Crop-Livestock Integration Improves Physical Soil, Agronomic and Environmental Aspects in Soybean Cultivation.

Soybean is one of the most widely grown crops in the world and technologies are increasingly needed to increase productivity without impacting environmental degradation. In this context, the aim was to evaluate the action of forage plants of the genus Brachiaria sp. in crop-livestock integration on physical soil, agronomic and environmental aspects of soybean cultivation. The experiment was conducted in a subdivided plot design with seven integrated systems corresponding to the previous cultivation of Paiaguas palisadegrass, Xaraes palisadegrass and Ruziziensis grass in monocropping and intercropped with maize, as well as maize in monocropping. In the subplots, two grass management systems were evaluated: free growth and a grazing simulation cut. The bulk density and least limiting water range were assessed using soil samples and, after the pastures were desiccated when the soybean crop was planted, straw decomposition and plantability. A soil physics diagnosis by the bulk density and least limiting water range showed that the Paiaguas palisadegrass and Xaraes palisadegrass improved the soil environment due to biological soil loosening. The remaining mulch biomass did not affect soybean sowing and the adoption of Brachiaria sp. grass in the off-season, in addition to contributing to the provision of environmental services, and did not compromise grain productivity in succession.

Evaluation of the RDW Index (Red Cell Distribution Width) in Women with Breast Cancer Treated with Doxorubicin in a One-Year Follow-Up Study.

Breast cancer is the most common cancer and the most frequent cause of death in women. Doxorubicin, an anthracycline, is an important drug due to its efficacy in treating solid cancers, especially breast cancer. However, this drug is often responsible for cardiotoxicity that may affect more than 25% of patients. This study aimed to evaluate the red cell distribution width (RDW) in women with breast cancer to monitor adverse events associated with the use of doxorubicin. A prospective study of 80 women with breast malignancy undergoing neoadjuvant doxorubicin-based chemotherapy was conducted. The patients were evaluated at baseline (T0), just after the last cycle of chemotherapy with doxorubicin (T1), and 1 year after the treatment (T2). There was a significant increase over the time points for the RDW (p < 0.001). There was a negative correlation between the RDW and C-reactive protein (CRP) levels at T1. The RDW did not show a significant difference between the groups classified according to cardiotoxicity. Based on these results, the RDW is a cost-effective test that shows a relationship with the doxorubicin response, but not with cardiotoxicity. It is a potential biomarker to evaluate patients with breast cancer after they receive chemotherapy with doxorubicin.

Germline Mutations Landscape in a Cohort of the State of Minas Gerais, Brazil, in Patients Who Underwent Genetic Counseling for Gynecological and Breast Cancer.

OBJECTIVE: The present study evaluated the profile of germline mutations present in patients who underwent genetic counseling for risk assessment for breast cancer (BC), ovarian cancer (OC), and endometrial cancer (EC) with a possible hereditary pattern. METHODS: Medical records of 382 patients who underwent genetic counseling after signing an informed consent form were analyzed. A total of 55.76% of patients (213/382) were symptomatic (personal history of cancer), and 44.24% (169/382) were asymptomatic (absence of the disease). The variables analyzed were age, sex, place of birth, personal or family history of BC, OC, EC, as well as other types of cancer associated with hereditary syndromes. The Human Genome Variation Society (HGVS) nomenclature guidelines were used to name the variants, and their biological significance was determined by comparing 11 databases. RESULTS: We identified 53 distinct mutations: 29 pathogenic variants, 13 variants of undetermined significance (VUS), and 11 benign. The most frequent mutations were BRCA1 c.470_471delCT, BRCA1 c.4675 + 1G > T, and BRCA2 c.2T> G. Furthermore, 21 variants appear to have been described for the first time in Brazil. In addition to BRCA1/2 mutations, variants in other genes related to hereditary syndromes that predispose to gynecological cancers were found. CONCLUSION: This study allowed a deeper understanding of the main mutations identified in families in the state of Minas Gerais and demonstrates the need to assess the family history of non-gynecological cancer for risk assessment of BC, OC, and EC. Moreover, it is an effort that contributes to population studies to evaluate the cancer risk mutation profile in Brazil.

OBJETIVO: O presente estudo avaliou o perfil de mutações germinativas presentes em pacientes submetidas a aconselhamento genético para avaliação de risco para câncer de mama (CM), câncer de ovário (OC) e câncer de endométrio (CE) com possível padrão hereditário. MéTODOS: Foram analisados os prontuários de 382 pacientes que realizaram aconselhamento genético após consentimento informado. Um total de 55,76% dos pacientes (213/382) eram sintomáticos (história pessoal de câncer), e 44,24% (169/382) eram assintomáticos (ausência da doença). As variáveis analisadas foram idade, sexo, naturalidade, história pessoal ou familiar de CM, OC, CE bem como outros tipos de câncer associados a síndromes hereditárias. As diretrizes de nomenclatura da Human Genome Variation Society (HGVS) foram usadas para nomear as variantes e seu significado biológico foi determinado pela comparação de 11 bancos de dados. RESULTADOS: Identificamos 53 mutações distintas: 29 variantes patogênicas, 13 variantes de significado indeterminado e 11 benignas. As mutações mais frequentes foram BRCA1 c.470_471delCT, BRCA1 c.4675 + 1G > T e BRCA2 c.2T > G. Além disso, 21 variantes parecem ter sido descritas pela primeira vez no Brasil. Além das mutações BRCA1/2, foram encontradas variantes em outros genes relacionados a síndromes hereditárias que predispõem a cânceres ginecológicos. CONCLUSãO: Este estudo permitiu conhecer melhor as principais mutações identificadas nas famílias do estado de Minas Gerais e demonstra a necessidade de avaliar a história familiar de câncer não ginecológico para avaliação do risco de CM, OC e CE. Além disso, é um esforço que contribui com estudos populacionais para avaliar o perfil de mutações de risco para câncer no Brasil.

Germline Mutations Landscape in a Cohort of the State of Minas Gerais, Brazil, in Patients Who Underwent Genetic Counseling for Gynecological and Breast Cancer / Cenário de mutações germinativas em uma coorte do estado de Minas Gerais, Brasil, em pacientes submetidas ao aconselhamento genético para câncer ginecológico e de mama

Abstract Objective The present study evaluated the profile of germline mutations present in patients who underwent genetic counseling for risk assessment for breast cancer (BC), ovarian cancer (OC), and endometrial cancer (EC) with a possible hereditary pattern. Methods Medical records of 382 patients who underwent genetic counseling after signing an informed consent form were analyzed. A total of 55.76% of patients (213/382) were symptomatic (personal history of cancer), and 44.24% (169/382) were asymptomatic (absence of the disease). The variables analyzed were age, sex, place of birth, personal or family history of BC, OC, EC, as well as other types of cancer associated with hereditary syndromes. The Human Genome Variation Society (HGVS) nomenclature guidelines were used to name the variants, and their biological significance was determined by comparing 11 databases. Results We identified 53 distinct mutations: 29 pathogenic variants, 13 variants of undetermined significance (VUS), and 11 benign. The most frequent mutations were BRCA1 c.470_471delCT, BRCA1 c.4675 + 1G > T, and BRCA2 c.2T> G. Furthermore, 21 variants appear to have been described for the first time in Brazil. In addition to BRCA1/2 mutations, variants in other genes related to hereditary syndromes that predispose to gynecological cancers were found. Conclusion This study allowed a deeper understanding of the main mutations identified in families in the state of Minas Gerais and demonstrates the need to assess the family history of non-gynecological cancer for risk assessment of BC, OC, and EC. Moreover, it is an effort that contributes to population studies to evaluate the cancer risk mutation profile in Brazil.

Resumo Objetivo O presente estudo avaliou o perfil de mutações germinativas presentes em pacientes submetidas a aconselhamento genético para avaliação de risco para câncer de mama (CM), câncer de ovário (OC) e câncer de endométrio (CE) com possível padrão hereditário. Métodos Foram analisados os prontuários de 382 pacientes que realizaram aconselhamento genético após consentimento informado. Um total de 55,76% dos pacientes (213/382) eram sintomáticos (história pessoal de câncer), e 44,24% (169/382) eram assintomáticos (ausência da doença). As variáveis analisadas foram idade, sexo, naturalidade, história pessoal ou familiar de CM, OC, CE bem como outros tipos de câncer associados a síndromes hereditárias. As diretrizes de nomenclatura da Human Genome Variation Society (HGVS) foram usadas para nomear as variantes e seu significado biológico foi determinado pela comparação de 11 bancos de dados. Resultados Identificamos 53 mutações distintas: 29 variantes patogênicas, 13 variantes de significado indeterminado e 11 benignas. As mutações mais frequentes foram BRCA1 c.470_471delCT, BRCA1 c.4675 + 1G > T e BRCA2 c.2T > G. Além disso, 21 variantes parecem ter sido descritas pela primeira vez no Brasil. Além das mutações BRCA1/2, foram encontradas variantes em outros genes relacionados a síndromes hereditárias que predispõem a cânceres ginecológicos. Conclusão Este estudo permitiu conhecer melhor as principais mutações identificadas nas famílias do estado de Minas Gerais e demonstra a necessidade de avaliar a história familiar de câncer não ginecológico para avaliação do risco de CM, OC e CE. Além disso, é um esforço que contribui com estudos populacionais para avaliar o perfil de mutações de risco para câncer no Brasil.

Low-dose melittin is safe for intravitreal administration and ameliorates inflammation in an experimental model of uveitis.

Uveitis is a group of sight-threatening ocular inflammatory disorders, whose mainstay of therapy is associated with severe adverse events, prompting the investigation of alternative treatments. The peptide melittin (MEL) is the major component of Apis mellifera bee venom and presents anti-inflammatory and antiangiogenic activities, with possible application in ophthalmology. This work aims to investigate the potential of intravitreal MEL in the treatment of ocular diseases involving inflammatory processes, especially uveitis. Safety of MEL was assessed in retinal cells, chick embryo chorioallantoic membranes, and rats. MEL at concentrations safe for intravitreal administration showed an antiangiogenic activity in the chorioallantoic membrane model comparable to bevacizumab, used as positive control. A protective anti-inflammatory effect in retinal cells stimulated with lipopolysaccharide (LPS) was also observed, without toxic effects. Finally, rats with bacille Calmette-Guerin- (BCG) induced uveitis treated with intravitreal MEL showed attenuated disease progression and improvement of clinical, morphological, and functional parameters, in addition to decreased levels of proinflammatory mediators in the posterior segment of the eye. These effects were comparable to the response observed with corticosteroid treatment. Therefore, MEL presents adequate safety profile for intraocular administration and has therapeutic potential as an anti-inflammatory and antiangiogenic agent for ocular diseases.

microRNA miR-133a as a Biomarker for Doxorubicin-Induced Cardiotoxicity in Women with Breast Cancer: A Signaling Pathway Investigation.

Cardiovascular toxicity is the main adverse effect of Doxorubicin (DOX) in cancer patients. microRNAs (miRNAs) are promising biomarkers to identify cardiac injury induced by DOX in breast cancer patients during the subclinical phase. Using RT-qPCR, we compared the expression of circulating miR-208a5p, miR-133a, miR-499a5p, miR-15a, miR-133b, and miR-49a3p in serum samples from DOX-induced cardiotoxicity (case) compared to the non-cardiotoxicity group (control). To further explore the potential roles of these circulating miRNA in cardiotoxicity, we searched the miRTarBase for experimentally validated miRNA-target interactions and performed a functional enrichment analysis based on those interactions. miR-133a was significantly upregulated in case compared to control group. The most relevant pathway regulated by miR-133a was ErbB2 signaling, whose main genes involved are EGFR, ERBB2, and RHOA, which are possibly downregulated by miR133a. The other miRNAs did not show significant differential expression when compared on both groups. The data suggest that miR-133a is associated with DOX-based cardiotoxicity during chemotherapy in breast cancer patients through ErbB2 signaling pathway. Moreover, miR-133a may be a future marker of DOX-induced cardiotoxicity in women with breast cancer.

Markers to sensibility and relapse on IMR-32 neuroblastoma cell line cultured in monolayer (2D) and neurosphere (3D) models cisplatin-treated.

The complexity of different components of tumor stroma poses huge challenges for therapies targeting the neuroblastoma (NB) microenvironment. The present study aimed to evaluate platinum-based response in IMR-32 neuroblastoma cell line cultured in monolayer (2D) and neurosphere (3D) models. For this, we evaluated mRNA expression of heat shock proteins HSPA1A, HSPB1, TRAP1, HSPA1AL, HSPD1, and DNA damage repair gene ERCC1. After treatment, residual cells were grafted on CAM (chicken chorioallantoic membrane) to evaluate the growth capability and histological paraffin sections were made to assess Ki-67 and HER-2 proteins by immunofluorescence. Our results showed that cisplatin induces mRNA downregulation of Heat Shock Proteins and ERCC1 in IMR-32 cells cultured in 2D or 3D models. In addition, the cisplatin-treatment approach increased HER-2 expression in residual IMR-32 cells grafted on the CAM. Therefore, these insights provide many advances in neuroendocrine tumor biology and knowledge about cisplatin-response in neuroblastoma.

Identification of best housekeeping genes for the normalization of RT-qPCR in human cell lines.

The identification of the best reference gene is a critical step to evaluate the relative change in mRNA expression of a target gene by RT-qPCR. In this work, we evaluated nineteen genes of different functional classes using Real Time Human Reference Gene Panel (Roche Applied Sciences), to identify the internal housekeeping genes (HKGs) most suitable for gene expression normalization data in human cell lines. Normal cell lines CCD-19LU (lung fibroblast), HEK-293 (epithelial cell of embryonic kidney), WI-26 VA4 (lung fibroblast), and human cancer cells, BT-549 (breast cancer), Hs 578T (breast cancer), MACL-1 (breast cancer), HeLa (cervical carcinoma), U-87 MG (glioblastoma/astrocytoma), RKO-AS45-1 (colorectal carcinoma), and TOV-21G (ovarian adenocarcinoma) were cultivated according to manufacturer's protocol. Twelve candidate reference genes were commonly expressed in five cell lines (CCD-19Lu, HEK-293, RKO-AS45-1, TOV-21G, and U-87 MG). To verify the expression stability, we used the RefFinder web tool, which integrates data from the computational programs Normfinder, BestKeeper, geNorm, and the comparative Delta-Ct method. The ACTB was the most stable reference gene to the CCD-19Lu and HEK-293 cells. The best combination of HKGs for the RKO-AS45-1 and TOV-21G cell lines were B2M/GAPDH and PBGD/B2M, respectively. For the U-87 MG cells, GAPDH and IPO8 were the most suitable HKGs. Thus, our findings showed that it is crucial to use the right HKGs to precise normalize gene expression levels in cancer studies, once a suitable HKG for one cell type cannot be to the other.

Interleukin-10 Levels are Associated with Doxorubicin-Related Cardiotoxicity in Breast Cancer Patients in a One-Year Follow-Up Study.

BACKGROUND: Myocardial toxicity is a common side effect of doxorubicin (DOXO) therapy in breast cancer patients. We hypothesized that DOXO-induced cardiotoxicity may be related to the release of inflammatory cytokines in response to the treatment. This study aimed to assess changes in plasma levels of interleukin (IL)-1ß, IL-6, IL-10 and tumor necrosis factor (TNF) after chemotherapy and to correlate these levels with cardiac biomarkers and clinical data. METHODS: Sixty-four patients with breast cancer treated with DOXO were included. Twenty-two subjects (cases) developed cardiotoxicity until one year after the end of DOXO treatment. Cytokines and cardiac markers were evaluated before starting chemotherapy (T0), up to 7 days after the last infusion (T1) and 12 months after the last infusion (T2). RESULTS: Higher IL-10 levels were observed in the case group compared to controls at T1 (p = .006) and T2 (p = .046). The IL-1ß, IL-6 and TNF levels did not change during treatment in each group (p > .05), nor between the case and control groups. The IL-10 levels were higher at T1 than at T0 and T2 (p < .05 for both) in the cardiotoxicity group. A correlation between IL-10 and N-terminal pro-brain natriuretic peptide (NT-proBNP) levels at T0 and T2 in the cardiotoxicity group was observed (p = .048 and p = .004, respectively). CONCLUSION: Our study demonstrated that DOXO induced an increase in plasma IL-10 levels in patients who presented cardiotoxicity after treatment, which correlated with NT-proBNP levels.

A vivência da guarda compartilhada após o divórcio e/ou dissolução conjugal / The experience of joint custody after divorce and/or marital dissolution

O objetivo desta revisão integrativa foi investigar, em estudos acadêmicos publicados entre 2010 e 2020, como pais e filhos vivenciam a guarda compartilhada após o processo de divórcio e/ou dissolução conjugal. As bases utilizadas foram: LILACS, SciELO, MEDLINE e PsycINFO. Os descritores elencados foram: Guarda Compartilhada, Cuidados Parentais, Custódia da Criança, Divórcio, Educação Infantil, Família, Parentalidade, Poder Familiar, Relações Familiares, Relações Pais-Filho. Após a realização das buscas (N= 381), procedeu-se com a exclusão de estudos por repetição (N=121), título (N=83) e resumo (N=18). Os artigos remanescentes foram lidos na íntegra e (N=16) foram recuperados e compõem esta revisão. Os estudos apontam a dificuldade de difusão da modalidade de guarda compartilhada no Brasil, destacando fatores favoráveis e contrários à sua aplicação. Evidencia-se a necessidade de mais estudos empíricos na área, a fim de consolidar inferências consonantes em grande parte dos estudos que se mostraram relevantes na composição deste trabalho de revisão.

The objective of this integrative review was to explore how parentes and children experience joint custody after divorce and/or marital dissolution, in studies from 2015 to 2020. The data bases LILACS, SciELO, MEDLINE and PsycINFO were used. The descriptors listed were Joint Custody, Child Custody, Divorce, Child Rearing, Family, Parenting, Family Relations, Parent-Child Relations. After performing the searches (n=381), we proceeded to the exclusion of repetitive studies (n=121), title (n=83) and summary (n=18). The remaining articles were read in full and 16 were retrieved and make up this review. Studies explore the difficulty of diffusion of the modality in Brasil and shows factors considered as favorable and adversary to its application. We emphasize the importance of more studies in the area, with the aim of consolidating consonant inferences in most of the studies that proved to be relevant in the composition of this integrative review.

Phenotypic changes on central nervous system (CNS) tumor cell lines cultured in vitro 2D and 3D models and treated with cisplatin.

Despite aggressive therapy, most patients with brain tumors present disease relapse due to the cellular and molecular nature of these tumors. One of the models that best explains the heterogeneity observed in CNS tumors is the presence of cancer stem cells (CSCs). In this paper, we evaluated platinum-based response in brain tumor U-87 MG, LN-18, and KELLY cell lines cultured in monolayer (2D) and neurosphere (CSC enrichment- 3D) models. We evaluated mRNA expression of heat shock proteins (HSPA1A, HSPB1, HSPA1AL, TRAP1, and HSPD1), and DNA repair gene ERCC1. Changes in cell cycle and glycosylation profile were assessed by flow cytometry. After treatment with cisplatin, we found that the mRNA expression of HSPs markedly increased in the U-87 MG and LN-18 neurosphere cells. In KELLY monolayer cells, cisplatin induced upregulation of all genes. In KELLY neurosphere cells, only the HSPA1A, HSPB1, TRAP1, and HSPD1 genes were upregulated. The proportion of cells in the G0/G1 phase was significantly higher in U-87 MG neurosphere cisplatin-treated cells. A trend towards a greater proportion of cells in the S phase of U-87 MG monolayer cisplatin-treated cells was also observed. On the other hand, a significant decrease in the number of cells in the S phase and an increase in G2/M was observed in LN-18 monolayer cisplatin-treated cells. Glycosylation analysis using lectins showed a higher surface binding for PNA in the U-87 MG treated monolayer and a lower binding for Concanavalin A in the treated neurospheres. The binding of Isolectin GS-IB4, GSII, and SBA in KELLY monolayer cisplatin-treated cells was lower whereas the proportion of cells labeled with Concanavalin A was higher. In the KELLY neurosphere cisplatin-treated cells, the binding of Concanavalin A was lower than nontreated cells. Thus, our findings strongly supported the idea that definitions of phenotypic characteristics may help to establish better therapeutic strategies for brain tumors.

Neurological manifestations due to dengue virus infection in children: clinical follow-up.

The aim was to assess neurological complications in children with an invasive neurological disease by dengue virus (DENV) and the time to resolve symptoms after hospital discharge. A prospective study was conducted at a referral hospital for infectious diseases in Brazil between March 2014 and July 2019. All children hospitalized with neurologic manifestations and DENV RNA detected by real-time reverse transcription-polymerase chain reaction (RT-qPCR) in cerebrospinal fluid (CSF) were followed up until complete resolution of neurological complications. On average, they were followed up for 16 months. Among 56 DENV-positive children, 39% had some neurologic complications after hospital discharge and found that 19.6% were discharged with anticonvulsants due to seizures, 10.7% developed motor complications (e.g. muscle weakness, paresis, ataxia, and walking disability), 5.4% had headaches, and 14.3% had sleep disorders. Among the 56 children, only three had a clinical diagnosis of dengue because the symptoms are nonspecific and 35% showed no change in cerebrospinal fluid (CSF). The average time to resolve complications was 5.9 months (ranging from 1 m to 32 m). These results should alert physicians to the difficulties of a clinical diagnosis of an infection that causes neurological complications after discharge in a significant number of children. RT-qPCR's etiological diagnosis of DENV infection enabled better clinical follow-up for early intervention in children with neurological complications.

Neurologic Manifestations of Noncongenital Zika Virus in Children.

We evaluated neurologic complications following noncongenital Zika virus infection in 11 children who presented with central nervous system signs. Zika virus RNA was detected by real-time reverse transcription-polymerase chain reaction in cerebrospinal fluid. Approximately one-quarter of patients required antiepileptic medication in follow-up, and 2 children progressed to learning difficulties or developmental delay.

Identification of Clinical and Laboratory Variables Associated with Cardiotoxicity Events Due to Doxorubicin in Breast Cancer Patients: A 1-Year Follow-Up Study.

Cardiovascular adverse events in patients with breast cancer undergoing chemotherapy (CT) are frequent due to the high cardiotoxic potential of treatments, especially doxorubicin (DOXO). This study aimed to evaluate the association of plasma levels of various biomarkers with cardiotoxicity in women with breast cancer on DOXO-based chemotherapy. In this single center prospective cohort, 80 breast cancer patients who used DOXO as a first-line treatment for cancer were evaluated. Patients were assessed at three time points: before CT (T0), 1 week after (T1) and 12 months after DOXO treatment (T2). The predominant histological classification was ductal carcinoma, n = 72 (90.0%); the most frequent molecular classification was Human epidermal growth factor receptor-type 2 positive (HER2+), n = 34 (43.0%). In patients submitted to complementary treatment with trastuzumab (n = 23), there was no association with cardio-specific biomarkers. Evaluating the clinical variables and the laboratory parameters in T1 and T2 in relation to T0, the reduction any time of N-terminal-pro hormone B-type natriuretic peptide (NT-proBNP), triglycerides and hematocrit levels showed an association with higher cardiotoxicity risk. In addition, increased levels of troponin I (cTnI) and glycated hemoglobin (HbA1c) showed an independent association with the occurrence of cardiotoxicity. These results suggest that the evaluation of these laboratory tests should be included routinely to identify breast cancer patients under DOXO treatment at cardiotoxicity risk.

Interaction of Biomphalaria cells in primary cultures with Schistosoma mansoni sporocysts.

INTRODUCTION: Biomphalaria snails may display varying levels of susceptibility to Schistosoma mansoni infection. We have been developing an in vitro model to study the interaction between the snail and the parasite, using tissue-derived cell cultures from Biomphalaria. METHODS: The digestive gland- and kidney-derived cells from primary cultures of resistant (B. tenagophila Taim) and susceptible (B. tenagophila HM and B. glabrata BH) strains of Biomphalaria were exposed to S. mansoni sporocysts. RESULTS: S. mansoni sporocysts were surrounded and encapsulated exclusively by cells derived from the digestive gland (DG) of B. tenagophila Taim. The process was followed by a marked decrease in the number of free sporocysts in the culture medium. The morphological characteristics of DG-derived cells in culture have been described. CONCLUSIONS: Cells derived from DG (but not SK) primary cultures of B. tenagophila Taim may participate in S. mansoni sporocyst control.

Epithelial-to-mesenchymal transition markers are differentially expressed in epithelial cancer cell lines after everolimus treatment.

The epithelial-to-mesenchymal transition (EMT) is a phenomenon during which cancer epithelial cells undergo changes in plasticity and lose cell-cell adhesion with consequent remodeling of the extracellular matrix and development of mesenchymal characteristics. Long non-coding RNAs (lncRNAs) have been described as EMT modulation markers, becoming a promising target in the development of new therapies for cancer. The present study aimed to investigate the role of everolimus at 100 nM as inductor of the EMT phenomenon in cell lines derived from human breast (BT-549), colorectal (RKO-AS45-1) and ovary (TOV-21G) cancer. The integrity of cellular junctions was monitored using an in vitro model of epithelial resistance. The results demonstrated that the EMT genes ZEB1, TWIST1 and TGFB1 were differentially expressed in cells treated with everolimus compared with in untreated cells. lncRNA HOTAIR was upregulated post-treatment only in BT-549 cells compared with in untreated cells. After treatment with everolimus, the intensity of fluorescence of P-cadherin decreased, and that of fibronectin increased in RKO-AS45-1 and TOV-21G cells compared with control cells. The transepithelial electrical resistance at the RKO-AS45-1 monolayer treated with everolimus started to decrease at 48 h. The changes in the gene expression and epithelial resistance may confirm the role of everolimus in EMT.

Central nervous system (CNS) tumor cell heterogeneity contributes to differential platinum-based response in an in vitro 2D and 3D cell culture approach.

One of the models that best explains the cellular heterogeneity observed in central nervous system (CNS) tumors is the presence of cancer stem cells (CSCs). CSCs can originate from differentiated adult cells that return to an undifferentiated stage through the mechanism known as epithelial-mesenchymal transition (EMT). In this paper, we evaluated cellular and molecular heterogeneity and the participation of the epithelial-mesenchymal transition (EMT) in glioblastoma (U-87 MG and LN-18) and neuroblastoma (KELLY and IMR-32) cell lines cultured in monolayer (2D) and neurosphere (CSC enrichment- 3D) models. For this, after treatment with cisplatin, we studied different cell subpopulations by immunophenotyping using neural stem cell/progenitor markers (ALDH, CD24, CD56, and CD133), mesenchymal stem cell markers (CD73, CD90, CD105, and CD146) and hematopoietic markers (CD14, CD19, CD34, CD45, and HLA-DR) and mRNA expression profiles of genes related to EMT, such as ZEB1, TWIST1, TGFB1, STAT3, and lncRNA HOTAIR. In addition, we evaluated the growth capacity of residual cells when treated with cisplatin using the chorioallantoic membrane (CAM) model to study disease relapse. After treatment with cisplatin, we found that the expression of STAT3 and TGFB1 genes markedly increased in the neurosphere of the IMR-32 cell line, and TWIST1 was upregulated in the neurosphere of LN-18. Only the nontreated monolayer of LN-18, KELLY, and IMR-32 amplified the lncRNA HOTAIR. The IMR-32 cell line exhibited an enrichment of CD24+/ALDH+ and this cell subset decreased after cisplatin treatment. We observed the loss of CD146+/CD73+ cell subpopulations in U-87 MG monolayer and neurosphere models, after cisplatin treatment, while in LN-18 monolayer cisplatin-treated cells, CD73+/CD90+ cell subpopulations increased. Neuroblastoma cell lines showed CD14+/HLA-DR- cell subpopulations representative of myeloid-derived suppressor cells (MDSCs). Tumors generated from residual cells, after exposure to cisplatin, grafted on CAM showed patterns of organization different from those of the controls. Thus, our findings strongly supported the idea that definitions of tumor phenotypic characteristics may help to establish better therapeutic strategies for the development of new drug targets.

Rosmarinic Acid Intravitreal Implants: A New Therapeutic Approach for Ocular Neovascularization.

Rosmarinic acid, a plant-derived compound with antiangiogenic activity, can be applied for the treatment of ocular diseases related to neovascularization, such as diabetic retinopathy, macular edema, and age-related macular degeneration. These diseases represent the leading causes of blindness worldwide if they are not properly treated. Intravitreal devices allow for localized drug delivery to the posterior segment, increasing the drug bioavailability and promoting extended release, thus, reducing side effects and enhancing the patient's compliance to the treatment. In this work, rosmarinic acid-loaded poly lactic-co-glycolic acid intraocular implants were developed with a view for the treatment of ocular neovascularization. Physical-chemical, biocompatibility, and safety studies of the implants were carried out in vitro and in vivo as well as an evaluation of the antiangiogenic activity in a chorioallantoic membrane assay. Data obtained showed that rosmarinic acid released from the implants was quantified in the vitreous for 6 weeks, while when it was in the solution formulation, after 24 h, no drug was found in the vitreous. The delivery device did not show any sign of toxicity after clinical evaluation and in electroretinographic findings. Histological analysis showed normal eye tissue. Rosmarinic acid released from implants reduced 30% of new vessel's formation. The intravitreal implant successfully allowed for the prolonged release of rosmarinic acid, was safe to rabbits eyes, and demonstrated activity in vessel reduction, thus demonstrating potential in preventing neovascularization in ophthalmic diseases.

Deep sequencing of small RNAs reveals the repertoire of miRNAs and piRNAs in Biomphalaria glabrata.

BACKGROUND: Biomphalaria glabrata snails are widely distributed in schistosomiasis endemic areas like America and Caribe, displaying high susceptibility to infection by Schistosoma mansoni. After the availability of B. glabrata genome and transcriptome data, studies focusing on genetic markers and small non-coding RNAs have become more relevant. The small RNAs have been considered important through their ability to finely regulate the gene expression in several organisms, thus controlling the functions like cell growth, metabolism, and susceptibility/resistance to infection. OBJECTIVE: The present study aims on identification and characterisation of the repertoire of small non-coding RNAs in B. glabrata (Bgl-small RNAs). METHODS: By using small RNA sequencing, bioinformatics tools and quantitative reverse transcription polymerase chain reaction (RT-qPCR), we identified, characterised, and validated the presence of small RNAs in B. glabrata. FINDINGS: 89 mature miRNAs were identified and five of them were classified as Mollusk-specific. When compared to model organisms, sequences of B. glabrata miRNAs showed a high degree of conservation. In addition, several target genes were predicted for all the mature miRNAs identified. Furthermore, piRNAs were identified in the genome of B. glabrata for the first time. The B. glabrata piRNAs showed strong conservation of uridine as first nucleotide at 5' end, besides adenine at 10th position. Our results showed that B. glabrata has diverse repertoire of circulating ncRNAs, several which might be involved in mollusk susceptibility to infection, due to their potential roles in the regulation of S. mansoni development. MAIN CONCLUSIONS: Further studies are necessary in order to confirm the role of the Bgl-small RNAs in the parasite/host relationship thus opening new perspectives on interference of small RNAs in the organism development and susceptibility to infection.

Apoptosis-related gene expression can predict the response of ovarian cancer cell lines to treatment with recombinant human TRAIL alone or combined with cisplatin.

OBJECTIVES: The objectives of this study were to determine the sensitivity of ovarian cancer (OC) cell lines (TOV-21G and SKOV-3) to cisplatin and to the recombinant human TRAIL (rhTRAIL), and to evaluate the expression profile of TNFRSF10B, TNFRSF10C, TP53TG5, MDM2, BAX, BCL-2 and CASPASE-8 genes and their participation in the resistance/susceptibility mechanism of these tumor cell lines. METHODS: To determine the IC50 values associated with Cisplatin and rhTRAIL, inhibition of cell growth was observed using MTT assays in two human OC cell lines (SKOV-3 and TOV-21G). The analysis of gene expression was performed using quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: Both cell lines had different susceptibility profiles to the tested drugs. In the SKOV-3 cell line, the IC50 values for cisplatin and for rhTRAIL were 270.83 ug/mL and 196.5 ng/mL, respectively. The same concentrations were used for TOV-21G. Different gene expression profiles were observed in each tested cell line. CASPASE-8 and TNFRSF10B expression levels could predict the response of both the cell lines to rhTRAIL alone or the response to a combination of rhTRAIL and cisplatin. In addition, we observed a relationship between BCL-2 and BAX expression that may be helpful in estimating the proliferation rate of the OC cell lines. CONCLUSION: SKOV-3 and TOV-21G respond differently to cisplatin and rhTRAIL exposure, and expression of CASPASE-8 and TNFRSF10B are good predictors of responses to these treatments.