Salivary proteomic profile of response to different resistance training protocols: A case report.

Resistance training (RT) with blood flow restriction (BFR) or high intensity (HI) are effective to increase muscle mass. To understand this effect, techniques known as "omics" are used to identify possible biomarkers. This study analyzed the salivary proteomic profile of healthy individuals trained before and after two RT protocols both designed with eight exercises for upper- and lower-limbs, one performed at low percentage of one-maximum repetition (%1RM) with BFR technique, and other at high %1RM (HI) without BRF technique. Four healthy males between 18 and 28 years participated in the study. Stimulated saliva was collected before (BBFR/BHI) and immediately after (ABFR/AHI) the two RT protocols. All protein-related processing was performed using label-free proteomic. The difference in expression between groups was expressed as p < .05 for downregulated proteins and 1-p > .95 for upregulated proteins. There was difference in salivary flow between ABFR and BBFR (p = .005). For HI, 87 proteins were found after the practice and 119 before. Three hemoglobin isoforms were increased in AHI compared with BHI. In the BFR comparison, 105 proteins were identified after (ABFR) and 70 before (BBFR). Among those increased ABFR, we highlight five hemoglobin isoforms and Deleted in malignant brain tumors 1 protein. Between ABFR and AHI, 17 isoforms of histones, Transaldolase, Transketolase, Glyceraldehyde-3-phosphate dehydrogenase, and Antileukoproteinase were decreased ABFR. For HI, there was an increase in proteins related to oxidative stress and metabolism of the musculoskeletal system, compared with BFR. HI seems to induce higher anabolic signaling to muscle mass increase and antiatherosclerotic effects.

Statherin-Derived Peptide Protects against Intrinsic Erosive Enamel Wear in situ.

INTRODUCTION: This in situ study investigated the protective effect of a solution containing statherin-derived peptide (StatpSpS) against enamel intrinsic erosion (ERO). METHODS: Fifteen volunteers wore appliances containing 2 bovine specimens. The samples were subjected to ERO with HCl, mimicking dental ERO by intrinsic acid. The volunteers participated in 3 phases (double-blind and crossover): (1) deionized water (negative control); (2) commercial solution containing SnCl2/NaF/AmF (800 ppm Sn+2, 500 ppm F-, pH 4.5) (positive control); (3) solution containing 1.88 × 10-5m StatpSpS. Four times a day, the volunteers administered one drop of the solutions (50 µL, 1 min) on each specimen. After the treatment, erosive challenges were performed extraorally with 0.01 m HCl (pH 2.0, 4 times/day, 1 min, 150 mL). Enamel wear was assessed by profilometry. Data were analyzed by one-way RM-ANOVA/Bonferroni's tests (p < 0.05). RESULTS: In terms of the treatments, both the commercial solution (SnCl2/NaF/AmF) and StatpSpS significantly reduced the wear when compared to the negative control (p < 0.01), without significant differences between them (p > 0.05). CONCLUSION: The solution containing StatpSpS demonstrated protection against enamel intrinsic erosive wear. This study marks a significant advancement in the prevention of intrinsic erosive wear, utilizing StatpSpS in acquired pellicle engineering procedures.

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.

Proteomic profile of the acquired enamel pellicle of children with early childhood caries and caries-free children.

Acquired enamel pellicle plays an important role in the pathogenesis of early childhood caries (ECC), working as a protective interface between the tooth and the oral cavity. The aim of this cross-sectional in vivo proteomic study was to compare the acquired enamel pellicle protein profile of 3-5-year-old children with ECC (n = 10) and caries-free children (n = 10). Acquired enamel pellicle samples were collected and processed for proteomic analysis (nLC-ESI-MS/MS). In total, 241 proteins were identified. Basic salivary proline-rich protein 1 and 2, Cystatin-B, and SA were found only in the caries free group. When comparing caries free and ECC groups, lower protein levels were found in the caries free group for hemoglobin subunit beta, delta, epsilon, gamma-2, globin domain-containing protein and gamma-1, neutrophil defensin 3, serum albumin, protein S100-A8, and S100-A9. The proteins histatin-1, statherin, salivary acidic proline-rich phosphoprotein ½, proline-rich protein 4, submaxillary gland androgen-regulated protein 3B, alpha-amylase 1 and 2B were found at higher levels in the caries free group. The exclusive and the proteins found at higher levels in the caries free group might have protective functions that play a role in the prevention of caries, besides providing important insights to be evaluated in future studies for the possible development of new therapeutic strategies for ECC.

Teeth with acute apical abscess vs. teeth with chronic apical periodontitis: a quantitative and qualitative proteomic analysis.

OBJECTIVE: To quantitatively and qualitatively analyze the proteomic profile of teeth with acute apical abscesses (AAA) compared with teeth with chronic apical periodontitis (CAP) and to correlate the expression of detected human proteins with their main biological functions. MATERIALS AND METHODS: Samples were obtained from root canals of 9 patients diagnosed with AAA and 9 with CAP. Samples were analyzed by reversed-phase liquid chromatography coupled to mass spectrometry. Label-free quantitative proteomic analysis was performed by Protein Lynx Global Service software. Differences in protein expression were calculated using the t-test (p < 0.05). RESULTS: In total, 246 human proteins were identified from all samples. Proteins exclusively found in the AAA group were mainly associated with the immunoinflammatory response and oxidative stress response. In the quantitative analysis, 17 proteins were upregulated (p < 0.05) in the AAA group, including alpha-1-acid glycoprotein, hemopexin, fibrinogen gamma chain, and immunoglobulin. Additionally, 61 proteins were downregulated (p < 0.05), comprising cathepsin G, moesin, gelsolin, and transketolase. Most of the proteins were from the extracellular matrix, cytoplasm, and nucleus. CONCLUSIONS: The common proteins between the groups were mainly associated with the immune response at both expression levels. Upregulated proteins mostly belonged to the acute-phase proteins, while the downregulated proteins were associated with DNA/RNA regulation and repair, and structural function. CLINICAL RELEVANCE: The host response is directly related to the development of apical abscesses. Thus, understanding the behavior of human proteins against the endodontic pathogens involved in this condition might contribute to the study of new approaches related to the treatment of this disease.

Saliva proteomic profile of early childhood caries and caries-free children.

OBJECTIVE: Saliva plays an important antimicrobial role and it is related to the pathogenesis of early childhood caries (ECC). The aim of this study was to compare the proteomic profile of unstimulated saliva of children aged 3-5 years who had ECC and caries-free (CF) children. MATERIALS AND METHODS: After the saliva collection from 20 children (ECC: n = 10; CF: n = 10), the samples were processed for proteomic analysis on a mass spectrometer. RESULTS: 1638 proteins were identified, of which 355 were present in both groups. A total of 579 proteins were exclusively identified in the CF group and included Leucine-rich alpha-2-glycoprotein, Protein S100-A5, Protein S100-A8 and Mucin-2. Moreover, 704 proteins were exclusively identified in the ECC group, including Enamelin. The differential expression analysis revealed that 112 proteins were up-regulated in the CF group. Among these proteins, we highlighted Hemoglobin subunit gamma-1 (343-fold increase), gamma-2 (336-fold increase) and alpha (40-fold increase). CONCLUSIONS: The proteomic profile of the saliva varied substantially between the groups. Hemoglobin subunit gamma-1, gamma-2 and alpha may play a protective role in children with ECC. These proteins should be evaluated in future studies, because they may be possible good candidates to be included in anti-caries dental products.

Proteomic analysis of infected root canals with apical periodontitis in patients with type 2 diabetes mellitus: A cross-sectional study.

AIM: This study aimed to quantitatively and qualitatively determine the proteomic profile of apical periodontitis (AP) in type 2 diabetes mellitus (T2DM) patients in comparison with systemically noncompromised patients and to correlate the protein expression of both groups with their biological functions. METHODOLOGY: The sample consisted of 18 patients with asymptomatic AP divided into two groups according to the presence of T2DM: diabetic group-patients with T2DM (n = 9) and control group-systemically healthy patients (n = 9). After sample collection, the root canal samples were prepared for proteomic analysis using reverse-phase liquid chromatography-mass spectrometry. Label-free quantitative proteomic analysis was performed by Protein Lynx Global Service software. Differences in protein expression between groups were calculated using t-test (p < .05). Biological functions were analysed using the Homo sapiens UniProt database. RESULTS: A total of 727 human proteins were identified in all samples. Among them, 124 proteins common to both groups were quantified, out of which 65 proteins from the diabetic group showed significant differences compared with the control: 43 upregulated (p < .05) and 22 downregulated (p < .05) proteins. No significant differences in protein expression were seen for the remaining 59 proteins (p > .05). Most proteins with differences in expression were related to immune/inflammatory response. Neutrophil gelatinase-associated lipocalin, Plastin-2, Lactotransferrin and 13 isoforms of immunoglobulins were upregulated. In contrast, Protein S100-A8, Protein S100-A9, Histone H2B, Neutrophil defensin 1, Neutrophil defensin 3 and Prolactin-inducible protein were downregulated. CONCLUSIONS: Quantitative differences were demonstrated in the expression of proteins common to diabetic and control groups, mainly related to immune response, oxidative stress, apoptosis and proteolysis. These findings revealed biological pathways that provide the basis to support clinical findings on the relationship between AP and T2DM.

Radiotherapy changes the salivary proteome in head and neck cancer patients: evaluation before, during, and after treatment.

OBJECTIVES: Salivary glands are affected during radiotherapy in the head and neck region, leading to a reduction in salivary flow and changes its composition. Besides negatively affecting the oral soft tissues, this can also lead to dental impairment. Thus, we evaluated the effect of radiotherapy in the proteomic profile of the saliva in patients with head and neck cancer (HNC). MATERIALS AND METHODS: HNC patients had their saliva collected before (BRT), during (2-5 weeks; DRT), and after (3-4 months; ART) radiotherapy. Saliva was also collected from healthy volunteers (control; C). Samples were processed for proteomic analysis. RESULTS: In total, 1055 proteins were identified, among which 46 were common to all groups, while 86, 86, 286, and 395 were exclusively found in C, BRT, DRT, and ART, respectively. Remarkably, alpha-enolase was increased 35-fold DRT compared with BRT, while proline-rich proteins were decreased. ART there was a 16-fold increase in scaffold attachment factor-B1 and a 3-fold decrease in alpha-enolase and several cystatins. When compared with C, salivary proteins of BRT patients showed increases cystatin-C, lysozyme C, histatin-1, and proline-rich proteins CONCLUSION/CLINICAL REVELANCE: Both HNC and radiotherapy remarkably change the salivary protein composition. Altogether, our results, for the first time, suggest investigating alpha-enolase levels in saliva DRT in future studies as a possible biomarker and strategy to predict the efficiency of the treatment. Moreover, our data provide important insights for designing dental products that are more effective for these patients and contribute to a better understanding of the progressive changes in salivary proteins induced by radiotherapy. Graphical abstract.

Proteomic profile of the acquired enamel pellicle of professional wine tasters with erosive tooth wear.

The aim of this study was to compare the acquired enamel pellicle protein profile of professional wine tasters with mild and moderate erosive tooth wear. Twelve professional wine tasters participated (3 from a low tooth wear group; 9 from a high tooth wear group). Acquired enamel pellicle samples were collected and processed for proteomic analysis (nLC-ESI-MS/MS). The acquired enamel pellicle proteomic profile was different between the groups. The proteins found exclusively in the low tooth wear group were histatins 1 and 3 and mucins 7 and 21. When comparing the wear groups, proteins with higher levels in the low tooth wear group included neutrophil defensins (1 and 3), lysozyme C, lysozyme, myeloperoxidase, and squalene monooxygenase. In conclusion, the findings indicate that the proteins found at higher levels in the low tooth wear group and proteins exclusively found in the low tooth wear group might be protective and, therefore, could be good candidates for further studies regarding their potential to be added to dental products to protect professional wine tasters from extrinsic erosive tooth wear.

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.

A new role for resveratrol: Protection of enamel against erosion.

OBJECTIVE: The aim of this study was to determine the effect of different concentrations of resveratrol in protecting enamel against initial dental erosion in vitro. METHODS: Ninety bovine enamel samples (4 × 4 mm) were divided into six groups: Phosphate buffered saline (negative control; PBS), Commercial solution (Elmex Erosion Protection™; positive control) and resveratrol at 4 different concentrations (1, 10, 100 or 400 µg/mL). Initially, the samples were incubated in saliva for the formation of the acquired pellicle (250 µL, 1 h, 37 °C, 250 rpm). Afterward, the samples were incubated in the respective treatments (250 µL, 1 min, 37 °C, 250 rpm) and then reincubated in saliva (250 µL, 1 h, 37 °C, 250 rpm). Finally, the samples were subjected to an erosive challenge by incubating in 1 % citric acid (1 mL, pH 3.5, 1 min, 25 °C, 250 rpm). The percentage surface microhardness change (% SMC) was assessed using a microhardness tester. Data were analyzed by Kruskal-Wallis and Dunn's tests (p < 0.05). RESULTS: The treatments with Elmex™ and resveratrol (1, 10 and 100 µg/mL) significantly protected enamel compared to the negative control, without significant differences among them. However, the group treated with the highest resveratrol concentration (400 µg/mL) did not show a significant difference from the negative control. CONCLUSIONS: Resveratrol at concentrations ranging from 1 to 100 µg/ml was effective in preventing loss of enamel surface microhardness. CLINICAL SIGNIFICANCE: This result suggests a potential new direction for the development of dental products based on resveratrol for the prevention of dental erosion.

In vivo modification of the enamel pellicle and saliva resveratrol levels after use of resveratrol-containing orodispersible capsules.

OBJECTIVES: To evaluate in vivo 1) the bioavailability of trans-resveratrol when administered through sublingual capsules; 2) the effect of resveratrol on the protein composition of the acquired enamel pellicle (AEP). DESIGN: Ten volunteers received a sublingual capsule containing 50 mg of trans-resveratrol. Unstimulated saliva was then collected after 0, 30, 60, and 120 min and AEP was collected after 120 min following administration of the capsule. In the next week, the volunteers received a placebo sublingual capsule, and saliva and AEP were collected again. Saliva samples were analyzed for free trans-resveratrol using high-performance liquid chromatopgraphy (HPLC), and AEP samples were subjected to proteomic analysis (nLC-ESI-MS/MS). RESULTS: Trans-resveratrol was detected in saliva at all the time points evaluated, with the peak at 30 min. A total of 242 proteins were identified in both groups. Ninety-six proteins were increased and 23 proteins were decreased in the Resveratrol group. Among the up-regulated proteins, isoforms of cystatins, PRPs, Mucin-7, Histatin-1, Lactotrasnferrin and Lysozyme-C were increased and the isoforms of Protein S100, Neutrophil defensins, Albumin, PRPs, and, Statherin were decreased in Resveratrol group. CONCLUSION: The sublingual capsule is effective at increasing the bioavailability of trans-resveratrol in saliva. Several proteins involved in important processes to maintain systemic and oral health homeostasis were identified. These proteins differently expressed due to the presence of trans-resveratrol deserve attention for future studies, since they have important functions, mainly related to antimicrobial action.

Optimized protocol for shotgun label-free proteomic analysis of pancreatic islets.

Pancreatic islets are crucial in diabetes research. Consequently, this protocol aims at optimizing both the protein-extraction process and the proteomic analysis via shotgun methods for pancreatic islets. Six protocols were tested, combining three types of chemical extraction with two mechanical extraction methods. Furthermore, two protocols incorporated a surfactant to enhance enzymatic cleavage. The steps involved extraction and concentration of protein, protein quantification, reduction, alkylation, digestion, purification and desalination, sample concentration to ∼1 µl, and proteomic analysis using the mass spectrometer. The most effective protocol involves either a milder chemical extraction paired with a more intensive mechanical process, or a more robust chemical extraction paired with a gentle mechanical process, tailored to the sample's characteristics. Additionally, it was observed that the use of a surfactant proved ineffective for these types of samples. Protocol 5 was recently used with success to examine metabolic changes in pancreatic islets of non-obese diabetic mice exposed to low doses of fluoride ions (F-) and the primary pathways altered by the treatment.

Exploring the effects of vanillin and divanillin on murine osteosarcoma cells: evaluation of cellular response and proteomic analysis.

The objective of this study was to evaluate the antitumor properties of vanillin and divanillin in murine bone tumour cells. The action of the compounds on the cell viability of the normal (MC3T3-E1) and the tumour cell line (UMR-106) was evaluated. Action of the compounds in colony formation, migration, and production of reactive oxygen species (ROS) in tumour cells were evaluated along with proteomic analysis. Both compounds affected the cell viability of normal and tumour cell lines, being divanillin the more effective. For UMR-106, both compounds reduced the cell viability by less than 50%. Vanillin inhibited the migration process, and divanillin decreased ROS production (p < 0.05). The proteomic analysis showed that both compounds acted in the expression of proteins involved in tumour progression. Our results suggest that vanillin and divanillin are effective drugs against murine bone tumour cells. They can be a promising alternative for the adjuvant treatment of bone cancer.

Is There a Difference in the Proteomic Profile of Stimulated and Unstimulated Saliva Samples from Pregnant Women with/without Obesity and Periodontitis?

This study aimed to compare the proteomic profile of stimulated and unstimulated saliva samples from pregnant women with/without obesity and periodontitis. Pregnant women were allocated into four groups: with obesity and periodontitis (OP); with obesity but without periodontitis (OWP); with normal BMI but with periodontitis (NP); with normal BMI and without periodontitis (NWP). Stimulated saliva (SS) and unstimulated saliva (US) samples were collected, and salivary proteins were extracted and individually processed by proteomic analysis (nLC-ESI-MS/MS). Proteins involved with the immune response process, antioxidant activity, and retina homeostasis were decreased or absent in SS samples from all groups (i.e., Antileukoproteinase, Lysozyme C, Alpha-2-macroglobulin-like protein 1, Heat shock proteins-70 kDa 1-like, 1A, 1B, 6, Heat shock-related 70 kDa protein 2, Putative Heat shock 70 kDa protein 7, Heat shock cognate 71 kDa). Additionally, proteins related to the carbohydrate metabolic process and glycolytic and glucose metabolic process were absent in SS, mainly from OP and OWP (i.e., Frutose-bisphosphate aldose A, Glusoce-6-phosphate isomerase, Pyruvate kinase). Saliva stimulation decreased important proteins involved with immune response and inflammation process in all groups. Unstimulated salivary samples seem to be the best choice for the proteomic approach in pregnant women.

New insights into the protective effect of statherin-derived peptide for different acquired enamel pellicle formation times on the native human enamel surfaces.

OBJECTIVES: This study evaluated the protective impact of acquired enamel pellicle (AEP) engineering with statherin-derived peptide (StatpSpS), considering different AEP formation times. DESIGN: A total of 120 native human enamel specimens were divided into 2 main groups: 1) No AEP engineering and 2) AEP engineering with StatpSpS (pretreatment for 1 min; 37 °C, under agitation). Each group was further divided into 4 subgroups: No pellicle, or 1, 60-and-120 min AEP formation times (human saliva; 37 °C). The specimens were then subjected to an erosive challenge (1% citric acid; pH 3.6; 1 min; 25 °C). This procedure was repeated for 5 cycles. Relative surface reflection intensity (%SRI) was measured and scanning electron microscopy (SEM) of the enamel surface was done. RESULTS: All AEP engineering groups protected against initial dental erosion in comparison with No pellicle (p < 0.001), likewise all groups with AEP, independent of engineering or formation times (p 0.001). Furthermore, engineering with StatpSpS even without the presence of AEP protected the enamel when compared to the No engineering/No pellicle group (p < 0.0001). No difference was observed regarding the protection from the different AEP formation times (p > 0.05). Regarding the SEM analysis, in the "No AEP engineering & No AEP" group, a more severe effect of citric acid was observed, with more enamel prism heads and scratches on the surface when compared with the other groups. CONCLUSIONS: AEP provides almost instant protection at formation times even as short as 1 min, protecting the native enamel against erosion. Treatment with StatpSpS by itself provides similar protection as the AEP.

Use of Reflectometer Optipen to assess the preventive effect of a sugarcane cystatin on initial dental erosion in vivo.

The sugarcane cystatin 5 (CaneCPI-5) showed protection against erosion and erosive tooth wear (ETW) under several protocols. However, evaluating these conditions in vivo is hard due to the lack of a suitable device. The aim of this study was to use clinically the relative surface reflection intensity (%SRI) by the Reflectometer Optipen to assess the acquired pellicle engineering with CaneCPI-5 rinse for the prevention of initial erosion in vivo. Nine volunteers were distributed in three cross-over phases, according to the rinse used, as follows: 1) Deionized water (negative control); 2) Elmex® (800 ppm Sn2+, 500 ppm F-; positive control); 3) 0.1 mg/mL CaneCPI-5. The following experimental steps were performed: Initially, the volunteers received prophylaxis and the initial %SRI was performed. Subsequently, they rinsed with the solutions (10 mL; 1min), followed by the formation of the acquired enamel pellicle (AEP; 120min). After, the erosive challenge with citric acid 1%, pH 2.5 (10 µL; 10s) was performed (in isolation) on the buccal surface of the maxillary central incisors (right and left). The calcium present in the acid was analyzed by Arsenazo III method. Finally, the final %SRI was performed. Data were analyzed by Kruskal-Wallis/Dunn's tests and Spearman's correlation were used (p < 0.05). For both variables, the negative control led to significantly less protection (lower reflectivity and higher calcium release) in comparison with the other groups. The best protection (higher reflectivity and lower calcium release) was observed for the Elmex® and the CaneCPI-5 groups, with no significant differences between them (p < 0.05). There was a significant correlation between both analyzes. The Reflectometer Optipen demonstrated to be a good device to assess clinically. Moreover, CaneCPI-5 rinse proved effective through acquired pellicle engineering against initial erosion in vivo.

Maquiberry Cystatins: Recombinant Expression, Characterization, and Use to Protect Tooth Dentin and Enamel.

Phytocystatins are proteinaceous competitive inhibitors of cysteine peptidases involved in physiological and defensive roles in plants. Their application as potential therapeutics for human disorders has been suggested, and the hunt for novel cystatin variants in different plants, such as maqui (Aristotelia chilensis), is pertinent. Being an understudied species, the biotechnological potential of maqui proteins is little understood. In the present study, we constructed a transcriptome of maqui plantlets using next-generation sequencing, in which we found six cystatin sequences. Five of them were cloned and recombinantly expressed. Inhibition assays were performed against papain and human cathepsins B and L. Maquicystatins can inhibit the proteases in nanomolar order, except MaquiCPIs 4 and 5, which inhibit cathepsin B in micromolar order. This suggests maquicystatins' potential use for treating human diseases. In addition, since we previously demonstrated the efficacy of a sugarcane-derived cystatin to protect dental enamel, we tested the ability of MaquiCPI-3 to protect both dentin and enamel. Both were protected by this protein (by One-way ANOVA and Tukey's Multiple Comparisons Test, p < 0.05), suggesting its potential usage in dental products.

Metabolic effect of low fluoride levels in the islets of NOD mice: integrative morphological, immunohistochemical, and proteomic analyses.

OBJECTIVES: Fluoride (F) has been widely used to control dental caries, and studies suggest beneficial effects against diabetes when a low dose of F is added to the drinking water (10 mgF/L). This study evaluated metabolic changes in pancreatic islets of NOD mice exposed to low doses of F and the main pathways altered by the treatment. METHODOLOGY: In total, 42 female NOD mice were randomly divided into two groups, considering the concentration of F administered in the drinking water for 14 weeks: 0 or 10 mgF/L. After the experimental period, the pancreas was collected for morphological and immunohistochemical analysis, and the islets for proteomic analysis. RESULTS: In the morphological and immunohistochemical analysis, no significant differences were found in the percentage of cells labelled for insulin, glucagon, and acetylated histone H3, although the treated group had higher percentages than the control group. Moreover, no significant differences were found for the mean percentages of pancreatic areas occupied by islets and for the pancreatic inflammatory infiltrate between the control and treated groups. Proteomic analysis showed large increases in histones H3 and, to a lesser extent, in histone acetyltransferases, concomitant with a decrease in enzymes involved in the formation of acetyl-CoA, besides many changes in proteins involved in several metabolic pathways, especially energy metabolism. The conjunction analysis of these data showed an attempt by the organism to maintain protein synthesis in the islets, even with the dramatic changes in energy metabolism. CONCLUSION: Our data suggests epigenetic alterations in the islets of NOD mice exposed to F levels comparable to those found in public supply water consumed by humans.

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.