of 10 mg/kg KHK4083 was similar in healthy Japanese and Caucasian subjects. Of 8 UC patients, a clinical response was observed in 3 patients (37.5%) and clinical remission in 2 patients (25.0%) in week 6. Our study demonstrated the safety and tolerability of single and multiple administrations of KHK4083 in healthy Japanese and Caucasian subjects and Japanese patients with moderate to severe UC. It also indicated favorable pharmacological properties of the drug.

Clinical characteristics of patients with not well-controlled severe asthma in Japan: Analysis of the Keio Severe Asthma Research Program in Japanese population (KEIO-SARP) registry.

BACKGROUND: Multiple phenotypes exist within the classification of severe asthma. However, characteristics of patients with not well-controlled severe asthma have not been well identified. METHODS: Japanese patients with asthma (age ≥ 20 years) were enrolled at the Keio University Hospital and its affiliated hospitals in this observational study (Keio Severe Asthma Research Program). Among them, patients with severe asthma (those undergoing Global Initiative for Asthma [GINA] 2018 step 4 or 5 treatment) were included in this analysis and investigated clinical characteristics based on asthma control status. RESULTS: Of the 154 patients (men, 46.8%; age, 60.1 ± 14.9 years), 87 (56.5%) had not well-controlled (partly controlled and uncontrolled) asthma (GINA step 4, 42 patients; step 5, 45 patients). Overall, there were no significant differences in clinical characteristics between patients with well-controlled and not well-controlled asthma. However, cluster analysis revealed that distinct 5 clusters (cluster 1, well-controlled; cluster 2, eosinophilic; cluster 3, non-type 2 inflammation; cluster 4, high periostin; and cluster 5, late-onset type 2 inflammation), and clusters 2-5 were not well-controlled. Among them, cluster 3 was characterized by low eosinophil counts, low periostin levels, and less frequent olfactory disturbance, and this cluster had the worst asthma control. CONCLUSIONS: Japanese patients with severe asthma were divided into well-controlled and not-well controlled asthma, and we confirmed heterogeneity of not well-controlled severe asthma. These patients, especially non-type 2 phenotype, require a further therapeutic approach. (University Hospital Medical Information Network Clinical Trials Registry, UMIN000002980).

Crystal structure of α-glucosyl transfer enzyme XgtA from Xanthomonas campestris WU-9701.

The α-glucosyl transfer enzyme XgtA is a novel type α-Glucosidase (EC 3.2.1.20) produced by Xanthomonas campestris WU-9701. One of the unique properties of XgtA is that it shows extremely high α-glucosylation activity toward alcoholic and phenolic -OH groups in compounds using maltose as an α-glucosyl donor and allows for the synthesis of various useful α-glucosides with high yields. XgtA shows no hydrolytic activity toward sucrose and no α-glucosylation activity toward saccharides to produce oligosaccharides. In this report, the crystal structure of XgtA was solved at 1.72 Å resolution. The crystal belonged to space group P22121, with unit-cell parameters a = 73.07, b = 83.48, and c = 180.79 Å. The ß→α loop 4 of XgtA, which is proximal to the catalytic center, formed a unique structure that is not observed in XgtA homologs. Furthermore, XgtA was found to contain unique amino acid residues around its catalytic center. The unique structure of XgtA provides an insight into the mechanism for the regulation of substrate specificity in this enzyme.

The outcomes of anatomical lung resection for nontuberculous mycobacterial lung disease.

BACKGROUND: The number of cases of nontuberculous mycobacterial (NTM) lung disease has been increasing in recent years, and the efficacy of surgical treatment has been recognized. We investigated the clinical characteristics and behavior of NTM lung disease and analyzed the outcomes of surgery. METHODS: The data of 25 patients who underwent anatomical resection for NTM lung disease in our institution between January 2004 and December 2014 were retrospectively examined. RESULTS: The patients included 10 men and 15 women (mean age, 63.1 years). Twenty patients had Mycobacterium avium, and 5 had Mycobacterium intracellular. The indications for lung resection in 20 definitively diagnosed patients included a remaining or worsening lesion despite medical treatment (n=16), massive hemoptysis or bloody sputum (n=5), and prolonged smear positivity (n=1); multiple reasons were allowed. In five cases without a definitive diagnosis, surgery was performed due to the suspicion of lung cancer. The surgical procedures included pneumonectomy, n=4; lobectomy, n=13; and segmentectomy, n=8. Complete resection was achieved in 10 cases (40.0%). Video-assisted thoracoscopic surgery (VATS) was performed in 17 cases (68.0%), especially in 6 of 8 cases (75.0%) that underwent segmentectomy and in 10 of 11 cases (90.9%) that received simple lobectomy. There was one case of hospital mortality. Among the 22 patients who were followed at our institution, relapse occurred in 4 patients, and new infection occurred in 1 patient. NTM lung disease was controlled in 17 patients (77.3%). In the four cases that relapsed, the median relapse-free interval was 29.5 months. CONCLUSIONS: Surgical resection was a feasible treatment for NTM lung disease and was associated with favorable outcomes, although there was 1 case of hospital mortality. VATS procedures were considered adequate for the treatment of NTM lung disease; however, the surgical indications must be carefully considered.

Severe Mediastinitis and Pericarditis After Transbronchial Needle Aspiration.

Endobronchial ultrasonographically guided transbronchial needle aspiration (EBUS-TBNA) is now widely performed for mediastinal lymph node staging of lung cancer. Although this procedure is less invasive than mediastinoscopy, some infectious complications have been reported. We report the successful use of pericardial and mediastinal drainage in a case of acute severe mediastinitis with pericarditis after EBUS-TBNA.

Does oral care contribute to brain activation?: One case of functional near-infrared spectroscopy study in patients with a persistent disturbance of consciousness.

KEY CLINICAL MESSAGE: We used functional near-infrared spectroscopy (fNIRS) to measure cerebral blood flow during oral care in a patient with persistent disturbance of consciousness. We experienced that cerebral blood flow to frontal area increased during oral care, suggesting that oral care may have a potential role in rehabilitation for the brain.