Standardization of a protocol for shotgun proteomic analysis of saliva

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.

Liver proteome of mice with different genetic susceptibilities to the effects of fluoride

ABSTRACT A/J and 129P3/J mice strains have been widely studied over the last few years because they respond quite differently to fluoride (F) exposure. 129P3/J mice are remarkably resistant to the development of dental fluorosis, despite excreting less F in urine and having higher circulating F levels. These two strains also present different characteristics regardless of F exposure. Objective In this study, we investigated the differential pattern of protein expression in the liver of these mice to provide insights on why they have different responses to F. Material and Methods Weanling male A/J and 129P3/J mice (n=10 from each strain) were pared and housed in metabolic cages with ad libitum access to low-F food and deionized water for 42 days. Liver proteome profiles were examined using nLC-MS/MS. Protein function was classified by GO biological process (Cluego v2.0.7 + Clupedia v1.0.8) and protein-protein interaction network was constructed (PSICQUIC, Cytoscape). Results Most proteins with fold change were increased in A/J mice. The functional category with the highest percentage of altered genes was oxidation-reduction process (20%). Subnetwork analysis revealed that proteins with fold change interacted with Disks large homolog 4 and Calcium-activated potassium channel subunit alpha-1. A/J mice had an increase in proteins related to energy flux and oxidative stress. Conclusion This could be a possible explanation for the high susceptibility of these mice to the effects of F, since the exposure also induces oxidative stress.

Atividade antibacteriana in vitro de pastas de hidróxido de cálcio associadas à bioprodutos frente a linhagens gram positivas e gram negativas / Antibacterial activity in vitro of calcium hydroxide paste associated to bioproducts against on Gram-positive and Gram-negative strains

Introdução: o hidróxido de cálcio Ca(OH)2 em endodontia, tem sido utilizado em pulpotomias, tratamento de perfurações radiculares, como componente de cimentos obturadores e como medicação intracanal, sendo que quando utilizado nesta última situação, é associado a um veículo com a finalidade de se obter a consistência de pasta. Assim, diferentes veículos têm sido propostos para associação ao Ca(OH)2. A atividade antimicrobiana do Ca(OH)2 está relacionada a liberação de íons hidroxila. Apesar de sua ampla utilização, esta substância não tem demonstrado eficácia sobre algumas cepas de micro-organismos in vivo. Objetivo: o propósito da presente pesquisa foi avaliar a atividade antibacteriana in vitro de várias pastas de Ca(OH)2 associadas com bioprodutos contra linhagens ATCC de Enterococcus. faecalis, Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeruginosa e Escherichia coli. Métodos: os testes de susceptibilidade bacteriana frente às pastas, aos bioprodutos na forma de extratos e aos géis foram realizados pelo método da difusão, sobre ágar Mueller-Hinton. Os dados foram submetidos a análise estatística, empregando-se o teste Kruskal-Wallis com nível de significância de 5 por cento. Resultados e Discussão: a clorexidina, tanto a 1 por cento como a 2 por cento, mostrou grande atividade antibacteriana pura, na forma de gel e associada como veículo ao Ca(OH)2, estatisticamente significante (p<0,05). Todas as pastas de Ca(OH)2 revelaram efetividade contra todos os micro-organismos testados com exceção da pasta cujo veículo foi o óleo de alho. Frente ao St. pyogenes, somente as pastas de clorexidina 1 por cento e 2 por cento revelaram efetividade. As pastas de Ca(OH)2 cujos veículos puros revelaram atividade antibacteriana, não foram potencializadas para atividade antibacteriana.

Introduction: calcium hydroxide Ca(OH)2 is a highly alkaline white powder that has been used in Endodontics in pulpotomies, treatment of root perforations, as component of sealers and intracanal medication; when used for the latter purpose, it is associated with a vehicle to achieve a paste consistency. Thus, different vehicles have been proposed for association with Ca(OH)2. The antimicrobial activity of Ca(OH)2 is related to the release of hydroxyl ions. Despite its wide utilization, this substance has not been demonstrated to be effective against some microorganism stains in vivo. Objective: this study evaluated the in vitro antibacterial activity of several Ca (OH)2 pastes associated with bioproducts against ATCC strains of E. faecalis, Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeruginosa and Escherichia coli. Methods: thebacterial susceptibility test to the pastes of bioproducts in extract andgel was analyzed by the paste diffusion method on Mueller-Hinton agar. Data were statistically analyzed by the Kruskal-Wallis test at a significance level of 5 per cent. Results and Discussion: chlorhexidine, both at 1 per cent and 2 per cent, presented wide antibacterial activity both pure, in gel and associated as vehicle to Ca(OH)2, with statistical significance (p<0.05). All Ca(OH)2 pastes were effective against all microorganisms tested, except for the paste with garlic oil as vehicle. Concerning the St. pyogenes, only 1 per cent and 2 per cent chlorhexidine pastes were effective. The Ca(OH)2 pastes whose pure vehicles presented antibacterial activity were not strengthened for the antibacterial activity.