SOX4 reversibly induces phenotypic changes by suppressing the epithelial marker genes in human keratinocytes.

BACKGROUND: SOX4 is a transcription factor belonging to the SOX (Sry-related High Mobility Group [HMG] box) family and plays a pivotal role in various biological processes at various stages of life. SOX4 is also expressed in the skin in adults and has been reported to be involved in wound healing, tumor formation, and metastasis. METHODS AND RESULTS: In this study, we investigated the role of SOX4 in keratinocyte phenotypic changes. We generated a SOX4-overexpressing keratinocyte cell line that expresses SOX4 in a doxycycline (DOX)-inducible manner. DOX treatment induced a change from a paving stone-like morphology to a spindle-like morphology under microscopic observation. Comprehensive gene analysis by RNA sequencing revealed increased expression of genes related to anatomical morphogenesis and cell differentiation as well as decreased expression of genes related to epithelial formation and keratinization, suggesting that SOX4 induced EMT-like phenotype in keratinocytes. Differentially expressed genes (DEGs) obtained by RNA-seq were confirmed using qRT-PCR. DOX-treated TY-1 SOX4 showed a decrease in the epithelial markers (KRT15, KRT13, KRT5, and CLDN1) and an increase in the mesenchymal marker FN1. Protein expression changes by Western blotting also showed a decrease in the epithelial marker proteins keratin 15, keratin 13, and claudin 1, and an increase in the mesenchymal marker fibronectin. Removal of DOX from DOX-treated cells also restored the epithelial and mesenchymal markers altered by SOX4. CONCLUSION: Our results indicate that SOX4 reversibly induces an EMT-like phenotype in human keratinocytes via suppression of epithelial marker genes.

SNAI2 is induced by transforming growth factor-ß1, but is not essential for epithelial-mesenchymal transition in human keratinocyte HaCaT cells.

Epithelial-mesenchymal transition (EMT) is a cellular process in which epithelial cells lose their epithelial traits and shift to the mesenchymal phenotype, and is associated with various biological events, such as embryogenesis, wound healing and cancer progression. The transcriptional program that promotes phenotype switching is dynamically controlled by transcription factors during EMT, including Snail (SNAI1), twist family bHLH transcription factor (TWIST) and zinc finger E-box binding homeobox 1 (ZEB1). The present study aimed to investigate the molecular mechanisms underlying EMT in squamous epithelial cells. Western blot analysis and immunocytochemical staining identified Slug (SNAI2) as a transcription factor that is induced during transforming growth factor (TGF)-ß1-mediated EMT in the human keratinocyte cell line HaCaT. The effect of SNAI2 overexpression and knockdown on the phenotypic characteristics of HaCaT cells was evaluated. Filamentous actin staining and western blot analysis revealed that the overexpression of SNAI2 did not induce the observed EMT-related phenotypic changes. In addition, SNAI2 knockdown demonstrated almost no impact on the EMT phenotypes induced by TGF-ß1. Notably, DNA microarray analysis followed by comprehensive bioinformatics analysis revealed that the differentially expressed genes upregulated by TGF-ß1 were significantly enriched in cell adhesion and extracellular matrix binding, whereas the genes downregulated in response to TGF-ß1 were significantly enriched in the cell cycle. No enriched gene ontology term and biological pathways were identified in the differentially expressed gene sets of SNAI2-overexpressing cells. In addition, the candidates for master transcription factors regulating the TGF-ß1-induced EMT were identified using transcription factor enrichment analysis. In conclusion, the results of study demonstrated that SNAI2 does not play an essential role in the EMT of HaCaT cells and identified candidate transcription factors that may be involved in EMT-related gene expression induced by TGF-ß1. These findings may enhance the understanding of molecular events in EMT and contribute to the development of a novel therapeutic approach against EMT in cancers and wound healing.

Monitoring Chilecomadia valdiviana (Lepidoptera: Cossidae) Using Sex Pheromone-Baited Traps in Apple Orchards in Chile.

Chilecomadia valdiviana (Philippi) (Lepidoptera: Cossidae) is a native xylophagous pest in apple orchards in Chile. A series of experiments evaluated the efficacy of trap type, sex pheromone (Z7,Z10-16:Ald) dose, and trap location in the apple tree canopy on trap catch of male adults. Bucket traps (6 L), with and without roof and cross vane spacers, together with bucket traps (20 L) without roof and spacers, showed higher catches among the four types of traps evaluated. In a second experiment, the UNI-trap and Delta trap showed higher catches than Multipher, wing, and bucket traps (6 L). Male catches were not affected by height when tested at 0, 1.5, and 3 m in the canopy. A 300 µg dose of Z7,Z10-16:Ald showed higher catch than the control treatment. This dose allowed monitoring of male flight of C. valdiviana for at least five weeks in apple orchards in Chile. Based on relative trap costs, we propose the use of 6 L bucket traps for male mass trapping, while Delta traps can be used for monitoring of male flight. We found that male flight of C. valdiviana occurred mainly from mid-August to late November, reaching its maximum in mid-September.

Association between Occupational Dysfunction and Metabolic Syndrome in Community-Dwelling Japanese Adults in a Cross-Sectional Study: Ibara Study.

The purpose of this study was to investigate the relationship between occupational dysfunction and metabolic syndrome (MetS) and its component factors in community-dwelling Japanese adults (N = 1,514). Self-reported lifestyle behaviors, Classification and Assessment of Occupational Dysfunction (CAOD) scores, and metabolic traits were measured. CAOD levels were divided into tertiles (low, moderate, and high), and their associations with MetS and its components were evaluated through logistic regression analysis. The association of MetS with CAOD was demonstrated in the total number of individuals [OR = 1.92 (95% CI 1.17⁻3.17)] and in older individuals [OR = 1.90 (95% CI 1.04⁻3.46)]. The association of dyslipidemia and CAOD was evident for overweight individuals [OR = 2.08 (95% CI 1.17⁻3.68)]. A higher association of high blood pressure with CAOD was evidenced in younger individuals [OR = 2.02 (95% CI 1.05⁻3.89)] who belonged to the highest-CAOD-score group in comparison to those who registered the lowest-CAOD-score group. The evaluation of MetS and interventions related to its prevention may be more effective if the viewpoint of occupational dysfunction is taken into account.

Influence of Age on Cardiorespiratory Kinetics During Sinusoidal Walking in Humans.

We sought to determine the influence of age on cardiorespiratory kinetics during sinusoidal walking in two groups: 13 healthy young subjects (YG; 7 men and 6 women, age 21 ± 2 years) and 15 healthy elderly subjects (ELD; 9 men and 6 women, age 67 ± 5 years). A treadmill's speed was sinusoidally changed between 3 and 6 km h-1 in the YG and between 3 and 5 km h-1 in the ELD during periods of 1, 2, 5, and 10 min, and in a stepwise manner. We compared the groups' heart rate (HR), ventilation ( V˙E), and gas exchange (CO2 output ( V˙ CO2) and O2 uptake ( V˙ O2)) responses. We determined the phase shift (PS) and the normalized amplitude (Amp) ratio of these kinetics in relation to the sinusoidal change in walking speed in response to the magnitude from the maximum to minimum speeds as revealed by a Fourier analysis in all cardiorespiratory variables. Both the Amp ratio and PS in the V˙E, V˙ CO2, and V˙ O2 responses were very similar between the ELD and YG, and being independent of the periods of sinusoidal oscillations. In marked contrast, the PS of the HR kinetics was significantly slowed in the ELD compared to the YG. The Amp ratio of HR was not related to the covariance variation of HR (CVHR) at standing rest in the ELD. The HR kinetics during sinusoidal walking may not be attributable to parasympathetic nerve activity into the heart in the ELD. The slope of the Amp of V˙E related to the Amp of V˙ CO2 ( V˙E/ V˙ CO2 slope) was steeper in the ELD (0.0258) compared to the YG (0.0132), suggesting that exercise hyperpnea could be greatly induced during walking in the ELD. These findings suggest that aging influences the alterations of autonomic nervous system-dependent slower HR kinetics and exercise hyperpnea during walking in the ELD.

TRPV2 channel inhibitors attenuate fibroblast differentiation and contraction mediated by keratinocyte-derived TGF-ß1 in an in vitro wound healing model of rats.

BACKGROUND: Keratinocytes release several factors that are involved in wound contracture and scar formation. We previously reported that a three-dimensional reconstruction model derived from rat skin represents a good wound healing model. OBJECTIVE: We characterized the role of transient receptor potential (TRP) channels in the release of transforming growth factor (TGF)-ß1 from keratinocytes and the differentiation of fibroblasts to identify possible promising pharmacological approaches to prevent scar formation and contractures. METHODS: The three-dimensional culture model was made from rat keratinocytes seeded on a collagen gel in which dermal fibroblasts had been embedded. RESULTS: Among the TRP channel inhibitors tested, the TRPV2 inhibitors SKF96365 and tranilast attenuated most potently keratinocyte-dependent and - independent collagen gel contraction due to TGF-ß signaling as well as TGF-ß1 release from keratinocytes and α-smooth muscle actin production in myofibroblasts. Besides the low amounts detected in normal dermis, TRPV2 mRNA and protein levels were increased after fibroblasts were embedded in the gel. TRPV2 was also expressed in the epidermis and keratinocyte layers of the model. Both inhibitors and TRPV2 siRNA attenuated the intracellular increase of Ca2+ induced by the TRPV agonist 2-aminoethoxydiphenyl borate in TGF-ß1-pretreated fibroblasts. CONCLUSION: This is the first study to show that compounds targeting TRPV2 channels ameliorate wound contraction through the inhibition of TGF-ß1 release and the differentiation of dermal fibroblasts in a culture model.

Keratin 13 gene is epigenetically suppressed during transforming growth factor-ß1-induced epithelial-mesenchymal transition in a human keratinocyte cell line.

Epithelial-mesenchymal transition (EMT) is a biological event in which epithelial cells lose their polarity and cell-cell adhesions and concomitantly acquire mesenchymal traits, and is thought to play an important role in pathological processes such as wound healing and cancer progression. In this study, we evaluated transforming growth factor (TGF)-ß1-treated human keratinocyte HaCaT cells as an in vitro model of EMT. HaCaT cells were changed into an elongated fibroblast-like morphology, which is indicative of EMT in response to TGF-ß1. Phalloidin staining demonstrated the formation of actin stress fibers in TGF-ß1-treated cells. Quantitative RT-PCR analysis revealed that TGF-ß1 increased the mRNA levels of EMT transcription factors (SNAI2, TWIST1, and ZEB1) and mesenchymal markers (CDH2, VIM, and FN1), while it decreased the transcripts of epithelial phenotypic genes (CLDN1, OCLN, KRT5, KRT15, KRT13, and TGM1). Furthermore, we found that KRT13 was drastically suppressed through the reduction of RNA polymerase II occupancy of its promoter, which was accompanied by a decrease in active histone marks (H3K4me3 and H3K27ac) and an increase in a repressive mark (H3K27me3) during EMT. These findings indicate that the TGF-ß1-induced EMT program regulates a subset of epithelial and mesenchymal marker genes, and that KRT13 is transcriptionally suppressed through the modulation of the chromatin state at the KRT13 promoter in HaCaT cells.

Use of Mixture Designs to Investigate Contribution of Minor Sex Pheromone Components to Trap Catch of the Carpenterworm Moth, Chilecomadia valdiviana.

Field experiments were carried out to study responses of male moths of the carpenterworm, Chilecomadia valdiviana (Lepidoptera: Cossidae), a pest of tree and fruit crops in Chile, to five compounds previously identified from the pheromone glands of females. Previously, attraction of males to the major component, (7Z,10Z)-7,10-hexadecadienal, was clearly demonstrated while the role of the minor components was uncertain due to the use of an experimental design that left large portions of the design space unexplored. We used mixture designs to study the potential contributions to trap catch of the four minor pheromone components produced by C. valdiviana. After systematically exploring the design space described by the five pheromone components, we concluded that the major pheromone component alone is responsible for attraction of male moths in this species. The need for appropriate experimental designs to address the problem of assessing responses to mixtures of semiochemicals in chemical ecology is described. We present an analysis of mixture designs and response surface modeling and an explanation of why this approach is superior to commonly used, but statistically inappropriate, designs.

The effect of trunk coordination exercise on dynamic postural control using a Core Noodle.

OBJECTIVE: To investigate the influence of trunk coordination exercise on dynamic postural control relative to postural sway. METHOD: The effects of trunk coordination exercises were examined using a Core Noodle for the postural sway in healthy students who were assigned to an exercise or control group. The independent variable was the extent of exposure to Core Noodle exercise, and the dependent variable was dynamic postural control. A stabilometer, which measures dynamic postural control, was used to evaluate the effectiveness of the exercises. In addition, center of gravity movements were assessed using a Gravicorder G-620 stabilometer in which the subject was asked to shift their center of gravity between 2 circles on a computer monitor. Pre- and post-intervention dynamic postural control was statistically evaluated between the exercise group and control group using the Mann-Whitney test. Finally, we investigated the application of these exercises for a stroke patient. RESULTS AND CONCLUSION: For post-intervention, the envelop area, mean length of the pathways between 2 circles, and the number of circles were significantly higher in the exercise group. Trunk coordination exercise performed Core Noodle may be used to enhance the dynamic postural balance of healthy young adults, and it can also be adapted for stroke patients.

Core exercises elevate trunk stability to facilitate skilled motor behavior of the upper extremities.

OBJECTIVE: The purpose of this study was to investigate the influence of core exercises on upper extremity function relative to skilled motor behavior and postural sway. METHOD: We examined the effects of core exercises on the skilled motor behavior and postural sway of 40 healthy students who were assigned randomly to the core exercise group or the control group. Independent variable is extent of exposure to core exercise and dependent variables are skilled motor behavior and postural sway. A Purdue pegboard which measures skilled motor behavior and a stabilometer which measures postural sway were used to evaluate the influence of core exercises. Pre-intervention and post-intervention skilled motor behavior and postural sway were compared between the core exercise group and control group using the Wilcoxon rank sum test; a significance level of α = 0.05 was considered statistically significant. Also, we investigated the application of core exercises in a clinical setting for one patient with cerebral vascular disease. RESULTS: The post intervention skilled motor behavior (p = 0.04) and postural sway, LNG (p = 0.05), LNG/TIME (p = 0.04) and X LNG (p = 0.02) were significantly higher in the core exercise group than control group. In the case report, there were good results; function of the upper extremity improved after doing the exercises. There were positive changes in some daily living activities. DISCUSSION: Core exercises are likely to enhance trunk stabilization to improve upper extremity function. It is possible for core exercises to be adapted for patients.

[Examination of onset cases in contact investigation].

OBJECTIVES: To analyze and evaluate onset cases of tuberculosis detected in contact investigations and to apply the results to future countermeasures. METHODS: Index and secondary cases in contact investigations in which the secondary cases occurred in Osaka City between 2005 and 2008 were enrolled. The tuberculin skin test or QFT (QuantiFERON-TB Gold) was conducted to diagnose whether the contacts were infected with tuberculosis. X-ray examination of the chest was conducted to determine whether tuberculosis had developed, immediately or 6 months, 1 year or 2 years after the contact investigation. RESULTS: (1) Index cases: Index cases followed by secondary cases numbered 131 patients. Regarding the chest X-ray findings, a cavity was observed in 67.7% of the index cases, and a sputum smear of 3 + was observed in 51.5% of the index cases. A 3-month or longer delay in index case-finding was noted in 50.4% of the index cases. (2) Secondary cases: Secondary cases numbered 177 patients, consisting of 107 patients who showed an onset of less than 6 months after their last contact with index cases. Of 70 secondary cases in whom tuberculosis was detected in the investigation conducted 6 months to 2 years after the contact investigation, 50% of them were not tested for infection due to their older age, consisting of 11 patients in their 40's, 8 in their 50's, and 16 in their 60's or over, and 17.1% of them refused or discontinued the treatment for latent tuberculosis infection, leading to onset. DISCUSSION: Many secondary cases were detected immediately after the contact investigation, suggesting the importance of reducing the delay in index case-finding. Regarding the onset of secondary cases who showed an onset 6 months after the last contact with index cases, many cases showed an onset without being tested for infection due to their older age. More attention should be paid to the cases who refused or discontinued the treatment for latent tuberculosis infection, leading to onset.

[Comparative performance of tuberculin skin test and QuantiFERON TB-gold in contact investigations for tuberculosis].

OBJECTIVES: To evaluate the performances of the QuantiFERON TB-Gold assay (QFT) and tuberculin skin test (TST) and to examine how a latent tuberculosis infection (LTBI) should be diagnosed in contact investigations of children aged 6 to 17 years. METHODS: A total of 232 boys and girls aged 6 to 17 years who were in contact with 134 culture-confirmed pulmonary tuberculosis patients (index cases) were examined both with QFT and TST. Factors influencing the results of the tests and their interactions were evaluated with multivariate analyses. RESULTS: Two variables (whether household contact and with/without contact with a cavitary disease patient) were found to significantly predict a positive QFT result. Positive TST defined with erythema being either greater than 20 mm or 30mm correlated significantly with two variables (whether household contact and with/without contact with a smear positive patient). There was moderate agreement between QFT and TST (positivity defined as with erythema greater than 30mm), with kappa=0.49, for contacts aged 6 to 11 years. Among contacts aged 6 to 11 years, 14 had a negative QFT result and TST with erythema greater than 30 mm. Of these 14, 7 contacts (50%) of smear positive index case were not indicated for LTBI treatment. CONCLUSIONS: When diagnosing LTBI among contacts aged 6 to 11 years who show negative QFT and strong TST reactions, we should take into consideration the factors related with a higher probability to the risk of infection. Because a history of past BCG vaccination is more likely to affect TST results in those aged 12 to 17 years than in younger subjects, greater care must be taken when evaluating the TST of these contacts.

[Examination of latent tuberculosis infection treatment status].

OBJECTIVE: To clarify factors involved in the refusal or discontinuation of treatment for latent tuberculosis infection (LTBI), we reviewed LTBI treatment. METHOD: The subjects were 193 patients for whom LTBI treatment was indicated on a family contact investigation in 2006. We examined the subjects' backgrounds and reasons for treatment refusal or discontinuation. In addition, we investigated the incidence of the onset of tuberculosis within 2 years after the final contact with the source of infection. RESULTS: i) Patient background: The state of treatment could be evaluated in 185 patients. Of these, drug therapy was completed in 138 (75%), whereas 47 (25%) refused or discontinued treatment. The mean ages of the former and latter were 21.0 and 26.2 years, respectively, showing a significant difference. Concerning the state of contact, 9 (8%) of 114 patients who had lived with and 12 (17%) of 71 who had lived apart from the source of infection refused treatment, showing a significant difference. ii) Onset within 2 years: We analyzed 180 patients for whom follow-up was possible. Drug therapy was completed in 137 patients. No patient developed tuberculosis onset. On the other hand, drug therapy was not completed in 43 patients, and 6 (14%) developed tuberculosis onset. iii) The reasons were investigated in 47 patients who did not complete drug therapy. There were 15 episodes associated with side effects, followed by 14 related to personal circumstances, such as being busy, disliking medicines, and the absence of confidence regarding the completion of drug therapy, and 6 associated with either insufficient explanations regarding the diagnosis or an absence of symptoms. CONCLUSION: Treatment refusal/discontinuation was more frequent in the more advanced age groups and patients who had lived apart from the source of infection. It may therefore be necessary to more closely explain the importance of treatment to these patients. Most reasons for treatment refusal/discontinuation were associated with side effects or insufficient explanations, suggesting the necessity of providing sufficient information to aid in patient understanding.