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.

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.

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.

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.

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.

Reduced FOXO1 expression accelerates skin wound healing and attenuates scarring.

The forkhead box O (FOXO) family has been extensively investigated in aging and metabolism, but its role in tissue-repair processes remains largely unknown. Herein, we clarify the molecular aspect of the FOXO family in skin wound healing. We demonstrated that Foxo1 and Foxo3a were both up-regulated during murine skin wound healing. Partial knockout of Foxo1 in Foxo1(+/-) mice throughout the body led to accelerated skin wound healing with enhanced keratinocyte migration, reduced granulation tissue formation, and decreased collagen density, accompanied by an attenuated inflammatory response, but we observed no wound phenotype in Foxo3a(-/-) mice. Fibroblast growth factor 2, adiponectin, and notch1 genes were significantly increased at wound sites in Foxo1(+/-) mice, along with markedly altered extracellular signal-regulated kinase 1/2 and AKT phosphorylation. Similarly, transient knockdown of Foxo1 at the wound site by local delivery of antisense oligodeoxynucleotides enhanced skin wound healing. The link between FOXO1 and scarring extends to patients, in particular keloid scars, where we see FOXO1 expression markedly increased in fibroblasts and inflammatory cells within the otherwise normal dermis. This occurs in the immediate vicinity of the keloid by comparison to the center of the mature keloid, indicating that FOXO1 is associated with the overgrowth of this fibrotic response into adjacent normal skin. Overall, our data indicate that molecular targeting of FOXO1 may improve the quality of healing and reduce pathological scarring.

NPY antagonism reduces adiposity and attenuates age-related imbalance of adipose tissue metabolism.

An orexigenic hormone, neuropeptide Y (NPY), plays a role not only in the hypothalamic regulation of appetite, but also in the peripheral regulation of lipid metabolism. However, the intracellular mechanisms triggered by NPY to regulate lipid metabolism are poorly understood. Here we report that NPY deficiency reduces white adipose tissue (WAT) mass and ameliorates the age-related imbalance of adipose tissue metabolism in mice. Gene expression involved in adipogenesis/lipogenesis was found to decrease, whereas proteins involved in lipolysis increased in gonadal WAT (gWAT) of NPY-knockout mice. These changes were associated with an activated SIRT1- and PPARγ-mediated pathway. Moreover, the age-related decrease of de novo lipogenesis in gWAT and thermogenesis in inguinal WAT was inhibited by NPY deficiency. Further analysis using 3T3-L1 cells showed that NPY inhibited lipolysis through the Y1 receptor and enhanced lipogenesis following a reduction in cAMP response element-binding protein (CREB) and SIRT1 protein expression. Therefore, NPY appears to act as a key regulator of adipose tissue metabolism via the CREB-SIRT1 signaling pathway. Taken together, NPY deficiency reduces adiposity and ameliorates the age-related imbalance of adipose tissue metabolism, suggesting that antagonism of NPY may be a promising target for drug development to prevent age-related metabolic diseases.

Cancer understanding among Japanese students based on a nationwide survey.

OBJECTIVE: The objective of this study was to determine cancer understanding among Japanese primary and secondary school students. METHODS: The study design was a cross-sectional nationwide survey using a self-administered questionnaire. The prefecture with the lowest student population was set to 1, and that with the highest student population was set to 18 for elementary schools and 19 for junior high and high schools based on the ratio of the student population. In this way, 213 elementary schools, 222 junior high schools, and 208 high schools were selected from all 47 prefectures in Japan, and questionnaires were sent to each school. The questionnaire listed the names of 15 cancers and asked respondents to choose one answer from three: "Never heard of," "Heard of/Don't understand," or "Heard of/Understand." RESULTS: Response rates for schools were 44.1 % (n = 94) for elementary schools, 46.4 % (n = 103) for junior high schools, and 55.8 % (n = 116) for high schools. A total of 8,876 questionnaires were used for the analysis. Our survey suggests that the most commonly understood types of cancer differed by grade, with lung cancer the most commonly understood in elementary school, leukemia in junior high schools, and breast cancer in high schools. Girls tended to demonstrate greater cancer understanding than boys, with particularly large differences by gender in rates of understanding of breast and uterine cancer at each assessed grade level. CONCLUSIONS: Here, we examined Japanese primary and secondary school students. Marked differences in cancer recognition by grade and gender suggest that educational efforts are needed at various grade levels and gender-specific cancer education. Further, more than 50 % of students at any school level were not familiar with most cancers. It suggests that cancer education is deficient.

Tunneling nanotube-driven complete regeneration of murine fetal skin.

This study investigated the three-dimensional (3D) cellular interactions and tunneling nanotubes (TNTs) during fetal mouse skin regeneration on embryonic days 13 (E13) and 15 (E15). We aimed to understand spatial relationships among cell types involved in skin regeneration and assess the potential role of TNTs. Full-thickness skin incisions were performed in E13 and E15 embryos. Wound sites were collected, embedded in epoxy resin, processed for 3D reconstruction (1 µm thickness sections), and subjected to whole-mount immunostaining. We conducted in vitro co-culture experiments with fetal macrophages and fibroblasts to observe TNT formation. To assess the effect of TNTs on skin regeneration, an inhibiting agent (cytochalasin B) was administered to amniotic fluid. Results revealed that E13 epidermal keratinocytes interacted with dermal fibroblasts and macrophages, facilitating skin regrowth. TNT structures were observed at the E13-cell wound sites, among macrophages, and between macrophages and fibroblasts, confirmed through in vitro co-culture experiments. In vitro and utero cytochalasin B administration hindered those formation and inefficient skin texture regeneration at E13 wound sites. This emphasizes the necessity of 3D cellular interactions between epidermal and dermal cells during skin regeneration in mouse embryos at E13. The prevalence of TNT structures indicated their involvement in achieving complete skin texture restoration.

Release of TNF-α from macrophages is mediated by small GTPase Rab37.

Activated macrophages at wound sites release many cytokines which positively affect skin wound healing. However, the molecular mechanisms controlling cytokine secretion from macrophages have not been elucidated. In the present study, we performed an RT-PCR analysis and found that 19 small GTPase Rab isoforms were expressed at skin wound sites, with six of them (i.e. Rab3B, Rab27B, Rab30, Rab33A, Rab37, and Rab40C) being upregulated during the inflammation and proliferation/migration phase of skin repair. We also found that gene expression of Rab37 in murine primary and RAW264.7 macrophages was significantly induced after stimulation with LPS. Overexpression of wild type and constitutively active Rab37 in RAW264.7 cells significantly increased TNF-α secretion, whereas knockdown of Rab37 by siRNA significantly decreased it. We also identified 29 putative Rab37-interacting proteins, including the membrane fusion regulating Munc13-1, using liquid chromatography/linear ion trap mass spectrometry (LC-MS/MS). Immunocytochemical analysis further revealed that TNF-α-containing vesicles were colocalized with both Rab37 and Munc13-1 in activated macrophages. Knockdown of Munc13-1 by siRNA significantly decreased TNF-α secretion. Taken together, these findings demonstrate that Rab37 interacts with Munc13-1 to control TNF-α secretion from activated macrophages.

Protein reporter bioassay systems for the phenotypic screening of candidate drugs: a mouse platform for anti-aging drug screening.

Recent drug discovery efforts have utilized high throughput screening (HTS) of large chemical libraries to identify compounds that modify the activity of discrete molecular targets. The molecular target approach to drug screening is widely used in the pharmaceutical and biotechnology industries, because of the amount of knowledge now available regarding protein structure that has been obtained by computer simulation. The molecular target approach requires that the structure of target molecules, and an understanding of their physiological functions, is known. This approach to drug discovery may, however, limit the identification of novel drugs. As an alternative, the phenotypic- or pathway-screening approach to drug discovery is gaining popularity, particularly in the academic sector. This approach not only provides the opportunity to identify promising drug candidates, but also enables novel information regarding biological pathways to be unveiled. Reporter assays are a powerful tool for the phenotypic screening of compound libraries. Of the various reporter genes that can be used in such assays, those encoding secreted proteins enable the screening of hit molecules in both living cells and animals. Cell- and animal-based screens enable simultaneous evaluation of drug metabolism or toxicity with biological activity. Therefore, drug candidates identified in these screens may have increased biological efficacy and a lower risk of side effects in humans. In this article, we review the reporter bioassay systems available for phenotypic drug discovery.

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.

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.

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.

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.

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.

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.

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.

Development of a bioassay to screen for chemicals mimicking the anti-aging effects of calorie restriction.

Suppression of the growth hormone/insulin-like growth factor-I pathway in Ames dwarf (DF) mice, and caloric restriction (CR) in normal mice extends lifespan and delays the onset of age-related disorders. In combination, these interventions have an additive effect on lifespan in Ames DF mice. Therefore, common signaling pathways regulated by DF and CR could have additive effects on longevity. In this study, we tried to identity the signaling mechanism and develop a system to assess pro-longevity status in cells and mice. We previously identified genes up-regulated in the liver of DF and CR mice by DNA microarray analysis. Motif analysis of the upstream sequences of those genes revealed four major consensus sequence motifs, which have been named dwarfism and calorie restriction-responsive elements (DFCR-REs). One of the synthesized sequences bound to hepatocyte nuclear factor-4α (HNF-4α), an important transcription factor involved in liver metabolism. Furthermore, using this sequence information, we developed a highly sensitive bioassay to identify chemicals mimicking the anti-aging effects of CR. When the reporter construct, containing an element upstream of a secreted alkaline phosphatase (SEAP) gene, was co-transfected with HNF-4α and its regulator peroxisome proliferator-activated receptor (PPAR) γ coactivator-1α (PGC-1α), SEAP activity was increased compared with untransfected controls. Moreover, transient transgenic mice established using this construct showed increased SEAP activity in CR mice compared with ad libitum-fed mice. These data suggest that because of its rapidity, ease of use, and specificity, our bioassay will be more useful than the systems currently employed to screen for CR mimetics, which mimic the beneficial effects of CR. Our system will be particularly useful for high-throughput screening of natural and synthetic candidate molecules.