The potential of mRNA expression evaluation in predicting HER2 positivity in gastroesophageal cancer

Gastroesophageal cancer (GEC) is an aggressive disease characterized by a high frequency of metastasis and poor overall survival rates. GEC presents HER2 overexpression in 5 to 25% of tumors eligible for HER2-targeted therapy. HER2 evaluation requires protein levels and copy number alteration analyses by immunohistochemistry (IHC) and in situ hybridization (FISH or SISH), respectively. These are semiquantitative methodologies that need an expert and well-trained pathologist. Therefore, the use of new surrogate methods for HER2 evaluation in cancer, such as gene expression analysis, might improve GEC HER2 classification. We evaluated HER2 positivity in GEC through conventional IHC and SISH analyses and investigated the potential application of HER2 mRNA expression by quantitative PCR to categorize GEC samples as HER2-positive or HER2-negative. Among 270 GEC samples, 10.9% were HER2-positive by IHC and SISH analyses. HER2 mRNA was overexpressed in HER2-positive GEC samples and presented high accuracy in distinguishing those tumors from HER2-negative GEC. Nevertheless, HER2 mRNA analysis was not capable of classifying HER2-equivocal GEC samples into HER2-positive or -negative according to SISH data. Quantitative PCR analysis showed HER2 overexpression in HER2-positive GEC samples. Nevertheless, HER2 mRNA analysis failed to classify HER2-equivocal GEC according to SISH data.

MET overexpression and intratumor heterogeneity in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is among the ten most frequent and deadly cancers, without effective therapies for most patients. More recently, drugs targeting deregulated growth factor signaling receptors have been developed, such as HGF-MET targeted therapy. We assessed MET and HGF genetic alterations and gene and protein expression profiles in ESCC patients from the Brazilian National Cancer Institute and publicly available datasets, as well as the intratumor heterogeneity of the alterations found. Our analyses showed that HGF and MET genetic alterations, both copy number and mutations, are not common in ESCC, affecting 5 and 6% of the cases, respectively. HGF showed a variable mRNA expression profile between datasets, with no alterations (GSE20347), downregulation (GSE45670), and upregulation in ESCC (our dataset and GSE75241). On the other hand, MET was found consistently upregulated in ESCC compared to non-tumor surrounding tissue, with median fold-changes of 5.96 (GSE20347), 3.83 (GSE45670), 6.02 (GSE75241), and 5.0 (our dataset). Among our patients, 84% of the tumors showed at least a two-fold increase in MET expression. This observation was corroborated by protein levels, with 55% of cases exhibiting positivity in 100% of the tumor cells. Intratumor heterogeneity was evaluated in at least four tumor biopsies from five patients and two cases showed a consistent increase in MET expression (at least two-fold) in all tumor samples. Our data suggested that HGF-MET signaling pathway was likely to be overactivated in ESCC, representing a potential therapeutic target, but eligibility for this therapy should consider intratumor heterogeneity.

Parâmetros genéticos e efeitos de sexo e cruzamento recíproco sobre características de interesse econômico em aves F2 / Genetic parameters and effects of sex and reciprocal cross on economic traits in F2 broilers

Este estudo teve por objetivo estimar os parâmetros genéticos de características de interesse econômico, mensuradas em populações F2 desenvolvidas pela Embrapa Suínos e Aves utilizando cruzamento recíproco entre linhagens de corte e de postura. Ainda, foram avaliados os efeitos de sexo e de cruzamento recíproco sobre as características em estudo. Os pesos com um, 35 e 42 dias de idade; ganho de peso; consumo de ração e conversão alimentar entre 35 e 41 dias de idade; pesos dos pulmões, fígado, coração, moela, peito, pernas, carcaça, dorso, asas, cabeça, pés e gordura abdominal, além do comprimento do intestino, foram os fenótipos estudados. Foram estimados os componentes de variâncias genética aditiva e residual, além dos coeficientes de herdabilidade e das correlações genética e fenotípica. Os machos apresentaram maior peso para todas as características estudadas, nos dois cruzamentos recíprocos, exceto para gordura abdominal na população oriunda do cruzamento de machos de postura com fêmeas de corte. Os animais oriundos do cruzamento de machos de postura com fêmeas de corte foram mais pesados que os recíprocos, para todas as idades, além de apresentarem maior comprimento de intestino e maiores pesos de moela, carcaça, dorso, peito e cabeça. Os coeficientes de herdabilidade foram altos para consumo de ração e para os pesos ao nascimento, da moela e da gordura abdominal. As correlações fenotípicas estimadas foram, em sua maioria, baixas ou moderadas, contudo muitas correlações genéticas altas foram observadas. Ressalta-se que houve expressiva diferença nos coeficientes de herdabilidade de algumas características em função do cruzamento recíproco estudado, o que pode ser devido a efeitos materno, citoplasmático, ligados ao sexo ou imprinting.(AU)

This study aimed to estimate genetic parameters for several economic traits in F2 populations developed by Embrapa Swine and Poultry National Research Center, through reciprocal crosses of broilers and layer lines. Furthermore, sex and reciprocal cross effects were evaluated. Weights at 1, 35 and 42 days of age; weight gain, feed intake and feed conversion between 35 and 41 days of age; weights of the lungs, heart, liver, gizzard, breast, thighs, carcass, back, wings, head, legs and abdominal fat; and intestine length were studied. Residual and genetic additive variance components, heritability coefficients, and genetic and phenotypic correlations were estimated. Males were heavier than females for all studied traits in both reciprocal crosses, except for abdominal fat weight in chickens from the male layer x female broiler cross. Chickens from male layer x female broiler cross were heavier than those from its reciprocal cross, also having larger intestine length and gizzard, carcass, back, breast and head weights. The heritability coefficients were high for feed intake and body weight at birth, gizzard, and abdominal fat. The estimated phenotypic correlations were mostly lower or moderate, however, most high genetic correlations were observed. We found significant differences in heritability coefficients for some traits due to the reciprocal cross, which may be due maternal, cytoplasmic, sex-linked or imprinting effects.(AU)

Histomorfometria do epitélio ruminal de cabritos 1/2 sangue Boer submetidos a dietas com tortas oriundas da produção do biodiesel / Histomorphometrics of the ruminal epithelium of 1/2 blood Bôer goats submitted to diets with pies from the production of biodiesel

Objetivou-se determinar o melhor nível de inclusão e de substituição das tortas de dendê e amendoim, respectivamente, em dietas para cabritos 1/2 sangue Boer, por meio da avaliação histomorfométrica das papilas ruminais. Foram utilizados 40 cabritos 1/2 sangue Boer, para cada período experimental, machos, não castrados, com idade aproximada de três meses e com peso inicial de 15,01±1,76kg. Os períodos experimentais constaram de 75 dias e 72 dias. As dietas consistiram de volumoso feno de Tifton-85 e de ração em mistura completa, contendo níveis de inclusão da torta de dendê nas proporções de 0,0; 7,0; 14,0; e 21,0% com base na matéria seca; e de mistura completa, contendo níveis de substituição do farelo de soja pela torta de amendoim nas proporções de 0,0; 33,0; 66,0; e 100%, constituindo-se os tratamentos. No epitélio do rúmen, foram avaliadas altura e largura das papilas, assim como densidade papilar e espessura da parede muscular do rúmen. Entre estas, a altura das papilas, no experimento com torta de amendoim, foi a única a sofrer efeito linear decrescente (P<0,0014); as demais variáveis, tanto dos animais alimentados com torta de amendoim quanto daqueles alimentados com torta de dendê, não foram afetadas pela dieta. As médias de altura encontradas foram de 2,6 e 2,3mm para as tortas de amendoim e dendê, respectivamente. Portanto, a inclusão de até 21% de torta de dendê e a substituição de 100% de torta de amendoim no concentrado de cabritos não alteraram a morfometria das papilas ruminais.

The aim of this study was to determine the best level of pies and replacement of palm oil and peanut, respectively, in diets of 1/2 blood Bôer goats from the histomorphometry of the rumen papillae. A total of 40 1/2 blood male, unneutered, aged approximately three months and initial weight of 15.01±1.76kg Bôer goats were used in each experimental period. The experimental periods consisted of 75 days and 72 days. The diets consisted of roughage hay Tifton-85 and total mixed ration containing levels of inclusion of palm kernel cake in the proportions of 0.0, 7.0, 14.0 and 21.0% based on dry matter; and the second experiment consisted of a complete mixture containing substitution levels of soybean meal by groundnut cake in the proportions of 0.0, 33.0, 66.0 and 100%. Epithelial cells were evaluated in the rumen height and width of the papillae, and density and thickness of the papillary muscle of the rumen. Among these, the height of the papillae in the experiment with peanut butter pie was the only one to suffer a negative linear effect (P<0.0014), the other variables, both in animals fed groundnut cake and palm oil, were not affected by the diet. The average heights found were 2.6 and 2.3, for pies, peanut and palm oil, respectively. Therefore, the inclusion of up to 21% palm kernel cake and replacement of 100% peanut cake in the concentrate of kids did not alter the morphology of the rumen papillae.

Histomorfometria do epitélio ruminal de cabritos 1/2 sangue Boer submetidos a dietas com tortas oriundas da produção do biodiesel / Histomorphometrics of the ruminal epithelium of 1/2 blood Bôer goats submitted to diets with pies from the production of biodiesel

Objetivou-se determinar o melhor nível de inclusão e de substituição das tortas de dendê e amendoim, respectivamente, em dietas para cabritos 1/2 sangue Boer, por meio da avaliação histomorfométrica das papilas ruminais. Foram utilizados 40 cabritos 1/2 sangue Boer, para cada período experimental, machos, não castrados, com idade aproximada de três meses e com peso inicial de 15,01±1,76kg. Os períodos experimentais constaram de 75 dias e 72 dias. As dietas consistiram de volumoso feno de Tifton-85 e de ração em mistura completa, contendo níveis de inclusão da torta de dendê nas proporções de 0,0; 7,0; 14,0; e 21,0% com base na matéria seca; e de mistura completa, contendo níveis de substituição do farelo de soja pela torta de amendoim nas proporções de 0,0; 33,0; 66,0; e 100%, constituindo-se os tratamentos. No epitélio do rúmen, foram avaliadas altura e largura das papilas, assim como densidade papilar e espessura da parede muscular do rúmen. Entre estas, a altura das papilas, no experimento com torta de amendoim, foi a única a sofrer efeito linear decrescente (P<0,0014); as demais variáveis, tanto dos animais alimentados com torta de amendoim quanto daqueles alimentados com torta de dendê, não foram afetadas pela dieta. As médias de altura encontradas foram de 2,6 e 2,3mm para as tortas de amendoim e dendê, respectivamente. Portanto, a inclusão de até 21% de torta de dendê e a substituição de 100% de torta de amendoim no concentrado de cabritos não alteraram a morfometria das papilas ruminais.(AU)

The aim of this study was to determine the best level of pies and replacement of palm oil and peanut, respectively, in diets of 1/2 blood Bôer goats from the histomorphometry of the rumen papillae. A total of 40 1/2 blood male, unneutered, aged approximately three months and initial weight of 15.01±1.76kg Bôer goats were used in each experimental period. The experimental periods consisted of 75 days and 72 days. The diets consisted of roughage hay Tifton-85 and total mixed ration containing levels of inclusion of palm kernel cake in the proportions of 0.0, 7.0, 14.0 and 21.0% based on dry matter; and the second experiment consisted of a complete mixture containing substitution levels of soybean meal by groundnut cake in the proportions of 0.0, 33.0, 66.0 and 100%. Epithelial cells were evaluated in the rumen height and width of the papillae, and density and thickness of the papillary muscle of the rumen. Among these, the height of the papillae in the experiment with peanut butter pie was the only one to suffer a negative linear effect (P<0.0014), the other variables, both in animals fed groundnut cake and palm oil, were not affected by the diet. The average heights found were 2.6 and 2.3, for pies, peanut and palm oil, respectively. Therefore, the inclusion of up to 21% palm kernel cake and replacement of 100% peanut cake in the concentrate of kids did not alter the morphology of the rumen papillae.(AU)

Utilização de modelos unicaracterística e multicaracterística na estimação de parâmetros genéticos na raça Nelore / Utilization of single-trait and multi-trait models applied to genetic parameter estimation in Nellore Cattle

Estimativas dos parâmetros genéticos e dos componentes de covariância para peso ao nascimento (PN), peso à desmama (PD), circunferência escrotal (CE), ganho de peso pós-desmama (GP) e musculosidade (MUSC) foram obtidas com modelos unicaracterística, bicaracterística e multicaracterística, utilizando-se um banco de dados de 69.525 animais da raça Nelore. Os parâmetros foram estimados pelo método da máxima verossimilhança restrita, com modelos que incluíram o efeito genético aditivo direto e materno, além do grupo de manejo a desmama e de ambiente permanente da vaca, todos como aleatórios, e do efeito fixo de grupo de contemporâneo. Os coeficientes de herdabilidade estimados para as características pós-desmama com o modelo multicaracterística (0,45; 0,31 e 0,25, respectivamente, para CE, GP e MUSC) foram maiores que as estimativas obtidas com os modelos unicaracterística (0,42; 0,21 e 0,22) e bicaracterística (0,39; 0,25 e 0,24). Da mesma forma, as correlações genéticas estimadas foram, em grande parte, maiores para o modelo multicaracterística. Estes resultados indicam que a redução do viés, proporcionada pelo modelo multicaracterística, aumenta as estimativas dos parâmetros genéticos e demonstra que os resultados com análises unicaracterística e bicaracterística são subestimados. Os resultados da resposta à seleção demonstraram que a musculosidade apresentou grande potencial seletivo, e o cálculo das respostas correlacionadas de musculosidade e demais características estudadas demonstrou que sua utilização em programas de seleção pode ser vantajosa aos criadores...

Genetic parameters and components of (co)variance for birth weight (BW), weaning weight (WW), scrotal circumference (SC), weight gain at post-weaning (WG), and muscle score (MUSC) were obtained with univariate, bivariate and multivariate models, using a dataset of 69,525 Nellore cattle. The parameters were estimated by restricted maximum likelihood method. Direct and maternal additive genetic effects, group of management, and maternal permanent environmental effect were adjusted as random effects, while contemporary group was adjusted as fixed effect. Multivariate approach allowed higher estimate heritability for postweaning traits (0.45; 0.31 and 0.25, respectively for SC, WG and MUSC) than univariate (0,42; 0,21 and 0,22) and bivariate models (0,39; 0,25 and 0,24). Similarly, the genetic correlations were higher in the multivariate approach. These results indicate that reducing the bias provided by the mutivariate model improves the estimates of genetic parameters and shows that the results of bivariate and univarite models are underestimated. Selection for muscle score presented great potential for correlated response in other traits and its use in selection programs can be a useful tool for breeders...

Fracionamento de carboidratos e degradabilidade ruminal da cana-de-açúcar tratada com óxido de cálcio / Fractioning Fractioning of carbohydrates and rumen degradability of sugar cane treated with calcium oxide

Avaliaram-se o fracionamento de carboidratos e a degradabilidade in situ da matéria seca (MS) e da fibra em detergente neutro (FDN) da cana-de-açúcar tratada com óxido de cálcio (CaO). Foram testadas as porcentagens de 0; 0,75; 1,5; 2,25; 3,0; 3,75 e 4,5% de CaO, e o material testemunha (cana-de-açúcar in natura) para a avaliação do fracionamento de carboidratos, e as porcentagens de 0; 1,5; 3,0 e 4,5% de CaO para a avaliação da degradabilidade in situ da MS e da FDN. O CaO foi adicionado em pó na cana-de-açúcar, em porcentagem da matéria natural, por 24 horas. A porcentagem de inclusão de CaO na cana-de-açúcar proporcionou valores mais baixos para teores de carboidratos totais (CT) comparados aos valores da cana-de-açúcar in natura. Observou-se crescimento linear das frações A+B1 e B2, e redução na fração C da cana-de-açúcar em função da porcentagem de CaO. Os maiores valores de fração insolúvel potencialmente degradável da MS e menores de fração indigestível (Ip) da FDN foram observados na cana-de-açúcar com 3,0 e 4,5% de CaO. A adição de 3,0 e 4,5% de CaO na cana-de-açúcar promove diminuição da fração indigestível dos carboidratos e melhores taxas de degradação ruminal tanto da MS quanto da FDN.

Were evaluated the fractioning of carbohydrates and in situ dry matter (DM) and neutral detergent fiber (NDF) of sugarcane treated with calcium oxide (CaO). Levels 0, 0.75, 1.5, 2.25, 3.0, 3.75 and 4.5% CaO, and the material witness (sugarcane in nature) were tested for the assessment of fractioning of carbohydrates and levels 0, 1.5, 3.0 and 4.5% CaO for the evaluation of in situ DM and NDF. The levels of CaO were added in powder form, as a percentage of natural matter in sugarcane for 24 hours. The inclusion levels of CaO in sugarcane showed lower values for the total carbohydrates (TC) when compared with sugarcane in natura. A linear growth of the fractions A + B1 and B2 was observed, as well as a decrease in C fraction of sugarcane depending on the levels of CaO. The highest values of insoluble fraction potentially degradable in rumen and the lower values of NDF indigestible fraction (Ip) were observed in sugarcane with 3.0 and 4.5% CaO. The addition of 3.0 and 4.5% CaO to sugarcane decreased the indigestible carbohydrates fraction and promotes better rates of ruminal degradation of both DM and NDF.

CYP2A6 and CYP2E1 polymorphisms in a Brazilian population living in Rio de Janeiro

Cytochrome P450 (CYP) is a superfamily of enzymes involved in the metabolism of endogenous compounds and xenobiotics. CYP2A6 catalyzes the oxidation of nicotine and the activation of carcinogens such as aflatoxin B1 and nitrosamines. CYP2E1 metabolizes ethanol and other low-molecular weight compounds and can also activate nitrosamines. The CYP2A6 and CYP2E1 genes are polymorphic, altering their catalytic activities and susceptibility to cancer and other diseases. A number of polymorphisms described are ethnic-dependent. In the present study, we determined the genotype and allele frequencies of the main CYP2A6 and CYP2E1 polymorphisms in a group of 289 volunteers recruited at the Central Laboratory of Hospital Universitário Pedro Ernesto. They had been residing in the city of Rio de Janeiro for at least 6 months and were divided into two groups according to skin color (white and non-white). The alleles were determined by allele specific PCR (CYP2A6) or by PCR-RFLP (CYP2E1). The frequencies of the CYP2A6*1B and CYP2A6*2 alleles were 0.29 and 0.02 for white individuals and 0.24 and 0.01 for non-white individuals, respectively. The CYP2A6*5 allele was not found in the population studied. Regarding the CYP2E1*5B allele, we found a frequency of 0.07 in white individuals, which was statistically different (P < 0.05) from that present in non-white individuals (0.03). CYP2E1*6 allele frequency was the same (0.08) in both groups. The frequencies of CYP2A6*1B, CYP2A6*2 and CYP2E1*6 alleles in Brazilians are similar to those found in Caucasians and African-Americans, but the frequency of the CYP2E1*5B allele is higher in Brazilians.

Expression of CYP2A3 mRNA and its regulation by 3-methylcholanthrene, pyrazole, and ß-ionone in rat tissues

Cytochrome P450 (CYP) 2A enzymes are involved in the metabolism of numerous drugs and hormones and activate different carcinogens. Human CYP2A6, mouse CYP2A5 and rat CYP2A3 are orthologous enzymes that present high similarity in their amino acid sequence and share substrate specificities. However, different from the human and mouse enzyme, CYP2A3 is not expressed in the rat liver. There are limited data about expression of CYP2A3 in extrahepatic tissues and its regulation by typical CYP inducers. Therefore, the objective of the present study was to analyze CYP2A3 mRNA expression in different rat tissues by RT-PCR, and to study the influence of 3-methylcholanthrene, pyrazole and ß-ionone treatment on its expression. Male Wistar rats were divided into four groups of 5 rats each, and were treated ip for 4 days with 3-methylcholanthrene (25 mg/kg body weight), pyrazole (150 mg/kg body weight), ß-ionone (1 g/kg body weight), or vehicle. Total RNA was extracted from tissues and CYP2A3 mRNA levels were analyzed by semiquantitative RT-PCR. CYP2A3 mRNA was constitutively expressed in the esophagus, lung and nasal epithelium, but not along the intestine, liver, or kidney. CYP2A3 mRNA levels were increased in the esophagus by treatment with 3-methylcholanthrene and pyrazole (17- and 7-fold, respectively), in lung by pyrazole and ß-ionone (3- and 4-fold, respectively, although not statistically significant), in the distal part of the intestine and kidney by 3-methylcholanthrene and pyrazole, and in the proximal part of the intestine by pyrazole. CYP2A3 mRNA was not induced in nasal epithelium, liver or in the middle part of the intestine. These data show that, in the rat, CYP2A3 is constitutively expressed in several extrahepatic tissues and its regulation occurs through a complex mechanism that is essentially tissue specific

Genotoxic evaluation of the organophosphorous pesticide temephos

The chemical compound temephos (0,0,0',0'-tetrametyl-0,0'-thiodi-p-phenylene phosphorothioate) is an organophosphorous pesticide that has been used in Brazil since 1967 in control campaigns against the mosquito Aedes aegypti, the vector of dengue and yellow fever. We used single cell gel electrophoresis (SCGE), SOS/umu and Ames/Salmonella assays to test the toxicity and mutagenicity of temephos. Temephos was genotoxic in the SCGE assay, inducing severe DNA lesions (type IV lesions) at doses above 1.34 micro M. It was mutagenic, but not toxic, in the SOS/umu assay to Escherichia coli strain PQ37, but not to PQ35, at concentrations above 1.33 micro M, particularly when the S9 mixture was not used in the assay. Temephos was not mutagenic in the Ames assay with S. typhimurium strains TA97, TA98, TA100 and TA102, both with and without metabolic activation. However, temephos at concentrations above 3.33 micro M was mutagenic to TA98NR, YG7104 and YG7108, both with and without metabolic activation. In conclusion, temephos was genotoxic and mutagenic in all the three tests used, and in two of them at concentrations similar to those routinely used to combat Aedes aegypti