Liquid Chromatography-Tandem Mass Spectrometry Method for Detection and Quantification of Meloxicam and 5'-Carboxymeloxicam in Oral Fluid Samples.

A sensitive, selective and particularly fast method of liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated for the determination of meloxicam and its main metabolite, 5'-carboxymeloxicam, in oral fluid samples. Meloxicam and its major metabolite were separated using a Shim-Pack XR-ODS 75 L × 2.0 column and C18 pre-column at 40 °C using a mixture of methanol and 10 mM ammonium acetate (80:20, v/v) with an injection flow rate of 0.3 mL/min. The total time of the analytical run was 5 min. Sixteen volunteers had oral fluid samples collected sequentially before and after taking a meloxicam tablet (15 mg) for up to 96 h. With the concentrations obtained, the pharmacokinetic parameters were determined using the Phoenix WinNonlin software. The parameters evaluated for meloxicam and 5'-carboxymeloxicam in the oral fluid samples showed linearity, accuracy, precision, medium-quality control (MQC-78.12 ng/mL), high-quality control (HQC-156.25 ng/mL), lower limits of quantification (LLOQ-0.6103 ng/mL), low-quality control (LQC-2.44 ng/mL), stability and dilution. Prostaglandin E2 (PGE2) was also detected and quantified in the oral fluid samples, demonstrating the possibility of a pharmacokinetic/pharmacodynamic (PK/PD) study with this methodology. All the parameters evaluated in the validation of the methodology in the oral fluid samples proved to be stable and within the possible variations in each of the described parameters. Through the data presented, the possibility of a PK/PD study was demonstrated, detecting and quantifying meloxicam, its main metabolite and PGE2 in oral fluid samples using LC-MS/MS.

Analysis of Plasma Proteins Involved in Inflammation, Immune Response/Complement System, and Blood Coagulation upon Admission of COVID-19 Patients to Hospital May Help to Predict the Prognosis of the Disease.

The development of new approaches allowing for the early assessment of COVID-19 cases that are likely to become critical and the discovery of new therapeutic targets are urgently required. In this prospective cohort study, we performed proteomic and laboratory profiling of plasma from 163 COVID-19 patients admitted to Bauru State Hospital (Brazil) between 4 May 2020 and 4 July 2020. Plasma samples were collected upon admission for routine laboratory analyses and shotgun quantitative label-free proteomics. Based on the course of the disease, the patients were divided into three groups: (a) mild (n = 76) and (b) severe (n = 56) symptoms, whose patients were discharged without or with admission to an intensive care unit (ICU), respectively, and (c) critical (n = 31), a group consisting of patients who died after admission to an ICU. Based on our data, potential therapies for COVID-19 should target proteins involved in inflammation, the immune response and complement system, and blood coagulation. Other proteins that could potentially be employed in therapies against COVID-19 but that so far have not been associated with the disease are CD5L, VDBP, A1BG, C4BPA, PGLYRP2, SERPINC1, and APOH. Targeting these proteins' pathways might constitute potential new therapies or biomarkers of prognosis of the disease.

Influence of genetic polymorphisms on mechanical pain sensitivity and endogenous pain modulation of trigeminal and spinal areas.

BACKGROUND: Previous evidence indicates significant association between genetic polymorphisms and phenotypes related to pain sensitivity in patients with temporomandibular disorders (TMD). Despite the important advances in cataloguing diverse factors such as sleep disorders, anxiety and depression, the interrelated mechanisms of painful TMD aetiopathogenesis still need investigation. OBJECTIVES: This case-control study aimed to evaluate the influence of genetic polymorphisms (rs6296, rs6295, rs1799971, rs4680, rs4633, rs4818) and psychosocial factors on the mechanical pain sensitivity and endogenous pain modulation in women with painful TMD and asymptomatic controls. METHODS: We evaluated six independent variables: anxiety levels, depression, stress, sleep quality, pain catastrophising and genetic polymorphisms, and four dependent variables: mechanical pain threshold (MPT), pressure pain threshold (PPT), wind-up ratio (WUR) and conditioned pain modulation (CPM) collected at masseter (trigeminal) and hand (spinal) areas in a sample of 95 painful TMD patients and 85 controls. A regression model was used to test the possible effect of the independent variables on dependent variables. RESULTS: The regression model was significant for MPT (F11,168  = 9.772; R2  = .390). Painful TMD diagnoses and sleep quality were associated with trigeminal MPT (B coefficient = -.499; and B coefficient = -.211, respectively). WUR was associated with rs6295 and rs6746030, respectively, for the spinal and the trigeminal area. CONCLUSION: Genetic polymorphisms had a slight contribution to endogenous pain modulation as indicated by the significant association with WUR but did not contribute to mechanical pain sensitivity. On the other hand, the presence of painful TMD and the sleep quality contributed significantly to mechanical pain sensitivity.

Effect of 10% Proanthocyanidin gel on demineralized organic matrix degradation: ELISA method / Efeito do gel de Proantocianidina a 10% na degradação da matriz orgânica desmineralizada: método ELISA

Objective: This study evaluated Proanthocyanidin protective effect on dentin subjected to erosion and its inhibition on degradation of the demineralized organic matrix (DOM). Material and Methods: The tested groups were: G1 - 10% Proanthocyanidin gel (test group), G2 - 1.23% NaF (positive control 1), G3 - 0.012% Chlorhexidine (positive control 2) and G4 ­ Placebo (negative control with no active compound) and two methodologies were performed: contact profilometry and ICTP ELISA method. To quantify dentin wear, profilometry was performed. Data were submitted to Analysis of Variance followed by Fisher's LSD Test. To assess the collagen degradation, ICTP ELISA method was performed. Data were submitted to the Kruskal-Wallis followed by the Dunn ́s test. Simple linear regression and Pearson Correlation test were also performed (p<0.05). Results: The profilometry showed significantly lower wear of G1 when compared to other groups and G2, G3 and G4, which did not present significant difference among them. In the ICTP ELISA analysis, G1 and G4 did not show significant differences and the same happened between G2 and G3. However, G1 and G4 had lower values of collagen degradation compared to groups G2 and G3. Data showed that degraded DOM is a significant predictor to explain the values obtained through the ICTP ELISA. Conclusions: The results allow to verify that 10% proanthocyanidin provided less tooth wear and decreased degradation of the DOM, suggesting a good ability to prevent dentin erosion. The regression analysis also suggests that contact profilometry is a good strategy to quantify dentin wear (AU)

Objetivo: Este estudo avaliou o efeito protetor da proantocianidina na dentina submetida à erosão e sua inibição na degradação da matriz orgânica desmineralizada (MOD). Material e Métodos: Os grupos testados foram: G1 - gel de Proantocianidina 10% (grupo teste), G2 - NaF 1,23% (controle positivo 1), G3 - Clorexidina 0,012% (controle positivo 2) e G4 - Placebo (controle negativo sem princípio ativo) e duas metodologias foram realizadas: perfilometria de contato e método ICTP ELISA. Para quantificar o desgaste da dentina, a perfilometria foi realizada. Os dados foram submetidos à Análise de Variância seguida do Teste LSD de Fisher. Para avaliar a degradação do colágeno, foi realizado o método ICTP ELISA. Resultados: Os dados foram submetidos ao teste de Kruskal-Wallis seguido do teste de Dunn. Regressão linear simples e teste de correlação de Pearson também foram realizados (p<0,05). A perfilometria mostrou desgaste significativamente menor do G1 quando comparado aos outros grupos e G2, G3 e G4, que não apresentaram diferença significativa entre si. Na análise ICTP ELISA, G1 e G4 não apresentaram diferenças significativas e o mesmo ocorreu entre G2 e G3. No entanto, G1 e G4 apresentaram valores menores de degradação do colágeno em relação aos grupos G2 e G3. Os dados mostraram que a MOD degradada é um preditor significativo para explicar os valores obtidos pelo ICTP ELISA. Conclusão: Os resultados permitem verificar que a proantocianidina a 10% proporcionou menor desgaste dentário e diminuição da degradação da MOD, sugerindo uma boa capacidade de prevenir a erosão dentinária. Também sugere que a perfilometria de contato é uma boa estratégia para quantificar o desgaste da dentina (AU)

Genetic recurrence and molecular markers of dyslexia in the Brazilian population

ABSTRACT Purpose: to investigate genetic recurrence and molecular markers for dyslexia in two candidate genes in the Brazilian population. Methods: a cross-sectional, case-control, observational study, with five single nucleotide polymorphisms (SNPs) studied in DYX1C1 and KIAA0319 genes in 86 subjects with dyslexia and 66 controls, matched for gender and age. SNPs were genotyped using the polymerase chain reaction technique in real time, and distribution of genotypic and allelic frequencies between the groups was analyzed. Results: it was determined that 68% of the subjects with dyslexia present a family history of learning difficulties. The DYX1C1 gene did not demonstrate an association with dyslexia, which was found regarding the rs9461045 marker of the KIAA0319 gene. Conclusion: a family history of learning problems was present in more than two-thirds of the group with dyslexia, indicating that this is an important risk factor. An association with dyslexia in the rs9461045 marker was noted, making the study the first one to show an association of the KIAA0319 gene with dyslexia, in Latin America.

Simultaneous separation of naproxen and 6-O-desmethylnaproxen metabolite in saliva samples by liquid chromatography-tandem mass spectrometry: Pharmacokinetic study of naproxen alone and associated with esomeprazol-Results.

After performing liquid-liquid extraction with ethyl acetate and HCl, samples from 12 volunteers who performed sequential collections after taking a tablet of naproxen alone (n = 6) or associated with esomeprazole (n = 6) were analyzed in a triple quadrupole mass spectrometer 8040 LC MS/MS Shimadzu. Separation of naproxen and its main metabolite 6-O-desmethylnaproxen was performed in a Shim-Pack XR-ODS 75Lx2.0 column and C18 pre-column at 40°C using a mixture of methanol and ammonium acetate 10 mM (70:30, v/v) with an injection rate of 0.3 ml/min. The total analytical run time for each sample was 5 min. The association of naproxen with esomeprazole take considerably longer time to reach the maximum concentration [Tmax 0.17 h (interquartile range, 0.13-1.95) for naproxen alone and 13.18*h (interquartile range, 10.12-27.15) for naproxen with esomeprazole, p = 0.002], also to be eliminated [T1/2 0.12 h (interquartile range, 0.09-1.35) for naproxen alone and 9.16*h (interquartile range, 7.16-41.40) for naproxen with esomeprazole, p = 0.002] and lower maximum concentrations (Cmax 4.6 ± 2.5 ug/mL for naproxen alone and 2.04 ± 0.78* µg/mL, p = 0.038). The association of naproxen with esomeprazole showed increased values of AUC0-t [82.06* h*µg/mL (interquartile range, 51.90-157.00) with esomeprazole and 2.97 h*µg/mL (interquartile range, 1.82-7.84) naproxen alone, p = 0.002] in drug concentrations in relation to the naproxen tablet alone, probably, such differences are due to the delay in the absorption of naproxen when it is associated with the drug proton pump inhibitor, esomeprazole. As well as reduced values of full clearance when naproxen is combined with esomeprazole (0.07* µg/h (interquartile range, 0.005-0.01) with esomeprazole and 7.29 µg/h (interquartile range, 3.17-16.23) in naproxen alone, p = 0.002). Both naproxen and 6-O-desmethylnaproxen in saliva samples can be effectively quantified using LC-MS/MS, this methodology proved to be rapid, sensitive, accurate and selective for each drug and allows for the analysis of their pharmacokinetic parameters, in both situations.

CYP2C9 Polymorphism Influence in PK/PD Model of Naproxen and 6-O-Desmethylnaproxen in Oral Fluid.

Polymorphisms in CYP2C9 can significantly interfere with the pharmacokinetic (PK) and pharmacodynamic (PD) parameters of nonsteroidal anti-inflammatory drugs (NSAIDs), including naproxen. The present research aimed to study the PK/PD parameters of naproxen and its metabolite, 6-O-desmethylnaproxen, associated with allelic variations of CYP2C9. In our study, a rapid, selective, and sensitive Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) method was developed and validated for the determination of naproxen and its main metabolite, 6-O-desmethylnaproxen, in oral fluid. Naproxen and its main metabolite were separated using a Shim-Pack XR-ODS 75L × 2.0 column and C18 pre-column at 40 °C using a mixture of methanol and 10 mM ammonium acetate (70:30, v/v), with an injection flow of 0.3 mL/min. The total analytical run time was 3 min. The volunteers, previously genotyped for CYP2C9 (16 ancestral­CYP2C9 *1 and 12 with the presence of polymorphism­CYP2C9 *2 or *3), had their oral fluids collected sequentially before and after taking a naproxen tablet (500 mg) at the following times: 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6 8, 11, 24, 48, 72 and 96 h. Significant differences in the PK parameters (* p < 0.05) of naproxen in the oral fluid were: Vd/F (L): 98.86 (55.58−322.07) and 380.22 (261.84−1097.99); Kel (1/h): 0.84 (0.69−1.34) and 1.86 (1.09−4.06), in ancestral and mutated CYP2C9 *2 and/or *3, respectively. For 6-O-desmethylnaproxen, no PK parameters were significantly different between groups. The analysis of prostaglandin E2 (PGE2) proved to be effective and sensitive for PD parameters analysis and showed higher levels in the mutated group (p < 0.05). Both naproxen and its main metabolite, 6-O-desmethylnaproxen, and PGE2 in oral fluid can be effectively quantified using LC-MS/MS after a 500 mg oral dose of naproxen. Our method proved to be effective and sensitive to determine the lower limit of quantification of naproxen and its metabolite, 6-O-desmethylnaproxen, in oral fluid (2.4 ng/mL). All validation data, such as accuracy, precision, and repeatability intra- and inter-assay, were less than 15%. Allelic variations of CYP2C9 may be considered relevant in the PK of naproxen and its main metabolite, 6-O-desmethylnaproxen.

Detection and quantification of prostaglandin E2 in saliva by liquid chromatography-tandem mass spectrometry using microextraction by packed sorbent.

The detection of eicosanoids in saliva samples can assist pharmacokinetic/pharmacodynamic studies due to the facility of obtaining samples, minimal discomfort and high adherence of volunteers to the study. The present study enabled determine prostaglandin E2 concentrations in saliva samples, using a microextraction by packed sorbent methodology and subsequent detection in liquid chromatography-tandem mass spectrometry. Twelve volunteers underwent scaling and coronary-radicular polishing of the upper molars and sequential saliva collections: 0.25-96 h after ingestion of a 600 mg ibuprofen tablet, to quantify prostaglandin E2 concentrations. There was an increase in the level of prostaglandin E2 with a significant difference after the dental procedure (0.25 h) compared to 11, 24, 48 and 72 h (*p < 0.05). After taking the drug, these levels begin to decrease up to 5 h, returning to normal in the subsequent hours. The method was developed and validated with linearity between 2.4 and 1250 ng/mL and r2 above 0.9932. The limit of quantitation was about 2.4 ng/mL. The coefficients of variation and the relative standard errors of the accuracy and precision analyzes were < 15%. The proposed extraction and analysis methodology proved to be efficient, fast and promising for pharmacokinetic/pharmacodynamic assays after using anti-inflammatory drugs.

An international, interlaboratory ring trial confirms the feasibility of an extraction-less "direct" RT-qPCR method for reliable detection of SARS-CoV-2 RNA in clinical samples.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is used worldwide to test and trace the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). "Extraction-less" or "direct" real time-reverse transcription polymerase chain reaction (RT-PCR) is a transparent and accessible qualitative method for SARS-CoV-2 detection from nasopharyngeal or oral pharyngeal samples with the potential to generate actionable data more quickly, at a lower cost, and with fewer experimental resources than full RT-qPCR. This study engaged 10 global testing sites, including laboratories currently experiencing testing limitations due to reagent or equipment shortages, in an international interlaboratory ring trial. Participating laboratories were provided a common protocol, common reagents, aliquots of identical pooled clinical samples, and purified nucleic acids and used their existing in-house equipment. We observed 100% concordance across laboratories in the correct identification of all positive and negative samples, with highly similar cycle threshold values. The test also performed well when applied to locally collected patient nasopharyngeal samples, provided the viral transport media did not contain charcoal or guanidine, both of which appeared to potently inhibit the RT-PCR reaction. Our results suggest that direct RT-PCR assay methods can be clearly translated across sites utilizing readily available equipment and expertise and are thus a feasible option for more efficient COVID-19 coronavirus disease testing as demanded by the continuing pandemic.

Ganoderma lucidum polysaccharides inhibit in vitro tumorigenesis, cancer stem cell properties and epithelial-mesenchymal transition in oral squamous cell carcinoma.

ETHNOPHARMACOLOGICAL RELEVANCE: The polysaccharides of the millenary mushroom Ganoderma lucidum (GL) have been shown for decades to present anti-tumor activities, but few studies evaluated its importance on cancer stem cells and EMT process. Cancer stem cells (CSC) drive the development of carcinoma and are also involved in cancer treatment failure, being a good target for treatment success. Also, the process of epithelial-mesenchymal transition (EMT) is involved in metastasis and cancer relapse. Besides that, the increasing incidence worldwide of oral squamous cell carcinoma (OSCC) became a public health issue with a high rate of metastasis and poor quality of life for patients during and after treatment. AIM OF THE STUDY: to evaluate G. lucidum polysaccharides (GLPS) in vitro effects on OSCC, focusing on hallmarks associated with tumorigenesis using the SCC-9, a squamous cells carcinoma lineage from the tongue. MATERIALS AND METHODS: SCC-9 cells were treated in vitro for 72h with different GLPS concentrations. The controls cells were maintained with culture media only and cisplatin was used as treatment control. After the treatment period, the cells were evaluated. RESULTS: GLPS treatment changed cell morphology and granularity, delayed migration, decreased colony, and impaired sphere formation, thereby leading to a non-invasive and less proliferative behavior of tumoral cells. Additionally, GLPS downregulated CSC, EMT, and drug sensitivity (ABC) markers. CONCLUSIONS: These results show that the natural product GLPS has the potential to be an important ally for tongue squamous cell carcinoma treatment, bringing the millenary compound to modern therapy, providing a basis for future studies and the improvement of life quality for OSCC patients.

Profile of host cell responses to exposure to stressed bacteria in planktonic; dislodged, and intact biofilm mode.

The host defense response to microbial challenge emerging from the root canal system leads to apical periodontitis. The aim of this study was to evaluate the expression of inflammatory cytokines and Nitric Oxide (NO) by macrophages after interaction with Enterococcus faecalis in the: plankton and dislodged biofilm mode; intact biofilm mode stimulated by calcium hydroxide (CH), CH and chlorhexidine (CHX) or Triple Antibiotic Paste (TAP). For this purpose, culture of macrophages from monocytes in human peripheral blood (N=8) were exposed to the different modes of bacteria for 24 hours. Subsequently, the cytokines, such as, Tumor Necrotic Factor- alfa (TNF-α), interleukin (IL)-1ß, IL-6, IL-10; and NO were quantified by Luminex xMAP and Greiss reaction, respectively. In addition to the potential therapeutic effects of the intracanal medication, their antimicrobial activity against Enterococcus faecalis biofilm were also tested in vitro by confocal microscopy. The experiments` data were analyzed by the Kruskal-Wallis test with the Dunn post hoc test (α < 0.05). Bacteria in dislodged biofilm mode were shown to be more aggressive to the immune system than bacteria in plankton mode and negative control, inducing greater expression of NO and TNF-α. Relative to bacteria in intact biofilm mode, the weakest antimicrobial activity occurred in Group CH. In Groups CH/CHX and TAP the percentage of dead bacteria was significantly increased to the same extent. Interestingly, the biofilm itself did not induce the release of pro-inflammatory cytokines - except for NO - while the biofilm treated with TAP and CH based pastes enhanced the levels of IL-6 and TNF-α; and IL-1 ß, respectively. In contrast, the levels of a potent anti-inflammatory (IL-10) were increased in Group TAP.

Association between endothelial nitric oxide synthase and the renin-angiotensin-aldosterone system polymorphisms, blood pressure and training status in normotensive/pre-hypertension and hypertensive older adults: a pilot study.

Introduction:Variations in blood pressure (BP) are, in part, genetically determined and some polymorphisms of renin-angiotensin- aldosterone system (RAAS) and synthase of endothelial nitric oxide (eNOS) have been related to hypertension (HT). Conversely, physical exercise is considered a non-pharmacological tool for HT control, treatment, and prevention.Objective: The purpose of this study is to investigate the relationship between eNOS and RAAS polymorphisms, their epistatic interaction, and the respective humoral factors in the BP control in normotensive/pre-hypertension and hypertensive older adults and how this relationship can be modulated by training status (TS) level.Methods:A total of 155 older adults (66.94 ± 6.83 years old) performed the following evaluations: AAHPERD battery test to determine the general functional fitness index (GFFI), systolic and diastolic blood pressure (SBP and DBP), blood collection for DNA extraction, analysis of eNOS gene polymorphisms rs2070744; rs61722009 and rs1799983 and RAAS polymorphisms rs699; rs1799752 and rs5186, and quantification of ACE activity (Fluorimetric Assay) and nitrite concentration (Chemiluminescence Method).Results and Conclusion:Good TS level appears to exert greater influence on SBP for G2 and G3 (G1: 125.79 ± 14.03/ G2: 119.91 ± 11.72/G3: 119.71 ± 10.85) and on NO2 for G3 (G1: 0.42 ± 0.25/ G2: 0.54 ± 0.45/ G3: 0.71 ± 0.52). No associations were observed between eNOS and RAAS polymorphisms, but the epistasis was identified between eNOS polymorphism, rs2070744, and RAAS polymorphism, rs699, revealing a statistically significant interaction (p = .0235) with training score of 0.63, a training test accuracy of 0.61 and a cross-validation consistency of 10/10. This result suggests an increased risk of hypertension.

Profile of host cell responses to exposure to stressed bacteria in planktonic; dislodged, and intact biofilm mode

Abstract The host defense response to microbial challenge emerging from the root canal system leads to apical periodontitis. The aim of this study was to evaluate the expression of inflammatory cytokines and Nitric Oxide (NO) by macrophages after interaction with Enterococcus faecalis in the: plankton and dislodged biofilm mode; intact biofilm mode stimulated by calcium hydroxide (CH), CH and chlorhexidine (CHX) or Triple Antibiotic Paste (TAP). For this purpose, culture of macrophages from monocytes in human peripheral blood (N=8) were exposed to the different modes of bacteria for 24 hours. Subsequently, the cytokines, such as, Tumor Necrotic Factor- alfa (TNF-α), interleukin (IL)-1β, IL-6, IL-10; and NO were quantified by Luminex xMAP and Greiss reaction, respectively. In addition to the potential therapeutic effects of the intracanal medication, their antimicrobial activity against Enterococcus faecalis biofilm were also tested in vitro by confocal microscopy. The experiments` data were analyzed by the Kruskal-Wallis test with the Dunn post hoc test (α < 0.05). Bacteria in dislodged biofilm mode were shown to be more aggressive to the immune system than bacteria in plankton mode and negative control, inducing greater expression of NO and TNF-α. Relative to bacteria in intact biofilm mode, the weakest antimicrobial activity occurred in Group CH. In Groups CH/CHX and TAP the percentage of dead bacteria was significantly increased to the same extent. Interestingly, the biofilm itself did not induce the release of pro-inflammatory cytokines - except for NO - while the biofilm treated with TAP and CH based pastes enhanced the levels of IL-6 and TNF-α; and IL-1 β, respectively. In contrast, the levels of a potent anti-inflammatory (IL-10) were increased in Group TAP.

Resumo A resposta de defesa do hospedeiro ao desafio microbiano que emerge do sistema de canais radiculares leva à periodontite apical. Os objetivos deste estudo foram avaliar a expressão de citocinas pró e anti-inflamatórias e Óxido Nítrico (NO) por macrófagos após interação com Enterococcus faecalis no modo: planctônio e de biofilme desalojado; biofilme intacto estimulado por hidróxido de cálcio (CH), CH e clorexidina ou Pasta Tri Antibiótica (TAP). Para isto, a cultura de macrófagos originados de monócitos do sangue periférico de humanos (N=8) foi exposta aos diferentes tipos de bactéria por 24 horas. Então, a quantificação da produção de of Fator de Necrose Tumoral- alfa (TNF-α), interleucina (IL)-1β, IL-6, IL-10 e NO por macrófagos se deu por meio do Luminex xMAP e reação de Greiss, respectivamente. Além dos potenciais efeitos terapêuticos desses compostos, sua atividade antimicrobiana contra E. faecalis também foi testada através microscopia confocal. Os dados dos experimentos foram analisados através do teste de Kruskal-Wallis com Dunn`s post hoc (α < 0.05). Bactéria em modo de biofilme desalojado se mostrou mais agressivo ao sistema imune que as bactérias planctônicas e controle negativo induzindo a maior excreção de NO e TNF-α. Em relação ao biofilme intacto, a atividade antimicrobiana mais fraca ocorreu no grupo de CH. Os grupos CHX e TAP aumentaram significativamente a porcentagem de bactérias mortas na mesma extensão. Interessantemente, o biofilme por ele mesmo não induziu a liberação de citocinas pro-inflamatórias - exceto por NO - enquanto que o biofilme tratado com TAP ou pastas a base de CH aumentaram os níveis de IL-6; e TNF-α e IL-1 β respectivamente. Em contraste, os níveis da potente citocina anti-inflamatória (IL-10) foram aumentados pelo grupo TAP.

Multifocal Analysis of Acute Pain After Third Molar Removal.

Background: To analyze the pain modulation capacity profile in a Brazilian population, the relationship between opioid receptor (OPRM1) and Catechol-O-methyltransferase (COMT) 1polymorphisms and pain modulation capacity was determined through preoperative pain modulation tests and acute postoperative pain control evaluation, swelling, and trismus in 200 volunteers undergoing lower third molar removal. Methods: Psychologic and clinical parameters were measured. Patient DNA was sequenced for single nucleotide polymorphisms in OPRM1 and COMT, and the salivary concentration of interleukin (IL)-2 (IL)-6, interferon (IFN)-γ and tumor necrosis factor (TNF)-α was evaluated. Primary outcomes were the influence of all predictors on the fluctuation of pain intensity using a visual analogue scale (VAS), and swelling and trismus on the 2nd and 7th postoperative days. Preoperative pain modulation capacity (CPM), pain catastrophizing scale (PCS), body mass index (BMI), and surgery duration and difficulty were evaluated. Results: Salivary concentration of IFN-γ and IL-2 as well as the duration of surgery influenced the fluctuation of postoperative pain in the VAS, and in the sum of the differences in pain intensity test at 8, 48, and 96 h. BMI influenced swelling, while both BMI and COMT haplotype influenced trismus on the 2nd postoperative day. Conclusion: Polymorphisms in COMT, salivary concentrations of IL-2 and IFN-γ, BMI, and duration of surgery were predictors for pain fluctuation, swelling, and trismus on the 2nd day after lower third molar extraction. This therapy was effective in controlling inflammatory symptomatology after lower third molar extraction and ibuprofen was well tolerated by patients. Clinical Trial Registration: www.ClinicalTrials.gov, identifier NCT03169127.

Could the photobiomodulation therapy induce angiogenic growth factors expression from dental pulp cells?

This study aimed to evaluate the effect of different photobiomodulation (PBM) radiant exposures on the viability, proliferation, and gene expression of pulp fibroblasts from human primary teeth (HPF) involved in the pulp tissue repair. HPF were irradiated with Laser InGaAlP (Twin Flex Evolution, MMOptics®) at 660-nm wavelength (red); single time, continuous mode, 0.04-cm2 laser tip area, and 0.225-cm laser tip diameter, keeping the distance of 1 mm between the laser beam and the cell culture. The doses used were between 1.2 and 6.2 J/cm2 and were evaluated at the 6 h, 12 h, and 24 h after PBM. MTT and crystal violet assays evaluated the cell viability and proliferation. RT-PCR verified VEGF and FGF-2 mRNA expression. A blinded examiner analyzed the data through two-way ANOVA followed by Tukey test (p < 0.05). The groups with higher powers (10 mW, 15 mW, 20 mW, and 25 mW), shortest application periods (10 s), and radiant exposures between 2.5 and 6.2 J/cm2 exhibited statistically higher viability than that of the groups with small power (5 mW), longer application period (50 s), and radiant exposure of 6.2 J/cm2 (p < 0.05). VEGF and FGF-2 mRNA expression were observed at the three evaluated periods (6 h, 12 h, and 24 h) and the highest expression was in the shortest period (p < 0.05). All radiant exposures maintained HPF viable. The period of 6 h after irradiation showed statistically greater gene expression for both growth factors than other periods. VEGF mRNA had no differences among the dosimetries studied. The best radiant exposures for FGF-2 gene expression were 2.5 J/cm2 and 3.7 J/cm2.

Pharmacogenetic and Pharmacokinetic Assays from Saliva Samples Can Guarantee Personalized Drug Prescription.

Saliva is widely used for clinical and laboratory analysis. This study proposed to use DNA extracted from saliva for genotyping and pharmacokinetics of piroxicam. A fast and efficient genotyping method was used to determine relevant allelic variants of CYP2C9 (*2 and *3), since genetic factors can influence in non-steroidal anti-inflammatory drugs (NSAIDs) metabolization. DNA Extract All Reagents Kit® was used for DNA extraction and genotyping was performed using TaqMan® GTXpress™ Master Mix, SNP genotyping assays and a Viia7 Real-Time PCR system. Volunteers performed sequential collections of saliva samples before and after taking a single dose of piroxicam (0.25 to 72 h) which were used for pharmacokinetics assays. Piroxicam concentrations were analyzed using LC-MS/MS. Sixty-six percent of volunteers were ancestral homozygous (CYP2C9*1/*1), and 34% showed one or both polymorphisms. Of these 34%, 22 individuals showed CYP2C9*2 polymorphism, 8 CYP2C9*3, and 4 CYP2C9*2/*3. Piroxicam pharmacokinetics were performed in 5 subjects. Areas under the curve (AUC0-t(h*ng/mL)) for CYP2C9*1/*1, *1/*2 and *1/*3 were, respectively, 194.33±70.93, 166 and 303. Maximum concentrations (Cmax(ng/mL)) for these genotypes were respectively 6.46±2.56, 4.3 and 10.2. Saliva sampling was a very effective matrix for both pharmacogenetic and pharmacokinetic tests, ensuring the speed of the procedure and the well-being and agreement of the participants. Once having the knowledge about the slow and fast metabolizers, it is possible to make an adequate prescription in order to avoid the adverse effects of the medication and to guarantee greater analgesic comfort to the patients respectively.

Acute Exercise, Plasma Nitric Oxide, and Blood Pressure in Older Adults With Different Levels of Training Status: The Influence of Polymorphisms of Endothelial Nitric Oxide Synthase.

BACKGROUND: This study aimed to analyze the acute effect of physical exercise on nitric oxide concentration and blood pressure (BP) in older adults with different levels of training status (TS) and verified the influence of endothelial nitric oxide synthase polymorphisms on these variables. METHODS: A total of 145 older adults were divided into good TS (G1) and weak TS (G2). Participants were subjected to a 40-minute treadmill walk (40%-60% of maximum oxygen consumption) with BP measurements and blood collections for plasma nitrite and oxidative stress biomarkers at pretest and posttest moments. Data were analyzed by 2-way repeated-measures with Sidak post hoc test (P < .05) and multivariate linear analysis. RESULTS: After acute exercise, G2 showed an increase in oxidative stress biomarkers (P = .008), and both groups showed an increase in systolic BP (P < .001). Polymorphisms 894G > T and intron 4b/a had no association with nitrite and BP. However, -786T > C polymorphism showed an association with reduced systolic and diastolic BP (TT genotype) and increased diastolic BP (TC genotype). Higher TS level was also associated with lower BP. CONCLUSION: The maintenance of good TS levels may have a protective effect on cardiovascular risks regardless of the genetic profile.

In vitro effect of curcumin-mediated antimicrobial photodynamic therapy on fibroblasts: viability and cell signaling for apoptosis.

Although it was demonstrated that curcumin-mediated antimicrobial photodynamic therapy (aPDT) is effective for reducing the viability of microbial cells and the vitality of oral biofilms, the cytotoxicity of this therapeutic approach for host cells has not been yet elucidated. Hence, the aim of this study was to evaluate the cytotoxicity and apoptotic effects of curcumin-mediated aPDT on mouse fibroblasts. Cells were treated with 0.6 or 6 µmol.L-1 curcumin combined with 0.075 or 7.5 J.cm-2 LED at 455 nm. Cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and crystal violet (CV) assays, while quantitative reverse transcriptase-PCR (qRT-PCR) was used to assess the expression of Bax, Bad, Bcl-2, VDAC-1, cytochrome C, and Fas-L genes for apoptosis. The differences between groups were detected by Kruskal-Wallis and post hoc Dunn's tests for MTT and CV assays and by ANOVA and post hoc Tukey test for qRT-PCR (P < 0.05). The effect of 0.6 µmol.L-1 curcumin plus 0.075 J.cm-2 LED (minimum parameter) did not differ statistically from control group; however, the combination of 0.6 µmol.L-1 curcumin plus 7.5 J.cm-2 LED reduced viable cells in 34%, while the combinations of 6 µmol.L-1 curcumin plus 0.075 and 7.5 J.cm-2 LED reduced viable cells in 47% and 99%, respectively. aPDT increased significantly the relative expression of Bax/Bcl-2, cytochrome C, VDAC-1, and Fas-L genes, without influence on the ratio Bad/Bcl-2. Therefore, curcumin-mediated aPDT activated Bcl-2 apoptosis signaling pathways in mouse fibroblasts regarding present conditions, reducing the viability of cells with the increase of curcumin concentrations and light energies.

Simultaneous separation of naproxen and 6-O-desmethylnaproxen metabolite in saliva samples by liquid chromatography-tandem mass spectrometry: Pharmacokinetic study of naproxen alone and associated with esomeprazole.

Naproxen is a widely used non-steroidal anti-inflammatory drug for the control of postoperative inflammatory signs and symptoms in dentistry. Its association with esomeprazole has been widely studied and has yielded good results for the control of acute pain, even with the delayed absorption of naproxen owing to the presence of esomeprazole. To further understand the absorption, distribution, and metabolism of this drug alone and in combination with esomeprazole, we will analyze the pharmacokinetic parameters of naproxen and its major metabolite, 6-O-desmethylnaproxen, in saliva samples. A rapid, sensitive, and selective liquid chromatography-tandem mass spectrometric method for the simultaneous determination of naproxen and 6-O-desmethylnaproxen in saliva will be developed and validated. Sequential saliva samples from six patients will be analyzed before and 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6 8, 11, 24, 48, 72, and 96 h after the ingestion of one naproxen tablet (500 mg) and esomeprazole-associated naproxen tablets (500 + 20 mg), at two different times. After liquid-liquid extraction with ethyl acetate and HCl, the samples will be analyzed using an 8040 Triple Quadrupole Mass Spectrometer (Shimadzu, Kyoto, Japan). Separation of naproxen and its major metabolic products will be performed using a Shim-Pack XR-ODS 75Lx2.0 column and C18 pre-column (Shimadzu, Kyoto, Japan) at 40°C using a mixture of methanol and 10 mM ammonium acetate (70:30, v/v) with an injection flow of 0.3 mL/min. The total analytical run time will be 5 min. The detection and quantification of naproxen and its metabolite will be validated, which elucidate the pharmacokinetics of this drug, thereby contributing to its proper prescription for the medical and dental interventions that cause acute pain.

Molecular Response of Pulp Fibroblasts after Stimulation with Pulp Capping Materials.

This in vitro study evaluated cell viability and metabolism, nitric oxide release and production of two chemokines and one cytokine by cultured human dental pulp fibroblasts (HDPF) in contact with two glass ionomer cements (Ketac Molar-KM and Vitrebond-VB), Single Bond (SB) and calcium hydroxide (Dycal-DY). Cultures of HDPF were established by means of an explant technique. The specimens were prepared under sterile conditions and in disks measuring 5 mm x 2 mm obtained from a prefabricated mold and placed on a permeable membrane to avoid direct contact with the cells. Cytotoxicity was assessed by Trypan Blue exclusion method and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Nitric oxide release in cell supernatant was detected by the Griess Method whereas stromal derived factor-1 alpha (SDF-1α or CXCL12), chemokine (C-X-C motif) ligand 8 [Interleukin 8 (IL-8 or CXCL8)] and interleukin-6 (IL-6) were detected by ELISA. RT-qPCR was employed for gene expression analysis. Statistical analyses were performed by One-way ANOVA followed by Tukey's post hoc test for materials independent of the time, and Two-way ANOVA followed by Bonferroni correction test for the comparisons between materials and experimental time (p<0.05). Cytotoxic tests showed significant differences only for DY. Protein levels and mRNA expression were significantly increased for IL-8 for both periods of time. IL-6 production increased when fibroblasts were stimulated by KM. SDF-1α protein production and mRNA expression were not affected by any of the materials. There was a decrease in nitrate/nitrite levels only for KM. Although DY caused intense cell death and did not stimulate the production of the inflammatory mediators evaluated in this work, it is known that this event seems to be fundamental for the process of repair of the pulp tissue and formation of mineralized barrier. KM and VB increased production of proteins related to the inflammatory process, thus favoring tissue repair. Therefore, although these glass ionomer cements did not lead to large cell death, they should be used with caution.