Maria Carolina Martins Mussi

Bacteriostatic effect of copaiba oil (Copaifera officinalis) against Streptococcus mutans

This study evaluated the inhibitory activity of copaiba oil (Copaifera officinalis against the cariogenic microorganism, Streptococcus mutans. For such purpose, a minimum inhibition concentration test of copaiba oil against S. mutans was performed, using the serial dilution in broth technique, with a negative control, a positive control (0.12% chlorhexidine) and a 10% copaíba oil solution as a test. A minimum bactericidal concentration test with tubes presenting microbial inhibition was also conduced. In the minimum inhibitory concentration test, copaiba oil showed inhibition of bacterial growth at all concentrations tested up to 0.78 µL/mL of the 10% copaiba oil solution in the broth. In addition, the negative control had no inhibition, and the 0.12% chlorhexidine solution was effective up to 6.25 µL/mL in the broth. Copaiba oil showed a bacteriostatic activity against S. mutans at low concentrations, and could be a an option of phytotherapic agent to be used against cariogenic bacteria in the prevention of caries disease.

Efeitos clínicos e microbiológicos do óleo de copaíba (Copaifera officinalis) sobre bactérias formadoras de placa dental em cães

The copaiba oil (Copaifera officinalis) potential was evaluated in preventing periodontal disease and reducing its etiology. For that 18 mongrel dogs were homogeneously distributed in three groups: test (copaiba oil), positive control (chlorexidine) and negative control. The treatments were carried out three times a day, during eight days. On the 9 th day, the animals were tested with a 0.5% basic solution of fuchsin for the detection of biofilm. Changes in halitosis and gingivitis were daily observed. In addition, the following laboratory tests were done: inhibition of the adherence of Streptococcus mutans, and plaque forming bacteria antimicrobial assays by the agar diffusion method. The results of the fuchsin test showed that dental plaque reached areas of 53.4±8.8%, 28.5±5.4%, and 22.3±5.3% in the negative control, positive control, and test groups, respectively, showing differences between dogs from the negative control group and dogs from the other two groups (P<0.05). Furthermore, halitosis and gingivitis decreased in the copaiba group animals when compared with the negative group (P<0.05). The results of the attachment inhibition and agar diffusion tests showed that copaiba induced better effects against the microorganisms as compared to the results of the other groups (P<0.05). These findings suggest that copaiba oil may effectively replace chlorexidine for oral antimicrobial therapy and prevention of periodontal disease.

Identification of acid-resistant proteins in acquired enamel pellicle

OBJECTIVES This study characterized the proteome profile of the acquired pellicle formed in vivo on enamel. Changes in this proteome profile after exposure to lactic or citric acid were also evaluated. METHODS Volunteers (n=8) were subjected to dental prophylaxis. After 2 h to allow the formation of the acquired pellicle, the teeth were isolated with cotton rolls and 1 mL of citric acid (1%, pH 2.5) or lactic acid (0.1 M pH 4.8) or deionized water was gently applied with a pipette on the anterior teeth (both maxillary and mandibular) for 10 s. In sequence, the pellicle was collected with an electrode filter paper soaked in 3% citric acid. This procedure was repeated for two additional days following a crossover protocol. Proteins were subjected to reverse phase liquid chromatography coupled to mass spectrometry (nLC-ESI-MS/MS). MS/MS data were processed and submitted to Proteome Discoverer software. Searches were done using SWISS-PROT and TrEMBL databases for human proteins. RESULTS In total, seventy-two proteins were present in all groups and were submitted to quantitative analysis (SIEVE). Some of these proteins were increased more than two-fold after exposure to the acids. Among them, cystatin-B was increased 20- and 13-fold after exposure to citric and lactic acids, respectively. Additionally, some proteins were identified in only one of the groups (18, 5, and 11 proteins for deionized water, citric and lactic acids, respectively). CONCLUSIONS Our results open new insights regarding potentially acid-resistant proteins that could be added to dental products to prevent acidic dissolution of the teeth.

The Impact of Stannous, Fluoride Ions and Its Combination on Enamel Pellicle Proteome and Dental Erosion Prevention

Objectives To compare the effects of stannous (Sn) and fluoride (F) ions and their combination on acquired enamel pellicle (AEP) protein composition (proteome experiment), and protection against dental erosion (functional experiment). Methods In the proteome experiment, bovine enamel specimens were incubated in whole saliva supernatant for 24h for AEP formation. They were randomly assigned to 4 groups (n=10), according to the rinse treatment: Sn (800ppm/6.7mM, SnCl2), F (225ppm/13mM, NaF), Sn and F combination (Sn+F) and deionized water (DIW, negative control). The specimens were immersed 3× in the test rinses for 2min, 2h apart. Pellicles were collected, digested, and analyzed for protein content using liquid chromatography electrospray ionization tandem mass spectrometry. In the functional experiment, bovine enamel specimens (n=10) were similarly treated for pellicle formation. Then, they were subjected to a five-day erosion cycling model, consisting of 5min erosive challenges (15.6 mM citric acid, pH 2.6, 6×/d) and 2min treatment with the rinses containing Sn, F or Sn+F (3×/d). Between the treatments, all specimens were incubated in whole saliva supernatant. Surface loss was determined by profilometry. Results Our proteome approach on bovine enamel identified 72 proteins that were common to all groups. AEP of enamel treated with Sn+F demonstrated higher abundance for most of the identified proteins than the other groups. The functional experiment showed reduction of enamel surface loss for Sn+F (89%), Sn (67%) and F (42%) compared to DIW (all significantly different, p<0.05). Conclusion This study highlighted that anti-erosion rinses (e.g. Sn+F) can modify quantitatively and qualitatively the AEP formed on bovine enamel. Moreover, our study demonstrated a combinatory effect that amplified the anti-erosive protection on tooth surface.

Hydroxyapatite Growth Inhibition Effect of Pellicle Statherin Peptides

In our recent studies, we have shown that in vivo–acquired enamel pellicle is a sophisticated biological structure containing a significant portion of naturally occurring salivary peptides. From a functional aspect, the identification of peptides in the acquired enamel pellicle is of interest because many salivary proteins exhibit functional domains that maintain the activities of the native protein. Among the in vivo–acquired enamel pellicle peptides that have been newly identified, 5 peptides are derived from statherin. Here, we assessed the ability of these statherin pellicle peptides to inhibit hydroxyapatite crystal growth. In addition, atomistic molecular dynamics (MD) simulations were performed to better understand the underlying physical mechanisms of hydroxyapatite growth inhibition. A microplate colorimetric assay was used to quantify hydroxyapatite growth. Statherin protein, 5 statherin-derived peptides, and a peptide lacking phosphate at residues 2 and 3 were analyzed. Statherin peptide phosphorylated on residues 2 and 3 indicated a significant inhibitory effect when compared with the 5 other peptides (P < 0.05). MD simulations showed a strong affinity and fast adsorption to hydroxyapatite for phosphopeptides, whereas unphosphorylated peptides interacted weakly with the hydroxyapatite. Our data suggest that the presence of a covalently linked phosphate group (at residues 2 and 3) in statherin peptides modulates the effect of hydroxyapatite growth inhibition. This study provides a mechanism to account for the composition and function of acquired enamel pellicle statherin peptides that will contribute as a base for the development of biologically stable and functional synthetic peptides for therapeutic use against dental caries and/or periodontal disease.

Histatin 5 inhibits adhesion of C. albicans to Reconstructed Human Oral Epithelium.

Candida albicans is the most pathogenic fungal species, commonly colonizing on human mucosal surfaces. As a polymorphic species, C. albicans is capable of switching between yeast and hyphal forms, causing an array of mucosal and disseminated infections with high mortality. While the yeast form is most commonly associated with systemic disease, the hyphae are more adept at adhering to and penetrating host tissue and are therefore frequently observed in mucosal fungal infections, most commonly oral candidiasis. The formation of a saliva-derived protein pellicle on the mucosa surface can provide protection against C. albicans on oral epithelial cells, and narrow information is available on the mucosal pellicle composition. Histatins are one of the most abundant salivary proteins and presents antifungal and antibacterial activities against many species of the oral microbiota, however, its presence has never been studied in oral mucosa pellicle. The objective of this study was to evaluate the potential of histatin 5 to protect the Human Oral Epithelium against C. albicans adhesion. Human Oral Epithelial Tissues (HOET) were incubated with PBS containing histatin 5 for 2 h, followed by incubation with C. albicans for 1 h at 37°C. The tissues were then washed several times in PBS, transferred to fresh RPMI and incubated for 16 h at 37°C at 5% CO2. HOET were then prepared for histopathological analysis using light microscopy. In addition, the TUNEL assay was employed to evaluate the apoptosis of epithelial cells using fluorescent microscopy. HOET pre-incubated with histatin 5 showed a lower rate of C. albicans growth and cell apoptosis when compared to the control groups (HOET alone and HOET incubated with C. albicans). The data suggest that the coating with histatin 5 is able to reduce C. albicans colonization on epithelial cell surfaces and also protect the basal cell layers from undergoing apoptosis.

Zika virus infection spread through saliva--a truth or myth?

In this Point-of-view article we highlighted some features related to saliva and virus infection, in special for zika virus. In addition, we pointed out the potential oral problems caused by a microcephaly originated by a zika virus infection. In the end the, we demonstrated the importance of a more comprehensive exploration of saliva and their components as a fluid for diagnostic and therapeutic approaches on oral and systemic diseases.

Salivary parameters and oral health in the Moebius syndrome.

PURPOSE To evaluate salivary characteristics, such as flow rate, buffering capacity and amylase activity, and associate these parameters with oral health in individuals with Moebius syndrome (MS) and controls. METHODS The researchers assessed caries (ICDAS), periodontal disease (PSR), and plaque (Silness Löe) indexes in both groups. Unstimulated, stimulated, and bilateral parotid saliva were also collected. The salivary flow (milliliters/minute) and α-amylase activity were measured in all samples. The α-amylase activity was determined by maltose production. The buffering capacity was measured in stimulated saliva by titration of 0.01N HCl. RESULTS The occurrence of carious lesions, related to cut-off 2, as well as the occurrence of periodontal disease, was significantly higher (p > 0.05) in participants with MS when compared to the control group. Also, MS individuals presented decreased salivary flow, decreased buffering capacity, and amylase activity. CONCLUSIONS MS individuals present salivary alterations, which can be related to higher risk for caries.

In Vitro Identification of Histatin 5 Salivary Complexes

With recent progress in the analysis of the salivary proteome, the number of salivary proteins identified has increased dramatically. However, the physiological functions of many of the newly discovered proteins remain unclear. Closely related to the study of a protein’s function is the identification of its interaction partners. Although in saliva some proteins may act primarily as single monomeric units, a significant percentage of all salivary proteins, if not the majority, appear to act in complexes with partners to execute their diverse functions. Coimmunoprecipitation (Co-IP) and pull-down assays were used to identify the heterotypic complexes between histatin 5, a potent natural antifungal protein, and other salivary proteins in saliva. Classical protein–protein interaction methods in combination with high-throughput mass spectrometric techniques were carried out. Co-IP using protein G magnetic Sepharose TM beads suspension was able to capture salivary complexes formed between histatin 5 and its salivary protein partners. Pull-down assay was used to confirm histatin 5 protein partners. A total of 52 different proteins were identified to interact with histatin 5. The present study used proteomic approaches in conjunction with classical biochemical methods to investigate protein–protein interaction in human saliva. Our study demonstrated that when histatin 5 is complexed with salivary amylase, one of the 52 proteins identified as a histatin 5 partner, the antifungal activity of histatin 5 is reduced. We expected that our proteomic approach could serve as a basis for future studies on the mechanism and structural-characterization of those salivary protein interactions to understand their clinical significance.

Doing more with less: the challenging diagnosis of polymorphous low-grade adenocarcinoma in incisional biopsy samples.

The diagnosis of polymorphous low‐grade adenocarcinoma (PLGA) remains difficult for general pathologists, particularly in cases of small biopsy samples. We aimed to characterize the histopathological spectrum and immunohistochemical aspects by using an accessible immunohistochemical panel of cytoskeletal proteins in limited samples of PLGA.