Stoichioproteomics study of differentially expressed proteins and pathways in head and neck cancer

Abstract Hypoxia is a prominent feature of head and neck cancer. However, the oxygen element characteristics of proteins and how they adapt to hypoxia microenvironments of head and neck cancer are still unknown. Human genome sequences and proteins expressed data of head and neck cancer were retrieved from pathology atlas of Human Protein Atlas project. Then compared the oxygen and carbon element contents between proteomes of head and neck cancer and normal oral mucosa-squamous epithelial cells, genome locations, pathways, and functional dissection associated with head and neck cancer were also studied. A total of 902 differentially expressed proteins were observed where the average oxygen content is higher than that of the lowly expressed proteins in head and neck cancer proteins. Further, the average oxygen content of the up regulated proteins was 2.54% higher than other. None of their coding genes were distributed on the Y chromosome. The up regulated proteins were enriched in endocytosis, apoptosis and regulation of actin cytoskeleton. The increased oxygen contents of the highly expressed and the up regulated proteins might be caused by frequent activity of cytoskeleton and adapted to the rapid growth and fast division of the head and neck cancer cells. The oxygen usage bias and key proteins may help us to understand the mechanisms behind head and neck cancer in targeted therapy, which lays a foundation for the application of stoichioproteomics in targeted therapy and provides promise for potential treatments for head and neck cancer.

Resumo A hipóxia é uma característica proeminente do câncer de cabeça e pescoço. No entanto, as características do elemento oxigênio das proteínas e como elas se adaptam aos microambientes de hipóxia do câncer de cabeça e pescoço ainda são desconhecidas. Sequências do genoma humano e dados expressos de proteínas de câncer de cabeça e pescoço foram recuperados do atlas de patologia do projeto Human Protein Atlas. Em seguida, comparou o conteúdo do elemento de oxigênio e carbono entre proteomas de câncer de cabeça e pescoço, e células epiteliais escamosas da mucosa oral normal, localizações do genoma, vias e dissecção funcional associada ao câncer de cabeça e pescoço também foram estudadas. Um total de 902 proteínas expressas diferencialmente foi observado onde o conteúdo médio de oxigênio é maior do que as proteínas expressas de forma humilde em proteínas de câncer de cabeça e pescoço. Além disso, o conteúdo médio de oxigênio das proteínas reguladas positivamente foi 2,54% maior do que das outras. Nenhum de seus genes codificadores foi distribuído no cromossomo Y. As proteínas reguladas positivamente foram enriquecidas em endocitose, apoptose e regulação do citoesqueleto de actina. O conteúdo aumentado de oxigênio das proteínas altamente expressas e reguladas pode ser causado pela atividade frequente do citoesqueleto e adaptado ao rápido crescimento e divisão das células cancerosas de cabeça e pescoço. O viés do uso de oxigênio e as proteínas-chave podem nos ajudar a entender os mecanismos por trás do câncer de cabeça e pescoço na terapia direcionada, o que estabelece uma base para a aplicação da estequioproteômica na terapia direcionada e oferece uma promessa para potenciais tratamentos para o câncer de cabeça e pescoço.

Stoichioproteomics study of differentially expressed proteins and pathways in head and neck cancer / Estudo estequioproteômico de proteínas e vias expressas diferencialmente no câncer de cabeça e pescoço

Hypoxia is a prominent feature of head and neck cancer. However, the oxygen element characteristics of proteins and how they adapt to hypoxia microenvironments of head and neck cancer are still unknown. Human genome sequences and proteins expressed data of head and neck cancer were retrieved from pathology atlas of Human Protein Atlas project. Then compared the oxygen and carbon element contents between proteomes of head and neck cancer and normal oral mucosa-squamous epithelial cells, genome locations, pathways, and functional dissection associated with head and neck cancer were also studied. A total of 902 differentially expressed proteins were observed where the average oxygen content is higher than that of the lowly expressed proteins in head and neck cancer proteins. Further, the average oxygen content of the up regulated proteins was 2.54% higher than other. None of their coding genes were distributed on the Y chromosome. The up regulated proteins were enriched in endocytosis, apoptosis and regulation of actin cytoskeleton. The increased oxygen contents of the highly expressed and the up regulated proteins might be caused by frequent activity of cytoskeleton and adapted to the rapid growth and fast division of the head and neck cancer cells. The oxygen usage bias and key proteins may help us to understand the mechanisms behind head and neck cancer in targeted therapy, which lays a foundation for the application of stoichioproteomics in targeted therapy and provides promise for potential treatments for head and neck cancer.

A hipóxia é uma característica proeminente do câncer de cabeça e pescoço. No entanto, as características do elemento oxigênio das proteínas e como elas se adaptam aos microambientes de hipóxia do câncer de cabeça e pescoço ainda são desconhecidas. Sequências do genoma humano e dados expressos de proteínas de câncer de cabeça e pescoço foram recuperados do atlas de patologia do projeto Human Protein Atlas. Em seguida, comparou o conteúdo do elemento de oxigênio e carbono entre proteomas de câncer de cabeça e pescoço, e células epiteliais escamosas da mucosa oral normal, localizações do genoma, vias e dissecção funcional associada ao câncer de cabeça e pescoço também foram estudadas. Um total de 902 proteínas expressas diferencialmente foi observado onde o conteúdo médio de oxigênio é maior do que as proteínas expressas de forma humilde em proteínas de câncer de cabeça e pescoço. Além disso, o conteúdo médio de oxigênio das proteínas reguladas positivamente foi 2,54% maior do que das outras. Nenhum de seus genes codificadores foi distribuído no cromossomo Y. As proteínas reguladas positivamente foram enriquecidas em endocitose, apoptose e regulação do citoesqueleto de actina. O conteúdo aumentado de oxigênio das proteínas altamente expressas e reguladas pode ser causado pela atividade frequente do citoesqueleto e adaptado ao rápido crescimento e divisão das células cancerosas de cabeça e pescoço. O viés do uso de oxigênio e as proteínas-chave podem nos ajudar a entender os mecanismos por trás do câncer de cabeça e pescoço na terapia direcionada, o que estabelece uma base para a aplicação da estequioproteômica na terapia direcionada e oferece uma promessa para potenciais tratamentos para o câncer de cabeça e pescoço.

Stoichioproteomics study of differentially expressed proteins and pathways in head and neck cancer / Estudo estequioproteômico de proteínas e vias expressas diferencialmente no câncer de cabeça e pescoço

Abstract Hypoxia is a prominent feature of head and neck cancer. However, the oxygen element characteristics of proteins and how they adapt to hypoxia microenvironments of head and neck cancer are still unknown. Human genome sequences and proteins expressed data of head and neck cancer were retrieved from pathology atlas of Human Protein Atlas project. Then compared the oxygen and carbon element contents between proteomes of head and neck cancer and normal oral mucosa-squamous epithelial cells, genome locations, pathways, and functional dissection associated with head and neck cancer were also studied. A total of 902 differentially expressed proteins were observed where the average oxygen content is higher than that of the lowly expressed proteins in head and neck cancer proteins. Further, the average oxygen content of the up regulated proteins was 2.54% higher than other. None of their coding genes were distributed on the Y chromosome. The up regulated proteins were enriched in endocytosis, apoptosis and regulation of actin cytoskeleton. The increased oxygen contents of the highly expressed and the up regulated proteins might be caused by frequent activity of cytoskeleton and adapted to the rapid growth and fast division of the head and neck cancer cells. The oxygen usage bias and key proteins may help us to understand the mechanisms behind head and neck cancer in targeted therapy, which lays a foundation for the application of stoichioproteomics in targeted therapy and provides promise for potential treatments for head and neck cancer.

Resumo A hipóxia é uma característica proeminente do câncer de cabeça e pescoço. No entanto, as características do elemento oxigênio das proteínas e como elas se adaptam aos microambientes de hipóxia do câncer de cabeça e pescoço ainda são desconhecidas. Sequências do genoma humano e dados expressos de proteínas de câncer de cabeça e pescoço foram recuperados do atlas de patologia do projeto Human Protein Atlas. Em seguida, comparou o conteúdo do elemento de oxigênio e carbono entre proteomas de câncer de cabeça e pescoço, e células epiteliais escamosas da mucosa oral normal, localizações do genoma, vias e dissecção funcional associada ao câncer de cabeça e pescoço também foram estudadas. Um total de 902 proteínas expressas diferencialmente foi observado onde o conteúdo médio de oxigênio é maior do que as proteínas expressas de forma humilde em proteínas de câncer de cabeça e pescoço. Além disso, o conteúdo médio de oxigênio das proteínas reguladas positivamente foi 2,54% maior do que das outras. Nenhum de seus genes codificadores foi distribuído no cromossomo Y. As proteínas reguladas positivamente foram enriquecidas em endocitose, apoptose e regulação do citoesqueleto de actina. O conteúdo aumentado de oxigênio das proteínas altamente expressas e reguladas pode ser causado pela atividade frequente do citoesqueleto e adaptado ao rápido crescimento e divisão das células cancerosas de cabeça e pescoço. O viés do uso de oxigênio e as proteínas-chave podem nos ajudar a entender os mecanismos por trás do câncer de cabeça e pescoço na terapia direcionada, o que estabelece uma base para a aplicação da estequioproteômica na terapia direcionada e oferece uma promessa para potenciais tratamentos para o câncer de cabeça e pescoço.

Stoichioproteomics study of differentially expressed proteins and pathways in head and neck cancer / Estudo estequioproteômico de proteínas e vias expressas diferencialmente no câncer de cabeça e pescoço

Hypoxia is a prominent feature of head and neck cancer. However, the oxygen element characteristics of proteins and how they adapt to hypoxia microenvironments of head and neck cancer are still unknown. Human genome sequences and proteins expressed data of head and neck cancer were retrieved from pathology atlas of Human Protein Atlas project. Then compared the oxygen and carbon element contents between proteomes of head and neck cancer and normal oral mucosa-squamous epithelial cells, genome locations, pathways, and functional dissection associated with head and neck cancer were also studied. A total of 902 differentially expressed proteins were observed where the average oxygen content is higher than that of the lowly expressed proteins in head and neck cancer proteins. Further, the average oxygen content of the up regulated proteins was 2.54% higher than other. None of their coding genes were distributed on the Y chromosome. The up regulated proteins were enriched in endocytosis, apoptosis and regulation of actin cytoskeleton. The increased oxygen contents of the highly expressed and the up regulated proteins might be caused by frequent activity of cytoskeleton and adapted to the rapid growth and fast division of the head and neck cancer cells. The oxygen usage bias and key proteins may help us to understand the mechanisms behind head and neck cancer in targeted therapy, which lays a foundation for the application of stoichioproteomics in targeted therapy and provides promise for potential treatments for head and neck cancer.(AU)

A hipóxia é uma característica proeminente do câncer de cabeça e pescoço. No entanto, as características do elemento oxigênio das proteínas e como elas se adaptam aos microambientes de hipóxia do câncer de cabeça e pescoço ainda são desconhecidas. Sequências do genoma humano e dados expressos de proteínas de câncer de cabeça e pescoço foram recuperados do atlas de patologia do projeto Human Protein Atlas. Em seguida, comparou o conteúdo do elemento de oxigênio e carbono entre proteomas de câncer de cabeça e pescoço, e células epiteliais escamosas da mucosa oral normal, localizações do genoma, vias e dissecção funcional associada ao câncer de cabeça e pescoço também foram estudadas. Um total de 902 proteínas expressas diferencialmente foi observado onde o conteúdo médio de oxigênio é maior do que as proteínas expressas de forma humilde em proteínas de câncer de cabeça e pescoço. Além disso, o conteúdo médio de oxigênio das proteínas reguladas positivamente foi 2,54% maior do que das outras. Nenhum de seus genes codificadores foi distribuído no cromossomo Y. As proteínas reguladas positivamente foram enriquecidas em endocitose, apoptose e regulação do citoesqueleto de actina. O conteúdo aumentado de oxigênio das proteínas altamente expressas e reguladas pode ser causado pela atividade frequente do citoesqueleto e adaptado ao rápido crescimento e divisão das células cancerosas de cabeça e pescoço. O viés do uso de oxigênio e as proteínas-chave podem nos ajudar a entender os mecanismos por trás do câncer de cabeça e pescoço na terapia direcionada, o que estabelece uma base para a aplicação da estequioproteômica na terapia direcionada e oferece uma promessa para potenciais tratamentos para o câncer de cabeça e pescoço.(AU)

Coenzyme Q-10 improves preservation of mitochondrial functionality and actin structure of cryopreserved stallion sperm

Abstract Coenzyme Q-10 (CoQ-10) is a cofactor for mitochondrial electron transport chain and may be an alternative to improve sperm quality of cryopreserved equine semen. This work aimed to improve stallion semen quality after freezing by adding CoQ-10 to the cryopreservation protocol. Seven saddle stallions were utilized. Each animal was submitted to five semen collections and freezing procedures. For cryopreservation, each ejaculate was divided in three treatments: 1) Botucrio® diluent (control); 2) 50 μmol CoQ-10 added to Botucrio® diluent; 3) 1 mmol CoQ-10 added to Botucrio® diluent. Semen batches were analyzed for sperm motility characteristics (CASA), plasma and acrosomal membranes integrity and mitochondrial membrane potential (by fluorescence probes propidium iodide, Hoechst 33342, FITC-PSA and JC-1, respectively), alterations in cytoskeletal actin (phalloidin-FITC) and mitochondrial function (diaminobenzidine; DAB). The 1 mmol CoQ-10 treatment presented higher (P<0.05) amount (66.8%) of sperm cells with fully stained midpiece (indicating high mitochondrial activity) and higher (P<0.05) amount (81.6%) of cells without actin reorganization to the post-acrosomal region compared to control group (60.8% and 76.0%, respectively). It was concluded that the addition of 1 mmol CoQ-10 to the freezing diluent was more effective in preserving mitochondria functionality and cytoskeleton of sperm cells submitted to cryopreservation process.

Escherichia coli vacuolating factor, involved in avian cellulitis, induces actin contraction and binds to cytoskeleton proteins in fibroblasts

Avian pathogenic Escherichia coli (APEC) isolated from avian cellulitis lesions produces a toxin, named Escherichia coli vacuolating factor (ECVF), that causes cell vacuolization and induces inflammatory response in broiler chicken. Methods We investigated the intracellular activities of ECVF in avian fibroblasts using fluorescence staining, electron microscopy, MTT and LDH measurements. As ECVF act specifically in avian cells, we performed blotting assay followed by mass spectrometry to better understand its initial intracellular protein recognition. Results ECVF induced actin contraction, mitochondrial damage and membrane permeability alterations. Ultrastructural analysis showed intracellular alterations, as nuclear lobulation and the presence of degraded structures inside the vacuoles. Moreover, ECVF induced cell death in fibroblasts. ECVF-biotin associates to at least two proteins only in avian cell lysates: alpha-actinin 4 and vinculin, both involved in cytoskeleton structure. Conclusion These findings demonstrated that ECVF plays an important role in avian cellulitis, markedly in initial steps of infection. Taken together, the results place this toxin as a target for drug and/or vaccine development, instead of the use of large amounts antibiotics.(AU)

Escherichia coli vacuolating factor, involved in avian cellulitis, induces actin contraction and binds to cytoskeleton proteins in fibroblasts

Avian pathogenic Escherichia coli (APEC) isolated from avian cellulitis lesions produces a toxin, named Escherichia coli vacuolating factor (ECVF), that causes cell vacuolization and induces inflammatory response in broiler chicken. Methods We investigated the intracellular activities of ECVF in avian fibroblasts using fluorescence staining, electron microscopy, MTT and LDH measurements. As ECVF act specifically in avian cells, we performed blotting assay followed by mass spectrometry to better understand its initial intracellular protein recognition. Results ECVF induced actin contraction, mitochondrial damage and membrane permeability alterations. Ultrastructural analysis showed intracellular alterations, as nuclear lobulation and the presence of degraded structures inside the vacuoles. Moreover, ECVF induced cell death in fibroblasts. ECVF-biotin associates to at least two proteins only in avian cell lysates: alpha-actinin 4 and vinculin, both involved in cytoskeleton structure. Conclusion These findings demonstrated that ECVF plays an important role in avian cellulitis, markedly in initial steps of infection. Taken together, the results place this toxin as a target for drug and/or vaccine development, instead of the use of large amounts antibiotics.(AU)

Vitamin and mineral supplementation improves erythrocytic recovery and productive performance of infected by gastrointestinal parasites / A suplementação de vitaminas e minerais melhora a recuperação eritrocítica e o desempenho produtivo de cordeiros infectados por parasitas gastrointestinais

The objective of the present study was to evaluate the effect of a vitamin and mineral complex, associated with the application of an efficient anthelmintic, in parasitized lambs, with characteristic signs of gastrointestinal nematode infection, on hematological clinical parameters of clinical improvementand weight gain. 60 lambs Australian Merino breed, with 8 and 9 months of age, were segregated in four groups: control (no supplemetation); formula 1 (Iron Dextran, Organic Phosphorus, Cyanocobalamin and Vitamin k); formula 2 (Iron Dextran, Organic Phosphorus and Cyanocobalamin); and formula 3 (Vitamin k). Hematocrit, prothrombin time, total plasma proteins, color of the conjunctiva and weight gain were analyzed. The results were submitted to analysis of variance (ANOVA) through the repeated measures test. There was no statistical difference between treatments for the variables of hematocrit (P = 0.564), prothrombin time (P = 0.911) and plasma proteins (P = 0.6), for the conjunctiva color variable there was a difference (P = 0.052 ), with greater results for the groups supplemented with Vitamin K, Butafosfan, Cyanocobalamin and Iron (groups F1 and F3), as well as those same groups reached higher body weight at D35 (P = 0.023). It can be concluded that the administration of vitamin and mineral complex, associated with efficient anthelmintic, promoted a better performance in parasitized lambs.(AU)

O objetivo do presente estudo foi avaliar o efeito da aplicação de um complexo vitamínico e mineral, aplicado junto com a administração de um anti-helmíntico eficiente, em cordeiros parasitados, com sinais característicos de infecção por nematóide gastrointestinal, sobre os parâmetros clínicos hematológicos de melhora clínica e ganho de peso. Foram utilizados 60 cordeiros da raça Merino Australiano, com 8 e 9 meses de idade, segregados em quatro grupos: controle (não suplementado); fórmula 1 (Ferro Dextrano, Fósforo Orgânico, Cianocobalamina e Vitamina k); fórmula 2 (Ferro Dextrano, Fósforo Orgânico e Cianocobalamina); e fórmula 3 (Vitamina k). Foram analisados hematócrito, tempo de protrombina, proteínas plasmáticas totais (PPT), coloração da conjuntiva e ganho de peso. Os resultados foram submetidos à análise de variância (ANOVA) através do teste de medidas repetidas. Não houve diferença estatísticas entre os tratamentos para as variáveis de hematócrito (P=0,564), tempo de protrombina (P=0,911) e PPT (P=0,6), para a variável de coloração da conjuntiva houve diferença (P=0,052), com resultados superiores para os grupos suplementados com Vitamina K, Butafosfan, Cianocobalamina e Ferro (grupos F1 e F3), assim como esses mesmos grupos atingiram peso vivo no D35 superior (P=0,023). Podendo concluir que a administração de complexo vitamínico e mineral, administrado em conjunto com anti-helmíntico eficiente, promoveu um melhor desempenho em cordeiros parasitados.(AU)

Vitamin and mineral supplementation improves erythrocytic recovery and productive performance of infected by gastrointestinal parasites / A suplementação de vitaminas e minerais melhora a recuperação eritrocítica e o desempenho produtivo de cordeiros infectados por parasitas gastrointestinais

The objective of the present study was to evaluate the effect of a vitamin and mineral complex, associated with the application of an efficient anthelmintic, in parasitized lambs, with characteristic signs of gastrointestinal nematode infection, on hematological clinical parameters of clinical improvementand weight gain. 60 lambs Australian Merino breed, with 8 and 9 months of age, were segregated in four groups: control (no supplemetation); formula 1 (Iron Dextran, Organic Phosphorus, Cyanocobalamin and Vitamin k); formula 2 (Iron Dextran, Organic Phosphorus and Cyanocobalamin); and formula 3 (Vitamin k). Hematocrit, prothrombin time, total plasma proteins, color of the conjunctiva and weight gain were analyzed. The results were submitted to analysis of variance (ANOVA) through the repeated measures test. There was no statistical difference between treatments for the variables of hematocrit (P = 0.564), prothrombin time (P = 0.911) and plasma proteins (P = 0.6), for the conjunctiva color variable there was a difference (P = 0.052 ), with greater results for the groups supplemented with Vitamin K, Butafosfan, Cyanocobalamin and Iron (groups F1 and F3), as well as those same groups reached higher body weight at D35 (P = 0.023). It can be concluded that the administration of vitamin and mineral complex, associated with efficient anthelmintic, promoted a better performance in parasitized lambs.

O objetivo do presente estudo foi avaliar o efeito da aplicação de um complexo vitamínico e mineral, aplicado junto com a administração de um anti-helmíntico eficiente, em cordeiros parasitados, com sinais característicos de infecção por nematóide gastrointestinal, sobre os parâmetros clínicos hematológicos de melhora clínica e ganho de peso. Foram utilizados 60 cordeiros da raça Merino Australiano, com 8 e 9 meses de idade, segregados em quatro grupos: controle (não suplementado); fórmula 1 (Ferro Dextrano, Fósforo Orgânico, Cianocobalamina e Vitamina k); fórmula 2 (Ferro Dextrano, Fósforo Orgânico e Cianocobalamina); e fórmula 3 (Vitamina k). Foram analisados hematócrito, tempo de protrombina, proteínas plasmáticas totais (PPT), coloração da conjuntiva e ganho de peso. Os resultados foram submetidos à análise de variância (ANOVA) através do teste de medidas repetidas. Não houve diferença estatísticas entre os tratamentos para as variáveis de hematócrito (P=0,564), tempo de protrombina (P=0,911) e PPT (P=0,6), para a variável de coloração da conjuntiva houve diferença (P=0,052), com resultados superiores para os grupos suplementados com Vitamina K, Butafosfan, Cianocobalamina e Ferro (grupos F1 e F3), assim como esses mesmos grupos atingiram peso vivo no D35 superior (P=0,023). Podendo concluir que a administração de complexo vitamínico e mineral, administrado em conjunto com anti-helmíntico eficiente, promoveu um melhor desempenho em cordeiros parasitados.

Coenzyme Q-10 improves preservation of mitochondrial functionality and actin structure of cryopreserved stallion sperm

Coenzyme Q-10 (CoQ-10) is a cofactor for mitochondrial electron transport chain and may be an alternative to improve sperm quality of cryopreserved equine semen. This work aimed to improve stallion semen quality after freezing by adding CoQ-10 to the cryopreservation protocol. Seven saddle stallions were utilized. Each animal was submitted to five semen collections and freezing procedures. For cryopreservation, each ejaculate was divided in three treatments: 1) Botucrio® diluent (control); 2) 50 μmol CoQ-10 added to Botucrio® diluent; 3) 1 mmol CoQ-10 added to Botucrio® diluent. Semen batches were analyzed for sperm motility characteristics (CASA), plasma and acrosomal membranes integrity and mitochondrial membrane potential (by fluorescence probes propidium iodide, Hoechst 33342, FITC-PSA and JC-1, respectively), alterations in cytoskeletal actin (phalloidin-FITC) and mitochondrial function (diaminobenzidine; DAB). The 1 mmol CoQ-10 treatment presented higher (P<0.05) amount (66.8%) of sperm cells with fully stained midpiece (indicating high mitochondrial activity) and higher (P<0.05) amount (81.6%) of cells without actin reorganization to the post-acrosomal region compared to control group (60.8% and 76.0%, respectively). It was concluded that the addition of 1 mmol CoQ-10 to the freezing diluent was more effective in preserving mitochondria functionality and cytoskeleton of sperm cells submitted to cryopreservation process.(AU)

Antitumoral effects of Amblyomma sculptum Berlese saliva in neuroblastoma cell lines involve cytoskeletal deconstruction and cell cycle arrest / Efeito antitumoral da saliva do carrapato Amblyomma sculptum Berlese em células de neuroblastoma envolve desconstrução do citoesqueleto e parada do ciclo celular

The antitumor properties of ticks salivary gland extracts or recombinant proteins have been reported recently, but little is known about the antitumor properties of the secreted components of saliva. The goal of this study was to investigate the in vitro effect of the saliva of the hard tick Amblyomma sculptum on neuroblastoma cell lines. SK-N-SK, SH-SY5Y, Be(2)-M17, IMR-32, and CHLA-20 cells were susceptible to saliva, with 80% reduction in their viability compared to untreated controls, as demonstrated by the methylene blue assay. Further investigation using CHLA-20 revealed apoptosis, with approximately 30% of annexin-V positive cells, and G0/G1-phase accumulation (>60%) after treatment with saliva. Mitochondrial membrane potential (m) was slightly, but significantly (p 0.05), reduced and the actin cytoskeleton was disarranged, as indicated by fluorescent microscopy. The viability of human fibroblast (HFF-1 cells) used as a non-tumoral control decreased by approximately 40%. However, no alterations in cell cycle progression, morphology, and m were observed in these cells. The present work provides new perspectives for the characterization of the molecules present in saliva and their antitumor properties.(AU)

As propriedades antitumorais de extratos de glândulas salivares de carrapatos ou proteínas recombinantes foram relatadas recentemente, mas pouco se sabe sobre as propriedades antitumorais dos componentes secretados da saliva. O objetivo deste estudo foi investigar o efeito in vitro da saliva bruta do carrapato duro Amblyomma sculptum sobre as linhagens celulares de neuroblastoma. Células SK-N-SK, SH-SY5Y, Be(2)-M17, IMR-32 e CHLA-20 foram suscetíveis à saliva, com redução de 80% na sua viabilidade em comparação com controles não tratados, como demonstrado pelo ensaio de Azul de Metileno. Investigações posteriores utilizando CHLA-20 revelaram apoptose, com aproximadamente 30% de células positivas para anexina-V, e G0/G1 (> 60%) após tratamento com saliva. O potencial de membrana mitocondrial (m) foi reduzido significativamente (p 0,05), e o citoesqueleto de actina foi desestruturado, como indicado pela microscopia de fluorescência. A viabilidade do fibroblasto humano (células HFF-1), usado como controle não tumoral, diminuiu em aproximadamente 40%. No entanto, não foram observadas alterações na progressão do ciclo celular, morfologia e m nestas células. O presente trabalho fornece novas perspectivas para a caracterização das moléculas presentes na saliva e suas propriedades antitumorais.(AU)

Antitumoral effects of Amblyomma sculptum Berlese saliva in neuroblastoma cell lines involve cytoskeletal deconstruction and cell cycle arrest / Efeito antitumoral da saliva do carrapato Amblyomma sculptum Berlese em células de neuroblastoma envolve desconstrução do citoesqueleto e parada do ciclo celular

The antitumor properties of ticks salivary gland extracts or recombinant proteins have been reported recently, but little is known about the antitumor properties of the secreted components of saliva. The goal of this study was to investigate the in vitro effect of the saliva of the hard tick Amblyomma sculptum on neuroblastoma cell lines. SK-N-SK, SH-SY5Y, Be(2)-M17, IMR-32, and CHLA-20 cells were susceptible to saliva, with 80% reduction in their viability compared to untreated controls, as demonstrated by the methylene blue assay. Further investigation using CHLA-20 revealed apoptosis, with approximately 30% of annexin-V positive cells, and G0/G1-phase accumulation (>60%) after treatment with saliva. Mitochondrial membrane potential (m) was slightly, but significantly (p 0.05), reduced and the actin cytoskeleton was disarranged, as indicated by fluorescent microscopy. The viability of human fibroblast (HFF-1 cells) used as a non-tumoral control decreased by approximately 40%. However, no alterations in cell cycle progression, morphology, and m were observed in these cells. The present work provides new perspectives for the characterization of the molecules present in saliva and their antitumor properties.(AU)

As propriedades antitumorais de extratos de glândulas salivares de carrapatos ou proteínas recombinantes foram relatadas recentemente, mas pouco se sabe sobre as propriedades antitumorais dos componentes secretados da saliva. O objetivo deste estudo foi investigar o efeito in vitro da saliva bruta do carrapato duro Amblyomma sculptum sobre as linhagens celulares de neuroblastoma. Células SK-N-SK, SH-SY5Y, Be(2)-M17, IMR-32 e CHLA-20 foram suscetíveis à saliva, com redução de 80% na sua viabilidade em comparação com controles não tratados, como demonstrado pelo ensaio de Azul de Metileno. Investigações posteriores utilizando CHLA-20 revelaram apoptose, com aproximadamente 30% de células positivas para anexina-V, e G0/G1 (> 60%) após tratamento com saliva. O potencial de membrana mitocondrial (m) foi reduzido significativamente (p 0,05), e o citoesqueleto de actina foi desestruturado, como indicado pela microscopia de fluorescência. A viabilidade do fibroblasto humano (células HFF-1), usado como controle não tumoral, diminuiu em aproximadamente 40%. No entanto, não foram observadas alterações na progressão do ciclo celular, morfologia e m nestas células. O presente trabalho fornece novas perspectivas para a caracterização das moléculas presentes na saliva e suas propriedades antitumorais.(AU)

Histopathological pattern recognition of cresty neck in horses in Spain

The aim of this study was to describe patterns of histopathological recognition of the cresty neck in Horses in Spain. A total of 250 horses were studied in Andalusia and Extremadura, Spain. Seventy-six percent of horses present cresty neck. The damage of the cresty neck in horses was categorized as Grade 0 - Muscle fibers are observed, no fat vacuoles are observed (24% of the horses). Grade 1 - Scarce adipose deposit was observed. An unaltered muscle tissue is observed (21% of the horses). Grade 2 - fat vacuoles are evident in muscle tissue intermyofibrillar space and prone to coalescence (23% of the horses). Grade 3 - Abundant fat vacuoles in the intermyofibrillar space, with tendency to coalesce, and low fat infiltration in muscle tissue (moderate lipomatosis) (16% of the horses). Grade 4 - Abundant fat vacuoles in the intermyofibrillar space, with tendency to coalesce, and fatty infiltration in muscle tissue (marked lipomatosis) (8% of the horses). Grade 5 - Only fat vacuoles are observed, without muscle tissue (severe lipomatosis) (8% of the horses). The results for desmin antibody (1: 100 dilution) was positive (++) in grades 0-2, and negative (-) in grades 3-5. These results suggest that as fat/lipomatosis infiltration increases (progresses in grades 3, 4 and 5), the intercellular space (intermyofibrillar) increases and therefore the cell cytoskeleton is lost, with loss of the bands Z, so the negative response to this antibody. Inconclusion, we describe histopathological pattern recognition of cresty neck in horses in Spain.

Histopathological pattern recognition of cresty neck in horses in Spain

The aim of this study was to describe patterns of histopathological recognition of the cresty neck in Horses in Spain. A total of 250 horses were studied in Andalusia and Extremadura, Spain. Seventy-six percent of horses present cresty neck. The damage of the cresty neck in horses was categorized as Grade 0 - Muscle fibers are observed, no fat vacuoles are observed (24% of the horses). Grade 1 - Scarce adipose deposit was observed. An unaltered muscle tissue is observed (21% of the horses). Grade 2 - fat vacuoles are evident in muscle tissue intermyofibrillar space and prone to coalescence (23% of the horses). Grade 3 - Abundant fat vacuoles in the intermyofibrillar space, with tendency to coalesce, and low fat infiltration in muscle tissue (moderate lipomatosis) (16% of the horses). Grade 4 - Abundant fat vacuoles in the intermyofibrillar space, with tendency to coalesce, and fatty infiltration in muscle tissue (marked lipomatosis) (8% of the horses). Grade 5 - Only fat vacuoles are observed, without muscle tissue (severe lipomatosis) (8% of the horses). The results for desmin antibody (1: 100 dilution) was positive (++) in grades 0-2, and negative (-) in grades 3-5. These results suggest that as fat/lipomatosis infiltration increases (progresses in grades 3, 4 and 5), the intercellular space (intermyofibrillar) increases and therefore the cell cytoskeleton is lost, with loss of the bands Z, so the negative response to this antibody. Inconclusion, we describe histopathological pattern recognition of cresty neck in horses in Spain.(AU)

Isolation and identification of proteins from swine sperm chromatin and nuclear matrix

The aim of this study was to perform a proteomic analysis to isolate and identify proteins from the swine sperm nuclear matrix to contribute to a database of swine sperm nuclear proteins. We used pre-chilled diluted semen from seven boars (19 to 24 week old) from the commercial lineLandrace x Large Whitex Pietran. The semen was processed to separate the sperm heads and extract the chromatin and nuclear matrix for protein quantification and analysis by mass spectrometry, by LTQ Orbitrap ELITE Mass spectrometer (Thermo-Finnigan) coupled to a nanoflow chromatography system (LC-MS/MS). We identified 222 different proteins in the sample; a total of 159 (71.6%) were previously described as present in the somatic or sperm nuclei of other species, 41 (18.5%) did not have a previously reported nuclear presence and 22(9.9%) had not been characterized. The most abundant family of proteins corresponded to ribosomal (13.1%), followed by cytoskeleton (12.2%), uncharacterized (9.9%), histones (5.4%), proteasome subunits (3.6%)and heat shock (1.8%). The other proteins clustered in other families accounted for 54% of the total proteins. The protein isolation of the nuclear matrix of the swine spermatozoa was satisfactory, thus demonstrating that the protocol used was efficient. Several proteins were identified and described. However, it was not possible to identify some protein structures. Therefore,this study helps to establish a starting point for future proteomic studies comparing fertile and sub-fertile animals.

Isolation and identification of proteins from swine sperm chromatin and nuclear matrix

The aim of this study was to perform a proteomic analysis to isolate and identify proteins from the swine sperm nuclear matrix to contribute to a database of swine sperm nuclear proteins. We used pre-chilled diluted semen from seven boars (19 to 24 week old) from the commercial lineLandrace x Large Whitex Pietran. The semen was processed to separate the sperm heads and extract the chromatin and nuclear matrix for protein quantification and analysis by mass spectrometry, by LTQ Orbitrap ELITE Mass spectrometer (Thermo-Finnigan) coupled to a nanoflow chromatography system (LC-MS/MS). We identified 222 different proteins in the sample; a total of 159 (71.6%) were previously described as present in the somatic or sperm nuclei of other species, 41 (18.5%) did not have a previously reported nuclear presence and 22(9.9%) had not been characterized. The most abundant family of proteins corresponded to ribosomal (13.1%), followed by cytoskeleton (12.2%), uncharacterized (9.9%), histones (5.4%), proteasome subunits (3.6%)and heat shock (1.8%). The other proteins clustered in other families accounted for 54% of the total proteins. The protein isolation of the nuclear matrix of the swine spermatozoa was satisfactory, thus demonstrating that the protocol used was efficient. Several proteins were identified and described. However, it was not possible to identify some protein structures. Therefore,this study helps to establish a starting point for future proteomic studies comparing fertile and sub-fertile animals.(AU)

Recelularização da matriz extracelular de músculo de cães saudáveis e distróficos

A Distrofia Muscular de Duchenne (DMD) é uma doença hereditária de caráter progressivo causada por mutação no gene produtor da proteína distrofina, responsável por ligar o citoesqueleto da célula muscular a matriz extracelular (MEC). Além de humanos, outros animais podem apresentar distrofias musculares, podendo ser utilizados como modelos para o estudo de distrofias musculares em humanos. O modelo animal mais utilizado é o modelo de Distrofia Muscular do Golden Retriever (GRMD). Os estudos de distrofias musculares podem ser associados a diversas áreas, como a Bioengenharia de tecidos, área que visa a utilização de MEC descelularizadas (scaffolds) para reparação/substituição de órgãos e tecidos, porém estes scaffolds também podem servir como ferramenta para estudo de MEC em doenças. Portando, o objetivo deste trabalho foi a caracterização de alguns componentes da MEC distrófica, sugerir se uma MEC distrófica pode ser um possível biomaterial para medicina regenerativa e a recelularização de MEC distróficas para observação da relação célula- matriz. Para realização deste trabalho, músculos bíceps femorais distróficos e não distróficos foram descelularizados com protocolo de utilização de SDS a 1% e passaram por análises de quantificação de DNA e fluorescência por DAPI para validação da descelularização e análises histológicas (colorações), imunohistoquímica e de Microscopia Eletrônica de Varredura para avaliação da composição, ultraestrutura e preservação das MEC distróficas e não distróficas. Como resultados, obtivemos as quantificações de DNA que alcançaram quantidade inferior a 50ng de DNA por mg de tecido e na fluorescência por DAPI não foram visualizados núcleos celulares nos tecidos descelularizados. Pelas análises histológicas foi possível observar a ausência de células nos tecidos descelularizados e a preservação de fibras colágenas da MEC. Pela imunohistoquímica, não foram observadas grandes diferenças nos componentes da MEC de músculos distróficos quando comparados com músculos não distróficos. Quando observadas, as células utilizadas para recelularização dos scaffolds apresentaram maior taxa de proliferação quando em contato com o scaffold muscular e o protocolo de esterilização do scaffold foi eficiente, já que os testes para detecção de micoplasma foram negativos. Foi observado o aumento da quantidade de DNAg após a recelularização dos scaffolds e pela MEV são visíveis camadas de células sobre a matriz extracelular. Concluímos que a MEC de músculo distrófico não interfere na diferenciação e adesão celular quando comparadas às MEC de músculo não distrófico. Embora o aumento de distrofina tenha sido observado em alguns processos de recelularização, mais estudos precisam ser realizados para comprovação deste dado.

Duchenne Muscular Dystrophy (DMD) is a progressive hereditary disease caused by a mutation in the gene producing the dystrophin protein, responsible for linking the cytoskeleton of the muscle cell to the extracellular matrix (ECM). In addition to humans, other animals may have muscular dystrophies and can be used as models for the study of muscular dystrophies in humans. The most used animal model is the Golden Retriever Muscular Dystrophy (GRMD) model. Muscular dystrophy studies can be associated with several areas, such as tissue bioengineering, an area that aims to use decellularized ECM (scaffolds) for repairing / replacing organs and tissues, but these scaffolds can also serve as a tool for studying the ECM in diseases. Therefore, the objective of this work was to characterize some components of the dystrophic ECM, to suggest whether a dystrophic ECM may be a possible biomaterial for regenerative medicine and the recellularization of dystrophic ECM to observe the cell-matrix relationship. To perform this work, dystrophic and non-dystrophic femoral biceps muscles were decellularized using a 1% SDS protocol and underwent DNA quantification and fluorescence analyzes by DAPI to validate decellularization and histological (staining), immunohistochemistry and Scanning Electron microscopy analyzes to evaluate the composition, ultrastructure and preservation of dystrophic and non-dystrophic ECM. As a result, we obtained the DNA quantifications that reached a quantity of less than 50ng of DNA per mg of tissue and in the fluorescence by DAPI, cell nuclei were not seen in the decellularized tissues. Through histological analyzes it was possible to observe the absence of cells in the decellularized tissues and the preservation of collagen fibers of the ECM. Due to immunohistochemistry, no major differences were observed in the ECM components of dystrophic muscles when compared with non-dystrophic muscles. When observed, the cells used to recellularize the scaffolds showed a higher proliferation rate when in contact with the muscle scaffold and the scaffold sterilization protocol was efficient, since the tests for mycoplasma detection were negative. An increase in the amount of DNAg was observed after the scaffolds were recellularized and by SEM, layers of cells are visible on the extracellular matrix. We conclude that the ECM of dystrophic muscle does not interfere with cell differentiation and adhesion when compared to the ECM of non-dystrophic muscle. Although the increase in dystrophin has been observed in some recellularization processes, more studies need to be carried out to prove this data.

Interação entre células mesenquimais adipoderivadas e scaffold de poli (ácido lático-coglicólico) e poli (isopreno) epox confeccionado a partir de centrifugal spinning: estudo in vitro

A engenharia de tecidos compreende o uso de células tronco mesenquimais associadas com biomateriais, tendo como finalidade o reparo, regeneração e restauração de tecidos lesados. O objetivo desse trabalho foi avaliar a interação entre células mesenquimais adipoderivadas de humanos com scaffold de PLGA/PI epox, confeccionado através da técnica de centrifugal spinning. Preconizou-se, a partir de testes in vitro, o estabelecimento da melhor densidade de plaqueamento entre 0,325x105, 0,65x105 e 1,3x105 e melhor tempo de cultivo entre 24, 48 ou 72 horas. A partir das características fibroblastóides, aderência a garrafas plásticas de cultivo celular, diferenciação em linhagens adipogênicas e osteogênicas e imunofenotipagem positiva para CD90 e CD105 e negativa para CD34 e CD45, as células utilizadas neste estudo foram consideradas tronco mesenquimais. A integração das células à matriz fibrosa foi observada por meio de microscopia eletrônica de varredura, onde foi possível evidenciar a emissão de pseudópodes celulares. As fibras confeccionadas pela técnica de centrifugal spinning apresentaram-se fibrosas e porosas, com diâmetro médio de 3,61±2,81m, tendo uma maior representatividade de fibras com 1m (n=113). A atividade metabólica das células cultivadas junto ao biomaterial foi similar aos seus respectivos controles (células aderidas no fundo da placa, padrão ouro) (p>0,05). A exceção evidenciada entre os grupos testes e controle, perante ao teste de MTT, foi notória no cultivo de 1,3x105 em 72 horas, onde as células cultivadas junto ao scaffold mostraram-se metabolicamente mais ativas que o controle (2,87±0,30; 2,18±0,15, p>0,05), demonstrando a não interferência do biomaterial na viabilidade celular. A menor absorbância foi no grupo de 0,325x105 ADSCs (0,69±0,19; 1,27±0,46; 1,25±0,43, diferindo em todos os tempos de avaliação em relação ao grupo de 1,3x105 células (1,96±0,53; 2,45±0,26; 2,87±0,30, p<0,05), porém foi homogênea ao plaqueamento de 0,65x105 (1,02±0,24; 2,07±0,71; 1,77±0,56, p>0,05). Esse último, apresentou menor absorbância quando contrastado ao grupo de maior densidade nas avaliações de 24 e 72 horas (p<0,05). Perante a coloração do citoesqueleto celular, não foram demonstradas alterações quando as células foram cultivadas junto as matrizes fibrosas, independente do tempo de incubação e densidade. A contagem celular foi realizada por meio da coloração nuclear com DAPI, onde a maior média obtida por campo foi no plaqueamento de 1,3x105 células, durante 24 horas (529±81,70). Porém, há uma tendência ao descolamento celular com o passar das horas, podendo se justificar pelas forças moleculares atuantes entre o scaffold e as células e por ter um número alto de células aderidas incialmente. Porém, as células retornam a proliferar em 72 horas de avaliações. Fundamentado nos achados descritos acima, constatamos que o plaqueamento de 1,3x105 por 24 horas foi o mais eficaz. Ademais, o uso do biomaterial junto ao cultivo durante três dias de avaliações, não interferiu na viabilidade e morfologia das células. Segundo esses resultados, associação entre PLGA/PI epox e células mesenquimais adipoderivadas podem ser consideradas para futuros estudos in vivo na engenharia de tecidos

Tissue engineering comprises the use of mesenchymal stem cells associated with biomaterials, with the purpose of repairing, regenerating and restoring damaged tissues. The aim of this work was to evaluate the interaction between human adipoderivated mesenchymal cells with PLGA/PI epox scaffold, made using the centrifugal spinning technique. It was recommended, based on in vitro tests, the establishment of the best plating density between 0.325x105, 0.65x105 and 1.3x105 and the best culture time between 24, 48 or 72 hours. From fibroblastoid characteristics, adherence to plastic bottles of cell culture, differentiation into adipogenic and osteogenic lines and positive immunophenotyping for CD90 and CD105 and negative for CD34 and CD45, the cells used in this study were considered mesenchymal stem. The integration of cells in the fibrous matrix was observed by means of scanning electron microscopy, where it was possible to evidence the emission of cellular pseudopods. The fibers made by the spinning centrifugal technique were fibrous and porous, with an average diameter of 3.61±2.81m, with a greater representation of fibers with 1m (n = 113). The metabolic activity of cells grown next to the biomaterial was similar to their respective controls (cells adhered to the bottom of the plate, gold standard) (p> 0.05). The exception shown between the test and control groups, compared to the MTT test, was evident in the cultivation of 1.3x105 in 72 hours, where cells cultured next to the scaffold were more metabolically active than the control (2.87±0.30; 2.18±0.15 p>0.05), demonstrating the non-interference of the biomaterial in cell viability. The lowest absorbance was in the group of 0.325x105 ADSCs (0.69±0.19; 1.27±0.46; 1.25±0.43, differing at all times in relation to the 1.3x105 group cells (1.96±0.53; 2.45±0.26; 2.87±0.30, p <0.05), but it was homogeneous at 0.65x105 (1.02± 0.24; 2.07±0.71; 1.77±0.56, p> 0.05) .The latter showed lower absorbance when compared to the group with higher density in the 24 and 72 hour evaluations (p<0.05). Due to the staining of the cell cytoskeleton, no changes were demonstrated when the cells were cultured with fibrous matrices, regardless of the incubation time and density. Cell count was performed by means of nuclear staining with DAPI, where the highest average obtained by field was in the plating of 1.3x105 cells for 24 hours (529±81.70). However, there is a tendency for cell detachment over time, which may be justified by the molecular forces acting between the scaffold and the cells and for having a high number of cells adhered initially. However, the cells return to proliferate in 72 hours of evaluations. Based 8 on the findings described above, we found that the plating of 1.3x105 for 24 hours was the most effective. In addition, the use of biomaterial with the culture during three days of evaluations, did not interfere in the viability and morphology of the cells. According to these results, an association between PLGA/PI epox and adipoderivated mesenchymal cells can be considered for future in vivo studies in tissue engineering

COMPARAÇÃO DOS TRANSCRIPTOMAS DE SUÍNOS AFETADOS COM HÉRNIA UMBILICAL E HÉRNIA ESCROTAL

A hérnia é considerada um dos defeitos congênitos mais comuns encontrados em suínos. As mais observadas são escrotais (HE), inguinais (HI) e umbilicais (HU). As hérnias causam prejuízo para a suinocultura mundial devido a redução da eficiência produtiva, além de afetar negativamente o bem-estar do animal. Vários estudos genômicos já foram conduzidos, porém, ainda não foi possível identificar os genes responsáveis pela formação das hérnias em suínos, dificultando a seleção contra essas características. Dessa forma, buscou-se comparar o perfil de expressão dos transcriptomas relacionados às hérnias escrotal e umbilical e identificar genes candidatos aos dois tipos de hérnia, utilizando análises de RNA-Seq. Coletaram-se amostras biológicas de anel inguinal e umbilical de suínos com HE, HU e livres destes defeitos, as quais passaram pela extração do RNA. Após, foram preparadas bibliotecas de cDNA e estas sequenciadas na plataforma Illumina. As sequências passaram por análise de controle de qualidade e foram alinhadas e mapeadas contra o genoma de referência do suíno (Sus scrofa, v11.1). Posteriormente, foram identificados os genes expressos nos tecidos e os genes diferencialmente expressos (DE) quando comparados os grupos controle e afetados. O perfil de expressão dos transcriptomas relacionados à HE e HU foi comparado para identificar genes DE nos dois tipos de hérnia. Realizaram-se análises para a descoberta de polimorfismos nesses genes com posterior anotação daqueles encontrados para as duas hérnias estudadas. Em cada grupo, compararam-se os genes DE e foi verificado se estes estavam em regiões de QTL (Quantitative trait loci) já relatadas para suínos. Após comparação dos dois transcriptomas (HE e HU), observou-se que 94,91% dos genes encontrados estavam contidos em ambos os grupos. Quando comparadas amostras de animais afetados com aquelas de seus respectivos grupos controle, identificaram-se 627 genes DE para HE e 199 para HU, dos quais 35 genes estavam DE em ambos os grupos. Estes genes participam de 108 processos biológicos que envolvem desde o sistema imunológico até a organização celular. Dos genes DE em ambos os grupos, dois (ACAN e BCHE) estão em regiões de QTL já relatadas para hérnia escrotal. Considerou-se os genes MAP1LC3C, VIT, ACAN, ACER2, KCNMA1 e SYNPO2 candidatos ao surgimento dos dois tipos de defeito por apresentarem expressão equivalente em ambas as hérnias e participarem nos processos de adesão celular, organização do citoesqueleto, produção de colágeno, relaxamento muscular e autofagia. Identificaram-se 67 polimorfismos no tecido do anel inguinal e 76 no anel umbilical dos quais 11 e 14 são novos, respectivamente. Além disso, foi observada uma variante com função deletéria localizada no gene ITGAM, que participa do processo biológico de diferenciação celular ectodérmica. Considera-se que o perfil da expressão desses genes possa interferir no desenvolvimento normal do tecido, causar enfraquecimento e diminuir a resistência do local, podendo levar a formação de ambas as hérnias em suínos. Assim, avançou-se no conhecimento dos genes relacionados ao surgimento da HE e HU, contribuindo para a compreensão do mecanismo genético relacionado aos dois tipos de hérnia em suínos.

Hernia is considered one of the main birth defects found in pigs. The most common are scrotal (SH), inguinal (IH) and umbilical (UH). Hernias cause losses in pig production worldwide due to reduced production efficiency, also negatively affecting the animal's welfare. Several genomic studies have already been conducted; however, it has not yet been possible to identify the genes responsible for the formation of hernias in pigs, hindering the selection against these characteristics. Thus, we aimed to compare the expression profile of transcriptomes related to scrotal and umbilical hernias and to identify candidate genes for both types of hernia, using RNA-Seq analyses. Biological samples of inguinal and umbilical rings from pigs with HE, HU and without any of the defects were collected and submitted to RNA extraction. The cDNA libraries were prepared and sequencing was performed using Illumina platform. After the reads quality control, they were aligned and mapped against the swine reference genome (Sus scrofa, v11.1). Subsequently, the genes expressed in each tissue were identified as well as the DE genes between the control and affected groups. The expression profile of SH and UH-related transcriptomes was then compared in order to identify genes that were DE in both types of hernia. Analyses were carried out to discover polymorphisms in these genes with further annotation of those found in both hernias studied. The DE genes of each group were compared and verified if they were located in QTL regions (Quantitative trait loci) already reported for pigs. After comparing the two transcriptomes (HE and HU), 94.91% of the genes found were contained in both groups. When samples of affected animals were compared with those of their respective control groups, 627 DE genes were identified for SH and 199 for UH, of which 35 genes were DE in both groups. These genes participate in 108 biological processes (BP) that involve since the immune system to cellular organization. From the DE genes in both groups, two genes (ACAN and BCHE) were in QTL regions already reported for scrotal hernia. Furthermore, the MAP1LC3C, VIT, ACAN, ACER2, KCNMA1 and SYNPO2 genes were considered candidates to the appearance of both types of defect for having equivalent expression in the two types of hernia and participating in cell adhesion, cytoskeleton organization, collagen production, muscle relaxation and autophagy BP. A total of 67 polymorphisms were identified in the inguinal ring and 76 in the umbilical ring tissues, of which 11 and 14 were new, respectively. Also, a variant with deleterious function was found in the ITGAM gene, which participates in the BP of ectodermal cell differentiation. The expression profile of these genes possibly interferes with the normal development of the tissue, causing weakness and decreasing the resistance of the site, which can lead to the formation of both hernias in pigs. Therefore, progress has been made in the knowledge of genes related to the emergence of SH and UH contributing to a better understanding of the genetic mechanism related to both types of hernia in pigs.

Development of experimental in vitro burn model

To propose an experimental burn model in NIH-3T3 cell line. Induction of thermal injury in cultures of mouse fibroblast - NIH-3T3- cell line and determination of cell viability by MTT and imunofluorescence. The heating of the Petri dish increased proportionally to the temperature of the base and the time of exposure to microwave. In this in vitro burn model, using the cell line NIH-3T3 was observed drastic cellular injury with significant changes in cell viability and activity. It showed drastically modified cell morphology with altered membrane, cytoskeleton and nucleus, and low cellularity compared to the control group. The burn model in vitro using the cell line NIH-3T3 was reproductive and efficient. This burn model was possible to determine significant changes in cell activity and decreased viability, with drastic change in morphology, cell lysis and death.(AU)