The influence of ions on the lubricative abilities of mucin and the role of sialic acids.

Mucus reduces friction between epithelial surfaces by providing lubrication in the boundary and mixed regime. Mucins, the main macromolecule, are heavily glycosylated proteins that polymerise and retain water molecules, resulting in a hydrated biogel. It is assumed that positively charged ions can influence mucin film structure by screening the electrostatic repulsions between the negatively charged glycans on mucin moieties and draw in water molecules via hydration shells. The ionic concentration can vary significantly in different mucus systems and here we show that increasing the ionic concentration in mucin films leads to an increase in lubrication between two polydimethylsiloxane surfaces at sliding contact in a compliant oral mimic. Mucins were found to bind sodium ions in a concentration-dependent manner and increased ionic concentration appears to cause mucin films to swell when assessed by Quartz Crystal hiMicrobalance with Dissipation (QCM-D) analysis. Furthermore, we determined that the removal of negatively charged sialic acid moieties by sialidase digestion resulted in reduced adsorption to hydrophilic surfaces but did not affect the swelling of mucin films with increasing ionic concentrations. Moreover, the coefficient of friction was increased with sialic acid removal, but lubrication was still increased with increasing ionic concentrations. Taken together this suggests that sialic acids are important for lubrication and may exert this through the sacrificial layer mechanism. Ionic concentration appears to influence mucin films and their lubrication, and sialic acids, at least partly, may be important for ion binding.

Novel bacterial proteolytic and metabolic activity associated with dental erosion-induced oral dysbiosis.

BACKGROUND: Dental erosion is a disease of the oral cavity where acids cause a loss of tooth enamel and is defined as having no bacterial involvement. The tooth surface is protected from acid attack by salivary proteins that make up the acquired enamel pellicle (AEP). Bacteria have been shown to readily degrade salivary proteins, and some of which are present in the AEP. This study aimed to explore the role of bacteria in dental erosion using a multi-omics approach by comparing saliva collected from participants with dental erosion and healthy controls. RESULTS: Salivary proteomics was assessed by liquid-chromatography mass spectrometry (LC-MS) and demonstrated two altered AEP proteins in erosion, prolactin inducible protein (PIP), and zinc-alpha-2 glycoprotein (ZAG). Immunoblotting further suggested that degradation of PIP and ZAG is associated with erosion. Salivary microbiome analysis was performed by sequencing the bacterial 16S rRNA gene (V1-V2 region, Illumina) and showed that participants with dental erosion had a significantly (p < 0.05) less diverse microbiome than healthy controls (observed and Shannon diversity). Sequencing of bacterial mRNA for gene expression (Illumina sequencing) demonstrated that genes over-expressed in saliva from erosion participants included H + proton transporter genes, and three protease genes (msrAB, vanY, and ppdC). Salivary metabolomics was assessed using nuclear magnetic resonance spectrometry (NMR). Metabolite concentrations correlated with gene expression, demonstrating that the dental erosion group had strong correlations between metabolites associated with protein degradation and amino acid fermentation. CONCLUSIONS: We conclude that microbial proteolysis of salivary proteins found in the protective acquired enamel pellicle strongly correlates with dental erosion, and we propose four novel microbial genes implicated in this process. Video Abstract.

Salivary peptidome analysis and protease prediction during orthodontic treatment with fixed appliances.

Orthodontic tooth movement (OTM) occurs through proteolytic remodelling within the periodontium following the application of external force to the tooth. This study describes the first characterization of the salivary peptidome and protease profile during the alignment stage of fixed appliance orthodontic treatment. Unstimulated whole mouth saliva from 16 orthodontic patients (10 males, 6 females, mean (SD) age 15.2 (1.6) years) was collected prior to fixed appliance placement (T1), 1-h (T2), 1-week (T3) following fixed appliance placement and on completion of mandibular arch alignment (T4). Salivary peptides were extracted using filtration followed by mass spectrometry to identify amino acid sequences. Protease prediction was carried out in silico using Proteasix and validated with gelatin zymography and enzyme-linked immunosorbent assay. A total of 2852 naturally-occurring peptides were detected, originating from 436 different proteins. Both collagen and statherin-derived peptide levels were increased at T2. Proteasix predicted 73 proteases potentially involved in generating these peptides, including metalloproteinases, calpains and cathepsins. Changes in predicted activity of proteases over time were also observed, with most metalloproteinases showing increased predicted activity at T2-T3. Increased gelatinolytic activity and MMP8/MMP9 levels were detected at T3. Collectively, multiple protein targets and changes in protease-predicted activity during OTM have been identified.

Duration of canine retraction with fixed appliances: A systematic review and meta-analysis.

INTRODUCTION: Space closure is a challenging and time-consuming phase of orthodontic treatment with fixed appliances. This systematic review evaluated canine retraction duration using fixed appliances after maxillary first premolar extraction. METHODS: Unrestricted systematic literature searches were conducted in 8 databases for randomized clinical trials, assessing the duration and rate of maxillary canine retraction using fixed appliances with or without treatment adjuncts published up to July 2021. Study selection, data extraction, and risk of bias evaluation were conducted independently and in duplicate. Random-effects meta-analyses of average rates or mean differences (MD) and 95% confidence intervals (CI) were conducted at α = 5%, followed by sensitivity and Grading of Recommendations Assessment, Development, and Evaluation analysis. RESULTS: Fifty randomized clinical trials (6 parallel and 44 split-mouth designs) covering 811 participants (mean age 19.9 years; 34% male) were included. The estimated average pooled duration to achieve complete canine retraction was 4.98 months (2 trials; 95% CI, -2.9 to 12.88 months). Pooled average canine retraction was 0.97 mm at months 0-1 (23 trials; 95% CI, 0.79-1.16), 1.83 mm at months 0-2 (20 trials; 95% CI, 1.52-2.14), 2.44 mm at months 0-3 (23 trials; 95% CI, 2.10-2.79), 3.49 mm at months 0-4 (6 trials; 95% CI, 1.81-5.17) and 4.25 mm at months 0-5 (2 trials; 95% CI, 0.36-8.14). Surgically-assisted orthodontics was associated with greater canine retraction at all time points: months 0-1 (10 trials; MD, 0.52 mm; P = 0.004), months 0-2 (8 trials; MD, 0.53 mm; P = 0.04), months 0-3 (8 trials; MD, 0.67 mm; P = 0.01), and months 0-4 (3 trials; MD, 1.13 mm; P = 0.01), whereas subgroup analyses indicated significant effects of anchorage reinforcement method and bracket slot size on canine retraction. CONCLUSIONS: The average time to achieve complete retraction of the maxillary canine using fixed appliances was around 5.0 months. Most studies used split-mouth randomization to investigate canine retraction for around 1-3 months, with substantial heterogeneity across studies. At 3 months of treatment, high-quality evidence supported greater canine retraction with surgically-assisted orthodontics.

Salivary bacterial community profile in normal-weight and obese adolescent patients prior to orthodontic treatment with fixed appliances.

OBJECTIVES: The aim of this study was to compare the intra-oral bacterial profile of normal-weight and obese adolescents prior to orthodontic treatment with fixed appliances. MATERIALS AND METHODS: Nineteen adolescent patients were recruited into two groups based upon body mass index (BMI) and classified as normal-weight or obese. Unstimulated whole mouth saliva was obtained for 5 minutes. Bacterial DNA extraction was performed from saliva, and 16S rRNA gene sequencing of the V1-2 variable regions was undertaken followed by analysis using the mothur pipeline. RESULTS: Saliva from a total of 19 adolescent patients with mean (SD) age 15.6 (1.8) years were divided into 10 normal-weight with mean BMI of 19.4 (2.2) kg/m2 and 9 obese with mean BMI of 30.2 (3.5) kg/m2 . A total of 156 783 sequences were obtained from the 19 samples with no significant differences in richness or diversity between sample groups by obesity status or gender (AMOVA). The bacterial community in both groups was dominated by bacterial genera characteristic of the human mouth, which included Streptococcus, Porphyromonas, Veillonella, Gemella, Prevotella, Fusobacterium and Rothia. CONCLUSION: There were no differences in alpha or beta diversity of oral bacterial communities between normal-weight and obese orthodontic patients. Obese adolescents attending for orthodontic treatment had a similar microflora to their normal-weight counterparts.

Molecular and cellular insight into Escherichia coli SslE and its role during biofilm maturation.

Escherichia coli is a Gram-negative bacterium that colonises the human intestine and virulent strains can cause severe diarrhoeal and extraintestinal diseases. The protein SslE is secreted by a range of pathogenic and commensal E. coli strains. It can degrade mucins in the intestine, promotes biofilm maturation and it is a major determinant of infection in virulent strains, although how it carries out these functions is not well understood. Here, we examine SslE from the commensal E. coli Waksman and BL21 (DE3) strains and the enterotoxigenic H10407 and enteropathogenic E2348/69 strains. We reveal that SslE has a unique and dynamic structure in solution and in response to acidification within mature biofilms it can form a unique aggregate with amyloid-like properties. Furthermore, we show that both SslE monomers and aggregates bind DNA in vitro and co-localise with extracellular DNA (eDNA) in mature biofilms, and SslE aggregates may also associate with cellulose under certain conditions. Our results suggest that interactions between SslE and eDNA are important for biofilm maturation in many E. coli strains and SslE may also be a factor that drives biofilm formation in other SslE-secreting bacteria.

Duration of tooth alignment with fixed appliances: A systematic review and meta-analysis.

INTRODUCTION: A key goal of orthodontic treatment with fixed appliances is alignment of the dentition, and this remains a commonly selected outcome in clinical studies investigating orthodontic tooth movement. This systematic review has evaluated treatment duration to achieve alignment of the mandibular dentition using fixed appliances. METHODS: Systematic literature searches without restrictions were undertaken in 9 databases for randomized clinical trials (RCTs) assessing duration and rate of tooth alignment using fixed appliances with or without treatment adjuncts published up to January 2021. After duplicate study selection, data extraction, and risk of bias assessment according to Cochrane, random-effects meta-analyses of aggregate data, and individual patient data were conducted. RESULTS: Thirty-five trials were included with 2258 participants (39% male; mean age 17.8 years), giving a pooled duration to achieve whole-arch alignment of the mandibular dentition of 263.0 days (4 trials; 95% confidence interval [CI], 186.7-339.4 days) and incisor alignment in the mandibular arch of 100.7 days (9 trials; 95% CI, 84.1-117.4 days). Surgical-assisted orthodontics was associated with reduced duration of incisor alignment: mean difference of 44.3 days less (4 trials; 95% CI, 20.0-68.9 days; P <0.001; high quality of evidence), whereas subgroup and meta-regression analyses indicated significant effects of baseline crowding and premolar extractions. Individual patient data analysis from 3 RCTs indicated that for each additional participant age year, whole-arch alignment of the mandibular dentition took 13.7 days longer (3 trials; 95% CI, 7.7-17.7 days; P <0.001) and for each additional mm of irregularity, 17.5 days more were needed (2 trials; 95% CI, 9.8-25.2 days; P <0.001). CONCLUSIONS: Patient and treatment-related characteristics can significantly affect the duration of tooth alignment and should be taken into account both clinically and when designing trial outcomes. Future research studies investigating rates of orthodontic tooth alignment would benefit from adequate sample sizes and a more consistent methodology in outcome assessment. Data in this systematic review provides a basis for appropriate trial design for future RCTs investigating the rate of orthodontic tooth alignment with fixed appliances.

The impact of obesity on orthodontic treatment outcome in adolescents: a prospective clinical cohort study.

INTRODUCTION: This prospective clinical cohort study investigated the potential influence of obesity on orthodontic treatment outcome. METHODS: A prospective cohort of adolescent patients undergoing routine fixed appliance treatment were recruited into normal-weight or obese groups based upon body mass index (BMI) centile and followed up until the completion of treatment. Primary outcome was treatment duration, and secondary outcomes included treatment outcome (occlusal change measured using peer assessment rating [PAR]), appointment characteristics, and compliance measures. RESULTS: A total of 45 patients mean age 14.8 (1.6) years were included in the final analysis. The normal-weight group included 23 patients with mean BMI 19.4 (2.4) kg/m2 and the obese group 22 patients with mean BMI 30.5 (3.8) kg/m2. There were no significant differences in baseline demographics between groups, except for BMI and pre-treatment PAR. The normal-weight group had a mean pre-treatment PAR of 25.6 (8.3) and the obese 33.3 (11.8) giving the obese group a more severe pre-treatment malocclusion (P = 0.02). There were no significant differences in treatment duration between groups (P = 0.36), but obese patients needed less time per each additional baseline PAR point compared to normal weight (P = 0.02). Obese patients also needed less appointments compared to normal-weight patients (P = 0.02). There were no significant differences between groups for appointment characteristics or compliance. Finally, obese patients were more likely to experience a great PAR reduction than normal-weight patients (relative risk = 2.6; 95% confidence interval = 1.2-4.2; P = 0.02). CONCLUSIONS: There were no significant differences in treatment duration between obese and normal-weight patients. Obesity does not appear to be a risk factor for negative orthodontic treatment outcome with fixed appliances.

Evidence for Proline Utilization by Oral Bacterial Biofilms Grown in Saliva.

Within the mouth bacteria are starved of saccharides as their main nutrient source between meals and it is unclear what drives their metabolism. Previously oral in vitro biofilms grown in saliva have shown proteolytic degradation of salivary proteins and increased extracellular proline. Although arginine and glucose have been shown before to have an effect on oral biofilm growth and activity, there is limited evidence for proline. Nuclear magnetic resonance (NMR) spectroscopy was used to identify extracellular metabolites produced by bacteria in oral biofilms grown on hydroxyapatite discs. Biofilms were inoculated with stimulated whole mouth saliva and then grown for 7 days using sterilized stimulated whole mouth saliva supplemented with proline, arginine or glucose as a growth-medium. Overall proline had a beneficial effect on biofilm growth-with significantly fewer dead bacteria present by biomass and surface area of the biofilms (p < 0.05). Where arginine and glucose significantly increased and decreased pH, respectively, the pH of proline supplemented biofilms remained neutral at pH 7.3-7.5. SDS-polyacrylamide gel electrophoresis of the spent saliva from proline and arginine supplemented biofilms showed inhibition of salivary protein degradation of immature biofilms. NMR analysis of the spent saliva revealed that proline supplemented biofilms were metabolically similar to unsupplemented biofilms, but these biofilms actively metabolized proline to 5-aminopentanoate, butyrate and propionate, and actively utilized glycine. This study shows that in a nutrient limited environment, proline has a beneficial effect on in vitro oral biofilms grown from a saliva inoculum.

Reduced Salivary Mucin Binding and Glycosylation in Older Adults Influences Taste in an In Vitro Cell Model.

BACKGROUND: Taste loss is a significant problem in older adults, affecting quality of life and nutrition. Altered salivary rheology and loss of mucin function may contribute to taste loss by reducing mucosal defences in the oral cavity, impairing sensitivity to oral stimulants. This study aimed to investigate the effects of salivary rheology on taste loss in ageing. Salivary mucin glycosylation and binding to the oral epithelium was investigated in older and younger adults. A cell-based model was utilised to consider the role of saliva in taste loss. METHODS: Human subjects aged >60 years (n = 25) and 18-30 (n = 30) provided saliva samples which were analysed for viscosity, mucin composition and mucin binding to oral epithelial cells (TR146/MUC1). Oral epithelial cells (TR146/MUC1 and SCC090) provided models for taste receptor activation. RESULTS: Reduced levels and sialylation of MUC7 were evident in saliva of older adults which may lead to reduced viscoelasticity, while viscosity is unaffected. Impaired muco-adhesion of saliva from older adults was also observed. Saliva from older adults facilitated the bitter taste receptor activation less well than saliva from younger adults. The causes of taste dysfunction in older adults are unknown, but this study supports a role of saliva in facilitating the activation of taste receptors.

Mixed aerobic-anaerobic incubation conditions induce proteolytic activity from in vitro salivary biofilms.

Oral biofilms have not been studied using both metabolome and protein profiling concurrently. Bacteria produce proteases that lead to degradation of functional salivary proteins. The novel protocol described here allows for complete characterisation of in vitro oral biofilms, including proteolytic, metabolic, and microbiome analysis. Biofilms were grown on hydroxyapatite discs from whole mouth saliva, using sterilised saliva as a growth-medium, in different growth environments. Salivary protein degradation was assessed from spent saliva growth-medium using SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and metabolic activity by nuclear magnetic resonance (NMR). Discs were assessed for depth and coverage of biofilms by confocal laser scanning microscopy (CLSM), and biofilms were collected at the end of the experiment for 16S rRNA gene sequence analysis. There was a significant difference in biofilm viability, salivary protein degradation, and metabolites identified between biofilms grown aerobically and biofilms exposed to an anaerobic environment. Bacterial 16S rRNA gene sequencing showed the predominant genus in the 7-day aerobic biofilms was Streptococcus, in aerobic-anaerobic and anaerobic 7-day biofilms Porphyromonas, and in aerobic-anaerobic and anaerobic 13-day biofilms Fusobacterium. This data suggests new growth requirements and capabilities for analysing salivary biofilms in vitro, which can be used to benefit future research into oral bacterial biofilms.

Anatomical stability of human fungiform papillae and relationship with oral perception measured by salivary response and intensity rating.

Fungiform papillae house taste buds on the anterior dorsal tongue. Literature is inconclusive as to whether taste perception correlates with fungiform papillae density (FPD). Gustatory reflexes modulate the amount and composition of saliva subsequently produced, and thus may be a more physiologically objective measure of tastant-receptor interactions. Taste perception fluctuates with time but the stability of individual fungiform papillae is unclear. This study followed ten healthy volunteers longitudinally at baseline, one and six months. FPD, diameter and position were measured and participants rated intensity perception of sucrose, caffeine, menthol and capsaicin solutions. Salivary flow rate, protein concentration and relative changes in protein composition were measured following each tastant. FPD, diameter and position were unchanged at six months. FPD did not correlate with intensity rating for any taste. FPD did correlate with changes in salivary protein output following sucrose (ρ = 0.72, p = 0.02) and changes in levels of proline-rich protein and mucin 7 following capsaicin (ρ = 0.71, p = 0.02, ρ = 0.68, p = 0.04, respectively). These results suggest that over six months fungiform papillae are anatomically stable, playing a greater role in mediating the physiological salivary response to stimuli rather than determining the perceived intensity of taste.

Targeting macrophages and their recruitment in the oral cavity using swellable (+) alpha tocopheryl phosphate nanostructures.

The phosphorylation of (+) alpha tocopherol produces adhesive nanostructures that interact with oral biofilms to restrict their growth. The aim of this work was to understand if these adhesive (+) alpha tocopheryl phosphate (α-TP) nanostructures could also control macrophage responses to the presence of oral bacteria. The (+) α-TP planar bilayer fragments (175 nm ±â€¯21 nm) formed in a Trizma®/ethanol vehicle swelled when exposed to the cell lines (maximum stabilized size = 29 µm). The swelled (+) α-TP aggregates showed selective toxicity towards THP-1 macrophages (LD50 = 304 µM) compared to human gingival fibroblasts (HGF-1 cells; LD50 > 5 mM), and they inhibited heat killed bacteria stimulated MCP-1 production in both macrophages (control 57.3 ±â€¯18.1 pg/mL vs (+) α-TP 6.5 ±â€¯3.2 pg/mL) and HGF-1 cells (control 673.5 ±â€¯133 pg/mL vs (+) α-TP - 463.9 ±â€¯68.9 pg/mL).

A cross-sectional cohort study of gingival crevicular fluid biomarkers in normal-weight and obese subjects during orthodontic treatment with fixed appliances.

OBJECTIVES: To investigate the effects of obesity on biomarker levels within lower incisor gingival crevicular fluid (GCF) in subjects undergoing routine fixed appliance orthodontic treatment. MATERIALS AND METHODS: This was a cross-sectional clinical cohort study. GCF was collected from normal-weight and obese subjects at completion of alignment at least 1 month after placement of 0.019 × 0.025-inch stainless-steel archwires. The primary outcome was the difference in GCF biomarker levels between groups. Secondary outcomes included differences in clinical parameters of plaque and gingival indices, unstimulated whole-mouth saliva, and GCF flow rates. RESULTS: Thirty-eight subjects (18 male, 20 female) with a mean age of 25.6 (SD, 6.3) years and mean body mass index (BMI) of 22.6 (1.6) in normal-weight and 32.4 (2.2) kg/m2 in obese groups were investigated. Apart from BMI (P < .0001), there were no statistically significant differences in essential demographics between groups. Significantly increased levels of the adipokine leptin (P < .009) and the tissue-remodeling biomarker matrix metalloproteinase-9 (MMP9; P < .020) were identified in the obese cohort. For the remainder of the biomarkers, including the RANKL bone-remodeling marker and several inflammatory markers, there were no significant differences between groups. No correlation was observed between plaque index or gingival index for any GCF biomarker for either group (P = .07-1.00). CONCLUSIONS: This study investigated the GCF biochemical profile of obese and normal-weight subjects undergoing fixed-appliance orthodontic treatment. Significantly increased levels of the adipokine leptin and the tissue-remodeling biomarker MMP9 were identified in the obese group. These data provide evidence of differences in GCF biochemistry between obese and normal-weight subjects undergoing fixed appliance orthodontic treatment.

Predictive value of ultrasound scoring in relation to clinical and histological parameters in xerostomia patients.

BACKGROUND: Salivary gland dysfunction is one of the main clinical features of Sjögren's syndrome (SS), manifested by xerostomia with subsequent complications and well-established effects on the person's quality of life. OBJECTIVES: To determine firstly whether selected tests of salivary gland function and structure, unstimulated whole salivary flow rate (UWSFR), parotid flow rate (PFR), clinical oral dryness score (CODS) and ultrasound score (USS), can discriminate SS from non-SS sicca patients and secondly whether these tests can differentiate between patients in different subgroups of SS. METHOD: Unstimulated whole salivary flow rate, PFR, CODS and USS were determined in 244 patients comprised of SS patients (n = 118), SS patients at higher risk of lymphoma (n = 30) or with lymphoma (n = 26), and non-SS sicca disease controls (n = 70). RESULTS: All assessments showed a significant difference between the overall SS group and the disease control group, attributed mainly to the lymphoma subgroups of SS (p < 0.0001 for all parameters). There was a significant correlation (Spearman r = 0.7, p value <0.0001) and 87.3% agreement between USS and the histology focus scores of 119 patients. CONCLUSION: The results suggest that salivary gland tests including USS can aid in differentiating between SS and non-SS dry mouth, especially the subgroups of SS with lymphoma or at higher risk of developing lymphoma.

Diminishing biofilm resistance to antimicrobial nanomaterials through electrolyte screening of electrostatic interactions.

The extracellular polymer substances (EPS) generated by biofilms confers resistance to antimicrobial agents through electrostatic and steric interactions that hinder molecular diffusion. This resistance mechanism is particularly evident for antibacterial nanomaterials, which inherently diffuse more slowly compared to small organic antibacterial agents. The aim of this study was to determine if a biofilm's resistance to antibacterial nanomaterial diffusion could be diminished using electrolytes to screen the EPS's electrostatic interactions. Anionic (+) alpha-tocopherol phosphate (α-TP) liposomes were used as the antimicrobial nanomaterials in the study. They self-assembled into 700 nm sized structures with a zeta potential of -20 mV that were capable of killing oral bacteria (S. oralis growth inhibition time of 3.34 ± 0.52 h). In a phosphate (-ve) buffer the -ve α-TP liposomes did not penetrate multispecies oral biofilms, but in a Tris (hydroxymethyl)aminomethane (+ve) buffer they did (depth - 12.4 ± 3.6 µm). The Tris did not modify the surface charge of the α-TP nanomaterials, rather it facilitated the α-TP-biofilm interactions through electrolyte screening (Langmuir modelled surface pressure increase of 2.7 ± 1.8 mN/ m). This data indicated that EPS resistance was mediated through charge repulsion and that this effect could be diminished through the co-administration of cationic electrolytes.

Determining bacterial and host contributions to the human salivary metabolome.

BACKGROUND: Salivary metabolomics is rapidly advancing. AIM AND METHODS: To determine the extent to which salivary metabolites reflects host or microbial metabolic activity whole-mouth saliva (WMS), parotid saliva (PS) and plasma collected contemporaneously from healthy volunteers were analysed by 1H-NMR spectroscopy. Spectra underwent principal component analysis and k-means cluster analysis and metabolite quantification. WMS samples were cultured on both sucrose and peptide-enriched media. Correlation between metabolite concentration and bacterial load was assessed. RESULTS: WMS contained abundant short-chain fatty acids (SCFAs), which were minimal in PS and plasma. WMS spectral exhibited greater inter-individual variation than those of PS or plasma (6.7 and 3.6 fold, respectively), likely reflecting diversity of microbial metabolomes. WMS bacterial load correlated strongly with SCFA levels. Additional WMS metabolites including amines, amino acids and organic acids were positively correlated with bacterial load. Lactate, urea and citrate appeared to enter WMS via PS and the circulation. Urea correlated inversely with WMS bacterial load. CONCLUSIONS: Oral microbiota contribute significantly to the WMS metabolome. Several WMS metabolites (lactate, urea and citrate) are derived from the host circulation. WMS may be particularly useful to aid diagnosis of conditions reflective of dysbiosis. WMS could also complement other gastrointestinal fluids in future metabolomic studies.

Salivary S100A8/A9 in Sjögren's syndrome accompanied by lymphoma.

BACKGROUND: Sjögren's syndrome (SS) is an autoimmune inflammatory disease that affects the exocrine glands. The absence of early diagnostic markers contributes to delays in its diagnosis. Identification of changes in the protein profile of saliva is considered one of the promising strategies for the discovery of new biomarkers for SS. OBJECTIVE: To identify salivary protein biomarkers with potential for use in discriminating between different lymphoma risk subgroups of SS. METHOD: Parotid and whole mouth saliva samples were collected from patients with SS, including those in subgroups at higher risk of developing or with confirmed lymphoma, non-SS sicca disease controls and healthy subjects. An initial proteomics analysis by mass spectrometry (LCMSMS) identified S100A8/A9 as a biomarker and was followed by validation with an enzyme-linked immunosorbent assay (ELISA). RESULTS: Significant differences were found in levels of S100A8/A9 in parotid saliva but not whole mouth saliva between patients with SS compared with healthy and disease control subjects (P = 0.001 and 0.031, respectively). Subgroups of patients with SS based on lymphoma risk showed significant differences in salivary levels of S100A8/A9. CONCLUSION: The results suggest that salivary levels of S100A8/A9 can aid in differentiating between SS, disease control and healthy control subjects, especially the subgroups of SS with lymphoma or at higher risk of lymphoma.

Developing and Standardizing a Protocol for Quantitative Proton Nuclear Magnetic Resonance (1H NMR) Spectroscopy of Saliva.

Metabolic profiling by 1H NMR spectroscopy is an underutilized technology in salivary research, although preliminary studies have identified promising results in multiple fields (diagnostics, nutrition, sports physiology). Translation of preliminary findings into validated, clinically approved knowledge is hindered by variability in protocol for the collection, storage, preparation, and analysis of saliva. This study aims to evaluate the effects of differing sample pretreatments on the 1H NMR metabolic profile of saliva. Protocol considerations are highly varied in the current literature base, including centrifugation, freeze-thaw cycles, and different NMR quantification methods. Our findings suggest that the 1H NMR metabolite profile of saliva is resilient to any change resulting from freezing, including freezing of saliva prior to centrifuging. However, centrifugation was necessary to remove an unidentified broad peak between 1.24 and 1.3 ppm, the intensity of which correlated strongly with saliva cellular content. This peak obscured the methyl peak from lactate and significantly affected quantification. Metabolite quantification was similar for saliva centrifuged between 750 g to 15 000 g. Quantification of salivary metabolites was similar whether quantified using internal phosphate-buffered sodium trimethylsilyl-[2,2,3,3-2H4]-propionate (TSP) or external TSP in a coaxial NMR tube placed inside the NMR tube containing the saliva sample. Our results suggest that the existing literature on salivary 1H NMR will not have been adversely affected by variations of the common protocol; however, use of TSP as an internal standard without a buffered medium appears to affect metabolite quantification, notably for acetate and methanol. We include protocol recommendations to facilitate future NMR-based studies of saliva.

The effect of obesity on orofacial pain during early orthodontic treatment with fixed appliances: a prospective cohort study.

Introduction: We have investigated orofacial pain in a prospective cohort of obese and normal-weight subjects undergoing fixed-appliance orthodontic treatment. Methods: Fifty-five subjects (27 males, 28 females) mean age 15.1 (1.6) years and mean body mass index 30.2 (3.5) in obese and 19.4 (2.2) kg/m2 in normal-weight groups were followed for 1 week after appliance placement. Primary outcome was maximum-pain measured using a 100-mm visual analogue scale. Secondary outcomes included mean pain and oral analgesic consumption. Results: Mean maximum pain for the total sample was 73.7 (standard deviation 14.8; 95% confidence interval 69.8-77.7) mm with no significant differences among groups (P = 0.247). However, mean maximum pain was higher at all time-points for the obese group and significant at 72 hours (P = 0.034). Total analgesia consumed by the obese group was also significantly higher than normal weight (P = 0.041). Multivariable regression found the only significant predictor for mean pain was time. After adjusting for confounding, obesity was associated with higher (+4.47 mm) mean pain at each time-point (P = 0.018). A significant association existed between obesity and total analgesic consumption (univariable-analysis, P = 0.035; multivariable analysis, P = 0.023). After accounting for confounders, obese patients were associated with taking a higher quantity of oral analgesics. Conclusions: We found a trend towards increased mean pain and an association with increased analgesic consumption in obese subjects during the first week following fixed-appliance placement.