Lupus erythematosus Glycine, serine and threonine metabolism Jak-STAT signaling pathway Vascular smooth muscle contraction Arhythmogenic right ventricular cardiomyopathy (ARVC)aTotal genes in pathway 256 267 190 50 31 46 178 56 140 31 155 115Unique genes in pathwaya 19 18 10 5 5 4 7 4 6 3 6 5Adj p valueb 8.18e-10 6.75e-09 0.0004 0.0014 0.0022 0.0103 0.0160 0.0161 0.0168 0.0196 0.0229 0.0271 0.Lists of uniquely expressed genes within the enriched pathways can be found in Table S1. adj p value indicates the significance of the enrichment, (adj p,0.05). doi:10.1371/journal.pone.0046440.tbcategory Lixisenatide web biological process was used for the functional annotation analysis.RNA-SequencingWe sequenced cDNA from 10 periodontitis-affected and 10 healthy gingival tissues, with an average of 15 million reads of 100 bp in length per sample. A pairwise approach, where each periodontitis-affected biopsy had a healthy counterpart from the same individual, was used to eliminate the background noise of individual-specific gene transcription, enabling acquisition of more relevant data from the cohort. Aligning the sequence reads against the human genome yielded a median of 68 of uniquely aligned reads across all samples. The expression pattern, based on RNASeq reads, of well-known inflammatory mediators IL-1b, IL-6, IL8, TNFa, Regulated upon Activation, Normal T-cell Expressed, and Secreted (RANTES) and Monocyte Chemotactic Protein-1 (MCP-1) were analyzed in all the tissue samples. The expression (log2 fold change) of these mediators was shown to be higher in the majority of the periodontitis-affected gingival tissue compared to healthy gingival tissue from the same patient (Fig. 2).Results Patients and gingival tissuesA total of 10 patients, six males and four females, with a mean age of 5068, were included in the study. For each patient, a total of four gingival biopsies of about the same 
size were obtained from periodontitis-affected and healthy gingiva, with two biopsies from each site. Bleeding status, probing depth and degree of inflammation in the gingival tissues for each of the two gingival sites was recorded (Table 1). To assess the degree of gingival inflammation in the periodontitis-affected and healthy tissues, histological and immunohistochemistry staining was performed using H E and 
anti-CD3 (Fig. 1). Scoring of the degree of inflammatory cell infiltration, assessed by H E staining, and the amount of CD3 positive cells showed significantly higher inflammation in tissue from periodontitis-affected sites (p,0.01 for H E and p,0.05 for CD3; Table 1).Table 3. Enriched regulated (KEGG) biological pathways among unique genes in healthy tissues.Unique genes in pathwaya 11 3 4 3 2 2 Adj p valueb 8.18e-10 6.75e-09 0.0004 0.0014 0.0022 0.Pathway Neuroactive ligand-receptor PS-1145 web interaction Glycolysis/Gluconeogenesis Calcium signaling pathway Gap junction Pyruvate metabolism Tryptophan metabolismaTotal genes in pathway 256 62 178 90 40Lists of uniquely expressed genes within the enriched pathways can be found in Table S1. adj p value indicates the significance of the enrichment, (adj p,0.05). doi:10.1371/journal.pone.0046440.tbGene Expression in PeriodontitisFigure 4. Clustering dendrogram and heatmap of periodontitis-affected and healthy biopsies. Clustering of all samples was based on gene transcripts with a median read above three times the background noise. The length of the branch between two biopsies and the colors of the heatmap correspond to degree of si.Lupus erythematosus Glycine, serine and threonine metabolism Jak-STAT signaling pathway Vascular smooth muscle contraction Arhythmogenic right ventricular cardiomyopathy (ARVC)aTotal genes in pathway 256 267 190 50 31 46 178 56 140 31 155 115Unique genes in pathwaya 19 18 10 5 5 4 7 4 6 3 6 5Adj p valueb 8.18e-10 6.75e-09 0.0004 0.0014 0.0022 0.0103 0.0160 0.0161 0.0168 0.0196 0.0229 0.0271 0.Lists of uniquely expressed genes within the enriched pathways can be found in Table S1. adj p value indicates the significance of the enrichment, (adj p,0.05). doi:10.1371/journal.pone.0046440.tbcategory Biological process was used for the functional annotation analysis.RNA-SequencingWe sequenced cDNA from 10 periodontitis-affected and 10 healthy gingival tissues, with an average of 15 million reads of 100 bp in length per sample. A pairwise approach, where each periodontitis-affected biopsy had a healthy counterpart from the same individual, was used to eliminate the background noise of individual-specific gene transcription, enabling acquisition of more relevant data from the cohort. Aligning the sequence reads against the human genome yielded a median of 68 of uniquely aligned reads across all samples. The expression pattern, based on RNASeq reads, of well-known inflammatory mediators IL-1b, IL-6, IL8, TNFa, Regulated upon Activation, Normal T-cell Expressed, and Secreted (RANTES) and Monocyte Chemotactic Protein-1 (MCP-1) were analyzed in all the tissue samples. The expression (log2 fold change) of these mediators was shown to be higher in the majority of the periodontitis-affected gingival tissue compared to healthy gingival tissue from the same patient (Fig. 2).Results Patients and gingival tissuesA total of 10 patients, six males and four females, with a mean age of 5068, were included in the study. For each patient, a total of four gingival biopsies of about the same size were obtained from periodontitis-affected and healthy gingiva, with two biopsies from each site. Bleeding status, probing depth and degree of inflammation in the gingival tissues for each of the two gingival sites was recorded (Table 1). To assess the degree of gingival inflammation in the periodontitis-affected and healthy tissues, histological and immunohistochemistry staining was performed using H E and anti-CD3 (Fig. 1). Scoring of the degree of inflammatory cell infiltration, assessed by H E staining, and the amount of CD3 positive cells showed significantly higher inflammation in tissue from periodontitis-affected sites (p,0.01 for H E and p,0.05 for CD3; Table 1).Table 3. Enriched regulated (KEGG) biological pathways among unique genes in healthy tissues.Unique genes in pathwaya 11 3 4 3 2 2 Adj p valueb 8.18e-10 6.75e-09 0.0004 0.0014 0.0022 0.Pathway Neuroactive ligand-receptor interaction Glycolysis/Gluconeogenesis Calcium signaling pathway Gap junction Pyruvate metabolism Tryptophan metabolismaTotal genes in pathway 256 62 178 90 40Lists of uniquely expressed genes within the enriched pathways can be found in Table S1. adj p value indicates the significance of the enrichment, (adj p,0.05). doi:10.1371/journal.pone.0046440.tbGene Expression in PeriodontitisFigure 4. Clustering dendrogram and heatmap of periodontitis-affected and healthy biopsies. Clustering of all samples was based on gene transcripts with a median read above three times the background noise. The length of the branch between two biopsies and the colors of the heatmap correspond to degree of si.
