RNA-seq analysis of the response of plant-pathogenic oomycete Phytophthora parasitica to the fungicide dimethomorph / Análisis RNA-seq de la respuesta del oomiceto fitopatógeno Phytophthora parasitica al fungicida dimetomorf

Phytophthora parasitica is an important oomycete that causes disease in a variety of plants, dimethomorph fungicides being specific for oomycetes. The aim of this study was to use RNA-seq to rapidly discover the mechanism by which dimethomorph acts in the treatment of P. parasitica. We found that the expression of 832 genes changed significantly after the dimethomorph treatment, including 365 up-regulated genes and 467 down-regulated genes. According to the Gene Ontology (GO) enrichment analysis, pathway enrichment and verification test results, the following conclusions are obtained: (i) the treatment of P. parasitica with dimethomorph causes changes in the expression levels of genes associated with the cell wall and cell wall synthesis; (ii) dimethomorph treatment results in reduced permeability of the cell membrane and changes in the expression of certain transport-related proteins; (iii) dimethomorph treatment increased reactive oxygen species and reduced the expression of genes related to the control of oxidative stress.

Phytophthora parasitica es un importante oomiceto que origina enfermedades en una variedad de plantas; el fungicida dimetomorf es específico contra oomicetos. El objetivo de este estudio fue utilizar la tecnología de RNA-seq para descubrir rápidamente el mecanismo por el que el dimetomorf actúa en el tratamiento de P. parasitica. Descubrimos que la expresión de 832 genes se modificaba significativamente tras el tratamiento con dimetomorf, incluyendo 365 genes que son sobrerregulados y 467 genes que son subrregulados. El análisis de enriquecimiento de ontología de genes (GO), análisis de enriquecimiento de las vías y pruebas de verificación permitieron extraer las conclusiones siguientes: 1) el tratamiento de P. parasitica con dimetomorf origina cambios en los niveles de expresión de los genes relacionados con la pared celular y su síntesis; 2) el tratamiento con dimetomorf origina una reducción de la permeabilidad de la membrana celular, así como cambios en la expresión de ciertas proteínas relacionadas con el transporte, y 3) el tratamiento con dimetomorf incrementó las especies reactivas del oxígeno y redujo la expresión de los genes relacionados con el control del estrés oxidativo.

The m-rna, expression of serca2 and ncx1 in the process of pharmacological cell protection in experimental acute pancreatitis induced by taurocholate / Expressão do rna-m, da serca2 e do ncx1 no processo de proteção celular farmacológica na pancreatite aguda experimental induzida por taurocolato

ABSTRACT Background: Intracellular calcium overload is known to be a precipitating factor of pancreatic cell injury in acute pancreatitis (AP). Intracellular calcium homeostasis depends of Plasmatic Membrane Calcium ATPase (PMCA), Sarcoplasmic Endothelial Reticulum Calcium ATPase 2 (SERCA 2) and the Sodium Calcium Exchanger (NCX1). The antioxidant melatonin (Mel) and Trisulfate Disaccharide (TD) that accelerates NCX1 action could reduce the cell damage determined by the AP. Aim: To evaluate m-RNA expressions of SERCA2 and NCX1 in acute pancreatitis induced by sodium taurocholate in Wistar rats pre-treated with melatonin and/or TD. Methods: Wistar rats were divided in groups: 1) without AP; 2) AP without pre-treatment; 3) AP and Melatonin; 4) AP and TD; 5) AP and Melatonin associated to TD. Pancreatic tissue samples were collected for detection of SERCA2 and NCX1 m-R NA levels by polymerase chain reaction (PCR). Results: Increased m-RNA expression of SERCA2 in the melatonin treated group, without increase of m-RNA expression of the NCX1. The TD did not affect levels of SERCA2 and NCX1 m-RNA expressions. The combined melatonin and TD treatment reduced the m-RNA expression of SERCA2. Conclusions: The effect of melatonin is restricted to increased m-RNA expression of SERCA2. Although TD does not affect gene expression, its action in accelerating calcium exchanger function can explain the slightest expression of SERCA2 m-RNA when associated with Melatonin, perhaps by a joint action of drugs with different and but possibly complementary mechanisms.

RESUMO Racional: A lesão celular da pancreatite aguda (PA) envolve sobrecarga de cálcio, regulada pela atividade da Cálcio ATPase de membrana (PMCA), Cálcio ATPase do Retículo (SERCA2) e pelo Trocador Sódio Cálcio (NCX1). A melatonina (antioxidante) e o Dissacarídeo Trissulfatado (acelerador do NCX1) poderiam reduzir a lesão celular na PA. Objetivo: Avaliar a expressão do RNAm da SERCA2 e NCX1 em modelo animal de pancreatite aguda tratados com melatonina e/ou dissacarídeo trissulfatado (DT). Método: Ratos Wistar foram divididos em grupos: 1) sem pancreatite aguda; 2) com pancreatite aguda por taurocolato; 3) PA e Melatonina; 4) PA e DT; 5) PA e Melatonina com DT. Amostras de tecido foram colhidas para detecção dos níveis de RNAm da SERCA2 e NCX1 por PCR. Resultados: Houve aumento da expressão do RNAm da SERCA2 no grupo com PA tratados com Melatonina, porém sem aumento de expressão do NCX1. O DT não afetou os níveis de SERCA2 e NCX1. O tratamento conjunto com Melatonina e DT diminuiu a expressão da SERCA2. Conclusões: O efeito da Melatonina é restrito ao aumento da expressão da SERCA2. O DT não tem ação na expressão gênica, porém sua ação na aceleração do trocador na retirada do cálcio pode explicar a menor expressão da SERCA2 quando associado à Melatonina, pela ação conjunta de drogas com mecanismos diferentes e possivelmente complementares.

Efecto de las alteraciones genéticas y epigenéticas de la cadherina E y su expresión en la transcripción en la propensión al cáncer de mama / Effect of genetics, epigenetics and variations in the transcriptional expression of cadherin-E in breast cancer susceptibility

RESUMEN Introducción. La cadherina E (CDH1) cumple un papel importante en la transición epitelio-mesénquima y está relacionada con la invasión y las metástasis en varios tipos de carcinomas. Sin embargo, el efecto de las mutaciones y 'epimutaciones' germinales en la propensión al cáncer de mama no es claro. Objetivo. Evaluar el polimorfismo rs5030625, los cambios en el patrón de metilación del promotor y la expresión en la transcripción del gen CDH1 en pacientes con cáncer de mama. Materiales y métodos. Se tomaron muestras de sangre periférica de 102 pacientes con cáncer de mama y 102 mujeres de control. La genotipificación del polimorfismo rs5030625 se hizo mediante reacción en cadena de la polimerasa (PCR) y análisis de polimorfismos de longitud del fragmento de restricción; la PCR y el análisis de disociación de alta resolución sensible a metilación se emplearon para determinar el estado y el nivel de metilación del promotor del CDH1; por último, el nivel de expresión en la transcripción del CDH1 se evaluó mediante PCR cuantitativa con transcripción inversa. Resultados. Los resultados no evidenciaron asociación entre el polimorfismo rs5030625 y el cáncer de mama. Se encontraron perfiles aberrantes de metilación del promotor del CDH1 en las pacientes con cáncer de mama relacionados con las primeras etapas de desarrollo del cáncer. La disminución de la expresión del CDH1 se asoció con la presencia de metástasis y el estado de metilación del promotor. Conclusión. Las alteraciones en el CDH1 se asociaron con la invasión y las metástasis en el cáncer de mama. Se proporcionó evidencia adicional sobre la relevancia del CDH1 en el desarrollo y la progresión del cáncer de mama.

ABSTRACT Introduction: Cadherin-E (CDH1) is an important regulator of epithelial-mesenchymal transition, invasion and metastasis in many carcinomas. However, germinal epimutations and mutations effect in breast cancer susceptibility is not clear. Objective: To evaluate rs334558 polymorphism, promoter methylation status and CDH1 expression profile in breast cancer patients. Materials and methods: We collected peripheral blood samples from 102 breast cancer patients and 102 healthy subjects. The identification of rs334558 polymorphism was performed using PCR-RFLP, while methylation-specific PCR (MSP) and methylation-sensitive high-resolution melting (MS-HRM) were used to explore CDH1 methylation status; finally, CDH1 transcriptional expression profile was evaluated using RT-qPCR. Results: We found no association between rs334558 polymorphism and breast cancer. Aberrant promoter methylation profile was found in breast cancer patients and it was related with early cancer stages. CDH1 down-regulation was significantly associated with metastasis and promoter methylation. Conclusion: CDH1 alterations were associated with invasion and metastasis in breast cancer. Our results offer further evidence of CDH1 relevance in breast cancer development and progression.

Efeito da suplementação de colecalciferol nos RNA mensageiros urinários associados ao podócito em pacientes com doença renal crônica / Effect of cholecalciferol supplementation on urine podocyte-associated messenger RNAs in patients with chronic kidney disease

RESUMO Introdução: A vitamina D reduz a albuminúria em pacientes com doença renal crônica (DRC), mas o seu efeito sobre os podócitos glomerulares ainda não é claro. Objetivos: Avaliar se a suplementação de colecalciferol reduz os RNAm urinários associados ao podócito em pacientes com DRC. Métodos: Vinte e sete pacientes com DRC estágios 2 a 4 e níveis sub-ótimos de 25-hidroxi-vitamina D [25(OH)D] sérica foram tratados com colecalciferol por seis meses. Foram medidos antes e após a intervenção a 25(OH)D sérica e o RNAm urinário da nefrina, podocina, podocalixina, receptor transitório potencial do canal de cátions, subfamília C, membro 6 (TRPC6), fator A de crescimento do endotélio vascular (VEGF-A) e fator de crescimento transformador beta (TGF-β1). Resultados: A TFGe reduziu em média 4,71 mL/min/1,73 m2 (p = 0,010 vs. basal), sendo 28 ± 16 mL/min/1,73 m2 aos seis meses. Os RNAm dos produtos do podócito na urina não tiveram alteração significativa após o tratamento. Entretanto, pacientes que atingiram níveis de 25(OH)D ≥ 20 ng/mL aos 6 meses tiveram tendência de redução do RNAm da nefrina e da podocina na urina; nos pacientes em que a 25(OH)D permaneceu < 20 ng/mL houve aumento significativo da podocalixina, e tendência de maior expressão do RNAm da nefrina e da podocina. Conclusão: A reposição de colecalciferol por seis meses não teve efeito sobre os RNAm associados ao podócito nestes pacientes com DRC avançada. O efeito protetor da vitamina D ou seus análogos sobre o podócito glomerular deve ser investigado em estágios mais precoces da DRC e com maior tempo de tratamento.

ABSTRACT Introduction: Vitamin D reduces albuminuria in patients with chronic kidney disease (CKD) but its effects on glomerular podocytes are not entirely understood. Objective: To evaluate if cholecalciferol supplementation reduces the levels of podocyte-associated urine mRNAs in patients with CKD. Methods: A total of 27 patients with stages 2 to 4 CKD and suboptimal serum vitamin D [25(OH)D] levels were treated with cholecalciferol for 6 months. Serum 25(OH)D level, estimated glomerular filtration rate (eGFR), proteinuria, and urine mRNA of nephrin, podocin, podocalyxin, transient receptor potential cation channel 6, vascular endothelial growth factor A, and transforming growth factor beta were assessed before and after intervention. Results: eGFR declined at an average rate of -4.71 mL/min/1.73 m2 (p = 0.010 vs. baseline), being 28 ± 16 mL/min/1.73 m2 at six months. No changes in proteinuria or mineral and bone metabolism parameters were observed after cholecalciferol supplementation. Urinary podocyte-associated mRNAs did not change significantly after treatment. However, patients who achieved 25(OH)D level > 20 ng/mL at six months showed a trend of reduction of urinary nephrin and podocin mRNA levels; in patients with 25(OH)D that remained < 20 ng/mL there was a significant increase in urinary podocalyxin, and a trend of higher expression of urinary nephrin and podocin mRNA. Conclusion: Six months of cholecalciferol supplementation had no effect on urine podocyte-associated mRNA profile of patients with advanced CKD. The protective effect of vitamin D or its analogues on the glomerular podocyte should be investigated in early stages of CKD with a longer treatment period.

Health inequalities by gradients of access to water and sanitation between countries in the Americas, 1990 and 2010 / Gradientes de acceso a agua y saneamiento y desigualdades en salud entre los países de la Región de las Américas, 1990 y 2010

OBJECTIVE: To explore distributional inequality of key health outcomes as determined by access coverage to water and sanitation (WS) between countries in the Region of the Americas. METHODS: An ecological study was designed to explore the magnitude and change-over-time of standard gap and gradient metrics of environmental inequalities in health at the country level in 1990 and 2010 among the 35 countries of the Americas. Access to drinking water and access to improved sanitation facilities were selected as equity stratifiers. Five dependent variables were: total and healthy life expectancies at birth, and infant, under-5, and maternal mortality. RESULTS: Access to WS correlated with survival and mortality, and strong gradients were seen in both 1990 and 2010. Higher WS access corresponded to higher life expectancy and healthy life expectancy and lower infant, under-5, and maternal mortality risks. Burden of life lost was unequally distributed, steadily concentrated among the most environmentally disadvantaged, who carried up to twice the burden than they would if WS were fairly distributed. Population averages in life expectancy and specific mortality improved, but whereas absolute inequalities decreased, relative inequalities remained mostly invariant. CONCLUSIONS: Even with the Region on track to meet MDG 7 on water and sanitation, large environmental gradients and health inequities among countries remain hidden by Regional averages. As the post-2015 development agenda unfolds, policies and actions focused on health equity-mainly on the most socially and environmentally deprived-will be needed in order to secure the right for universal access to water and sanitation.

OBJETIVO:Explorar la desigualdad distributiva de resultados clave en salud determinada por la cobertura de acceso a agua y saneamiento (AS) entre países en la Región de las Américas. MÉTODOS: Se diseñó un estudio ecológico para explorar la magnitud y el cambio en el tiempo de métricas estándar de brecha y gradiente de desigualdades ambientales en salud a nivel país en 1990 y 2010 entre los 35 países de las Américas. El acceso a agua potable y el acceso a instalaciones sanitarias mejoradas fueron seleccionados como estratificadores de equidad. Las cinco variables dependientes fueron: expectativa de vida al nacer total y saludable, mortalidad infantil, en menores de cinco años y materna. RESULTADOS: El acceso a AS se correlacionó con la supervivencia y mortalidad y se observaron intensos gradientes tanto en 1990 como en 2010. Un acceso a AS más alto se correspondió con más alta expectativa de vida al nacer total y saludable y con más bajos riesgos de muerte infantil, en menores de 5 años y materna. La carga de vida perdida se distribuyó inequitativamente, concentrándose de manera sostenida entre los más desaventajados ambientalmente, quienes acarrearon hasta dos veces la carga que hubieran acarreado si el acceso a AS hubiese estado equitativamente distribuido. Los promedios poblacionales en la expectativa de vida y la mortalidad específica mejoraron pero, mientras que las desigualdades absolutas se redujeron, las desigualdades relativas se mantuvieron esencialmente invariantes. CONCLUSIONES: Aún cuando la Región está en curso para alcanzar el ODM 7 sobre agua y saneamiento, los promedios regionales siguen ocultando grandes gradientes ambientales y desigualdades en salud entre países. A medida que se despliega la agenda de desarrollo post-2015, serán necesarias políticas y acciones orientadas a la equidad en salud -principalmente hacia aquellos con mayor privación social y ambiental- a fin de asegurar el derecho por el acceso universal al agua y saneamiento.

Eupatilin Ameliorates Collagen Induced Arthritis

Eupatilin is the main active component of DA-9601, an extract from Artemisia. Recently, eupatilin was reported to have anti-inflammatory properties. We investigated the anti-arthritic effect of eupatilin in a murine arthritis model and human rheumatoid synoviocytes. DA-9601 was injected into collagen-induced arthritis (CIA) mice. Arthritis score was regularly evaluated. Mouse monocytes were differentiated into osteoclasts when eupatilin was added simultaneously. Osteoclasts were stained with tartrate-resistant acid phosphatase and then manually counted. Rheumatoid synoviocytes were stimulated with TNF-alpha and then treated with eupatilin, and the levels of IL-6 and IL-1beta mRNA expression in synoviocytes were measured by RT-PCR. Intraperitoneal injection of DA-9601 reduced arthritis scores in CIA mice. TNF-alpha treatment of synoviocytes increased the expression of IL-6 and IL-1beta mRNAs, which was inhibited by eupatilin. Eupatilin decreased the number of osteoclasts in a concentration dependent manner. These findings, showing that eupatilin and DA-9601 inhibited the expression of inflammatory cytokines and the differentiation of osteoclasts, suggest that eupatilin and DA-9601 is a candidate anti-inflammatory agent.

Incremento en la expresión del ARNm del receptor c-Met en el cáncer gástrico / Increased expression of the c-Met receptor mRNA in gastric cancer

El cáncer gástrico es una de las patologías malignas más frecuentes en el mundo. En las últimas décadas la atención se ha centrado en posibles alteraciones de factores genéticos que incluyen la activación de proto-oncogenes y/o la inactivación de genes supresores tumorales. El producto del C-MET proto-oncogen y su ligando, el factor de crecimiento del hepatocito (HGF), han sido implicados en la proliferación y migración celular en el cáncer gástrico. En este estudio se analizó a nivel molecular la amplificación del ARNm del receptor c-Met a partir del tejido tumoral gástrico de pacientes a quienes se les practicó gastrectomías, utilizando el método del ácido guanidina-tiocianato-fenol-cloroformo, y el método semicuantitativo de la Reacción en Cadena de la Polimerasa con Transcriptasa Reversa (RT-PCR), encontrándose que los elevados niveles del ARNm del receptor c-Met en las muestras tumorales de los pacientes están relacionados con mayor invasión en la profundidad de la pared gástrica (r = 0,762, p<0,01), incremento en la metástasis a los ganglios linfáticos (r = 0,766, p<0,01), alta frecuencia en tumores pocos diferenciados o indiferenciados (r = 0,912, p<0,001), aumento en el estadiaje del cáncer gástrico (r = 0,838, p<0,001), y en la sobreexpresión por el método inmunohistoquímico (IHQ) de la estreptavidina-biotina marcada de su receptor a nivel proteico (r = 0,858, p<0,001). La amplificación del ARNm y/o la sobreexpresión a nivel proteico del receptor c-Met, pudieran ser utilizados como factores pronósticos en el cáncer gástrico.

Gastric cancer is one of the most common malignancies in the world. In the last decades, the attention has been focused in possible alterations of genetic factors that include proto-oncogene activation and/or the tumor suppressor gene inactivation. The product of the proto-oncogene c-MET and its ligand, hepatocyte growth factor (HGF), have been implicated in cell proliferation and migration in gastric cancer. In this study we analyzed at the molecular level, the amplification of c-Met receptor mRNA from gastric tumor tissue of patients who underwent gastrectomy, using the acid guanidinium-thiocyanate-phenol-chloroform method and the semiquantitative Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) method. It was found that high levels of c-Met receptor mRNA in tumor samples from patients are associated with greater depth of invasion in the gastric wall (r = 0.762, p<0.01), increase in metastases to lymph nodes (r = 0.766, p<0.01), high frequency of poorly differentiated or undifferentiated tumors (r = 0.912, p<0.001), increase in the gastric cancer staging (r = 0.838, p<0.001), and the overexpression, by the immunohistochemistry method (IHC) of the labeled streptavidin-biotin, of the c-Met receptor at the protein level (r = 0.858, p<0.001). The amplification of mRNA and/or protein level overexpression of the c-Met receptor could be used as prognostic factors in gastric cancer.

Expresión diferencial de proteínas en Leishmania (Viannia) panamensis asociadas con mecanismos de resistencia a antimoniato de meglumina / Differential expression of proteins in Leishmania (Viannia) panamensis associated with mechanisms of resistance to meglumine antimoniate.

Introducción. Los mecanismos de resistencia al antimonio pentavalente conocidos hasta el momento, se han descrito ampliamente en cepas del subgénero Leishmania, pero poco se sabe sobre las proteínas involucradas en los mecanismos de resistencia presentes en cepas del subgénero Viannia, como Leishmania panamensis. Objetivo. Identificar proteínas diferencialmente expresadas entre las cepas de L. panamensis (UA140), sensible y resistente al antimonio pentavalente, y analizar el posible papel de estas proteínas en mecanismos de resistencia. Materiales y métodos. Las proteínas de las cepas, sensible y resistente al antimonio pentavalente, se compararon usando electroforesis bidimensional. Las proteínas con aumento de la expresión fueron aisladas e identificadas por espectrometría de masas mediante MALDI-TOF/TOF (Matrix Assisted Laser Desorption Ionization/Time of Flight). La expresión del ARNm de cinco de estas proteínas se cuantificó mediante PCR en tiempo real. Resultados. Los geles bidimensionales de las cepas sensible y resistente detectaron 532±39 y 541±43 manchas proteicas. Se encontraron 10 manchas con aumento de la expresión en la cepa resistente, identificadas como proteínas de choque térmico (Hsp60 mitocondrial, Hsp70 mitocondrial y citosólica), isomerasa de disulfuro, proteasa de cisteína, enolasa, factor de elongación 5-α, la subunidad 5-α del proteasoma y dos proteínas hipotéticas nombradas como Sp(2) y Sp(25). Conclusión. Este es el primer estudio llevado a cabo con una cepa resistente al antimonio pentavalente en L. panamensis, en el cual se han identificado proteínas que están relacionadas con el mecanismo de resistencia del parásito frente al medicamento, abriendo el camino para futuros estudios de estas proteínas como blancos terapéuticos.

Introduction. The well-known drug resistance mechanisms to pentavalent antimony have been widely described in strains of the Leishmania subgenus, but little is known about the mechanisms of resistance and the proteins associated with it in strains of the Viannia subgenus such as Leishmania panamensis. Objective. Differentially expressed proteins were identified between pentavalent antimonial sensitive and resistant L. panamensis (UA140) strains, and the role of these proteins was analyzed as possible resistance mechanisms. Materials and methods. The protein lysates of pentavalent antimony sensitive and resistant strains were separated by two-dimensional gel electrophoresis,and the protein patterns compared. The proteins identified as overexpressed were separated and analyzed using MALDI-TOF/TOF (Matrix Assisted Laser Desorption Ionization/Time of Flight). The level of mRNA expression of five of these proteins was quantified using real-time PCR. Results. On the 2-dimensional gels, 532 ± 39 protein spots were identified for the sensitive strains, and 541 ± 43 spots for the resistant strains. Ten spots were overexpressed in the resistant strain and identified as heat shock protein (Hsp60 mitochondrial, Hsp70 cytosolic and mitochondrial), disulfide isomerase, cysteine protease, enolase, elongation factor 5-alpha, the proteasome alpha-5 subunit and two hypothetical proteins named as Sp(2) and Sp(25). Conclusion. This is the first proteomic study conducted with a L. panamensis resistant strain where several proteins were identified and related with the parasite resistance mechanism to pentavalent antimony. This opens the way for future studies aimed at modulating the drug resistance or at evaluating these proteins as therapeutic targets.

Estradiol regulates miR-135b and mismatch repair gene expressions via estrogen receptor-beta in colorectal cells

Estrogen has anti-colorectal cancer effects which are thought to be mediated by mismatch repair gene (MMR) activity. Estrogen receptor (ER) expression is associated with microRNA (miRNA) expression in ER-positive tumors. However, studies of direct link between estrogen (especially estradiol E2), miRNA expression, and MMR in colorectal cancer (CRC) have not been done. In this study, we first evaluated the effects of estradiol (E2) and its antagonist ICI182,780 on the expression of miRNAs (miR-31, miR-155 and miR-135b) using COLO205, SW480 and MCF-7 cell lines, followed by examining the association of tissue miRNA expression and serum E2 levels using samples collected from 18 colorectal cancer patients. E2 inhibited the expressions of miRNAs in COLO205 cells, which could be reversed by E2 antagonist ICI 182.780. The expression of miR-135b was inversely correlated with serum E2 level and ER-beta mRNA expression in CRC patients' cancer tissues. There were significant correlations between serum E2 level and expression of ER-beta, miR-135b, and MMR in colon cancer tissue. This study suggests that the effects of estrogen on MMR function may be related to regulating miRNA expression via ER-beta, which may be the basis for the anti-cancer effect in colorectal cells.

El aumento de la expresión del ARNm de la enzima convertidora de angiotensina I homóloga (ECA-2) inducido por atorvastatina se asocia a menor fibrosis e hipertrofia ventricular izquierda en un modelo de cardiomiopatía diabética / Atorvastatin induced increase in homologous angiotensin i converting enzyme (ACE2) mRNA is associated to decreased fibrosis and decreased left ventricular hypertrophy in a rat model of diabetic cardiomyopathy

Objetivos. Evaluar el efecto de atorvastatina sobre la progresión del remodelado cardiaco y la expresión de ECA-2 en el miocardio de ratas diabéticas. Materiales y métodos. La diabetes fue inducida en ratas Holtzman con una inyección intraperitoneal de estreptozotocina. Los animales fueron divididos en tres grupos: (1) ratas control, (2) ratas diabéticas y (3) ratas diabéticas tratadas con atorvastatina (50 mg/kg/día). Después de ocho semanas de tratamiento, los corazones fueron extraídos para el análisis morfométrico, la cuantificación de colágeno y la determinación de los niveles de ARNm de ECA y ECA-2. Resultados. El índice de hipertrofia ventricular y el depósito de colágeno se incrementaron significativamente en las ratas diabéticas. La administración de atorvastatina previno estos cambios sin modificar los niveles de colesterol. La hiperglicemia produjo un incremento significativo en los niveles del ARNm de ECA y una marcada disminución en la expresión de ECA-2 en el miocardio de ratas diabéticas. La administración de atorvastatina indujo la expresión del ARNm de ECA-2 e inhibió la sobreexpresión del ARNm de ECA en el miocardio de las ratas diabéticas. Conclusiones. Nuestros resultados indican que la atorvastatina, independientemente de su capacidad para disminuir el colesterol, normaliza la relación de la expresión de ECA/ECA-2 y atenúa el desarrollo del remodelado adverso en el corazón diabético.

Objectives. This study has investigated the effect of atorvastatin on the progression of cardiac remodelling and ACE- 2 expression in diabetic myocardium in rats. Materials and Methods. Diabetes was induced in Holtzman rats with an intraperitoneal injection of streptozotocin. The animals were divided into 3 groups: (1) normal control rats, (2) diabetic rats and (3) diabetic rats treated orally with atorvastatin (50 mg/kg/day). After eight weeks of treatment, the hearts were removed for morphometric studies, collagen content assay and genetic expressions of ACE and ACE2 mRNA. Results. Myocardial hypertrophy index and collagen deposition were increased in diabetic rats, but not in the treated-diabetic rats, without producing changes in cholesterol levels. Myocardial ACE mRNA levels were increased while ACE2 mRNA levels were decreased in diabetic rats. Atorvastatin administration attenuated overexpression of ACE mRNA and overexpression of ACE-2 mRNA in diabetic rats. Conclusions. Our results indicate that atorvastatin, independently of its cholesterol-lowering capacity, lowers the ACE/ACE2 ratio to normal values and attenuates the development of adverse remodeling in the diabetic heart.

Inhibition of carrageenan-induced expression of tissue and plasma prekallikreins mRNA by low level laser therapy in a rat paw edema model / Inibição da expressão de RNA mensageiro de pré-calicreínas tecidual e plasmática pela laserterapia em modelo de edema de pata induzido pela carragenina em ratos

BACKGROND: Low level laser therapy (LLLT) has been used clinically in order to treat inflammation, where tissue and plasma prekallikrein have crucial importance. Plasma prekallikrein (PPK) is synthesized by the hepatocytes and secreted into the bloodstream, where it participates in the surface-dependent activation of blood coagulation, fibrinolysis, kinin generation and inflammation. Tissue prekallikrein is associated with important disease states (including cancer, inflammation, and neurodegeneration) and has been utilized or proposed as clinically important biomarker or therapeutic target of interest. OBJECTIVE: To evaluate if LLLT modulates tissue and plasma prekallikreins mRNA expression in the carrageenan-induced rat paw edema. METHODS: Experimental groups were assigned as followed: A1 (Control-saline), A2 (Carrageenan-only), A3 (laser 660nm only) and A4 (Carrageenan + laser 660nm). Edema was measured by a plethysmometer. Subplantar tissue was collected for the quantification of prekallikreins mRNA by Real time-Polymerase Chain Reaction. RESULTS: A significantly decrease in the edema was observed after laser irradiation. Expression of prekallikreins increased after carrageenan injection. Tissue and plasma prekallikrein mRNA expression significantly decreased after LLLT's 660nm wavelength. CONCLUSION: These results suggest that expression of tissue and plasma prekallikreins is modulated by LLLT, which can be used in clinical practice due to its anti-inflammatory effects.

CONTEXTUALIZAÇÃO: A laserterapia de baixa potência tem sido usada para o tratamento de processos inflamatórios diversos em que a calicreína tecidual e a plasmática possuem participação ativa. A pré-calicreína plasmática (PPK) é sintetizada pelos hepatócitos e secretada na corrente sanguínea, onde participa da ativação da coagulação, fibrinólise, geração de cininas e inflamação. A pré-calicreína tecidual está associada com importantes doenças (incluindo câncer, inflamação e neurodegeneração) e tem sido utilizada ou sugerida clinicamente como importante biomarcador ou alvo terapêutico. OBJETIVO: Avaliar se a laserterapia altera a expressão gênica da pré-calicreína tecidual e da plasmática no modelo de inflamação aguda induzida pela carragenina. MÉTODOS: Quarenta ratos foram separados em quatro grupos experimentais: A1 (controle), A2 (carragenina, apenas), A3 (laser 660nm, apenas) e A4 (Carragenina + laser 660nm). O edema foi medido por um pletismômetro. Tecido subplantar foi coletado para a quantificação de RNA mensageiro (RNAm) de pré-calicreínas tecidual e plasmática por PCR em tempo real. RESULTADOS: Observou-se uma diminuição significativa no volume de edema após irradiação com laser 660nm. A expressão de RNAm de pré-calicreínas tecidual e plasmática aumentou após a inoculação de carragenina, entretanto a expressão gênica das pré-calicreínas diminuiu significantemente após laserterapia de baixa potência de 660nm. CONCLUSÃO: Os resultados sugerem que a expressão de RNAm das pré-calicreínas tecidual e plasmática é modulada pela laserterapia de baixa potência, podendo ser alvo terapêutico para tratamento de processos inflamatórios.

Genes Associated with Recurrence of Hepatocellular Carcinoma: Integrated Analysis by Gene Expression and Methylation Profiling

Gene expression is suppressed by DNA methylation. The goal of this study was to identify genes whose CpG site methylation and mRNA expression are associated with recurrence after surgical resection for hepatocellular carcinoma (HCC). Sixty-two HCCs were examined by both whole genome DNA methylation and transcriptome analysis. The Cox model was used to select genes associated with recurrence. A validation was performed in an independent cohort of 66 HCC patients. Among fifty-nine common genes, increased CpG site methylation and decreased mRNA expression were associated with recurrence for 12 genes (Group A), whereas decreased CpG site methylation and increased mRNA expression were associated with recurrence for 25 genes (Group B). The remaining 22 genes were defined as Group C. Complement factor H (CFH) and myosin VIIA and Rab interacting protein (MYRIP) in Group A; proline/serine-rich coiled-coil 1 (PSRC1), meiotic recombination 11 homolog A (MRE11A), and myosin IE (MYO1E) in Group B; and autophagy-related protein LC3 A (MAP1LC3A), and NADH dehydrogenase 1 alpha subcomplex assembly factor 1 (NDUFAF1) in Group C were validated. In conclusion, potential tumor suppressor (CFH, MYRIP) and oncogenes (PSRC1, MRE11A, MYO1E) in HCC are reported. The regulation of individual genes by methylation in hepatocarcinogenesis needs to be validated.

Cathepsin L derived from skeletal muscle cells transfected with bFGF promotes endothelial cell migration

Gene transfer of basic fibroblast growth factor (bFGF) has been shown to induce significant endothelial migration and angiogenesis in ischemic disease models. Here, we investigate what factors are secreted from skeletal muscle cells (SkMCs) transfected with bFGF gene and whether they participate in endothelial cell migration. We constructed replication-defective adenovirus vectors containing the human bFGF gene (Ad/bFGF) or a control LacZ gene (Ad/LacZ) and obtained conditioned media, bFGF-CM and LacZ-CM, from SkMCs infected by Ad/bFGF or Ad/LacZ, respectively. Cell migration significantly increased in HUVECs incubated with bFGF-CM compared to cells incubated with LacZ-CM. Interestingly, HUVEC migration in response to bFGF-CM was only partially blocked by the addition of bFGF-neutralizing antibody, suggesting that bFGF-CM contains other factors that stimulate endothelial cell migration. Several proteins, matrix metalloproteinase-1 (MMP-1), plasminogen activator inhibitor-1 (PAI-1), and cathepsin L, increased in bFGF-CM compared to LacZ-CM; based on 1-dimensional gel electrophoresis and mass spectrometry. Their increased mRNA and protein levels were confirmed by RT-PCR and immunoblot analysis. The recombinant human bFGF protein induced MMP-1, PAI-1, and cathepsin L expression in SkMCs. Endothelial cell migration was reduced in groups treated with bFGF-CM containing neutralizing antibodies against MMP-1 or PAI-1. In particular, HUVECs treated with bFGF-CM containing cell-impermeable cathepsin L inhibitor showed the most significant decrease in cell migration. Cathepsin L protein directly promotes endothelial cell migration through the JNK pathway. These results indicate that cathepsin L released from SkMCs transfected with the bFGF gene can promote endothelial cell migration.

Induction of apoptotic lesions in liver and lymphoid tissues and modulation of cytokine mRNA expression by acute exposure to deoxynivalenol in piglets

Six 1-month-old piglets were intravenously injected with deoxynivalenol (DON) at the concentration of 1 mg/kg body weight, with three pigs each necropsied at 6 and 24 h post-injection (PI) for investigation of hepatotoxicity and immunotoxicity with special attention to apoptotic changes and cytokine mRNA expression. Histopathological examination of the DON-injected pigs revealed systemic apoptosis of lymphocytes in lymphoid tissues and hepatocytes. Apoptosis of lymphocytes and hepatocytes was confirmed by the TdT-mediated dUTP-biotin nick end-labeling (TUNEL) method and immunohistochemical staining against single-stranded DNA and cleaved caspase-3. The number of TUNEL-positive cells in the thymus and Peyer's patches of the ileum was increased at 24 h PI compared to 6 h PI, but the peak was at 6 h PI in the liver. The mRNA expression of interleukin (IL)-1beta, IL-6, IL-18, and tumor necrosis factor (TNF)-alpha in the spleen, thymus and mesenteric lymph nodes were determined by semi-quantitative RT-PCR, and elevated expression of IL-1beta mRNA at 6 h PI and a decrease of IL-18 mRNA at 24 h PI were observed in the spleen. IL-1beta and IL-6 mRNA expressions increased significantly at 6 h PI in the thymus, but TNF-alpha decreased at 6 h PI in the mesenteric lymph nodes. These results show the apoptosis of hepatocytes suggesting the hepatotoxic potential of DON, in addition to an immunotoxic effect on the modulation of proinflammatory cytokine genes in lymphoid organs with extensive apoptosis of lymphocytes induced by acute exposure to DON in pigs.

Transcriptional and translational expression of calbindin-D9k in the duodenum, kidney and uterus of a female canine model

Calbindin-D9k (CaBP-9k) is a cytosolic calcium-binding protein expressed in tissues in the intestine, uterus, placenta, kidney, pituitary gland and bone. Its exact function is unknown, but it is considered to regulate intracytoplasmic concentration and transport of free ions (Ca2+). CaBP-9k protein is involved in intestinal calcium absorption in the intestine and in the regulation of myometrial activity by intracellular calcium in the uterus. Renal CaBP-9k protein is expressed at the site of calcium re-absorption in the kidney and expressed in distal convoluted tubules, where it is thought to facilitate calcium re-absorption. Expression of the CaBP-9k gene has been explored in most mammalians except in a canine model. Presently, we elucidated the expression of CaBP-9k mRNA and protein in the duodenum, kidney and uterus in a canine model involving two adult (2.5-year-old) female beagles. To collect tissues, the dogs were euthanized and then the abdominal cavity was exposed by midline incision. The proximal duodenum, cortex of kidney and uterine horn were collected. Expression of CaBP-9k mRNA was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR. CaBP-9k protein expression and localization were ascertained by Western blot analysis and immunohistochemistry, respectively. CaBP-9k mRNA was detected in the duodenum, but not in the kidney and uterus. Its protein was expressed only in the enterocytes of the duodenum. Taken together, the results indicate that CaBP-9k mRNA and protein are highly expressed in the enterocytes of the duodenum of a canine model, consistent with findings in other mammalian species.

O sistema nervoso central como alvo das ações anti-hipertensivas de um peptídeo rico em resíduos de prolina do veneno da Bothrops jararaca / The central nervous system as target for anti-hypertensive actions of proline-rich peptide from Bothrops jararaca venom

Os peptídeos potenciadores da bradicinina (BPPs) presentes no veneno da serpente Bothrops jararaca são oligopeptídeos ricos em prolinas. Eles foram os primeiros inibidores naturais da enzima conversora de angiotensina (ECA) descritos. As propriedades bioquímicas e farmacológicas desses peptídeos foram essenciais para o desenvolvimento do captopril, o primeiro inibidor sítio-dirigido da ECA, usado para tratar a hipertensão humana. Recentes dados têm sugerido que a atividade farmacológica dos BPPs não pode ser explicada somente pela ação inibitória da atividade da ECA e que os efeitos dos BPPs devem envolver a participação do sistema nervoso central (SNC). Nesse trabalho foi caracterizada a sinalização de Ca2+ induzida pelo BPP-lOc [

A novel beta-glucan produced by Paenibacillus polymyxa JB115 induces nitric oxide production in RAW264.7 macrophages

The effect of extracellular beta-(1-->3), (1-->6)-glucan, produced by Paenibacillus polymyxa JB115, on nitric oxide (NO) production in RAW264.7 macrophages was investigated. beta-glucan induced the production of NO by RAW264.7 macrophages in a concentration- and time-dependent manner. Moreover, beta-glucan stimulation increased the mRNA expression of iNOS, COX-2 and IL-6 in RAW264.7 macrophages in a concentration-dependent manner.

Dietary cholesterol and estrogen administration elevate brain apolipoprotein E in mice by different mechanisms.

Apolipoprotein (apo) E plays an important role in the whole body cholesterol homeostasis. Recent studies suggest that it may also be involved in the local cholesterol transport in the brain, and influence the pathogenesis of Alzheimer's disease (AD) by interacting with the beta-amyloid protein and brain lipoprotein receptors. Since apoE expression is highest in the brain, next only to the liver and associated with the pathogenesis of AD, we hypothesized that dietary and hormonal intervention, known to regulate hepatic apoE expression may also regulate brain apoE and thereby influence local cholesterol transport. To test this hypothesis, groups of male C57BL mice were fed either regular rodent chow or high fat (HF) and high cholesterol enriched diet for 3 weeks. In a separate study, groups of male mice were administered pharmacological doses of 17-beta estradiol for 5 consecutive days and sacrificed on the 6th day. As expected, HF diet elevated liver apoE mRNA and apoE synthesis. Similar to liver, brain apoE mRNA and synthesis also increased, following HF feeding. Estradiol administration increased liver apoE synthesis without affecting apoE mRNA. Interestingly, estradiol administration also increased the brain apoE synthesis, but without altering the brain apoE mRNA. These studies suggested that dietary cholesterol and estrogen administration elevated the brain apoE by different mechanisms.

Amilóide sérica A: efeitos biológicos sobre células mononucleares / Serum amyloid A: biological effects on mononuclear cells

Nos últimos anos, nosso grupo de pesquisa vem descrevendo vários efeitos da SAA em células do sistema imune no que diz respeito à expressão e liberação de citocinas pró-inflamatórias. Neste estudo centramos nossa atenção na verificação dos efeitos da SAA sobre células mononucleares. Para isto, usamos três modelos experimentais. Em murinos, descrevemos a habilidade da SAA induzir a produção de NO por macrófagos peritoneais e, com uso de animais, Knockout para TLR4, sugerimos que SAA seja um ligante endógeno do TLR4. Em células mononucleares de sangue periférico humano, a SAA induz a expressão e liberação de CCL20, uma quimiocina importante na transição da resposta imune inata para adaptativa, bem como a expressão dos fatores M-CSF e VEGF. Em células THP-1, mostramos a cinética de fosforilação de proteínas tirosina quinases promovida pela SAA e comparamos com LPS, um estímulo pró-inflamatório clássico. Ainda em células THP-1 mostramos que a SAA induz a fosforilação de duas proteínas importantes no processo inflamatório por induzirem a ativação de NFқB; a p38 e a ERK1/2. Com este estudo contribuímos com o conhecimento a respeito do papel regulatório da SAA em células mononucleares. A ação da SAA sobre estas células torna-se importante, pois estas são cruciais na resposta imune inata e também atuam como células acessórias na resposta imune adaptativa. Desta forma, evidencia-se que, no processo de fase aguda, a expressão e a síntese de SAA resultam na modulação de etapas que controlam este processo e sua progressão...

TNF/TNFR1 signaling up-regulates CCR5 expression by CD8+ T lymphocytes and promotes heart tissue damage during Trypanosoma cruzi infection: beneficial effects of TNF-á blockade

In Chagas disease, understanding how the immune response controls parasite growth but also leads to heart damage may provide insight into the design of new therapeutic strategies. Tumor necrosis factor-alpha (TNF-á) is important for resistance to acute Trypanosoma cruzi infection; however, in patients suffering from chronic T. cruzi infection, plasma TNF-á levels correlate with cardiomyopathy. Recent data suggest that CD8-enriched chagasic myocarditis formation involves CCR1/CCR5-mediated cell migration. Herein, the contribution of TNF-á, especially signaling through the receptor TNFR1/p55, to the pathophysiology of T. cruzi infection was evaluated with a focus on the development of myocarditis and heart dysfunction. Colombian strain-infected C57BL/6 mice had increased frequencies of TNFR1/p55+ and TNF-á+ splenocytes. Although TNFR1-/- mice exhibited reduced myocarditis in the absence of parasite burden, they succumbed to acute infection. Similar to C57BL/6 mice, Benznidazole-treated TNFR1-/- mice survived acute infection. In TNFR1-/- mice, reduced CD8-enriched myocarditis was associated with defective activation of CD44+CD62Llow/- and CCR5+ CD8+ lymphocytes. Also, anti-TNF-á treatment reduced the frequency of CD8+CCR5+ circulating cells and myocarditis, though parasite load was unaltered in infected C3H/HeJ mice. TNFR1-/- and anti-TNF-á-treated infected mice showed regular expression of connexin-43 and reduced fibronectin deposition, respectively. Furthermore, anti-TNF-á treatment resulted in lower levels of CK-MB, a cardiomyocyte lesion marker. Our results suggest that TNF/TNFR1 signaling promotes CD8-enriched myocarditis formation and heart tissue damage, implicating the TNF/TNFR1 signaling pathway as a potential therapeutic target for control of T. cruzi-elicited cardiomyopathy.