circRNAs May Be Involved in Dysfunction of Neutrophils of Type 2 Diabetic Mice through Regulation of Specific miRNAs.

Diabetes is known to delay wound healing, and this delay is attributed to prolonged inflammation. We found that microRNAs (miRNAs) might be involved in the dysfunction of diabetic-derived neutrophils, and dynamics of neutrophil and chronic inflammation might be initiated by miRNA-regulated genes. Moreover, studies of miRNA function in nephropathy have suggested that circular RNAs (circRNAs), which function as sponges of miRNA to regulate their expression, are potential biomarkers and new therapeutic targets for the diagnosis of diabetic nephropathy. Accordingly, to investigate the molecular mechanism of the regulation of inflammation in diabetic-derived neutrophils, we identified circRNAs in diabetic-derived neutrophils obtained from BKS.Cg-Dock7m +/+ Leprdb/J (Leprdb/db and Leprdb/+) mice using microarrays. Neutrophils from pooled bone marrow of three diabetic and three non-diabetic mice were isolated and total RNA was extracted. Microarray analysis was performed using the Arraystar Mouse Circular RNA Array. The results showed that three circRNAs were significantly increased and six circRNAs were significantly decreased in diabetic-derived neutrophils compared with non-diabetic-derived neutrophils. The expressions of some circRNAs in diabetic-derived neutrophils were more than double those in non-diabetic-derived neutrophils. The circRNAs contain binding sites of miRNAs, which were differentially expressed in diabetic-derived neutrophils. Our results suggest that circRNAs may be involved in the regulation of inflammation in diabetic-derived neutrophils.

Astaxanthin Exerts Immunomodulatory Effect by Regulating SDH-HIF-1α Axis and Reprogramming Mitochondrial Metabolism in LPS-Stimulated RAW264.7 Cells.

Astaxanthin (AX) is a carotenoid that exerts potent antioxidant activity and acts in cell membranes and mitochondria, which consist of the bilayer molecules. Targeting mitochondria to ameliorate inflammatory diseases by regulating mitochondrial metabolism has become possible and topical. Although AX has been shown to have anti-inflammatory effects in various cells, the mechanisms are quite different. In particular, the role of AX on mitochondrial metabolism in macrophages is still unknown. In this study, we investigated the effect of AX on mitochondria-mediated inflammation and its mechanisms in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. AX attenuated the mitochondrial O2- production and maintained the mitochondrial membrane potential, implying that AX preserved mitochondrial homeostasis to avoid LPS stimulation-induced mitochondrial dysfunction. Additionally, AX prevented the decrease in mitochondrial complexes I, II, and III, which were caused by LPS stimulation. Especially, AX inhibited the reduction in mitochondrial succinate dehydrogenase (SDH; complex II) activity and upregulated the protein and mRNA level of SDH complex, subunit B. Furthermore, AX blocked the IL-1ß expression by regulating the SDH-HIF-1α axis and suppressed the energy shift from an OXPHOS phenotype to a glycolysis phenotype. These findings revealed important effects of AX on mitochondrial enzymes as well as on mitochondrial energy metabolism in the immune response. In addition, these raised the possibility that AX plays an important role in other diseases caused by SDH mutation and metabolic disorders.

rno-miR-203a-3p and Mex3B contribute to cell survival of iliopsoas muscle via the Socs3-Casp3 axis under severe hypothermia in rats.

Forensic diagnosis of fatal hypothermia is considered difficult because no specific findings, such as molecular markers, have been identified. Therefore, determining the molecular mechanism in hypothermia and identifying novel molecular markers to assist in diagnosing fatal hypothermia are important. This study aimed to investigate microRNA (miRNA) and mRNA expression in iliopsoas muscle, which plays a role in homeostasis in mammals, to resolve the molecular mechanism in hypothermia. We generated rat models of mild, moderate, and severe hypothermia, then performed body temperature-dependent miRNA and mRNA expression analysis of the iliopsoas muscle using microarray and next-generation sequencing. Analysis showed that rno-miR-203a-3p expression was lower with decreasing body temperature, while Socs3 expression was significantly increased only by severe hypothermia. Luciferase reporter assays suggested that Socs3 expression is regulated by rno-miR-203a-3p. Socs3 and Mex3B small interfering RNA-mediated knockdown showed that suppressing Mex3B could induce the activation of Socs3, followed by a change in caspase 3/7 activity and adenosine triphosphate levels in iliopsoas muscle cells. These findings indicate that rno-miR-203a-3p and Mex3B are deactivated by a decrease in body temperature, whereby it contributes to suppressing apoptosis by accelerating Socs3. Accordingly, the rno-miR-203a-3p-Socs3-Casp3 or Mex3B-Socs3-Casp3 axis may be the part of the biological defense response to maintain homeostasis under extreme hypothermia.

Effectiveness of a Comprehensive Mental Health Literacy Educational Programme for Junior High School Students: A Randomised Controlled Trial Examining Changes in Their Knowledge, Attitudes, and Behaviour.

BACKGROUND: This study evaluated the effectiveness of a comprehensive mental health literacy (MHL) educational programme known as "Sanita" for improving junior high school students' knowledge of mental illness, attitudes towards people with mental health problems, and help-seeking behaviour. METHODS: A randomised controlled trial with a parallel-group design was conducted. A total of 125 students (51 in the intervention group and 74 in the control group) received three 50-min classes and completed self-report questionnaires (Mental Illness and Disorder Understanding Scale, MIDUS; Reported and Intended Behaviour Scale, RIBS-J; and an original questionnaire investigating help-seeking behaviour) before and after the programme and three months later. RESULTS: Regarding MIDUS, the post-test and 3-month follow-up test results showed a significant main effect of time-by-group interactions in a linear mixed model. Regarding RIBS-J, the post-test results showed a significant main effect of time-by-group interactions; however, the 3-month follow-up test showed no significant effect. No significant effects of time-by-group interactions were seen in the post-test and 3-month follow-up test results for help-seeking behaviour in a logistic regression-mixed model. CONCLUSIONS: The Sanita MHL educational programme was longitudinally effective at improving junior high school students' knowledge of mental illness, although improvements in attitudes and help-seeking behaviour were insufficient.

Anticancer agent α-sulfoquinovosyl-acylpropanediol enhances the radiosensitivity of human malignant mesothelioma in nude mouse models.

Malignant pleural mesothelioma (MPM) is a highly malignant disease that develops after asbestos exposure. Although the number of MPM cases is predicted to increase, no effective standard therapies have been established. The novel radiosensitizer α-sulfoquinovosyl-acylpropanediol (SQAP) enhances the effects of γ-radiation in human lung and prostate cancer cell lines and in animal models. In this study, we explored the radiosensitizing effect of SQAP and its mechanisms in MPM. The human MPM cell lines MSTO-211H and MESO-4 were implanted subcutaneously into the backs and thoracic cavities of immunodeficient KSN/Slc mice, then 2 mg/kg SQAP was intravenously administered with or without irradiation with a total body dose of 8 Gy. In both the orthotopic and ectopic xenograft murine models, the combination of irradiation plus SQAP delayed the implanted human MSTO-211H tumor growth. The analysis of the changes in the relative tumor volume of the MSTO-211H indicated a statistically significant difference after 8 Gy total body combined with 2 mg/kg SQAP, compared to both the untreated control (P = 0.0127) and the radiation treatment alone (P = 0.0171). After the treatment in each case, immunostaining of the harvested tumors revealed decreased cell proliferation, increased apoptosis and normalization of tumor blood vessels in the SQAP- and irradiation-treated group. Furthermore, hypoxia-inducible factor (HIF) 1 mRNA and protein expression were decreased, indicating reoxygenation in this group. In conclusion, SQAP improved hypoxic conditions in tumor tissue and may elicit a radiosensitizing effect in malignant mesothelioma models.

A case of tocilizumab-refractory idiopathic multicentric Castleman's disease successfully treated with sirolimus.

mTOR signaling may be a new therapeutic target for IL-6 inhibitor refractory iMCD-NOS.

The Role of Neuropeptide Y in Adipocyte-Macrophage Crosstalk during High Fat Diet-Induced Adipose Inflammation and Liver Steatosis.

Obesity is associated with an increased risk of non-alcoholic fatty liver disease (NAFLD), which is initiated by adipocyte-macrophage crosstalk. Among the possible molecules regulating this crosstalk, we focused on neuropeptide Y (NPY), which is known to be involved in hypothalamic appetite and adipose tissue inflammation and metabolism. In this study, the NPY-/- mice showed a marked decrease in body weight and adiposity, and lower free fatty acid and adipose inflammation without food intake alteration during a high fat diet (HFD). Moreover, NPY deficiency increased the thermogenic genes expression in brown adipose tissue. Notably, NPY-mRNA expression was upregulated in macrophages from the HFD mice compared to that from the mice on a standard diet. The NPY-mRNA expression also positively correlated with the liver mass/body weight ratio. NPY deletion alleviated HFD-induced adipose inflammation and liver steatosis. Hence, our findings point toward a novel intracellular mechanism of NPY in the regulation of adipocyte-macrophage crosstalk and highlight NPY antagonism as a promising target for therapeutic approaches against obesity and NAFLD.

Itpr1 regulates the formation of anterior eye segment tissues derived from neural crest cells.

Mutations in ITPR1 cause ataxia and aniridia in individuals with Gillespie syndrome (GLSP). However, the pathogenic mechanisms underlying aniridia remain unclear. We identified a de novo GLSP mutation hotspot in the 3'-region of ITPR1 in five individuals with GLSP. Furthermore, RNA-sequencing and immunoblotting revealed an eye-specific transcript of Itpr1, encoding a 218amino acid isoform. This isoform is localized not only in the endoplasmic reticulum, but also in the nuclear and cytoplasmic membranes. Ocular-specific transcription was repressed by SOX9 and induced by MAF in the anterior eye segment (AES) tissues. Mice lacking seven base pairs of the last Itpr1 exon exhibited ataxia and aniridia, in which the iris lymphatic vessels, sphincter and dilator muscles, corneal endothelium and stroma were disrupted, but the neural crest cells persisted after completion of AES formation. Our analyses revealed that the 218-amino acid isoform regulated the directionality of actin fibers and the intensity of focal adhesion. The isoform might control the nuclear entry of transcriptional regulators, such as YAP. It is also possible that ITPR1 regulates both AES differentiation and muscle contraction in the iris.

Increased In Vitro Intercellular Barrier Function of Lung Epithelial Cells Using Adipose-Derived Mesenchymal Stem/Stromal Cells.

With the emergence of coronavirus disease-2019, researchers have gained interest in the therapeutic efficacy of mesenchymal stem/stromal cells (MSCs) in acute respiratory distress syndrome; however, the mechanisms of the therapeutic effects of MSCs are unclear. We have previously reported that adipose-derived MSCs (AD-MSCs) strengthen the barrier function of the pulmonary vessels in scaffold-based bioengineered rat lungs. In this study, we evaluated whether AD-MSCs could enhance the intercellular barrier function of lung epithelial cells in vitro using a transwell coculture system. Transepithelial electrical resistance (TEER) measurements revealed that the peak TEER value was significantly higher in the AD-MSC coculture group than in the AD-MSC non-coculture group. Similarly, the permeability coefficient was significantly decreased in the AD-MSC coculture group compared to that in the AD-MSC non-coculture group. Immunostaining of insert membranes showed that zonula occuldens-1 expression was significantly high at cell junctions in the AD-MSC coculture group. Moreover, cell junction-related gene profiling showed that the expression of some claudin genes, including claudin-4, was upregulated in the AD-MSC coculture group. Taken together, these results showed that AD-MSCs enhanced the barrier function between lung epithelial cells, suggesting that both direct adhesion and indirect paracrine effects strengthened the barrier function of lung alveolar epithelium in vitro.

Cancer Awareness and Understanding of Students in Japan: What Do Students Having Close Relatives with Cancer Think About the Disease?

Students have become more familiar with cancer because of media, such as television or the Internet, reporting on celebrity cancer cases. Moreover, with Japan's increasing age and cancer rates, the number of students whose parents/relatives develop cancer is likely to increase. This study examined cancer awareness and understanding among students aged 10 to 16 or more. A cross-sectional nationwide survey was conducted using a self-administered questionnaire. Cancer awareness and cancer understanding were assessed using a self-administered questionnaire. We collected a total of 9139 questionnaires and excluded those with missing data. Thus, we analyzed the responses of 8701 students: 2135, 2902, and 3664 from elementary, junior, and high school, respectively. Data were analyzed using a multivariable model adjusted for gender and grade. Approximately 30% of respondents had parents/relatives with cancer. In addition, there was a significant association between having parents/relatives with cancer and cancer awareness; however, students having parents/relatives with cancer had more negative awareness (i.e., "I think cancer is scary," "I think I will get cancer in the future," and "I think cancer is preventable"). Furthermore, there was a significant association between cancer understanding and awareness. These findings suggest that cancer education could have a desirable effect on students whose parents/relatives have cancer. Further, cancer education offers benefits to students who are naive about cancer and ill prepared to cope when a family member discloses a cancer diagnosis.

Innovative approach to adolescent mental health in Japan: School-based education about mental health literacy.

AIMS: Improving mental health literacy through school-based education may encourage mental health promotion, prevention and care and reduce stigma in adolescents. In Japan, instruction about mental illness has been formulated in a Course of Study that reflects governmental curriculum guidelines, which will be enforced from 2022 to promote an understanding of current issues of adolescent health. Educational resources available to schoolteachers have been developed. This article describes the development processes and contents of these resources. METHODS: Our collaborating team, consisting of mental health professionals and schoolteachers, developed educational resources, based on feedback from high school students in general and young people who had experienced mental health problems. RESULTS: The new Course of Study covers: (1) mechanisms of mental illness, prevalence, age at onset, risk factors and treatability; (2) typical symptoms of mental health problems and illnesses; (3) self-help strategies for prevention of and recovery from mental illness; (4) enhancing help-seeking and helping behaviour and (5) decreasing stigma associated with people with mental health problems. The educational strategy is targeted at high school students (grades 10-12) and is conducted by teachers of health and physical education. The educational resources include short story animated films, filmed social contact and educators' manuals, which are freely available through the internet and open to all concerned including schoolteachers in Japan. CONCLUSIONS: Our efforts are expected to help implement mental health education of the public throughout Japan and other countries and promote the practice of early intervention and prevention of mental illnesses in adolescents.

Does Calorie Restriction Modulate Inflammaging via FoxO Transcription Factors?

Calorie restriction (CR) has been shown to extend lifespan and retard aging-related functional decline in animals. Previously, we found that the anti-neoplastic and lifespan-extending effects of CR in mice are regulated by forkhead box O transcription factors (FoxO1 and FoxO3), located downstream of growth hormone (GH)-insulin-like growth factor (IGF)-1 signaling, in an isoform-specific manner. Inflammaging is a term coined to represent that persistent low-level of inflammation underlies the progression of aging and related diseases. Attenuation of inflammaging in the body may underlie the effects of CR. Recent studies have also identified cellular senescence and activation of the nucleotide-binding domain, leucine-rich-containing family, pyrin-domain-containing-3 (NLRP3) inflammasome as causative factors of inflammaging. In this paper, we reviewed the current knowledge of the molecular mechanisms linking the effects of CR with the formation of inflammasomes, particularly focusing on possible relations with FoxO3. Inflammation in the brain that affects adult neurogenesis and lifespan was also reviewed as evidence of inflammaging. A recent progress of microRNA research was described as regulatory circuits of initiation and propagation of inflammaging. Finally, we briefly introduced our preliminary results obtained from the mouse models, in which Foxo1 and Foxo3 genes were conditionally knocked out in the myeloid cell lineage.

NPY Deficiency Prevents Postmenopausal Adiposity by Augmenting Estradiol-Mediated Browning.

The orexigenic hormone neuropeptide Y (NPY) plays a pivotal role in the peripheral regulation of fat metabolism. However, the mechanisms underlying the effects of sex on NPY function have not been extensively analyzed. In this study, we examined the effects of NPY deficiency on fat metabolism in male and female mice. Body weight was slightly decreased, whereas white adipose tissue (WAT) mass was significantly decreased as the thermogenic program was upregulated in NPY-/- female mice compared with that in wild-type mice; these factors were not altered in response to NPY deficiency in male mice. Moreover, lack of NPY resulted in an increase in luteinizing hormone (LH) expression in the pituitary gland, with concomitant activation of the estradiol-mediated thermogenic program in inguinal WAT, and alleviated age-related modification of adiposity in female mice. Taken together, these data revealed a novel intracellular mechanism of NPY in the regulation of fat metabolism and highlighted the sexual dimorphism of NPY as a promising target for drug development to reduce postmenopausal adiposity.

Effects of rikkunshito supplementation on resistance to oxidative stress and lifespan in mice.

AIM: Caloric restriction (CR), which limits the caloric intake to 60-70% of ad libitum (AL) amounts in various experimental animals, delays aging and extends the lifespan. We previously showed that neuropeptide Y (NPY), an appetite-stimulating peptide, is essential for the anti-oxidative and life-extending effects of CR. Here, we investigated whether a Japanese traditional herbal medicine, rikkunshito (RKT), which induces NPY activation, has CR-like life-extending effects. METHODS: First, we evaluated the life-extending activity of RKT by examining the effect of long-term RKT administration on wild-type and NPY knockout mice. Furthermore, we tested whether RKT enhances CR-mediated beneficial effects under AL conditions with a normal diet and under mild CR conditions with a high-fat diet. We then used 3-nitropropionic acid or doxorubicin to induce oxidative stress, and analyzed the differences in survival rate, weight loss, gene expression and cellular oxidative damage among groups. RESULTS: RKT administration did not extend the lifespan of wild-type or NPY knockout mice. In the oxidative stress models, RKT treatment upregulated anti-oxidative gene expression in the liver. Furthermore, RKT administration reduced the oxidative damage in the liver compared to the CR conditions alone. However, on induction of oxidative stress by 3-nitropropionic acid or doxorubicin, RKT administration did not affect the survival rate. CONCLUSIONS: These results show that RKT administration only partially mimics the effects of CR at the cellular level, but not at the organismal level to increase the lifespan of mice. Geriatr Gerontol Int 2019; ••: ••-••.

Identification of Specific miRNAs in Neutrophils of Type 2 Diabetic Mice: Overexpression of miRNA-129-2-3p Accelerates Diabetic Wound Healing.

Neutrophils are involved in the first stage of acute inflammation. After injury, they are mobilized and recruited to the injured tissue. In diabetes, wound healing is delayed and aberrant, leading to excessive recruitment and retention of neutrophils that fail to promote angiogenesis and prolong inflammation. However, the exact pathological mechanisms of diabetic-derived neutrophils in chronic inflammation remain unclear. Here, miRNA profiling of neutrophils from bone marrow in type 2 diabetic mice was performed using a microarray. miRNAs regulate the posttranscriptional expression of target mRNAs and are important in countering inflammation-related diseases. Our study revealed that miRNAs exhibit differential expression in diabetic-derived neutrophils compared with non-diabetic-derived neutrophils, especially miR-129 family members. miR-129-2-3p directly regulated the translation of Casp6 and Ccr2, which are involved in inflammatory responses and apoptosis. Furthermore, miR-129-2-3p overexpression at the wound site of type 2 diabetic mice accelerated wound healing. These results suggest possible involvement of miR-129-2-3p in diabetic-derived neutrophil dysfunction and that retention kinetics of neutrophils and chronic inflammation may be initiated through miR-129-2-3p-regulated genes. This study characterizes changes in global miRNA expression in diabetic-derived neutrophils and systematically identifies critical target genes involved in certain biological processes related to the pathology of diabetic wound healing.

Mechanisms of Calorie Restriction: A Review of Genes Required for the Life-Extending and Tumor-Inhibiting Effects of Calorie Restriction.

This review focuses on mechanisms of calorie restriction (CR), particularly the growth hormone (GH)/insulin-like growth factor-1 (IGF-1) axis as an evolutionary conserved signal that regulates aging and lifespan, underlying the effects of CR in mammals. Topics include (1) the relation of the GH-IGF-1 signal with chronic low-level inflammation as one of the possible causative factors of aging, that is, inflammaging, (2) the isoform specificity of the forkhead box protein O (FoxO) transcription factors in CR-mediated regulation of cancer and lifespan, (3) the role for FoxO1 in the tumor-inhibiting effect of CR, (4) pleiotropic roles for FoxO1 in the regulation of disorders, and (5) sirtuin (Sirt) as a molecule upstream of FoxO. From the evolutionary view, the necessity of neuropeptide Y (Npy) for the effects of CR and the pleiotropic roles for Npy in life stages are also emphasized. Genes for mediating the effects of CR and regulating aging are context-dependent, particularly depending on nutritional states.

Targeting miR-223 in neutrophils enhances the clearance of Staphylococcus aureus in infected wounds.

Argonaute 2 bound mature microRNA (Ago2-miRNA) complexes are key regulators of the wound inflammatory response and function in the translational processing of target mRNAs. In this study, we identified four wound inflammation-related Ago2-miRNAs (miR-139-5p, miR-142-3p, miR-142-5p, and miR-223) and show that miR-223 is critical for infection control. miR-223Y/- mice exhibited delayed sterile healing with prolonged neutrophil activation and interleukin-6 expression, and markedly improved repair of Staphylococcus aureus-infected wounds. We also showed that the expression of miR-223 was regulated by CCAAT/enhancer binding protein alpha in human neutrophils after exposure to S. aureus peptides. Treatment with miR-223Y/--derived neutrophils, or miR-223 antisense oligodeoxynucleotides in S. aureus-infected wild-type wounds markedly improved the healing of these otherwise chronic, slow healing wounds. This study reveals how miR-223 regulates the bactericidal capacity of neutrophils at wound sites and indicates that targeting miR-223 might be of therapeutic benefit for infected wounds in the clinic.

Identification and functional analysis of inflammation-related miRNAs in skin wound repair.

Inflammation at a wound site is essential for preventing infection. However, misregulated inflammation leads to pathologies of the healing process, including chronic non-healing wounds and scarring. MicroRNAs (miRNAs) are key regulators of the inflammatory response and tissue repair, acting by translational processing of target mRNAs. In the final step of miRNA processing, Argonaute 2 (Ago2)-bound mature miRNA complexes bind to target mRNAs and inhibit their translation. A variety of wound healing-related miRNAs have been identified and their misregulation likely contributes to wound pathologies, including scarring and chronic healing. Recently, we have developed an Ago2-bound mature miRNA purification system that uses Ago2 antibody to analyze the expression of miRNAs from wound tissues by microarray and next generation sequencing. We have identified several wound inflammation-related miRNAs via Ago2-target immunoprecipitation assays and next generation sequencing of wound tissues from wild-type and PU.1 knockout mice, which exhibit no inflammatory response because of a lack of immune cell lineages. We demonstrated that miR-142, an identified inflammation-related miRNA, is essential role for neutrophilic chemotaxis via inhibition of small GTPase translation; its misregulation leads to susceptibility to infection against Staphylococcus aureus at skin wound sites. In this review, we summarize recent advances of miRNA studies in skin wound healing, introduce our miRNA purification system using an immunoprecipitation assay method, and discuss the function of miR-142 in skin wound healing.

A Cross-sectional Investigation of Cancer-Screening Intentions, Sources of Information, and Understanding of Cancer in Japanese Adolescents.

The purpose of this study was to describe the cancer-screening intention, sources of cancer information, and cancer understanding among Japanese adolescents. A cross-sectional nationwide survey involving a self-administered questionnaire was conducted. Response rates of the target schools were 46.4 % (n = 103) for junior high schools and 55.8 % (n = 116) for high schools. From these, we analyzed the data of 2960 junior high school students (1520 males, 1440 females) and 3703 high school students (1546 males, 2157 females) to examine the association between cancer-screening intention and sources of cancer-related information and understanding. A significant association between cancer-screening intention and sources of cancer information and cancer understanding was observed. The screening intention group identified more sources of cancer information than the no-screening intention group did. Understanding about cancer was reported by a higher proportion of students in the screening intention group compared with the no-screening intention group. Recognition that healthy people must take part in cancer screening was significantly associated with screening intention in both junior high (odds ratio (OR), 1.859; 95 % confidence interval (CI), 1.582-2.185; P < 0.001) and high school (OR, 2.485; 95 % CI, 2.139-2.887; P < 0.001) students. Health education at school was indicated by a high proportion of students as a source of cancer-related information, although the association was not significant. The present survey indicated that those in of our sample who intended to undergo future cancer screening (67.8 %) had more sources of information and understanding regarding cancer. Thus, schools should enrich health education curricula with more information and understanding about cancer to promote cancer-screening intention among Japanese adolescents.