Targeting the Neuropilin-1 receptor with Ovatodiolide and progress in using periodontal ligament organoids for COVID-19 research and therapy.

The discovery of SARS-CoV-2 RNA in the periodontal tissues of patients who tested positive for COVID-19, 24 days post the initial symptom onset, indicates the oral cavity could serve as a viral reservoir. This research aims to investigate the antiviral capabilities of Ovatodiolide, introducing a novel periodontal ligament organoid model for the study of SARS-CoV-2. We have successfully established a reliable and expandable organoid culture from the human periodontal ligament, showcasing characteristics typical of epithelial stem cells. This organoid model enables us to delve into the lesser-known aspects of dental epithelial stem cell biology and their interactions with viruses and oral tissues. We conducted a series of in vitro and ex vivo studies to examine the inhibitory impacts of Ova on SARS-CoV-2. Our findings indicate that Ovatodiolide molecules can bind effectively to the NRP1 active domain. Our study identifies potential interaction sites for Ovatodiolide (OVA) within the b1 domain of the NRP1 receptor. We generated point mutations at this site, resulting in three variants: Y25A, T44A, and a double mutation Y25A/T44A. While these mutations did not alter the binding activity of the spike protein, they did impact the concentration of OVA required for inhibition. The inhibitory concentrations for these variants are 15 µM for Y25A, 15.2 µM for T44A, and 25 µM for the double mutant Y25A/T44A. In addition, in vitro inhibition experiments demonstrate that the EC50 of Ova against the main protease (Mpro) of the SARS-CoV-2 virus is 7.316 µM. Our in vitro studies and the use of the periodontal ligament organoid model highlight Ovatodiolide's potential as a small molecule therapeutic agent that impedes the virus's ability to bind to the Neuropilin-1 receptor on host cells. The research uncovers various pathways and biochemical strategies through which Ovatodiolide may function as an effective antiviral small molecule drug.

Polymicrobial periodontal disease triggers a wide radius of effect and unique virome.

Periodontal disease is a microbially-mediated inflammatory disease of tooth-supporting tissues that leads to bone and tissue loss around teeth. Although bacterially-mediated mechanisms of alveolar bone destruction have been widely studied, the effects of a polymicrobial infection on the periodontal ligament and microbiome/virome have not been well explored. Therefore, the current investigation introduced a new mouse model of periodontal disease to examine the effects of a polymicrobial infection on periodontal ligament (PDL) properties, changes in bone loss, the host immune response, and the microbiome/virome using shotgun sequencing. Periodontal pathogens, namely Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, and Fusobacterium nucleatum were used as the polymicrobial oral inoculum in BALB/cByJ mice. The polymicrobial infection triggered significant alveolar bone loss, a heightened antibody response, an elevated cytokine immune response, a significant shift in viral diversity and virome composition, and a widening of the PDL space; the latter two findings have not been previously reported in periodontal disease models. Changes in the PDL space were present at sites far away from the site of insult, indicating that the polymicrobial radius of effect extends beyond the bone loss areas and site of initial infection and wider than previously appreciated. Associations were found between bone loss, specific viral and bacterial species, immune genes, and PDL space changes. These findings may have significant implications for the pathogenesis of periodontal disease and biomechanical properties of the periodontium. This new polymicrobial mouse model of periodontal disease in a common mouse strain is useful for evaluating the features of periodontal disease.

Retroviral transduction of human periodontal cells with a temperature-sensitive SV40 large T antigen.

The periodontal ligament (PDL) is considered to contain subpopulations of cells responsible for the development, repair and regeneration of the periodontium. Cell cultures have been used as model systems in order to understand the complex cellular and biochemical events underlying these processes. In order to obtain long-term cultures of these cells that can be cloned and characterized, primary cultures of PDL and gingival cells were infected with an amphotropic retroviral construct encoding a temperature-sensitive SV40 large T antigen (tsT). After selection for drug resistance, the cells expressed the T antigen and proliferated at 34 degrees C for more than 40 passages. However, when the T antigen was inactivated by incubation at 39 degrees C, the cultures became growth-arrested and the granularity of the cells increased, possibly as a result of differentiation. Reverse transcribed-polymerase chain reaction and flow cytometry showed that the tsT-transduced cells expressed a number of soft and hard connective-tissue antigens, including osteocalcin, osteonectin, osteopontin, collagen type I and alkaline phosphatase. Moreover, incubation of the transduced PDL cells at 39 degrees C was found to upregulate the expression of osteocalcin, osteopontin and collagen type I, but downregulate osteonectin. At this temperature, the presence of the dexamethasone downregulated type I collagen, while vitamin D3 had no effect on the expression of any of the antigens examined. Under all culture conditions, antigen expression was far higher in the transduced PDL cells than the gingival cells. The findings thus show that growth of the tsT-transduced PDL and gingival cells is temperature-dependent and that the presence of the T antigen increases their lifespan but does not ablate the expression of certain of their characteristic phenotypic and functional features.