Evaluation of SRPK silencing by CRISPR/Cas9 and its interaction with EFHD2 in murine metastatic melanoma

As proteínas cinases serina/arginina (SRPK) são classicamente relacionadas à regulação do splicing do pré-mRNA pela fosforilação de fatores de splicing ricos em dipeptídeos serina e arginina, as proteínas SR. É evidente a importância de estudos que visem elucidar os mecanismos moleculares da atuação das SRPK em diversas vias de sinalização, splicing do pré-mRNA e outros processos celulares, sobretudo relacionados ao desenvolvimento do câncer e formação de metástase, nos quais essas cinases já foram identificadas superexpressas. Neste trabalho, avaliamos o impacto do silenciamento de SRPK em melanoma metastático murino. O silenciamento de SRPK2 na linhagem celular de melanoma, B16F10, prejudicou o desenvolvimento do tumor subcutâneo e a formação de nódulos pulmonares in vivo e diminuiu a proliferação e a invasão celular in vitro. A supressão da expressão de SRPK1 e SRPK2, simultaneamente, diminuiu a capacidade de formação de colônias de B16F10. O mecanismo de ação de SRPK2 no desenvolvimento do melanoma parece estar relacionado à sua atuação na dinâmica de polimerização do citoesqueleto de Actina, pois o silenciamento de SRPK2 diminuiu a formação de F-actina nas células B16F10. Um ensaio de duplo-híbrido em leveduras, realizado anteriormente pelo nosso grupo de pesquisa, mostrou a possível interação entre a região espaçadora de SRPK2 e proteínas relacionadas à regulação do citoesqueleto de Actina. Dentre elas, EFhd2 está envolvida na regulação da dinâmica de polimerização do citoesqueleto de actina e formação de metástase em melanoma. Neste trabalho a interação entre SRPK2 e EFhd2 foi confirmada pelos ensaios de co-imunoprecipitação e GST-pulldown. Ensaios de fosforilação in vitro mostraram que EFhd2 pode ser fosforilada por SRPK2. Portanto, com os resultados obtidos até o momento, é possível sugerir que a diminuição da capacidade metastática de B16F10 em decorrência da supressão da expressão de SRPK2 pode envolver, pelo menos parcialmente, alterações na dinâmica do citoesqueleto de Actina via EFhd2. Esses dados criam novas perspectivas sobre o entendimento da relação funcional entre SRPK2 e a dinâmica do citoesqueleto de Actina no contexto do melanoma metastático e potencialmente em outros tipos de tumores. Palavras-chave: Serine/arginine protein kinase. Melanoma. Metástase.Serine/arginine protein kinases (SRPK) are involved in the regulation of pre-mRNA splicing by the phosphorylation of splicing factors rich in serine and arginine dipeptides (SR proteins). It is clear the importance of studies that aim to elucidate the molecular mechanisms of SRPK action in several signaling pathways, pre-mRNA splicing and other cellular processes. This is especially related to the development of cancer and metastasis formation, in which these overexpressed kinases have already been identified. In this study, we evaluated the effect of SRPK silencing on murine metastatic melanoma. The silencing of SRPK2 in the melanoma cell line B16F10 impaired the development of a subcutaneous tumor and the formation of pulmonary nodules in vivo and decreased the proliferation and the cell invasion in vitro. The simultaneous suppression of the expression of SRPK1 and SRPK2 decreased the colony- forming ability of B16F10 cells. The mechanism of action of SRPK2 in the development of melanoma seems to be related to its role in the polymerization of the Actin cytoskeleton, since the silencing of SRPK2 decreased the formation of F-actin in B16F10 cells. A yeast two- hybrid assay previously performed by our research group showed a possible interaction between the SRPK2 spacer region and some proteins related to the regulation of the Actin cytoskeleton. Among the proteins found, EFhd2 is involved in the regulation of the polymerization dynamics of the Actin cytoskeleton and in the formation of metastases in melanoma. In this study, the interaction between SRPK2 and EFhd2 was confirmed by performing co-immunoprecipitation and GST pull-down assays. In vitro phosphorylation assays have demonstrated that EFhd2 can be phosphorylated by SRPK2. Therefore, from the results obtained so far, it is possible to suggest that the decrease in the metastatic ability of B16F10 cells due to the suppression of SRPK2 expression, may involve at least partially, changes to the dynamics of the Actin cytoskeleton via EFhd2. These data create new perspectives on the understanding of the functional relationship between SRPK2 and the dynamics of the Actin cytoskeleton in the context of metastatic melanoma and potentially in other types of tumors. Keywords: Serine/arginine protein kinase. Melanoma. Metastasis.Coordenação de Aperfeiçoamento de Pessoal de Nível Superio

Sapucaia nuts (Lecythis pisonis) modulate the hepatic inflammatory and antioxidant metabolism activity in rats fed high-fat diets

Lecythis pisonis Cambess is commonly known as “sapucaia” nut. Previous studies show that it is rich in unsaturated fatty acids and in antioxidant minerals. The aim of the present study was to assess the antioxidant and anti-inflamatory effects of this nut after its introduction into a control (AIN-93G) or highfat diet in Wistar rats. The animals were divided into four groups: a control diet, the same control diet supplemented with sapucaia nuts, a high-fat diet or the high-fat diet supplemented with sapucaia nuts and were fed with these diets for 14 or 28 days. The gene expression of the markers tumor necrosis factor (TNF)-α NFκB (p65) zinc superoxide dismutase (ZnSOD) and heat shock protein 72 (HSP72) was determined by the chain reaction to the quantitative reverse transcription polymerase chain reaction (q- PCR). The antioxidant activity was also measured as thiobarbituric acid reactive substances (TBARS) through the activity of the SOD enzyme. The groups treated with “sapucaia” nuts presented reduced lipid peroxidation values and increased ZnSOD and HSP72 gene expression, as well as decreased TNF- α and NFκB (p65) gene expression levels. The significant results showed that “sapucaia” could serve as a potential source of antioxidants and as a protector agent for the examined animals.Keywords: Sapucaia nuts, inflammation, oxidative stress, gene expressio

Iron oxide polyaniline-coated nanoparticles modulate tumor microenvironment in breast cancer: an in vitro study on the reprogramming of tumor-associated macrophages

Abstract Background Breast cancer is the neoplastic disease with the highest incidence and mortality in the female population worldwide. Treatment remains challenging due to various factors. Therefore, it is of great importance to develop new therapeutic strategies that promote the safe destruction of neoplastic cells without compromising patients' quality of life. Among advances in the treatment of breast cancer, immunotherapy stands out as a promising trend. Recent studies have demonstrated the potential of iron oxide nanoparticles in promoting the reprogramming of M2 macrophages (pro-tumor phenotype) into M1 macrophages (anti-tumor phenotype) within the tumor microenvironment, resulting in potent antitumor effects. In this study, the effect of polyaniline-coated iron oxide nanoparticles (Pani/y-Fe2O3) on macrophage polarization and breast cancer cell death was investigated. Methods The non-cytotoxic concentration of nanoparticles was determined using the MTT assay. For in vitro co-culture experiments, breast cancer cell lines MCF -7 and MDA-MB -231 and macrophages THP-1 were co-cultured in a Transwell system and then the effects of Pani/y-Fe2O3 on cell viability, gene expression, cytokine profile, and oxidative stress markers were investigated. Results The results showed that Pani/y-Fe2O3 nanoparticles induced M2-to-M1 macrophage polarization in both cell lines through different pathways. In MCF -7 and THP-1 macrophage co-culture, the study showed a decrease in cytokine levels IL -1β, upregulation of M1-associated genes (IL-12, TNF-α) in macrophages, resulting in increased MCF -7 cell death by apoptosis (caspase 3/7+). In MDA-MB -231 co-cultures, increases in cytokines IL -6, IL -1β, and oxidative stress markers were observed, as well as upregulation of the inducible nitric oxide synthase (iNOS) gene in macrophages, leading to tumor cell death via apoptosis-independent pathways (Sytox+). Conclusions These findings highlight the potential of Pani/y-Fe2O3 as a promising therapeutic approach in the context of breast cancer treatment by effectively reprogramming M2 macrophages into an anti-tumor M1 phenotype, Pani/y-Fe2O3 nanoparticles demonstrated the ability to elicit antitumor effects in both MCF-7 and MDA-MB-231 breast cancer cell lines

Table1_Impaired expression of serine/arginine protein kinase 2 (SRPK2) affects melanoma progression.PDF

Melanoma is one of the most aggressive tumors, and its lethality is associated with the ability of malignant cells to migrate and invade surrounding tissues to colonize distant organs and to generate widespread metastasis. The serine/arginine protein kinases 1 and 2 (SRPK1 and SRPK2) are classically related to the control of pre-mRNA splicing through SR protein phosphorylation and have been found overexpressed in many types of cancer, including melanoma. Previously, we have demonstrated that the pharmacological inhibition of SRPKs impairs pulmonary colonization of metastatic melanoma in mice. As the used compounds could target at least both SRPK1 and SRPK2, here we sought to obtain additional clues regarding the involvement of these paralogs in melanoma progression. We analyzed single-cell RNA sequencing data of melanoma patient cohorts and found that SRPK2 expression in melanoma cells is associated with poor prognosis. Consistently, CRISPR-Cas9 genome targeting of SRPK2, but not SRPK1, impaired actin polymerization dynamics as well as the proliferative and invasive capacity of B16F10 cells in vitro. In further in vivo experiments, genetic targeting of SRPK2, but not SRPK1, reduced tumor progression in both subcutaneous and caudal vein melanoma induction models. Taken together, these findings suggest different functional roles for SRPK1/2 in metastatic melanoma and highlight the relevance of pursuing selective pharmacological inhibitors of SRPK2.</p

Image1_Impaired expression of serine/arginine protein kinase 2 (SRPK2) affects melanoma progression.pdf

Melanoma is one of the most aggressive tumors, and its lethality is associated with the ability of malignant cells to migrate and invade surrounding tissues to colonize distant organs and to generate widespread metastasis. The serine/arginine protein kinases 1 and 2 (SRPK1 and SRPK2) are classically related to the control of pre-mRNA splicing through SR protein phosphorylation and have been found overexpressed in many types of cancer, including melanoma. Previously, we have demonstrated that the pharmacological inhibition of SRPKs impairs pulmonary colonization of metastatic melanoma in mice. As the used compounds could target at least both SRPK1 and SRPK2, here we sought to obtain additional clues regarding the involvement of these paralogs in melanoma progression. We analyzed single-cell RNA sequencing data of melanoma patient cohorts and found that SRPK2 expression in melanoma cells is associated with poor prognosis. Consistently, CRISPR-Cas9 genome targeting of SRPK2, but not SRPK1, impaired actin polymerization dynamics as well as the proliferative and invasive capacity of B16F10 cells in vitro. In further in vivo experiments, genetic targeting of SRPK2, but not SRPK1, reduced tumor progression in both subcutaneous and caudal vein melanoma induction models. Taken together, these findings suggest different functional roles for SRPK1/2 in metastatic melanoma and highlight the relevance of pursuing selective pharmacological inhibitors of SRPK2.</p

Table_1_Retrospective cohort study to evaluate the continuous use of anticholesterolemics and diuretics in patients with COVID-19.DOCX

PurposeThe purpose of this study is to evaluate the interference of the continuous use of drug classes in the expression of biomarkers during the first week of hospitalization and in the prognosis of patients with COVID-19.MethodsThe patients diagnosed with COVID-19 and confirmed with SARS-CoV-2 by RT-qPCR assay underwent the collection of fasting whole blood samples for further analysis. Other data also extracted for this study included age, sex, clinical symptoms, related comorbidities, smoking status, and classes of continuous use. Routine serum biochemical parameters, including alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, C-reactive protein, N-terminal fragment of B-type natriuretic peptide, and cardiac troponin, were measured.ResultsIn this cross-sectional study, a total of 176 patients with COVID-19 hospitalizations were included. Among them, 155 patients were discharged (88.5%), and 21 patients died (12%). Among the drug classes evaluated, we verified that the continuous use of diuretic 4.800 (1.853–11.67) (p = 0.0007) and antihypercholesterolemic 3.188 (1.215–7.997) (p = 0.0171) drug classes presented a significant relative risk of death as an outcome when compared to the group of patients who were discharged. We evaluated biomarkers in patients who used continuous antihypercholesterolemic and diuretic drug classes in the first week of hospitalization. We observed significant positive correlations between the levels of CRP with cardiac troponin (r = 0.714), IL-6 (r = 0.600), and IL-10 (r = 0.900) in patients who used continuous anticholesterolemic and diuretic drug classes and were deceased. In these patients, we also evaluated the possible correlations between the biomarkers AST, NT-ProBNP, cardiac troponin, IL-6, IL-8, and IL-10. We observed a significantly negative correlations in AST levels with NT-ProBNP (r = −0.500), cardiac troponin (r = −1.00), IL-6 (r = −1.00), and IL-10 (r = −1.00) and a positive correlation with IL-8 (r = 0.500). We also observed significant negative correlation in the levels of NT-ProBNP with IL-10 (r = −0.800) and a positive correlation with cardiac troponin (r = 0.800). IL-6 levels exhibited positive correlations with cardiac troponin (r = 0.800) and IL-10 (r = 0.700).ConclusionIn this study, we observed that hospitalized COVID-19 patients who continued using anticholesterolemic and diuretic medications showed a higher number of correlations between biomarkers, indicating a poorer clinical prognosis. These correlations suggest an imbalanced immune response to injuries caused by SARS-CoV-2.</p

Image_1_Retrospective cohort study to evaluate the continuous use of anticholesterolemics and diuretics in patients with COVID-19.TIF

PurposeThe purpose of this study is to evaluate the interference of the continuous use of drug classes in the expression of biomarkers during the first week of hospitalization and in the prognosis of patients with COVID-19.MethodsThe patients diagnosed with COVID-19 and confirmed with SARS-CoV-2 by RT-qPCR assay underwent the collection of fasting whole blood samples for further analysis. Other data also extracted for this study included age, sex, clinical symptoms, related comorbidities, smoking status, and classes of continuous use. Routine serum biochemical parameters, including alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, C-reactive protein, N-terminal fragment of B-type natriuretic peptide, and cardiac troponin, were measured.ResultsIn this cross-sectional study, a total of 176 patients with COVID-19 hospitalizations were included. Among them, 155 patients were discharged (88.5%), and 21 patients died (12%). Among the drug classes evaluated, we verified that the continuous use of diuretic 4.800 (1.853–11.67) (p = 0.0007) and antihypercholesterolemic 3.188 (1.215–7.997) (p = 0.0171) drug classes presented a significant relative risk of death as an outcome when compared to the group of patients who were discharged. We evaluated biomarkers in patients who used continuous antihypercholesterolemic and diuretic drug classes in the first week of hospitalization. We observed significant positive correlations between the levels of CRP with cardiac troponin (r = 0.714), IL-6 (r = 0.600), and IL-10 (r = 0.900) in patients who used continuous anticholesterolemic and diuretic drug classes and were deceased. In these patients, we also evaluated the possible correlations between the biomarkers AST, NT-ProBNP, cardiac troponin, IL-6, IL-8, and IL-10. We observed a significantly negative correlations in AST levels with NT-ProBNP (r = −0.500), cardiac troponin (r = −1.00), IL-6 (r = −1.00), and IL-10 (r = −1.00) and a positive correlation with IL-8 (r = 0.500). We also observed significant negative correlation in the levels of NT-ProBNP with IL-10 (r = −0.800) and a positive correlation with cardiac troponin (r = 0.800). IL-6 levels exhibited positive correlations with cardiac troponin (r = 0.800) and IL-10 (r = 0.700).ConclusionIn this study, we observed that hospitalized COVID-19 patients who continued using anticholesterolemic and diuretic medications showed a higher number of correlations between biomarkers, indicating a poorer clinical prognosis. These correlations suggest an imbalanced immune response to injuries caused by SARS-CoV-2.</p

Table_2_Retrospective cohort study to evaluate the continuous use of anticholesterolemics and diuretics in patients with COVID-19.DOCX

PurposeThe purpose of this study is to evaluate the interference of the continuous use of drug classes in the expression of biomarkers during the first week of hospitalization and in the prognosis of patients with COVID-19.MethodsThe patients diagnosed with COVID-19 and confirmed with SARS-CoV-2 by RT-qPCR assay underwent the collection of fasting whole blood samples for further analysis. Other data also extracted for this study included age, sex, clinical symptoms, related comorbidities, smoking status, and classes of continuous use. Routine serum biochemical parameters, including alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, C-reactive protein, N-terminal fragment of B-type natriuretic peptide, and cardiac troponin, were measured.ResultsIn this cross-sectional study, a total of 176 patients with COVID-19 hospitalizations were included. Among them, 155 patients were discharged (88.5%), and 21 patients died (12%). Among the drug classes evaluated, we verified that the continuous use of diuretic 4.800 (1.853–11.67) (p = 0.0007) and antihypercholesterolemic 3.188 (1.215–7.997) (p = 0.0171) drug classes presented a significant relative risk of death as an outcome when compared to the group of patients who were discharged. We evaluated biomarkers in patients who used continuous antihypercholesterolemic and diuretic drug classes in the first week of hospitalization. We observed significant positive correlations between the levels of CRP with cardiac troponin (r = 0.714), IL-6 (r = 0.600), and IL-10 (r = 0.900) in patients who used continuous anticholesterolemic and diuretic drug classes and were deceased. In these patients, we also evaluated the possible correlations between the biomarkers AST, NT-ProBNP, cardiac troponin, IL-6, IL-8, and IL-10. We observed a significantly negative correlations in AST levels with NT-ProBNP (r = −0.500), cardiac troponin (r = −1.00), IL-6 (r = −1.00), and IL-10 (r = −1.00) and a positive correlation with IL-8 (r = 0.500). We also observed significant negative correlation in the levels of NT-ProBNP with IL-10 (r = −0.800) and a positive correlation with cardiac troponin (r = 0.800). IL-6 levels exhibited positive correlations with cardiac troponin (r = 0.800) and IL-10 (r = 0.700).ConclusionIn this study, we observed that hospitalized COVID-19 patients who continued using anticholesterolemic and diuretic medications showed a higher number of correlations between biomarkers, indicating a poorer clinical prognosis. These correlations suggest an imbalanced immune response to injuries caused by SARS-CoV-2.</p

Brazilian Flora 2020: Leveraging the power of a collaborative scientific network

International audienceThe shortage of reliable primary taxonomic data limits the description of biological taxa and the understanding of biodiversity patterns and processes, complicating biogeographical, ecological, and evolutionary studies. This deficit creates a significant taxonomic impediment to biodiversity research and conservation planning. The taxonomic impediment and the biodiversity crisis are widely recognized, highlighting the urgent need for reliable taxonomic data. Over the past decade, numerous countries worldwide have devoted considerable effort to Target 1 of the Global Strategy for Plant Conservation (GSPC), which called for the preparation of a working list of all known plant species by 2010 and an online world Flora by 2020. Brazil is a megadiverse country, home to more of the world's known plant species than any other country. Despite that, Flora Brasiliensis, concluded in 1906, was the last comprehensive treatment of the Brazilian flora. The lack of accurate estimates of the number of species of algae, fungi, and plants occurring in Brazil contributes to the prevailing taxonomic impediment and delays progress towards the GSPC targets. Over the past 12 years, a legion of taxonomists motivated to meet Target 1 of the GSPC, worked together to gather and integrate knowledge on the algal, plant, and fungal diversity of Brazil. Overall, a team of about 980 taxonomists joined efforts in a highly collaborative project that used cybertaxonomy to prepare an updated Flora of Brazil, showing the power of scientific collaboration to reach ambitious goals. This paper presents an overview of the Brazilian Flora 2020 and provides taxonomic and spatial updates on the algae, fungi, and plants found in one of the world's most biodiverse countries. We further identify collection gaps and summarize future goals that extend beyond 2020. Our results show that Brazil is home to 46,975 native species of algae, fungi, and plants, of which 19,669 are endemic to the country. The data compiled to date suggests that the Atlantic Rainforest might be the most diverse Brazilian domain for all plant groups except gymnosperms, which are most diverse in the Amazon. However, scientific knowledge of Brazilian diversity is still unequally distributed, with the Atlantic Rainforest and the Cerrado being the most intensively sampled and studied biomes in the country. In times of “scientific reductionism”, with botanical and mycological sciences suffering pervasive depreciation in recent decades, the first online Flora of Brazil 2020 significantly enhanced the quality and quantity of taxonomic data available for algae, fungi, and plants from Brazil. This project also made all the information freely available online, providing a firm foundation for future research and for the management, conservation, and sustainable use of the Brazilian funga and flora