Systematic elucidation of the second coordination sphere effect on the structure and properties of a blue copper protein, pseudoazurin.

The rational structural and computational studies of a blue copper protein, pseudoazurin (PAz), and its Met16X (X = Phe, Leu, Val, Ile) variants gave clear functional meanings of the noncovalent interaction (NCI) through the second coordination sphere. The high-resolution X-ray crystal structures of Met16X PAz demonstrated that the active site geometry is significantly affected by the substitution of Met16, which is located within the NCI distance from the His81 imidazole ring at the copper active site. The computational chemistry calculations based on the crystal structure analyses confirmed that the NCI of S-π/CH-π (wild-type), π-π (Met16Phe), double CH-π (Met16Leu), and single CH-π (Met16Val and Met16Ile). The estimated interaction energies for the NCI demonstrated that the fine-tuning of the protein stability and Cu site properties form the second coordination sphere of PAz.

Effectiveness of distributing pocket cards in improving the behavior, attitude, and knowledge regarding proper medication use among junior high school students in Japan.

Objective: This study aimed to explore the effectiveness of distributing pocket cards with summaries of key information on appropriate medication usage after the implementation of a structured school-based medication education program for junior high school students in Japan. Methods: A total of 227 3rd-grade high school students participated in the intervention. Students who received the program without the provision of pocket cards in 2022 were included in the comparison group, and students who took the program with the provision of pocket cards in 2023 were included in the intervention group. After propensity score matching, the final sample of N = 116 comprised n = 58 comparison group participants and n = 58 intervention group participants. Questionnaires were administered at baseline, end-of-class, and 3-month follow-up to assess the changes in behavior, attitude, and knowledge scores. Results: The matched intervention group showed significantly lower scores at the 3-month follow-up than the matched comparison group. The results of the multiple linear regression analysis showed that for both groups, only the attitude scores were significantly correlated with the behavior scores. In addition, regardless of the baseline scores, the matched intervention group demonstrated smaller or negative changes in scores at the 3-month follow-up. Conclusion: Overall, the results of this study did not support the effectiveness of distributing pocket cards after in-class intervention. However, the usefulness of medication education intervention was confirmed. These results emphasize the need to explore other supplemental teaching tools to further enhance the impact of structured medication education programs.

Ingestion of Indigestible Cacao Proteins Promotes Defecation and Alters the Intestinal Microbiota in Mice.

Background: In animals, the health effects of ingested cacao proteins are unknown because the proteins are difficult to extract and purify from cacao beans. Objectives: This study aimed to develop an extraction and purification method for cacao proteins and reveal the effect of ingestion of cacao proteins on defecation and intestinal microbiota in mice. Methods: Three groups of mice were fed a control diet (AIN-93 G), a cacao lignin diet (AIN-93 G containing 1.22% cacao lignin), or a cacao protein and lignin diet (AIN-93 G containing 1.97% cacao proteins and 1.22% cacao lignin) by pair-feeding for 8 d. Feces were collected as 2 bulked samples from days 1 to 4 and days 5 to 8 on each diet. The collected feces were weighed and the intestinal microbiota was analyzed by next-generation sequencing-based 16S rRNA. Results: A new extraction and purification method for cacao proteins has been developed, then found that the proteins are resistant to digestive enzymes. However, the cacao protein powder made by this method contained 34.9% of lignin in addition to 56.4% of proteins. Therefore, to reveal the effect by cacao proteins alone, the fecal weight and intestinal microbiota of mice fed the cacao protein and lignin diet were compared with those of mice fed the cacao lignin diet. The fecal weight of mice fed the cacao protein and lignin diet was significantly greater than of mice fed the cacao lignin diet. The relative abundance of Lactococcus and Mucispirillum species in mice fed the cacao protein and lignin diet was significantly higher than in mice fed the cacao lignin diet, but the relative abundance of Anaerotruncus, Oscillospira, and Roseburia species in mice fed the cacao protein and lignin diet was significantly lower than in mice fed the cacao lignin diet. Conclusions: Ingestion of indigestible cacao proteins promoted defecation and altered the intestinal microbiota such as Lactococcus, Mucispirillum, Anaerotruncus, Oscillospira, and Roseburia species in mice.

Endoplasmic reticulum stress promotes nuclear translocation of calmodulin, which activates phenotypic switching of vascular smooth muscle cells.

BACKGROUND AND AIMS: Increased endoplasmic reticulum (ER) stress is strongly associated with the phenotypic switching of vascular smooth muscle cells (VSMCs) in atherosclerosis. Depletion of the ER Ca2+ content is one of the leading causes of increased ER stress in VSMCs. The ryanodine receptor (RyR) is a major Ca2+ release channel in the sarcoplasmic reticulum membrane. Calmodulin (CaM), which binds to RyR (CaM-RyR), stabilizes the closed state of RyR in the resting state in normal cells. Defective CaM-RyR interactions can cause abnormal Ca2+ leakage through RyR, resulting in decreased Ca2+ content, indicating that defective CaM-RyR interactions may be a cause of increased ER stress. Herein, we used a mouse VSMCs to assess whether CaM-RyR plays a pivotal role in VSMCs phenotypic switching, which is caused by ER stress, and whether dantrolene, which enhances the binding affinity of CaM to RyR, affects VSMCs phenotypic switching. METHODS AND RESULTS: Tunicamycin was used to mimic ER stress in vitro. Tunicamycin-induced ER stress caused CaM to dissociate from the RyR and translocate to the nucleus, which stimulated phenotypic switching through the activation of MEF2 and KLF5. Dantrolene suppressed tunicamycin-induced apoptosis, ER stress (restoring ER Ca2+ content), and phenotypic switching of VSMCs. Suramin, which directly unbinds CaM from RyR, promoted nuclear CaM accumulation with parallel VSMCs phenotypic switching, and dantrolene prevented these effects. CONCLUSIONS: We observed that ER stress causes CaM translocation to the nucleus and drives the phenotypic switching of VSMCs. Thus, restoration of the binding affinity of CaM to RyR may be a therapeutic target for atherosclerosis.

Dantrolene reduces platelet-derived growth factor (PDGF)-induced vascular smooth muscle cell proliferation and neointimal formation following vascular injury in mice.

Dantrolene is a ryanodine receptor blocker that is used clinically for treatment of malignant hyperthermia. This study was conducted using murine aortic vascular smooth muscle cells (MOVAS) and a mouse arterial injury model to investigate the inhibitory effect of dantrolene on smooth muscle cell proliferation and migration. We investigated whether dantrolene suppressed platelet-derived growth factor (PDGF)-induced vascular smooth muscle cell proliferation and migration in vitro. The effect of dantrolene on smooth muscle phenotype was evaluated using immunostaining. In addition, smooth muscle cell proliferation and phenotype switching were tested by applying dantrolene around blood vessels using a mouse arterial injury model. Dantrolene inhibited PDGF-induced cell proliferation and migration of MOVAS. Dantrolene also inhibited the switch from contractile to synthetic phenotype both in vitro and in vivo. Dantrolene is effective at inhibiting vascular smooth muscle cell proliferation, migration, and neointimal formation following arterial injury in mice.

Herpud1 suppress angiotensin II induced hypertrophy in cardiomyocytes.

Purpose: The purpose of this study was to analyze the role of homocysteine-responsive endoplasmic reticulum-resident ubiquitin-like domain member 1 (Herpud1) gene in the development of cardiomyocyte hypertrophy. Method: In order to examine the effect of suppressing Herpud1 expression, Herpud1 small interfering RNA (siRNA) was introduced into H9C2 cells, which are cell lines derived from rat myocardium, and the degree of Herpud1 protein expression and cell hypertrophy in the Herpud1 siRNA-transfected group and the control group was compared by immunostaining 48 h after Herrpud1 siRNA introduction. To examine whether hypertrophy induced by angiotensin II (Ang II) can be suppressed by the overexpression of Herpud1, the green fluorescent protein (GFP)-Herpud1 plasmid was introduced into H9C2 cells, and the degree of cell hypertrophy was examined in the GFP-Herpud1-and control groups for 48 h. Nuclear translocation of nuclear factor of activated T-cells, cytoplasmic 4 (NFATc4), a transcription factor for hypertrophic genes, was also examined. Results: [1] Herpud1 siRNA-transfected cells showed decreased Herpud1 protein expression and hypertrophy formation compared to control cells [2]; Overexpression of Herpud1 suppresses Ang II-induced cell hypertrophy; and [3] Overexpression of Herpud1 inhibits nuclear translocation of NFATc4. Discussion: It was suggested that Herpud1 might be an anti-hypertrophic gene in Ang II induced cardiomyocytes hypertrophy.

[Survey of Label Information of Foods with Function Claims].

The consumption of health food products, such as Foods with Function Claims, has grown in Japan. Significant information, such as possible side effects or drug interactions, are expected to be described on the packaging to help consumers to make an informed choice about products. In this study, we checked the items described on the packaging of Foods with Function Claims containing eicosapentaenoic acid (EPA) and/or docosahexaenoic acid (DHA), Salacinol/Fagomine/Neokotalanol, or Varyl-Tyrosine/Lactotripeptide. We found that the label information on the package have issues that need to be addressed; for example, the description about a warning for concomitant use with antithrombotic drugs was found in only 29.7% of EPA and/or DHA containing products (44 out of 148). Providing information for safe usage of products to consumers is pivotal. Therefore, improving product labeling, and further pharmaceutical support in case of taking health foods, should be considered.

Factors Influencing Medicine Use Behavior in Adolescents in Japan Using a Bayesian Network Analysis.

Background: Medicine education in Japan was introduced to junior high schools in 2012. However, the effectiveness of existing education programs is limited. In order to develop more effective programs for high school students, the present study investigated the variables that directly influence medicine use behavior and the magnitude of their influence, using a Bayesian network analysis. Methods: A national cross-sectional survey was conducted in 2017. Eighty-three public high schools across Japan were randomly selected, and questionnaires were administered to 15-16 years old 10th grade students. The number of valid responses was 17,437 (effective response rate was 98.46%). Responses were analyzed to measure students' behavior toward, attitudes regarding, and knowledge of medicines, and awareness of their prior medicine education. Results: Students' "attitude score" and "awareness of a class" directly influenced their "behavior score." The "score on attitude," which had a large influence on "score on behavior," was directly influenced by "score on knowledge of proper use" and "awareness of class." Conclusion: The present study argues that acquiring knowledge of appropriate medicine use leads to the acquisition of favorable attitudes, which may result in behavioral change. Therefore, for medicine education, it is expected that incorporating content related to knowledge acquisition for changing attitudes will be important for promoting behavioral change.

[Questionnaire Survey on Collaboration with Medical Facilities/Nursing Home Care Facilities at Community Pharmacies].

Construction of regional inclusive care system is being promoted and pharmacists are required to implement multidisciplinary collaboration in order to provide appropriate pharmaceutical care to patients. However, there are few studies on collaboration between medical facilities and community pharmacies. In this study, we conducted a questionnaire survey of community pharmacies in Gifu City, which assessed the experience and attitudes regarding collaboration with other medical facilities. The survey items are: I. Participation in regional care meetings, II. Case discussion conferences, III. Joint workshops/study conferences, IV. Community service, V. Sharing information through medical cooperation network, and VI. Accompanying community pharmacists at home medical care. For the implementation of collaboration, the percentage of "not implemented" were as high as 70% or more in II, IV, V and VI. Regarding the attitudes toward collaboration, more than half of pharmacies answered that they wanted to implement in all items. In the comparison by the number of pharmacists, pharmacies with two or more pharmacists had significantly higher implementation ratios than pharmacies with one pharmacist in IV and V. Regarding the attitudes toward collaboration, pharmacies with two or more pharmacists had significantly higher ratios of considering implementation than pharmacies with one pharmacist in I and VI. Based on the results of this survey, there were many items that were not implemented as collaboration with other medical facilities at community pharmacies. However, many community pharmacies are planning to collaborate with other medical facilities in the future.

[Survey of the Distribution of Community Pharmacists Using Government Statistics].

　In Japan, within the background of a progressively aging society, a community general support system is gradually being established. Under this system, community pharmacists are expected to expand their activities in local communities. Here, we surveyed the distribution of community pharmacists in Japan by using government statistics. We found that there are 153 towns/villages without community pharmacists, which is about six times the number of towns without physicians (26 towns/villages). The number of community pharmacists per 100000 population was correlated with the population of the municipality. There was a significant difference in the number of community pharmacists per 100000 population between depopulated and non-depopulated areas. A multiple regression analysis revealed that population, financial capability index, and number of physicians per 100000 population were positively associated with the number of community pharmacists per 100000 population in a given municipality. We hope that the survey provides useful information about future issues facing community pharmacy in a community general support system.

Changes in the quality of medicines during storage under LED lighting and consideration of countermeasures.

BACKGROUND: In recent years, the popularity of LED lighting has rapidly increased, owing to its many advantages, including economic benefits. We examined the change in the quality of drugs during storage under LED and fluorescent lighting and found that some medicines exhibited a different degree of color change depending on the light source. The purpose of this study was to investigate the effects of different plastic storage bags on the color change over time when various medicines were stored under LED and fluorescent lighting conditions. METHODS: Photostability tests were conducted on several types of target drugs. Subsequently, subjective evaluation by ten evaluators and objective evaluation by image analysis software were carried out regarding color change. RESULTS: A similar change in color tone was observed after all types of illumination. Subjective evaluation by 10 evaluators revealed that "change in color tone" occurred in the order of bulb-color LED lighting < daylight-color LED lighting < fluorescent lighting, regardless of the type of plastic bags. A similar tendency was observed also in objective evaluation. In this study, it was considered that a brown light-shielding plastic bag was more effective than a normal plastic bag for the prevention of the color change of medicines stored under LED lighting. CONCLUSIONS: The above results suggested that the most appropriate combination of plastic bag and light source for medicine storage was a brown light-shielding plastic bag and bulb-color LED lighting.

[Survey of Description on Package Inserts of OTC Drugs].

　The "self-medication tax deduction" system began in Japan in January 2017, allowing people to encourage the use of OTC drugs. Package inserts contain important information for consumers regarding their use. In this study, we first checked whether the items, as required in the notifications of the Japanese Ministry of Health, Labour and Welfare, are described in the package inserts of cold remedies and analgesic antipyretics in OTC drugs. The descriptions of almost all packages checked in this study were based on the notifications, but those of a small number of them were not. Next, we examined the description of the items, unrequired in the notification, but worthy for proper use of drugs; e.g., the description of prohibition for use by "patients with severe hypertension" in case of ibuprofen-containing products, and the description was found in only seven of 180 products. Manufactures should make package inserts along with notifications, including the description for proper use of drugs.

A new member of the family Totiviridae associated with arboreal ants (Camponotus nipponicus).

A putative new member of the family Totiviridae was identified in arboreal ants (Camponotus nipponicus). The viral dsRNA consisted of 5,713 nt with two overlapping open reading frames (ORFs). ORF1 encodes a putative capsid protein. ORF2 encodes a viral RNA-dependent RNA polymerase (RdRp). ORF2 could be translated as a fusion protein with the ORF1 product through a -1 frameshift in the overlapping ORF1. Phylogenetic analysis based on the RdRp revealed that the virus from C. nipponicus is closely related to Camponotus yamaokai virus, a member of the family Totiviridae, from another ant species. The name Camponotus nipponicus virus (CNV) is proposed for the new virus.

Identification, characterization and full-length sequence analysis of a novel dsRNA virus isolated from the arboreal ant Camponotus yamaokai.

A novel dsRNA virus was identified from the arboreal ant Camponotus yamaokai. The complete nucleotide sequence analysis of the virus revealed that the virus consisted of 5704 bp with two ORFs. ORF1 (3084 nt) encoded a putative capsid protein. ORF2 (1977 nt) encoded a viral RNA-dependent RNA polymerase (RdRp). ORF2 could be translated as a fusion with the ORF1 product by a - 1 frameshift in the overlapping ORF1. Phylogenetic analyses based on the RdRp revealed that the virus from C. yamaokai was most likely a novel totivirus, but it was not closely related to the previously known totiviruses in arthropods. Transmission electron microscopy revealed isometric virus particles of ~30 nm diameter in the cytoplasm, which was consistent with the characteristics of the family Totiviridae. The virus was detected by reverse transcription-PCR in all caste members and developmental stages of ants, including eggs, larvae, pupae, adult workers, alates (male and female) and queens. To our knowledge, this is the first report of a member of the family Totiviridae in a hymenopteran; the virus was designated Camponotus yamaokai virus.

Placental extracts induce the expression of antioxidant enzyme genes and suppress melanogenesis in B16 melanoma cells.

One of the activities of placental extracts (PEs) is skin-whitening effect, but the physiological and genetic mechanism for this effect has not yet been clarified. Here, we focus on PE as a regulator of antioxidant enzyme genes. Porcine PE was prepared, and its activity was investigated in B16 melanoma cells. PE treatment decreased the melanin content of UV-irradiated B16 cells in a dose-dependent manner. PE directly reduced the enzyme activity of tyrosinase in a cell-free assay. In addition, PE treatment increased the gene expression of cytosolic superoxide dismutase (SOD-1), extracellular SOD (SOD-3) and catalase but did not affect the expression of tyrosinase. Moreover, PE protected the B16 cells from H2O2-induced cell death. Taken together, our data suggest that PEs could play a role not only as a suppressor of melanin synthesis but also as a regulator of antioxidant genes and might protect the skin against oxidative stress.

Impairment of endothelial-mesenchymal transformation during atrioventricular cushion formation in Tmem100 null embryos.

BACKGROUND: Endothelial-mesenchymal transformation (EndMT) is essential for endocardial cushion formation during cardiac morphogenesis. We recently identified Tmem100 as an endothelial gene indispensable for vascular development. In this study, we further investigated its roles for EndMT during atrioventricular canal (AVC) cushion formation. RESULTS: Tmem100 was expressed in AVC endocardial cells, and Tmem100 null embryos showed severe EndMT defect in the AVC cushions. While calcineurin-dependent suppression of vascular endothelial growth factor (VEGF) expression in the AVC myocardium is important for EndMT, significant up-regulation of Vegfa expression was observed in Tmem100 null heart. EndMT impaired in Tmem100 null AVC explants was partially but significantly restored by the expression of constitutively-active calcineurin A, suggesting dysregulation of myocardial calcineurin-VEGF signaling in Tmem100 null heart. Moreover, Tmem100 null endocardial cells in explant culture did not show EndMT in response to the treatment with myocardium-derived growth factors, transforming growth factor ß2 and bone morphogenetic protein 2, indicating involvement of an additional endocardial-specific abnormality in the mechanism of EndMT defect. The lack of NFATc1 nuclear translocation in endocardial cells of Tmem100 null embryos suggests impairment of endocardial calcium signaling. CONCLUSIONS: The Tmem100 deficiency causes EndMT defect during AVC cushion formation possibly via disturbance of multiple calcium-related signaling events.

An important role of endothelial hairy-related transcription factors in mouse vascular development.

The Hairy-related transcription factor family of Notch- and ALK1-downstream transcriptional repressors, called Hrt/Hey/Hesr/Chf/Herp/Gridlock, has complementary and indispensable functions for vascular development. While mouse embryos null for either Hrt1/Hey1 or Hrt2/Hey2 did not show early vascular phenotypes, Hrt1/Hey1; Hrt2/Hey2 double null mice (H1(ko) /H2(ko) ) showed embryonic lethality with severe impairment of vascular morphogenesis. It remained unclear, however, whether Hrt/Hey functions are required in endothelial cells or vascular smooth muscle cells. In this study, we demonstrate that mice with endothelial-specific deletion of Hrt2/Hey2 combined with global Hrt1/Hey1 deletion (H1(ko) /H2(eko) ) show abnormal vascular morphogenesis and embryonic lethality. Their defects were characterized by the failure of vascular network formation in the yolk sac, abnormalities of embryonic vascular structures and impaired smooth muscle cell recruitment, and were virtually identical to the H1(ko) /H2(ko) phenotypes. Among signaling molecules implicated in vascular development, Robo4 expression was significantly increased and activation of Src family kinases was suppressed in endothelial cells of H1(ko) /H2(eko) embryos. The present study indicates an important role of Hrt1/Hey1 and Hrt2/Hey2 in endothelial cells during early vascular development, and further suggests involvement of Robo4 and Src family kinases in the mechanisms of embryonic vascular defects caused by the Hrt/Hey deficiency.