Salivary proteomic profile of dogs with and without dental calculus

“In mammals, the main saliva functions are lubrication and protection of the buccal tissue, buffer capacity, maintenance of teeth integrity, and antimicrobial effect [3].”

“Mass spectrometry is an indicated method for studies focused on protein identification, characterization and quantification [20].”

“Abbreviations AEP: Acquired enamel pellicle; PRPs: Proline-rich proteins; WSS: Whole saliva supernatants; Micro BCA: Micro Bicinchoninic Acid; S1P1: Sphingosine-1Phosphate Receptor Protein 1; TNF: Tumor necrosis factor; MMPs: Matrixmetalloproteinases; TGFB1: Transforming growth factor beta; C8B: Complement C8 beta cha…”

“The C-type lysozymes, as the ones identified, are the main lysozymes produced by vertebrates [44].”

“TGFB1 was previously correlated to chronic periodontitis [55], its importance lies in the fact that this growth factor stimulate tissue remodeling and wound healing through increasing fibroblast proliferation, angiogenesis, and extracellular matrix production [56], and by inhibiting MMPs [57].”

“Other than constituents involved in the formation of dental calculus, saliva possesses chemical and physical properties that may serve to detect evidence, through proteomic analysis, of systemic diseases [2].”

“This protein is highly expressed in humans in endothelial cells, brain, heart and immune system cells [46, 47]; it is coupled to G protein and binds to sphingosine-1-phosphate, which is a bioactive sphingolipid that behaves as an intracellular messenger of some cytokines and also as an autocrine and paracrine extracel…”

“It is deemed that proteins Ttype calcium channel play important roles in neuronal activity and have been described in studies with rats [52] and humans [53]; but there are no reports in the literature relating these proteins to the dogs’ saliva.”

“Between then it can be pointed out the presence of fragments of the Polymeric immunoglobulin receptor protein, which has been described in human saliva having the function of transporter of IgA, the main salivary antibody [39].”

“In addition, sphingosine-1-phosphate stimulates events of intracellular signaling, such as activation of phospholipase C, increased cytoplasmic calcium concentration, regulation of adenylate cyclase, activation of the MAP kinase pathway and the Rho cascade [49].”

“Dental calculus formation is always preceded by the development of a saliva protein layer on the tooth enamel’s surface, called acquired enamel pellicle (AEP), which serves both to protect dental enamel, reduce friction between teeth and oral mucosa [11], and as basis to formation and maturation of dental”

“In addition, the breeds studied here are similar in appearance and the genetic investigation presented a close relationship between them [34].”

“The organic constituents are predominantly salivary proteins [1].”

“When bound, they participate in several cellular processes such as proliferation, differentiation, adhesion, motility, angiogenesis, apoptosis, migration, morphogenesis and changes in the cytoskeleton [50].”

“This analysis is available both for humans [21, 22] and animals [23–25].”

“Furthermore, another abundant protein detected by this research was the Calcium-sensing receptor (CaSR), whose function is calcium (Ca) transportation and regulation [54].”

“Dental calculus consists in a mixture of calcium carbonate and calcium phosphate, presenting a rough surface that increases the occurrence of periodontal disease [8], due to the calcification of more dental biofilm, bringing it closer to the soft tissues [14].”

“Dental calculus consists in a mixture of calcium carbonate and calcium phosphate, presenting a rough surface that increases the occurrence of periodontal disease [8], due to the calcification of more dental biofilm, bringing it closer to the soft tissues [14].”

“A visual oral inspection was carried out by a veterinary, where each participant of the study was categorized based on the criteria adopted to evaluate the dental calculus: 0 for absence of dental calculus; 1 for supragingival calculus covering not more than one-third of the exposed surface of the examined tooth; 2 fo…”

“Search results were filtered at a false discovery rate of 1% using a reverse database search strategy [26, 27].”

“The total protein concentration of WSS was measured by Micro BCA Protein Assay Kit (Thermo Scientific Pierce, Rockford, IL, USA) with bovine serum albumin used as the standard and it was stored at − 80 °C until further analysis [69, 70].”

“The total protein concentration of WSS was measured by Micro BCA Protein Assay Kit (Thermo Scientific Pierce, Rockford, IL, USA) with bovine serum albumin used as the standard and it was stored at − 80 °C until further analysis [69, 70].”

“This lack of amylase in dog’s saliva was also observed by other studies [4, 36, 37].”

“This lack of amylase in dog’s saliva was also observed by other studies [4, 36, 37].”

“biofilm, which constitutes the organic matrix to its possible calcification [12].”

“Rho GTPases can modulate the effects on human periodontal ligament cells of TNFB1, an important cytokine already known by its role in periodontal pathologies [61].”

“In some species, some saliva components represent an essential part on the enzymatic digestion assisting mastication and deglutition [5].”

“In other words, they promote cell lysis in bacteria [40], since its biochemically function hydrolyze beta-1,4 bond between N-acetyl glucosamine and N-acetyl muramic acid residues of bacterial peptidoglycan, which is an essential part of bacterial cell wall and also promotes structural stiffness and neutralize osmotic…”

“In Sousa Pereira study [23], the samples were collected in mixed breed while in our study two specific breeds were selected.”

“[25] identified 249 proteins on a mixed breed group and Torres et al.”

“Concerning the analyses in the two different groups, the most abundant proteins in the group of dogs without dental calculus participated in metabolism functions, whereas in the group with dental calculus there were major proteins related to immune response, enzymatic regulation and uncharacterized proteins, potential…”

“This analysis is available both for humans [21, 22] and animals [23–25].”

“As described previously [26], after identification of the proteome profile, the most abundant proteins had their biological functions verified through the accession number using the database www.”

“Quantification of electrolytes such as calcium and phosphate, which participate in the formation of the dental calculus and processes of demineralization and remineralization of dental enamel, should also be performed, not forgetting to mention sodium, potassium, zinc and magnesium, that are important in the metabolis…”

“It is also important to note that in the kidney, the formation of stones is associated with changes in calcium reabsorption, causing hypercalcemia [59].”

“compared the salivary proteome between dogs and humans, and observed that alpha amylase was present in dogs saliva, but in lower levels than in humans saliva; however, the presence of this protein in dogs in that study may be due the fact that in Thailand these animals are often fed with rice and starch [35].”

“This lack of amylase in dog’s saliva was also observed by other studies [4, 36, 37].”

“Since our main goal is to study dental calculus, we selected the breeds Shih Tzu and Lhasa Apso that possesses brachycephalic skulls and tend to show malocclusion, dental crowding and rotation [31, 32], which facilitates accumulation of dental calculus.”

“In salivary glands, it was showed that functional CaSR proteins can be stimulated by Ca concentration, that is, CaSR can serve as a Ca sensor in the luminal membrane of salivary gland ducts and regulate reabsorption of Ca from the saliva via transient receptor potential canonical 3 (TRPC3), thus contributing to mainte…”

“TGFB1 was previously correlated to chronic periodontitis [55], its importance lies in the fact that this growth factor stimulate tissue remodeling and wound healing through increasing fibroblast proliferation, angiogenesis, and extracellular matrix production [56], and by inhibiting MMPs [57].”

“A visual oral inspection was carried out by a veterinary, where each participant of the study was categorized based on the criteria adopted to evaluate the dental calculus: 0 for absence of dental calculus; 1 for supragingival calculus covering not more than one-third of the exposed surface of the examined tooth; 2 fo…”

“Finally, aliquots from each sample were dried again in a rotary evaporator, de-salted by C18 Pipette Tips (Millipore, USA) and finally subjected to mass spectrometry analyses [71].”

“Foremost, differently from previous studies where the collection of saliva in dogs was performed under anesthesia with stimulation with acid [28, 29], we have opted for a noninvasive collection without any other stimulation mechanism or general anesthesia.”

“S1P1 proteins were also described in memory T cells and a cell immunophenotyping revealed that humans secrete CD4(+) T cells in saliva [51].”

“showed that TGFB1 can induce proliferation and cytoskeletal rearrangement in periodontal ligament cells via Rho GTPase-dependent pathways [64].”

“In dogs, the formation of dental calculus is typical since the first year of life and it appears as granular, yellow– brown masses on the buccal surfaces of molar teeth of the upper jaw near salivary duct orifices [7, 13].”

“In other words, they promote cell lysis in bacteria [40], since its biochemically function hydrolyze beta-1,4 bond between N-acetyl glucosamine and N-acetyl muramic acid residues of bacterial peptidoglycan, which is an essential part of bacterial cell wall and also promotes structural stiffness and neutralize osmotic…”

“This protein is highly expressed in humans in endothelial cells, brain, heart and immune system cells [46, 47]; it is coupled to G protein and binds to sphingosine-1-phosphate, which is a bioactive sphingolipid that behaves as an intracellular messenger of some cytokines and also as an autocrine and paracrine extracel…”

“[29], in which there was no major differences between genders.”

“5 [6, 7], a range that facilitates dental calculus formation, through calcification of dental biofilm present on teeth cervical region [8–10].”

“Similar to humans, there is a link in dogs between the calculus presence with the increasing rate of periodontal diseases, which in some cases may lead to the animal starvation as a result of feeding difficulties [13, 15, 16].”

“In dogs, very few studies have reported spectral libraries using saliva [75] and tears [76].”

“Because these proteins have distinct origins, the whole amount of saliva is composed of food particles, compounds produced by oral bacteria, and gingival crevicular fluid (which contains plasma proteins) [39].”

“For protein identification, varying false discovery rate (FDR) threshold values were used including ≤ 1% [50], 1% [18, 23, 48, 51], and 5% [52] (Table 1).”

“Proteomic markers have also been used to differentiate dogs with and without dental calculus, aiding in periodontitis detection [11].”

“The increased salivaryALS activity observed in dogs with gingivitis in this study is consistent with previous findings in dogs with dental calculus (associated with periodontal disease), where proteomic analysis identified fructose-bisphosphate ALS as an abundant salivary protein 34 .”