Proteomic analysis of the acquired enamel pellicle formed on human and bovine tooth: a study using the Bauru in situ pellicle model (BISPM)

The acquired enamel pellicle (AEP) is an organic film, bacteria-free, formed in vivo as a result of the selective adsorption of salivary proteins and glycoproteins to the solid surfaces exposed to the oral environment. Objective: This study aimed to compare the proteomic profile of AEP formed in situ on human and bovine enamel using a new intraoral device (Bauru in situ pellicle model – BISPM). Material and Methods: One hundred and eight samples of human and bovine enamel were prepared (4x4 mm). Nine subjects with good oral conditions wore a removable jaw appliance (BISPM) with 6 slabs of each substrate randomly allocated. The AEP was formed during the morning, for 120 minutes, and collected with an electrode filter paper soaked in 3% citric acid. This procedure was conducted in triplicate and the pellicle collected was processed for analysis by LC-ESI-MS/MS. The obtained mass spectrometry MS/MS spectra were searched against human protein database (SWISS–PROT). Results: The use of BISPM allowed the collection of enough proteins amount for proper analysis. A total of 51 proteins were found in the AEP collected from the substrates. Among them, 15 were common to both groups, 14 were exclusive of the bovine enamel, and 22 were exclusive of the human enamel. Proteins typically found in the AEP were identified, such as Histatin-1, Ig alpha-1, Ig alpha 2, Lysozyme C, Statherin and Submaxillary gland androgen-regulated protein 3B. Proteins not previously described in the AEP, such as metabolism, cell signaling, cell adhesion, cell division, transport, protein synthesis and degradation were also identified. Conclusion: These results demonstrate that the proteins typically found in the AEP appeared in both groups, regardless the substrate. The BISPM revealed to be a good device to be used in studies involving proteomic analysis of the AEP

SENESCÊNCIA DO APARELHO CARDIOVASCULAR E SUAS IMPLICAÇÕES FISIOPATOLÓGICAS

O tema envelhecimento é amplamente discutido, posto que diversos fatores como o avanço da medicina, a população acarretam maior longevidade à população. Com isso, vários temas relacionados aos idosos têm ganhado importância, entre eles a qualidade de vida e principalmente a saúde dessa população. Objetivos: Explicar sobre a senescência do sistema cardiovascular e o impacto disso na saúde do idoso, ou seja, as doenças cardiovasculares e suas prevalências nas pessoas acima dos 65 anos. Metodologia: Foram utilizados artigos publicados no SCIELO e Google Acadêmico, os descritores utilizados foram, idoso, doenças, senescência e aparelho cardiovascular. Sistema Cardiovascular; Envelhecimento, Doenças Cardiovasculares, no período de 2008 a 2016. Resultados: Os resultados remetem à relação íntima entre o envelhecimento e o surgimento de doenças no aparelho cardiovascular. Essas doenças decorrem aumento da pressão arterial, espessamento e enrijecimento da parede dos vasos sanguíneos, diminuição do calibre venoso, ente outros. Conclusão: A saúde do sistema cardiovascular deve ser acompanhada de perto, pois o envelhecimento traz mudanças significativas em todo o aparelho cardiovascular e tem como consequência o aumento nos casos de doenças nos idosos, tornando-se assim a principal causa de morte nessa população

Advanced circulatory support: artificial heart use in patients with heart failure / Suporte circulatório avançado: uso do coração artificial em pacientes com Insuficiência cardíaca

Objectives: To analyze recent clinical trials about mechanical circulatory support and other adjuvant therapies for the treatment of heart failure. Methods: Articles were selected from the Pubmed and Embase databases published between the years 2015 and 2020, randomized controlled trial or not. The descriptors used were: assisted circulation AND artificial heart AND heart failure, and the descriptor artificial heart was not used for research at EMBASE with 2 articles as results.  And 40 at PubMed with the filters: Controlled Clinical Trial, published in the last 5 years, Humans. Results: Several therapies have been proposed as management alternatives for critically ill patients with heart failure. Among them, we can highlight the HeartMate III implant, which has been shown to have fewer adverse effects compared to HeartMate II; the implantation of an atrial bypass device, which has been shown to decrease the pressure of pulmonary artery occlusion in 1 month compared to the control group, but has shown no long-term effects; and the use of mesenchymal precursor cells, which has not been shown to be significantly effective. Final considerations: It is suggested to carry out further studies to improve the indication criteria, making it possible to allocate resources in hospitalized patients

Acquired enamel pellicle protects gastroesophageal reflux disease patients against erosive tooth wear

Abstract The objective of this study was to compare the protein profile of the acquired enamel pellicle (AEP) formed in vivo in patients with or without gastroesophageal reflux disease (GERD), and with or without erosive tooth wear (ETW). Twenty-four volunteers were divided into 3 groups: 1) GERD and ETW; 2) GERD without ETW; and 3) control (without GERD). The AEP formed 120 min after prophylaxis was collected from the lingual/palatal surfaces. The samples were subjected to mass spectrometry (nLC-ESI-MS/MS) and label-free quantification by Protein Lynx Global Service software. A total of 213 proteins were identified, or 119, 92 and 106 from each group, respectively. Group 2 showed a high number of phosphorylated and calcium-binding proteins. Twenty-three proteins were found in all the groups, including 14-3-3 protein zeta/delta and 1-phosphatidylinositol. Several intracellular proteins that join saliva after the exfoliation of oral mucosa cells might have the potential to bind hydroxyapatite, or participate in forming supramolecular aggregates that bind to precursor proteins in the AEP. Proteins might play a central role in protecting the dental surface against acid dissolution

Efeito da frequência de ingestão e quantidade de fluoreto no leite sobre cárie de esmalte e dentina: estudo in situ

This study analysed the effect of frequency of intake and amount of fluoride in milk on the remineralisation of artificial enamel and dentine caries lesions in situ. Predemineralised bovine enamel and dentine slabs were randomly allocated to 5 in situ phases. Twenty-three subjects wore removable appliances with 2 enamel and 2 dentine slabs for 7 days each phase (separated by a 7-day washout period), following a crossover double-blind protocol. In each phase, treatment was performed with milk containing 2.5 ppm fluoride (F) everyday (T1), 2.5 ppm F every other day (T2), 5.0 ppm F every day (T3), 5.0 ppm F every other day (T4) or no treatment (control; T5). The subjects were instructed to immerse the appliance in 100 ml of milk for 5 minutes and then drank 200 ml of the respective milk. The enamel alterations were quantified by surface hardness (%SHR) and transversal microradiography (TMR, Z) and dentine by TMR only. Data were analysed by repeated-measures ANOVA/Tukey´s tests (p<0.05). For enamel, the highest %SHR was found for groups treated with fluoridated milk every day compared to control, without significant differences between T1 and T3. All groups showed positive values of Z, except for T4; significant differences were seen between T1/T3 and T4. For dentine, the only group that presented remineralisation was T2. Fluoridated milk every day seems to have better remineralising effect on enamel than its use every other day, but no dose-response effect was seen. Dentine, however, does not seem to benefit from every day use of fluoridated milk.O presente estudo teve como objetivo analisar o efeito do leite fluoretado com concentrações e frequências diferentes na remineralização de lesões de cárie de esmalte e dentina produzidas artificialmente in situ. Blocos de esmalte e dentina bovino previamente desmineralizados foram distribuídos aleatoriamente em cinco grupos. Vinte e três indivíduos usaram aparelhos removíveis contendo 2 blocos de esmalte e 2 blocos de dentina por 7 dias em cada fase (separadas por um período de washout de 7 dias), seguindo um protocolo duplo-cego, cruzado. Em cada fase, o tratamento realizado foi com leite contendo 2,5 ppm de flúor (F), todos os dias (T1), 2,5 ppm de F em dias alternados (T2), 5,0 ppm F todos os dias (T3), 5,0 ppm F em dias alternados (T4) ou sem tratamento (T5). Os sujeitos foram instruídos a mergulhar o aparelho em 100 ml de leite por 5 minutos e, em seguida, beberam 200 ml do mesmo leite. As alterações no esmalte foram quantificadas por dureza superficial e microradiografia transversal (TMR), e dentina apenas por microradiografia transversal. Os dados foram analisados por medidas repetidas ANOVA / Tukey (p <0,05). Para o esmalte, o mais alto valor de porcentagem de recuperação de dureza superficial foi encontrado para os grupos tratados com leite fluoretado todos os dias em relação ao controle, sem diferenças significativas entre T1 e T3. Todos os grupos apresentaram valores positivos de Z, com exceção de T4; foram observadas diferenças significativas entre T1/T3 e T4. Para a dentina, o único grupo que apresentou remineralização foi T2. O leite fluoretado todos os dias parece ter melhor efeito remineralizante sobre o esmalte do que seu uso em dias intercalados, mas nenhum efeito dose-resposta foi visto. A dentina, no entanto, não parece se beneficiar do uso diário de leite fluoretado

Efeito protetor do leite contra a desmineralização dentária: compreensão dos mecanismos

This study aimed to answer the following questions: 1) does whole fluoridated milk protect more against enamel and dentin erosion than fat-free fluoridated milk? 2) does the protective effect of fluoridated milk against erosion follow a dose-response relationship? 3) is the treatment with whole or fat-free fluoridated milk before the first erosive challenge more protective against enamel and dentin erosion? 4) does the fat content of milk change the proteomic profile of the acquired enamel pellicle (AEP)? This study was divided into 2 parts. The first part analyzed in vitro the effect of milk against dental erosion, considering three factors: type of bovine milk (whole/fat-free), presence of different fluoride concentrations (0- 10.0 ppm) and time of application (before/after erosive challenge). Bovine enamel (n=15/group) and root dentin (n=12/group) specimens were submitted to the following treatments: 0.9% NaCl solution (negative control)( after first erosive challenge); whole milk with 0, 2.5, 5.0, 10.0 ppm F; fat-free milk with 0, 2.5, 5.0, 10.0 ppm F; 0.05% NaF solution (positive control) (before or after first erosive challenge). Specimens were submitted to demineralization - remineralization regimes, 4 times/ day, for 5 days. The response variables were enamel and dentin loss, evaluated by profilometry (m). Data were analyzed using KruskalWallis/Dunns test (p<0.05). The presence of fluoride, especially at 10 ppm, was the most important factor in reducing dental erosion. The second part detected changes in protein profile of AEP formed in vivo after rinsing with whole milk, fat-free milk or water. Nine subjects with good oral conditions participated. The AEP was formed in the morning, for 120 min, after prophylaxis with pumice. In sequence, the volunteers rinsed with 10 mL of whole milk, fat-free milk or deionized water for 30 s, following a blind, crossover protocol. After 60 min, the AEP was collected with filter paper soaked in 3% citric acid and processed for analysis by liquid chromatography-electrospray ionization tandem mass spectrometry (LCESI- MS/MS). The obtained MS/MS spectra were searched against human protein database (SWISSPROT). The proteomic data related to protein quantification were analyzed using the PLGS software. A total of 260 proteins were successfully identified in the AEP samples collected in all groups. Forty-nine were common to the 3 groups, while 72, 62 and 49 were specific for groups treated with whole milk, fat-free milk and water, respectively. Some were typical components of the AEP, such as Cystatin-B, Lysozyme C, Histatin-1, Statherin and Lactotransferrin. Other proteins are not commonly described as part of the AEP but could act in the defense of the organism against pathogens. Distinct proteomic profiles were found in the AEP after rinsing with whole or fat-free milk, which could have an impact in bacterial adhesion and tooth dissolution. The use of fat-free milk could favorably modulate the adhesion of bacteria in the AEP and the biofilm formation in comparison to whole milk.Este estudo objetivou responder as seguintes questões: 1) o leite integral fluoretado protege mais contra a erosão do esmalte e dentina do que o leite fluoretado desnatado? 2) o efeito protetor do leite fluoretado segue um padrão dose-resposta? 3) o tratamento com leite integral ou leite desnatado fluoretado antes do primeiro desafio erosivo protege mais contra a erosão do esmalte e dentina? 4) o leite contendo gordura altera o perfil proteico da película adquirida do esmalte (PAE)? O estudo foi dividido em 2 partes. Na primeira parte foi realizado um estudo in vitro, considerando três fatores: tipo de leite bovino (integral/ desnatado), diferentes concentrações de fluoreto e tempo de aplicação (antes/após desafio erosivo). Os espécimes de esmalte bovino (n=15 /grupo) e dentina radicular (n=12 /grupo) foram submetidos aos seguintes tratamentos: solução de NaCl a 0,9% (controle negativo)(após o desafio erosivo); Leite integral com 0, 2,5, 5,0, 10,0 ppm F Leite desnatado com 0, 2,5, 5,0, 10,0 ppm F 0,05% de solução de NaF (controle positivo) (antes ou após o primeiro desafio erosivo). Os espécimes foram submetidos a regimes de desmineralização e remineralização, 4 vezes/dia, durante 5 dias. As variáveis de resposta foram perda de esmalte e dentina, avaliadas por perfilometria (m). Os dados foram analisados usando o teste de Kruskal-Wallis / Dunn (p <0,05). A presença de fluoreto, especialmente na concentração de 10 ppm, demonstrou ser o fator mais importante na redução da erosão dentária. A parte II do estudo detectou alterações no perfil proteico da PAE formada in vivo após bochecho com leite integral, leite desnatado ou água. Nove indivíduos com boas condições de saúde bucal participaram. A PAE foi formada pela manhã, durante 120 minutos, após profilaxia com pedra-pomes. Em seguida, os voluntários bochecharam com 10 mL de leite integral, leite desnatado ou água deionizada durante 30 s, seguindo um protocolo cego e cruzado. Após 60 min, a película foi coletada com papel de filtro embebido em ácido cítrico a 3% e processada para análise por cromatografia líquida acoplada à espectrometria de massas com ionização por eletrospray (LC-ESI-MS / MS). Os espectros MS/MS obtidos foram confrontados com bases de dados de proteínas humanas (SWISSPROT). Os dados proteômicos relacionados à quantificação de proteínas foram analisados usando o software PLGS. Um total de 260 proteínas foi identificado nas amostras de PAE coletadas em todos os grupos. Quarenta e nove eram comuns aos 3 grupos, enquanto 72, 62 e 49 eram específicas para grupos tratados com leite integral, leite desnatado e água, respectivamente. Algumas proteínas encontradas são típicas da PAE, como Cistatina-B, Lisozima C, Histatina-1, Estaterina e Lactotransferrina. Outras proteínas não são comumente descritas como parte da PAE, mas podem atuar na defesa do organismo contra patógenos. Perfis proteômicos distintos foram encontrados na PAE após o bochecho com leite integral ou desnatado, o que poderia ter um impacto na adesão bacteriana e na dissolução dentária. O uso de leite desnatado pode modular favoravelmente a adesão de bactérias na PAE e a formação do biofilme em comparação com o leite integral

Proteomic analysis of the acquired enamel pellicle formed on human and bovine tooth: a study using the Bauru in situ pellicle model (BISPM)

Abstract The acquired enamel pellicle (AEP) is an organic film, bacteria-free, formed in vivo as a result of the selective adsorption of salivary proteins and glycoproteins to the solid surfaces exposed to the oral environment. Objective: This study aimed to compare the proteomic profile of AEP formed in situ on human and bovine enamel using a new intraoral device (Bauru in situ pellicle model - BISPM). Material and Methods: One hundred and eight samples of human and bovine enamel were prepared (4×4 mm). Nine subjects with good oral conditions wore a removable jaw appliance (BISPM) with 6 slabs of each substrate randomly allocated. The AEP was formed during the morning, for 120 minutes, and collected with an electrode filter paper soaked in 3% citric acid. This procedure was conducted in triplicate and the pellicle collected was processed for analysis by LC-ESI-MS/MS. The obtained mass spectrometry MS/MS spectra were searched against human protein database (SWISS-PROT). Results: The use of BISPM allowed the collection of enough proteins amount for proper analysis. A total of 51 proteins were found in the AEP collected from the substrates. Among them, 15 were common to both groups, 14 were exclusive of the bovine enamel, and 22 were exclusive of the human enamel. Proteins typically found in the AEP were identified, such as Histatin-1, Ig alpha-1, Ig alpha 2, Lysozyme C, Statherin and Submaxillary gland androgen-regulated protein 3B. Proteins not previously described in the AEP, such as metabolism, cell signaling, cell adhesion, cell division, transport, protein synthesis and degradation were also identified. Conclusion: These results demonstrate that the proteins typically found in the AEP appeared in both groups, regardless the substrate. The BISPM revealed to be a good device to be used in studies involving proteomic analysis of the AEP

Proteomics of acquired pellicle in gastroesophageal reflux disease patients with or without erosive tooth wear.

OBJECTIVES This in vivo study compared the protein profile of the acquired enamel pellicle (AEP) in volunteers 1) with gastroesophageal reflux disease (GERD) and erosive tooth wear (ETW) (BEWE ≥ 9; GE group); 2) with GERD without ETW (BEWE = 0; GNE group) and 3) control (without GERD and BEWE = 0; C group). MATERIALS AND METHODS Twenty-four subjects (8/group) participated. AEP was formed during 120 min and collected. After protein extraction, the samples were submitted to reverse phase liquid chromatography coupled to mass spectrometry. Label-free proteomic quantification was performed using Protein Lynx Global Service software. RESULTS In total, 458 proteins were identified. Seventy-six proteins were common to all the groups. The proteomic profile of the AEP was quite different among the distinct groups. The numbers of proteins exclusively found in the C, GE and GNE groups were 113, 110 and 81, respectively. Most of the proteins exclusively identified in the C and GNE groups bind metals, while those in the GE group are mainly membrane proteins. Many proteins were found exclusively in the reflux groups. In the quantitative analyses, when the GNE group was compared with the GE group, the proteins with the highest decreases were Lysozyme C, Antileukoproteinase, Cathepsin G, Neutrophil defensins and Basic salivary proline-rich proteins, while those with the highest increases were subunits of Hemoglobin, Albumin and isoforms of Cystatin. CONCLUSION Profound alterations in the proteomic profile of the AEP were seen in GNE compared with GE volunteers, which might play a role in the resistance to ETW seen in the first. CLINICAL SIGNIFICANCE This pioneer study compared the proteomic profile of the AEP of patients with GERD with or without ETW. Increased proteins in those without ETW might be protective and are good candidates to be added to dental products to protect against erosion caused by intrinsic acids

Salivary Hemoglobin Protects against Erosive Tooth Wear in Gastric Reflux Patients.

OBJECTIVE Saliva is the most important biological factor to protect against erosive tooth wear (ETW). Gastroesophageal reflux disease (GERD) patients have an increased risk of ETW due to the frequent presence of intrinsic acids in the oral cavity. Remarkably, not all GERD patients suffer from ETW, which might be due to differences in the composition of the saliva. METHODS This study compared the proteomic profile of saliva in patients (1) with GERD and ETW (basic erosive wear examination, BEWE, score ≥9; GE group) and (2) with GERD without ETW (BEWE = 0; GNE group) using shotgun label-free quantitative proteomic analysis nLC-ESI-MS/MS. The ability of hemoglobin (Hb) to protect against initial enamel erosion caused by a daily 10-s immersion of enamel specimens in 0.01 M HCl (pH 2.3) for 3 days was evaluated in vitro for proof of concept. Surface hardness change was used as response variable. RESULTS The differential expression of Hb subunits was significantly increased in the GNE group versus the GE group, in particular the Hb α-subunit that showed a >22-fold increase. Expressions of serum albumin (4.5-fold) and isoforms of cytoskeletal keratin type II (>3-fold) were also increased in the GNE group. Proteinase inhibitors, such as α1-antitrypsin and α2-macroglobulin, were only identified in the GNE group. In vitro, Hb (1.0 and 4.0 mg/mL) significantly reduced initial enamel erosion compared to a negative control after 3 days. CONCLUSIONS Our results indicate that many proteins, with special emphasis on Hb, may be involved in the resistance of GERD patients to the occurrence of ETW. These proteins may be candidates for inclusion in dental products to protect against ETW

Proteomic analysis of stimulated saliva in gastroesophageal reflux disease patients with and without erosive tooth wear: observational study.

OBJECTIVE To evaluate the difference in the proteomic profile of stimulated saliva in patients with gastroesophageal reflux disease (GERD) with (GE) and without (GNE) erosive tooth wear (ETW), regarding both human and bacterial proteins. METHODS Stimulated saliva (SS) was collected from 16 patients (8/group). Samples were centrifuged at 4.500g for 15 min under refrigeration to remove all debris. The supernatant from each saliva sample was taken and frozen at -80°C. After extracting the proteins, they were submitted to reverse phase liquid chromatography and mass spectrometry (nLC-ESI-MS/MS). Label-free proteomic quantification was performed using Protein Lynx Global Service (PLGS) software (p < 0.05) for human and bacterial proteins. RESULTS In total, 67 human proteins were common for GNE and GE groups. GNE group presented, compared to GE group, increase in proteins that confer antimicrobial and acid resistant properties, such as cystatins, histatin and immunoglobulins. However, GNE group had a marked decrease in subunits of hemoglobin (α, β and delta). Regarding bacterial proteins, for SS, 7 and 10 unique proteins were identified in the GE and GNE groups, respectively. They are related to protein synthesis and energy metabolism and interact with human proteins typically found in saliva and supramolecular complexes of the acquired pellicle. CONCLUSIONS Our data indicate that the stimulation of the salivary flow increases acid resistant and antimicrobial proteins in saliva, which might protect against ETW. CLINICAL SIGNIFICANCE This pioneer study showed important differences in the human and bacterial proteome of SS in patients with GERD with or without ETW