Análisis de posibles agentes de control biológico de <i>Lutzomyia longipalpis</i>, vector de leishmaniasis visceral

La leishmaniasis es una enfermedad transmitida por vectores que tienen una ecología y epidemiología complejas. Se manifiesta clínicamente de cuatro maneras principales de las cuales la leishmaniasis visceral (LV) es la más severa, ya que es fatal si no es tratada (Desjeux, 1996). Esta "Enfermedad de la Pobreza" es un serio problema de salud pública a nivel mundial y, recientemente, a nivel nacional. Actualmente, no existe una vacuna para su prevención, y el tratamiento con drogas es un procedimiento caro que acarrea efectos secundarios indeseables. La leishmaniasis es causada por un protista parásito, Leishmania spp., y transmitida a los humanos por insectos flebótomos. En Latinoamérica, LV es causada por Leishmania infantum (syn. chagasi) y transmitida por Lutzomyia longipalpis (WHO, 2010). Este flebótomo solamente se encuentra en el Nuevo Mundo, con una amplia distribución desde México hasta Argentina. Entre 1925 y 1989, en Argentina se reportaron 14 casos humanos de leishmaniasis pero ninguno fue atribuído a Le. infantum y, adicionalmente, sólo hubo 2 registros aislados de Lu. longipalpis sin reporte de LV (en 1951 y 2000). Sin embargo, debido al avance indiscriminado de la urbanización, que acarrea pobreza y condiciones insalubres, recientemente se describió el primer foco de LV en Argentina (Salomón et al., 2008). La mejor manera de interrumpir cualquier enfermedad transmitida por vectores consiste en reducir el contacto humano-vector. Las estrategias dirigidas a los vectores son especialmente atractivas debido a que la capacidad vectorial para transmitir enfermedades infecciosas a los humanos es proporcional a la densidad del vector y es exponencial a la supervivencia del vector. El control biológico es una manera efectiva de reducir o mitigar plagas a través del uso de agentes naturales, y es más amigable para el ambiente que los tratamientos tradicionales con insecticidas químicos. Sin embargo, hay muy poca información sobre el control biológico de flebótomos y potenciales agentes de control. En el laboratorio donde se desarrolló esta tesis, recientemente se utilizó la pirosecuenciación para analizar los taxones asociados a Lu. longipalpis de zonas endémica (ZE) y no endémica (ZNE) de LV en Argentina y Brasil, respectivamente (McCarthy et al., 2011), y para comparar los transcriptomas de estos especímenes (McCarthy et al., 2013). Estos análisis permitieron ensamblar un inventario abarcativo de los taxones asociados a Lu. longipalpis adultos de ZE y ZNE de LV, y proveyó el fundamento para analizar la significancia de posibles candidatos para el control biológico de este vector (McCarthy et al., 2011; McCarthy et al., 2013). En este contexto, el objetivo general de la presente tesis consistió en establecer la significancia de los taxones previamente encontrados en Lu. longipalpis adultos (entre ellos, protistas, hongos y bacterias) (McCarthy et al., 2011), con particular énfasis en los posibles agentes de control biológico y en los agentes etiológicos de importancia médica y veterinaria. Con este fin, se diseñaron y evaluaron cebadores diagnósticos para confirmar la presencia y asociación de los taxones identificados.Facultad de Ciencias Exacta

Yerba Mate Modulates Tumor Cells Functions Involved in Metastasis in Breast Cancer Models

Breast cancer (BC) is the most frequent cancer in women and tumor metastasis is a major cause of cancer-related deaths. Our aim was to evaluate anti-metastatic properties of yerba mate extract (YMe) in BC models. 4T1, F3II, MCF-7, and MDA-MB231 cell lines were used to perform in vitro assays. The F3II syngeneic mammary carcinoma model in BALB/c mice was used to evaluate tumor progression, BC metastasis and survival. Cells were inoculated subcutaneously into the flank for the heterotopic model and into the mammary fat pad for the orthotopic model. YMe was administered p.o. in a dose of 1.6 g/kg/day. In vitro YMe inhibited cell proliferation and reduced tumor cell adhesion, migration and invasion. These biological effects were cell-line dependent. In vivo YMe reduced tumor metastasis and increased mice survival in both models. Our preclinical results suggest that YMe could modulate tumor progression and metastasis in BC models.Fil: Garcia Lazaro, Rocio Soledad. Universidad Nacional de Quilmes; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Caligiuri, Lorena Gisel. Universidad Nacional de Quilmes; ArgentinaFil: Lorenzo Pérez, Norailys. Universidad Nacional de Quilmes; ArgentinaFil: Lamdan, Humberto. Universidad Nacional de Quilmes; ArgentinaFil: Alonso, Daniel Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes; ArgentinaFil: Farina, Hernán Gabriel. Universidad Nacional de Quilmes; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Anti-proliferative effects of a blueberry extract on a panel of tumor cell lines of different origin

Background: Blueberries are among the fruits with the highest antioxidant activity and have been recognized by their health promoting properties. Aim:In vitro study of the anti-proliferative effects of a blueberry extract on a panel of cancer cells from different origin. Materials and Methods: A blueberry extract was produced using ethanol as extracting solvent. The anti-proliferative activity of the extract was evaluated against seven tumor cell lines. The properties of blueberry extract to decrease cell adhesion and migration were also investigated. Results: Blueberry extract showed a dose-dependent inhibitory effect on cell proliferation for all cell lines. Non-cytotoxic concentrations of the extract decreased cell adhesion in five of seven cell lines studied and inhibited the migration of MDA-MB-231 and PC-3 tumor cells. Conclusion: This work provides additional evidence regarding the ability of blueberry extract to inhibit the growth and decrease cell adhesion and migration of different cancer cell lines in vitro. Key Words: anthocyanins, anti-proliferative, blueberry extract, cancer, polyphenols.Fil: Lamdan Ordas, Humberto. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; ArgentinaFil: Garcia Lazaro, Rocio Soledad. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Lorenzo Pérez, Norailys. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; ArgentinaFil: Caligiuri, Lorena Gisel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; ArgentinaFil: Alonso, Daniel Fernando. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Farina, Hernán Gabriel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Optimization of DNA Extraction from Individual Sand Flies for PCR Amplification

Numerous protocols have been published for extracting DNA from phlebotomines. Nevertheless, their small size is generally an issue in terms of yield, efficiency, and purity, for large-scale individual sand fly DNA extractions when using traditional methods. Even though this can be circumvented with commercial kits, these are generally cost-prohibitive for developing countries. We encountered these limitations when analyzing field-collected Lutzomyia spp. by polymerase chain reaction (PCR) and, for this reason, we evaluated various modifications on a previously published protocol, the most significant of which was a different lysis buffer that contained Ca2+ (buffer TESCa). This ion protects proteinase K against autolysis, increases its thermal stability, and could have a regulatory function for its substrate-binding site. Individual sand fly DNA extraction success was confirmed by amplification reactions using internal control primers that amplify a fragment of the cacophony gene. To the best of our knowledge, this is the first time a lysis buffer containing Ca2+ has been reported for the extraction of DNA from sand flies.Centro Regional de Estudios Genómico

Polymerase chain reaction-based assay for the detection and identification of sand fly gregarines in Lutzomyia longipalpis, vector of visceral leishmaniasis

Gregarines that parasitise phlebotomine sand flies belong to the genus Psychodiella and, even though they are highly host-specific, only five species have been described to date. Their most outstanding features include the unique localisation of the oocysts in the accessory glands of the female host, which ensures contamination of the egg surface during oviposition, and the fact that they naturally parasitise the vectors of Leishmania, causal agent of leishmaniasis. The type species, Ps. chagasi, was first described in Lutzomyia longipalpis, vector of visceral leishmaniasis (VL), from Brazil. We recently reported Ps. chagasi sequences in Lu. longipalpis from Posadas (Misiones, Argentina), an endemic VL location where this gregarine had not been previously recorded. In order to analyse the incidence of Ps. chagasi infections in Lu. longipalpis from this location, the aim of this study was to develop a diagnostic assay for sand fly gregarine parasites in Lu. longipalpis. For this, we designed primers using the Ps. chagasi sequences we previously identified and performed an in vitro validation by PCR amplification of the original sand fly samples. Their specificity and sensitivity as diagnostic primers were subsequently confirmed by PCR reactions using total DNA extracted from naturally infected Lu. longipalpis from the same location (Posadas, Argentina).Fil: Caligiuri, Lorena Gisel. Universidad Nacional de La Plata. Centro Regional de Estudios Genómicos; Argentina. Universidad Nacional del Noroeste de la Provincia de Buenos Aires; ArgentinaFil: Acardi, Soraya Alejandra. Universidad Nacional de Misiones. Facultad de Ciencias Exactas, Químicas y Naturales. Laboratorio de Biología Molecular Aplicada; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Santini, Maria Soledad. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Centro Nacional de Diagnóstico e Investigaciones Endemo-epidémicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Salomón, Oscar Daniel. Ministerio de Salud. Instituto Nacional de Medicina Tropical; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Mccarthy, Cristina Beryl. Universidad Nacional de La Plata. Centro Regional de Estudios Genómicos; Argentina. Universidad Nacional del Noroeste de la Provincia de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

In vitro and in vivo antitumor activity of Yerba Mate extract in colon cancer models

Yerba Mate (Ilex paraguariensis St. Hill. Aquifoliaceae) is a native South American tree and has a large amount of bioactive compounds. Colorectal cancer (CRC) is one of the so-called westernized diseases and is the third most common cancer in both men and women. Efficient strategies for the treatment of CRC are extensively being explored including dietary intervention. The objective of our research was to evaluate the effects of Yerba Mate extract on cell proliferation, invasive capacity of tumor cells, and angiogenesis. For this, in vitro and in vivo experimentation was carried out using CRC models. The extract was generated by aqueous extraction and prepared according to traditional American procedure of preparing mate infusion. In vitro results showed that the Yerba Mate extract inhibits CT26 and COLO 205 cell proliferation with IC50 values of 0.25 and 0.46 mg/mL, respectively. We demonstrated by TUNEL assay that one of the mechanisms by which Yerba Mate extract decreases cell proliferation is by induction of apoptosis. In a murine syngeneic tumor model, oral administration of Yerba Mate extract in a dose of 1.6 g/kg/day significantly inhibited angiogenesis and tumor growth without affecting biological parameters or body weight. Our findings suggest that Yerba Mate may be a promising agent for the treatment of colon cancer and could be used as an herbal medicine or functional food ingredient. Practical Application: Considering the chemical composition and presence of phenolic compounds with their free-radical scavenging activities and bioactivities against colon cancer cells, Yerba Mate can be a promising candidate as healthy food sources in human nutrition, and also be considered a natural source of potential antitumor agents. Taking into account the economic importance of Yerba Mate in Argentina, this vegetable would have a greater commercial value as a functional food.Fil: Garcia Lazaro, Rocio Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; ArgentinaFil: Lamdan Ordas, Humberto. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; ArgentinaFil: Caligiuri, Lorena Gisel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; ArgentinaFil: Lorenzo Pérez, Norailys. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; ArgentinaFil: Berengeno, Andrea Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Ortega, Hugo Hector. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Alonso, Daniel Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; ArgentinaFil: Farina, Hernán Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; Argentin

In vitro

Yerba Mate (Ilex paraguariensis St. Hill. Aquifoliaceae) is a native South American tree and has a large amount of bioactive compounds. Colorectal cancer (CRC) is one of the so-called westernized diseases and is the third most common cancer in both men and women. Efficient strategies for the treatment of CRC are extensively being explored including dietary intervention. The objective of our research was to evaluate the effects of Yerba Mate extract on cell proliferation, invasive capacity of tumor cells, and angiogenesis. For this, in vitro and in vivo experimentation was carried out using CRC models. The extract was generated by aqueous extraction and prepared according to traditional American procedure of preparing mate infusion. In vitro results showed that the Yerba Mate extract inhibits CT26 and COLO 205 cell proliferation with IC50 values of 0.25 and 0.46 mg/mL, respectively. We demonstrated by TUNEL assay that one of the mechanisms by which Yerba Mate extract decreases cell proliferation is by induction of apoptosis. In a murine syngeneic tumor model, oral administration of Yerba Mate extract in a dose of 1.6 g/kg/day significantly inhibited angiogenesis and tumor growth without affecting biological parameters or body weight. Our findings suggest that Yerba Mate may be a promising agent for the treatment of colon cancer and could be used as an herbal medicine or functional food ingredient. Practical Application: Considering the chemical composition and presence of phenolic compounds with their free-radical scavenging activities and bioactivities against colon cancer cells, Yerba Mate can be a promising candidate as healthy food sources in human nutrition, and also be considered a natural source of potential antitumor agents. Taking into account the economic importance of Yerba Mate in Argentina, this vegetable would have a greater commercial value as a functional food.Fil: Garcia Lazaro, Rocio Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; ArgentinaFil: Lamdan Ordas, Humberto. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; ArgentinaFil: Caligiuri, Lorena Gisel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; ArgentinaFil: Lorenzo Pérez, Norailys. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; ArgentinaFil: Berengeno, Andrea Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Ortega, Hugo Hector. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Alonso, Daniel Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; ArgentinaFil: Farina, Hernán Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; Argentin

CIGB-300 anticancer peptide regulates the protein kinase CK2-dependent phosphoproteome

Casein-kinase CK2 is a Ser/Thr protein kinase that fosters cell survival and proliferation of malignant cells. The CK2 holoenzyme, formed by the association of two catalytic alpha/alpha’ (CK2α/CK2α’) and two regulatory beta subunits (CK2β), phosphorylates diverse intracellular proteins partaking in key cellular processes. A handful of such CK2 substrates have been identified as targets for the substrate-binding anticancer peptide CIGB-300. However, since CK2β also contains a CK2 phosphorylation consensus motif, this peptide may also directly impinge on CK2 enzymatic activity, thus globally modifying the CK2-dependent phosphoproteome. To address such a possibility, firstly, we evaluated the potential interaction of CIGB-300 with CK2 subunits, both in cell-free assays and cellular lysates, as well as its effect on CK2 enzymatic activity. Then, we performed a phosphoproteomic survey focusing on early inhibitory events triggered by CIGB-300 and identified those CK2 substrates significantly inhibited along with disturbed cellular processes. Altogether, we provided here the first evidence for a direct impairment of CK2 enzymatic activity by CIGB-300. Of note, both CK2-mediated inhibitory mechanisms of this anticancer peptide (i.e., substrate- and enzyme-binding mechanism) may run in parallel in tumor cells and help to explain the different anti-neoplastic effects exerted by CIGB-300 in preclinical cancer models.Fil: Perera, Yasser. China-Cuba Biotechnology Joint Innovation Center; China. Centro de Ingeniería Genética y Biotecnología; CubaFil: Ramos, Yassel. Centro de Ingeniería Genética y Biotecnología; CubaFil: Padrón, Gabriel. Centro de Ingeniería Genética y Biotecnología; CubaFil: Caballero, Evelin. Centro de Ingeniería Genética y Biotecnología; CubaFil: Guirola, Osmany. Centro de Ingeniería Genética y Biotecnología; CubaFil: Caligiuri, Lorena Gisel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; ArgentinaFil: Lorenzo Pérez, Norailys. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; ArgentinaFil: Gottardo, María Florencia. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Farina, Hernán Gabriel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Filhol, Odile. Universite Grenoble Alpes; FranciaFil: Cochet, Claude. Universite Grenoble Alpes; FranciaFil: Perea, Silvio E.. Centro de Ingeniería Genética y Biotecnología; Cub