ABSTRACT
Background: Dental enamel formation is a complex process that is regulated by various genes. One such gene, Family With Sequence Similarity 83 Member H (Fam83h), has been identified as an essential factor for dental enamel formation. Additionally, Fam83h has been found to be potentially linked to the Wnt/ß-catenin pathway. Objectives: This study aimed to investigate the effects of the Fam83h knockout gene on mineralization and formation of teeth, along with mediators of the Wnt/ß-catenin pathway as a development aspect in mice. Materials and Methods: To confirm the Fam83h-KnockOut mice, both Sanger sequencing and Western blot methods were used. then used qPCR to measure the expression levels of genes related to tooth mineralization and formation of dental root, including Fam20a, Dspp, Dmp1, Enam, Ambn, Sppl2a, Mmp20, and Wnt/ß-catenin pathway mediators, in both the Fam83h-Knockout and wild-type mice at 5, 11 and 18 days of age. also the expression level of Fgf10 and mediators of the Wnt/ß-catenin pathway was measured in the skin of both Knockout and wild-type mice using qPCR. A histological assessment was then performed to further investigate the results. Results: A significant reduction in the expression levels of Ambn, Mmp20, Dspp, and Fgf10 in the dental root of Fam83h-Knockout mice compared to their wild-type counterparts was demonstrated by our results, indicating potential disruptions in tooth development. Significant down-regulation of CK1a, CK1e, and ß-catenin in the dental root of Fam83h-Knockout mice was associated with a reduction in mineralization and formation-related gene. Additionally, the skin analysis of Fam83h-Knockout mice revealed reduced levels of Fgf10, CK1a, CK1e, and ß-catenin. Further histological assessment confirmed that the concurrent reduction of Fgf10 expression level and Wnt/ß-catenin genes were associated with alterations in hair follicle maturation. Conclusions: The concurrent reduction in the expression level of both Wnt/ß-catenin mediators and mineralization-related genes, resulting in the disruption of dental mineralization and formation, was caused by the deficiency of Fam83h. Our findings suggest a cumulative effect and multi-factorial interplay between Fam83h, Wnt/Β-Catenin signaling, and dental mineralization-related genes subsequently, during the dental formation process.
ABSTRACT
BACKGROUND: Toxoplasma gondii is a widely-distributed parasite all over the world whose attributed severe afflicting complications in human necessitate the development of serodiagnostic tests and vaccines for it. Immunological responses to monovalent vaccines and the application of diagnostic reagents including single antigens are not optimally effective. Bioinformatics approaches were used to introduce these epitopes, predict their immunogenicity and preliminarily evaluate their potential as an effective DNA vaccine and for serodiagnostic goals. MATERIALS AND METHODS: A 3D structure of proteins was predicted by I-TASSER server, and linear and conformational B cell and T cell epitopes were predicted using the online servers. Then, the predicted epitopes were constructed and called Toxoeb, and their expression in the prokaryotic and eukaryotic cells was demonstrated using SDS-PAGE. In the next step, Western blotting with pooled sera of mice infected with T. gondii was done. RESULTS: The current in silico analysis revealed that the B cell epitopes with high immunogenicity for GRA4 protein were located in the residues 34-71, and 230-266, for GRA14 in 308-387, for SAG1 in 182-195, 261-278, and for GRA7 in residues 101-120, 160-176. The T cell epitopes were selected in overlapping regions with the B cell epitopes. The immunogenic region for GRA4 are in the residues 245-253, 50-58, and 40-54, for GRA14 in 307-315, 351-359, and 308- 322, for SAG1 261-269, and 259-267, and for GRA7 in the residues 103-112, and 167-175. The results of the western blotting showed that the expressed protein had immunogenicity. CONCLUSION: Our constructed multi-epitope of T. gondii could be considered as a candidate for diagnostic and vaccination purposes.