High glucose promotes the aging of human dental pulp cells through Wnt/beta-catenin signaling.

BACKGROUND: Diabetes is one of the most common metabolic diseases that disrupt the functioning of different body organs, including oral tissue. Some diabetic complications remain even after the control of the hyperglycemic condition. The adverse effect of hyperglycemia on the pulp structure and function has been shown previously. OBJECTIVES: The purpose of this study was to investigate the effect of the hyperglycemic state on the aging of pulp cells and evaluate the role of Wnt signaling as the underlying mechanism of this process. MATERIAL AND METHODS: The isolated pulp cells were cultured in the Minimum Essential Medium (MEM)-alpha for 7, 14 and 21 days under the influence of glucose at concentrations of 5 mM, 20 mM and 30 mM. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to evaluate cell viability, the beta-galactosidase test was applied to assess cellular senescence and gene expression was measured with quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: The results of this study showed that cell proliferation decreased following exposure to 20 and 30 mM glucose, which was accompanied by an increased number of senescent cells and an increased p21 expression. There was a significant increase in beta-catenin and Wnt1 expression in response to high glucose. Treatment with beta-catenin inducer enhanced cellular aging in the hyperglycemic state, while betacatenin inhibitor decreased the senescence response. CONCLUSIONS: The present study further confirmed the effect of the high-glucose condition on pulp cell aging and suggests a role for beta-catenin in the induction of cellular aging. Targeting the key regulators of cellular aging in pulp tissue might lead to the development of new therapies for the prevention and treatment of endodontic complications in diabetic patients.

Azacitidine, as a DNMT Inhibitor Decreases hTERT Gene Expression and Telomerase Activity More Effective Compared with HDAC Inhibitor in Human Head and Neck Squamous Cell Carcinoma Cell Lines.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is one of the most fatal malignancies worldwide and despite using various therapeutic strategies for the treatment of HNSCC, the surveillance rate is low. Telomerase has been remarked as the primary target in cancer therapy. Considering the key regulatory role of epigenetic mechanisms in controlling genome expression, the present study aimed to investigate the effects of two epigenetic modulators, a DNA methylation inhibitor and a histone deacetylase inhibitor on cell migration, proliferation, hTERT gene expression, and telomerase activity in HNSCC cell lines. METHODS: Human HNSCC cell lines were treated with Azacitidine and Trichostatin A to investigate their effects on telomerase gene expression and activity. Cell viability, migration, hTERT gene expression, and telomerase activity were studied using MTT colorimetric assay, scratch wound assay, qRT-PCR, and TRAP assay, respectively. RESULTS: Azacitidine at concentrations of ≤1µM and Trichostatin A at 0.1 to 0.3nM concentrations significantly decreased FaDu and Cal-27 cells migration. The results showed that Azacitidine significantly decreased hTERT gene expression and telomerase activity in FaDu and Cal-27 cell lines. However, there were no significant changes in hTERT gene expression at different concentrations of Trichostatin A in both cell lines. Trichostatin A treatment affected telomerase activity at the high dose of 0.3 nM Trichostatin A. CONCLUSION: The findings revealed that unlike histone deacetylase inhibitor, Azacitidine as an inhibitor of DNA methylation decreases telomerase expression in HNSCC cells. This might suggest the potential role of DNA methyltransferase inhibitors in telomerase-based therapeutic approaches in squamous cell carcinoma.

Analysis of long non-coding RNA expression in hemophilia A patients.

OBJECTIVES: Hemophilia A (HA) is an inherited and rare X-linked bleeding disorder which is caused by mutations of the factor VIII gene (FVIII). Long non-coding RNAs (lncRNAs) are non-protein coding sequence transcripts containing more than 200 nucleotides and have potential in diagnosis, prevention and treatment of cancers and inherited bleeding disorders like thalassemia. The goal of this study was to determine the relationship between the expression of lncRNAs and the incidence of hemophilia A in Iranian population. METHODS: In this study, after bioinformatics analysis and lncRNA identification, the expression of two lncRNAs including NONHSAT139215 and NONHSAT139219 was investigated among the blood samples of severe hemophilia A and healthy (non-hemophilia) subjects using the relative qRT-PCR technique. RESULTS: The melting curve analysis confirmed the specificity of primers. Also, the standard curve showed that the efficiency of reactions for ß2-microglobulin (B2M), NONHSAT139215 and NONHSAT139219 was 1.91, 1.96 and 2.01, respectively. On the other hand, the statistical analysis using REST software indicated that the expression of NONHSAT139219 and NONHSAT139215 in severe HA patients was significantly down-regulated as compared to control group (p < .05). DISCUSSION: Our results demonstrated that a decrease in the expression of two lncRNAs located in FVIII gene may play an important role for the development of severe hemophilia A for the first time. CONCLUSIONS: Briefly, the abnormal expression levels of lncRNAs in hemophilia A cases may be correlated with disease intensity. However, further studies of these lncRNAs are required to provide a useful insight into hemophilia biology.

Electrospun poly-l-lactic acid/polyvinyl alcohol nanofibers improved insulin-producing cell differentiation potential of human adipose-derived mesenchymal stem cells.

Combination of adipose-derived mesenchymal stem cells (ADSCs) and synthetic materials in terms of pancreatic tissue engineering can be considered as a treatment of diabetes. This study aimed to evaluate the differentiation of human ADSCs to pancreatic cells on poly-l-lactic acid/polyvinyl alcohol (PLLA/PVA) nanofibers as a three-dimensional (3D) scaffold. Mesenchymal stem cells (MSCs) were characterized for mesenchymal surface markers by flow cytometry. Then ADSCs were seeded on 3D scaffolds and treated with pancreatic differentiation medium. Immunostaining assay showed that ADSCs were very efficiently differentiated into a relatively homogeneous population of insulin-producing cells. Moreover, real-time RT-PCR results revealed that pancreas-specific markers were highly expressed in 3D scaffolds compared with their expression in tissue culture plates and this difference in expression level was significant. In addition, insulin and C-peptide secreted in response to varying concentrations of glucose in the 3D scaffold group was significantly higher than that in 2D culture. The results of the present study confirmed that PLLA/PVA scaffold seeded with ADSCs could be a suitable option in pancreatic tissue engineering.

Flaxseed Oil Supplementation Improve Gene Expression Levels of PPAR-γ, LP(a), IL-1 and TNF-α in Type 2 Diabetic Patients with Coronary Heart Disease.

This study was carried out to determine the effects of flaxseed oil administration on gene expression levels related to insulin, lipid and inflammation in overweight diabetic patients with coronary heart disease (CHD). This randomized double-blind, placebo-controlled trial was conducted among 60 diabetic patients with CHD. Subjects were randomly allocated into two groups to intake either 1000 mg n-3 fatty acid from flaxseed oil containing 400 mg α-Linolenic acid [ALA (18:3n-3)] (n = 30) or placebo (n = 30) twice a day for 12 weeks. Gene expression related to insulin, lipid and inflammation were quantified in peripheral blood mononuclear cells (PBMC) of diabetic patients with CHD with RT-PCR method. Results of RT-PCR demonstrated that after the 12-week intervention, compared with the placebo, flaxseed oil supplementation could up-regulate gene expression of peroxisome proliferator-activated receptor gamma (PPAR-γ) (P = 0.02) in PBMC of diabetic patients with CHD. In addition, compared with the placebo, taking flaxseed oil supplements down-regulated gene expression levels of lipoprotein(a) [LP(a)] (P = 0.001), interleukin-1 (IL-1) (P = 0.001) and tumor necrosis factor alpha (TNF-α) (P = 0.02) in PBMC of diabetic patients with CHD. We did not observe any significant effect of flaxseed oil supplementation on gene expression levels of low-density lipoprotein receptor (LDLR), IL-8 and transforming growth factor beta (TGF-ß) in PBMC of diabetic patients with CHD. Overall, flaxseed oil supplementation for 12 weeks in diabetic patients with CHD significantly improved gene expression levels of PPAR-γ, LP(a), IL-1 and TNF-α, but did not influence LDLR, IL-8 and TGF-ß.

Enhancement of catalysis and functional expression of a bacterial laccase by single amino acid replacement.

Structure-function relationships underlying laccases properties are very limited that makes these enzymes interesting for protein engineering approaches. Therefore in the current study, a thermostable laccase that was isolated from Bacillus sp. HR03 with the ability of bilirubin oxidation besides its laccase and tyrosinase activity is used. The extensive application of this enzyme is limited by its low expression level in Escherichia coli. Based on sequence alignments and structural studies, three single amino acid substitutions, D500G, D500E, D500S and a glycine insertion, are introduced using site-directed mutagenesis to evaluate the role of Asp(500) located in the C-terminal segment close to the T1 copper center. Substitution of aspartic acid with less sterically hindered, conserved residue such as glycine increase kcat (2.3 fold) and total activity (7.3 fold) which is accompanied by a significant increase in the expression level up to 3 fold. Biochemical characterization and structural studies using far-UV CD and fluorescence spectroscopy reveal the importance of C-terminal copper-binding loop in the laccase functional expression and catalytic efficiency. Kinetic characterization of the purified mutants toward 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), syringaldazine (SGZ) and bilirubin, shows that substrate specificity is left unchanged.

1484insG polymorphism of the PTPN1 gene is associated with insulin resistance in an Iranian population.

BACKGROUND: Protein tyrosine phosphatase 1B (PTP1B), encoded by the PTPN1 gene, efficiently dephosphorylates the insulin receptor, and attenuates insulin signaling. Recently, a 1484insG variant of the PTPN1 gene has been associated with an increased risk of metabolic syndrome in an Italian population that has not been confirmed in the subsequent studies. The purpose of this study was to investigate the association of 1484insG polymorphism of the PTPN1 with obesity, insulin resistance, type 2 diabetes and other cardiovascular-related traits in an Iranian population. METHODS: The genotypes of 1484insG variant were determined by PCR-RFLP method in 696 unrelated subjects including 412 subjects with normal glucose tolerance and normal fasting glucose and 284 type 2 diabetic patients. RESULTS: The allelic frequency of 1484insG polymorphism among type 2 diabetic patients and non-diabetic subjects was 4.9 and 4.1%, respectively (p = 0.475). In type 2 diabetic patients, among quantitative traits, no significant difference in anthropometric and biochemical parameters was seen between the wild-type and heterozygous 1484insG genotypes in male and female groups and in non-diabetic subjects, male carriers of 1484insG allele had significantly higher fasting insulin (p = 0.003), cholesterol (p = 0.012), LDL-C (p = 0.037), apo B (p = 0.015), and HOMA-IR (p = 0.011) levels compared to the individuals carrying the wild-type genotype. Among non-diabetic female individuals, only body mass index was significantly higher in 1484insG subjects compared to the wild-type individuals (p = 0.007). CONCLUSIONS: Our results from a sample of Iranian type 2 diabetes cases and controls provide evidence that the 1484insG genotype of the PTPN1 gene may be associated with insulin resistance and other cardiovascular risk factors in non-diabetic male subjects.