Acquired enamel pellicle and biofilm engineering with a combination of acid-resistant proteins (CaneCPI-5, StN15, and Hemoglobin) for enhanced protection against dental caries - in vivo and in vitro investigations.

OBJECTIVE: This study was designed in two-legs. In the in vivo, we explored the potential of a rinse solution containing a combination (Comb) of 0.1 mg/mL CaneCPI-5 (sugarcane-derive cystatin), 1.88 × 10- 5M StN15 (statherin-derived peptide) and 1.0 mg/mL hemoglobin (Hb) to change the protein profile of the acquired enamel pellicle(AEP) and the microbiome of the enamel biofilm. The in vitro, was designed to reveal the effects of Comb on the viability and bacterial composition of the microcosm biofilm, as well as on enamel demineralization. MATERIALS AND METHODS: In vivo study, 10 participants rinsed (10mL,1 min) with either deionized water (H2O-control) or Comb. AEP and biofilm were collected after 2 and 3 h, respectively, after rinsing. AEP samples underwent proteomics analysis, while biofilm microbiome was assessed via 16 S-rRNA Next Generation Sequencing(NGS). In vitro study, a microcosm biofilm protocol was employed. Ninety-six enamel specimens were treated with: 1)Phosphate-Buffered Solution-PBS(negative-control), 2)0.12%Chlorhexidine, 3)500ppmNaF and 4)Comb. Resazurin, colony-forming-units(CFU) and Transversal Microradiography(TMR) were performed. RESULTS: The proteomic results revealed higher quantity of proteins in the Comb compared to control associated with immune system response and oral microbial adhesion. Microbiome showed a significant increase in bacteria linked to a healthy microbiota, in the Comb group. In the in vitro study, Comb group was only efficient in reducing mineral-loss and lesion-depth compared to the PBS. CONCLUSIONS: The AEP modification altered the subsequent layers, affecting the initial process of bacterial adhesion of pathogenic and commensal bacteria, as well as enamel demineralization. CLINICAL RELEVANCE: Comb group shows promise in shaping oral health by potentially introducing innovative approaches to prevent enamel demineralization and deter tooth decay.

Effect of a metal artifact reduction algorithm on dehiscence and fenestration detection around zirconia implants with cone beam computed tomography.

OBJECTIVE: To assess the efficacy of the metal artifact reduction algorithm (MARA) of the Cranex 3D cone beam computed tomography (CBCT) device in the detection of peri-implant dehiscence and fenestration around zirconia implants. STUDY DESIGN: In total, 60 implants were placed in bovine ribs. Dehiscence and fenestration defects were created around the implants, after which 60 CBCT images were obtained with and 60 without activation of MARA. Three radiologists examined the images for the presence of defects. The area under the curve (AUC) from receiver operating characteristic analysis, sensitivity, and specificity were calculated to assess the ability to discriminate the presence vs absence of bone defects. One-way analysis of variance was employed to analyze outcome measures. The significance level was established at 5% (α = 0.05). RESULTS: AUC values indicated excellent discrimination of dehiscence on images with MARA activation and an excellent to outstanding range of discrimination with MARA deactivation. For fenestration, MARA activation and deactivation both led to outstanding discrimination. Sensitivity and specificity values revealed that activation of MARA was helpful in distinguishing the presence vs. absence of dehiscence, while both MARA conditions were helpful for fenestration. However, there were no statistically significant differences between MARA activation and deactivation for any outcome measure (P >.05). CONCLUSION: CBCT is suitable for detecting peri-implant defects, but MARA application does not significantly affect peri-implant dehiscence and fenestration detection.

Increase in plasma resveratrol levels and in acid-resistant proteins in the acquired enamel pellicle after use of resveratrol-containing orodispersible tablets.

OBJECTIVE: This study evaluated the effect of administration of trans-resveratrol-containing orodispersible tablets on the protein composition of the AEP and on blood plasma trans-resveratrol concentrations. METHODS: Ten volunteers participated in two crossover double-blind phases. In each phase, after dental prophylaxis, they received a trans-resveratrol (15 mg) orodispersible tablet, or a placebo tablet (without actives). The AEP formed after 120 min was collected with electrode filter papers soaked in 3 % citric acid. Blood samples were collected 30, 45, 60 and 120 min after the use of the tablet. After protein extraction, AEP samples were analyzed by shotgun labelfree quantitative proteomics and plasma samples were analyzed by high-performance liquid chromatography (HPLC). RESULTS: Eight hundred and two proteins were identified in the AEP. Among them, 336 and 213 were unique to the trans-resveratrol and control groups, respectively, while 253 were common to both groups. Proteins with important functions in the AEP had increased expression in the trans-resveratroltreated group, such as neutrophil defensins, S100 protein isoforms, lysozyme C, cystatin-D, mucin-7, alphaamylase, albumin, haptoglobin and statherin. Trans-resveratrol was detected in the plasma at all the times evaluated, with the peak at 30 min. CONCLUSIONS: The administration of trans-resveratrol in sublingual orodispersible tablets was effective both to increase the bioavailability of the polyphenol and the expression of antibacterial and acid-resistant proteins in the AEP, which might benefit oral and general health.

Hemoglobin Protects Enamel against Intrinsic Enamel Erosive Demineralization.

INTRODUCTION: This study investigated the changes in the acquired enamel pellicle (AEP) proteome when this integument is formed in vivo after treatment with sugarcane-derived cystatin (CaneCPI-5), hemoglobin (HB), and a statherin-derived peptide (StN15), or their combination and then exposed to an intrinsic acid challenge. The effectiveness of these treatments in preventing intrinsic erosion was also evaluated. METHODS: Ten volunteers, after prophylaxis, in 5 crossover phases, rinsed with the following solutions (10 mL, 1 min): control (deionized water-H2O) - group 1, 0.1 mg/mL CaneCPI-5 - group 2, 1.0 mg/mL HB - group 3, 1.88 × 10-5M StN15 - group 4, or a blend of these - group 5. Following this, AEP formation occurred (2 h) and an enamel biopsy (10 µL, 0.01 m HCl, pH 2.0, 10 s) was conducted on one incisor. The biopsy acid was then analyzed for calcium (Arsenazo method). The vestibular surfaces of the other teeth were treated with the same acid. Acid-resistant proteins in the residual AEP were then collected and analyzed quantitatively via proteomics. RESULTS: Compared to control, treatment with the proteins/peptide, mixed or isolated, markedly enhanced acid-resistant proteins in the AEP. Notable increases occurred in pyruvate kinase PKM (11-fold, CaneCPI-5), immunoglobulins and submaxillary gland androgen-regulated protein 3B (4-fold, StN15), Hb, and lysozyme C (2-fold, StN15). Additionally, a range of proteins not commonly identified in the AEP but known to bind calcium or other proteins were identified in groups treated with the tested proteins/peptide either in isolation or as a mixture. The mean (SD, mM) calcium concentrations released from enamel were 3.67 ± 1.48a, 3.11 ± 0.72a, 1.94 ± 0.57b, 2.37 ± 0.90a, and 2.38 ± 0.45a for groups 1-5, respectively (RM-ANOVA/Tukey, p < 0.05). CONCLUSIONS: Our findings demonstrate that all treatments, whether using a combination of proteins/peptides or in isolation, enhanced acid-resistant proteins in the AEP. However, only HB showed effectiveness in protecting against intrinsic erosive demineralization. These results pave the way for innovative preventive methods against intrinsic erosion, using "acquired pellicle engineering" techniques.

Xylitol associated or not with fluoride: Is the action the same on de- and remineralization?

OBJECTIVES: This study evaluated the effect of xylitol combined or not with fluoride (F) on reduction of demineralization and increase of remineralization of shallow and deep artificial enamel lesions. METHODS: Bovine enamel samples were allocated to the following solutions groups: no xylitol (negative control), 5% xylitol, 10% xylitol, 20% xylitol, 500 ppm F (as NaF), 5% xylitol+F, 10% xylitol+F or 20% xylitol+F (n = 12-15). For the demin study, a pH-cycling model (demineralization-6 h, pH 4.7/remineralization 18 h, pH 7.0) was employed for 7 days. Treatments were applied 2 × 1 min. In the remin study, specimens were pre-demineralized for 2, 5 or 10 days. Afterwards, a pH-cycling protocol was conducted (2 h demineralizing and 22 h remineralizing solution/day for 8 days) and the same treatments were done. The response variables were percentage surface hardness loss (%SHL) and transverse microradiography. Data were analyzed by RM ANOVA/Tukey or Kruskal-Wallis/Dunn (p < 0.05) RESULTS: F and Xylitol combined with F reduced the %SHL (23-30%) compared to the negative control (61.5%). The integrated mineral loss and the lesion depth were not reduced by any treatment. Surface hardness recovery was seen only for shallow lesions in case of 20% xylitol+F compared to negative control. No lesion depth recovery, but significant mineral recovery was seen for F (2-days and 10-days lesion). CONCLUSIONS: All concentrations of xylitol+F reduced enamel surface demineralization, while only 20% xylitol+F improved surface remineralization of shallow lesions in vitro. CLINICAL SIGNIFICANCE: Our results suggest that while F or any concentration of xylitol + F reduces surface demineralization, only 20% xylitol+F improves surface remineralization of shallow lesions in vitro. Therefore, xylitol may be added into oral products, combined to F, to control dental caries.

Proteomic analysis of stimulated saliva in gastroesophageal reflux disease patients with and without erosive tooth wear: Observational study.

OBJECTIVE: To evaluate the difference in the proteomic profile of stimulated saliva in patients with gastroesophageal reflux disease (GERD) with (GE) and without (GNE) erosive tooth wear (ETW), regarding both human and bacterial proteins. METHODS: Stimulated saliva (SS) was collected from 16 patients (8/group). Samples were centrifuged at 4.500 g for 15 min under refrigeration to remove all debris. The supernatant from each saliva sample was taken and frozen at -80 °C. After extracting the proteins, they were submitted to reverse phase liquid chromatography and mass spectrometry (nLC-ESI-MS/MS). Label-free proteomic quantification was performed using Protein Lynx Global Service (PLGS) software (p < 0.05) for human and bacterial proteins. RESULTS: In total, 67 human proteins were common for GNE and GE groups. GNE group presented, compared to GE group, increase in proteins that confer antimicrobial and acid resistant properties, such as cystatins, histatin and immunoglobulins. However, GNE group had a marked decrease in subunits of hemoglobin (α, ß and delta). Regarding bacterial proteins, for SS, 7 and 10 unique proteins were identified in the GE and GNE groups, respectively. They are related to protein synthesis and energy metabolism and interact with human proteins typically found in saliva and supramolecular complexes of the acquired pellicle. CONCLUSIONS: Our data indicate that the stimulation of the salivary flow increases acid resistant and antimicrobial proteins in saliva, which might protect against ETW. CLINICAL SIGNIFICANCE: This pioneer study showed important differences in the human and bacterial proteome of SS in patients with GERD with or without ETW.

Acquired enamel pellicle protects gastroesophageal reflux disease patients against erosive tooth wear.

The objective of this study was to compare the protein profile of the acquired enamel pellicle (AEP) formed in vivo in patients with or without gastroesophageal reflux disease (GERD), and with or without erosive tooth wear (ETW). Twenty-four volunteers were divided into 3 groups: 1) GERD and ETW; 2) GERD without ETW; and 3) control (without GERD). The AEP formed 120 min after prophylaxis was collected from the lingual/palatal surfaces. The samples were subjected to mass spectrometry (nLC-ESI-MS/MS) and label-free quantification by Protein Lynx Global Service software. A total of 213 proteins were identified, or 119, 92 and 106 from each group, respectively. Group 2 showed a high number of phosphorylated and calcium-binding proteins. Twenty-three proteins were found in all the groups, including 14-3-3 protein zeta/delta and 1-phosphatidylinositol. Several intracellular proteins that join saliva after the exfoliation of oral mucosa cells might have the potential to bind hydroxyapatite, or participate in forming supramolecular aggregates that bind to precursor proteins in the AEP. Proteins might play a central role in protecting the dental surface against acid dissolution.

Estimated Dietary Fluoride Intake by 24-Month-Olds from Chocolate Bars, Cookies, Infant Cereals, and Chocolate Drinks in Brazil.

The use of fluoride (F) in the prevention of dental caries is established. However, a high amount of F intake during tooth development can cause dental fluorosis The aim of this study was to analyze variations in F concentrations in chocolate bars (CB), chocolate cookies (CC), infant cereals (IC), and chocolate milk drinks (CD) to determine the daily intake of F from different sources by children at the age of risk for developing dental fluorosis. Distinct brands of CB, CC, IC, and CD were analyzed. Fluoride was separated by hexamethyldisiloxane-facilitated diffusion. Analysis was made in triplicate with an F ion-specific electrode. F ingestion (mg/kg body weight) was evaluated with the suggested consumption (0.05-0.07 mg/kg/day) for children aged 24 months (12 kg). The concentrations for all the analyzed products ranged from 0.025 to 1.827 µg/g F. The mean (range) F concentrations were CB= 0.210 ± 0.205 µg/g (0.073-0.698, n = 8), CC = 0.366 ± 0.416 µg/g (0.320-1.827, n = 9), IC = 0.422 ± 0.395 µg/g (0.073-1.061, n = 5), and CD = 0.169 ± 0.170 µg/mL (0.025-0.443, n = 12). The products that had the highest concentration in the categories CB, CC, IC, and CD, respectively, were Nescau-Ball (0.698 µg/g), Passatempo (1.827 µg/g), Milnutri (1.061 µg/g), and Toddynho (0.443 µg/mL). The consumption of only one unit of Toddynho (CD) is equivalent to more than 11% of the maximum suggested daily intake for a 24-month-old child (0.07 mg/kg body weight). When one product from each category is consumed together only once a day, this consumption is equivalent to approximately 24% of the suggested daily intake of fluoride for a 24-month-old child. The presence of high levels of fluoride in certain products suggests that they play a significant role in overall fluoride intake. It is crucial to closely monitor the fluoride content of food and drinks that are consumed by children who are at risk for dental fluorosis, and for product labels to clearly display the fluoride concentrations.

Acquired enamel pellicle protects gastroesophageal reflux disease patients against erosive tooth wear

Abstract The objective of this study was to compare the protein profile of the acquired enamel pellicle (AEP) formed in vivo in patients with or without gastroesophageal reflux disease (GERD), and with or without erosive tooth wear (ETW). Twenty-four volunteers were divided into 3 groups: 1) GERD and ETW; 2) GERD without ETW; and 3) control (without GERD). The AEP formed 120 min after prophylaxis was collected from the lingual/palatal surfaces. The samples were subjected to mass spectrometry (nLC-ESI-MS/MS) and label-free quantification by Protein Lynx Global Service software. A total of 213 proteins were identified, or 119, 92 and 106 from each group, respectively. Group 2 showed a high number of phosphorylated and calcium-binding proteins. Twenty-three proteins were found in all the groups, including 14-3-3 protein zeta/delta and 1-phosphatidylinositol. Several intracellular proteins that join saliva after the exfoliation of oral mucosa cells might have the potential to bind hydroxyapatite, or participate in forming supramolecular aggregates that bind to precursor proteins in the AEP. Proteins might play a central role in protecting the dental surface against acid dissolution.

Protein-based engineering of the initial acquired enamel pellicle in vivo: Proteomic evaluation.

OBJECTIVE: To study the proteomic alterations in the initial AEP after rinsing with CaneCPI-5, StN15 or Hb or their combination. MATERIALS AND METHODS: In five crossover phases, after prophylaxis, 10 volunteers in 5 consecutive days, rinsed (10 mL, 1 min) with the following solutions: deionized water (H2O- negative control- 1), 0.1 mg/mL CaneCPI-5 (2), 1.88×10-5 M StN15 (3), 1.0 mg/mL Hb (4) or their combination (5). The AEP formed after 3 min was collected with electrode filter papers soaked in 3% citric acid. After protein extraction, samples were analyzed by quantitative shotgun label-free proteomics. RESULTS: Rinsing with the proteins/peptide increased the amounts of proteins in the AEP. The total numbers of proteins identified after rinsing with CaneCPI-5, StN15, Hb or their combination versus water, were 131, 167, 148 and 142, respectively. The treatment with the proteins/peptide or their combination increased proteins that bind calcium, phosphate and interact with distinct proteins, as well as proteins with antimicrobial and acid-resistant properties, such as, Cornifin-B (7.7, 12.6, and 4.3-fold for CaneCPI-5, StN15 and Hb, respectively), isoforms of Cystatin (2.2-2.4-fold for CaneCPI-5 and StN15), Proline-rich-protein 4 (4.3-fold; StN15), Histatin-1 (2.8-fold; StN15) and Hemoglobin (7.7-25-fold for Hb and Combination). Immunoglobulin, Keratin and Histone were exclusively identified upon treatment with the proteins/peptide, alone or combined. CONCLUSION: Rinsing with proteins/peptide, alone or combined, increased protective proteins in the initial AEP. CLINICAL RELEVANCE: Our results suggest that rinsing with the proteins/peptide or their combination increases the proteins capable of enhancing the protective function of the basal layer of AEP.

Rinsing with Statherin-Derived Peptide Alters the Proteome of the Acquired Enamel Pellicle.

Changes in the proteomic profile of the acquired enamel pellicle (AEP) formed for 3 min or 2 h after rinsing with a peptide containing the 15 N-terminal residues of statherin, with serines 2 and 3 phosphorylated (StatpSpS), were evaluated. Nine volunteers participated in 2 consecutive days. Each day, after professional tooth cleaning, they rinsed for 1 min with 10 mL of phosphate buffer containing 1.88 × 10-5 M StatpSpS or phosphate buffer only (control). The acquired pellicle formed on enamel after 3 min or 2 h was collected with electrode filter papers soaked in 3% citric acid. After protein extraction, samples were analyzed by quantitative shotgun label-free proteomics. In the 3-min AEP, 19 and 131 proteins were uniquely identified in the StatpSpS and control groups, respectively. Proteins typically found in the AEP were only found in the latter. Only 2 proteins (neutrophil defensins) were increased upon treatment with StatpSpS, while 65 proteins (among which are several typical AEP proteins) were decreased. In the 2-h AEP, 50 and 108 proteins were uniquely found in StatpSpS and control groups, respectively. Hemoglobin subunits and isoforms of keratin were only found in the StatpSpS group, while cystatin-C, cathepsin D, and cathepsin G, isoforms of heat shock 70 and protocadherin were exclusively found in the control group. In addition, 23 proteins were increased upon treatment with StatpSpS, among which are histatin-1, serum albumin, and isoforms of neutrophil defensin and keratin, while 77 were decreased, most of them were typical AEP proteins. In both evaluated periods, rinsing with StatpSpS profoundly changed the proteomic profile of the AEP, which might impact the protective role of this integument against carious or erosive demineralization. This study provides important insights on the dynamics of the protein composition of the AEP along time, after rinsing with a solution containing StatpSpS.

Effect of a sugarcane cystatin on the profile and viability of microcosm biofilm and on dentin demineralization.

To analyze the effect of a sugarcane cystatin (CaneCPI-5) on the microbial profile and viability, as well as on the prevention of dentin demineralization using a microcosm biofilm model. Ninety bovine dentine specimens were divided into five experimental groups according with the solution they were treated for 60 s: (1) PBS (negative control), (2) 0.12% chlorhexidine (positive control), (3) Fluoride (500 ppm F, as NaF), (4) 0.025 mg/ml CaneCPI-5, and (5) 0.05 mg/ml CaneCPI-5. Specimens were incubated with inoculum (McBain's saliva plus human saliva) in the first 8 h, and from then on, they were exposed to McBain saliva containing sucrose and daily treated (60 s) with the solutions for 5 days. Resazurin and colony-forming unit counting assays were performed. Dentin demineralization was measured by transverse micro-radiography (TMR). 0.12% chlorhexidine significantly reduced the metabolic activity of the microcosm biofilm in relation to the negative control and treated groups (p < 0.01). CHX and F significantly reduced the counts of total microorganisms, mutans group streptococci, and lactobacilli when compared with the negative control. None of the treatments was able to significantly reduce dentin demineralization in comparison with the negative control. In the model evaluated, CaneCPI-5 neither altered the microcosm biofilm profile and viability nor protected dentin against demineralization.

Increase of complex I and reduction of complex II mitochondrial activity are possible adaptive effects provoked by fluoride exposure.

Fluoride (F) can induce changes in the expression of several liver proteins, most of them localized in the mitochondria and its effect is dose- and time-dependent. This study analyzed the effect of distinct F concentrations and exposure periods on the mitochondrial activity of complex I-III and II-III in the liver. Thirty-six 21-day-old male Wistar rats were divided into 2 groups (n = 18) according to the duration of the treatment (20 or 60 days). They were subdivided into 3 subgroups (n = 6) according to the concentration of F (0 mg/L, 15 mg/L or 50 mg/L). After the experimental periods, the animals were anesthetized, liver mitochondria were isolated and stored for activity analyses. The determination of complexes II-III and I-III was based on the reduction of cytochrome c3+ to cytochrome c2+ performed spectrophotometrically. Bioinformatics analyses were performed using data from a previous study (Pereira et al., 2018). The mitochondrial complex I-III was significantly activated in the groups treated with 50 mgF/L for 20 days and 15 mgF/L for 60 days. The complex II-III was significantly reduced in the group treated with the higher F dose for 60 days. The networks indicated more changes in mitochondrial proteins in the group treated with the higher dose for 20 days; the reduction is probably linked to the activation of the complex I-III. The reduction in the complex II-III upon exposure to the higher F dose in the long term might be part of an adaptative mechanism of the body to counteract the deleterious effects of this ion on the energy metabolism.

A sugarcane cystatin (CaneCPI-5) alters microcosm biofilm formation and reduces dental caries.

The antimicrobial and anticaries effects of CaneCPI-5 were evaluated. Ninety bovine enamel samples were treated for 60 s with either phosphate-buffered-saline (PBS), 0.12% chlorhexidine (CHX), 0.05 mg ml-1 CaneCPI-5, 0.1 mg ml-1 CaneCPI-5 or 0.5 mg ml-1 CaneCPI-5. They were incubated with inoculum (human saliva + McBain's saliva) for the first 8 h. From then until the end of the experiment, the enamel was exposed to McBain saliva with sucrose and, once a day, for 5 days, they were treated with the solutions. At the end of the experimental period, resazurin and viable plate count assays were performed. Enamel demineralization was also measured. All concentrations of CaneCPI-5 and CHX significantly reduced the activity of biofilms compared with PBS. For viable plate counts, all treatments similarly reduced the lactobacilli and total streptococci; for the mutans streptococci, 0.05 mg ml-1 CaneCPI-5 performed better than CHX. All CaneCPI-5 concentrations significantly reduced the integrated mineral loss. This study represents the first step regarding the use of CaneCPI-5 within the concept of acquired enamel pellicle and biofilm engineering to prevent dental caries.

Intestinal changes associated with fluoride exposure in rats: Integrative morphological, proteomic and microbiome analyses.

Gastrointestinal signs and symptoms are the first signs of toxicity due to exposure to fluoride (F). This suggests the possibility that lower levels of subchronic F exposure may affect the gut. The aim of this study was to evaluate changes in the morphology, proteome and microbiome of the ileum of rats, after subchronic exposure to F. Male rats ingested water with 0, 10, or 50 mgF/L for thirty days. Treatment with F, regardless of the dose, significantly decreased the density of HuC/D-IR neurons, whereas CGRP-IR and SP-IR varicosities were significantly increased compared to the control group. Increased VIP-IR varicosities were significantly increased only in the group treated with 50 mgF/L. A significant increase in thickness of the tunica muscularis, as well as in the total thickness of the ileum wall was observed at both F doses when compared to controls. In proteomics analysis, myosin isoforms were increased, and Gastrotopin was decreased in F-exposed mice. In the microbiome metagenomics analysis, Class Clostridia was significantly reduced upon exposure to 10 mgF/L. At the higher F dose of 50 mg/L, genus Ureaplasma was significantly reduced in comparison with controls. Morphological and proteomics alterations induced by F were marked by changes associated with inflammation, and alterations in the gut microbiome. Further studies are needed to determine whether F exposure increases inflammation with secondary effects of the gut microbiome, and/or whether primary effects of F on the gut microbiome enhance changes associated with inflammation.

Acquired pellicle protein-based engineering protects against erosive demineralization.

OBJECTIVES: To evaluate, in vivo: 1) proteomic alterations in the acquired enamel pellicle (AEP) after treatment with sugarcane-derived cystatin (CaneCPI-5), hemoglobin (HB), statherin-derived peptide (StN15) or their combination before the formation of the AEP and subsequent erosive challenge; 2) the protection of these treatments against erosive demnineralization. MATERIALS AND METHODS: In 5 crossover phases, after prophylaxis, 10 volunteers rinsed (10 mL, 1 min) with: deionized water-1, 0.1 mg/mL CaneCPI-5-2, 1.0 mg/mL HB-3, 1.88 × 10-5 M StN15-4 or their combination-5. AEP was formed (2 h) and enamel biopsy (10 µL, 1%citric acid, pH 2.5, 10 s) was performed on one incisor for calcium analysis. The same acid was applied on the vestibular surfaces of the remaining teeth. The acid-resistant proteins within the remaining AEP were collected. Samples were quantitatively analyzed by label-free proteomics. RESULTS: Treatment with the proteins/peptide, isolated or combined, increased several acid-resistant proteins in the AEP, compared with control. The highest increases were seen for PRPs (32-fold, StN15), profilin (15-fold, combination), alpha-amylase (9-fold; StN15), keratins (8-fold, CaneCPI-5 and HB), Histatin-1 (7-fold, StN15), immunoglobulins (6.5-fold, StN15), lactotransferrin (4-fold, CaneCPI-5), cystatins, lysozyme, protein S-100-A9 and actins (3.5-fold, StN15), serum albumin (3.5-fold, CaneCPI-5 and HB) and hemoglobin (3-fold, StN15). Annexin, calmodulin, keratin, tubulin and cystatins were identified exclusively upon treatment with the proteins/peptide, alone or combined. Groups 2, 3 and 4 had significantly lower Ca released from enamel compared to group 1 (Kruskal-Wallis/Dunn's, p < 0.05). CONCLUSIONS: Treatment with CaneCPI-5, HB or StN15 remarkably increases acid-resistant proteins in the AEP, protecting against erosion. CLINICAL SIGNIFICANCE: Our results show, for the first time, that treatment with proteins/peptide remarkably increases acid-resistant proteins in the AEP, protecting against erosive demineralization. These findings open an avenue for a new preventive approach for erosive demineralization, employing acquired pellicle engineering procedures that may in the future be incorporated into dental products.

Effects of acute fluoride exposure on the jejunum and ileum of rats: Insights from proteomic and enteric innervation analysis.

Fluoride (F) is largely employed in dentistry, in therapeutic doses, to control caries. However, excessive intake may lead to adverse effects in the body. Since F is absorbed mostly from the gastrointestinal tract (GIT), gastrointestinal symptoms are the first signs following acute F exposure. Nevertheless, little is known about the mechanistic events that lead to these symptoms. Therefore, the present study evaluated changes in the proteomic profile as well as morphological changes in the jejunum and ileum of rats upon acute exposure to F. Male rats received, by gastric gavage, a single dose of F containing 0 (control) or 25 mg/Kg for 30 days. Upon exposure to F, there was a decrease in the thickness of the tunic muscularis for both segments and a decrease in the thickness of the wall only for the ileum. In addition, a decrease in the density of HuC/D-IR neurons and nNOS-IR neurons was found for the jejunum, but for the ileum only nNOS-IR neurons were decreased upon F exposure. Moreover, SP-IR varicosities were increased in both segments, while VIP-IR varicosities were increased in the jejunum and decreased in the ileum. As for the proteomic analysis, the proteins with altered expression were mostly negatively regulated and associated mainly with protein synthesis and energy metabolism. Proteomics also revealed alterations in proteins involved in oxidative/antioxidant defense, apoptosis and as well as in cytoskeletal proteins. Our results, when analyzed together, suggest that the gastrointestinal symptoms found in cases of acute F exposure might be related to the morphological alterations in the gut (decrease in the thickness of the tunica muscularis) that, at the molecular level, can be explained by alterations in the gut vipergic innervation and in proteins that regulate the cytoskeleton.

Saliva as a diagnostic tool for dental caries, periodontal disease and cancer: is there a need for more biomarkers?

Introduction: A biomarker is a biological indicator of normal or pathogenic processes. Identification of biomarkers is useful for the prevention, diagnosis and prognosis of diseases as well as for monitoring the progression of pathological disorders. Several types of molecules present in biological fluids can act as biomarkers such as DNA, coding and non-coding RNA, lipids, metabolites, proteins and even microbes. In this context, saliva emerges as a useful diagnostic tool for the detection of biomarkers involved with oral and systemic diseases, since it reflects the pathophysiological conditions of the organism and allows early, rapid, practical and noninvasive detection of biomarkers.Areas covered: This review discusses the properties of saliva as a diagnostic tool and addresses the main identified biomarkers related to dental caries, periodontal disease, head and neck cancer and other types of cancer of considerable incidence among the world population.Expert commentary: Despite extensive efforts which have been directed toward the identification of one or a combination of biomarkers with good predictive values for the early detection of dental caries, periodontal disease and cancer, these biomarkers still need validation before chairside point-of-care devices can be widely used in the clinic.

Changes in energy metabolism induced by fluoride: Insights from inside the mitochondria.

The mechanisms involved in changes in energy metabolism caused by excessive exposure to fluoride (F) are not completely understood. The present study employed proteomic tools to investigate the molecular mechanisms underlying the dose- and time-dependency of the effects of F in the rat liver mitochondria. Thirty-six male Wistar rats received water containing 0, 15 or 50 mgF/L (as NaF) for 20 or 60 days. Rat liver mitochondria were isolated and the proteome profiles were examined using label-free quantitative nLC-MS/MS. PLGS software was used to detect changes in protein expression among the different groups. The bioinformatics analysis was done using the software CYTOSCAPE® 3.0.7 (Java®) with the aid of ClueGo plugin. The dose of 15 mgF/L, when administered for 20 days, reduced glycolysis, which was counterbalanced by an increase in other energetic pathways. At 60 days, however, an increase in all energy pathways was observed. On the other hand, the dose of 50 mgF/L, when administered for 20 days, reduced the enzymes involved in all energetic pathways, indicating a lower rate of energy production, with less generation of ROS and consequent reduction of antioxidant enzymes. However, when the 50 mgF/L dose was administered for 60 days, an increase in energy metabolism was seen but in general no changes were observed in the antioxidant enzymes. Except for the group treated with 50 mgF/L for 20 days, all the other groups had alterations in proteins in attempt to maintain calcium homeostasis and avoid apoptosis. The results suggest that the organism seems to adapt to the effects of F over time, activating pathways to reduce the toxicity of this ion. Ultimately, our findings corroborate the safety of the use of fluoride for caries control.

Effect of Duration of Exposure to Fluoride and Type of Diet on Lipid Parameters and De Novo Lipogenesis.

The effect of duration of chronic treatment with fluoride (F, 50 mg/L as NaF) on the lipid profile, lipid droplets and triglycerides (TG) in liver was evaluated in mice with nonalcoholic fatty liver disease (NAFLD) previously induced by hyperlipidic diet and in animals fed normocaloric diet. In addition, the effect of F administered for a short period (20 days) was evaluated on de novo lipogenesis, by nuclear magnetic resonance. GRP78, Apo-E, and sterol regulatory element-binding protein (SREBP) were quantified by Western blotting. Our data indicate that F interferes in lipid metabolism and lipid droplets, having a different action depending on the exposure time and type of diet administered. F improved lipid parameters and reduced steatosis only when administered for a short period of time (up to 20 days) to animals fed normocaloric diet. However, when NAFLD was already installed, lipid parameters were only slightly improved at 20 days of treatment, but no effect was observed on the degree of steatosis. In addition, lipid profile was in general impaired when the animals were treated with F for 30 days, regardless of the diet. Moreover, F did not alter de novo lipogenesis in animals with installed NAFLD. Furthermore, hyperlipidic diet increased F accumulation in the body. GRP78 increased, while Apo-E and SREBP decreased in the F-treated groups. Our results provide new insights on how F affects lipid metabolism depending on the available energy source.