Comparison of rRNA-based reverse transcription PCR and rDNA-based PCR for the detection of streptococci in root canal infections

Objective: The rDNA-based method is unable to distinguish between alive and dead cells. Alternatively, bacterial viability can be assessed by molecular methods based on ribosomal RNA (rRNA). Therefore, this study aimed to detect viable streptococci in root canal samples using rRNA-based reverse transcription polymerase chain reaction (RT-PCR), compared to an rDNA-based PCR assay. Methodology: Microbiological root canal samples were obtained from 32 teeth with primary endodontic infections before (S1) and after chemomechanical preparation (S2), and after removal of intracanal medication (S3). RNA and DNA were extracted, and complementary DNA (cDNA) was synthesized from RNA using RT reaction. cDNA and genomic DNA were subjected to PCR with primers complementary to the 16S rRNA sequences of Streptococcus spp. McNemar’s test was used to compare the detection rate of both assays (P<0.05). Results: Streptococci were detected in 28.12% (9/32) and 37.5% (12/32) of S1 samples using rRNA- and rDNA-based PCR assays, respectively. In contrast, they were detected in only 6.25% (2/32) of S2 samples using rRNA-based RT-PCR, compared to 15.62% (5/32) using rDNA-based PCR. Finally, in S3 samples, streptococci were not detected by rRNA, whereas rDNA-based PCR still detected the bacteria in 12.5% (4/32) of the samples. The total number of PCR-positive reactions in the rDNA-based PCR was higher than in the rRNA-based assay (P<0.05). Conclusions: The rRNA-based RT-PCR showed a lower detection rate of streptococci when compared to the rDNA-based PCR, suggesting that the latter may have detected dead cells of streptococci in root canal samples

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear un derstanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5–7 vast areas of the tropics remain understudied.8–11 In the American tropics, Amazonia stands out as the world’s most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepre sented in biodiversity databases.13–15 To worsen this situation, human-induced modifications16,17 may elim inate pieces of the Amazon’s biodiversity puzzle before we can use them to understand how ecological com munities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple or ganism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region’s vulnerability to environmental change. 15%–18% of the most ne glected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lostinfo:eu-repo/semantics/publishedVersio

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear understanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5,6,7 vast areas of the tropics remain understudied.8,9,10,11 In the American tropics, Amazonia stands out as the world's most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepresented in biodiversity databases.13,14,15 To worsen this situation, human-induced modifications16,17 may eliminate pieces of the Amazon's biodiversity puzzle before we can use them to understand how ecological communities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple organism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region's vulnerability to environmental change. 15%–18% of the most neglected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lost

Evaluation of bacterial and endotoxin levels after root canal treatment with primary apical periodontitis teeth and its correlation with pro-inflammatory cytokines induction by human fibroblasts culture

Este estudo clínico avaliou a quantidade de bactérias e endotoxinas, antes e após o tratamento endodôntico utilizando ativação ultrassônica de solução de hidróxido de cálcio e sua correlação com a indução de citocinas pró-inflamatórias por fibroblastos humanos. Vinte e três pacientes com dentes com periodontite apical primária foram submetidos ao tratamento endodôntico. O preparo químico-cirúrgico (PQC) foi realizado com instrumentos reciprocantes, utilizando NaOCl 2,5% durante o preparo e EDTA 17% para remoção do magma dentinário; complementado com a ativação ultrassônica de uma solução de hidróxido de cálcio. Os canais radiculares foram preenchidos com pasta de hidróxido de cálcio por 14 dias e obturados. Foram realizadas coletas microbiológicas dos canais antes (S1) e após o PQC (S2), após o protocolo de ativação ultrassônica (S3) e após a medicação intracanal (S4). As amostras foram analisadas por: PCR quantitativo (qPCR) utilizando iniciadores universais para quantificação de bactérias após extração de DNA; teste turbidimétrico de LAL para quantificação de endotoxinas; e ELISA para quantificação de citocinas (IL-1?, IL-6, CCL2) em cultura de fibroblastos humanos após indução com o conteúdo do canal radicular. Os dados foram analisados pelos seguintes testes estatísticos: teste de Wilcoxon e Q de Cochran para análise quantitativa de bactérias e endotoxinas; ANOVA e teste de Tukey, para quantificação dos níveis de citocinas; e teste de Pearson para correlação entre os níveis de endotoxinas e citocinas (todos com nível de significância de 5%). Bactérias e endotoxinas foram observadas em todas as amostras iniciais com uma mediana de 3,61 x 104 e 109 EU/ml, respectivamente. Após o preparo químico-cirúrgico, houve uma redução significativa (p 0,05). Houve diminuição significativa de IL-6 e CCL-2 entre S1 e S2 (p 0,05). Conclui-se que o PQC foi eficaz na redução de bactérias e endotoxinas dos canais radiculares; enquanto a medicação intracanal influenciou apenas na redução de endotoxina. As amostras após o PQC induziram menores níveis de IL-6 e CCL-2 em fibroblastos humanos do que as amostras antes do tratamento endodôntico. Por outro lado, as demais etapas do tratamento não foram capazes de promover uma redução adicional dessas citocinas.This clinical study analyzed bacteria and endotoxins levels before and after endodontic treatment using ultrasonic activation of a calcium hydroxide solution and an intracanal medication and its correlation with pro-inflammatory cytokines induction by human fibroblasts. Twenty-three patients with teeth with primary apical periodontitis were submitted to endodontic treatment. The root canal preparation of all the teeth was carried out with reciprocating files and 2.5% NaOCl during preparation; and 17% EDTA for smear layer removal, supplemented with the ultrasonic activation of a solution of calcium hydroxide. The root canals were medicated with calcium hydroxide for 14 days and then obturated. Microbiological sampling were performed before (S1) and after the root canal preparation (S2), after ultrasonic activation protocol (S3) and after intracanal medication (S4). The samples were analyzed by quantitative PCR (qPCR) using universal primers quantification of bacteria after DNA extraction; LAL turbidimetric test for detection and determination of endotoxin and ELISA for evaluate the cytokine production (IL-1?, IL-6, CCL2) by humam fibroblasts culture. Data was analyzed by Wilcoxon and Q cochran tests for the quantitative analysis of bacteria and endotoxins. ANOVA and tukey test, with significance level of 5%, were used for quantification of cytokine levels and Pearson\'s test for correlation between endotoxins and cytokine levels. Bacteria and endotoxins were observed in all initial samples with a median of 3.61 x 104 and 109 EU/ml respectively. After root canal preparation, there was a significant reduction (p0.05). There was a significant decrease in the presence of the proinflammatory cytokine IL-6 and CCL-2 between the initial stage of endodontic treatment (S1) and the next step (S2) (p 0.05). It was concluded that the root canal preparation was effective in reducing bacteria and endotoxins, and intracanal medication only influenced in endotoxin reduction. Samples after the root canal preparation induced lower levels of IL-6 and CCL-2 in human fibroblasts than samples before endodontic treatment, whereas other stages of the endodontic treatment were not able to promote an additional reduction

Evaluation of bacterial and endotoxin levels after root canal treatment with primary apical periodontitis teeth and its correlation with pro-inflammatory cytokines induction by human fibroblasts culture

Este estudo clínico avaliou a quantidade de bactérias e endotoxinas, antes e após o tratamento endodôntico utilizando ativação ultrassônica de solução de hidróxido de cálcio e sua correlação com a indução de citocinas pró-inflamatórias por fibroblastos humanos. Vinte e três pacientes com dentes com periodontite apical primária foram submetidos ao tratamento endodôntico. O preparo químico-cirúrgico (PQC) foi realizado com instrumentos reciprocantes, utilizando NaOCl 2,5% durante o preparo e EDTA 17% para remoção do magma dentinário; complementado com a ativação ultrassônica de uma solução de hidróxido de cálcio. Os canais radiculares foram preenchidos com pasta de hidróxido de cálcio por 14 dias e obturados. Foram realizadas coletas microbiológicas dos canais antes (S1) e após o PQC (S2), após o protocolo de ativação ultrassônica (S3) e após a medicação intracanal (S4). As amostras foram analisadas por: PCR quantitativo (qPCR) utilizando iniciadores universais para quantificação de bactérias após extração de DNA; teste turbidimétrico de LAL para quantificação de endotoxinas; e ELISA para quantificação de citocinas (IL-1?, IL-6, CCL2) em cultura de fibroblastos humanos após indução com o conteúdo do canal radicular. Os dados foram analisados pelos seguintes testes estatísticos: teste de Wilcoxon e Q de Cochran para análise quantitativa de bactérias e endotoxinas; ANOVA e teste de Tukey, para quantificação dos níveis de citocinas; e teste de Pearson para correlação entre os níveis de endotoxinas e citocinas (todos com nível de significância de 5%). Bactérias e endotoxinas foram observadas em todas as amostras iniciais com uma mediana de 3,61 x 104 e 109 EU/ml, respectivamente. Após o preparo químico-cirúrgico, houve uma redução significativa (p 0,05). Houve diminuição significativa de IL-6 e CCL-2 entre S1 e S2 (p 0,05). Conclui-se que o PQC foi eficaz na redução de bactérias e endotoxinas dos canais radiculares; enquanto a medicação intracanal influenciou apenas na redução de endotoxina. As amostras após o PQC induziram menores níveis de IL-6 e CCL-2 em fibroblastos humanos do que as amostras antes do tratamento endodôntico. Por outro lado, as demais etapas do tratamento não foram capazes de promover uma redução adicional dessas citocinas.This clinical study analyzed bacteria and endotoxins levels before and after endodontic treatment using ultrasonic activation of a calcium hydroxide solution and an intracanal medication and its correlation with pro-inflammatory cytokines induction by human fibroblasts. Twenty-three patients with teeth with primary apical periodontitis were submitted to endodontic treatment. The root canal preparation of all the teeth was carried out with reciprocating files and 2.5% NaOCl during preparation; and 17% EDTA for smear layer removal, supplemented with the ultrasonic activation of a solution of calcium hydroxide. The root canals were medicated with calcium hydroxide for 14 days and then obturated. Microbiological sampling were performed before (S1) and after the root canal preparation (S2), after ultrasonic activation protocol (S3) and after intracanal medication (S4). The samples were analyzed by quantitative PCR (qPCR) using universal primers quantification of bacteria after DNA extraction; LAL turbidimetric test for detection and determination of endotoxin and ELISA for evaluate the cytokine production (IL-1?, IL-6, CCL2) by humam fibroblasts culture. Data was analyzed by Wilcoxon and Q cochran tests for the quantitative analysis of bacteria and endotoxins. ANOVA and tukey test, with significance level of 5%, were used for quantification of cytokine levels and Pearson\'s test for correlation between endotoxins and cytokine levels. Bacteria and endotoxins were observed in all initial samples with a median of 3.61 x 104 and 109 EU/ml respectively. After root canal preparation, there was a significant reduction (p0.05). There was a significant decrease in the presence of the proinflammatory cytokine IL-6 and CCL-2 between the initial stage of endodontic treatment (S1) and the next step (S2) (p 0.05). It was concluded that the root canal preparation was effective in reducing bacteria and endotoxins, and intracanal medication only influenced in endotoxin reduction. Samples after the root canal preparation induced lower levels of IL-6 and CCL-2 in human fibroblasts than samples before endodontic treatment, whereas other stages of the endodontic treatment were not able to promote an additional reduction

Cytokine profile, proliferation and phosphorylation of ERK1/2 and Akt in circulating mononuclear cells from individuals during the chronic intestinal phase of <it>Schistosomiasis mansoni</it> infection

Abstract Background The immune response to Schistosoma mansoni is characterized by a granulomatous reaction around the parasite eggs that are trapped in the host liver, and this reaction modulates the immune response during the chronic phase of the disease. The typical peripheral blood mononuclear cell (PBMC) response of patients during the chronic intestinal phase of infection is characterized by a decreased response to an S. mansoni soluble egg antigen. To obtain a greater understanding of Schistosoma infections, this study investigated the effects of the soluble egg antigen (SEA) and soluble adult worm antigen (SWAP) of S. mansoni on cellular proliferation, cytokine production, and ERK1/2 and Akt phosphorylation in PBMCs from infected (XTO) and egg-negative (NI) individuals living in the same endemic area. Methods The activation status was evaluated by cell immunophenotypic staining (cytometry). The cell proliferation assay was by CFSE method. Cytokine detection assay (Th1 and Th2) was by Cytometric Bead and Array phosphorylation status was by ELISA. Results The XTO, NI and BD (blood donor) individuals from an area not endemic for schistosomiasis were compared. The CD4+ T lymphocyte proliferation rate was lower in the XTO group, but not the NI group, after SEA stimulation compared to the BD group. The CD8+ T cell proliferation rate was lower in the XTO group in the unstimulated cultures and after both SEA and SWAP stimulation compared to the BD group. Cytokine analysis after either SEA or SWAP stimulation showed a balanced cytokine pattern in the XTO and NI groups. ERK1/2 and Akt phosphorylation were only marginally detected in all groups; however, a decrease in ERK 1/2 phosphorylation was observed in the SWAP-stimulated XTO group compared to both the NI and BD groups. Conclusions The data indicate that SEA-stimulated CD4+ T cells from infected patients have a lower proliferation rate than the same cells from the NI group. Furthermore, we observed that SWAP stimulation influences ERK1/2 phosphorylation in the XTO group.</p

O papel do conselheiro municipal de saúde na fiscalização do orçamento público

Este estudo tem por objetivo analisar o conhecimento de conselheiros municipais de saúde sobre os seus papéis na fiscalização do orçamento público. É um estudo descritivo-exploratório, realizado com seis conselheiros do município de Jequié-BA. Os dados foram coletados através de questionário e entrevista semiestruturada, e analisados pela técnica de análise de conteúdo de Bardin (2009). Da análise, emergiram quatro categorias temáticas, através das quais se verificou que os conselheiros demonstraram consciência dos seus papéis de fiscalizar e acompanhar os recursos financeiros do Sistema Único de Saúde (SUS) e de favorecer a efetivação do controle social na fiscalização do orçamento público

O papel do conselheiro municipal de saúde na fiscalização do orçamento público

Este estudo tem por objetivo analisar o conhecimento de conselheiros municipais de saúde sobre os seus papéis na fiscalização do orçamento público. É um estudo descritivo-exploratório, realizado com seis conselheiros do município de Jequié-BA. Os dados foram coletados através de questionário e entrevista semiestruturada, e analisados pela técnica de análise de conteúdo de Bardin (2009). Da análise, emergiram quatro categorias temáticas, através das quais se verificou que os conselheiros demonstraram consciência dos seus papéis de fiscalizar e acompanhar os recursos financeiros do Sistema Único de Saúde (SUS) e de favorecer a efetivação do controle social na fiscalização do orçamento público