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

Quality-Quantitative Identification of Dangerous Waste From FOB-Usp: Waste Management

Hazardous waste is defined by the Environmental Protection Agency of the United States (EPA) as any waste that because of its quantity, concentration, physical, chemical or infectious characteristics, may cause or significantly contribute to increased mortality, serious irreversible or incapacitating reversible diseases, or pose a present substantial or potential hazard to human health and the environment when improperly managed. Aiming to comply with the laws, the Bauru Dental School (FOB) in 2003, installed its first Laboratory of Chemical Waste (LRQ). However due to increased demand and new waste generated, there was the need to build a new laboratory. Therefore, this study conducted a qualitative and quantitative survey of hazardous waste generated in different sectors of the FOB, Hospital for Rehabilitation of Craniofacial Anomalies (HRAC) and Prefecture of Campus, beyond its current mode of handling by responsible. This survey was conducted through a questionnaire. After analyzing the data, we saw that were generated 433 liters of waste in 2013, mainly: developer liquid, fixer liquid, alcohol, ethylenediaminetetraacetic acid (EDTA), xylene and formaldehyde. In addition, we found the key points for a better management of waste. This work was important to quantify and qualify the hazardous waste on the USP campus of Bauru. The implementation of a specialized laboratory in management and treatment of waste on campus, for the recovery of these, beyond to proper disposal, is important for both environment and people

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

Fluoride Intensifies Hypercaloric Diet-Induced ER Oxidative Stress and Alters Lipid Metabolism.

BACKGROUND:Here, we evaluated the relationship of diet and F-induced oxidative stress to lipid metabolism in the liver of rats eating normocaloric or hypercaloric diets for two time periods (20 or 60 days). METHODS:Seventy-two 21-day-old Wistar rats were divided into 2 groups (n = 36) based on the type of diet they were eating; each of these groups was then further divided into another two groups (n = 18) based on the time periods of either 20 or 60 days, for a total of four groups. Each of these was divided into 3 subgroups (n = 6 animals/subgroup), dependent on the dose of F administered in the drinking water (0 mg/L(control), 15 mg/L or 50 mg/L). After the experimental period, blood samples and the liver were collected. Plasma samples were analyzed for HDL, cholesterol and triglycerides. Western blots were performed to probe for GRP78, Erp29, SOD2, Apo-E and SREBP in hepatic tissues. RESULTS:As expected,the expression of target proteins involved in oxidative stress increased in the F-treated groups, especially in liver tissue obtained from animals eating a hypercaloric diet. Most changes in the lipid levels and pathological conditions were seen earlier in the time period, at day 20. The morphometric analyses showed a reduction in steatosis in groups on ahypercaloric diet and treated with 50 mg F/L compared to the control, while no changes were obtained in normocaloric-fed rats. Accordingly, plasma TG was reduced in the F-treated group. The reduced expression of Apo-E in a time- and diet-dependent pattern may account for the particular decrease in steatosis in hypercaloric-fed F-treated rats. CONCLUSIONS:These results suggest that F changes liver lipid homeostasis, possibly because of the induction of oxidative stress, which seems to be higher in animals fed hypercaloric diets

Chronic Exposure to Sodium Fluoride Triggers Oxidative Biochemistry Misbalance in Mice: Effects on Peripheral Blood Circulation

The excessive fluoride (F) exposure is associated with damage to cellular processes of different tissue types, due to changes in enzymatic metabolism and breakdown of redox balance. However, few studies evaluate doses of F compatible with human consumption. Thus, this study evaluated the effects of chronic exposure to sodium fluoride (NaF) on peripheral blood of mice from the evaluation of biochemical parameters. The animals were divided into three groups (n=10) and received three concentrations of NaF in the drinking water for 60 days: 0 mg/L F, 10 mg/L F, and 50 mg/L F. The blood was then collected for trolox equivalent antioxidant capacity (TEAC), thiobarbituric acid reactive substances (TBARS), concentrations of nitric oxide (NO), superoxide dismutase (SOD), catalase (CAT), and reduced glutathione (GSH). The results showed that doses of 10 mg/L F and 50 mg/L F were able to increase TBARS concentration and decrease NO levels and CAT activity in the blood, but there was no statistical difference for SOD levels. The 50 mg/L F group showed an increase in TEAC levels and a decrease in the GSH content when compared to the control group. In this way, oxidative changes in blood from chronic exposure to F, especially at the highest dose, indicate that F may be a toxic agent and, therefore, the long-term exposure to excessive doses should be avoided

Proposed mechanism by which fluoride affects lipid metabolism depending on the type of diet consumed and time of exposure.

<p>The diet-dependency effects of fluoride in lipid metabolism are observed strictly at earlier periods and are related with ER oxidative stress. Upon consumption of normocaloric diet, fluoride-induced oxidative stress leads to increase of GRP78, which in turn reduces SREBP, thus deactivating TG synthesis. However, in the presence of hypercaloric diet, fluoride exposition induces oxidative stress highlighted by GRP78, ERp29 and SOD2 increases, which in turn, by undetermined mechanism, leads to reduction of Apo-E, which diminishes precursors of Acetyl–Coa and leads to less formation of liver TG.</p

Representative expression of proteins SREBP, and of the constitutive protein β-tubulin ou α-tubulin in samples of individual animals (n = 3) from each group.

<p>Densitometric analysis was performed for 6 animals per group. <b>A.</b> Protein expression in the liver of rats in the group treated with the hypercaloric diet for 20 and 60 days; <b>B.</b> Protein expression in the liver of rats treated with the normocaloric diet for 20 and 60 days. Densitometry was analyzed using the software <i>Image Studio Lite</i>. Vertical bars represent standard deviations. Distinct superscript denote significant differences among the groups (ANOVA and Tukey’s test, p<0.05, n = 6).</p

Representative expression of proteins GRP-78, APO-E, SOD2, ERP-29 and of the constitutive protein β-tubulin in samples of individual animals (n = 3) from each group.

<p>Densitometric analysis was performed for 6 animals per group. <b>A.</b> Protein expression in the liver of rats in the group treated with the hypercaloric diet for 20 days; <b>B.</b> Protein expression in the liver of rats treated with the normocaloric diet for 20 days; <b>C.</b> Protein expression in the liver of rats in in the group treated with the hypercaloric diet for 60 days; <b>D.</b> Protein expression in the liver of rats treated with the normocaloric diet for 60 days. Densitometry was analyzed using the software <i>Image Studio Lite</i>. Vertical bars represent standard deviations. For each condition, distinct superscripts denote significant differences among the groups (ANOVA and Tukey’s test, p<0.05, n = 6).</p