TOR as a regulatory target in Rhipicephalus microplus embryogenesis

Embryogenesis is a metabolically intensive process carried out under tightly controlled conditions. The insulin signaling pathway regulates glucose homeostasis and is essential for reproduction in metazoan model species. Three key targets are part of this signaling pathway: protein kinase B (PKB, or AKT), glycogen synthase kinase 3 (GSK-3), and target of rapamycin (TOR). While the role of AKT and GSK-3 has been investigated during tick embryonic development, the role of TOR remains unknown. In this study, TOR and two other downstream effectors, namely S6 kinase (S6K) and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), were investigated in in vitro studies using the tick embryonic cell line BME26. First, we show that exogenous insulin can stimulate TOR transcription. Second, TOR chemical inhibition led to a decrease in BME26 cell viability, loss of membrane integrity, and downregulation of S6K and 4E-BP1 transcription. Conversely, treating BME26 cells with chemical inhibitors of AKT or GSK-3 did not affect S6K and 4E-BP1 transcription, showing that TOR is specifically required to activate its downstream targets. To address the role of TOR in tick reproduction, in vivo studies were performed. Analysis of relative transcription during different stages of tick embryonic development showed different levels of transcription for TOR, and a maternal deposition of S6K and 4E-BP1 transcripts. Injection of TOR double-stranded RNA (dsRNA) into partially fed females led to a slight delay in oviposition, an atypical egg external morphology, decreased vitellin content in eggs, and decreased larval hatching. Taken together, our data show that the TOR signaling pathway is important for tick reproduction, that TOR acts as a regulatory target in Rhipicephalus microplus embryogenesis and represents a promising target for the development of compounds for tick control

Perceptual analysis of heterogeneous stakeholders on the impact of the Rio 2016 games in the territory of Lagoa Rodrigo de Freitas

Highlights: Development of partnership with local entities for integrated social actions; Actions and projects are developed as close as possible to the public audience; Participation in meetings and community councils; Establishment of constant dialog between social management and community leaders; Dialog with public entities; Work communication with all the impacts caused by the implementation of the megaproject, according to the affected area. Goal: Analyze, through a case study, what were the effects resulting from the Rio 2016 Olympic Games in the territory of Lagoa Rodrigo de Freitas, and how they were perceived by the stakeholders during and after the performance. Design / Methodology / Approach: On-site open and participant observation, semi-structured interviews, and on-site questionnaires and descriptive statistics Results: A series of consequences were triggered, affecting each of the different stakeholders in different ways. Limitations of the investigation: Megaprojects promote mutually positive and negative impacts concerning the local stakeholders and their multiple and different perceptions. Thus, results cannot be generalized. Practical implications: Fragmentation of the megaproject, heterogeneity, and perceptions of different stakeholders Originality / Value: Practices that are sensitive to the stakeholders’ engagement and impacts perceived

Non-invasive delivery of dsRNA into de-waxed tick eggs by electroporation

RNA interference-mediated gene silencing was shown to be an efficient tool for validation of targets that may become anti-tick vaccine components. Here, we demonstrate the application of this approach in the validation of components of molecular signaling cascades, such as the Protein Kinase B (AKT) / Glycogen Synthase Kinase (GSK) axis during tick embryogenesis. It was shown that heptane and hypochlorite treatment of tick eggs can remove wax, affecting corium integrity and but not embryo development. Evidence of AKT and GSK dsRNA delivery into de-waxed eggs of via electroporation is provided. Primers designed to amplify part of the dsRNA delivered into the electroporated eggs dsRNA confirmed its entry in eggs. In addition, it was shown that electroporation is able to deliver the fluorescent stain, 4',6-diamidino-2-phenylindole (DAPI). To confirm gene silencing, a second set of primers was designed outside the dsRNA sequence of target gene. In this assay, the suppression of AKT and GSK transcripts (approximately 50% reduction in both genes) was demonstrated in 7- day-old eggs. Interestingly, silencing of GSK in 7-day-old eggs caused 25% reduction in hatching. Additionally, the effect of silencing AKT and GSK on embryo energy metabolism was evaluated. As expected, knockdown of AKT, which down regulates GSK, the suppressor of glycogen synthesis, decreased glycogen content in electroporated eggs. These data demonstrate that electroporation of de-waxed R. microplus eggs could be used for gene silencing in tick embryos, and improve the knowledge about arthropod embryogenesis

The modulation of the symbiont/host interaction between wolbachia pipientis and aedes fluviatilis embryos by glycogen metabolism

Wolbachia pipientis, a maternally transmitted bacterium that colonizes arthropods, may affect the general aspects of insect physiology, particularly reproduction. Wolbachia is a natural endosymbiont of Aedes fluviatilis, whose effects in embryogenesis and reproduction have not been addressed so far. In this context, we investigated the correlation between glucose metabolism and morphological alterations during A. fluviatilis embryo development in Wolbachia-positive (W+) and Wolbachia-negative (W2) mosquito strains. While both strains do not display significant morphological and larval hatching differences, larger differences were observed in hexokinase activity and glycogen contents during early and mid-stages of embryogenesis, respectively. To investigate if glycogen would be required for parasite-host interaction, we reduced Glycogen Synthase Kinase-3 (GSK-3) levels in adult females and their eggs by RNAi. GSK-3 knock-down leads to embryonic lethality, lower levels of glycogen and total protein and Wolbachia reduction. Therefore, our results suggest that the relationship between A. fluviatilis and Wolbachia may be modulated by glycogen metabolism

Redox imbalance induces remodeling of glucose metabolism in Rhipicephalus microplus embryonic cell line

Carbohydrate metabolism not only functions in supplying cellular energy but also has an important role in maintaining physiological homeostasis and in preventing oxidative damage caused by reactive oxygen species. Previously, we showed that arthropod embryonic cell lines have high tolerance to H²O² exposure. Here, we describe that Rhipicephalus microplus tick embryonic cell line (BME26) employs an adaptive glucose metabolism mechanism that confers tolerance to hydrogen peroxide at concentrations too high for other organisms. This adaptive mechanism sustained by glucose metabolism remodeling promotes cell survival and redox balance in BME26 cell line after millimolar H²O² exposure. The present work shows that this tick cell line could tolerate high H²O² concentrations by initiating a carbohydrate-related adaptive response. We demonstrate that gluconeogenesis was induced as a compensation strategy that involved, among other molecules, the metabolic enzymes NADP-ICDH, G6PDH, and PEPCK. We also found that this phenomenon was coupled to glycogen accumulation and glucose uptake, supporting the pentose phosphate pathway to sustain NADPH production and leading to cell survival and proliferation. Our findings suggest that the described response is not atypical, being also observed in cancer cells, which highlights the importance of this model to all proliferative cells. We propose that these results will be useful in generating basic biological information to support the development of new strategies for disease treatment and parasite control

Aedes fluviatilis cell lines as new tools to study metabolic and immune interactions in mosquito‑Wolbachia symbiosis

In the present work, we established two novel embryonic cell lines from the mosquito Aedes fluviatilis containing or not the naturally occurring symbiont bacteria Wolbachia, which were called wAflu1 and Aflu2, respectively. We also obtained wAflu1 without Wolbachia after tetracycline treatment, named wAflu1.tet. Morphofunctional characterization was performed to help elucidate the symbiont-host interaction in the context of energy metabolism regulation and molecular mechanisms of the immune responses involved. The presence of Wolbachia pipientis improves energy performance in A. fluviatilis cells; it affects the regulation of key energy sources such as lipids, proteins, and carbohydrates, making the distribution of actin more peripheral and with extensions that come into contact with neighboring cells. Additionally, innate immunity mechanisms were activated, showing that the wAflu1 and wAflu1.tet cells are responsive after the stimulus using Gram negative bacteria. Therefore, this work confirms the natural, mutually co-regulating symbiotic relationship between W. pipientis and A. fluviatilis, modulating the host metabolism and immune pathway activation. The results presented here add important resources to the current knowledge of Wolbachia-arthropod interactions

Non-classical gluconeogenesis-dependent glucose metabolism in Rhipicephalus microplus embryonic cell line BME26

In this work we evaluated several genes involved in gluconeogenesis, glycolysis and glycogen metabolism, the major pathways for carbohydrate catabolism and anabolism, in the BME26 Rhipicephalus microplus embryonic cell line. Genetic and catalytic control of the genes and enzymes associated with these pathways are modulated by alterations in energy resource availability (primarily glucose). BME26 cells in media were investigated using three different glucose concentrations, and changes in the transcription levels of target genes in response to carbohydrate utilization were assessed. The results indicate that several genes, such as glycogen synthase (GS), glycogen synthase kinase 3 (GSK3), phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6 phosphatase (GP) displayed mutual regulation in response to glucose treatment. Surprisingly, the transcription of gluconeogenic enzymes was found to increase alongside that of glycolytic enzymes, especially pyruvate kinase, with high glucose treatment. In addition, RNAi data from this study revealed that the transcription of gluconeogenic genes in BME26 cells is controlled by GSK-3. Collectively, these results improve our understanding of how glucose metabolism is regulated at the genetic level in tick cells

Theoretical and practical approach of spirituality in institutionalized patients.

A rela??o entre medicina e espiritualidade ? alvo de estudos da atualidade, cujos resultados evidenciam associa??o positiva com comportamentos saud?veis. No entanto, h? uma lacuna de tal abordagem na forma??o em sa?de. O presente projeto de extens?o objetiva construir com estudantes de medicina substrato te?rico e viv?ncias pr?ticas na abordagem da espiritualidade de pacientes institucionalizados atrav?s de question?rios validados como o FICA. As interven??es com as atividades registradas e discutidas com os professores visam ofertar cuidado humanizado e valorizar a integralidade dos sujeitos. A an?lise qualitativa dos dados revelou aus?ncia de abordagem pr?via da espiritualidade dos pacientes, cuja maioria manifestou interesse nessa interven??o. ? relevante para o m?dico saber o momento e a forma adequada dessa abordagem visando ? singularidade de cada caso.The relationship between medicine and spirituality is subject of current studies, whose results show a positive association with healthy behaviors. However, there is a lack of such approach in healthcare education. The objectives of this extension project were to build together with medical students theoretical and practical experiences in approaching the spirituality of institutionalized patients through validated questionnaires, such as FICA. The interventions with the activities record and discussion with professors aim to offer humanized care and to value the integrality of these individuals. Qualitative data analysis revealed a lack of previous approach to the spirituality of the patients, whom (most of them) expressed interest in this intervention. It is relevant for the physician to know the timing and appropriate way of this approach aiming at the uniqueness of each case

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