Novel compounds isolated from health food products containing beni-koji (red yeast rice) with adverse event reports.

Recently, health hazards, such as kidney damage, have been reported owing to the ingestion of a health food product, so-called "foods with functional claims (FFC)'', containing beni-koji (red yeast rice). Although not an expected compound in the FFC, the detection of puberulic acid has also been reported. Further investigations of these health food products, such as the identification of other unintended compounds and clarifying the health impacts of puberulic acid, are required. To clarify the causes of these health issues, we investigated the presence of unintended compounds in the FFC containing beni-koji using comprehensive instrumental analyses. Using differential analysis, novel compounds 1 and 2 were detected as unexpected components between the samples with and without adverse event reports. Although limited to the samples available for analyses in this study, both compounds 1 and 2 were detected in all the samples that also contained puberulic acid. Compounds 1 and 2, with molecular formulas of C23H34O7 and C28H42O8, respectively, may be lovastatin derivatives. Their structures were confirmed using NMR analyses and are novel natural compounds. For definitive confirmation, we are in the process of synthesizing compounds 1 and 2 from lovastatin. The route of contamination of these compounds are currently under investigation. The findings of this study could be used to address the growing health hazards associated with health food products.

Psychometrics of rating scales for externalizing disorders in Japanese outpatients: The ADHD-Rating Scale-5 and the Disruptive Behavior Disorders Rating Scale.

OBJECTIVES: This study validated the Japanese version of the Attention-Deficit/Hyperactivity Disorder-Rating Scale-5 (ADHD-RS-5) and the Disruptive Behavior Disorders Rating Scale. We extended the ADHD-RS-5 by adding the oppositional defiant disorder and conduct disorder subscales to compare the two rating scales psychometrically. METHODS: We examined the internal consistency, test-retest reliability, construct validity and criterion validity of the two rating scales in 135 Japanese outpatients aged 6-18 years. RESULTS: The internal consistency and test-retest reliability were good for all the subscales of the two rating scales except for the conduct disorder subscale of the ADHD-RS-5 extended. Good construct validity was revealed by expected correlational patterns between subscales from the two rating scales and the Children Behavior Checklist. The criterion validity was good for all the subscales of the two rating scales rated by parents, while teacher-ratings revealed substantially lower predictive ability for all the subscales. Agreement between parent- and teacher-ratings of the two rating scales was generally moderate and using predictive ratings alone of both ratings showed the best predictive ability among the integration methods examined. CONCLUSION: The two rating scales have sound psychometric properties and will aid in screening and severity assessment of externalizing disorders in Japanese clinical settings.

Validity and reliability of the Japanese versions of the coronavirus anxiety scale for adolescents and obsession with COVID-19 scale for adolescents.

Background: The coronavirus disease 2019 (COVID-19) has caused mental health issues in both adults and adolescents. The Coronavirus Anxiety Scale (CAS) and Obsession with COVID-19 Scale (OCS) questionnaires measure anxiety and persistent and disturbed thoughts (also known as obsessions) related to COVID-19. We developed Japanese versions of the CAS (i.e., CAS-JA) and OCS (i.e., OCS-JA) questionnaires to make them suitable for adolescents and validated the characteristics of these scales. Methods: Two online surveys were administered to high school students aged 15-18 years. A total of 263 students participated in the first survey and almost half of them participated in the second survey. In the first survey, participants responded to the CAS-JA, OCS-JA, generalized anxiety and obsessive-compulsive subscales of the Spence Children's Anxiety Scale (SCAS), and Kessler 6 Scale (K6). The SCAS and K6 were used to verify discriminant validity and inter-scale correlations. In the second survey, the participants completed the CAS-JA and OCS-JA again to verify test-retest reliability. We performed a confirmatory factor analysis and calculated the model fit indices. Additionally, we examined the internal consistency reliability, convergent validity, and inter-item correlations of the CAS-JA and OCS-JA. Moreover, differences in CAS-JA and OCS-JA responses by gender and region of residence (state of emergency and non-emergency areas) were examined. Results: The results of the single-factor model confirmatory factor analysis of model fit indices were above the threshold. The required criteria for internal consistency reliability, test-retest reliability, and discriminant and convergent validity were met in both the CAS-JA and OCS-JA. No statistically significant differences attributed to residence and gender were found in both questionnaires. Conclusions: The results indicate that the CAS-JA and OCS-JA questionnaires are useful in measuring COVID-19-related anxiety, and persistent and disturbed thoughts in Japanese adolescents.

Psychometrics of the kiddie schedule for affective disorders and schizophrenia present and lifetime version for DSM-5 in Japanese outpatients.

OBJECTIVE: The Schedule for Affective Disorders and Schizophrenia for School-Age Children-Present and Lifetime version (K-SADS-PL) is a widely used semi-structured diagnostic interview in child and adolescent psychiatry. However, the psychometric properties of its updated version, the K-SADS-PL for DSM-5, have scarcely been examined, especially for criterion validity. This study was designed to examine the inter-rater reliability, criterion validity and construct validity of the K-SADS-PL for DSM-5 in 137 Japanese outpatients. METHODS: Two of 12 experienced clinicians independently performed the K-SADS interview for each patient in a conjoint session, and the resulting consensus diagnosis was compared with a "best-estimate" diagnosis made by two of eight experienced clinicians using all available information for the patient. RESULTS: The inter-rater reliability was excellent, as shown by κ > 0.75 for all disorders, with the exception of current separation anxiety disorder. The criterion validity was fair to good, as shown by κ > 0.40 for all disorders, with the exception of current and lifetime agoraphobia. The construct validity was also good, as shown by theoretically expected associations between the K-SADS-PL diagnoses and subscales of the child behavior checklist. CONCLUSION: The K-SADS-PL for DSM-5, now available in Japanese, generates valid diagnoses in child and adolescent psychiatry.

Guided internet-based cognitive behavioral therapy for obsessive-compulsive disorder: A multicenter randomized controlled trial in Japan.

Few studies have compared the effectiveness of internet-based cognitive behavior therapy (ICBT) for obsessive-compulsive disorder (OCD) with treatment as usual (TAU). We investigated the effectiveness of guided ICBT for patients with OCD. This prospective, randomized, controlled, assessor-blinded, multicenter clinical trial was conducted at three facilities in Japan from January 2020 to March 2021. Thirty-one patients with OCD as the primary diagnosis participated in the trial and were randomly assigned to either the intervention group or the control group. The primary outcome was the Yale-Brown obsessive-compulsive scale score; the assessors were blinded. Results of the analysis of covariance among the groups were significantly different between the groups (p < 0.01, effect size Cohen's d = 1.05), indicating the superiority of guided ICBT. The results suggest that guided ICBT is more effective than TAU for treating OCD. RCT REGISTRATION: UMIN Clinical Trials Registry (UMIN000039375).

Discovery of a novel 9-position modified second-generation anti-HCV candidate via bioconversion and semi-synthesis of FR901459.

Evidence that hepatitis C virus (HCV) utilizes cellular cyclophilin proteins in the virus replication cycle has increased attention on cyclophilin inhibitors as attractive therapeutic targets in the treatment of HCV. Previous reports have described a number of non-immunosuppressive cyclophilin inhibitors, most of which require many synthetic steps for their preparation. Sasamura et al. have previously reported the isolation of bioconversion derivative 4. This analog is a convenient starting point for optimization due to the presence of the readily modifiable primary hydroxyl group and because it shows moderate anti-HCV activity and decreased immunosuppressive activity. We have also established an efficient C-alkylation reaction at the 3-position. Through a detailed structure-activity relationship study, we discovered a new type of clinical candidate 14 which requires a short synthetic process and has potent anti-HCV activity and reduced immunosuppressive activity, as well as improved aqueous solubility and pharmacokinetics.

Discovery of ASP5286: A novel non-immunosuppressive cyclophilin inhibitor for the treatment of HCV.

Evidence indicates that hepatitis C virus (HCV) utilizes cellular cyclophilin proteins in its replication, and cyclophilin inhibitors represent a new class of anti-HCV agents. We have established an efficient synthetic methodology to generate FR901459 derivatives via N, O-acyl migration reaction while avoiding total synthesis. Through a detailed structure-activity relationship study, we improved anti-HCV activity while decreasing immunosuppressive activity. Additionally, we discovered the importance of substitution at the 3 position for not only improving anti-HCV activity but also pharmacokinetic profile. Finally, by striking an appropriate balance between potency, solubility, and permeability, we discovered ASP5286 (13) as a potential clinical candidate for anti-HCV therapy.

Development of an efficient semisynthetic modification of FR901459 via a novel regioselective N, O-acyl migration.

HCV utilizes cellular protein cyclophilins in the virus replication cycle and cyclophilin inhibitors have become a new class of anti-HCV agents. In our screening of natural products, we identified a unique cyclosporin analogue, FR901459, as a cyclophilin inhibitor with potent anti-HCV activity. In this work, we developed an efficient synthetic methodology to prepare FR901459 derivatives via an N, O-acyl migration reaction. This method allows us to efficiently manipulate the amino acid residues at the 3 position while avoiding lengthy total synthesis for each compound. By using this methodology, we discovered 4, which has superior anti-HCV activity and decreased immunosuppressive activity compared to FR901459.

Guided Internet-Based Cognitive Behavioral Therapy in Japanese Patients With Obsessive-Compulsive Disorder: Protocol for a Randomized Controlled Trial.

BACKGROUND: Cognitive behavioral therapy for obsessive-compulsive disorder has been established, but access to this therapy in Japan is limited. Internet-based cognitive behavioral therapy may improve treatment accessibility and sufficiently improve obsessive-compulsive symptoms. There are few randomized controlled trials examining the effectiveness of internet-based cognitive behavioral therapy in patients with obsessive-compulsive disorder. We designed a randomized controlled trial protocol to assess the effectiveness of guided internet-based cognitive behavioral therapy in Japanese patients with obsessive-compulsive disorder. OBJECTIVE: We aimed to develop a protocol for a randomized controlled trial of internet-based cognitive behavioral therapy in Japanese patients with obsessive-compulsive disorder. METHODS: The randomized controlled trial will compare internet-based cognitive behavioral therapy treatment and usual care groups, each consisting of 15 participants (n=30) diagnosed with obsessive-compulsive disorder. We will evaluate the effectiveness of a 12-week intervention. The primary outcome of symptom severity will be measured using the Yale-Brown Obsessive-Compulsive Scale. Secondary outcomes will be assessed with the Obsessive-Compulsive Inventory, Beck Anxiety Inventory, Patient Health Questionnaire-9, Generalized Anxiety Disorder-7, Working Alliance Inventory-Short Form, and the Euro Qol - 5 Dimension. All measures will be assessed at weeks 0 (baseline) and 12 (follow-up). In the statistical analysis comparing treatment effects, the least-squares means and their 95% CIs will be estimated by analysis of covariance with the change in total outcomes scores at week 12. All comparisons are planned, and all P values will be two-sided, with values <.05 considered statistically significant. RESULTS: The study will be performed from January 2020 to March 2021, and results are expected to be available in mid-2021. CONCLUSIONS: The trial will demonstrate whether internet-based cognitive behavioral therapy improves access and is more effective than more usual care for patients with obsessive-compulsive disorder in Japan. TRIAL REGISTRATION: University Hospital Medical Information Network (UMIN) 000039375; https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000044422. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/18216.

Regio- and stereospecific hydroxylation of various steroids at the 16α position of the D ring by the Streptomyces griseus cytochrome P450 CYP154C3.

Cytochrome P450 monooxygenases (P450s), which constitute a superfamily of heme-containing proteins, catalyze the direct oxidation of a variety of compounds in a regio- and stereospecific manner; therefore, they are promising catalysts for use in the oxyfunctionalization of chemicals. In the course of our comprehensive substrate screening for all 27 putative P450s encoded by the Streptomyces griseus genome, we found that Escherichia coli cells producing an S. griseus P450 (CYP154C3), which was fused C terminally with the P450 reductase domain (RED) of a self-sufficient P450 from Rhodococcus sp., could transform various steroids (testosterone, progesterone, Δ(4)-androstene-3,17-dione, adrenosterone, 1,4-androstadiene-3,17-dione, dehydroepiandrosterone, 4-pregnane-3,11,20-trione, and deoxycorticosterone) into their 16α-hydroxy derivatives as determined by nuclear magnetic resonance and high-resolution mass spectrometry analyses. The purified CYP154C3, which was not fused with RED, also catalyzed the regio- and stereospecific hydroxylation of these steroids at the same position with the aid of ferredoxin and ferredoxin reductase from spinach. The apparent equilibrium dissociation constant (Kd) values of the binding between CYP154C3 and these steroids were less than 8 µM as determined by the heme spectral change, indicating that CYP154C3 strongly binds to these steroids. Furthermore, kinetic parameters of the CYP154C3-catalyzed hydroxylation of Δ(4)-androstene-3,17-dione were determined (Km, 31.9 ± 9.1 µM; kcat, 181 ± 4.5 s(-1)). We concluded that CYP154C3 is a steroid D-ring 16α-specific hydroxylase which has considerable potential for industrial applications. This is the first detailed enzymatic characterization of a P450 enzyme that has a steroid D-ring 16α-specific hydroxylation activity.

Biocatalytic synthesis of flavones and hydroxyl-small molecules by recombinant Escherichia coli cells expressing the cyanobacterial CYP110E1 gene.

BACKGROUND: Cyanobacteria possess several cytochrome P450s, but very little is known about their catalytic functions. CYP110 genes unique to cyanaobacteria are widely distributed in heterocyst-forming cyanobacteria including nitrogen-fixing genera Nostoc and Anabaena. We screened the biocatalytic functions of all P450s from three cyanobacterial strains of genus Nostoc or Anabaena using a series of small molecules that contain flavonoids, sesquiterpenes, low-molecular-weight drugs, and other aromatic compounds. RESULTS: Escherichia coli cells carrying each P450 gene that was inserted into the pRED vector, containing the RhFRed reductase domain sequence from Rhodococcus sp. NCIMB 9784 P450RhF (CYP116B2), were co-cultured with substrates and products were identified when bioconversion reactions proceeded. Consequently, CYP110E1 of Nostoc sp. strain PCC 7120, located in close proximity to the first branch point in the phylogenetic tree of the CYP110 family, was found to be promiscuous for the substrate range mediating the biotransformation of various small molecules. Naringenin and (hydroxyl) flavanones were respectively converted to apigenin and (hydroxyl) flavones, by functioning as a flavone synthase. Such an activity is reported for the first time in prokaryotic P450s. Additionally, CYP110E1 biotransformed the notable sesquiterpene zerumbone, anti-inflammatory drugs ibuprofen and flurbiprofen (methylester forms), and some aryl compounds such as 1-methoxy and 1-ethoxy naphthalene to produce hydroxylated compounds that are difficult to synthesize chemically, including novel compounds. CONCLUSION: We elucidated that the CYP110E1 gene, C-terminally fused to the P450RhF RhFRed reductase domain sequence, is functionally expressed in E. coli to synthesize a robust monooxygenase, which shows promiscuous substrate specificity (affinity) for various small molecules, allowing the biosynthesis of not only flavones (from flavanones) but also a variety of hydroxyl-small molecules that may span pharmaceutical and nutraceutical industries.

Observation of microstructural changes in polymer-coated Kompeito-type platinum particles by in situ heating TEM.

We have successfully prepared Kompeito-like platinum particles by hydrogen reduction of Pt4+ in the presence of sodium polyacrylate. We performed in situ TEM observation of these platinum particles at high temperatures. At 300 degrees C, a thin carbon layer due to polyacrylate formed on the particle surface. The detailed structure of the particles did not change with temperatures up to 700 degrees C. Continuous heating up to 800 degrees C blunted the particles' edges and also sintering of the particles was observed. This result strongly indicates that the shape change and sintering of platinum particles are exclusively controlled by the carbon layer, which is generated from a surface stabilizer polymer.

Characterization of cyanobacterial carotenoid ketolase CrtW and hydroxylase CrtR by complementation analysis in Escherichia coli.

The pathway from beta-carotene to astaxanthin is a crucial step in the synthesis of astaxanthin, a red antioxidative ketocarotenoid that confers beneficial effects on human health. Two enzymes, a beta-carotene ketolase (carotenoid 4,4'-oxygenase) and a beta-carotene hydroxylase (carotenoid 3,3'-hydroxylase), are involved in this pathway. Cyanobacteria are known to utilize the carotenoid ketolase CrtW and/or CrtO, and the carotenoid hydroxylase CrtR. Here, we compared the catalytic functions of CrtW ketolases, which originated from Gloeobacter violaceus PCC 7421, Anabaena (also known as Nostoc) sp. PCC 7120 and Nostoc punctiforme PCC 73102, and CrtR from Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120 and Anabaena variabilis ATCC 29413 by complementation analysis using recombinant Escherichia coli cells that synthesized various carotenoid substrates. The results demonstrated that the CrtW proteins derived from Anabaena sp. PCC 7120 as well as N. punctiforme PCC 73102 (CrtW148) can convert not only beta-carotene but also zeaxanthin into their 4,4'-ketolated products, canthaxanthin and astaxanthin, respectively. In contrast, the Anabaena CrtR enzymes were very poor in accepting either beta-carotene or canthaxanthin as substrates. By comparison, the Synechocystis sp. PCC 6803 CrtR converted beta-carotene into zeaxanthin efficiently. We could assign the catalytic functions of the gene products involved in ketocarotenoid biosynthetic pathways in Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120 and N. punctiforme PCC 73102, based on the present and previous findings. This explains why these cyanobacteria cannot produce astaxanthin and why only Synechocystis sp. PCC 6803 can produce zeaxanthin.