CRISPR/Cas9-Induced Fam83h Knock-out Leads to Impaired Wnt/ß-Catenin Pathway and Altered Expression of Tooth Mineralization Genes in Mice.

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.

Feeding role of mouse embryonic fibroblast cells is influenced by genetic background, cell passage and day of isolation.

Mouse embryonic fibroblast (MEF) cells are commonly used as feeder cells to maintain the pluripotent state of stem cells. MEFs produce growth factors and provide adhesion molecules and extracellular matrix (ECM) compounds for cellular binding. In the present study, we compared the expression levels of Fgf2, Bmp4, ActivinA, Lif and Tgfb1 genes at the mRNA level and the level of Fgf2 protein secretion and Lif cytokine secretion at passages one, three and five of MEFs isolated from 13.5-day-old and 15.5-day-old embryos of NMRI and C57BL/6 mice using real-time PCR and enzyme-linked immunosorbent assay. We observed differences in the expression levels of the studied genes and secretion of the two growth factors in the three passages of MEFs isolated from 13.5-day-old and 15.5-day-old embryos, respectively. These differences were also observed between the NMRI and C57BL/6 strains. The results of this study suggested that researchers should use mice embryos that have different genetic backgrounds and ages, in addition to different MEF passages, when producing MEFs based on the application and type of their study.

An amplified comparative fluorescence resonance energy transfer immunosensing of CA125 tumor marker and ovarian cancer cells using green and economic carbon dots for bio-applications in labeling, imaging and sensing.

CA125, is a marker in the clinical diagnosis of several cancers and currently is the best serum-based tumor marker for ovarian cancer. Here, we developed an ultrasensitive antibody-ssDNA aptamer sandwich-type fluorescence immunosensor for CA125 detection. Based on a novel signal amplification strategy the carbon dots (CDs) functionalized with aptamer (CD-aptamer) used as detection probe and PAMAM-Dendrimers/AuNPs was used for covalent attachment of CA125-antibody and completing the sandwich assay method. By measuring of fluorescence resonance energy transfer (FRET) signals between CDs and AuNPs as nanoquenchers, the fluorescence signal quenched during sandwich complex formed between anti-CA125, CA125 and CDs-Aptamer and decreasing of fluorescence response signal is related to CA125 concentrations. Under optimal conditions, the immunosensor exhibited an extremely low calculated detection limit of 0.5fg/mL with wide linear range 1.0fg/mL to 1.0ng/mL of CA 125. The application of the immunosensor for CA125 detection in serum samples and measuring of ovarian-cancer cells was also investigated. The immunosensor revealed good sensitivity and specificity with ovarian cell concentrations from 2.5×103 to 2×104cells/mL with correlation coefficient of 0.9937 and detection limit of 400cells/mL (4 cell in 10µL), indicating potential application of immunosensor in clinical monitoring of tumor biomarkers. Furthermore, the cell viability was not changed upon treatment with CDs probe during 24h, showing the low cytotoxicity of the probe. More importantly, CDs-antibody hybrid was achieved in selective imaging of the cancer cells over the OVCAR-3 line cells, implying its potential applications in biosensing, as well as in cancer diagnosis.