DOI: 10.5061/dryad.f4qrfj6sn
title: Rapamycin rejuvenates oral health in aging mice

description: Periodontal disease is an age-associated disorder clinically defined by
 periodontal bone loss, inflammation of the specialized tissues that
 surround and support the tooth, and microbiome dysbiosis. Currently, there
 is no therapy for reversing periodontal disease, and treatment is
 generally restricted to preventive measures or tooth extraction. The
 FDA-approved drug rapamycin slows aging and extends lifespan in multiple
 organisms, including mice. Here we demonstrate that short-term treatment
 with rapamycin rejuvenates the aged oral cavity of elderly mice, including
 regeneration of periodontal bone, attenuation of gingival and periodontal
 bone inflammation, and revertive shift of the oral microbiome toward a
 more youthful composition. This provides a geroscience strategy to
 potentially rejuvenate oral health and reverse periodontal disease in the
 elderly.
description: DNA extraction Mandible
 samples were cryogrinded and homogenized using Evolution with bead-beating
 tubes and ceramic beads (Bertin Instruments, Rockville, MD). Bacterial
 genomic DNA was extracted using the QIAamp DNA Microbiome Kit (Qiagen,
 Hilden, Germany) and further purified and concentrated using DNA Clean and
 Concentrator Kit (Zymo Research, Irvine, CA, USA) according to the
 manufacturer’s protocol, then stored at - 80°C until all samples were
 collected Sequencing The V3-V4 variable region of the 16s ribosomal RNA
 gene was amplified using gene-specific primers with Illumina adapter
 overhang sequences (5’-
 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3’ and 5’-
 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3’). Each reaction
 mixture contained 2.5 µl of genomic DNA, 5 µl of each 1 µM primer, and
 12.5 µl of KAPA HiFi HotStart ReadyMix. Amplicon PCR was carried out as
 follows: denaturation at 95°C for 3 minutes, 35-40 cycles at 95°C for 30
 sec, 55°C for 30 sec, 72°C for 30 sec, followed by a final extension step
 at 72°C for 5 min. PCR products were verified using gel electrophoresis
 (1% agarose gel) and cleaned with AMPure XP beads (Agencourt, Beckman
 Coulter Inc., Pasadena, CA, USA). Amplicons were then indexed using the
 Nextera XT Index Kit V2 set A and set D (Illumina) and purified again with
 AMPure XP beads to remove low molecular weight primers and primer-dimer
 sequences. DNA concentrations were concentration of 1-2nM using the
 SequalPrep<sup>™</sup> Normalization Kit (Invitrogen). Samples
 were pooled into a single library which was analyzed using the TapeStation
 4200 High Sensitivity D1000 assay (Agilent Technologies, Waldbronn,
 Germany) and Qubit High Sensitivity dsDNA assay (Thermo Fischer
 Scientific) to assess DNA quality and quantity. The final pooled library
 was then loaded on to an Illumina MiSeq sequencer with 10% PhiX spike,
 which served as an internal control to balance for possible low diversity
 and base bias present in the 16S amplicon samples, and was run for 478
 cycles and generated a total of 5.68 million paired-end reads (2×239
 bp). Bioinformatics Raw paired-end sequences were imported in to Qiime2
 (v. 2019.1) and were trimmed by 15 nt from the 5’ end and truncated to 239
 nt for the 3’ end for both the forward and reverse reads respectively. The
 trimmed reads were then demultiplexed and denoised using the DADA2
 package. Forward reads were only used in our analysis. Taxonomy was then
 assigned using the feature-classifier suite trained on the and the Human
 Oral Microbiome Database (HOMD v. 15.1). Samples were then filtered for
 taxonomic contaminants excluded samples with less than 10,000 reads. Alpha
 and Beta diversity as well as other analysis were done in R-Studio using
 the Phyloseq, Clustvis, ggplot2,  ampvis2, vegan, ade4 packages as part of
 the R suite. Supplemental methods Taxonomy filtered from samples was determined by
 analysis of kit controls with no template and zymo sequencing controls of
 know diversity and abundance (the QIAamp DNA Microbiome Kit (Qiagen,
 Hilden, Germany) DNA Clean and Concentrator Kit (Zymo Research). The
 following taxonomic assignments were removed as part of the dada2
 workflow: Unassigned, Cyanobacteria, acidovorans, pestis, coli,
 flavescens, sakazakii, durans, diminuta, anthropi, monocytogenes,
 parasanquinis_clade_411, otitidis, subtilis, aeruginosa,
 fermentum. Statistical Analysis Results for the 16s rRNA sequencing, to identify
 statistically significant differences among agglomerated and normalized
 amplicon sequence variants (ASV) between samples as well as differences in
 alpha and beta diversity measures, we applied both the unpaired Wilcoxon
 rank-sum test as well as the two-tailed paired t-test – both with a 95%
 confidence interval (α = 0.05). Alpha diversity was assessed measuring
 Shannon, Chao1, Observed (ASV), and Fisher diversity measures. Beta
 diversity was measured using weighted Unifrac distances. Statistical
 analysis for the microbiome analysis was completed in R (v.
 3.5.3).
description: Workflow materials and R Markdown are provided in detail on the
 McLean Lab Github repository: https://github.com/kkerns85/Rapamycin_rejuvenates_oral_health_in_aging_mice A web version of the R Markdown is available at: https://rpubs.com/kkerns85/Rapamycin_Rmrkdown Raw read data is available on the European Nucleotide Archive (ENA) under study accession no. PRJEB35672