Orofacial cleft (OFC) is a relatively common birth defect that has major impacts on affected individuals and their families. Underlying causes of OFC include genetics and environmental influences. Genome-wide association studies (GWAS) and linkage analyses have revealed genes in which DNA variants are enriched in OFC cases relative to in unaffected individuals in the same ethnic group. Only a portion of the genetic causes have been identified. Here we focus on ARHGAP29, which was identified in several GWAS of OFC. To uncover the role of this gene in craniofacial morphogenesis, and to identify other members of its regulatory pathway, we are working on deleting a paralog of this gene, arhgap29b, in zebrafish embryos. To this end, we have designed four CRISPR guide RNAs that target specific exons in the arhgap29b gene and injected them into zebrafish embryos. We predict that such embryos will a) harbor mutations in the arhgap29b gene, which we plan to test with PCR and sequencing, and b) display abnormal morphogenesis of the face, which we plan to test by microscopy. Alternatively, we may observe a) but not b). In this event we would simultaneously disrupt the other paralog, arhgap29a. These findings will advance our understanding of genes associated with orofacial cleft, hopefully leading to improved diagnosis and underpinning the design of therapies for this disorder.

Cellular senescence is one hallmark in the biology of aging in which cells stop growing and accumulate over time. These cells secrete factors called SASP, or senescence-associated secretory phenotype, which may be involved in inflammation and cancer. A cancer that most often occurs with age is cancer of the tongue. Investigating tongue cancer has been primarily in animal models that have induced cancer, thereby losing the opportunity to study the impact of aging on cancer. This project evaluated cellular senescence in aged male and female mice tongues and tested the effect of inhibiting mTOR via rapamycin, an intervention that improves the health and extends lifespan in animal models. RNA from tongue specimens was isolated and Real-Time PCR was used to quantify the levels of SASP expression. Histology was completed to visualize the tongue structure. The results show differences in aged male and female mice in senescence markers p16 (Cdkn2a) and p21 (Cdkn1a). There were also significant increases in major markers such as IL-1A, IL-1B, and TNF-a. Treatment with rapamycin decreased SASP expressions in older mice. Further research is needed to test how these SASP markers relate to aging and oral cancer. Future experiments include conducting protein assays and overexpressing these SASPs to determine if they increase the risk for oral cancer in mice. This project is important because these markers can become valuable tools for clinicians to diagnose oral cancer in older adults before its onset. This will provide more time for them to implement preventative measures and treatments, increasing the survival rates in the fight against cancer and age-related diseases.

Previous work has shown that short-term treatment of rapamycin (an mTOR inhibitor) in aged mice can increase lifespan, improve heart function, attenuate inflammation in gingiva and periodontal bone, and remodel the oral microbiome toward a more youthful state. Nevertheless, important questions arise of whether the beneficial effects of rapamycin persists. Here, we demonstrate that rapamycin continues attenuating periodontitis in old mice even following an 8-week cessation in treatment. Quantitative Real-Time PCR analysis was used to determine expression levels of inflammation markers attributed to periodontitis. Quantitation and analysis were completed by first standardizing target genes relative to ß-actin and GAPDH expression. Data were analyzed where appropriate using one-way analysis of variance (ANOVA) with post-hoc Tukey test for multiple comparisons, where p-values <0.05 were considered statistically significant. Statistical analysis was completed on GraphPad Prism 10.0. Our findings show elevated levels of inflammatory cytokines IL-1a and IL-1b and bone turnover markers NFATc1 and MMP13 in aged alveolar bone were decreased after rapamycin treatment, and the attenuation persisted. Additionally, PI3K-α and PI3K-γ expression, both upstream of mTOR, associated with periodontitis, and elevated in old mice, were inhibited to levels of young mice, and results persisted. These findings demonstrate that the potential therapeutic effect of rapamycin persists in attenuating periodontitis and suggest that a drug dosage regimen can potentially be optimized.

Periodontal disease is a prevalent oral disease most frequently found in older adults, and left untreated can lead to inflammation, bone loss, and patients may lose their teeth. Treatment of periodontitis includes repeated maintenance by a dentist and surgical procedures. These treatments can be invasive and time-consuming and do not target the underlying molecular changes that occur during the aging process. Previously, the mTOR pathway was shown to decrease inflammation and bone loss during periodontal disease in aged mice. Here, we investigate if targeting a marker upstream of mTOR, called colony-stimulating factor 1 receptor (CSF-1R), can recapitulate the effects of targeting mTOR. CSF1-R plays a role in inflammatory diseases by regulating myeloid cell lineages such as macrophages and osteoclasts. We hypothesize that inhibiting CSF-1R will decrease the expression of inflammation markers associated with periodontal disease in aged mice. Real-time quantitative PCR was used to identify relative expression levels of various inflammatory markers involved during aging and periodontal disease, including IL1-a and IL1a. Quantitation and analysis were completed by first standardizing target genes relative to ß-actin and GAPDH expression. Data was analyzed where appropriate using one-way analysis of variance (ANOVA) with post-hoc Tukey test for multiple comparisons, where p-values <0.05 were considered statistically significant. Statistical analysis was completed on GraphPad Prism 10.0. Our analysis demonstrates that CSF-1R inhibition decreases multiple cytokines involved during periodontal disease and major isoforms of the PI3K pathway. These findings indicate the importance of the CSF1R signaling along the mTOR pathway and possible implications for future research. This study was supported by the VitaDAO/Molecule Longevity Fund to Dr. Jonathan An, Oral Health Sciences

Tooth decay is a public health challenge that disproportionately impacts Indigenous populations. The main risk factor for pediatric tooth decay is added sugar intake. This study was part of a community-based clinical trial aimed at reducing sugared fruit drink intake among Yup’ik Alaska Native children by introducing families to sugar-free fruit drinks, and sought to collect caregiver feedback on various components of the intervention. Our team administered a 14-item semi-structured interview script to caregivers during their 6-month and 12-month study visits. I inductively analyzed the data, classified the data into categories, and further organized these categories into domains: opinions about sugar-free Kool-Aid (SFKA), accessing SFKA, and thoughts about the SFKA program among the intervention arm. This feedback will be used to redesign the intervention for the delayed-treatment control group, which is currently receiving a modified version of the intervention. The long-term goal is to develop a sustainable, scalable intervention that can help reduce sugar intake in Alaska Native communities.