Insights into the proteomic profile and gene expression of Lutzomyia longipalpis-derived Lulo cell line

BACKGROUND Lutzomyia longipalpis-derived cell line (Lulo) has been suggested as a model for studies of interaction between sandflies and Leishmania. OBJECTIVES Here, we present data of proteomic and gene expression analyses of Lulo cell related to interactions with Leishmania (Viannia) braziliensis. METHODS Lulo cell protein extracts were analysed through a combination of two-dimensional gel electrophoresis and mass spectrometry and resulting spots were further investigated in silico to identify proteins using Mascot search and, afterwards, resulting sequences were applied for analysis with VectorBase. RESULTS Sixty-four spots were identified showing similarities to other proteins registered in the databases and could be classified according to their biological function, such as ion-binding proteins (23%), proteases (14%), cytoskeletal proteins (11%) and interactive membrane proteins (9.5%). Effects of interaction with L. (V.) braziliensis with the expression of three genes (enolase, tubulin and vacuolar transport protein) were observed after an eight-hour timeframe and compared to culture without parasites, and demonstrated the impact of parasite interaction with the expression of the following genes: LLOJ000219 (1.69-fold), LLOJ000326 (1.43-fold) and LLOJ006663 (2.41-fold). CONCLUSIONS This set of results adds relevant information regarding the usefulness of the Lulo cell line for studies with Leishmania parasites that indicate variations of protein expression.

Comparative assessment of the replication efficiency of dengue, yellow fever, and chikungunya arboviruses in some insect and mammalian cell lines

Abstract INTRODUCTION: Insect cell cultures play an essential role in understanding arboviral replication. However, the replicative efficiency of some of these viruses such as dengue (DENV), yellow fever (YFV), and chikungunya (CHIKV) in a new cellular substrate (Lulo) and in the other two recognized cell lines has not been comparatively assessed. METHODS: Vero, C6/36, and Lulo cell lines were infected with DENV, YFV, and CHIKV. The viral progeny was quantified through plaque assays and quantitative reverse transcription-polymerase chain reaction, while for DENV2, the findings were confirmed by immunofluorescence antibody assay. RESULTS: The higher DENV2 titer (from multiplicity of infection 0.001) was obtained on day four post-infection in C6/36 and on day six in Vero cells, while the Lulo cell line was almost impossible to infect under the same conditions. However, C6/36 showed the highest values of viral RNA production compared to Vero cells, while the quantification of the viral RNA in Lulo cells showed high levels of viral genomes, which had no correlation to the infectious viral particles. CONCLUSIONS: C6/36 was the most efficient cell line in the alpha and flavivirus production, followed by Vero cells. Thus, Lulo cells may be a useful substrate to study the mechanisms by which cells evade viral replication.

EXTRACCIÓN DE ENZIMAS PÉCTICAS DEL EPICARPIO DE LULO (Solanum quitoense Lam) INVOLUCRADAS EN EL PROCESO DE ABLANDAMIENTO / Exytraction of Pectic Enzymes from of Lulo(Solanum quitoense Lam) Involved in Softening

Durante el periodo de poscosecha el principal problema de deterioro del lulo (Solanum quitoense Lam) es el ablandamiento que es generado principalmente por actividad de enzimas pécticas que atacan la red estructural de la pared celular. Esta investigación se basó en la búsqueda de las mejores condiciones de extracción y medida de actividad de las enzimas pectinesterasa, poligalacturonasa y pectato liasa; herramientas necesarias para estudiar posteriormente el rol de estas enzimas en el deterioro por ablandamiento sufrido por el fruto debido a diversos cambios metabólicos. Se encontró que las dos primeras enzimas pueden ser extraídas simultáneamente con buffer fosfatos 20 mM pH 7,0 + NaCl 0,06 M y 60 min de extracción, relación 1:2 (material vegetal: buffer de extracción), a su vez, pectato liasa se extrajo con buffer fosfatos 20 mM pH 7,0 + cisteína 20 mM y 30 min de extracción, relación 1:3. Para la cuantificación de la actividad pectinesterasa es necesario incubar 15 min a 42 °C 2.500 µL de extracto enzimático crudo (EE) en buffer fosfatos 20 mM pH 7,0 + NaCl 0,15 M y 1,6% de pectina cítrica como sustrato, con valores de Km aparente de 3,78% de PC y Vmax 17,95 µmolH+/min*mg prot. Para la cuantificación de la actividad poligalacturonasa es necesario incubar 15 min a 37 °C 30 µL (EE) en buffer acetatos 200 mM pH 4,5 + NaCl 0,25 M y 1,0% de APG como sustrato, con valores de Km aparente 0,141% de APG y Vmax 28,46 nKat/s*mg prot. Para la cuantificación de la actividad pectato liasa es necesario incubar 2 min a 17 °C 100 µL (EE) en buffer TRIS:HCl 50 Mm pH 8,5 + CaCl2 4 mM y 0,1% de APG como sustrato, con valores de Km aparente 0,0865% de APG y Vmax 82,75 µg/s*mg prot.

The main problem of post-harvest deterioration of lulo (Solanum quitoense Lam) is the softening is the main problem of post-harvest deteriorarion of Lulo, that is generated mainly by the activity of pectic enzymes, which attack the structural network of the cell wall. This research was based on finding the best conditions structural cell wall network for extraction and measurement of enzyme activity pectinesterase (PE), polygalacturonase (PG) and pectato liasa (PL); tools needed to study the further role of these enzymes in the deterioration of pectatelyase fruit softening, due to various metabolic changes. It was found that the first two enzymes can be extracted simultaneously with 20 mM phosphate buffer pH 7.0, 0.06 M NaCl and 60 minutes of extraction, ratio 1:2 (plant material: extraction buffer), pectatelyase extracted with 20 mM phosphate buffer pH 7.0, 20 mM cysteine and 30 minutes of extraction, ratio 1:3. For quantification of pectinesterase activity is necessary to incubate 15 minutes at 42 ° C, 2500 µL of crude enzyme extract (EE) in 20 mM phosphate buffer pH 7.0, to 0.15 M NaCl and 1.6% citrus pectin as (CP) substrate with apparent Km values of 3.78% CP and Vmax 17.95 mol H+/min * mg prot. For the quantification of pectinesterase activity is necessary to incubate 15 minutes to 42 °C 2500 µL of crude enzyme extract (EE) in 20 mM phosphate buffer pH 7.0, 0.15 M NaCl and 1.6% citrus pectin as substrate with apparent Km values of 3.78% CP and 17.95 µ Vmax mol H+/min*mg prot. For the quantification of polygalacturonase activity is necessary to incubate 15 minutes to 37 °C 30 µL (EE) in 200 mM Acetate buffer pH 4.5, 0.25 M NaCl and 1.0% of APG as substrate, with apparent Km values 0.141% of APG and Vmax 28.46 nKat/s*mg prot. For the quantification of the pectatelyase activity is necessary to incubate 2 minutes to 17 °C, 100 µL (EE) in buffer TRIS: HCl pH 8.5, 50 mM 4 mM CaCl2 and 0.1% PGA as substrate, with apparent Km values 0.0865% of APG and Vmax 82.75 µg/s*mg prot.

INDUCCIÓN DE FENILALANINA AMONIO LIASA Y VARIACIÓN EN EL CONTENIDO DE COMPUESTOS FENÓLICOS EN FRUTOS DE LULO (Solanum quitoense Lam) INFECTADOS CON Colletotrichum acutatum. / Induction of phenylalanine ammonia lyase and variation in phenolic compounds content in Lulo fruits (Solanum quitoense Lam) infected by Colletotrichum acutatum)

Se evaluó la dinámica de la actividad fenilalanina amonio liasa (PAL) en corteza de frutos de lulo (Solanum quitoense Lam) con el fin de determinar su participación en respuestas bioquímicas hacia Colletotrichum acutatum. Se establecieron como mejores condiciones para la extracción de la enzima, buffer ácido bórico-borato de sodio 0.1M pH 8.8, 1% SDS, 3% PVPP y para medir la actividad, sustrato L-fenilalanina 5 mM , pH 8,0, 20°C , 30 ΜL de extracto y 45 min. Se realizó un ensayo in vivo usando frutos en tres estados de madurez, los cuales fueron inoculados con el patógeno o tratados con agua estéril. A cinco tiempos (hpi = horas post-infección) se determinó la actividad PAL y el contenido total de fenoles, encontrándose que hay una respuesta diferencial de la enzima por efecto del patógeno y por el estado de madurez. Para frutos en el estado pintón se obtuvo el mayor aumento de PAL, el que perduró hasta 48 hpi, al compararlo con los controles y con los otros dos estados de madurez. Este aumento mostró relación con un marcado incremento en el contenido total de fenoles y con el desarrollo más tardío de síntomas característicos de antracnosis, observado para los frutos pintones. Estos resultados permiten postular, una posible relación positiva entre inducción de PAL, aumento de fenólicos y respuesta de tolerancia a C. acutatum. Para lulos en estado verde y maduro se observó aumento de PAL a 12 y 24 hpi que coincidió también con incremento en el contenido de fenoles totales, aunque para estos dos últimos estados dicho contenido disminuyó significativamente a tiempos mayores.

Phenylalanine ammonia lyase (PAL) activity induction was evaluated in lulo fruits to determine the role of this enzyme in biochemical responses towards the pathogen Colletotrichum acutatum. We studied the experimental conditions to obtain the enzyme, using lulo peel, and found that the best conditions for extraction were buffer of boric acid-sodium borate 0.1M pH 8.8, 1% SDS, 3% PVPP and, for measuring the enzymatic activity: L- phenylalanine 5mM, pH 8.0, 20°C , 30 ΜL of extract and incubation during 45 min. Then, we performed an in vivo assay using lulo fruits in three maturity stages, which were inoculated either with the fungus or sterile water. Enzymatic induction was studied at five post-inoculation times, and it was found that there is a differential response as a consequence of the presence of the pathogen and the maturity stage. In semi-mature lulos, a bigger increase in PAL was obtained among 12-48 hpi which is related to a higher content of phenolic compounds and to the latest development in the characteristic symptoms of anthracnose. These results allow postulating, in a preliminary way, a possible positive relationship between inductions of PAL, phenolics and responses of tolerance to C. acutatum. For green and mature lulos we observed as well, an increase of PAL activity but only at 12 and 24 hpi which showed relation with an increase in the total phenolic content. However, for these stages the phenolics were significantly lower at higher times.

Genetic variation in the Solanaceae fruit bearing species lulo and tree tomato revealed by Conserved Ortholog (COSII) markers

The Lulo or naranjilla (Solanum quitoense Lam.) and the tree tomato or tamarillo (Solanum betaceum Cav. Sendt.) are both Andean tropical fruit species with high nutritional value and the potential for becoming premium products in local and export markets. Herein, we present a report on the genetic characterization of 62 accessions of lulos (n = 32) and tree tomatoes (n = 30) through the use of PCR-based markers developed from single-copy conserved orthologous genes (COSII) in other Solanaceae (Asterid) species. We successfully PCR amplified a set of these markers for lulos (34 out of 46 initially tested) and tree tomatoes (26 out of 41) for molecular studies. Six polymorphic COSII markers were found in lulo with a total of 47 alleles and five polymorphic markers in tree tomato with a total of 39 alleles in the two populations. Further genetic analyses indicated a high population structure (with F ST > 0.90), which may be a result of low migration between populations, adaptation to various niches and the number of markers evaluated. We propose COSII markers as sound tools for molecular studies, conservation and the breeding of these two fruit species.

INDUCCIÓN DE ACTIVIDAD PEROXIDASA Y DE FENOLES TOTALES COMO RESPUESTA DEL FRUTO DE LULO (Solatium quitoense L.) AL PATÓGENO CAUSAL DE LA ANTRACNOSIS / INDUCTION OF PEROXIDASE AND TOTAL PHENOLS AS A RESPONSE OF LULO (Solanum quitoense L.) FRUIT TO PATHOGEN CAUSING ANTHRACNOSE / INDUÇÃO DA ACTMDADE PEROXIDASA E FENÓIS TOTAIS COMO RESPOSTA DO FRUTO DE LULO (Solanum quitoense L.) AO PATÓGENO QUE CAUSA ANTRACNOSE

Se evaluó la inducción de peroxidasa en frutos de lulo con el fin de determinar su participación en las respuestas bioquímicas hacia el patógeno Colletotrichum acutatum, causante de la antracnosis. Se establecieron como mejores condiciones para su extracción y para determinación de la actividad: buffer fosfatos 100 mM pH 7, 1% SDS y 1 % PVPP; sustrato guayacol 15 mM, peróxido de hidrógeno 10 mM, pH 6,5, 55 °C y 30 µL de extracto. Se realizó un ensayo in vivo usando frutos verdes, pintones y maduros, inoculados con el hongo o con agua estéril. Se determinó la actividad peroxidasa a diferentes horas a partir de la inoculación, encontrándose una respuesta diferencial con el tiempo por efecto de la presencia del patógeno, y según el estado de madurez de los frutos. En lulos verdes inoculados con el hongo se observó aumento en la actividad al cabo de 6 y 144 horas. En lulos pintones no se observó efecto notable, mientras que en maduros el aumento en actividad fue prácticamente a todos los tiempos. Los resultados del contenido de fenoles totales mostraron que hubo acumulación a 96 y 144 horas por efecto del patógeno, para lulos en estado verde y maduro, mientras que para pintones, en los que se presentaron más rápido y con mayor severidad los síntomas de la antracnosis, no se observó aumento a ninguno de los tiempos. En los frutos más enfermos, el cambio en actividad peroxidasa y en contenido total de fenoles fue menos evidente, por lo que se sugiere una relación inversa de estos con el desarrollo de la antracnosis.

The induction of peroxidase in lulo fruits was evaluated in order to determine its participation in the biochemical responses towards the pathogen Colletotrichum acutatum, which causes the antrachnosis disease. For extraction and activity determination, these conditions were established as the best: buffer phosphates 100 mM pH 7, 1% SDS y 1 % PVPP; substrate guaiacol 15 mM, hydrogen peroxide 10 mM, pH 6,5, 55 °C and 30 µL of the extract. An in vivo test was developed using unripe, semi-ripe and ripe fruits, inoculated with the fungus or sterile water. The peroxidase activity was measured at different hours starting from the inoculation, finding a time differential response caused by the pathogen presence and according to the maturity state of fruits; in unripe lulo fruits inoculated with the fungus, an increase of the activity was observed after 6 and 144 hours. In semi-ripe fruits no considerable effect was seen, while in ripe fruits the increase of the activity was found practically at all times. The results of the measured total phenol content, showed accumulation at 96 and 144 hours as a result of the pathogen presence in unripe and ripe fruits, while for semi-ripe fruits, in which the antrachnosis symptoms were noticed faster and more severely, no phenol increase was found at any time. The less evident changes seen in peroxidase and phenol content, using severely affected fruits by the disease, suggest an inverse relationship between these parameters and the development of the antrachnosis.

A indução da peroxidasa foi avaliada na casca de frutos de lulo com a finalidade de determinar a sua possível participação em reacções de defesa contra o patógeno Colletotrichum acutatum, causador da doença antracnose. Foram estabelecidas as melhores condições para a sua extracção e para medir a actividade da enzima extraída, encontrando-se que com tampão fosfato 100 mM pH 7, 1% SDS y 1% PVPP, substrato guaicol 15 mM, peróxido de hidrogeno 10 mM, pH 6,5, 55 °C y 30 µL de extracto enzimático se conseguiram as actividades enzimáticas mais elevadas. Foi realizado um ensaio in vivo usando frutos verdes, em processo de maduração e maduros, inoculados com o fungo ou com água estéril. A actividade peroxidasa foi determinada a diferentes horas a partir da inoculação encontrando-se uma resposta diferencial com o tempo pelo efeito da presença ao patógeno e de acordo ao estado de madurez dos frutos. Em lulos verdes observou-se um aumento da actividade nos frutos inoculados com o fungo ao fim de 6 e 144 horas. Em lulos em processo de maduração, o patógeno não teve maior efeito, enquanto que em lulos maduros o aumento na actividade da enzima, como resposta à presença do patógeno, foi praticamente a todos os tempos avaliados. Os resultados do conteúdo de fenóis totais mostraram que ouve uma acumulação significativa a 96 e 144 horas por efeito da inoculação com o patógeno para lulos em estado verde e maduro, enquanto que para os lulos em processo de maduração, nos quais se apresentaram mais rápido e com maior severidade os sintomas de antracnose, não se observou aumento em nenhum dos tempo avaliados. Nos frutos mais afectados pelos sintomas, a mudança na actividade POD e no conteúdo total de fenóis foi menos evidente, motivo pelo qual se sugere uma relação inversa de estes com o desenvolvimento de antracnose.

INDUCCIÓN DE POLIFENOLOXIDASA EN FRUTOS DE LULO (Solanum quitoense) COMO RESPUESTA A LA INFECCIÓN CON Colletotrichum acutatum / Polyphenol Oxidase Induction in Lulo Fruits (Solanum quitoense) Infected by Colletotrichum acutatum

Se evaluó la actividad polifenoloxidasa (PFO) en corteza de frutos de lulo con el fin de determinar su participación en respuestas hacia el patógeno Colletotrichum acutatum, causante de la antracnosis. Se estudiaron condiciones para la adecuada extracción de esta enzima, encontrándose que con buffer fosfatos 100 mM pH 7, 1% SDS y 1% PVPP se logran las mayores actividades. Se determinaron como mejores parámetros para medir la actividad de la enzima extraída, sustrato catecol 40 mM, pH 7,0, 23 °C y 30 µL de extracto. Para determinar su posible inducción en la interacción con el patógeno, se realizó un ensayo in vivo usando frutos verdes, pintones y maduros, inoculados con el hongo o con agua estéril. A nueve tiempos postinoculación se determinó la actividad PFO encontrándose que hay una respuesta diferencial con el tiempo y la madurez de los frutos y por efecto del patógeno. Se obtuvo aumento de actividad en lulos verdes a 48, 96 y 144 horas postinoculación (hpi) y en maduros a la mayoría de los tiempos evaluados, siendo éste estado en el que se presentó la respuesta más notable de inducción. En pintones aumentó solo a 72 y 144 hpi. Los mayores valores se registraron en general para frutos en estado verde. Los frutos respondieron al estrés ocasionado por la herida activando también esta enzima. La inducción de actividad se presentó a tiempos más rápidos en los frutos menos afectados por la enfermedad (verdes y maduros), por lo que se puede postular una relación positiva entre inducción de PFO y respuesta de tolerancia a la antracnosis.

Polyphenol oxidase (PFO) activity induction was evaluated in lulo fruits to determine the role of this enzyme in resistance responses towards the pathogen Colletotrichum acutatum which causes anthracnose disease. We studied the experimental conditions to obtain the enzyme, using lulo peel, and found that extraction with phosphates buffer 100 mM pH 7, 1% SDS y 1% PVPP showed higher activities. The adequate parameters for activity measurement was also evaluated and established as cathecol 40 mM, pH 7,0, 23 °C and 30 µL of extract. Then, we performed an in vivo assay using lulo fruits in three maturity stages, green, semimature and mature, which were inoculated with the fungus or with sterile water. Enzymatic induction of this protein was studied at nine postinoculation times, and it was found that the induction was differential according to the time, the maturity stage, and as consequence of pathogen presence. The PFO activity increased in green fruits at 48, 96 y 144 (hours postinoculation (hpi), and in mature lulos for the majority of times studied, with the most significant induction response at this stage. In semimature lulo, the induction was observed only at 72 and 144 hpi. The highest nominal value of activity was found in green fruits. Fruits responded to incision with enzyme activation. The increase in the activity of the enzyme was faster in the fruits with the minor anthracnose symptoms than the ones that were more affected. Therefore, it is possible to postulate a positive relation between PFO induction and tolerance to anthracnose symptoms.