Standardization of a protocol for shotgun proteomic analysis of saliva

Saliva contains numerous proteins and peptides, each of them carries a number of biological functions that are very important in maintaining the oral cavity health and also yields information about both local and systemic diseases. Currently, proteomic analysis is the basis for large-scale identification of these proteins and discovery of new biomarkers for distinct diseases. Objective: This study compared methodologies to extract salivary proteins for proteomic analysis. Material and MethodsSaliva samples were collected from 10 healthy volunteers. In the first test, the necessity for using an albumin and IgG depletion column was evaluated, employing pooled samples from the 10 volunteers. In the second test, the analysis of the pooled samples was compared with individual analysis of one sample. Salivary proteins were extracted and processed for analysis by LC-ESI-MS/MS. Results: In the first test, we identified only 35 proteins using the albumin and IgG depletion column, while we identified 248 proteins without using the column. In the second test, the pooled sample identified 212 proteins, such as carbonic anhydrase 6, cystatin isoforms, histatins 1 and 3, lysozyme C, mucin 7, protein S100A8 and S100A9, and statherin, while individual analysis identified 239 proteins, among which are carbonic anhydrase 6, cystatin isoforms, histatin 1 and 3, lactotransferrin, lyzozyme C, mucin 7, protein S100A8 and S100A9, serotransferrin, and statherin. Conclusions: The standardization of protocol for salivary proteomic analysis was satisfactory, since the identification detected typical salivary proteins, among others. The results indicate that using the column for depletion of albumin and IgG is not necessary and that performing individual analysis of saliva samples is possible

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

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). Objectives: 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

Do commercial whitening dentifrices increase enamel erosive tooth wear?

Objective: This in vitro study evaluated the effect of commercial whitening dentifrices on erosive tooth wear (ETW) of bovine enamel samples, in comparison with commercial regular dentifrices. Methodology: Sixty bovine crowns were embedded in acrylic resin, polished and then had their baseline profile determined. They were randomly assigned to 5 groups (n=12/group), according to the type of commercial dentifrice to be tested: GI – Crest Anti-cavity Regular; GII – Crest 3D White; GIII – Colgate Total 12 Clean Mint; GIV – Colgate Optic White; GV – Placebo (negative control, fluoride-free dentifrice). The samples were submitted to daily erosive and abrasive challenges for 3 days. The erosive challenges were performed 3 times a day by immersing the specimens in 0.1% citric acid solution (pH 2.5) for 90 s. Each day after the first and last erosive challenges, the specimens were subjected to the abrasive challenge for 15 s, using a toothbrushing machine (Biopdi, São Carlos, SP, Brazil), soft toothbrushes and slurry (1:3 g/ml) of the tested toothpastes (1.5 N). The specimens were kept in artificial saliva between the challenges. The final profile was obtained and the ETW (µm) was calculated. Data were analyzed by Kruskal-Wallis and Dunn’s tests (p<0.05). Results: All dentifrices tested significantly reduced the enamel wear in comparison with the Placebo, except GIII. The median (95% CI) ETW was 1.35 (1.25-1.46)bc for GI, 1.17 (1.01-1.34)cd for GII, 1.36 (1.28-1.45)ab for GIII, 1.08 (1.04-1.14)d for GIV and 2.28 (2.18-2.39)a for GV. Conclusion: When dentifrices from the same manufacturer were compared, the whitening dentifrices led to similar or less wear than the regular ones

Standardization of a protocol for shotgun proteomic analysis of saliva

<div><p>Abstract Saliva contains numerous proteins and peptides, each of them carries a number of biological functions that are very important in maintaining the oral cavity health and also yields information about both local and systemic diseases. Currently, proteomic analysis is the basis for large-scale identification of these proteins and discovery of new biomarkers for distinct diseases. Objective This study compared methodologies to extract salivary proteins for proteomic analysis. Material and Methods Saliva samples were collected from 10 healthy volunteers. In the first test, the necessity for using an albumin and IgG depletion column was evaluated, employing pooled samples from the 10 volunteers. In the second test, the analysis of the pooled samples was compared with individual analysis of one sample. Salivary proteins were extracted and processed for analysis by LC-ESI-MS/MS. Results In the first test, we identified only 35 proteins using the albumin and IgG depletion column, while we identified 248 proteins without using the column. In the second test, the pooled sample identified 212 proteins, such as carbonic anhydrase 6, cystatin isoforms, histatins 1 and 3, lysozyme C, mucin 7, protein S100A8 and S100A9, and statherin, while individual analysis identified 239 proteins, among which are carbonic anhydrase 6, cystatin isoforms, histatin 1 and 3, lactotransferrin, lyzozyme C, mucin 7, protein S100A8 and S100A9, serotransferrin, and statherin. Conclusions The standardization of protocol for salivary proteomic analysis was satisfactory, since the identification detected typical salivary proteins, among others. The results indicate that using the column for depletion of albumin and IgG is not necessary and that performing individual analysis of saliva samples is possible.</p></div

Analysis of Plasma Proteins Involved in Inflammation, Immune Response/Complement System, and Blood Coagulation upon Admission of COVID-19 Patients to Hospital May Help to Predict the Prognosis of the Disease

The development of new approaches allowing for the early assessment of COVID-19 cases that are likely to become critical and the discovery of new therapeutic targets are urgently required. In this prospective cohort study, we performed proteomic and laboratory profiling of plasma from 163 COVID-19 patients admitted to Bauru State Hospital (Brazil) between 4 May 2020 and 4 July 2020. Plasma samples were collected upon admission for routine laboratory analyses and shotgun quantitative label-free proteomics. Based on the course of the disease, the patients were divided into three groups: (a) mild (n = 76) and (b) severe (n = 56) symptoms, whose patients were discharged without or with admission to an intensive care unit (ICU), respectively, and (c) critical (n = 31), a group consisting of patients who died after admission to an ICU. Based on our data, potential therapies for COVID-19 should target proteins involved in inflammation, the immune response and complement system, and blood coagulation. Other proteins that could potentially be employed in therapies against COVID-19 but that so far have not been associated with the disease are CD5L, VDBP, A1BG, C4BPA, PGLYRP2, SERPINC1, and APOH. Targeting these proteins’ pathways might constitute potential new therapies or biomarkers of prognosis of the disease