Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation.

Programmed necrosis is a form of caspase-independent cell death whose molecular regulation is poorly understood. The kinase RIP1 is crucial for programmed necrosis, but also mediates activation of the prosurvival transcription factor NF-kappaB. We postulated that additional molecules are required to specifically activate programmed necrosis. Using a RNA interference screen, we identified the kinase RIP3 as a crucial activator for programmed necrosis induced by TNF and during virus infection. RIP3 regulates necrosis-specific RIP1 phosphorylation. The phosphorylation of RIP1 and RIP3 stabilizes their association within the pronecrotic complex, activates the pronecrotic kinase activity, and triggers downstream reactive oxygen species production. The pronecrotic RIP1-RIP3 complex is induced during vaccinia virus infection. Consequently, RIP3(-/-) mice exhibited severely impaired virus-induced tissue necrosis, inflammation, and control of viral replication. Our findings suggest that RIP3 controls programmed necrosis by initiating the pronecrotic kinase cascade, and that this is necessary for the inflammatory response against virus infections.

Predictive System Implementation to Improve the Accuracy of Urine Self-Diagnosis with Smartphones: Application of a Confusion Matrix-Based Learning Model through RGB Semiquantitative Analysis.

Urinalysis, an elementary chemical reaction-based method for analyzing color conversion factors, facilitates examination of pathological conditions in the human body. Recently, considerable urinalysis-centered research has been conducted on the analysis of urine dipstick colors using smartphone cameras; however, such methods have a drawback: the problem of reproducibility of accuracy through quantitative analysis. In this study, to solve this problem, the function values for each concentration of a range of analysis factors were implemented in an algorithm through urine dipstick RGB semi-quantitative color analysis to enable real-time results. Herein, pH, glucose, ketones, hemoglobin, bilirubin, protein (albumin), and nitrites were selected as analysis factors, and the accuracy levels of the existing equipment and the test application were compared and evaluated using artificial urine. In the semi-quantitative analysis, the red (R), green (G), and blue (B) characteristic values were analyzed by extracting the RGB characteristic values of the analysis factors for each concentration of artificial urine and obtaining linear function values. In addition, to improve the reproducibility of detection accuracy, the measurement value of the existing test equipment was set to an absolute value; using a machine-learning technique, the confusion matrix, we attempted to stabilize test results that vary with environment.

Development of a Highly Sensitive Enzyme-Linked Immunosorbent Assay for Mouse Soluble Epoxide Hydrolase Detection by Combining a Polyclonal Capture Antibody with a Nanobody Tracer.

Enzyme-linked immunosorbent assays (ELISA) for the detection of soluble epoxide hydrolase (sEH), a key enzyme in the metabolism of fatty acids and a biomarker, may increasingly represent an important diagnostic tool. However, there is a lack of ELISAs for mouse sEH quantification, thus resulting in a bottleneck in understanding the pathogenesis of many diseases related to sEH based on mouse models. In this work, nanobodies recognizing mouse sEH were obtained through rebiopanning against mouse sEH in the previous phage display library of human sEH. Later, we developed four ELISAs involving a combination of anti-mouse sEH polyclonal antibodies (pAbs) and nanobodies. It was found that the double antibodies worked as dual filters and had a huge impact on both the sensitivity and selectivity of sandwich immunoassays. The switch from anti-human sEH pAbs to anti-mouse sEH pAbs led to over a 100-fold increase in the sensitivity and a dramatic decrease of the limit of detection to a picogram per milliliter range in format B (pAb/biotin-VHH/streptavidin-poly-horseradish peroxidase). Moreover, we found that the four sandwich ELISAs might demonstrate excellent selectivities to mouse sEH, despite the antibodies alone showing significant cross-reactivity to the matrix, indicating the enhanced selectivity of double antibodies as dual filters. Eventually, for the first time, the ELISA (format B) was successfully used to measure the mouse sEH level in cancer cells with ultralow abundances. The ELISAs proposed here represent a sensitive tool for tracking sEH in various biological processes and also provide deep insights into developing sandwich immunoassays against various targets in terms of both the sensitivity and selectivity.

Licochalcone D directly targets JAK2 to induced apoptosis in human oral squamous cell carcinoma.

Licochalcone (LC) families have been reported to have a wide range of biological function such as antioxidant, antibacterial, antiviral, and anticancer effects. Although various beneficial effects of LCD were revealed, its anticancer effect in human oral squamous cancer has not been identified. To examine the signaling pathway of LCD's anticancer effect, we determined whether LCD has physical interaction with Janus kinase (JAK2)/signal transducer and activator of transcription-3 (STAT3) signaling, which is critical in promoting cancer cell survival and proliferation. Our results demonstrated that LCD inhibited the kinase activity of JAK2, soft agar colony formation, and the proliferation of HN22 and HSC4 cells. LCD also induced mitochondrial apoptotic events such as altered mitochondrial membrane potential and reactive oxygen species production. LCD increased the expression of apoptosis-associated proteins in oral squamous cell carcinoma (OSCC) cells. Finally, the xenograft study showed that LCD significantly inhibited HN22 tumor growth. Immunohistochemical data supported that LCD suppressed p-JAK2 and p-STAT3 expression and induced cleaved-caspase-3 expression. These results indicate that the anticancer effect of LCD is due to the direct targeting of JAK2 kinase. Therefore, LCD can be used for therapeutic application against OSCC.

Licochalcone C induced apoptosis in human oral squamous cell carcinoma cells by regulation of the JAK2/STAT3 signaling pathway.

Oral cancer is of an aggressive malignancy that arises on oral cavity and lip, 90% of cancers histologically originated in the squamous cells. Licochalcone (LC)C has been known as natural phenolic chalconoid substances, and its origin is the root of Glycyrrhiza glabra or Glycyrrhiza inflata. LCC inhibited oral squamous cell carcinoma (OSCC) cell viability, mitochondrial function, and anchorage-independent growth in a dose-dependent manner. To investigate the ability of LCC to target Janus kinase 2 (JAK2), we performed pull-down binding assay, kinase assay, and docking simulation. The molecular docking studies were performed between JAK2 and the potent inhibitor LCC. It was shown that LCC tightly interacted with ATP-binding site of JAK2. In addition, LCC inhibited the JAK2/signal transducer and activator of transcription 3 pathway, upregulated p21, and downregulated Bcl-2, Mcl-1, and Survivin, while it disrupted mitochondrial membrane potential and subsequently caused cytochrome c release with activation of multi-caspase, eventually leading to apoptosis in HN22 and HSC4 cells. LCC elevated the protein levels of Bax, cleaved Bid and PARP, and increased Apaf-1, and this effect was reversed by LCC treatment. Our results demonstrated that treatment of OSCC cells with LCC induced the death receptor (DR)4 and DR5 expression level with the generation of reactive oxygen species and the upregulation of CHOP protein expression. Taken together, these results could provide the basis for clinical application as a new therapeutic strategy in the treatment of oral cancer.

Design of a Novel and Selective IRAK4 Inhibitor Using Topological Water Network Analysis and Molecular Modeling Approaches.

Protein kinases are deeply involved in immune-related diseases and various cancers. They are a potential target for structure-based drug discovery, since the general structure and characteristics of kinase domains are relatively well-known. However, the ATP binding sites in protein kinases, which serve as target sites, are highly conserved, and thus it is difficult to develop selective kinase inhibitors. To resolve this problem, we performed molecular dynamics simulations on 26 kinases in the aqueous solution, and analyzed topological water networks (TWNs) in their ATP binding sites. Repositioning of a known kinase inhibitor in the ATP binding sites of kinases that exhibited a TWN similar to interleukin-1 receptor-associated kinase 4 (IRAK4) allowed us to identify a hit molecule. Another hit molecule was obtained from a commercial chemical library using pharmacophore-based virtual screening and molecular docking approaches. Pharmacophoric features of the hit molecules were hybridized to design a novel compound that inhibited IRAK4 at low nanomolar levels in the in vitro assay.

Regional Differences of Proteins Expressing in Adipose Depots Isolated from Cows, Steers and Bulls as Identified by a Proteomic Approach.

Adipose tissue in the loin muscle area of beef cattle as a marbling factor is directly associated with beef quality. To elucidate whether properties of proteins involved in depot specific adipose tissue were sex-dependent, we analyzed protein expression of intramuscular adipose tissue (IMAT) and omental adipose tissue (OMAT) from Hanwoo cows, steers, and bulls of Korean native beef cattle by liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomic analysis, quantitative polymerase chain reaction (PCR) and western blot analysis. Two different adipose depots (i.e. intramuscular and omental) were collected from cows (n = 7), steers (n = 7), or bulls (n = 7). LC-MS/MS revealed a total of 55 and 35 proteins in IMAT and OMAT, respectively. Of the 55 proteins identified, 44, 40, and 42 proteins were confirmed to be differentially expressed in IMAT of cows, steers, and bulls, respectively. In OMAT of cows, steers, and bulls, 33, 33, and 22 were confirmed to be differentially expressed, respectively. Tropomyosin (TPM) 1, TPM 2, and TPM3 were subjected to verification by quantitative PCR and western blot analysis in IMAT and OMAT of Hanwoo cows, steers, and bulls as key factors closely associated with muscle development. Both mRNA levels and protein levels of TPM1, TPM2, and TPM3 in IMAT were lower in bulls compared to in cows or steers suggesting that they were positively correlated with marbling score and quality grade. Our results may aid the regulation of marbling development and improvement of meat quality grades in beef cattle.

Proteomic Assessment of the Relevant Factors Affecting Pork Meat Quality Associated with Longissimus dorsi Muscles in Duroc Pigs.

Meat quality is a complex trait influenced by many factors, including genetics, nutrition, feeding environment, animal handling, and their interactions. To elucidate relevant factors affecting pork quality associated with oxidative stress and muscle development, we analyzed protein expression in high quality longissimus dorsi muscles (HQLD) and low quality longissimus dorsi muscles (LQLD) from Duroc pigs by liquid chromatographytandem mass spectrometry (LC-MS/MS)-based proteomic analysis. Between HQLD (n = 20) and LQLD (n = 20) Duroc pigs, 24 differentially expressed proteins were identified by LC-MS/MS. A total of 10 and 14 proteins were highly expressed in HQLD and LQLD, respectively. The 24 proteins have putative functions in the following seven categories: catalytic activity (31%), ATPase activity (19%), oxidoreductase activity (13%), cytoskeletal protein binding (13%), actin binding (12%), calcium ion binding (6%), and structural constituent of muscle (6%). Silver-stained image analysis revealed significant differential expression of lactate dehydrogenase A (LDHA) between HQLD and LQLD Duroc pigs. LDHA was subjected to in vitro study of myogenesis under oxidative stress conditions and LDH activity assay to verification its role in oxidative stress. No significant difference of mRNA expression level of LDHA was found between normal and oxidative stress condition. However, LDH activity was significantly higher under oxidative stress condition than at normal condition using in vitro model of myogenesis. The highly expressed LDHA was positively correlated with LQLD. Moreover, LDHA activity increased by oxidative stress was reduced by antioxidant resveratrol. This paper emphasizes the importance of differential expression patterns of proteins and their interaction for the development of meat quality traits. Our proteome data provides valuable information on important factors which might aid in the regulation of muscle development and the improvement of meat quality in longissimus dorsi muscles of Duroc pigs under oxidative stress conditions.

Discovery of Tyk2 inhibitors via the virtual site-directed fragment-based drug design.

In this study, we synthesized compound 12 with potent Tyk2 inhibitory activity from FBDD study and carried out a cell-based assay for Tyk2/STAT3 signaling activation upon IFNα5 stimulation. Compound 12 completely suppressed the IFNα5-mediated Tyk2/STAT3 signaling pathway as well as the basal levels of pSTAT3. Stimulation with IFNα/ß leads to the tyrosine phosphorylation of the JAK1 and Tyk2 receptor-associated kinases with subsequent STATs activation, transmitting signals from the cell surface receptor to the nucleus. In conclusion, the potency of compound 12 to interrupt the signal transmission of Tyk2/STAT3 appeared to be equivalent or superior to that of the reference compound.

Synthesis and Biological Evaluation of N-Aryl-5-aryloxazol-2-amine Derivatives as 5-Lipoxygenase Inhibitors.

We describe the synthesis and biological evaluation of N-aryl-5-aryloxazol-2-amine derivatives that are able to inhibit 5-lipoxygenase (5-LOX), a key enzyme of leukotriene synthesis, for the treatment of inflammation-related diseases including asthma and rheumatoid arthritis. A novel structural moiety containing oxazole was initially identified from a chemical library using an in vitro enzymatic and cell-based assay, and its synthesized oxazole derivatives were further examined to develop a structure-activity relationship (SAR). SAR analysis demonstrated that a hydroxyl or amino group at the p-position on N-phenyl was essential for the 5-LOX-inhibitory activities of the derivatives, and that other halogen and methyl group-substituted derivatives affected the potency, positively or negatively. As a result, derivatives selected through first-round screening were further optimized using a cell-based assay and an in vivo assay to develop a potent, selective 5-LOX inhibitor. A final hit exhibited an improved efficacy in arachidonic acid-induced ear edema when applied topically but not orally. Moreover, it showed the additional advantage of sustainable antiinflammatory activity over a reference compound, zileuton. Taken together, chemical entities bearing an oxazole scaffold could be promising as therapeutic drugs for the treatment of chronic inflammatory skin disorders.

Heat shock protein 90 inhibitor regulates necroptotic cell death via down-regulation of receptor interacting proteins.

17-(Dimethylaminoethylamino)-17-demethoxygeldanamycin (DMAG) acts as an inhibitor of heat shock protein 90 (HSP 90), which serves as a nodal protein of diverse signaling networks leading to a variety of biological implications. HSP90 plays the role of a chaperone for a variety of client proteins including receptor interacting protein 1 (RIP1). Since RIP1 and RIP3 are, respectively, required for zVAD- and tumor necrosis factor alpha (TNFα)-mediated necrotic cell death, we pursued to address the effects of DMAG on receptor-and nonreceptor-mediated necroptotic cell death. DMAG facilitated the degradation of receptor interacting protein 3 (RIP3) as well as RIP1, a known client protein of HSP90, in L929 cells. Consequently, DMAG rendered cells more sensitive to TNFα stimulation while it rescued cells from necrotic cell death caused by zVAD. From this study, we propose that DMAG-downregulated RIP1 can shift cell death typing from necroptosis to apoptosis. In contrast, the protective effect of DMAG on zVAD-induced cytotoxicity could be partly explained by the fact that zVAD mediates cytotoxicity via a RIP1 -dependent route. In summary, functional disruption of HSP90 by DMAG destabilized necroptosis proteins RIP1 and RIP3, which in turn regulated zVAD- and TNFα-induced necroptosis. Therefore, pharmacological modulation of necroptotic cell death through HSP90 could be a promising strategy for overcoming cancer drug resistance or protecting ischemic cell death.

Protection of palmitic acid-mediated lipotoxicity by arachidonic acid via channeling of palmitic acid into triglycerides in C2C12.

BACKGROUND: Excessive saturated fatty acids have been considered to be one of major contributing factors for the dysfunction of skeletal muscle cells as well as pancreatic beta cells, leading to the pathogenesis of type 2 diabetes. RESULTS: PA induced cell death in a dose dependent manner up to 1.5 mM, but AA protected substantially lipotoxicity caused by PA at even low concentration of 62 µM, at which monounsaturated fatty acids including palmitoleic acid (POA) and oleic acid (OA) did not protect as much as AA did. Induction of cell death by PA was resulted from mitochondrial membrane potential loss, and AA effectively blocked the progression of apoptosis. Furthermore, AA rescued significantly PA-impaired glucose uptake and -signal transduction of Akt in response to insulin.Based on the observations that polyunsaturated AA generated competently cellular droplets at low concentration within the cytosol of myotubes compared with other monounsaturated fatty acids, and AA-driven lipid droplets were also enhanced in the presence of PA, we hypothesized that incorporation of harmful PA into inert triglyceride (TG) may be responsible for the protective effects of AA against PA-induced lipotoxicity. To address this assumption, C2C12 myotubes were incubated with fluorescent probed-PA analogue 4, 4-difluoro-5, 7-dimethyl-4-boro-3a,4a-diaza-s-indacene-3-hexadecanoic acid (BODIPY FL C16) in the presence of AA and their subsequent lipid profiles were analyzed. The analyses of lipids on thin layer chromatography (TLC) showed that fluorescent PA analogue was rapidly channeled into AA-driven TG droplets. CONCLUSION: Taken together, it is proposed that AA diverts PA into inert TG, therefore reducing the availability of harmful PA into intracellular target molecules.

Association between insulin resistance and periodontitis in Korean adults.

OBJECTIVE: Recent studies have proposed an association between periodontitis and metabolic abnormalities. We investigated the association between insulin resistance and periodontitis among Korean adults. METHODS: A cross-sectional analysis was conducted using the Korea National Health and Nutrition Examination Survey 2008-2010. A total of 16,720 non-diabetic subjects over 18 years old were evaluated (7060 men and 9660 women). Periodontal status was assessed by the Community Periodontal Index. Insulin resistance was measured using the homeostasis model assessment of insulin resistance (HOMA-IR). Participants in the highest and lowest quartile of HOMA-IR were defined as insulin-resistant and insulin-sensitive respectively. RESULTS: The prevalence of periodontitis increased significantly with higher HOMA-IR quartiles in post-menopausal women (p for linear association = 0.019). Among post-menopausal women, participants in the highest quartile of HOMA-IR were significantly more likely to have periodontitis compared to those in the lowest quartile [adjusted odds ratio (OR), 1.47; 95% confidence interval (CI), 1.07-2.01]. Moreover, obese post-menopausal women showed an increased association between insulin resistance and periodontitis (adjusted OR, 1.92; 95% CI,1.29-2.87). However, this association was not found in men or pre-menopausal women. CONCLUSIONS: Our results suggest that insulin resistance may be associated with periodontitis, especially when combined with obesity, among post-menopausal women in Korea.

Distinctive roles of receptor-interacting protein kinases 1 and 3 in caspase-independent cell death of L929.

Upon tumour necrosis factor alpha (TNFα) stimulation, cells respond actively by way of cell survival, apoptosis or programmed necrosis. The receptor-interacting proteins 1 (RIP1) and 3 (RIP3) are responsible for TNFα-mediated programmed necrosis. To delineate the differential contributions of RIP3 and RIP1 to programmed necrosis, L929 cells were stimulated with TNFα, carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (zVAD) or zVAD along with TNFα following RNA interference against RIP1 and RIP3, respectively. RIP1 silencing did not protect cells from TNFα-mediated cell death, while RIP3 down-regulation made them refractory to TNFα. The heat shock protein 90 inhibitor geldanamycin (GA) down-regulated both RIP1 and RIP3 expression, which rendered cells resistant to zVAD/TNFα-mediated cell death but not to TNFα-mediated cell death alone. Therefore, the protective effect of GA on zVAD/TNFα-stimulated necrosis might be attributed to RIP3, not RIP1, down-regulation. Pretreatment of L929 cells with rapamycin mitigated zVAD-mediated cell death, while the autophagy inhibitor chloroquine did not affect necrotic cell death. Meanwhile, necrotic cell death by zVAD and TNFα was caused by reactive oxygen species generation and effectively diminished by lipid-soluble butylated hydroxyanisole. Taken together, the results indicate that RIP1 and RIP3 can independently mediate death signals being transduced by two different death stimuli, zVAD and TNFα.

Cortisone induces insulin resistance in C2C12 myotubes through activation of 11beta-hydroxysteroid dehydrogenase 1 and autocrinal regulation.

The enzyme 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1) is known to catalyse inactive glucocorticoids into active forms, and its dysregulation in adipose and muscle tissues has been implicated in the development of metabolic syndrome. To delineate the molecular mechanism by which active cortisol has an antagonizing effect against insulin, we optimized the metabolic production of cortisol and its biological functions in myotubes (C2C12). Myotubes supplemented with cortisone actively catalysed its conversion into cortisol, which in turn abolished phosphorylation of Akt in response to insulin treatment. This led to diminished uptake of insulin-induced glucose. This was corroborated by the application of 11ß-HSD1 inhibitor glycyrrhetinic acid and a glucocorticoid receptor antagonist RU-486, which reversed completely the antagonizing effects of cortisol on insulin action. Therefore, development of specific inhibitors targeting 11ß-HSD1 might be a promising way to improve impaired insulin-stimulated glucose uptake.

Applying Machine Learning to Healthcare Operations Management: CNN-Based Model for Malaria Diagnosis.

The purpose of this study is to explore how machine learning technologies can improve healthcare operations management. A machine learning-based model to solve a specific medical problem is developed to achieve this research purpose. Specifically, this study presents an AI solution for malaria infection diagnosis by applying the CNN (convolutional neural network) algorithm. Based on malaria microscopy image data from the NIH National Library of Medicine, a total of 24,958 images were used for deep learning training, and 2600 images were selected for final testing of the proposed diagnostic architecture. The empirical results indicate that the CNN diagnostic model correctly classified most malaria-infected and non-infected cases with minimal misclassification, with performance metrics of precision (0.97), recall (0.99), and f1-score (0.98) for uninfected cells, and precision (0.99), recall (0.97), and f1-score (0.98) for parasite cells. The CNN diagnostic solution rapidly processed a large number of cases with a high reliable accuracy of 97.81%. The performance of this CNN model was further validated through the k-fold cross-validation test. These results suggest the advantage of machine learning-based diagnostic methods over conventional manual diagnostic methods in improving healthcare operational capabilities in terms of diagnostic quality, processing costs, lead time, and productivity. In addition, a machine learning diagnosis system is more likely to enhance the financial profitability of healthcare operations by reducing the risk of unnecessary medical disputes related to diagnostic errors. As an extension for future research, propositions with a research framework are presented to examine the impacts of machine learning on healthcare operations management for safety and quality of life in global communities.

Relationship between Racial Diversity in Medical Staff and Hospital Operational Efficiency: An Empirical Study of 3870 U.S. Hospitals.

Demand for foreign nurses and medical staff is rapidly increasing due to the severe labor shortage in U.S. hospitals triggered by the COVID-19 pandemic. However, empirical studies on the effect of the racial diversity of medical staff on hospital operations are still lacking. This research gap is thus investigated based on the foreign medical staff working in 3870 U.S. hospitals. Results show that workforce racial diversity has a significantly positive relationship with hospital operational efficiency regarding occupancy rate, manpower productivity, capacity productivity, and case mix index. Notably, this study empirically supports that increasing the ratio of foreign nurses positively affects the overall operational efficiency of hospitals. In addition, the study results also indicate that the hospital location, size, ownership, and teaching status act as significant control variables for the relationship between racial diversity and hospital efficiency. These results imply that hospitals with these specific operating conditions need to pay more attention to racial diversity in the workplace, as they are structurally more sensitive to the relationship between racial diversity and operational efficiency. In short, the findings of this study suggest that hospital efficiency can be operationally improved by implementing workforce ethnic diversity. For this reason, hospital stakeholders and healthcare policymakers are expected to benefit from this study's findings. Above all, the results of this study imply that if an organization adapts to extreme external environmental changes (e.g., the COVID-19 pandemic) through appropriate organizational restructuring (i.e., expanding the workforce racial diversity by hiring foreign medical staff), the organization can gain a competitive advantage, a claim that is supported by contingency theory. Further, investors are increasingly interested in ESG, especially companies that embody ethical and socially conscious workplaces, including a diverse and inclusive workforce. Thereby, seeking racial diversity in the workforce is now seen as a fundamental benchmark for organizational behavior that predicts successful ESG business practices, a claim that is supported by stakeholder theory. Therefore, in conclusion, the findings of this study suggest that workforce racial diversity is no longer an optional consideration but should be considered as one of the essential determinants of competitive advantage in organizations, particularly in the healthcare sector.

Zinc balance is critical for NFI-C mediated regulation of odontoblast differentiation.

Zinc is trace element essential for diverse metabolic and cellular signaling pathways for the growth, development, and maintenance. Zinc deficiency is involved in bone malformations and oral disease. Mice deficient in zinc transporter Zip13 show connective tissue and skeletal disorders, abnormal incisor teeth, and reduced root dentin formation in the molar teeth and share a morphologically similar phenotype to nuclear factor I-C (NFI-C)-deficient mice. However, the precise function of zinc in NFI-C signaling-mediated odontoblast differentiation and dentin formation remains unclear. Here, we show that zinc stimulated the expression of metal transcription factor-1, but decreased NFI-C expression in odontoblastic MDPC-23 cells. Zinc also enhanced the phosphorylation of Smad2/3 (p-Smad2/3) and increased the binding efficiency of NFI-C and p-Smad2/3 in the cytoplasm. In contrast, zinc deficiency resulted in the accumulation of NFI-C into nucleus. Consequently, NFI-C had the biologic properties of a transcription factor, including DNA binding affinity for metallothionein-1 and the dentin sialophosphoprotein (DSPP) promoter, and transcriptional activation of the DSPP gene. Furthermore, zinc deficiency condition promoted DSPP expression in odontoblasts and dentin mineralization, while zinc sufficiency condition decreased DSPP expression and slightly delayed dentin mineralization. These data suggest that zinc equilibrium is required for odontoblast differentiation and dentin formation during dentinogenesis through the nuclear accumulation and modulation of NFI-C.

Odontogenic ameloblasts-associated protein (ODAM), via phosphorylation by bone morphogenetic protein receptor type IB (BMPR-IB), is implicated in ameloblast differentiation.

To elucidate the function of the odontogenic ameloblast-associated protein (ODAM) in ameloblasts, we identified more than 74 proteins that interact with ODAM using protoarray. Of the identified proteins, bone morphogenetic protein receptor type-IB (BMPR-IB) was physiologically relevant in differentiating ameloblasts. ODAM and BMPR-IB exhibited similar patterns of expression in vitro, during ameloblast differentiation. ODAM and BMPR-IB interacted through the C-terminus of ODAM, which resulted in increased ODAM phosphorylation in the presence of bone morphogenetic protein 2 (BMP-2). Immunoprecipitation assays using Ser-Xaa-Glu (SXE) mutants of ODAM demonstrated that the phosphorylation of ODAM by BMPR-IB occurs at this motif, and this phosphorylation is required for the activation of MAPKs. ODAM phosphorylation was detected in ameloblasts during ameloblast differentiation and enamel mineralization in vitro and involved in the activation of downstream factors of MAPKs. Therefore, the BMP-2-BMPR-IB-ODAM-MAPK signaling cascade has important roles in ameloblast differentiation and enamel mineralization. Our data suggest that ODAM facilitates the progression of tooth development in cooperation with BMPR-IB through distinct domains of ODAM.

Protective effects of arachidonic acid against palmitic acid-mediated lipotoxicity in HIT-T15 cells.

Saturated fatty acids have been considered major contributing factors in type 2 diabetes, whereas unsaturated fatty acids have beneficial effects for preventing the development of diabetes. However, the effects of polyunsaturated fatty acids in pancreatic ß cells have not been reported. Here, we examined the effects of arachidonic acid (AA) on palmitic acid (PA)-mediated lipotoxicity in clonal HIT-T15 pancreatic ß cells. AA prevented the PA-induced lipotoxicity as indicated by cell viability, DNA fragmentation and mitochondrial membrane potential, whereas eicosatetraynoic acid (ETYA), a non-metabolizable AA, had little effect on PA-induced lipotoxicity. In parallel with its protective effects against PA-induced lipotoxicity, AA restored impaired insulin expression and secretion induced by PA. AA but not ETYA increased intracellular triglyceride (TG) in the presence of PA compared with PA alone, and xanthohumol, a diacylglycerol acyltransferase (DGAT) inhibitor, reversed AA-induced protection from PA. Taken together, our results suggest that AA protects against PA-induced lipotoxicity in clonal HIT-T15 pancreatic ß cells, and the protective effects may be associated with TG accumulation, possibly through sequestration of lipotoxic PA into TG.