Chemical characterization of Brazilian savannah Byrsonima species (muricis) and their impact on genomic instability and chemopreventive effects.

The identification of new drugs with few or no adverse effects is of great interest worldwide. In cancer therapy, natural products have been used as chemopreventive and chemotherapeutic agents. Plants from the Brazilian savannah belonging to the Byrsonima genus are popularly known as muricis and have attracted much attention due to their various pharmacological activities. However, there are currently no data on these plants concerning their use as chemopreventive or chemotherapeutic agents in human cell lines. The present study assessed the potential of B. correifolia, B. verbascifolia, B. crassifolia, and B. intermedia extracts as natural alternatives in the prevention and/or treatment of cancer. The chemical constituents present in each extract were analyzed by electrospray ionization-mass spectrometry (ESI-MSN). The mutagenic/antimutagenic (micronucleus assay), genotoxic/antigenotoxic (comet assay), apoptotic/necrotic (acridine orange/ethidium bromide uptake), and oxidative/antioxidative (CM-H2DCFDA) effects of the extracts and their influence on gene expression (RTqPCR) were investigated in nonmetabolizing gastric (MNP01) and metabolizing hepatocarcinoma (HepG2) epithelial cells to evaluate the effects of metabolism on the biological activities of the extracts. The genotoxicity, mutagenicity, and apoptotic effects observed in HepG2 cells with B. correifolia and B. verbascifolia extracts are probably associated with the presence of proanthocyanidins and amentoflavone. In MNP01 cells, none of the four extracts showed mutagenic effects. B. crassifolia and B. intermedia extracts exhibited strong antimutagenicity and enhanced detoxification in HepG2 cells and antioxidant capacities in both types of cells, possibly due to the presence of gallic and quinic acids, which possess chemopreventive properties. This study identifies for the first time B. correifolia and B. verbascifolia extracts as potential agents against hepatocarcinoma and B. crassifolia and B. intermedia extracts as putative chemopreventive agents.

A Synthetic Snake-Venom-Based Tripeptide Protects PC12 Cells from the Neurotoxicity of Acrolein by Improving Axonal Plasticity and Bioenergetics.

The synthetic peptide p-BTX-I is based on the native peptide (formed by glutamic acid, valine and tryptophan) isolated from Bothrops atrox venom. We have previously demonstrated its neuroprotective and neurotrophic properties in PC12 cells treated with the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium (MPP+). Now, we have investigated the neuroprotective effects and mechanisms of p-BTX-I against the toxicity of acrolein in PC12 cells. Studies have demonstrated that acrolein might play an important role in the etiology of Alzheimer's disease (AD), which is characterized by neuronal and synaptic loss. Our results showed that not only acrolein reduced cell differentiation and cell viability, but also altered the expression of markers of synaptic communication (synapsin I), energy metabolism (AMPK-α, Sirt I and glucose uptake), and cytoskeleton (ß-III-tubulin). Treatment with p-BTX-I increased the percentage of differentiation in cells treated with acrolein and significantly attenuated cell viability loss, besides counteracting the negative effects of acrolein on synapsin I, AMPK-α, Sirt I, glucose uptake, and ß-III-tubulin. Additionally, p-BTX-I alone increased the expression of apolipoprotein E (apoE) gene, associated with the proteolytic degradation of ß-amyloid peptide aggregates, a hallmark of AD. Taken together, these findings demonstrate that p-BTX-I protects against acrolein-induced neurotoxicity and might be a tool for the development of novel drugs for the treatment of neurodegenerative diseases.

Assessing the cytotoxic potential of glycoalkaloidic extract in nanoparticles against bladder cancer cells.

OBJECTIVE: This study proposed to use the nanotechnology to deliver glycoalkaloidic extract (AE) to bladder cancer cells, evaluating their activity in 2D and 3D models and the biological mechanism of cell death. METHODS: NPs were prepared by nanoprecipitation method using polylactic acid (PLA) and characterized considering their size, charge, particle concentration and stability. The cytotoxicity was evaluated in 2D and 3D model, and the apoptosis and cell cycle were investigated using flow cytometry. KEY FINDINGS: NPs loading AE (NP-AE) had diameter around 125 ± 6 nm (PdI <0.1) and negative charge. The encapsulation efficiency of SM and SS was higher than 85% for both compounds. The obtained formulation showed a significant in-vitro cytotoxic effect against RT4 cells in a dose-dependent manner with IC50 two fold lower than the free AE. The cytotoxic effect of NP-AE was mediated by apoptosis and cell cycle arrested in the S phase. RT4 cells cultured under 3D conditions exhibited a higher resistance to the treatments (IC50 ~ three fold higher than in 2D cell culture). CONCLUSION: The NP-AE might be a promising nanocarrier to load and deliver glycoalkaloids against bladder cancer.

Immunomodulatory actions and epigenetic alterations induced by proteases from Bothrops snake venoms in human immune cells.

The aim of this study was to evaluate the immunomodulatory effects of two toxins from Bothrops snake venoms (the P-I metalloprotease Batroxase and the thrombin-like serine protease Moojase) on human peripheral blood mononuclear cells (PBMC), also investigating changes in the expression of genes related to epigenetic alterations and their immunotherapeutic potential. After 24 h of PBMC stimulation, Batroxase (2 µg/mL) and Moojase (4 µg/mL) increased some cytokine levels (including IL-6 and IL-10), but did not promote cell death processes (apoptosis/necrosis) or alterations in the global DNA methylation levels. Gene expression experiments (RT-qPCR) showed that most of the genes with altered transcript levels encode enzymes that act on histones, such as acetyltransferases (HAT1), deacetylases (HDACs), methyltransferases (DOT1L) or demethylases (KDM5B), indicating that these toxins may alter gene regulation through epigenetic changes mainly related to histones and to methyl-CpG binding proteins (MECP2). Subsequently, the immunotherapeutic potential of these toxins was evaluated using in vitro cytotoxicity assays with NK cells and K562 leukemic cells. Both toxins were able to potentiate the NK cell cytotoxic effects against K562 tumor cells, and the effect of Batroxase was dependent on the concomitant stimulus with IL-2, whereas Moojase increased the NK cytotoxicity independently of IL-2. Thus, Batroxase and Moojase presented interesting immunomodulatory effects that could be explored for the development of new strategies in anticancer immunotherapies.

CR-LAAO causes genotoxic damage in HepG2 tumor cells by oxidative stress.

Snake venom L-amino acid oxidases (SV-LAAOs) are enzymes of great interest in research due to their many biological effects with therapeutic potential. CR-LAAO, an L-amino acid oxidase from Calloselasma rhodostoma snake venom, is a well described SV-LAAO with immunomodulatory, antiparasitic, microbicidal, and antitumor effects. In this study, we evaluated the genotoxic potential of this enzyme in human peripheral blood mononuclear cells (PBMC) and HepG2 tumor cells, as well as its interaction with these cells, its impact on the expression of DNA repair and antioxidant pathway genes, and reactive oxygen species (ROS)-induced intracellular production. Flow cytometry analysis of FITC-labelled CR-LAAO showed higher specificity of interaction with HepG2 cells than PBMC. Moreover, CR-LAAO significantly increased intracellular levels of ROS only in HepG2 tumor cells, as assessed by fluorescence. CR-LAAO also induced genotoxicity in HepG2 cells and PBMC after 4 h of stimulus, with DNA damages persisting in HepG2 cells after 24 h. To investigate the molecular basis underlying the genotoxicity attributed to CR-LAAO, we analyzed the expression profile (mRNA levels) of 44 genes involved in DNA repair and antioxidant pathways in HepG2 cells by RT2 Profiler polymerase chain reaction array. CR-LAAO altered the tumor cell expression of DNA repair genes, with two downregulated (XRCC4 and TOPBP1) and three upregulated (ERCC6, RAD52 and CDKN1) genes. In addition, two genes of the antioxidant pathway were upregulated (GPX3 and MPO), probably in an attempt to protect tumor cells from oxidative damage. In conclusion, our data suggest that CR-LAAO possesses higher binding affinity to HepG2 tumor cells than to PBMC, its genotoxic mechanism is possibly caused by the oxidative stress related to the production of H2O2, and is also capable of modulating genes related to the DNA repair system and antioxidant pathways.

Simulated medical consultations with standardized patients: In-depth debriefing based on dealing with emotions / Simulação de consultas médicas com pacientes padronizados: utilizando um debriefing aprofundado baseado nas emoções do paciente e do estudante

ABSTRACT In general, students have few opportunities to address their emotions under the guidance of an experienced physician, which can undermine their self-confidence to deal with real patients in stressful situations. Emotional detachment and cynicism are defense mechanisms, which can emerge as a consequence. The consolidation of a professional identity committed to patients' interests can become a challenge when medical students are not comfortable in their role as caregivers. In general, we consider that the undergraduate medical curriculum has been insufficient in providing appropriate environments for students to reflect on professional identity formation and on the future challenges of their profession. Objective: To develop an in-depth debriefing to address students' emotions and professional identity formation in the context of a simulation activity with simulated patients at a medical school in Brazil. Methods: The authors conducted a simulated medical consultation activity using standardized patients (SPs) with an in-depth debriefing based on the feelings of the patient and the student. During each encounter the formation and consolidation of professional identity was discussed. Fourth- and sixth-year medical students (n=551) participated and answered a questionnaire about the activity and the learning outcome. Results: The students felt comfortable during the activity, due to "openness to dialogue", "proximity with colleagues and teachers" and the "judgment-free environment". More than 90% reported that what they had learned would be useful in their professional and personal lives, providing a greater "understanding of emotions", "empathy", "ability to listen" and "ability to deal with conflicts". More than half of them were motivated to study, especially "doctor-patient relationship", "treatment", "common diseases" and "medicine in general". Students considered the activity important for retrieving the initial reasons that had led them to embarking on the medical profession in the first place. Conclusions: Reflecting on disease and its impact on patients' daily life may motivate learning in medicine, allowing for the recovery of the personal and social meaning of its practice. In-depth debriefing was important to nurture professional identity committed to empathy and patients' interests. Activities planned to discuss the influence and importance of emotions in medical practice can help students to reconcile personal and professional identities.

RESUMO Em geral, os estudantes de medicina têm poucas oportunidades para refletir sobre suas emoções guiados por um médico mais experiente, e isto pode levar a uma diminuição da sua autoconfiança para lidar com pacientes reais, particularmente em situações de estresse. Como consequência podem surgir o distanciamento emocional e o cinismo. Neste contexto, a consolidação de uma identidade profissional comprometida com os interesses do paciente pode ser um desafio se os estudantes não estiverem confortáveis em seus papéis de cuidadores. Muitas vezes, o currículo médico não cria oportunidades suficientes para refletir sobre o desenvolvimento da identidade profissional e sobre os desafios da prática médica futura. Objetivo: Desenvolver um debriefing estendido e profundo para abordar a dimensão afetiva das consultas médicas e a formação da identidade profissional no contexto de uma atividade de simulação com pacientes padronizados em uma escola médica no Brasil. Métodos: Os autores conduziram uma atividade de simulação de consultas médicas com paciente padronizado com um debriefing estendido baseado nas emoções do paciente e do estudante. Durante cada um dos encontros a formação e consolidação da identidade profissional foram abordadas. Alunos do quarto e sexto ano médicos (n=551) participaram das atividades e responderam um questionário sobre a atividade e sobre os objetivos alcançados. Resultados: Os estudantes sentiram-se confortáveis durante a atividade devido a "abertura para o diálogo", "proximidade com professores e colegas" e um "ambiente livre de julgamentos". Mais de 90% dos estudantes considerou que o aprendizado será aplicado tanto em suas vidas profissionais como em suas vidas pessoais, por um maior "entendimento das emoções", "empatia", "habilidade para ouvir" e "habilidade para lidar com conflitos". Mais da metade dos estudantes sentiu-se motivada a estudar, especialmente "relação médico-paciente", "tratamento", "doenças comuns" e "medicina em geral". A atividade foi considerada importante para resgatar a motivação inicial que os levaram a escolher o curso médico. Conclusões: Refletir sobre as doenças e seus impactos na vida dos pacientes pode motivar os estudantes a aprender medicina, permitindo o resgate do significado pessoal e social de sua prática. O aprofundamento do debriefing foi importante para nutrir uma identidade profissional comprometida com a empatia e com os interesses dos pacientes. Atividades planejadas para abordar a influência e a importância das emoções na prática médica podem ajudar os estudantes no processo de reconciliação entre suas identidades pessoal e profissional.

LDH, proliferation curves and cell cycle analysis are the most suitable assays to identify and characterize new phytotherapeutic compounds.

Brazilian flora biodiversity has been widely investigated to identify effective and safe phytotherapeutic compounds. Among the investigated plant species, the Byrsonima genus exhibits promising biological activities. This study aimed at evaluating the cytotoxicity of B. correifolia, B. verbascifolia, B. fagifolia and B. intermedia extracts using different assays in two cell lines (primary gastric and HepG2 cells). The different extract concentrations effects on cell viability were assayed using the MTT, aquabluer, neutral red and LDH assays. Non-cytotoxic concentrations were selected to generate cell proliferation curves and to assess cell cycle kinetics by flow cytometry. Byrsonima extracts differentially affected cell viability depending on the metabolic cellular state and the biological parameter evaluated. B. fagifolia and B. intermedia extracts exhibited lower cytotoxic effects than B. correifolia and B. verbascifolia in all assays. The results obtained with LDH and flow cytometry assays were more reliable, suggesting that they can be useful in the screening for herbal medicine and to further characterize these extracts as phytotherapeutic compounds.

A simplified ultrasound-based edema score to assess lung injury and clinical severity in septic patients.

BACKGROUND: Lung ultrasound (US) is an excellent tool to assess lung edema in a myriad of different clinical situations. We hypothesized that lung US might also be a good prognostic and management instrument in septic patients, regardless of disease severity. METHODS: This was a prospective observational cohort study at an urban academic emergency department (ED). Inclusion criteria were as follows: septic patients, at least 18 years old, admitted at the ED of a tertiary hospital. A simplified lung edema scoring system (SLESS) was developed, and 6 thoracic regions were evaluated. Four different lung US patterns were considered, from normal aeration to total consolidation. To evaluate disease severity, the SLESS was compared with the Mortality in Emergency Department Sepsis Score and the third version of the Simplified Acute Physiology Score scoring systems. Aiming to assess the effect of the lung edema in the gas exchange, the SLESS was compared with the Pao2/fraction of inspired oxygen ratio. RESULTS: Sixty-one patients were enrolled in a 3-month period. The SLESS had a good correlation with the Mortality in Emergency Department Sepsis Score and Simplified Acute Physiology Score (r = 0.53 and r = 0.55, respectively; P < .001 for both) and a negative correlation with the Pao2/fraction of inspired oxygen ratio (r = -0.62; P < .001). The SLESS also showed correlation with the respiratory rate (r = 0.45; P = .0003). The odds ratio for death related to the SLESS was 1.370 (95% confidence interval, 1.109-1.691; P = .0035). CONCLUSION: The SLESS is an easy and practical scoring system. It might be a useful tool to predict severity of disease in sepsis patients. The SLESS might also be able to be correlated with the oxygen exchange.