Post-mortem Interval and Its Relation with the RNA Degradation in the Dental Pulp in Submerged Teeth

Post-mortem interval is the time between death and the discovery of the body or human remains. Teeth are the most resistant structures of the human body, able to withstand extreme conditions such as high temperature, humidity, and post-mortem degradation. The objective of this study was to evaluate the applicability of the method of quantifying degradation of RNA extracted from dental pulps to estimate the post-mortem interval, by simulating drowning conditions with teeth submerged in fresh water and exposed to different time intervals. The sample consisted of 80 human teeth (third molars), divided into eight groups, and placed in the aquatic environment, for pre-established periods of three days, and 1, 2, 3, 4, 8, 12 and 16 weeks respectively. After the stipulated time, the teeth were removed and the RNA was extracted form the dental pulp. Finally, the RNA was electrophoresed and its Integrity Number (RIN) was calculated for each RNA pulp sample. After the analysis, significant amount of dental pulp degradation was observed showing a RNA RIN of 6.50. The 18S/28S ribosomal RNA ratio was null (with a value of zero), and only in sample, it was extremely low (0.8). The fact that the samples were submitt to the environment associated with that the low proportion the18S/28S ribosomal RNA found in the samples, may be essential factors to justify the results obtained. RNA degradation quantification method was not applicable, since it was not possible to establish a relation between the degradation of the RNA molecule and the estimation of the post-mortem interval

Efficacy of naproxen with or without esomeprazole for pain and inflammation in patients after bilateral third molar extractions : a double blinded crossover study

Using a double-blinded randomized crossover design, this study aimed to evaluate acute postoperative pain management, swelling and trismus in 46 volunteers undergoing extractions of the two lower third molars, in similar positions, at two different appointments who consumed a tablet of either NE (naproxen 500 mg + esomepraz ole 20 mg) or only naproxen (500 mg) every 12 hours for 4 days. Parameters were analyzed: self-reported pain intensity using a visual analog scale (VAS) pre- and postoperative mouth opening; incidence, type and severity of adverse reactions; total quantity consumed of rescue medication; and pre- and postoperative swelling. Female volunteers reported significantly more postoperative pain at 1, 1.5, 2, 3 and 4hrs after surgery while also taking their first rescue medication at a time significantly earlier when consuming NE when compared to naproxen (3.7hrs and 6.7hrs). Conversely, no differences were found between each drug group in males. In conclusion, throughout the entire study, pain was mild after using either drug in both men and women with pain scores on average well below 40mm (VAS), although in women naproxen improved acute postoperative pain management when compared to NE

Palatal mucosa derived fibroblasts present an adaptive behavior regarding cytokine secretion when grafted onto the gingival margin

BACKGROUND: Considering that grafted gingival tissue might have to be adapted to the receptor area and that fibroblasts have the ability to respond to bacterial stimuli through the release of various cytokines, this study investigated whether fibroblasts from the palatal mucosa behave differently when grafted onto the gingival margin regarding cytokine secretion. METHODS: Biopsies from the palatal mucosa were collected at the time of free gingival graft surgery, and after four months re-collection was performed upon surgery for root coverage. Fibroblasts were isolated by the explant technique, cultured and stimulated with Porphyromonas gingivalis (Pg) and Escherichia coli (Ec) LPS for 24 or 48 h for comparative evaluation of the secretion of cytokines and chemokines, such as IL-6, IL-8/CXCL8, MIP-1α/CCL3, TGF-β, VEGF and CXCL16. Unstimulated cells were used as the control group. Cells were tested for viability through MTT assay, and secretion of cytokines and chemokines was evaluated in the cell supernatants by Enzyme-Linked Immunosorbent Assay (ELISA). RESULTS: Fibroblasts from the palatal mucosa maintained the same secretion pattern of IL-6 when grafted onto the gingival margin. On the contrary, fibroblasts from the marginal gingival graft showed increased secretion of IL-8/CXCL8 even in the absence of stimulation. Interestingly, MIP-1α/CCL3 secretion by fibroblasts from the marginal gingival graft was significantly increased after 48 hours of stimulation with Pg LPS and after 24 h with Ec LPS. Only fibroblasts from the marginal gingival graft showed secretion of TGF-β. VEGF and CXCL16 secretion were not detected by both subsets of fibroblasts. CONCLUSION: Fibroblasts from the palatal mucosa seem to be adapted to local conditions of the site microenvironment when grafted onto the gingival marginal area. This evidence supports the effective participation of fibroblasts in the homeostasis of the marginal periodontium through secretion modulation of important inflammatory mediators

Polimorfismos genéticos e desempenho físico em jogadores de futebol das categorias de base do São Paulo Futebol Clube

Literature reports that genetic polymorphisms may determine important modulations on athletes phenotypes, such as height, cardiovascular adaptations, use of energy substrates as well as electrolyte and hormonal balance. It is possible that individuals who express the alpha actinin 3 gene (ACTN3; ancestral homozygous RR or heterozygous RX) may offer advantages in movements that require strength and fast twitch compared with individuals with XX genotype. ACTN3 is a sarcomeric Z line component, which is important for the actin filaments anchorage and myofibrillar arrangement maintenance. Regarding AMP deaminase (AMPD1) polymorphism, it has been reported that athletes with the mutant allele (allele T) may present disadvantages in intense and repetitive physical activities, since the enzyme encoded by this gene is responsible for the ATP resynthesis after intense muscle contractions. Polymorphisms in the angiotensin converting enzyme gene (ACE; deletion allele D) and angiotensinogen (AGT; mutated allele T) may favor athletes in activities requiring strength, due to the fact of higher Angiotensin (Ang) II circulating levels. The present study investigated whether polymorphisms in ACTN3, AMPD1, ACE and AGT genes, alone or in combination, may influence the hemodynamic and cardiac parameters as well as soccer players performance during physical tests such as jump, speed and endurance. Saliva from 220 young professional soccer players (14-20 years) from São Paulo Futebol Clube (Brazil) was collected. Then, total DNA was extracted from saliva and polymerase chain reaction (PCR) was used for genotyping of athletes. To provide more reliability to the study, athletes were also separated according to their age. Before this separation, the athletes with the mutation in the ACTN3 gene jumped lower heights in Squat Jump test (SJ) (RR/RX = 33.64 ± 5.31 vs XX = 30.81 ± 4.51 cm, p = 0.007), as well as in the Under (U)-15 (RR/RX = 34.88 ± 5.39 vs XX = 30.59 ± 4.07 cm, p = 0.04) and U- 17 (RR/RX = 35.82 ± 4.35 vs XX = 30.24 ± 5.16 cm, p = 0.01) categories. In the Counter Movement Jump test (CMJ), RR/RX jumped 37.26 ± 5.72 cm and XX 34.12 ± 4.84 cm (p = 0.005). In the U-17 category, RR/RX jumped 38.56 ± 5.69 cm and XX 32.90 ± 6.06 cm (p = 0.02). In the Counter Movement Jump with arms (CMJb) test, with all athletes, RR/RX jumped 43.85 ± 6.38 cm and XX 40.61 ± 5.06 cm (p = 0.009). The speed test (30 m) showed in the U-17 category that RR/RX were faster than the XX athletes (RR/RX = 4.13 ± 0.13 vs XX = 4.27 ± 0 17 s, p = 0.04). Regarding AMPD1 gene, no significant difference was found in the jumps and endurance tests, but in the speed test (10 m), CC athletes were faster than those with CT/TT genotypes (CC = 1.53 ± 0.19 vs CT/TT = 1.62 ± 0.16 s, p = 0.04). Athletes with DD genotype (ACE) jumped significantly higher in CMJb test compared with ID/II (DD = 44.37 ± 6.22 vs ID/II = 42.35 ± 6.23 cm, p = 0.02). In the U-17 category, DD athletes jumped higher in SJ (DD = 38.04 ± 5.00 vs ID/II = 33.16 ± 4.11 cm, p = 0.01), CMJ (DD = 41.03 ± 5.64 vs ID/II = 35.76 ± 4.26 cm, p = 0.01) and CMJb (DD = 48.62 ± 5.98 vs ID/II = 42.42 ± 4.81 cm, p = 0.007). In the endurance test, athletes from U-16 category with genotypes ID/II, traveled greater distances compared with DD (ID/II = 1.467 ± 63.70 vs DD =1.244 ± 64.25 m, p = 0.04). The DD genotype also favored athletes in speed test (30 m), either for players from U-14 category (DD = 4.29 ± 0.19 vs ID/II = 4.40 ± 0.16 s, p = 0.02) or for the U- 17 category (DD = 4.07 ± 0.15 vs ID/II = 4.20 ± 0.13 s, p = 0.04). AGT gene polymorphism did not influence the performance in the tests, but athletes with the mutant genotype (TT) showed greater left ventricle (LV) hypertrophy (114.6 ± 105.2 g/m2 for TT, 92.16 ± 18.88 g/m2 for MT and 94.78 ± 21.08 g/m2 for MM, p = 0.04) without any change in cardiac and other hemodynamic parameters. Greater LV hypertrophy (DD = 96.95 ± 19.96, ID = 90.14 ± 21.58 and II = 91.67 ± 21.09 g/m2, p = 0.04) and higher ejection fraction (DD = 71.73 ± 7.71, ID = 69.48 ± 6.51 and II = 68.59 ± 5.72 %, p = 0.02) were also found in the athletes with the DD genotype. The analysis of genes combination on athletic performance, when characteristics of strength and muscle fast twitch in the ranking by score were taken into account, showed that athletes with the highest scores (5-8) jumped higher than those with lower scores (1-4) in SJ test (score 5 to 8 = 33.80 ± 5.16 vs score 1 to 4 = 31.60 ± 5.22 cm, p = 0.01) and CMJ test (score 5 to 8 = 43.90 ± 6.85 vs score 1 to 4 = 41.87 ± 5.98 cm, p = 0.04). The present results suggest that RR/RX (ACTN3), DD (ACE) and CC (AMPD1) genotypes may benefit soccer players in activities requiring strength and fast twitch. In addition, ID/II genotypes seem to provide more resistance to athletes in endurance activity. In the future, the organization, standardization and ethical responsibility will be required in the management of these genetic markers for use in athletes training process.Universidade Federal de Minas GeraisHá relatos na literatura de que os polimorfismos genéticos podem determinar importantes modulações nos fenótipos dos atletas, como por exemplo, estatura, adaptações cardiovasculares, utilização dos substratos energéticos bem como balanço eletrolítico e hormonal. É possível que indivíduos que expressem o gene alfa actinina 3 (ACTN3; genótipos RR para homozigotos ancestrais ou RX para heterozigotos) possam apresentar vantagens em movimentos que exijam força e rápida contração muscular quando comparados aos indivíduos com genótipo XX. Isto pelo fato de a ACTN3 ser um componente da linha Z sarcomérica, o qual é importante para o ancoramento dos miofilamentos de actina e manutenção do arranjo miofibrilar. Com relação ao polimorfismo no gene AMP deaminase (AMPD1), tem sido relatado que os atletas que apresentam o alelo mutado (alelo T) possam apresentar desvantagens em atividades físicas intensas e repetitivas, uma vez que a enzima codificada por este gene é responsável pela ressíntese de ATP muscular após intensas contrações. Os polimorfismos nos genes da enzima conversora de angiotensina (ECA; alelo de deleção D) e angiotensinogênio (AGT; alelo T mutado) podem favorecer os atletas em atividades que requeiram força, isto por conta dos maiores níveis circulantes de Angiotensina (Ang) II. Este estudo investigou se os polimorfismos nos genes ACTN3, AMPD1, ECA e AGT, combinados ou não, podem influenciar nos parâmetros hemodinâmicos, cardíacos e no desempenho de jogadores de futebol em testes físico-motores tais como saltos, velocidade e endurance. Foi coletada a saliva de 220 jogadores jovens (14 a 20 anos) das categorias de base profissional do São Paulo Futebol Clube, Brasil. Em seguida, o DNA total foi extraído a partir da saliva e ensaios de reação em cadeia da polimerase (PCR) foram utilizados para a genotipagem dos atletas. Para conferir mais fidedignidade ao estudo, os atletas foram também separados de acordo com a idade. Antes desta separação, os atletas com a mutação no gene ACTN3 saltaram menos no teste Squat Jump (SJ) (RR/RX = 33,64 ± 5,31 vs XX = 30,81 ± 4,51 cm, p = 0,007), assim como nas categorias Sub-15 (RR/RX = 34,88 ± 5,39 vs XX = 30,59 ± 4,07 cm, p = 0,04) e Sub-17 (RR/RX = 35,82 ± 4,35 vs XX = 30,24 ± 5,16 cm, p = 0,01). No teste Counter Movement Jump (CMJ) os RR/RX saltaram 37,26 ± 5,72 cm e os XX 34,12 ± 4,84 cm (p = 0,005). Na categoria Sub-17, detectou-se que os RR/RX saltaram 38,56 ± 5,69 cm e os XX 32,90 ± 6,06 cm (p = 0,02). No teste Counter Movement Jump com os braços (CMJb), com todos os atletas, os RR/RX saltaram 43,85 ± 6,38 cm e os XX 40,61 ± 5,06 cm (p = 0,009). O teste de velocidade de deslocamento (30 m) revelou, na categoria Sub-17, que os RR/RX foram mais velozes que os atletas XX (RR/RX = 4,13 ± 0,13 vs XX = 4,27 ± 0,17 s, p = 0,04). Com relação ao gene AMPD1, nenhuma diferença significativa foi encontrada nos testes de saltos e endurance, porém no teste de velocidade de deslocamento (10 m), os atletas CC foram mais velozes comparados àqueles com genótipos CT/TT (CC = 1,53 ± 0,19 vs CT/TT = 1,62 ± 0,16 s, p = 0,04). Atletas com o genótipo DD (ECA) saltaram significativamente mais alto no teste CMJb comparados aos ID/II (DD = 44,37 ± 6,22 vs ID/II 42,35 ± 6,23 cm, p = 0,02). Na categoria Sub-17, os atletas DD saltaram mais nos testes SJ (DD = 38,04 ± 5,00 vs ID/II = 33,16 ± 4,11 cm, p = 0,01), CMJ (DD = 41,03 ± 5,64 vs ID/II = 35,76 ± 4,26 cm, p = 0,01) e CMJb (DD = 48,62 ± 5,98 vs ID/II = 42,42 ± 4,81 cm, p = 0,007). No teste de endurance, atletas da categoria Sub-16 com os genótipos ID/II, percorreram maiores distâncias comparados aos DD (ID/II = 1.467 ± 63,70 vs DD = 1.244 ± 64,25 m, p = 0,04). O genótipo DD do gene da ECA também favoreceu os atletas no teste de velocidade (30 m), pois jogadores da categoria Sub-14 com o referido genótipo foram mais velozes comparados aos ID/II (DD = 4,29 ± 0,19 vs ID/II = 4,40 ± 0,16 s, p = 0,02). O mesmo pôde ser visto para a categoria Sub-17 (DD = 4,07 ± 0,15 vs ID/II = 4,20 ± 0,13 s, p = 0,04). O polimorfismo no gene AGT parece não influenciar o desempenho nos testes propostos, porém atletas com o genótipo mutado (TT) apresentaram maior hipertrofia do ventrículo esquerdo (VE; 114,6 ± 105,2 g/m2 para TT; 92,16 ± 18,88 g/m2 para MT e 94,78 ± 21,08 g/m2 para MM, p = 0,04), sem qualquer outra alteração nos outros parâmetros cardíacos e hemodinâmicos. Maior hipertrofia do VE (DD = 96,95 ± 19,96; ID = 90,14 ± 21,58 e II = 91,67 ± 21,09 g/m2, p = 0,04) e maior fração de ejeção (DD = 71,73 ± 7,71; ID = 69,48 ± 6,51 e II = 68,59 ± 5,72 %, p = 0,02) também foram encontradas nos atletas com o genótipo DD. A análise da combinação dos genes no desempenho dos atletas, quando se privilegiaram as características de força e explosão muscular no ranqueamento por escore, revelou que os atletas com os escores mais altos (5 a 8) saltaram mais comparados àqueles com escores mais baixos (1 a 4) no teste SJ (Escore 5 a 8 = 33,80 ± 5,16 vs Escore 1 a 4 = 31,60 ± 5,22 cm, p = 0,01) e no teste CMJ (Escore 5 a 8 = 43,90 ± 6,85 vs Escore 1 a 4 = 41,87 ± 5,98 cm, p = 0,04). Os resultados do presente estudo sugerem que os genótipos RR/RX (ACTN3), DD (ECA) e CC (AMPD1) podem beneficiar os jogadores de futebol em atividades que requeiram força e rápida contração muscular. Além disso, os genótipos ID/II parecem proporcionar mais resistência aos atletas em atividade de endurance. Para o futuro, serão necessárias organização, padronização e responsabilidade ética no manejo desses dados genéticos para a utilização no processo de formação de atletas

Papel preventivo do exercício físico nas alterações observadas na via de sinalização insulínica induzidas pela Dexametasona

Peripheral insulin resistance is the major side effect after chronic dexamethasone (Dexa) treatment, which is widely used to control inflammatory diseases. On the other hand, exercise training increases insulin-dependent muscle glucose uptake and attenuates hyperglycemia and hyperinsulinemia induced by Dexa treatment. This study investigated whether exercise can modulate the insulin-dependent glucose uptake pathway in order to attenuate the peripheral insulin resistance induced by Dexa treatment. Eighty rats were distributed into 4 groups: sedentary control (SC), sedentary treated with Dexa (SD; 1 mg/kg per day, i.p.), trained control (TC) and trained treated with Dexa (TD). These rats underwent a training period where they were either submitted to a running protocol (60% of physical capacity, 5 days/week for 8 weeks) or kept sedentary. After this training period, the animals underwent Dexa treatment (10 days) concomitant with training. Body weight was measured weekly before treatment and daily during the treatment. Blood glucose was measured at the beginning, after five days and at the end of experimental protocol (one touch ultra test). An ipGTT was performed at the end of the experimental period. After an overnight fast, the rats were anesthetized. After collection of an unchallenged sample (time 0), a solution of 50% glucose (2.0 g/kg body weight) was administered into the peritoneal cavity. Blood samples were collected from the tail at 30, 60, 90 and 120 min for determination of glucose concentrations and calculations of the area under the glucose curve (AUC). The western blot technique was performed to identify IRS-1, PKC-α and p-AKT protein expression in the tibialis anterior (TA) muscle. During the training period, the increase of body weight was similar among the groups. Dexa treatment body weight and exercise did not attenuate this reduction. Exercise training did not alter fasting blood glucose. Dexa treatment significantly increased glycemia in both groups, sedentary animals (SD = +157%) and trained animals (TD = +98%). However, training attenuated this increase since TD was 22% lower than SD groups. The AUC was 39% higher for Dexa-treated rats, but exercise attenuated this peripheral insulin resistance. Dexa significantly reduced IRS-1 (-58%), PKC-α (-44%) and p- AKT (-48%) protein expression in the TA muscle. Moreover, exercise per si increased protein expression of IRS-1 (112%), PKC- α (17%) and p-AKT (93%). Also, exercise blocked these protein expression reductions after Dexa treatment. These results demonstrates for the first time that exercise training can prevent the reductions of IRS-1, p-AKT and PKC- α protein expressions induced by Dexa in the skeletal muscle. Therefore, exercise may be a good strategy to prevent dexamethasone-induced peripheral insulin resistance. Financial support: FAPESPUniversidade Federal de Minas GeraisA resistência periférica à insulina é um dos principais efeitos colaterais após o tratamento com dexametasona (Dexa), droga essa amplamente utilizada no controle de doenças inflamatórias. Por outro lado, o exercício físico aumenta a captação muscular de glicose, dependente e independente de insulina, o que atenua a hiperglicemia e hiperinsulinemia induzida pelo tratamento com Dexa. Este trabalho investigou como o exercício pode modular a via de captação de glicose, dependente de insulina, na tentativa de atenuar a resistência periférica à insulina induzida pela Dexa. Cento e quatro ratos foram distribuídos em 4 grupos: sedentário controle (SC), sedentários tratados com DEXA (SD; 1 mg / kg / dia, ip), treinado controle (TC) e treinados tratados com Dexa (TD). Estes ratos passaram por um período de treinamento, onde eram submetidos a um protocolo de corrida (60% da capacidade física, 5 dias / semana, por 8 semanas) ou mantidos sedentários. Após esse período de treinamento, os animais foram tratados por 10 dias com Dexa, concomitantemente ao treinamento. O peso corporal foi mensurado semanalmente antes do tratamento e diariamente durante o tratamento farmacológico. A glicose sanguínea foi mensurada no início, após cinco dias e no final do protocolo experimental (one touch ultra test). O ipGTT foi feito ao final do período experimental. Após o jejum de 12 horas, os ratos foram anestesiados. Logo em seguida, o sangue foi coletado da cauda (tempo 0) e uma solução a 50% de glicose (2 g/kg de peso corporal) foi injetada no peritônio. Amostras de sangue foram coletadas a partir da cauda do animal nos tempos 30, 60, 90 e 120 min para se determinar a concentração de glicose e calcular a área abaixo da curva glicêmica (AUC). A técnica de western blot foi utilizada para mensurar a expressão das proteínas IRS-1, PKC-α e p-AKT no músculo tibial anterior (TA). Durante o período de treinamento físico, o aumento de peso corporal foi semelhante em todos os grupos. O tratamento com Dexa determinou diminuição do peso corporal e o exercício não atenuou esta redução. O treinamento físico não alterou glicemia de jejum. O tratamento com Dexa aumentou significativamente a glicemia tanto nos animais sedentários (SD = +157%) como nos animais treinados (TD = +98%). No entanto, o treinamento atenuou este aumento uma vez que TD teve um aumento 22% menor que o SD. A AUC foi 39% maior nos ratos tratados com Dexa, mas o exercício atenuou essa resistência periférica à insulina. A Dexa reduziu significativamente as expressões do IRS-1 (-58%), PKC-α (-44%) e p-AKT (-48%) no músculo TA. Por outro lado, o exercício por si só aumentou a expressão do IRS-1 (112%), PKC-α (17%) e p-AKT (93%). Além disso, o exercício bloqueou a redução da expressão dessas proteínas após tratamento com Dexa. Estes resultados demonstram pela primeira vez que o exercício previne a redução da expressão do IRS-1, p-AKT e PKC-α no músculo esquelético após tratamento com Dexa. Portanto, o exercício pode ser uma boa estratégia para evitar a resistência periférica à insulina induzida pela Dexa. Apoio financeiro: FAPES

Angiotensin II Type 1 Receptor Knockdown Impairs Interleukin-1β-Induced Cytokines in Human Periodontal Fibroblasts

BACKGROUND: The renin-angiotensin (Ang) system (RAS) has been reported as an important modulator of inflammatory and immune responses. Evidence suggests an alternative Ang 1-7/Mas receptor axis as counter-regulatory to the classic RAS Ang II/Ang II Type 1 (AT1) receptor axis. It is known that periodontal pathogens elicit host-derived immune response due to release of cytokines such as interleukin (IL)-1β, and fibroblasts are among the most numerous sentinel cells that contribute to this production. The aim of this study is to determine whether AT1 receptor (AT1R) contributes to production of inflammatory cytokines that are important for periodontal pathogenesis using primary human gingival fibroblasts (HGFs) and human periodontal ligament fibroblasts (HPLFs) stimulated with IL-1β. METHODS: Through RNA interference or pharmacologic inhibition using AT1R antagonist losartan, HGF and HPLF were stimulated by IL-1β for 3 (messenger RNA [mRNA]) or 24 (protein) hours. RESULTS: IL-1β upregulated mRNA expression of AT1R, IL-1β, IL-6, IL-8, tumor necrosis factor-alpha, and osteoprotegerin (OPG) in HGF and HPLF. AT1R knockdown impaired IL-1β-induced IL-6 and IL-8 secretion in cultured HGF and HPLF. AT1R silencing also increased OPG gene expression in HGF only. Pharmacologic inhibition of AT1R through losartan modulated mRNA transcription of IL-6 and IL-8 in HPLF but not in HGF. In contrast, IL-1β-induced secretion of IL-6 and IL-8 was not influenced by losartan in HGF or HPLF. CONCLUSION: These results suggest that AT1R knockdown and AT1R pharmacologic blockade by losartan may differently control balance of inflammatory cytokines, such as IL-6 and IL-8, in primary human periodontal fibroblasts

Human DNA extraction from whole saliva that was fresh or stored for 3, 6 or 12 months using five different protocols

Saliva when compared to blood collection has the following advantages: it requires no specialized personnel for collection, allows for remote collection by the patient, is painless, well accepted by participants, has decreased risks of disease transmission, does not clot, can be frozen before DNA extraction and possibly has a longer storage time. Objective and Material and Methods This study aimed to compare the quantity and quality of human DNA extracted from saliva that was fresh or frozen for three, six and twelve months using five different DNA extraction protocols: protocol 1 – Oragene™ commercial kit, protocol 2 – QIAamp DNA mini kit, protocol 3 – DNA extraction using ammonium acetate, protocol 4 – Instagene™ Matrix and protocol 5 – Instagene™ Matrix diluted 1:1 using proteinase K and 1% SDS. Briefly, DNA was analyzed using spectrophotometry, electrophoresis and PCR. Results Results indicated that time spent in storage typically decreased the DNA quantity with the exception of protocol 1. The purity of DNA was generally not affected by storage times for the commercial based protocols, while the purity of the DNA samples extracted by the noncommercial protocols typically decreased when the saliva was stored longer. Only protocol 1 consistently extracted unfragmented DNA samples. In general, DNA samples extracted through protocols 1, 2, 3 and 4, regardless of storage time, were amplified by human specific primers whereas protocol 5 produced almost no samples that were able to be amplified by human specific primers. Depending on the protocol used, it was possible to extract DNA in high quantities and of good quality using whole saliva, and furthermore, for the purposes of DNA extraction, saliva can be reliably stored for relatively long time periods. Conclusions In summary, a complicated picture emerges when taking into account the extracted DNA’s quantity, purity and quality; depending on a given researchers needs, one protocol’s particular strengths and costs might be the deciding factor for its employment

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