Next Generation of Ovarian Cancer Detection Using Aptamers.

Ovarian cancer is among the seven most common types of cancer in women, being the most fatal gynecological tumor, due to the difficulty of detection in early stages. Aptamers are important tools to improve tumor diagnosis through the recognition of specific molecules produced by tumors. Here, aptamers and their potential targets in ovarian cancer cells were analyzed by in silico approaches. Specific aptamers were selected by the Cell-SELEX method using Caov-3 and OvCar-3 cells. The five most frequent aptamers obtained from the last round of selection were computationally modeled. The potential targets for those aptamers in cells were proposed by analyzing proteomic data available for the Caov-3 and OvCar-3 cell lines. Overexpressed proteins for each cell were characterized as to their three-dimensional model, cell location, and electrostatic potential. As a result, four specific aptamers for ovarian tumors were selected: AptaC2, AptaC4, AptaO1, and AptaO2. Potential targets were identified for each aptamer through Molecular Docking, and the best complexes were AptaC2-FXYD3, AptaC4-ALPP, AptaO1-TSPAN15, and AptaO2-TSPAN15. In addition, AptaC2 and AptaO1 could detect different stages and subtypes of ovarian cancer tissue samples. The application of this technology makes it possible to propose new molecular biomarkers for the differential diagnosis of epithelial ovarian cancer.

Use of an evidence-based health portal to improve teaching and learning in primary care: a mixed methods evaluation.

BACKGROUND: Worldwide, primary care is for most people the gateway into many health systems. Offering solutions to the demands of the communities served requires the constant preparation of professionals, especially doctors and medical undergraduate students. We analyze and propose ways to improve the teaching and learning processes facilitated by the Basic Family Health Units (BFHUs) based on the use of electronic portals with evidence-based medicine criteria. METHOD: First phase: The authors conducted a qualitative-quantitative study on students and instructors of primary care (PC) medicine by administering a survey of open- and closed-ended questions at medical schools. The closed-ended questions were studied with descriptive statistics, and open-ended questions were analyzed via the creation of categories. Perceptions of major teaching and learning problems were then identified. Second phase: Meetings were held with students and their instructors for 6 months and involved the use of electronic portals and the application of new questionnaires using a Likert scale for pre- and postevaluation. RESULTS: In the first phase, 40% of the students considered local instructor training levels a problem. A similar result was found regarding teachers' lectures, revealing a lack of adequate PC training and performance. Building on our results, we focused on BFHUs to apply new strategies for teaching and learning, such as the use of the Evidence-Based Health (SBE) Portal, which includes several databases with clinical evidence criteria. In the second phase, the authors identified an improvement in the quality of learning among instructors and students. This outcome improved safety in daily clinical practice in PC, possibly with better results for its users. CONCLUSIONS: The use of electronic portals can facilitate BFHU teaching and learning and promote the health of users.

A Hybrid Approach Combining Shape-Based and Docking Methods to Identify Novel Potential P2X7 Antagonists from Natural Product Databases.

P2X7 is an ATP-activated purinergic receptor implicated in pro-inflammatory responses. It is associated with the development of several diseases, including inflammatory and neurodegenerative conditions. Although several P2X7 receptor antagonists have recently been reported in the literature, none of them is approved for clinical use. However, the structure of the known antagonists can serve as a scaffold for discovering effective compounds in clinical therapy. This study aimed to propose an improved virtual screening methodology for the identification of novel potential P2X7 receptor antagonists from natural products through the combination of shape-based and docking approaches. First, a shape-based screening was performed based on the structure of JNJ-47965567, a P2X7 antagonist, using two natural product compound databases, MEGx (~5.8 × 103 compounds) and NATx (~32 × 103 compounds). Then, the compounds selected by the proposed shape-based model, with Shape-Tanimoto score values ranging between 0.624 and 0.799, were filtered for drug-like properties. Finally, the compounds that met the drug-like filter criteria were docked into the P2X7 allosteric binding site, using the docking programs GOLD and DockThor. The docking poses with the best score values were submitted to careful visual inspection of the P2X7 allosteric binding site. Based on our established visual inspection criteria, four compounds from the MEGx database and four from the NATx database were finally selected as potential P2X7 receptor antagonists. The selected compounds are structurally different from known P2X7 antagonists, have drug-like properties, and are predicted to interact with key P2X7 allosteric binding pocket residues, including F88, F92, F95, F103, M105, F108, Y295, Y298, and I310. Therefore, the combination of shape-based screening and docking approaches proposed in our study has proven useful in selecting potential novel P2X7 antagonist candidates from natural-product-derived compounds databases. This approach could also be useful for selecting potential inhibitors/antagonists of other receptors and/or biological targets.

Physiologic roles of P2 receptors in leukocytes.

Since their discovery in the 1970s, purinergic receptors have been shown to play key roles in a wide variety of biologic systems and cell types. In the immune system, purinergic receptors participate in innate immunity and in the modulation of the adaptive immune response. In particular, P2 receptors, which respond to extracellular nucleotides, are widely expressed on leukocytes, causing the release of cytokines and chemokines and the formation of inflammatory mediators, and inducing phagocytosis, degranulation, and cell death. The activity of these receptors is regulated by ectonucleotidases-expressed in these same cell types-which regulate the availability of nucleotides in the extracellular environment. In this article, we review the characteristics of the main purinergic receptor subtypes present in the immune system, focusing on the P2 family. In addition, we describe the physiologic roles of the P2 receptors already identified in leukocytes and how they can positively or negatively modulate the development of infectious diseases, inflammation, and pain.

Graph Neural Networks as a Potential Tool in Improving Virtual Screening Programs.

Despite the increasing number of pharmaceutical companies, university laboratories and funding, less than one percent of initially researched drugs enter the commercial market. In this context, virtual screening (VS) has gained much attention due to several advantages, including timesaving, reduced reagent and consumable costs and the performance of selective analyses regarding the affinity between test molecules and pharmacological targets. Currently, VS is based mainly on algorithms that apply physical and chemistry principles and quantum mechanics to estimate molecule affinities and conformations, among others. Nevertheless, VS has not reached the expected results concerning the improvement of market-approved drugs, comprising less than twenty drugs that have reached this goal to date. In this context, graph neural networks (GNN), a recent deep-learning subtype, may comprise a powerful tool to improve VS results concerning natural products that may be used both simultaneously with standard algorithms or isolated. This review discusses the pros and cons of GNN applied to VS and the future perspectives of this learnable algorithm, which may revolutionize drug discovery if certain obstacles concerning spatial coordinates and adequate datasets, among others, can be overcome.

Molecular Modeling Applied to the Discovery of New Lead Compounds for P2 Receptors Based on Natural Sources.

P2 receptors are a family of transmembrane receptors activated by nucleotides and nucleosides. Two classes have been described in mammals, P2X and P2Y, which are implicated in various diseases. Currently, only P2Y12 has medicines approved for clinical use as antiplatelet agents and natural products have emerged as a source of new drugs with action on P2 receptors due to the diversity of chemical structures. In drug discovery, in silico virtual screening (VS) techniques have become popular because they have numerous advantages, which include the evaluation of thousands of molecules against a target, usually proteins, faster and cheaper than classical high throughput screening (HTS). The number of studies using VS techniques has been growing in recent years and has led to the discovery of new molecules of natural origin with action on different P2X and P2Y receptors. Using different algorithms it is possible to obtain information on absorption, distribution, metabolism, toxicity, as well as predictions on biological activity and the lead-likeness of the selected hits. Selected biomolecules may then be tested by molecular dynamics and, if necessary, rationally designed or modified to improve their interaction for the target. The algorithms of these in silico tools are being improved to permit the precision development of new drugs and, in the future, this process will take the front of drug development against some central nervous system (CNS) disorders. Therefore, this review discusses the methodologies of in silico tools concerning P2 receptors, as well as future perspectives and discoveries, such as the employment of artificial intelligence in drug discovery.

Single-cell Microinjection for Cell Communication Analysis.

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a hemichannel by each neighboring cell to form the gap junction. In mammalian cells, the hemichannel is formed by six connexins, monomers with four transmembrane domains and a C and N terminal within the cytoplasm. Gap junctions permit the exchange of ions, second messengers, and small metabolites. In addition, they have important roles in many forms of cellular communication within physiological processes such as synaptic transmission, heart contraction, cell growth and differentiation. We detail how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells. It is expected that in functional gap junctions, the dye will diffuse from the loaded cell to the connected cells. It is a very useful technique to study gap junctions since you can evaluate the diffusion of the fluorescence in real time. We discuss how to prepare the cells and the micropipette, how to use a micromanipulator and inject a low molecular weight fluorescent dye in an epithelial cell line.

Role of P2 Receptors as Modulators of Rat Eosinophil Recruitment in Allergic Inflammation.

ATP and other nucleotides are released from cells through regulated pathways or following the loss of plasma membrane integrity. Once outside the cell, these compounds can activate P2 receptors: P2X ionotropic receptors and G protein-coupled P2Y receptors. Eosinophils represent major effector cells in the allergic inflammatory response and they are, in fact, associated with several physiological and pathological processes. Here we investigate the expression of P2 receptors and roles of those receptors in murine eosinophils. In this context, our first step was to investigate the expression and functionality of the P2X receptors by patch clamping, our results showed a potency ranking order of ATP>ATPγS> 2meSATP> ADP> αßmeATP> ßγmeATP>BzATP> UTP> UDP>cAMP. This data suggest the presence of P2X1, P2X2 and P2X7. Next we evaluate by microfluorimetry the expression of P2Y receptors, our results based in the ranking order of potency (UTP>ATPγS> ATP > UDP> ADP >2meSATP > αßmeATP) suggests the presence of P2Y2, P2Y4, P2Y6 and P2Y11. Moreover, we confirmed our findings by immunofluorescence assays. We also did chemotaxis assays to verify whether nucleotides could induce migration. After 1 or 2 hours of incubation, ATP increased migration of eosinophils, as well as ATPγS, a less hydrolysable analogue of ATP, while suramin a P2 blocker abolished migration. In keeping with this idea, we tested whether these receptors are implicated in the migration of eosinophils to an inflammation site in vivo, using a model of rat allergic pleurisy. In fact, migration of eosinophils has increased when ATP or ATPγS were applied in the pleural cavity, and once more suramin blocked this effect. We have demonstrated that rat eosinophils express P2X and P2Y receptors. In addition, the activation of P2 receptors can increase migration of eosinophils in vitro and in vivo, an effect blocked by suramin.

Johrei Effects on Water: A Pilot Study by Counting Drops.

BACKGROUND: Water is a key ingredient in the creation and sustainment of life. Moreover, water may be a key vehicle in the processes of energy healing, such as in the preparation of homeopathic remedies and spiritual treatments. Given these properties, the purpose of this study was to investigate whether the application of Johrei to water could lead to significant changes in the hydrodynamic behaviour of the fluid. METHODS: Four regular Johrei practitioners (P1, P2, P3 and P4) were selected for this study. Dripping water produced at the tip of a capillary was used as the hydrodynamic behaviour model. This behaviour was modelled mathematically, and tuning parameters φ4 and τ were used to assess significant differences in the dripping water samples that were subjected to Johrei compared with the samples that were not so treated. The tuning parameters were obtained using the Levenberg-Marquardt fitting algorithm. The data sets for each Johrei practitioner and the control experiment were analysed using ANOVA and a paired t-test. RESULTS: The mathematical model exhibited an excellent fit to our data, generating correlation coefficients (r) greater than or equal to 0.999. Significant differences were observed in both τ (P1 and P2, P < 0.05 and P < 0.01, respectively) and φ4 (P2, P < 0.01). As expected, no significant difference for the control experiment (without Johrei) was observed. CONCLUSIONS: Our results indicated a statistically significant change in the hydrodynamic behaviour of water correlated with Johrei treatment for 50% of the participating Johrei practitioners.

An Improved Method for P2X7R Antagonist Screening.

ATP physiologically activates the P2X7 receptor (P2X7R), a member of the P2X ionotropic receptor family. When activated by high concentrations of ATP (i.e., at inflammation sites), this receptor is capable of forming a pore that allows molecules of up to 900 Da to pass through. This receptor is upregulated in several diseases, particularly leukemia, rheumatoid arthritis and Alzheimer's disease. A selective antagonist of this receptor could be useful in the treatment of P2X7R activation-related diseases. In the present study, we have evaluated several parameters using in vitro protocols to validate a high-throughput screening (HTS) method to identify P2X7R antagonists. We generated dose-response curves to determine the EC50 value of the known agonist ATP and the ICs50 values for the known antagonists Brilliant Blue G (BBG) and oxidized ATP (OATP). The values obtained were consistent with those found in the literature (0.7 ± 0.07 mM, 1.3-2.6 µM and 173-285 µM for ATP, BBG and OATP, respectively) [corrected].The Z-factor, an important statistical tool that can be used to validate the robustness and suitability of an HTS assay, was 0.635 for PI uptake and 0.867 for LY uptake. No inter-operator variation was observed, and the results obtained using our improved method were reproducible. Our data indicate that our assay is suitable for the selective and reliable evaluation of P2X7 activity in multiwell plates using spectrophotometry-based methodology. This method might improve the high-throughput screening of conventional chemical or natural product libraries for possible candidate P2X7R antagonist or agonist.

Caracterização dos receptores P2 em eosinófilos de ratos e possíveis papéis funcionais / Characterization of the P2 receptors in rat eosinophil and possible functional roles

O ATP e outros nucleotídeos podem ser liberados das células para o meio extracelular, através de mecanismos regulados ou pela perda da integridade da membrana. Uma vez no meio extracelular, os nucleotídeos atuam via receptores P2, um grupo de receptores que abrange duas famílias: os receptores P2X que são canais iônicos ativados por ligantes e os receptores P2Y que são receptores acoplados à proteína G. Esses receptores foram caracterizados em diversas células do sistema imune de rato. Contudo, não há na literatura nenhuma caracterização descrita em eosinófilos. Estes são importantes por estarem envolvidos na asma e em diversos processos patológicos e fisiológicos. Por isso, fomos investigar a expressão dos receptores P2 nessas células, usando técnicas de eletrofisiologia, análise da variação de cálcio e imunofluorescência. Na primeira etapa do nosso trabalho, procuramos investigar a expressão de receptores P2X 9ionotrópico) por eletrofisiologia. Através da amplitude da corrente máxima normalizada, obtivemos a seguinte ordem de potência: ATP> ATPyS> 2meSATP> ADP> αβmeATP> βymeATP> BzATP> UTP> UDP> cAMP, sugerindo a presença dos receptores P2x2, P2X4, e P2X7. Nos experimentos de análise de variação de cálcio intracelular obtivemos a seguinte ordem de potência: UTP> ATPyS> ADP> UDP> ATP> BzATP> 2meSATP> αβmeATP> cAMP> Adenosina > βyme ATP, sugerindo a presença de P2X1, P2X2, PaX4, P2x4, P2Y1, P2Y2, P2Y4. Na imunofluorescência verificamos a expressão dos receptores P2Y2, P2Y4 e P2Y13. A partir dessa caracterização, fomos investigar a participação desses receptores na migração, in vitro, dos eosinófilos em sistema de transwell e observamos que o ATP foi capaz de induzir maior migração dos mesmos. Esse efeito foi bloqueado pela suramina um antagonista geral dos receptores P2. Além disso, empregamos o modelo de pleurisia alérgica em ratos para verificar se os receptores P2 poderiam estar implicados na migração dos eosinófilos para algum sítio de inflamação. Nossos resultados mostraram que a suramina diminuiu o número de células na cavidade pleural, indicando a participação de receptores P2, na resposta inflamatória. Nossos resultados demonstraram que eosinófilos de rato expressam receptores P2X e P2Y e que a ativação desses receptores pode aumentar a migração de eosinófilos in vitro e in vivo.

Caracterização dos receptores P2 em eosinófilos de ratos e possíveis papéis funcionais / Characterization of the P2 receivers in eosinophils of rats and possible functional papers

O ATP e outros nucleotídeos podem ser liberados das células para o meio extracelular, através de mecanismos regulados ou pela perda da integridade da membrana. Uma vez no meio extracelular, os nucleotídeos atuam via receptores P2, um grupo de receptores que abrange duas famílias: os receptores P2X que são canais iônicos ativados por ligantes e os receptores P2Y que são receptores acoplados à proteína G. Esses receptores foram caracterizados em diversas células do sistema imune de rato. Contudo, não há na literatura nenhuma caracterização descrita em eosinófilos. Estes são importantes por estarem envolvidos na asma e em diversos processos patológicos e fisiológicos. Por isso, fomos investigar a expressão dos receptores P2 nessas células, usando técnicas de eletrofisiologia, análise da variação de cálcio e imunofluorescência. Na primeira etapa do nosso trabalho, procuramos investigar a expressão de receptores P2X (ionotrópico) por eletrofisiologia. Através da amplitude da corrente máxima normalizada, obtivemos a seguinte ordem de potência: ATP (maior que) ATPyS (maior que) 2meSATP (maior que) ADP (maior que) alfabetameATP (maior que) BetaymeATP (maior que) BzATP (maior que) UTP (maior que) UDP (maior que) cAMP, sugerindo a presença dos receptores P2X(2), P2X(4) e P2X(7). Nos experimentos de análise de variação de cálcio intracelular obtivemos a seguinte ordem de potência: UTP (maior que) ATPyS (maior que) ADP (maior que) UDP (maior que) ATP (maior que) BzATP (maior que) 2meSATP (maior que) alfabetameATP (maior que) cAMP (maior que) Adenosina (maior que) BetaymeATP, sugerindo a presença de P2X(1), P2X(2) e P2X(4), P2Y(1), P2Y(2) e P2Y(4). Na imunofluorescência verificamos a expressão dos receptores P2Y(2), P2Y(4) e P2Y(13). A partir dessa caracterização, fomos investigar a participação desses receptores na migração, in vitro, dos eosinófilos em sistema de transwell e observamos que o ATP foi capaz de induzir maior migração dos mesmos. Esse efeito...in vivo.