DataSheet_1_Metabolic profiling of synovial fluid in human temporomandibular joint osteoarthritis.docx

IntroductionTemporomandibular joint (TMJ) osteoarthritis (OA) is a common TMJ degenerative disease with an unclear mechanism. Synovial fluid (SF), an important component of TMJ, contains various proteins and metabolites that may directly contribute to OA. The present study aimed to investigate the influence of SF in TMJOA at the metabolite level.MethodsUntargeted and widely targeted metabolic profiling were employed to identify metabolic changes in SF of 90 patients with different TMJOA grades according to TMJ magnetic resonance imaging.ResultsA total 1498 metabolites were detected. Most of the metabolites were amino acids and associated metabolites, benzene and substituted derivatives, and lipids. Among patients with mild, moderate and severe TMJOA, 164 gradually increasing and 176 gradually decreasing metabolites were identified, indicating that biosynthesis of cofactors, choline metabolism, mineral absorption and selenocompound metabolism are closely related to TMJOA grade. Combined metabolomics and clinical examination revealed 37 upregulated metabolites and 16 downregulated metabolites in patients with pain, of which 19 and 26 metabolites were positively and negatively correlated, respectively, with maximum interincisal opening. A model was constructed to diagnose TMJOA grade and nine biomarkers were identified. The identified metabolites are key to exploring the mechanism of TMJOA.DiscussionIn the present study, a metabolic profile was constructed and assessed using a much larger number of human SF samples from patients with TMJOA, and a model was established to contribute to the diagnosis of TMJOA grade. The findings expand our knowledge of metabolites in human SF of TMJOA patients, and provide an important basis for further research on the pathogenesis and treatment of TMJOA.</p

Post-translational modifications of TIPE1.

<p>(A) NetPhosK Server was performed to predict kinase specific phosphorylation sites in TIPE1. (B) NetPhos Server was performed to predict generic phosphorylation sites in TIPE1. X axis represents amino acid sequence from N- to C- terminal. Y axis represents values computed by the software. The values above the threshold mean the most potential of phosphorylation.</p

Protein-protein interaction analysis for TIPE1.

<p>STRING platform is used to predict protein interactions. FBXW5, caspase8, caspase10, POMP and et al. were predicted to be interacted with TIPE1.</p

Prediction of signal peptide and transmembrane domain of TIPE1.

<p>Signal peptide (A) and transmembrane domain (B) of TIPE1 are predicted by SignaIP Server and TMpred Server, respectively. X axis represents amino acid sequence from N- to C- terminal. Y axis represents scores computed by each server.</p

Prediction of hydrophilicity, accessibility, polarity, flexibility, mutability and bulkiness of TIPE1.

<p>The hydrophilicity (A), accessibility (B), polarity (C), flexibility (D), mutability (E) and bulkiness (F) of TIPE1 are predicted using Protscale Server of expasy platform. X axis represents amino acid sequence from N- to C- terminal. Y axis represents scores computed by each algorithm.</p

Homology modeling and evaluation of model stability.

<p>(A) Homology modeling was performed by Swiss-Model Server and the predicted 3D structure of TIPE1 protein was shown. (B) Model quality was evaluated using the method of Ramachandran plot and the results represent the acceptable stability of 3D structure of TIPE1 protein.</p

<i>P. gingivalis</i> strain W83 morphology and 16S rRNA gene analyzing.

<p>A, The colonial morphology of <i>P. gingivalis</i> strain W83 on the BHI agar plate. B, The PCR amplification result of 16s rRNA for <i>P. gingivalis</i>. Lane 1: The PCR amplification product; Lane 2: DL 2 000 DNA marker. C, The PCR amplification result of ragB from <i>P. gingivalis</i> strain W83. Lane 1: DL 2 000 DNA marker; Lane 2:The PCR amplification product. D, The oligonucleotide sequences of PCR primers for the genes of 16s rRNA and ragB from <i>P. gingivalis</i> strain W83.</p

Construction of recombinant plasmid pIRES<i>-ragB-mGITRL</i> for immunization.

<p>Amplified <i>ragB</i> and <i>mGITRL</i> genes were inserted into the eukaryotic expression vectors pIRES, respectively. CMV, human cytomegalovirus immediate-early promoter; IRES, internal ribosome entry site; SV40, simian virus 40; Amp^r, ampicillin resistance gene; Neo^r, neomycin resistance gene.</p

The OPG mRNA level in human placental tissues.

<p>The OPG mRNA level in severe preeclamptic group was much higher than those of the mild preeclamptic and normal control group.</p

Expression of <i>RagB</i> or <i>mGITRL</i> in COS-7 cells or muscle cells of mice.

<p>These data were representative of three independent experiments with similar results.</p