EXPLORING THE EFFECTS OF CANESTAT, A NOVEL HYBRID PROTEIN, ON ORAL MICROBIOME DYNAMICS AND COMPOSITION

• Grant number: 24/14291-1
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate (Direct)
• Start date: January 20, 2025
• End date: January 19, 2026
• Field of knowledge: Health Sciences - Dentistry - Periodontology
• Principal Investigator: Marília Afonso Rabelo Buzalaf
• Grantee: Carolina Ruis Ferrari
• Supervisor: Magda Feres Figueiredo
• Host Institution: Faculdade de Odontologia de Bauru (FOB). Universidade de São Paulo (USP). Bauru , SP, Brazil
• Institution abroad: Harvard University, Boston, United States
• Associated to the scholarship: 21/14161-2 - Recombinant production and functional evaluation of a hybrid recombinant protein with potential to interact with hydroxyapatite to protect against dental caries and erosion, BP.DD

Abstract

The study focuses on modifying the acquired pellicle (AP), a crucial factor in protecting against dental caries, erosive tooth wear, and biofilm modification. Advances in "omic" techniques have revealed acid-resistant proteins in AP, driving the "acquired pellicle engineering procedures". Thus, enrich this layer with proteins highly binding to hydroxyapatite, potentially influencing biofilm formation. CaneCPI-5, a sugarcane-derived cystatin, and StatpSpS, a 15-amino acid peptide from statherin, stand out among the proteins that were found in AP. The objective of this study is analyzing the impact of mouthwash containing a recombinant hybrid protein with CaneCPI-5 and StatpSpS, named "CaneStat" on the oral microbiome dynamics and composition, influence by AP. A total of forty-eight healthy and gingivitis patients will be recruited according to inclusion criteria. For the prior to the intervention, participants will undergo comprehensive dental prophylaxis using pumice, accompanied by detailed instructions on oral hygiene practices. This will be followed by a 5 mL rinse with either a placebo or a solution containing the CaneStat molecule 0.05 mg/mL (predetermined concentration in a pilot study - unpublished data) for 1 min. For subsequent visits at 24 and 48 hours, as well as at 1 week and 1 month, participants will receive additional oral hygiene instruction reinforcement. During these visits, biofilm samples will be collected for the Checkerboard DNA-DNA hybridization and Illumina sequencing (16S rRNA) analysis. The signals obtained for samples processed by Checkerboard DNA-DNA hybridization will be converted into absolute counts based on controls of 105 and 106 cells. These data will be expressed as counts of DNA probes at levels of (x 105) and computed per individual. Mean counts and standard deviations (SD) will be calculated for each bacterial species in each group. The significance of differences will be determined using the student's t-test for independent samples between healthy and gingivitis patients. Among the healthy and gingivitis in the different time points, the significance of differences will be determined by the Mann-Whitney U test. Variables of interest in the microbial profile will be evaluated through correlation analyses and the Chi-square test. In the Next-Generation Sequencing (NGS) results, we will conduct comprehensive bioinformatics investigations. The unfolding research will leverage alpha and beta diversity analyses to vividly portray nuances in differences or similarities across diverse groups. Furthermore, our investigation into differential abundance will utilize ANCOM-BC (Analysis of Compositions of Microbiomes with Bias Correction) and LefSe (Linear Discriminant Analysis Effect Size) analyses. The established criterion for statistical significance in this upcoming exploration is set at p < 0.05. This anticipatory research endeavors to unravel intricate microbial patterns, contributing valuable insights to the realm of microbiome analysis.