Proteomic Analysis of Epithelial to Mesenchymal Transition (EMT) Reveals Cross-talk between SNAIL and HDAC1 Proteins in Breast Cancer Cells.

Epithelial to mesenchymal transition (EMT)(1) occurs naturally during embryogenesis, tissue repair, cancer progression, and metastasis. EMT induces cellular and microenvironmental changes resulting in loss of epithelial and acquisition of mesenchymal phenotypes, which promotes cellular invasive and migratory capabilities. EMT can be triggered by extracellular factors, including TGF-ß, HGF, and EGF. Overexpression of transcription factors, such as SNAIL, SLUG, ZEB1/2, and TWIST1, also induces EMT and is correlated to cancer aggressiveness. Here, the breast adenocarcinoma cell line MCF7 was transduced with SNAIL to identify specific mechanisms controlled by this transcription factor during EMT. Overexpression of SNAIL led to EMT, which was thoroughly validated by molecular, morphological, and functional experiments. Subcellular proteome enrichment followed by GEL-LC-MS/MS was performed to provide extensive protein fractionation and in-depth proteomic analysis. Quantitative analysis relied on a SILAC strategy, using the invasive breast cancer cell line MDA-MB-231 as a reference for quantitation. Subsets of proteins enriched in each subcellular compartment led to a complementary list of 4289 proteins identified with high confidence. A subset of differentially expressed proteins was validated by Western blot, including regulation in specific cellular compartments, potentially caused by protein translocation. Protein network analysis highlighted complexes involved in cell cycle control and epigenetic regulation. Flow cytometry analysis indicated that SNAIL overexpression led to cell cycle arrest in G0/G1 phases. Furthermore, down-regulation of HDAC1 was observed, supporting the involvement of epigenetic processes in SNAIL-induced EMT. When HDAC1 activity was inhibited, MCF7 not only apparently initiated EMT but also up-regulated SNAIL, indicating the cross-talk between these two proteins. Both HDAC1 inhibition and SNAIL overexpression activated the AKT pathway. These molecular mechanisms appear to be essential to EMT and therefore for cancer metastasis. Specific control of such epigenetic processes might then represent effective approaches for clinical management of metastatic cancer.

Proteomics analysis reveals the role of ubiquitin specific protease (USP47) in Epithelial to Mesenchymal Transition (EMT) induced by TGFß2 in breast cells.

Epithelial to Mesenchymal Transition (EMT) is a normal cellular process that is also triggered during cancer progression and metastasis. EMT induces cellular and microenviromental changes, resulting in loss of epithelial features and acquisition of mesenchymal phenotypes. The growth factor TGFß and the transcription factor SNAIL1 (SNAIL) have been described as inducers of EMT. Here, we carried out an EMT model with non-tumorigenic cell line MCF-10A induced with the TGFß2 specific isoform of TGF protein family. The model was validated by molecular, morphological and functional experiments and showed correlation with the up-regulation of SNAIL. In order to identify additional regulators of EMT in this non-tumorigenic model, we explored quantitative proteomics, which revealed the Ubiquitin carboxyl-terminal hydrolase 47 (USP47) as one of the top up-regulated proteins. USP47 has a known role in cell growth and genome integrity, but not previously correlated to EMT. After validating USP47 alterations using MRM and antibody-based assays, we demonstrated that the chemical inhibition of USP47 with the inhibitor P5091 reduced expression of EMT markers and reverted morphological changes in MCF-10A cells undergoing EMT. These results support the involvement of USP47 in our EMT model as well as potential applications of deubiquitinases as therapeutic targets for cancer progression management. BIOLOGICAL SIGNIFICANCE: Metastasis is responsible for most cancer-associated mortality. Additionally, metastasis requires special attention, as the cellular transformations make treatment at this stage very difficult or occasionally impossible. Early steps in cancer metastasis involve the ability to detach from the solid tumor mass and invade the surrounding stromal tissues through cohesive migration, or a mesenchymal or amoeboid invasion. One of the first steps for metastatic cascade is denominated epithelial to mesenchymal transition (EMT), which can be triggered by different factors. Here, our efforts were directed to better understand this process and identify new pathways that contributes for acquisition of EMT, mainly focused on post translational modifications related to ubiquitin proteasome system. Our model of EMT induction by TGFß2 mimics early stage of metastatic cancer in epithelial breast cells and a proteomic study carried out for such model demonstrates that the deubiquitinase enzyme USP47 acts in SNAIL stabilization, one of the most important transcription factors for EMT phenotype acquisition and consequent metastasis. In addition, the inhibiton of USP47 with P5091, reverted the EMT phenotype. Together the knowledge of such processes of cancer progression and regulation can help in designing new strategies for combined therapies for control of cancer in early stages.

Profiling Cell Lines Nuclear Sub-proteome.

Proteins are very dynamic within the cell and their localization and trafficking between subcellular compartments are critical for their correct function. Indeed, the abnormal localization of a protein might lead to the pathogenesis of several diseases. The association of cell fractionation methods and mass spectrometry based proteomic methods allow both the localization and quantification of proteins in different sub-compartments. Here we present a detailed protocol for enrichment, identification, and quantitation of the nuclear proteome in cell lines combining nuclear subproteome enrichment by differential centrifugation and high-throughput proteomics.

The secretome analysis by high-throughput proteomics and multiple reaction monitoring (MRM).

The secretome is a sub-proteome of great interest in several fields of biomedical sciences, especially as a source of diagnostics and therapeutic targets. Proteomics has been contributing significantly to elucidate the secretome of a great diversity of cells, tissues, and organisms, turning profiles of thousands of proteins a usual practice. After elucidation of long protein lists, targeted proteomics also plays important roles in accurate quantification and validation of such secreted proteins. Here we present detailed protocols to explore and quantify the secretome of cancer cells, even though this protocol can be employed to any kind of biological material.

Programa de Pilates atua no estado de humor de mulheres no climatério / Pilates program influences the mood of women in the climacteric

Introdução: Pilates é um método de condicionamento que integra o corpo e a mente proporcionando bem-estar geral aos indivíduos. Objetivo: Avaliar o efeito do Pilates sobre o estado de humor de mulheres no climatério. Métodos: Participaram do estudo 10 mulheres sedentárias entre 41 e 64 anos, na fase do climatério. As participantes foram submetidas a um programa de treinamento de Pilates, composto por 10 sessões, uma vez por semana com duração de 50 minutos utilizando 7 exercícios. O estado de humor foi avaliado através do POMS (Profile of Mood States), composto por 42 adjetivos subdivididos em 6 estados de humor. Para avaliação dos sintomas do climatério foi aplicado o Índice de Kupperman e Blatt. O teste de Shapiro-Wilk foi realizado para confirmar a normalidade dos dados e teste t de Student pareado para analisar as diferenças entre as médias pré e pós-sessão. A significância de 5% foi utilizada para os testes estatísticos através do software SPSS. Resultados: Aumento significativo do Vigor (p< 0,01), diminuição da Fadiga (p<0,02) e redução da Perturbação Total de Humor (p<0,04). Conclusão: Pilates com frequência de uma vez por semana foi capaz de melhorar significativamente o estado de humor de mulheres sedentárias na fase do climatério. (AU)

Introduction: Pilates is a conditioning method that unites body and mind, providing general welfare to practitioners. Objective: To analyze the effects that Pilates has on climacteric women's mood state. Methods: 10 sedentary, climacteric women between 41 and 64 years old participated in this study. The group was submitted to Pilates training program, composed of 10 sessions of 50 minutes, once per week, using 7 exercises. In order to analyze women's mood, POMS (Profile of Mood States) was used, which consists of 42 adjectives divided in 6 states of mood. As evaluation for the climacteric symptoms the Blatt-Kupperman Index was applied. Furthermore, the ShapiroWilk test was applied to confirm the data reliability. The results were presented as the average and its standard deviation. It was also used a paired student t test to observe the differences between pre- and post-session. A significance of 5% was used for the statistical analysis using the software IBM SPSS. Results: It was observed a significant increase in Vigour levels (p< 0,01), reduction in Fatigue (p< 0,02) and in Total mood disturbance (p< 0,04). Conclusion: Pilates regularly (at least once a week) is capable of significantly improve sedentary, climacteric women's mood state. (AU)

Bacillus em dietas para alevinos de tilápia do nilo, variedade gift / Bacillus in diets for fingerlings of nile tilapia, gift variety

Os probióticos vêm se destacando na produção aquícola como promotores de crescimento, especialmente na tilapicultura, por serem capazes de sobreviver ao trato gastrointestinal, aderir células da parede intestinal, reduzir ou prevenir patógenos, não ser patogênico ao hospedeiro. Objetivou-se avaliar o desempenho produtivo, índices corpóreos, macro micronutrientes e composição química da carcaça de alevinos de tilápia do Nilo, variedade GIFT, alimentadas com ração formulada com probióticos Bacillus cereus var. Toyoi e Bacillus subtilis C-3102, durante alevinagem. Foram utilizados 720 pós-larvas com peso médio inicial de 26,6 ± 3,4 mg. Os peixes foram distribuídos aleatoriamente em um sistema de 24 aquários de 0,03 m³ num delineamento inteiramente casualizado composto por três tratamentos e oito repetições, constituído por dois probióticos e, tratamento ausente de probiótico. Os dados dos parâmetros analisados foram submetidos à ANOVA a 5%. Não houve diferenças significativas (p>0,05) ao final do cultivo para os parâmetros de peso final, ganho em peso, crescimento específico e consumo de ração. A biomassa final, ganho em biomassa e sobrevivência dos peixes foi afetada significativamente (p<0,05) pela adição de B. cereus ou B. subtilis em relação ao ausente. Não houve diferenças (p>0,05) nos índices corporais e composição centesimal com probióticos ou ausentes nas rações. A inclusão de quaisquer dos probióticos influenciou (p<0,05) sobre valores de cálcio na carcaça dos alevinos em relação aos peixes ausentes de probióticos. A utilização de B. subtilis ou B. cereus melhora o desempenho produtivo e não afetam negativamente os índices corpóreos, macro e micronutrientes na carcaça de alevinos de tilápia do Nilo, variedade GIFT.

Probiotics have gained prominence in aquaculture production as growth promoters, especially in tilapia culture, by being able to survive the gastrointestinal tract, adhere intestinal wall cells, reduce or prevent pathogens not be pathogenic to the host. The objective was to evaluate the performance, body indexes and macro and micronutrient composition of the carcass of the Nile tilapia, GIFT variety, fed with diets formulated with probiotic Bacillus cereus var. Toyoi and Bacillus subtilis C-3102, during nursery. There were used 720 post-larvae with initial average weight of 26.6 ± 3.4 mg. Fish were randomly distributed in a system of 24 aquaria of 0.03 m³ a completely randomized design consisting of three treatments and eight replications, consisting of two probiotics, and probiotic treatment absent. The data parameters were analyzed by ANOVA at 5%. There were no significant differences (p>0.05) in fish from all treatments for the final weight, weight gain, specific growth rate and feed intake. The final biomass, gain and survival of fish biomass was affected significantly (p<0.05) by addition of any one of the probiotics in relation to the absent. There were no differences (p>0.05) in body composition and with probiotics or absent in diets. The inclusion of any of probiotics was influenced (p<0.05) on calcium levels in the carcass of fish fingerlings in relation to the missing of probiotics. The use of B. subtilis or B.cereus improves performance and does not negatively affect the body indexes, macro and micronutrients in the carcass of Nile tilapia, GIFT variety.

Detection and characterization of human rotavirus in hospitalized patients in the cities of Ponta Grossa, Londrina and Assai - PR, Brazil.

UNLABELLED: Acute diarrheal disease is still one of the major public health problems worldwide. Rotaviruses (RV) are the most important viral etiologic agents and children under five years of age are the target population. OBJECTIVE: To investigate the rate of RV infection in hospitalized patients due to acute diarrhea in the cities of Ponta Grossa, Londrina and Assai - Paraná. METHODS: Latex agglutination (LA); immunochromatography (ICG); polyacrylamide gel electrophoresis (PAGE) and negative staining electron microscopy (ME) tests were used to detect the virus. For the genotyping, RT-PCR and RT-PCR-ELISA were used, respectively, for NSP4 and VP4/VP7. RESULT: Out of 124 samples there were 69 positive stool samples for RV, for at least one of the used tests, 67 of them being RV group A (RV-A). Overall, most of the RV positive stool samples came from children under thirteen years of age. However, 12 positive cases occurred in patients aged 13 years or above, including an 81-year old patient. CONCLUSION: The data showed similar electropherotypes and genotypes G, P and NSP4 of the inland wild circulating strains of RV.

Mananoligossacarídeo durante a reversão sexual de tilápia do nilo / Mannanoligosaccharidae during the sex reversal of nile tilapia

Os prebióticos são classificados como monossacarídeos, oligossacarídeos ou polissacarídeos e podem ser definidos como aditivos naturais não digeríveis por enzimas que afetam beneficamente o hospedeiro por meio do estímulo da ação microbiana. O Bio-Mos® é um prebiótico obtido a partir da parede celular de levedura (Saccharomyces cerevisiae) e em sua constituição encontram-se -mananos, -glicanos e glicoproteínas. Objetivou-se avaliar o efeito da adição do prebiótico mananoligossacarídeo (Bio-Mos®) na dieta durante a reversão de tilápia do Nilo sobre os parâmetros de desempenho, proporção sexual, uniformidade dos lotes e composição química da carcaça. Foram utilizadas 1.080 larvas com três dias de idade e peso médio inicial de 10,9 ± 1,2 mg. As larvas foram distribuídas em 36 aquários de 30 L, em um delineamento experimental casualizado com seis repetições e tratamentos (0, 0,2, 0,4, 0,6, 0,8 e 1,0% de inclusão do prebiótico). A alimentação foi realizada ad libitum, seis vezes ao dia, com dietas isoproteicas e isocalóricas, adicionadas de 60 mg kg-1 hormônio masculinizante 17--metiltestosterona. Ao final da reversão, os valores médios para peso, comprimento, sobrevivência, crescimento específico e fator de condição foram, 620 mg e 32,62 mm, 91,85%, 13,34% dia-1 e 1,31, respectivamente, não apresentando diferença significativa (p>0,05). A efetividade da reversão sexual foi de 100% e não houve diferença (p>0,05) na uniformidade dos lotes e na composição química dos peixes alimentados com diferentes níveis de inclusão de prebiótico na dieta. A inclusão até 1,0% de Bio-Mos® não prejudica os parâmetros de desempenho, proporção sexual, sobrevivência, uniformidade dos lotes e a composição química da carcaça de tilápia do Nilo durante a fase de reversão sexual.

The prebiotics are classified as monosaccharides, oligosaccharides or polysaccharides and can be defined as non-digestible natural additives by enzymes that beneficially affect the host by stimulating the microbial action. The Bio-Mos® is a prebiotic obtained from the cell wall of yeast (Saccharomyces cerevisiae) and in its constitution are - mannans, -glycans and glycoproteins. This study aimed to evaluate the effect of adding of prebiotic mannanoligosaccharides (Bio-Mos®) in the diet during the sexual reversion to larvae of Nile tilapia on zootechnical performance, sex ratio, uniformity of lots and chemical composition of carcass. One-thousand and eighty larvae with three days old and initial average weight of 10.9 ± 1.2 mg were used. The larvae were distributed in 36 aquaria with 30 L, in a completely randomized experimental design with six replications and treatments (0.0, 0.2, 0.4, 0.6, 0.8 and 1% for inclusion of prebiotic). The ad libitum feeding was carried out six times a day with diet isonutritive, isocaloric, and 60 mg kg kg-1 of hormone masculine 17--methyltestosterone. At the end of the reversion, the values of final average weight, length, survival, specific growth and condition factor were 620 mg, 32.62 mm, 91.85%, 13.34% day-1 and 1.31, respectively, and did not show significantly differences (p>0.05). The effectiveness of sex reversal was 100% and there was not difference (p>0.05) in uniformity of lots and in chemical composition of fish fed with different levels of inclusion of prebiotic in the diet. The inclusion up to 1.0% Bio-Mos® do not harm the zootechnical performance, sex ratio, survival, uniformity of lots and chemical composition of carcass of Nile tilapia during the sexual reversion.

Probióticos em dietas para tilápia do Nilo durante a reversão sexual / Probiotics in diets for Nile tilapia during the sex reversal

Objetivou-se avaliar o desempenho produtivo, sexagem fenotípica e microbiologia intestinal de tilápia do Nilo, linhagem GIFT, alimentadas com ração contendo Bacillus cereus var. Toyoi e Bacillus subtilis C-3102, durante a fase de reversão sexual. Os peixes (24,7±0,50mg) foram distribuídos aleatoriamente em 24 aquários, num delineamento inteiramente casualizado, composto por três tratamentos (dois probióticos e ausente de probiótico) e oito repetições. A utilização de B. cereus melhorou os parâmetros de peso final, ganho em peso, ganho em peso médio diário e crescimento específico dos peixes alimentados em relação à B. subtilis. Não houve associação entre a proporção de machos e fêmeas e a inclusão de probióticos. As contagens de bactérias totais e coliformes totais não foram influenciadas pela adição de probióticos nas rações. O probiótico Bacillus subtilis não melhora o desempenho produtivo das tilápias do Nilo. Na decisão entre os dois probióticos estudados, recomenda-se o probiótico contendo Bacillus cereus C-3102 por não afetar negativamente a efetividade sexual e parâmetros zootécnicos.

The objective was to evaluate the productive performance, phenotypic sexing, and intestinal microbiology of Nile tilapia, GIFT strain, fed with diets formulated with Bacillus cereus var. Toyoi and Bacillus subtilis C-3102, during sex reversal. Fish (24.7±0.50mg) were randomly distributed in 24 aquaria in a completely randomized design compound for three treatments (two probiotics and absent) and eight replications. The use of B. cereus and improved the final weight, weight gain, average daily weight gain and specific growth rate fed in relation to B. subtilis. There was no association between the proportion of males and females and inclusion of the probiotics. The counts of total bacteria and total coliforms were not influenced by the addition of probiotics. Bacillus subtilis did not improve the productive performance in Nile tilapia. Bacillus subtilis did not improve the productive performance of Nile tilapia. Among the two studied probiotics is recommended probiotic containing Bacillus cereus C-3102 not adversely affect the sex reversal effectiveness and performance zootechnical.

Detection and characterization of human rotavirus in hospitalized patients in the cities of Ponta Grossa, Londrina and Assai - PR, Brazil

Acute diarrheal disease is still one of the major public health problems worldwide. Rotaviruses (RV) are the most important viral etiologic agents and children under five years of age are the target population. OBJECTIVE: To investigate the rate of RV infection in hospitalized patients due to acute diarrhea in the cities of Ponta Grossa, Londrina and Assai - Paraná. METHODS: Latex agglutination (LA); immunochromatography (ICG); polyacrylamide gel electrophoresis (PAGE) and negative staining electron microscopy (ME) tests were used to detect the virus. For the genotyping, RT-PCR and RT-PCR-ELISA were used, respectively, for NSP4 and VP4/VP7. RESULT: Out of 124 samples there were 69 positive stool samples for RV, for at least one of the used tests, 67 of them being RV group A (RV-A). Overall, most of the RV positive stool samples came from children under thirteen years of age. However, 12 positive cases occurred in patients aged 13 years or above, including an 81-year old patient. CONCLUSION: The data showed similar electropherotypes and genotypes G, P and NSP4 of the inland wild circulating strains of RV.