Identification of Long Non-Coding RNA Profiles and Potential Therapeutic Agents for Fibrolamellar Carcinoma Based on RNA-Sequencing Data.

BACKGROUND: Fibrolamellar carcinoma (FLC) is a rare type of liver cancer that primarily affects adolescents and young adults without prior liver disease or viral infections. Patients with FLC generally have non-specific symptoms, are often diagnosed at a later stage, and experience a higher frequency of metastases compared to patients with other liver cancers. A fusion transcript of DNAJB1 and PRKACA, which can lead to increased activity of PKA and cellular proliferation, has been identified in all FLC patients, but the exact mechanism through which FLC develops remains unclear. In this study, we investigated common lncRNA profiles in various FLC samples using bioinformatics analyses. METHODS: We analyzed differentially expressed (DE) lncRNAs from three RNA sequencing datasets. Using lncRNAs and DE mRNAs, we predicted potential lncRNA target genes and performed Gene Ontology (GO) and KEGG analyses with the DE lncRNA target genes. Moreover, we screened for small-molecule compounds that could act as therapeutic targets for FLC. RESULTS: We identified 308 DE lncRNAs from the RNA sequencing datasets. In addition, we performed a trans-target prediction analysis and identified 454 co-expressed pairs in FLC. The GO analysis showed that the lncRNA-related up-regulated mRNAs were enriched in the regulation of protein kinase C signaling and cAMP catabolic processes, while lncRNA-related down-regulated mRNAs were enriched in steroid, retinol, cholesterol, and xenobiotic metabolic processes. The analysis of small-molecule compounds for FLC treatment identified vitexin, chlorthalidone, triamterene, and amiloride, among other compounds. CONCLUSIONS: We identified potential therapeutic targets for FLC, including lncRNA target genes as well as small-molecule compounds that could potentially be used as treatments. Our findings could contribute to furthering our understanding of FLC and providing potential avenues for diagnosis and treatment.

Comprehensive investigation of the expression profiles of common long noncoding RNAs during microglial activation.

Microglia, similar to peripheral macrophages, are the primary immune cells of the central nervous system (CNS). Microglia exist in the resting state in the healthy CNS, but can be activated and polarized into either M1 or M2 subtypes for immune defense and the maintenance of CNS homeostasis by multiple stimuli. Several long noncoding RNAs (lncRNAs) mediate human inflammatory diseases and neuropathologies by regulating their target genes. However, the function of common lncRNAs that contribute to microglial activation remains unclear. Thus, we used bioinformatic approaches to identify common lncRNAs involved in microglial activation in vitro. Our study identified several lncRNAs as common regulators of microglial activation. We identified 283 common mRNAs and 53 common lncRNAs during mouse M1 microglial activation processes, whereas 26 common mRNAs and five common lncRNAs were identified during mouse M2 microglial activation processes. A total of 648 common mRNAs and 274 common lncRNAs were identified during the activation of human M1 microglia. In addition, we identified 1,920 common co-expressed pairs in mouse M1 activation processes and 25 common co-expressed pairs in mouse M2 activation processes. Our study provides a comprehensive understanding of common lncRNA expression profiles in microglial activation processes in vitro. The list of common lncRNAs identified in this study provides novel evidence and clues regarding the molecular mechanisms underlying microglial activation.

Identification and Characterization of mRNA and lncRNA Expression Profiles in Age-Related Hearing Loss.

OBJECTIVES: Age-related hearing loss (ARHL), or presbycusis, is caused by disorders of sensory hair cells and auditory neurons. Many studies have suggested that the accumulation of mitochondrial DNA damage, the production of reactive oxygen species, noise, inflammation, and decreased antioxidant function are associated with subsequent cochlear senescence in response to aging stress. Long non-coding RNA (lncRNA) has been reported to play important roles in various diseases. However, the function of lncRNA in ARHL remains unclear. In this study, we analyzed the common expression profiles of messenger RNA (mRNA) and lncRNA through ARHL-related RNA-sequencing datasets. METHODS: We selected and downloaded three different sets of RNA-sequencing data for ARHL. We performed differential expression analysis to find common mRNA and lncRNA profiles in the cochleae of aged mice compared to young mice. Gene Ontology (GO) analysis was used for functional exploration. Real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was performed to validate mRNAs and lncRNAs. In addition, we performed trans target prediction analysis with differentially expressed mRNAs and lncRNAs to understand the function of these mRNAs and lncRNAs in ARHL. RESULTS: We identified 112 common mRNAs and 10 common lncRNAs in the cochleae of aged mice compared to young mice. GO analysis showed that the 112 upregulated mRNAs were enriched in the defense response pathway. When we performed qRT-PCR with 1 mM H2O2-treated House Ear Institute-Organ of Corti 1 (HEI-OC1) cells, the qRT-PCR. RESULTS: were consistent with the RNA-sequencing analysis data. lncRNA-mRNA networks were constructed using the 10 common lncRNAs and 112 common mRNAs in ARHL. CONCLUSION: Our study provides a comprehensive understanding of the common mRNA and lncRNA expression profiles in ARHL. Knowledge of ARHL-associated mRNAs and lncRNAs could be useful for better understanding ARHL and these mRNAs and lncRNAs might be a potential therapeutic target for preventing ARHL.

Effects of postpolymerization conditions on the physical properties, cytotoxicity, and dimensional accuracy of a 3D-printed dental restorative material.

STATEMENT OF PROBLEM: Although the introduction of high-speed 3-dimensional (3D) printing technology has significantly reduced printing time, the time required for postpolymerization is a speed-determining step because of the long wait time. How postpolymerization conditions affect material properties is unclear. PURPOSE: The purpose of this in vitro study was to assess the physical properties, accuracy, and biosafety of a 3D-printed dental restorative material according to postpolymerization conditions. MATERIAL AND METHODS: Specimens were prepared by 3D printing with a digital light processing 3D printer with 1 interim dental material (C&B MFH). All printed specimens underwent a postpolymerization process with 5 different postpolymerization devices and were designated as groups D1 (D102H), FO (Form Cure), LC (LC-3DPrintBox), ME (Medusa), and MP (MP100). The light intensity and temperature of each device were measured, and the Vickers hardness, flexural strength and modulus, degree of conversion (DC), cytotoxicity, and polymerization shrinkage were analyzed. Statistical analyses were conducted with 1-way analysis of variance, the Tukey post hoc test, and regression testing (α=.05). Scanning electron microscopy was used to assess the fracture surface characteristics of the specimens. RESULTS: Light intensity was strongest with the ME device, and the temperature inside the device during postpolymerization showed the highest increase with the LC device and the lowest increase with the D1 device. The LC group specimens showed the highest mean Vickers hardness, and the MP group showed the lowest. The flexural strength was ≥100 MPa in all groups, with a flexural modulus ranging from 1.17 to 1.5 GPa. The DC results were similar to the physical properties test results. The D1, FO, LC, and ME groups all showed ≥70% cell viability, indicating no toxicity. The FO group showed the highest shrinkage rate of 0.52%. CONCLUSIONS: When the light intensity was strong, the surface was sufficiently hard, and toxic substances were not eluted even after a short postpolymerization time, suggesting that light intensity modulation and time management can be used to improve the postpolymerization process.

Tumor-Suppressive Effect of Metformin via the Regulation of M2 Macrophages and Myeloid-Derived Suppressor Cells in the Tumor Microenvironment of Colorectal Cancer.

Myeloid-derived suppressor cells (MDSCs) and M2 macrophages in the tumor microenvironment contribute to tumor progression by inducing immune tolerance to tumor antigens and cancer cells. Metformin, one of the most common diabetes drugs, has shown anti-inflammatory and anti-tumor effects. However, the effects of metformin on inflammatory cells of the tumor microenvironment and its underlying mechanisms remain unclarified. In this study, we investigated the effect of metformin on M2 macrophages and MDSCs using monocyte THP-1 cells and a dextran sodium sulfate (DSS)-treated ApcMin/+ mouse model of colon cancer. Metformin decreased the fractions of MDSCs expressing CD33 and arginase, as well as M2 macrophages expressing CD206 and CD163. The inhibitory effect of metformin and rapamycin on MDSCs and M2 macrophages was reversed by the co-treatment of Compound C (an AMP-activated protein kinase (AMPK) inhibitor) or mevalonate. To examine the effect of protein prenylation and cholesterol synthesis (the final steps of the mevalonate pathway) on the MDSC and M2 macrophage populations, we used respective inhibitors (YM53601; SQLE inhibitor, FTI-277; farnesyl transferase inhibitor, GGTI-298; geranylgeranyl transferase inhibitor) and found that the MDSC and M2 populations were suppressed by the protein prenylation inhibitors. In the DSS-treated ApcMin/+ mouse colon cancer model, metformin reduced the number and volume of colorectal tumors with decreased populations of MDSCs and M2 macrophages in the tumor microenvironment. In conclusion, the inhibitory effect of metformin on MDSCs and M2 macrophages in the tumor microenvironment of colon cancers is mediated by AMPK activation and subsequent mTOR inhibition, leading to the downregulation of the mevalonate pathway.

Dental students' perceptions on a simulated practice using patient-based customised typodonts during the transition from preclinical to clinical education.

PURPOSE/OBJECTIVES: Dental students experience difficulties during the transition from preclinical to clinical curriculum. In order to help the students to adapt to the clinical education programme, a simulated practice using patient-based customised models was introduced in this study to prepare for their first clinical practice. METHODS: This study included 45 third-year predoctoral students (D3 students) who were about to perform the preparation of a single crown abutment on their first patient. After practicing abutment preparation using simulated models and providing the actual treatment to their own patient, the students were surveyed to investigate their perceptions on the simulated practice using the 3D-printed customised typodont model. The statistical analysis of the quantitative data and the thematic analysis of the qualitative data were conducted. RESULTS: Regarding this simulation, more than 80% of the students gave positive feedback on their practice of (a) operative positions and postures, (b) finger rest, (c) occlusal reduction, (d) axial reduction and (e) proximal reduction. Student responses on the open-ended questions about how they perceived the usefulness of this simulation were categorised as "First clinical case," "Patient-based model" and "Realistic simulation environment." In addition, a number of improvements of the simulation were also suggested by the students including the typodont and the manikin. CONCLUSIONS: This study gives insights into the significance of simulated practice using patient-based customised typodonts as a transitional education tool and its direction of development in the field of restorative treatments accompanied by irreversible tooth preparations.

Accuracy of Dental Implant Placement by a Novel In-House Model-Free and Zero-Setup Fully Guided Surgical Template Made of a Light-Cured Composite Resin (VARO Guide®): A Comparative In Vitro Study.

BACKGROUND: This in vitro study mainly aimed to compare VARO Guide® to the surgical guide fabricated by CAD/CAM (NAVI Guide®) in terms of accuracy and efficacy of the implant surgery held in the dentiform model. METHODS: Twenty surgeons, 10 dentists in the beginner group and 10 dentists in the expert group, participated in the study. Each surgeon conducted fully guided surgery in dentiform models twice, once with VARO Guide® (VG surgery) and the other time with a conventional type of templates, NAVI Guide® (NG surgery). Based on the superimposition of presurgical and postsurgical STL files, the positional deviations between the virtually planned and actually placed implants and the time spent on presurgical preparation and surgical procedures were estimated and compared. RESULTS: All dimensional deviations were similar between the two groups (p > 0.05), and there was no significant difference between the expert and beginner groups regardless of the guide system. The total procedure time (mean (median)) of the VG surgery (26.33 (28.58) min) was significantly shorter than that of the NG surgery (378.83 (379.35) min; p < 0.05). While the time spent only for the fully guided implant surgery (from the start of the surgical guide sitting onto the dentiform model to the final installation of the implant fixture) was comparable (p > 0.05), the presurgical preparation time spent on virtual implant planning and surgical guide fabrication in the VG surgery (19.63 (20.93) min) was significantly shorter compared to the NG surgery (372.93 (372.95) min; p < 0.05). CONCLUSIONS: Regardless of experience, both VG and NG surgery showed reliable positional accuracy; however, the total procedure time and the preparation time were much shorter in the VG surgery compared to the NG surgery.

Albumin inhibits the nuclear translocation of Smad3 via interleukin-1beta signaling in hepatic stellate cells.

Activation of quiescent hepatic stellate cells (HSCs) to myofibroblasts plays a key role in liver fibrosis. We had previously shown that albumin and its derivative, R-III (a retinol-binding protein-albumin domain III fusion protein), inhibited HSC activation by sequestering retinoic acid (RA) and that R-III administration reduced carbon tetrachloride (CCl4)-induced liver fibrosis. In this study, we aimed to elucidate the mechanism of action of albumin downstream of RA sequestration. Nuclear factor-κB p65 was evenly distributed in the cytoplasm in activated mouse HSCs, whereas albumin expression or R-III treatment (albumin/R-III) caused the nuclear translocation of p65, probably via RA sequestration, resulting in a dramatic increase in interleukin-1beta (IL-1ß) expression. Albumin/R-III in turn induced the phosphorylation of Smad3 at the linker region, inhibiting its nuclear import in an IL-1ß-dependent manner. Consistent with the in vitro results, the level of IL-1ß mRNA expression was higher in CCl4/R-III-treated livers than in CCl4-treated livers. These findings reveal that albumin/R-III inhibits the transforming growth factor-ß-Smad3 signaling as well as the retinoic acid receptor-mediated pathway, which probably contributes to the inhibition of HSC activation, and suggest that R-III may be an anti-fibrotic drug candidate.

Spherical Principal Curves.

This paper presents a new approach for dimension reduction of data observed on spherical surfaces. Several dimension reduction techniques have been developed in recent years for non-euclidean data analysis. As a pioneer work, (Hauberg 2016) attempted to implement principal curves on Riemannian manifolds. However, this approach uses approximations to process data on Riemannian manifolds, resulting in distorted results. This study proposes a new approach to project data onto a continuous curve to construct principal curves on spherical surfaces. Our approach lies in the same line of (Hastie and Stuetzle et al. 1989) that proposed principal curves for data on euclidean space. We further investigate the stationarity of the proposed principal curves that satisfy the self-consistency on spherical surfaces. The results on the real data analysis and simulation examples show promising empirical characteristics of the proposed approach.

Assessment of Compatibility between Various Intraoral Scanners and 3D Printers through an Accuracy Analysis of 3D Printed Models.

To assess the accuracy of various intraoral scanners (IOSs) and to investigate the existence of mutual compatibility that affects the accuracy between IOS and 3-dimensional (3D) printing using a scan quadrant model. For clinical implication, crown preparations and cavity design according to prosthetic diagnosis and treatment considerations must be acquired by a digital scanner. The selected typodont model was scanned using a reference scanner, from which reference (Ref) standard tessellation language (STL) data were created. Data obtained by scanning the typodont model with IOSs based on three different technologies were divided into three groups (CS3600, i500, and Trios3). Scanned data from the groups were divided into sub-groups of digital light processing (DLP), fused deposition modeling (FDM), and stereolithography apparatus (SLA), based on which 3D printed models (3DP) were fabricated. The 3DP dental models were scanned to obtain a total of 90 3DP STL datasets. The best-fit algorithm of 3D analysis software was used for teeth and arch measurements, while trueness was analyzed by calculating the average deviation among measured values based on superimposition of Ref and IOS and 3DP data. The differences between Ref and IOS (Ref-IOS), Ref and 3DP (Ref-IOS/3DP), and IOS and 3DP data (IOS-3DP) were compared and analyzed, while accuracy within each of the three main groups was assessed. For statistical analysis, the Kruskal-Wallis, Mann-Whitney U, and repeated measures ANOVA test were used (p < 0.05). The major finding is that the mutual relationships between IOSs and 3D printers vary depending on the combination. However, i500 intraoral scanner and DLP 3D printer was the combination that showed the best trueness value.

Metformin Suppresses Cancer Stem Cells through AMPK Activation and Inhibition of Protein Prenylation of the Mevalonate Pathway in Colorectal Cancer.

Metformin is a well-known AMPK (AMP-activated protein kinase) activator that suppresses cancer stem cells (CSCs) in some cancers. However, the mechanisms of the CSC-suppressing effects of metformin are not yet well understood. In this study, we investigated the CSC-suppressive effect of metformin via the mevalonate (MVA) pathway in colorectal cancer (CRC). Two colorectal cancer cell lines, HT29 and DLD-1 cells, were treated with metformin, mevalonate, or a combination of the two. We measured CSC populations by flow cytometric analysis (CD44+/CD133+) and by tumor spheroid growth. The expression of p-AMPK, mTORC1 (pS6), and key enzymes (HMGCR, FDPS, GGPS1, and SQLE) of the MVA pathway was also analyzed. We investigated the effects of metformin and/or mevalonate in xenograft mice using HT29 cells; immunohistochemical staining for CSC markers and key enzymes of the MVA pathway in tumor xenografts was performed. In both HT29 and DLD-1 cells, the CSC population was significantly decreased following treatment with metformin, AMPK activator (AICAR), HMG-CoA reductase inhibitor (simvastatin), or mTOR inhibitor (rapamycin), and was increased by mevalonate. The CSC-suppressing effect of these drugs was attenuated by mevalonate. The results of tumor spheroid growth matched those of the CSC population experiments. Metformin treatment increased p-AMPK and decreased mTOR (pS6) expression; these effects were reversed by addition of mevalonate. The expression of key MVA pathway enzymes was significantly increased in tumor spheroid culture, and by addition of mevalonate, and decreased upon treatment with metformin, AICAR, or rapamycin. In xenograft experiments, tumor growth and CSC populations were significantly reduced by metformin, and this inhibitory effect of metformin was abrogated by combined treatment with mevalonate. Furthermore, in the MVA pathway, CSC populations were reduced by inhibition of protein prenylation with a farnesyl transferase inhibitor (FTI-277) or a geranylgeranyl transferase inhibitor (GGTI-298), but not by inhibition of cholesterol synthesis with a squalene synthase inhibitor (YM-53601). In conclusion, the CSC-suppressive effect of metformin was associated with AMPK activation and repression of protein prenylation through MVA pathway suppression in colorectal cancer.

Changes in mucogingival junction after an apically positioned flap with collagen matrix at sites with or without previous guided bone regeneration: A prospective comparative cohort study.

AIM: To assess changes in the position of the mucogingival junction (MGJ) after an apically positioned flap (APF) with collagen matrix performed at sites with or without previous guided bone regeneration (GBR). MATERIALS AND METHODS: Dental implants were placed with or without GBR (group GBR or non-GBR) depending on the available ridge width in 30 patients with a limited width of keratinized mucosa (MGJ placed more coronally than the expected prosthetic margin). An apically positioned flap with collagen matrix was performed in both groups. Changes in the position of the MGJ from the day of an apically positioned flap up to 1, 3, and 12 months thereafter were assessed on digital scans (primary endpoint). Secondary endpoints were the width and thickness of the keratinized mucosa, and the position of the mucosal margin. RESULTS: The position of the MGJ changed significantly from baseline to the first month, by 5.25 ± 2.10 and 4.40 ± 1.41 mm in groups GBR and non-GBR, respectively. Thereafter, the position remained stable in both groups up to 1 year (changes from baseline of 5.46 ± 2.28 and 4.58 ± 1.92 mm, respectively; p = .34). The position of the mucosal margin did not differ between groups GBR and non-GBR (-1.57 ± 2.04 and -1.75 ± 2.08 mm, respectively; p = .84), nor did the width of the keratinized mucosa (1.20 ± 1.03 and 0.99 ± 0.66 mm, p = .91) or its thickness (1.28 ± 0.44 and 1.40 ± 0.78 mm, p = .87). CONCLUSION: Apically positioned flap combined with a collagen matrix results in a more apical position of the MGJ at sites with or without GBR. Following a coronal shift during the first month after the apical positioning of the flap, the level of the MGJ remained stable.

Clinical factors influencing implant positioning by guided surgery using a nonmetal sleeve template in the partially edentulous ridge: Multiple regression analysis of a prospective cohort.

OBJECTIVE: To determine the positional accuracy of implants placed with a three-dimensionally printed template having nonmetal sleeves and to determine the contributing factors to observed deviations. MATERIALS AND METHODS: One hundred and eighty-seven implants placed in 72 patients were analyzed. Presurgical intraoral scans and cone-beam computed tomography images obtained before and after surgery were superimposed, and vertical, angular, platform, and apex deviations were measured between the virtually planned and actually placed positions. A multiple linear regression model was designed for identifying the contributing factors. Statistical significance was set at p < .05, with Bonferroni correction if necessary (p < .0167). RESULTS: A total of 187 implants demonstrated deviations of 0.65 [0.56, 0.75] mm (mean [95% confidence interval]) vertically, 3.59° [3.30°, 3.89°] angularly, 1.16 [1.04, 1.28] mm at platform, and 1.50 [1.36, 1.65] mm at apex. Implants placed in the mandible showed larger angular, platform, and apex deviations compared with those in the maxilla (p = .049, p = .014 and p = .003, respectively). Implants placed at the third or fourth nearest sites from the most-distal tooth had larger deviations than those placed at the first or second nearest sites, in vertical, platform, and apical aspects (p = .015, p = .011 andp = .018, respectively). This was only applicable to free-ending-supported templates (p < .0167), and anchor pin-supported free-ending templates (p < .0167). CONCLUSION: Using a three-dimensionally printed surgical template with a nonmetal sleeve in the partial edentulous ridge resulted in larger deviations in implants placed in the mandible or distal free-end third or fourth nearest site.

Assessment of metal sleeve-free 3D-printed implant surgical guides.

OBJECTIVES: The aim of the present study was to investigate the adaptation and guide hole tolerance of metal sleeve-free computer-assisted implant surgical guides fabricated with 3D printers. METHODS: An implant surgical guide for full-guided implant placement was designed with a total of eight different guide holes. Ten implant surgical guides (n=10) were fabricated from the same design with each of five in-office 3D printers (D1, FOR, ONE, PER, and ZEN) using compatible printing materials. Ten surgical guides fabricated by the manufacturer of the implant company were used as the control group (CON). The adaptation of the surgical guides was evaluated by the replica technique. The tolerance of the guide holes was evaluated by measuring the degree of diversion with guide drills. RESULTS: CON and D1 showed superior internal adaptation with a gap distance of less than 1mm. The mean degree of diversion of the guide holes ranged from 3.45° for ZEN to 6.55° for PER. The tolerances of CON (4.70°) and D1 (4.50°) did not differ at the level of statistical significance at α=0.05. SIGNIFICANCE: The characteristics of implant surgical guides were evaluated per se. None of the 3D printers fabricated superior implant surgical guides to those produced by the manufacturer with regard to the internal fit and guide tolerance. However, the potential for the routine clinical use of in-office 3D printers was demonstrated. Further studies are required to determine how the guide hole tolerance and the angular deviation between the preplanned and actual implant positions are related.

UBE3A Suppresses Overnutrition-Induced Expression of the Steatosis Target Genes of MLL4 by Degrading MLL4.

Regulation of the protein stability of epigenetic regulators remains ill-defined despite its potential applicability in epigenetic therapies. The histone H3-lysine 4-methyltransferase MLL4 is an epigenetic transcriptional coactivator that directs overnutrition-induced obesity and fatty liver formation, and Mll4+/- mice are resistant to both. Here we show that the E3 ubiquitin ligase UBE3A targets MLL4 for degradation, thereby suppressing high-fat diet (HFD)-induced expression of the hepatic steatosis target genes of MLL4. In contrast to Mll4+/- mice, Ube3a+/- mice are hypersensitive to HFD-induced obesity and fatty liver development. Ube3a+/-;Mll4+/- mice lose this hypersensitivity, supporting roles of increased MLL4 levels in both phenotypes of Ube3a+/- mice. Correspondingly, our comparative studies with wild-type, Ube3a+/- and Ube3a-/- and UBE3A-overexpressing transgenic mouse livers demonstrate an inverse correlation of UBE3A protein levels with MLL4 protein levels, expression of the steatosis target genes of MLL4, and their decoration by H3-lysine 4-monomethylation, a surrogate marker for the epigenetic action of MLL4. Conclusion: UBE3A indirectly exerts an epigenetic regulation of obesity and steatosis by degrading MLL4. This UBE3A-MLL4 regulatory axis provides a potential therapeutic venue for treating various MLL4-directed pathogeneses, including obesity and hepatic steatosis.

Dlx1/2 and Otp coordinate the production of hypothalamic GHRH- and AgRP-neurons.

Despite critical roles of the hypothalamic arcuate neurons in controlling the growth and energy homeostasis, the gene regulatory network directing their development remains unclear. Here we report that the transcription factors Dlx1/2 and Otp coordinate the balanced generation of the two functionally related neurons in the hypothalamic arcuate nucleus, GHRH-neurons promoting the growth and AgRP-neurons controlling the feeding and energy expenditure. Dlx1/2-deficient mice show a loss-of-GHRH-neurons and an increase of AgRP-neurons, and consistently develop dwarfism and consume less energy. These results indicate that Dlx1/2 are crucial for specifying the GHRH-neuronal identity and, simultaneously, for suppressing AgRP-neuronal fate. We further show that Otp is required for the generation of AgRP-neurons and that Dlx1/2 repress the expression of Otp by directly binding the Otp gene. Together, our study demonstrates that the identity of GHRH- and AgRP-neurons is synchronously specified and segregated by the Dlx1/2-Otp gene regulatory axis.

Critical Roles of the Histone Methyltransferase MLL4/KMT2D in Murine Hepatic Steatosis Directed by ABL1 and PPARγ2.

The pathophysiologic continuum of non-alcoholic fatty liver disease begins with steatosis. Despite recent advances in our understanding of the gene regulatory program directing steatosis, how it is orchestrated at the chromatin level is unclear. PPARγ2 is a hepatic steatotic transcription factor induced by overnutrition. Here, we report that the histone H3 lysine 4 methyltransferase MLL4/KMT2D directs overnutrition-induced murine steatosis via its coactivator function for PPARγ2. We demonstrate that overnutrition facilitates the recruitment of MLL4 to steatotic target genes of PPARγ2 and their transactivation via H3 lysine 4 methylation because PPARγ2 phosphorylated by overnutrition-activated ABL1 kinase shows enhanced interaction with MLL4. We further show that Pparg2 (encoding PPARγ2) is also a hepatic target gene of ABL1-PPARγ2-MLL4. Consistently, inhibition of ABL1 improves the fatty liver condition of mice with overnutrition by suppressing the pro-steatotic action of MLL4. Our results uncover a murine hepatic steatosis regulatory axis consisting of ABL1-PPARγ2-MLL4, which may serve as a target of anti-steatosis drug development.

Verticine, ebeiedine and suchengbeisine isolated from the bulbs of Fritillaria thunbergii Miq. inhibited the gene expression and production of MUC5AC mucin from human airway epithelial cells.

BACKGROUND: The bulb of Fritillaria thunbergii has been utilised as mucoregulators and expectorants for controlling the airway inflammatory diseases in folk medicine. HYPOTHESIS/PURPOSE: We investigated whether verticine, ebeiedine and suchengbeisine isolated from the bulbs of Fritillaria thunbergii inhibit the gene expression and production of MUC5AC mucin from human airway epithelial cells. STUDY DESIGN: Confluent NCI-H292 cells were pretreated with verticine, ebeiedine or suchengbeisine for 30 min and then stimulated with EGF, PMA or TNF-α for 24h. The MUC5AC mucin gene expression was measured by RT-PCR. Production of MUC5AC mucin protein was measured by ELISA. RESULTS: (1) Verticine, ebeiedine or suchengbeisine inhibited the expression of MUC5AC mucin gene induced by EGF, PMA or TNF-α; (2) The production of MUC5AC mucin protein induced by EGF, PMA or TNF-α were also inhibited by treatment of verticine, ebeiedine or suchengbeisine. CONCLUSION: These results suggest that verticine, ebeiedine and suchengbeisine isolated from the bulbs of Fritillaria thunbergii inhibit the gene expression and production of MUC5AC mucin, by directly acting on airway epithelial cells, and the results are consistent with the traditional use of Fritillaria thunbergii as remedy for diverse inflammatory pulmonary diseases.

Inhibition of TNF-α-induced MUC5AC mucin gene expression and production by wogonin through the inactivation of NF-κB signaling in airway epithelial cells.

In this study, we investigated whether wogonin significantly affects MUC5AC mucin gene expression and production in human airway epithelial cells. Confluent NCI-H292 cells were pretreated with wogonin for 30 min and then stimulated with tumor necrosis factor-α (TNF-α) for 24 h or the indicated periods. The MUC5AC mucin gene expression and mucin protein production were measured by RT-PCR and ELISA, respectively. We found that incubation of NCI-H292 cells with wogonin significantly inhibited mucin production and down-regulated MUC5AC gene expression induced by TNF-α in a dose-dependent fashion. To elucidate the action mechanism of wogonin, effect of wogonin on TNF-α-induced NF-κB signaling pathway was investigated by western blot analysis. Wogonin inhibited NF-κB activation induced by TNF-α. Inhibition of IKK by wogonin led to the suppression of IκB phosphorylation and degradation, p65 nuclear translocation and NF-κB-regulated gene expression. This, in turn, led to the down-regulation of MUC5AC protein production in NCI-H292 cells. Wogonin also inhibited the gene products involved in cell survival (Bcl-2) and proliferation (cyclooxygenase-2). These results suggest that wogonin inhibits the NF-κB signaling pathway, which may explain its role in the inhibition of MUC5AC mucin gene expression and production.