V Symposium of Biochemistry and Molecular Biology

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Oct 10, 2018 - La Fisiología Vegetal es una especialidad que se encarga de analizar la .... alcohol-etílico al 96% por 24horas; ácido-sulfúrico al 75% por 5 y 10min.; ácido- .... Con base en nuestro trabajo anterior hemos propuesto a los ...... El estudio del equilibrio de distribución y de cinética de captura de los iones ...
V Symposium of Biochemistry and Molecular Biology

MIÉRCOLES/WEDNESDAY, OCT. 10th

SESSION: Physiology and Molecular Biology of Plants N-C 001

EL CAMBIO CLIMÁTICO Y LA FISIOLOGÍA VEGETAL EN CUBA Eduardo Ortega, Rodés R Laboratorio de Fisiología Vegetal, Dpto. Biología Vegetal, Facultad de Biología, Universidad de La Habana, Cuba. [email protected] El Cambio Climático además de ser una amenaza para el futuro es una realidad tangible. Los países se aprestan a preparar condiciones para mantener las actividades productivas que son vitales, aún en condiciones donde los valores de los indicadores climáticos cambien ostensiblemente. Cuba es un país insular, y los efectos de las altas temperaturas y la disminución y el aumento desmesurado de las lluvias en un corto período pueden afectar la actividad agrícola y otras. Por eso Cuba ha desarrollado de una manera ejemplar la Tarea Vida. La Fisiología Vegetal es una especialidad que se encarga de analizar la relación de las actividades fundamentales de las plantas con el ambiente. Entre ellos podemos citar como ejemplos las Relaciones Hídricas, la Nutrición Mineral y la Actividad Fotosintética. Para poder atenuar los efectos del estrés como son las altas temperaturas, la salinidad, la poca o excesiva lluvia, la Fisiología Vegetal puede contribuir a encontrar soluciones paliativas. Los Reguladores del Crecimiento Vegetal también pueden tener un papel relevante y en eso la UH ha desarrollado durante años los análogos de brasinoesteroides. Los autores abordarán los aspectos antes mencionados en el marco de las afectaciones que se esperan con el Cambio Climático.

N-C 002

EVIDENCE OF VX NERVE AGENT USE FROM CONTAMINATED WHITE MUSTARD PLANTS Christopher Timperley, Gravett M., Hopkins F., Self A., Webb A., Baker M.J. Defence Science and Technology Laboratory (Dstl), Porton Down, Salisbury, England [email protected] The Chemical Weapons Convention (CWC) prohibits the development, production, acquisition, stockpiling, retention, transfer or use of chemical weapons by Member States. Verification of compliance and investigations into allegations of use require accurate detection of chemical warfare agents (CWAs) and their degradation products. Detection of CWAs, such as organophosphorus nerve agents, in the environment relies mainly upon the analysis of soil.We now present a method for the detection of the nerve agent O-ethyl S-2-diisopropylaminoethyl methylphosphonothiolate (VX) and its hydrolysis products, O-ethyl methylphosphonic acid (EMPA) and methylphosphonic acid (MPA), by gas chromatography- and liquid chromatography-mass spectrometry of ethanol extracts of contaminated white mustard plants (Sinapis alba).Sinapis alba was grown in VX-, EMPA- and MPA-spiked (250 µg ml-1) clay, loam or sand. These substrates moderated the rate of initial uptake of VX but not the longer-term uptake: the duration the VX was detectable (sub 1 ng) in the plant was the same regardless of the soil type. The plants metabolised the VX to EMPA and MPA. The VX, EMPA and MPA were detected in the plants up to 45 days after sowing the white mustard seeds. The data suggest that the EMPA in the plants grown in soil contaminated with VX originated from metabolism of VX. The method described will help the Organisation for the Prohibition of Chemical Weapons (OPCW) and the CWC Member States confirm the presence of nerve agent residues in the environment. The use of localised samples and the simple extraction procedure will increase the probability of discovering nerve agent use. The ability of plants such as Sinapis alba to absorb nerve agents and their marker compounds protects against the removal of evidence of use, as CWAs can leach from soil over time. It also suggests that green manures might be useful for remediating nerve agent-polluted sites.

N-O 001

PROTEOMIC ANALYSIS OF APOPLAST FROM STEM WITH VASCULAR WILTING CAUSED BY Fusarium oxysporum f. sp. dianthi IN CARNATION Martínez-González A., Jorrín-Novo J., Castillejo-Sánchez M., López-Hidalgo C., MartínezPeralta S., Harold Duban Ardila-Barrantes Laboratory Research in Vegetal Metabolic Activities, Department of Chemistry, National, University of Colombia, Bogotá-Colombia [email protected] Vascular wilting caused by the fungus Fusarium oxysporum f. sp. dianthi (Fod) is one of the most devastating diseases affecting carnation (Dianthus caryophyllus L) production worldwide. The development of disease takes place in the stem host and differential studies on the dynamic changes of apoplastic protein profile, may reveal new insights into plant responses to biotic stress. In this direction, we present our current research on the proteomics analysis of the carnation apoplast, as the first defensive lane against Fod. Carnation cuttings from resistant (Golem) and susceptible (Solex) cultivars were inoculated using a conidial suspension of Fod. Washing apoplastic fluid was obtained by Vacuum-Infiltration-Centrifugation method, and TCA-acetone precipitation was made for obtaining protein extracts. A proteomic approach employing a label-free quantitative technique was applied to characterize the carnation stem secretome at 14 days after inoculation. We found 223 carnation secreted proteins from in planta systems using label-free proteomic approach (nHPLC-MS/MS) and bioinformatic programs to predict apoplastic proteins. According to Gene Ontology, the plant upregulated proteins in the incompatible interaction (42) are mainly related to metabolic processes, response to stress anddefense response. In the other hand, the upregulated proteins in the compatible interaction (22) are mainly related to metabolic processes and carbohydrate metabolic processes. These results demonstrate that plant resistance to Fodis correlated with activation of response at proteome level, including enhanced metabolic processes and production of proteins in the apoplast, related to defense and stress responses. Acknowledgements: This Project was supported by COLCIENCIAS (Project, No. 110165842786). Special thanks to Florval S.A.S - Sede QFC for cutting donation and, Proteomics Facility, University of Cordoba for prepared and analyzed the protein samples.

N-O 002

UNRAVELING PHYSIOLOGICAL TRAITS OF JATROPHA CURCAS, A BIODIESEL PLANT, TO OVERCOME THE CLIMATE CHANGE. Patricia Ortega-Rodes, Katherine Meirama Ross K., García D., Rodés R., Eduardo Ortega Laboratorio de Fisiología Vegetal, Dpto. Biología Vegetal, Facultad de Biología, Universidad de La Habana, Cuba. [email protected] Jatropha curcas is a promising crop for biofuel production with high oil seed content. Knowledge of responses for agricultural crops to stress conditions is crucial in the actual context, taking into account the environmental effects that bring the Climate Change. J. curcas has some reputation as moderately tolerant plant to salt stress; however, the physiological mechanisms to avoid damages induced by stress during the germination and the first development stage are not completely known. It could be a promise for countries like Cuba, which lacks traditional sources of energy, to take advantages of the biodiesel production by J. curcas. The germination of seeds from the cultivar Cabo Verde and the growth of plantlets under salinity were studied. The respiratory activity, the water uptake and the mitotic index of the seeds after the imbibition under salinity conditions was analyzed. Salinity values of 75 mM NaCl did not affect the seeds germination. The role of presence of testa, and the stem size of the plants after 6 and 40 days old were of the valuable traits unraveled; also, the water uptake capacity and the respiratory activity under salinity. The mitotic index was affected during the first hours of imbibition under saline conditions, it could be an additional reason for the lower growth at the highest NaCl tested. The testa of J. curcas´s seeds, acts as a reservoir of water, however, it constitutes a barrier to the passage of water during germination, slows the kinetics of its absorption and hinders the exchange of O2, negatively affecting germination.

N-O 003

GERMINACIÓN DE SEMILLAS DE “TOTORA” (Schoenoplectus californicus Cyperaceae) DE LOS LAGOS NORTE-ANDINOS: YAHUARCOCHA, CUICOCHA E IMBACOCHA, IMBABURA-ECUADOR Pabón Galo, Ortega E., Rodés R., Pérez L., Vásquez L. Universidad Técnica del Norte, Ibarra, Ecuador [email protected] La “totora” (Schoenoplectus californicus - Cyperaceae), especie acuática que se distribuye desde el sur de los EEUU, hasta el norte de Argentina, posee importancia ecológica debido a la absorción del exceso de nutrientes de los ecosistemas lacustres; e importancia económica por las artesanías que se elaboran a partir de sus tallos fibrosos, que constituye sustento económico de numerosas familias. Pese a esto, no existen reportes de plantas obtenidas a partir de semillas. La literatura considera que se trata de poblaciones híbridas entre las variedades californucus y totora, que producen semillas no viables. Para la germinación se utilizó semillas de tres lagos de la provincia de Imbabura (Yahurcocha, Cuicocha y Imbacocha), sometidas a distintos procedimientos: alcohol-etílico al 96% por 24horas; ácido-sulfúrico al 75% por 5 y 10min.; ácidoclorhídrico al 50% por 5 y 10min.; choque térmico: -10oC por 24horas + 80oC por 5min.; escarificación con arena extra fina (SiO₂). Las semillas fueron hidratadas con agua destilada y agua procedente del respectivo lago. Sólo el método de escarificación con SiO₂ logró germinar las semillas. Los porcentajes de germinación fueron de 69, 28 y 52 para Yahurcocha, Cuicocha e Imbacocha, respectivamente, y requirieron entre 26 y 53 días de hidratación para germinar. No hubo diferencias significativas en cuanto al tipo de agua usada, pero si determinaron diferencias en cuanto a la procedencia de las semillas. Pruebas de imbibición confirman que las semillas de “totora” presentan dormancia física (por la impermeabilidad de la testa) y dormancia fisiológica (evidenciada en la baja tasa de germinación). Se concluye que las semillas son viables, usando como método la escarificación, por lo que es improbable que se trate de poblaciones híbridas como reporta la literatura. Se está analizando los probables agentes escarificadores que pudieran existir de forma natural, y que podría tratarse de procesos microbianos.

N-O 005

AVANCES EN EL CONOCIMIENTO DE LA INTERACCIÓN CAÑA DE AZÚCARSPORISORIUM SCITAMINEUM: PAPEL DE LAS PROTEÍNAS DIRIGENTES

Roberto de Armas, Piñón, D., Sánchez-Elordi, E., Legaz, M.E., Vicente, C. Departamento de Biología Vegetal, Facultad de Biología, Universidad de La Habana, La Habana, Cuba. [email protected] Sporisorium scitamineum es un hongo causante de la enfermedad conocida como carbón de la caña de azúcar. Esta enfermedad produce grandes pérdidas en los rendimientos agrícolas del cultivo que pueden alcanzar valores superiores al 40% con afectaciones además en la calidad de los jugos. Debido a lo anterior son numerosos los estudios realizados en la dilucidación de los posibles mecanismos de resistencia de la planta al patógeno. La resistencia se asoció en un inicio a los niveles de flavonoides en yemas y de ceras acumuladas en la superficie por donde penetra el patógeno. Posteriormente se ha reportado la participación de poliaminas libres y conjugadas y la presencia de determinadas glicoproteínas que afectan la germinación de las esporas y la propagación de la infección. El presente trabajo profundiza en el papel de glicoproteínas extraídas del jugo de la caña de azúcar en cultivares resistentes y no, inoculados con esporidios conjugantes del patógeno. Se reporta la presencia en las glicoproteinas de arginasa y de varias enzimas hidrolíticas (quitinasa, β-1,3- glucanasa y β-1,4- glucanasa) y se discuten sus importancias en la interacción estudiada. Interés especial adquiere la presencia de proteínas dirigentes en el pool de glicoproteínas, no relacionadas hasta el momento con los posibles mecanismos de resistencia a la enfermedad. Se analiza la expresion diferencial del gen Sof DIR16 en ambos tipos de cultivares después de la inoculación con el patógeno, y se relacionan los resultados con la participación de estas proteínas en la dirección de la síntesis de lignina (componente de la pared celular) o de lignanos (moléculas implicadas en procesos defensivos en las plantas) y sus posibles efectos en los mecanismos de resistencia, relacionándolos con resultados previos obtenidos que involucran el metabolismo de los monolignoles en la respuesta de la planta durante la interacción caña de azúcar- S. scitamineum.

N-O 006

CONCENTRACIÓN DE METALES PESADOS (PLOMO Y CROMO) ASOCIADOS CON POBLACIONES DE TYPHA LATIFOLIA (L) EN LA LAGUNA DE YAHUARCOCHA, IMBABURA – ECUADOR. Jorge Renato Oquendo Andino

Universidad Técnica del Norte, Ibarra, Ecuador [email protected] Yahuarcocha comprende un ecosistema léntico de gran importancia ecológica, económica y escénica para la provincia de Imbabura. La contaminación por actividades antrópicas aledañas ha sido causante de su deterioro ecológico. El objetivo de la investi-gación fue cuantificar la concentración de los metales plomo y cromo en la raíz y nichos asociados a las poblaciones de Typha latifolia presentes en el sitio. Se recolectaron muestras de raíz, suelo, sedimentos y agua de cinco puntos en la laguna. Las mismas fueron procesadas según protocolos estandarizados y se cuantificaron mediante la técnica de espectrofotometría de absorción atómica modalidad horno de grafito. Las concentraciones de metales en la raíz y nichos asociados a T. latifolia tuvieron diferencias significativas entre las épocas lluviosa y seca. Los niveles más altos de contaminación en el sedimento, suelo y agua se registraron en los puntos más cercanas a actividades humanas. La contaminación por metales tiene su origen en los vertidos a los efluentes, las zonas de mayor contaminación de plomo corresponden a sitios donde se desarrollan actividades de recreación con motores, por su parte el cromo está asociado a actividades de curtido de cuero y teñido de telas en los centros poblados. Estudios similares muestran que los metales en agua obedecen a un proceso de sedimentación donde las formas menos solubles son acumuladas en la fase de sedimento. Por el hecho de ser una laguna urbana este cuerpo hídrico es el sumidero de varios contaminantes. Los metales señalados por su gran toxicidad y persistencia están presentes en la laguna y la relación de su mayor concentración se evidencia por su cercanía a actividades humanas. Typha latifolia está registrada en la literatura como un fitoremediador eficiente en la absorción de metales pesados, una alternativa viable para la recuperación de Yahuarcocha con opciones de humedales artificiales en zonas de mayor incidencia de estos contaminantes. .

N-O 007

¿CUÁL ES EL ROL DE LOS FACTORES TRANSCRIPCIONALES DE LA FAMILIA AGL DEL FRIJOL COMÚN (Phaseolusvulgaris) EN LA SIMBIOSIS CON Rhizobiumetli? Litzy Ayra, Fuentes S.I, Alfonso L., Salas P, Girard L. Hernández G.

Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México (UNAM), Cuernavaca, México [email protected] El frijol común (Phaseolusvulgaris) es la leguminosa más importante para el consumo humano y al igual que otras leguminosas, es capaz de asociarse con bacterias rhizobia que llevan a cabo la fijación simbiótica de N. Este es un proceso complejo y finamente regulado tanto por la planta como por la bacteria, pero aún desconocemos todos los reguladores de la planta involucrados. Con base en nuestro trabajo anterior hemos propuesto a los factores de transcripción de la familia AGL como posibles componentes de la vía de señalización: miR172c / AP2-1 que regula la simbiosis entre P. vulgaris y Rhizobiumetli. Para este proyecto se plantea analizar el perfil de expresión de genes AGL, por PCR en tiempo real, en raíces y nódulos de diferentes etapas de la simbiosis, para seleccionar aquellos con mayor respuesta en este proceso. Para definir el rol de los AGL seleccionados se usarán enfoques de genética reversa, utilizando la transformación de frijol con AgrobacteriumrhizogenesK599para obtener plantas compuestas (con raíces y nódulos transgénicos) que sobreexpresen o tengan silenciado cada AGL. Los genes AGL que hemos seleccionado los denominamos: PvSVP y PvCAL; estos se inducen en las raíces inoculadas desde tiempos tempranos y en nódulos maduros y jóvenes, respectivamente. Hemos construido los plásmidos (pOEPvSVP, pOE-PvCAL y pRNAi-AGL) para la sobreexpresión y el silenciamiento de cada gen y está en proceso el análisis fenotípico de las plantas compuestas en simbiosis. Este incluye aspectos iniciales (la deformación de pelos radicales y la formación de hilos infectivos) así como del desarrollo y función de los nódulos (su número, histología y actividad de la nitrogenasa). Nuestros resultados permitirán evidenciar la función específica que desempeñan estos factores transcripcionales.

N-O 008

DIVERSIDAD DE MORFOTIPOS DE CAMOTE Ipomoea batatas (Convolvulaceae) A NIVEL MORFOLÓGICO Y ECOGEOGRÁFICO; Y DETERMINACIÓN DE ÁREAS DE ALTA RIQUEZAEN ECUADOR Lucia Vazquez.1,2,Otero C., Tapia C., Pabón G. 1 Universidade de Santiago de Compostela, Santiago de Compostela, España

2

Universidad Técnica del Norte, Ibarra, Ecuador [email protected]

El Ecuador posee estudios sobre caracterización morfológica de camote(Ipomoea batatas); y la presente investigación adiciona información ecogeográfica, al mismo tiempo que fusiona los datos de estas dos fuentes para determinar áreas de alta diversidad de morfotipos de este cultivo en el país. Esta investigación fue desarrollada para conservar los recursos fitogenéticos, estudiando características en cuanto a requerimientos ambientales óptimos para eldesarrollo y distribución del camote. Los datos de la caracterización morfológica obtenidos de 48 descriptores; de los cuales 35 fueron cualitativos y 13 cuantitativos; se analizaron utilizando los complementos estadísticos para Microsoft Excel. Se efectuó el análisis de componentes principales, cálculo de la media aritmética, desviación estándar y análisis de correlación para establecer las relaciones entre datos cuantitativos de la caracterización morfológica; y el cálculo de la moda y fecuencia de caracteres cualitativos. Para el análisis espacial combinado se empleó datos de caracterización morfológica y ecogeográfica, más el uso de los Sistemas de Información Geográfica, se tomaron en cuenta variables consideradas relevantes para el cultivo según expertos. Se obtuvo los caracteres cuantitativos y cualitativos de alto poder discriminante, que permitieron identificar relaciones genéticas entre grupos y entradas de la colección. También, se identificaron posibles relaciones entre la distribución de los morfotipos de camote en el Ecuador y el ambiente donde es cultivado. Además, se establecieron las áreas de diversidad, definidas en base a la abundancia, riqueza de morfotipos, distancias morfológicas y ecogeográficas.

N-O 009

EVALUACIÓN DE LOS GENES QUE CODIFICAN PARA s’HSPs EN PLÁNTULAS DE ARROZ Rodolfo E. Arce, Martínez C. Universidad Distrital Francisco, Colombia [email protected], [email protected]

Se evaluó la expresión de los genes que codifican las proteínas de choque térmico sHsp’s, (Hsp16.9, Hsp18, Hsp26.7), en variedades de arroz Oryza sativa: Fedearroz 473, Fedearroz Mocarí y Línea avanzada Lv1645 irradiadas con 60Co. Se realizaron cultivos hidropónicos, una vez se alcanzó el estadio de plántula fueron sometidas a estrés térmico: tratamiento de estrés moderado (T1) a 35°C y un tratamiento de estrés alto (T2) a 42°C durante 4 horas. Por cada tratamiento se obtuvieron extracciones de ARN y proteínas totales en tres momentos diferentes: 11:10, 13:10 y 15:10. El ARN total se cuantifico a través de Thermo Scientific NanoDrop 2000c y por medio de q-RT-PCR se realizó una cuantificación absoluta de los genes de interés; para la cuantificación de proteínas se utilizó el método de Bradford y se hicieron geles de SDS-PAGE al 12.5%. Como resultado se obtuvo que, la expresión de los genes que codifican las proteínas de choque térmico small en los tres genotipos, incrementa a medida que aumenta la temperatura como mecanismo de protección de la plántula frente alteraciones que se puedan originar a nivel bioquímico, fisiológico y morfológico que comprometan el crecimiento, desarrollo y rendimiento del cultivo: por lo cual, la expresión de estos genes está relacionada con la tolerancia a altas temperaturas, siendo F473 tolerante, Mocarí resistente y Lv1645 susceptible. A nivel morfológico, los tres genotipos F473, Mocarí y Lv1645 no presentan diferencias significativas en el crecimiento de las plántulas durante las cuatro horas de exposición al estrés térmico, por otro lado una dosis de 60Co genera alteraciones físicas en las variedades Mocarí y Lv1645.

MIÉRCOLES/WEDNESDAY, OCT. 10th

N-O 010

CARACTERES MORFOMÉTRICOS, COMPONENTES DEL VENENO Y LA SECUENCIA DEL GEN CITOCROMO OXIDASA I EN Rhopalurus junceus (SCORPIONES: BUTHIDAE) DEL SECTOR MOANICUM DEL ORIENTE DE CUBA Georgina Espinosa-López, Rodríguez Ravelo R., Ruiz Urquiola A. Possani L. Universidad de La Habana, Cuba [email protected] La especie endémica de Cuba Rhopalurus junceus (Herbst, 1800) se encuentra distribuida por todo el país y las poblaciones del sector fitogeográfico Moanicum han sido poco estudiadas desde los puntos de vista genético, componentes del veneno y caracteres morfométricos. Se analizan especímenes de R. junceus provenientes de los distritos Nipense, Cristalense Moaënse, Baracoënse, Purialense y Yaterense del sector Moanicum, con el empleo de medidas de longitud de algunas partes del cuerpo, secuencias de 658 pb del gen Citocromo oxidasa I y componentes del veneno. Estos caracteres se compararon por métodos multivariados y análisis de secuencias. Los caracteres morfométricos no separan los distritos aunque si las clases sexuales. Las secuencias de DNA mostraron gran diversidad entre los distritos. Los componentes del veneno presentaron variación intraespecífica, entre clases sexuales y entre los distritos. Se ha informado gran variación genética y poca variación morfométrica en diferentes especies de alacranes Ojanguren-Affilastro y col 2016. Schaffratha y col, 2018 refieren la utilidad del mapeo peptídico para la identificación de especies de la familia Buthidae así como variación intraespecífica en algunas de las especies. Ojanguren-Affilastro y col 2016 Molecular Phylogenetics and Evolution 94 (2016) 159–170 Schaffratha y col, 2018 doi: 10.1016/j.toxicon.2018.02.004

N-O 011

PHYLODINAMICS OF PAPAYA RINGSPOT VIRUS ON CARICA PAPAYA L. IN CUBA Cabrera Mederos D., Giolitti F, Torres C., Orelvys Portal Departamento de Biología, Facultad de Ciencias Agropecuarias, Universidad Central “Marta Abreu” de Las Villas, Santa Clara, Cuba [email protected] Orchard and garden papaya crops grown in 47 Cuban municipalities were surveyed from 2008 to 2013, revealing the widespread distribution of Papaya ringspot virus (PRSV) in Cuba. Phylodynamic analyses performed with the coat protein partial gene of all Cuban PRSV-P isolates (34 sequences) and 107 sequences of isolates from the American continent and the Caribbean islands showed a most recent common ancestor in 1942 (HPD95% = 1911-1967). The substitution rate was estimated to be 7.7 × 10-4 substitutions per site per year (HPD95% = 4.6 × 10-4-1.1 × 10-3), which is equivalent to those detected in other RNA viruses. Demographic reconstruction of PRSV showed that viral diversity increased in the 1985-1990 period, which coincides with the implementation of extensive production practices. Moreover, in Cuba viral dispersion introductions were observed occurred from Mexico and other unknown ancestral localization. The spatio-temporal diffusion analysis proposed Mexico as an ancestral area for the origin of diversification in the American continent and suggests new dispersion events between American and Caribbean isolates. The observed widespread distribution, clear geographic grouping of Cuban isolates, virus growth and genetic diversity provide strong evidence of the PRSV dispersion patterns.

N-O 012

IDENTIFICATION AND VALIDATION OF A MOLECULAR MARKER FOR THE RAPID AND ACCURATE IDENTIFICATION OF Salmonella enterica Carolina Nathalie Resendiz-Nava, Esquivel-Hernandez Y., Gerardo M. Nava Universidad Autónoma de Querétaro. Querétaro, México. [email protected] In numerous laboratories, identification of Salmonella relies primarily on invA-PCR assays because they are rapid, reliable and low-cost; however, numerous recent studies have reported false-positive results using this molecular marker. To verify this problem, the objective of the present study was to evaluate the performance and specificity of published and validated PCR primers targeting the invA gene (invAnest1 + invAnest2, invA3F + invA3R, SA03 + SA04, invA139 + invA141, Salm 3 + Salm 4, invA1 + invA2 and SA01 + SA02). Additionally, other molecular markers, ttrBCA, 16S rRNA (16SF1 + 16SIII and MINf + MINr) and STM3098, were also included in these analyses.The performance and specificity of these eleven different PCR primer sets were evaluated using Salmonella enterica type-strains, Citrobacter spp. and Enterobacteriaceae isolates. Our study revealed that all published and validated invA primers generate false-positive signals (amplification of Citrobacter and other Enterobacteriaceae). Also, false-positive signals were observed using primers targeting 16S rRNA and STM3098 loci. Importantly, primers targeting the ttrBCA showed high specificity.These results indicate that identification of Salmonella enterica using primers targeting invA, 16S rRNA and STM3098 genes are not a reliable tool. It is recommended to modify PCR protocols and implement detection of Salmonella using the ttrBCA locus. Implementation of these PCR assay is simple, rapid and low-cost.

JUEVES/THURSDAY OCT. 11th SESSION: Proteins and Peptides in Biotechnology and Biomedicine N-C 003

THE SECRETS AND POTENTIAL OF A NOVEL CYCLIC NTIMICROBIAL PEPTIDE. Margitte Dathe, Scheinpflug K., Junkes Ch., Krylova O., Strahl H. Leibniz Research Institute of Molecular Pharmacology, Berlin, Germany [email protected] The development of antimicrobial peptides as antibiotic agents requires structural characterization and understanding of their diverse mechanisms of action. We investigated small cyclic anginine (R)- and tryptophan (W)-rich peptides characterised by variations in the amino acid position, exchange of R and W by other charged or aromatic residues, introduction of D-amino acid residues and reduction and enlargement of the ring size. The cyclic hexapeptide cycloRRRWFW (cWFW) revealed high antimicrobial activity and proved to be not toxic against eukaryotic cells. Its amphipathic structure and arginine content provide the prerequisites for membrane permeabilisation and translocation as modes of action [1-3]. Using a number of techniques to study peptide interaction with bacterial and eukaryotic cells and model membranes, we could show that the activity of cWFW is based on a novel antimicrobial mechanism. Strong interactions with the bacterial membrane lead to reduction in membrane fluidity and disturbance of the native lipid matrix. The formation of distinct lipid domains is related to a severe disturbance in the positioning of functional proteins [4]. Chemical modifications such as enhancement of the peptide hydrophobicity or enlargement of the cycle eliminated the bacterial selectivity and induced a membrane permeabilising mode of action. Although cWFW does not enter the cytoplasm of bacteria, it is rapidly internalized into human cells.The combination of cell penetrating properties with high antimicrobial activity and the novel mechanism of action render the cyclic hexapeptide an eligible compound with regard to the treatment of intracellular bacterial infections. References: 1. Scheinpflug, K. et al. (2015). PlosONE 10(4) e0125056. 2. Scheinpflug, K. et al. (2013) Pharmaceuticals 6:1130-1144. 3. Junkes, C. et al. (2011) Eur Biophys J 40: 515-528 4. Scheinpflug, K. et al. (2017) Scientific Reports 7:44332.

N-O 013

A CYCLIC AND TWO DIMERS DERIVED FROM THE PEPTIDE Cm-p5, ENHANCED ITS ANTIFUNGAL PROPERTIES AND IMPROVED ITS ANTIBACTERIAL ACTIVITY IN VITRO Anselmo J. Otero-González, Morales-Vicente F., González-García M., GarayPérez H., Wessjohann L., García-Rivera D., Grieshober M., Stenger S. Rodríguez A.A., Rosenau F., Standker L. Centre for Protein Studies, Faculty of Biology, Havana University, Cuba [email protected] Antimicrobial peptides are an essential part of the first line of defence against microbial pathogens in many organisms. Current treatments for fungal infections are limited by drug toxicity and pathogen resistance. Cm-p5 (SRSELIVHQRLF) has a significant fungistatic activity against pathogenic Candida albicans. Cm-p5 was characterized by circular dichroism and nuclear magnetic resonance revealed an a-helical structure in membranemimetic conditions and a tendency to random coil folding in aqueous solutions. Additional studies modeling Cm-p5 binding to a phosphatidylserine bilayer in silico and isothermal titration calorimetry using lipid monophases demonstrated that Cm-p5 has a high affinity for the phospholipids of fungal membranes (phosphatidylserine and phosphatidylethanolamine), only moderate interactions with a mammalian membrane phospholipid, low interaction with ergosterol, and no interaction with chitin. Adhesion of Cm-p5 to living C.albicans cells was confirmed by fluorescence microscopy with FITClabeled peptide. In a systemic candidiasis model in mice, intraperitoneal administration of Cm-p5 was unable to control the fungal kidney burden, although its low amphiphaticity could be modified to generate new derivatives with improved fungicidal activity and stability. Chemical and sequential derivatives have been synthetized to enhance the antimicrobial spectrum of Cm-p5. A cycled derivative (cys-cys Cm-p5) improved the minimal inhibitory concentration of the parental peptide from 10 to 5 µg/mL against Candida albicans. Cys-Cys CM-p5 was not toxic for human macrophages, the major host cell for the bacterial pathogen M. tuberculosis. Antimicrobial activity against extracellular, virulent M. tuberculosis reached >80% at 300 µg/ml concentration and was nearly as efficient as the first line antimicrobial drug rifampin. Acknowledgment: Proyecto del Ministerio Federal de Educación y Ciencia de Alemania (BMBF) Programa BMBF - WTZ - DLR para Cuba en Ciencias de la Vida, 2018-2020

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STICHOLYSINS, BEYOND THEIR ABILITY TO FORM PORES INTO MEMBRANES, CAN ENHANCE ANTIGEN SPECIFIC CYTOTOXIC T LYMPHOCYTES RESPONSE Maria E. Lanio , Laborde R., Cruz-Leal Y., Alvarez C., Pazos F., Luzardo M.C., Fernández A., Oliver L., Mesa C., Abreu-Butin L.., Longo-Maugéri I, Nogueira C.V., Grubaugha D., Higgins D., Fernández L.E. Centre for Protein Studies, Faculty of Biology, Havana University, Cuba [email protected] Sticholysins I and II (StI/II, Sts), two pore-forming proteins (PFPs) produced by the sea anemone Stichodactyla helianthus, are highly hemolytic cysteineless proteins exhibiting a preference for sphingomyelin-containing membranes. They are able to form oligomer pores of diameter 2 nm. Different strategies employing bacterial PFPs have been used to improve the antigen-specific cytotoxic T CD8+ lymphocytes (CTLs) response. We studied the enhancement of CTLs response by liposomes co-encapsulating Sts with ovalbumin as model antigen (Lp/OVA/Sts). Mice were immunized twice with Lp/OVA/Sts or Lp/OVA without Sts. SIINFEKL-specific B3Z CD8+ T and OVA-expressing EG-7 tumor cells were used to measure the antigen cross-presentation in vitro and antitumor activity in vivo, respectively. Lp/OVA/StII induced an OVA-specific CD8+ T-cell expansion superior to that observed with Lp/OVA and in vitro significantly enhanced activation of the SIINFEKL-specific B3Z CD8+ T cells as a consequence of antigen cross-presentation by macrophages, but not by dendritic cells. Interestingly, Lp/OVA/StII-induced activation was inhibited by lysosomes proteases inhibitors, but not proteasome inhibitor indicating that StII induces antigen cross-presentation by vacuolar pathway. The formulations Lp/OVA/Sts enhanced the OVA-specific CTLs response in vivo in comparison with Lp/OVA and also conferred a higher protection to mice challenged with OVA-expressing tumor cells. Additionally, CTLs activity induced by Lp/OVA/StII was independent of CD4+ T-cells, while anti-tumor response was strongly affected by CD8+ T-cells depletion. Curiously, free-Sts were able of inducing activation of DCs and it was dependent of TLR-4 and MyD88, suggesting that the effect of these proteins on the cellular immune response could be beyond their poreforming ability. The antigen-specific CTLs immune response enhanced by immunization of wild type mice with Lp/OVA/StII was significant reduced in TLR-4 knockout mice. Our results suggest the potentialities of Sts encapsulated into liposomes as adjuvant for enhancing effective CTLs mediated immune responses. Financial Support: FAPESP, CNPq, CAPES, IFS, DRLAC, Project of Lab UH-CIM

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STRUCTURAL STUDY OF THE PORE-FORMING PROTEIN STICHOLYSIN I BY SITE-DIRECTED SPIN LABELING AND EPR SPECTROSCOPY Yadira de la P. Hervis, Valle A., Dunkel S., Klare J., Canet L., Lanio M.E., Alvarez C., Pazos I.F., Steinhoff H.J. Centre for Protein Studies, Faculty of Biology, Havana University, Cuba [email protected] Sticholysin I (StI) is a water-soluble protein that forms oligomeric pores in membranes by inserting the N-terminal helical segment, leading to cell death. Three models have been proposed for actinoporins pore architecture: toroidal, conical and hybrid. The structural study of proteins in membrane has serious limitations. Site-directed spin-labeling (SDSL) combined with electron paramagnetic resonance (EPR) spectroscopy has emerged as a powerful technique to elucidate the structure of membrane proteins. The aim of this work is to investigate the topology of StI in membrane by SDSL-EPR. Single sitedirected Cys mutants of StI at positions relevant for the pore-forming activity were produced: N-terminal end (E2C, F15C), oligomerization interface (I59C, I161C) and membrane binding site (R52C, P80C, W111C). Also three double mutants were produced for inter-spin distance determination (F15C/I59C, F15C/P80C and I59C/I161C). All mutants were labeled with methanetiosulfonate spin label and analyzed in terms of secondary structure composition and pore-forming ability. Spin-labeled proteins were studied by EPR in terms of nitroxide mobility, accessibility to paramagnetic reagents, polarity of the microenvironment and distance between two spin-labeled positions. All Cys mutants were spin labeled with high efficiency and without significant modifications in the secondary structure composition according to circular dichroism. Reduction in pore-forming activity was observed for some positions implicated in protein-protein or protein-lipid interactions but all proteins retained their membrane binding capacity. The topology of StI in liposomes composed of POPC:SM (50:50) described deep penetration of position 111 into the lipid acyl-chains bilayer, hiding of positions 52, 59 and 80, and insertion of the N-terminal region. The intra and inter-spin distance determination revealed heterogeneous oligomer distribution for StI in membrane, with a combination of the three pore models proposed. The architecture of StI in liposomes was determined using a new technique, never used before for actinoporins, with similar results obtained with orthogonal techniques.

N-O 016

OMICS ANALYSIS OF MOLECULAR MECHANISMS ASSOCIATED TO DIFFERENT PHENOTYPES OF NS0 MYELOMA CELL LINE Katia R. de la Luz1,2, Rabasa Y. ,de los Santos C., Martínez D., Fernández O., Lao T., Morales O., Elvin M.,Dickson A. 1 Center of Molecular Immunology, Havana, Cuba 2 University of Manchester, UK [email protected] Introduction: The NS0 mouse myeloma cell line has become one of the most popular systems for large-scale heterologous protein expression, especially monoclonal antibodies. For reasons of regulatory compliance, cost, batch consistency, downstream processing, and material availability, industrial applications of NS0 has moved towards serum or protein-free media platforms. NS0 cells are naturally cholesterol-dependent; not only is their growth greatly facilitated by lipid supplementation, but is also dependent on provision of cholesterol. Materials and Methods: A quantitative study of proteins with differential expression levels in different media (host and recombinant NS0) is reported. The study is based on the use of the combination of proteomics and metabolomics technologies (GC- and LCMS/MS). Results: The molecular mechanisms of host and recombinant NS0 cell lines that could be related to the different culture conditions are studied in this work. Several proteins and metabolites with differential expression profile were characterized and quantified. Carbohydrate metabolism, protein synthesis and membrane transport were the principal pathways that change with the media change by proteomic and metabolite quantification analysis. The same results were obtained using bioinformatics tools with a murine metabolic network and selected media conditions. Conclusions: Taking into account the proteomic and metabolic results, a possible mechanism related with the recombinant NS0 myeloma cell line behaviour in different proteinfree media is proposed.

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APROXIMACIÓN PROTEÓMICA DE UNA NUEVA GLICOPROTEINA DE TIPO LECTINA (Lec-1) AISLADA DE SEMILLAS DE QUINUA (ChenopodiumquinoaWilld. variedad kankolla) José Antonio Valeriano-Zapana1,2, Reinoso-Rodríguez D.G., Vera Gonzales C., Segovia-Cruz F., Ponce-Soto L.A. 1 Universidad Nacional de Moquegua –Perú/Instituto de Investigación, Universidad José Carlos Mariátegui, Moquegua, Perú 2 Facultad de Ciencias de la Salud, Universidad José Carlos Mariátegui, Moquegua, Perú

[email protected] Las lectinas constituyen un amplio y heterogéneo grupo de proteínas que poseen la capacidad de conectarse reversiblemente a monosacáridos y oligosacáridos, pudiendo ser definidas como una clase de proteínas o glicoproteínas estructuralmente diversa. El presente trabajo se describe la purificación y caracterización de una nueva lectina aislado de Chenopodium quinoa Willd. variedad kankolla a través de 2 etapas cromatografías: Sephadex G-75 (3.5 x 75 cm) re-purificada en una columna Shim-pack CLC-ODS(M) (4.6mm i.d.x 25cm Shimatzu, preparativa) acoplada a un sistema de HPLC-PDA 991 (Waters), donde se evidenciaron tres picos (1Cq-a, 1Cq-b y 1Cq-c), registrándose en el pico Cq-b la actividad lectina el cual se denominó como Lec-1. El perfil de masa molecular de este pico en 2D-PAGE evidencia una masa molecular alrededor de 33 kDa y un pI=7.8, por espectrometría de masas electrospray (ESI) evidencia una masa de 33135.4 Da, y una secuencia peptídica de 195 aminoacidos: RYEEDSDEFSRPPYPTPSQPPFGAPFPSFGSQSHHEYGGSHHLESPNYPS YGHQSSYNESEQQHHESGGDEHRFRHGYESNEGGSLEGYFGNKKVVLA YSDPNDPTQHWYKDEKFSTKVKDEDGYPSFALVNKSTGQALKKPNVLDESILWTE SRDLGSGFRTIRMVNNVHLVLDAFNGDKKGDNQNQQWKLYPH La lectina (lec-1) mostró una menor actividad de hemaglutinación hacia los eritrocitos de tipo A y O humanos tripsinizados.

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OPTIMIZING PROTEIN PROPERTIES FOR IMPROVED MANUFACTURABILITY: DIRECTED EVOLUTION OF HIGHLY SOLUBLE SUPER-SECRETED INTERLEUKIN-2 VARIANTS Gertrudis Rojas, Carmenate T., Santo-Tomás J.F., Valiente PA., Becker M., Pérez-Riverón A., Tundidor Y., Ortiz Y., Fernandez de Cossio-Diaz J., Graça L., Dübel S., León K. Center of Molecular Immunology, La Habana, Cuba [email protected] Human Interleukin-2 (IL-2) is a pleiotropic cytokine with dual roles within the immune system, promoting either tolerance or effector responses depending on the physiological context. Such a complexity has been translated into a variety of therapeutic approaches to treat either cancer or autoimmune/inflammatory conditions. Despite a long history of therapeutic use, a strong aggregation propensity has limited IL-2 manufacturability and applications up to now. The current work took advantage of the high throughput potential of phage display to screen a large library of IL-2 variants. Panning on immobilized IL-2 receptor rendered novel molecules with recurrent mutations at position 35 (mainly K35E) associated to greatly enhanced display of the functional cytokine on filamentous phages. Moving to a totally different expression system closer to the ones used for manufacturing therapeutic proteins, the introduction of K35E was powerful enough to increase the secretion levels of IL-2-containing fusion proteins from human transfected host cells up to 20-fold. Super-secreted (K35E) IL-2/Fc is biologically active in vitro and in vivo, has anti-tumor activity and exhibits a remarkable reduction in its aggregation propensity. The molecular bases of this effect were explored in silico. Mutated IL-2 was predicted to be more flexible, soluble and stable than the wildtype protein. These properties could facilitate both its transit through the cell secretory pathway and its handling during downstream processing, giving rise to an improved product. Our findings illustrate the power of in vitro directed evolution to discover a rather simple solution (based on a single mutation) to optimize the properties of a typical poor manufacturability protein. This result will improve developability of the growing family of IL-2-derived immunotherapeutic agents. As IL-2 is a prototypic member of the family of four-alpha-helix bundle cytokines, this initial experience could be extended to the engineering of structurally related proteins of immunological interest.

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EFECTO WARBURG UN PUNTO DE PARTIDA PARA LA ACTIVIDAD ANTITUMORAL DE COMPLEJOS DITIOCARBAMICOS DE COBRE Maricela Viola-Rhenals Universidad de Cartagena, Facultad de Ciencias Exactas y Naturales, Grupo de Bioquímica y Biología Celular del Cáncer, Colombia [email protected] A nivel mundial el cáncer causa muchas muertes. Es una enfermedad debida al crecimiento estocástico de células anormales, con muchos efectos secundarios. Uno de las principales teoríaspara la búsqueda específica de antitumorales es el Efecto Warburg. Algunos de los mecanismos bioquímicos asociados son: (a) alterado ambiente redox celular; (b) variabilidad en las enzimas antioxidantes; (c) variación en enzimas NMNAT, (d) alterada expresión de sensores nucleares redox como NF- B, iNOS, HIF-1α entre otros, (e) rol preferencial de la mitocondria. Compuestos ditiocarbámicos son antitumorales, por su baja toxicidad y costo, empleados con metales como cobre, cobalto, zinc, y hierro, su toxicidad está asociada a la formación de complejos tóxicos. Las líneas celulares empleadas son cáncer de mama SKBR3, MDA-MB 231, melanoma C8161 y fibroblastos normales. Se adicionan tratamientos, se calcula el IC50 y a partir de esa concentración se elucida el mecanismo de acción, mediante western Blot, fluorescencia, ensayos clonogénicos y electroforesis en condiciones nativas para evidenciar actividad enzimática, así como pretratamientos antioxidantes. El IC50 para todas las líneas celulares está en el rango nanomolar, con pérdida de capacidad clonogénica y expresión diferencial de enzimas antioxidantes (mitocondrial MnSOD);incremento de iNOS y HIF-1α, inhibición de NF- B, patrón de fragmentación de ADN apoptótico, sin efectos sobre fibroblastos normales. El efecto es preferencial para cobre y no para otros metales. Los resultados sugieren la implicación de la mitocondria como foco del mecanismo de acción relacionado con la toxicidad mediada por complejos ditiocarbámicos de cobre, lo cual está de acuerdo con el rol de esta organela celular en el efecto Warburg. El efecto Warburg como una serie de eventos bioquímicos relacionados con el Cancer permite explicar la especificidad de ciertos eventos celulares y es un buen punto de partida para la búsqueda de nuevos fármacos antitumorales altamente eficaces.

N-C 004

PSEUDO-CATALYTIC NERVE AGENTS SCAVENGING ACETYLCHOLINESTERASE ASSISTED WITH ALDOXIMES Zrinka Kovarik Institute for Medical Research and Occupational Health, Zagreb, Croatia

BY

[email protected] Poisoning caused by organophosphates (OP) known as nerve agents calls for immediate treatment, which usually consists of a combined administration of an anticholinergic drug and an oxime as the reactivator of the enzyme acetylcholinesterase (AChE). Newly considered strategies in medical protection against nerve agents focus on the use of exogenously administered butyrylcholinesterase (BChE). The overall idea is to administer such an enzyme in combination with a specific oxime, to scavenge an organophosphate before it can reach and inhibit native AChE, thus helping organism detoxification from the excess OP. However, oxime antidotes commonly used to reactivate OP inhibited AChE are ineffective against soman and tabun, while the efficacy of the recommended nerve agent bioscavenger BChE is limited by strictly stoichiometric scavenging. Herein we demonstrated a feasible approach to the development of an oxime assisted catalytic bioscavenger of soman, tabun and VX based on human AChE mutants modified at the choline binding site (Y337A and an aging resistant Y337A/F338A) in combination with its efficient reactivator. Ultimately, the oxime assisted catalytic scavenging of the nerve agents in mice improved therapeutic outcomes preventing lethality and resulted in a delayed onset of toxicity symptoms. Acknowledgments: This work was supported by the Croatian Science Foundation (4307).

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DISCOVERY OF NOVEL NON-COMPETITIVE INHIBITORS OF MAMMALIAN NEUTRAL M1 AMINOPEPTIDASE (APN)

Isel Pascual, Valiente P.A., García G., Valdés-Tresanco M.E., Arrebola Y., Díaz L., Bounaadja L., Uribe R.M., Chappé-Pacheco M., Florent I., Sanchez B., Charli J.L. Center for Protein Studies, Faculty of Biology, University of Havana, Cuba [email protected] Neutral metallo-aminopeptidase (APN) catalyzes the cleavage of neutral and basic amino acids from the N-terminus of protein or peptide substrates. APN expression is dysregulated in inflammatory diseases as well as in several types of cancer. Therefore, inhibitors of APN may be effective against cancer and inflammation. By virtual screening and enzymatic assays, we identified three non-competitive inhibitors (α>1) of the porcine and human APN with Ki values in the µM range. These non-peptidic compounds lack the classical zinc-binding groups (ZBG) present in most of the APN inhibitors. Molecular docking simulations suggested the novel inhibitors suppress APN activity by an alternative mechanism to Zn coordination; they interacted with residues comprising the S1 and S5’ subsites of APN. Of note, these compounds also inhibited the porcine aminopeptidase A (pAPA) using a competitive inhibition mode. These results indicated differences in the binding mode of the compounds with APN and APA. Based on sequence and structural analyses, we predicted the significance of targeting human APN residues: Ala-351, Arg-442, Ala-474, Phe-896 and Asn-900 for improving the selectivity of the identified compounds. Remarkably, the intraperitoneal injection of compounds BTB07018 and JFD00064 inhibited APN activity in rat brain, liver and kidney indicating good bio-distribution of these inhibitors in vivo. These molecules had an anti-proliferative effect on tumor cell lines displaying APN activity at the cell surface. These data reinforce the idea of designing novel APN inhibitors based on lead compounds without ZBG. Acknowledgment: IFS grant 3276/3, Laboratorio UH-CIM

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SABELLASTARTE MAGNIFICA CARBOXYPEPTIDASE INHIBITOR: THE FIRST KUNITZ INHIBITOR SIMULTANEOUSLY INTERACTING WITH CARBOXYPEPTIDASES AND SERINE PROTEASES Reytor Gonzalez M.L., Maday Alonso-del-Rivero, Hedstrom L., Kuzmic P., Pires J.R. Center for Protein Studies, Faculty of Biology, University of Havana, Cuba [email protected] Multi-domain inhibitors capable to block the activity of different classes of proteases are not very common in nature. However, these kinds of molecules are attractive systems for biomedical or biotechnological applications, where two or more different targets need to be neutralized. SmCI, the Sabellastarte magnifica Carboxypeptidase Inhibitor, is a tri-domain BPTI-Kunitz inhibitor capable to inhibit serine proteases and A-like metallocarboxypeptidases. The BPTI-Kunitz family of proteins includes voltage gated channel blockers and inhibitors of serine proteases. SmCI is therefore, the only BPTI-Kunitz protein capable of inhibiting metallocarboxypeptidases. The X-ray structure of the SmCI carboxypeptidase A complex previously obtained by us, revealed that this enzyme interacts with SmCI N-tail. In the complex, the reactive loops for serine protease inhibition remain fully exposed to the solvent in each domain, suggesting SmCI can simultaneously interact with multiple serine proteases. The twofold goals of this study were: i) to establish serine proteases-SmCI binding stoichiometry, given that the inhibitor is comprised of three potential binding domains; and ii) to determine whether or not SmCI can simultaneously bind both classes of enzymes, to which it binds individually. Our experimental approach included a variety of techniques for the study of protein-protein interactions, using as model enzymes pancreatic trypsin, elastase and carboxypeptidase A. In particular, we combined information obtained from gel filtration chromatography, denaturing electrophoresis, nuclear magnetic resonance spectroscopy and enzyme inhibition assays. Our results show that SmCI is able to bind three trypsin molecules under saturating conditions, but only one elastase interacts with the inhibitor. Additionally, we demonstrated that SmCI can bind serine proteases and carboxypeptidases at the same time (at least in the ratio 1:1:1), becoming the first protease inhibitor that simultaneously blocks these two mechanistic classes of enzymes Acknowledgment: Authors thank Rafael Sánchez for helping during the rSmCI production and purification at the Center for Protein Studies. UNU-BIOLAC organization and IUBMB [Wood Whelan Research Fellowships and Mid Career Research Fellowship] to Mey Ling Reytor. This work was also supported by IFS [grant F/5109-1] and the bilateral funding program CAPES-Brazil/MES-Cuba (143/11).

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EL CANAL IONICO CATSPER SE REGULA POR FOSFORILACION MEDIADA POR PKA Carmén Beltrán, Singla, T., Rodríguez-Miranda E., Darszon, A. Instituto de Biotecnología-Universidad Nacional Autónoma de México (UNAM), México [email protected] CatSper, el canal catiónico de Ca2+ específico del espermatozoide, activable por pH intracelular (pHi) alcalino y despolarización de la membrana,es el responsable de los aumentos de Ca2+citosólicos indispensables para las funciones necesarias de la fecundación tanto en mamífero como en erizo de mar. Por lo anterior CatSper es un blanco excelente para anticonceptivos no hormonales para hombres y mujeres. En espermatozoides de erizo de mar (eem), la unión del péptidoregulador de la movilidad,speract (del óvulo),a su receptor en el flagelo, dispara una vía de señalización que involucra cambios rápidos en el potencial membranaly aumentos en elpHi que activan a CatSper induciendo entrada de Ca2+ y estimulan a la adenilil ciclasa soluble, aumentando el AMPc. Se ha propuesto que dicho influjo de Ca2+es clave para laquimiotaxis del eem. La movilidad de esta célula se regula también por AMPc, PKA, Ca2+ y por fosforilación mediada por PKA y PKC. En este trabajo exploramos si CatSper se regula por fosforilaciónmediada por PKA. Usando el indicador fluorescente de Ca2+, Fluo-4, evaluamos los aumentos de Ca2+ intracelular eneemStrongylocentrotuspurpuratus y Lytochinuspictusinducidos porsperact yNH4Cl, así comoagonistas de AMPc (db-AMPc y 8BrAMPc),isobutilmetilxantina (IBMX, inhibidor de fosfodiesterasas) y los efectos de bloqueadores de Catsper (NNC-0396 y Mibefradil) e inhibidores (H89 y PKI) de la PKA. Nuestros resultados sugieren que CatSper se modula al menos parcialmente por fosforilación mediada por PKA.Sin embargo, dado que la mayoría de los transportadores involucrados en la vía de señalización que dispara el speract tienen sitios probables de fosforilaciónpor PKA y PKC, la respuesta final a si CatSper se regula por fosforilación y cuáles cinasas involucra, se obtendrá registrando la corriente de CatSper directamente en la célula mediante experimentos de ¨patch clamp¨. *Este proyecto fue financiado por PAPIIT, DGAPA-UNAM IN206016 otorgado a CB e IN205516, a A.D, y por CONACyT Fronteras de la Ciencia 71 a A.D.

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PRODUCTION, PURIFICATION AND CHARACTERIZATION OF L-ASPARAGINASE PRODUCED BY STREPTOMYCES ISOLATED FROM THE ARAUCA RIVERBANK (COLOMBIA) Luis E. Díaz, Arévalo-Tristancho E., Cortázar-Gómez J. Facultad de Ingeniería, Universidad de La Sabana, Chía, Colombia [email protected] L-asparaginase, is known as an anti-cancer agent, mainly high performed on acute lymphoblastic leukemia (ALL) which prevents the proliferation of tumor cells by decreasing the level of asparagine in the blood. A morphological and molecular identification of Actinobacteria isolated from the Arauca riverbank (Colombia) for seven L-asparaginase producers strainswere made with 16S rRNA gene sequence. The effect of carbon and nitrogen sources, pH, temperature and agitation rate was determinated using Plackett-Burman design and Taguchi methodology, showing that lactose, malt extract/asparagine, at 32°C and 130 rpm for Streptomyces lacticiproducens were the best conditions to optimized activity. L-asparaginase was purified from the lyophilized broth, dialyzed and ion exchange chromatography using DEAE Sepharose. The kinetic studies of the purified enzyme were carried out, and the optimal pH and temperature for maximum L-asparaginase activity were found to be 6 and, 37 °C, respectively. The molecular weight of Lasparaginase was found to be approximately 37 kDa by SDS–PAGE and western blot analysis. The purified L-asparaginase showed a final specific activity of 113.484 U/mg. The cytotoxic potential of the purified enzyme was evaluated in THP-1 cell line (acute monocytic leukemia) resulting an IC50 of 36.74 µg/mL. S. lactici producens a new source of L-asparaginase with high activity that increases its availability as a drug and it is expected to reduce the side effects.

VIERNES/FRIDAY OCT. 12th

SESSION: Microbiology N 001

EXTREMOPHILES AND THEIR POTENTIAL FOR BIOREMEDIATION AND THE PRODUCTION OF BIOPLASTICS Michael Seeger, Méndez V., Villegas P., Orellana R., Barra B., Vega-Celedón P., Durán R., Hernández L., Álvarez-Santullano N., González A., Macaya C., González M., Rojas M., Carballo M.E. Departamento de Química & Centro de Biotecnología, Universidad Técnica Federico Santa María, Valparaíso, Chile [email protected] Extremophiles are organisms capable of survive or thrive in hostile habitats and are of increasing interest for biotechnology. Next generation sequencing techniques have allowed to sequence a high number of microbial genomes. Based on sequenced genomes, functional genomics may reveal novel metabolic pathways and potential biotechnological applications (1). The aims of this study are the isolation and characterization of environmental bacteria and their application in bioremediation and the production of bioplastics. Bacteriawere isolated in diverse zones in Chile that possess extreme conditions such as Altiplano, Central Chile, Andes Mountains, Patagonia, and La Habana region in Cuba.The isolates were screened for their capabilities to degrade organic pollutants and synthesize bioplastics. The genomes of selected environmental bacteria were sequenced and analyzed for metabolic reconstruction. Paraburkholderia xenovorans LB400 was used as model bacterium for the study of aromatic catabolic routes and the synthesis of polyhydroxyalkanoates (PHAs).Diverseisolates possess genes that encode metabolic routes for the degradation of organic pollutants and synthesis of PHAs. The functionality of key metabolic pathways were studied through biotransformation assays, metabolites identification, gene expression, protein pattern, enzymatic activities and heterologous expression of specific genes.Most promising strains were further studied. For example, Acinetobacter sp. DD78 and Alcaligenes aquatilis QD168that degrade a wide range of petroleum hydrocarbons.For PHA synthesis, Janthinobacterium sp. BmR6b and Pseudomonas sp. LC43that convert sugars into polyhydroxybutyrate and medium chain length PHAs were characterized. The molecular and physiological properties of diverse environmental bacteria isolated from extreme environments and their applications in bioremediation and bioplastic production will be discussed. Acknowledgements to RIABIN network (Programa Pablo Neruda, OEI), Conicyt fellowships (BB,PVC,RD,CM), Programa de Investigación Asociativa Anillo ACT172128 GAMBIO, FONDECYT 1151174 and USM grants. Seeger M., Padrón G. 2013. Genómica Funcional: Fundamentos y Aplicaciones, Editorial Universidad Técnica Federico Santa María, V

N-C 005

EXPLORING RESEARCH IN AQUATIC ECOSYSTEM: HISTORICAL POLLUTION AND DISSEMINATION OF MICROPOLLUTANTS, PATHOGENS AND ANTIBIOTIC RESISTANCE ACCORDING TO THE COUNTRY’S DEGREE OF DEVELOPMENT AND DIFFERENT CLIMATE CONDITIONS Devarajan N., Laffite A., Al Salah D. M., Sivalingam P., John Poté University of Geneva, Faculty of Sciences, Section of Earth and Environmental Science, Department F.-A. Forel for Environmental and Aquatic Sciences and Institute of Environmental Sciences, Geneva, Switzerland [email protected] Globally, contaminants such as antibiotics, antibiotic resistant bacteria (ARB),antibiotic resistance genes (ARGs), metals and persistence organic pollutants are of major environmental and human concern. In particular, the threat of antimicrobial resistance is rising at an alarming rate and the situation is aggravated in developing nations, where poor water quality and lack of sanitation infrastructure continues to pose serious threat to human health. The aquatic environment is a hot-spot for gene transfer and the sediments offer the opportunity for reconstructing the pollution history and evaluating the impacts using both quantitative and temporal data. In this context, water, surface and sediment core samples from three distinct geographical locations; Democratic Republic of the Congo, India and Switzerland were characterized to understand the time variation of these contaminants in river and lake ecosystems receiving untreated/partial treated urban and hospital effluents. Our results revealed that the presence of a high concentration of heavy metals, diverse Extended-Spectrum Beta-Lactamase and Carbapenemase-Producing Enterobacteriaceae in rivers and lakes depends to the degree of anthropogenic activities, sociocultural aspects and economic status of a country as well as climatic conditions. Remarkably, conjugative transfers of mobile genetic elements of antibiotic resistance plasmids in P. aeruginosa isolates from three geographic locations occurred more frequently under tropical temperatures (30 and 37 °C) than under temperate conditions (10 °C). Additionally, high level of genetic clonality was observed in E. coli strains carrying multiple resistance genes (ß-lactamases and carbapenamases) and virulence genes under tropical conditions. Although the epidemiology of antibiotic resistance can be geographically localised, the effects of cross border transmission can be global. Thus, our researches recommend strongly for the advancement in the treatment technologies for the wastewater to disinfect the bacterial load/ARB/ARGs in the effluents before being discharged to the receiving system and for the key measures to combat this escalating burden globally.

N-O 024

MICROORGANISMOS CON APLICACIÓN BIOTECNOLÓGICA EN LA BIOSORCIÓN DE METALES PESADOS: INTERACCIÓN BACTERIAS – CROMO Irina Salgado-Bernal, Larrea C., Pérez J.E., del Monte A., Castillo F., Martínez A., Pérez L., Liva M., Collazo O., Alleyne S., Cruz M., Carballo M.E. Facultad de Biología, Universidad de La Habana, Cuba [email protected] La concentración de cromo (Cr) en los ecosistemas se ha incrementado en las últimas décadas. Este elemento presenta dos formas de oxidación más estables en el ambiente, Cr (III) y Cr (VI), esta última es la más tóxica. La acumulada toxicidad y el efecto perjudicial que traen consigo los altos niveles de Cr (VI) hacen necesaria la búsqueda de alternativas sostenibles de remediación. Varias investigaciones se han enfocado en el uso de biomasas disponibles que puedan capturar los metales pesados, utilizando las características estructurales y metabólicas de los organismos vivos. Entre los retos relacionados con la biorremediación del cromo empleando bacterias se encuentra la profundización en los mecanismos de interacción biomasa metal; este trabajo está enfocado en esta temática. Para ello se seleccionaron 21 cepas, aisladas de un ecosistema acuático contaminado, resistentes al Cr (VI). La mayoría de ellas removieron el cromo. El comportamiento de las isotermas de biosorción demostró que la remoción del metal se incrementa con el aumento de la concentración inicial en la solución y que estas cepas pueden remover el metal a partir de soluciones que contengan hasta 1,6 mM, con niveles por encima de 60 mg.g-1; siendo la cepa TAN-125 la de mayores potencialidades. La coincidencia de los datos experimentales con el modelo matemático de Freundlich, así como la distribución heterogénea del cromo removido, demostró la implicación de la participación de una variedad de mecanismos para la remoción del Cr (VI). La obtención de derivados inmovilizados permitió contar con productos más activos en la remoción, en comparación con las bacterias libres, probablemente mediante un mecanismo predominante de biotransformación. Estos resultados tienen contribución en los estudios relacionados con los mecanismos de interacción microorganismos - cromo, como parte de las premisas para el empleo de estos agentes biológicos en el tratamiento de aguas contaminadas.

N-O 025

BIOLIXIVIACIÓN DE Ni Y Co A PARTIR DE COLAS Y ESCOMBROS LATERÍTICOS EMPLEANDO UNA BACTERIA ACIDÓFILA Alexander Govín-Sanjudo, Leal G., Sánchez M.I., Ramírez M.C., Pons J.A. Facultad de Biología, Universidad de La Habana, Cuba [email protected] La industria minera y metalúrgica de Moa genera grandes volúmenes de residuales sólidos que contienen concentraciones de metales pesados como Ni y Co. Estos una vez depositados en el ambiente afectan los ecosistemas y contribuyen a la pérdida de la biodiversidad. Los métodos convencionales no son eficientes para tratar estos residuales, por lo que los métodos biotecnológicos han ganado gran interés en este campo, debido a que constituye una alternativa eficiente y económica. Teniendo en cuenta esto el siguiente trabajo tuvo como objetivo: evaluar la solubilización de níquel y cobalto a partir de escombros y cola laterítica. Se empleó la cepa Acidithiobacillus thiooxidans DSM 14887 y tres minerales procedentes del yacimiento laterítico de Moa: dos escombros lateríticos (escombro de Camarioca norte Zona 12 y escombro de Ferroníquel) y la cola (cola de la presa vieja de la planta Pedro Soto Alba). Se determinó la influencia de la densidad de pulpa (5%, 10%, 20% p/v) en la solubilización de Ni y Co de los residuales en estudio, empleando un sistema discontinuo, además se evaluó la solubilización de Ni y Co empleando el bioácido producido por la cepa A. thiooxidans DSM 14887. El mejor porcentaje de recobrado de los metales se logró con 5% (p/v) de densidad de pulpa de los tres minerales (más del 40% p/v de Ni y más del 52% p/v de Co). Los recobrados de Ni empleando el bioácido producido por la cepa At. thiooxidans disminuyeron con respecto a la biolixiviación en presencia de la cepa. Mientras los porcentajes de extracción de Co empleando el bioácido fueron muy similares a los obtenidos en presencia. El empleo de la cepa At. thiooxidans DSM 14887 en la recuperación de metales constituye una alternativa interesante para la recuperación de metales de interés a partir de los minerales oxidados de baja ley.

N-O 026

MICROALGAS COMO BIOREMEDIADORAS DE METALES POTENCIALMENTE ANTICANCERIGENAS

Maricela Viola-Rhenals, Blanco S., Morelos L., Silgado J. Universidad de Cartagena, Facultad de Ciencias Exactas y Naturales, Grupo de Bioquímica y Biología Celular del Cáncer, Cartgena, Colombia [email protected] El cáncer una enfermedad con crecimiento descontrolado de células es una de las primeras causas de muerte en el mundo. La genética y el medio ambiente (epigenética) son importantes en la carcinogénesis. Los metales administrados junto con otros fármacos son importantes anticancerígenos, sin embargo, también son contaminantes que coadyuvan en el proceso carcinogénico, por lo que es importante mantener niveles notóxicos de metales, a fin de disminuir su incidencia.En este estudio, empleando como modelo microalgas se evaluó el efecto bioremediador de las cepas Nannochloropsis y Chlorella, expuestas a diferentes concentraciones de metales (cobre, zinc, cromo, hierro) cuantificando la viabilidad celular diariamente fluorimétricamente con Resazurim. La cepa promisoria para ser bioremediadora esChlorella que mostro inducción del crecimiento en agua suplementada todos los metales observándose toxicidad solo cuando era expuesta a hierro (35%), mientras que Nannochloropsis fue una cepa sensible observándose inhibición del crecimiento con hierro y dicromato, mucho más en presencia de cobre. Ambas cepas mantuvieron viabilidad celular cuando fueron expuestas a cuerpos de agua contaminada de la bahía (125% para NannochloropsisSpy 156% para Chlorella Vulgaris ). La viabilidad celular diminuída en NannochloropsisSp, puede deberse a la concentración de los metales y su proceso de absorción metabólica.Todo esto indica que la capacidadde biorremediación para estas cepas es indpendiente del metal. En el caso de la cepa de Chlorella Vulgaris los metales parecen ser ademas constiutyentes del médio de crecimiento lo que la hace uma buena microalga captadora de metales en ambientes tóxicos. Estos hallazgos sugieren que las microalgas pueden ser empleadas como bioremediadoras en cuerpos de agua contaminada con metales, tal vez debido a la cantidad de clorofila a, b y c que poseen y por ende ser captadoras de metales inductores de cáncer.

N-C 006

LECTIN-FUNCTIONALIZED COMPOSITE HYDROGELS FOR "CAPTURE-ANDKILLING" OF CARBAPENEM-RESISTANT Pseudomonas aeruginosa.

Bodenberger N., Kubiczek D., Wiese S., Ständker L., Stenger S., Francois Rosenau 1,2 1 Center for Peptide Pharmaceuticals, Facultyof Natural Science, Ulm University, Germany. 2 Synthesis of Macromolecules Department, Max-Planck-Institute for Polymer Research, Germany. [email protected] Infections with multiresistant pathogens are a leading cause for mortality worldwide. Just recently, the World Health Organization (WHO) increased the threatrating for multiresistant Pseudomonas aeruginosa to the highest possible level. With this background, it is crucial to develop novel material sand procedures in the fight against multiresistant pathogens. In thisstudy, we present a novel antimicrobial material, which could find applications as a wound dressingor antimicrobial coating. Lectins are multivalent sugar-binding proteins, which can be found in a variety of plants and bacteria, where they are associated with biofilm formation. By immobilizing lectin B on a proteinbased hydrogel surface, we provided the hydrogel with theability to immobilize ("catch") pathogens upon contact. Furthermore, another hydrogellayer was added which inhibits biofilm formation and releases a highly potent antimicrobial peptide to eradicate microorganisms ("kill"). The composite hydrogel showed a high antimicrobial activity against the reference strain Pseudomonas aeruginosa PAO1 as well as against a carbapenemresistant clinical isolate (multiresistant Gram-negative class 4) and may thus represent a novel material to develop a new type of antimicrobial wound dressings to prevent infections with this problematic pathogen of burn or other large wounds.

N-O 027

ROLE OF LONG-CHAIN FLAVODOXINS OF PARABURKHOLDERIA XENOVORANS LB400 DURING EXPOSURE TO OXIDIZING AGENTS Laura Rodríguez-Castro, Durán R., Méndez V., Seeger M. Laboratorio de Microbiología Molecular y Biotecnología Ambiental, Departamento de Química, Universidad Técnica Federico Santa María, Valparaíso, Chile [email protected] Paraburkholderia xenovorans LB400 is a model bacterium for the degradation of polychlorinated biphenyls and diverse aromatic compounds. The aerobic metabolism of aromatic compounds produces reactive oxygen species and therefore oxidative stress. Flavodoxins are small electronic transfer proteins expressed in conditions of oxidative stress for cell redox balance. The aims of this study were to identify and evaluate the protective effect of LB400 longchain flavodoxins during oxidative stress in Paraburkholderia xenovorans LB400. Bioinformatic tools were used to identify genes encoding flavodoxins in the genome of Paraburkholderia xenovoransLB400. Two genes encoding flavodoxins were identified at the major chromosome (FldA) and the minor chromosome (FldB). These genes were cloned in E. coli and transferredby biparental matingto Paraburkholderia xenovoransLB400 and the recombinant strains were checked by PCR. Survival assay in presence of 1 mM hydrogen peroxide (H2O2) and 1 mM paraquat were performed with the recombinant Paraburkholderia xenovorans strains. The protein carbonyl content was determined after exposure to these compounds for 1 h. To study the lipid peroxidation in recombinant strains, the thiobarbituric acid reactive substances (TBARS) were determined after exposure to 1 mM H2O2 and 1 mM paraquat. Recombinant strains overexpressing FldA and FldB showed a higher survival during exposure to paraquat compared to the control strain. Paraburkholderia xenovorans overexpressing FldA displayed less protein carbonylation in the presence of H2O2, while the strainoverexpressing FldB showed a lower carbonyl content during incubation with paraquat.Compared to control cells, the strains overexpressing both flavodoxins displayed a lower TBARS content after incubation with H2O2.In conclusion, the flavodoxins FldA and FldB of Paraburkholderia xenovorans LB400 confers protection during oxidative stress conditions. Acknowledgements to RIABIN network (Programa Pablo Neruda, OEI), Conicyt PhD fellowship (LR), Programa de Investigación Asociativa (PIA) Anillo ACT172128 GAMBIO, FONDECYT 1151174 and USM grants.

N-O 028

DIVERSITY AND ABUNDANCE OF BEAUVERIA BASSIANA IN SOILS, STINK BUGS AND PLANT TISSUES OF COMMON BEAN FROM ORGANIC AND CONVENTIONAL FIELDS Yordanys Ramos, Portal O., Lysøe E., Meyling N., Klingen I. Departamento de Agronomía, Universidad Central “Marta Abreu” de Las Villas, Santa Clara, Cuba [email protected] The aim of this study was to evaluate the natural occurrence of Beauveria spp. in soil, from infections in the stink bug Piezodorus guildinii, an important pest of common bean (Phaseolus vulgaris L.) and as endophytes in bean plant tissue. Twelve conventional and 12 organic common bean fields in the Villa Clara province, Cuba were sampled from September 2014 to April 2015. One hundred and fifty Beauveria isolates were obtained from soil samples, bean plant parts and stink bugs. The overall frequency of occurrence of Beauveria isolates in conventional fields (8.4%) was significantly lower than that in organic fields (23.6%). Beauveria were also obtained significantly more frequently from bean roots in organic fields (15.0%) compared to bean roots in conventional fields (3.3%). DNA sequencing of the intergenic Bloc region was performed for Beauveria species identification. All isolates where characterized as Beauveria bassiana (Balsamo-Crivelli) Vuillemin, and clustered with isolates of neotropical origin previously described as AFNEO_1. The Cuban B. bassiana isolates formed five clusters in the phylogeny. Isolates of two clusters originated from all four locations, organic and conventional fields, as well as soil, plants and stink bugs. Organic fields contained isolates of all five clusters while conventional fields only harbored isolates of the two most frequent ones. Mating type PCR assays revealed that mating type distribution was skewed, with MAT1/MAT2 proportion of 146/4, indicating limited potential for recombination. The present shows that B. bassiana occurs naturally in diverse environments of common bean fields, and constitutes a potential reservoir of natural enemies against pest insects particularly in organic fields.

N-O 029

MOLECULAR CHARACTERIZATION OF A POTATO VIRUS Y (PVY) ISOLATE INFECTING TOMATO IN ARTEMISA PROVINCE Acela Díaz de la Osa, Hernández Rodríguez A., Pons Ascaso F. Faculty of Biology, University of Havana, Cuba [email protected] Potyvirus genus is one of the most studied inside the plant virus group. It is worldwide distributed and affects different host. Potato Virus Y(PVY) is a pathogen of economic importance in tomato and other important crops species in the family Solanaceae. PVY is classified into three major strain groups: O, C, and N, on the basis of genome sequencing. The aims of this work was to report the first full-genome sequence of a PVY isolate (To31) infecting tomato from Artemisa province in Cuba. The complete genome sequence of To31 was assembled from overlapping RT-PCR products using MEGA 8 software. Two ORFs were identified at position 190 and 2921 of the sequence encoding the viral polyprotein and the frameshift translated protein P3NPIPO, respectively. RDP4 software confirmed one recombination breakpoints at position 9148 of the sequence, including the 3´end of Nib and CP protein. This isolate shared a high sequence similarity with the PVYO isolate EF026074 and it´s the first report of a complete genome sequence of a PVY isolate in Cuba.

N-O 030

MICOBIOTA Y DETERMINACIÓN DE FUMONISINAS EN PRODUCTOS ELABORADOS CON AVENA

Samuel R. Alvarez-López, Moreno-Lara J., Quezada-Viay M., Montiel-Sosa J.F. Universidad Nacional Autónoma de México, FES-Cuautitlán, Unidad de Investigación en Granos y Semillas, Cuautitlán Izcalli México, México [email protected] Las fumonisinas son micotoxinas producidas por el hongo Fusarium verticillioides y Fusarium proliferatum que pueden desarrollarse durante el cultivo de avena (Avena sativa), que es un grano altamente consumido por humanos y animales. El objetivo del presente trabajo fue determinar la micobiota y las fumonisinas presentes en diferentes productos elaborados con avena. Se analizaron 15 distintos productos de consumo humano que reportaron como ingrediente de elaboración la avena. Se determinó su calidad sanitaria evaluando la micobiota endógena. Se empleó como medio de cultivo Agar papa dextrosa para detectar la presencia de hongos de campo. El análisis de fumonisinas se realizó por la técnica preestablecida Vicamfumonitest ®, utilizando columnas de inmunoafinidad llevado a una lectura de fluorescencia identificando su concentración. Las pruebas se realizaron por triplicado en cada uno de los productos, empleando un análisis de varianza de una vía para su comparación. Los hongos Fusarium, Eurotium, Aspergillus flavus, Aspergillus sección nigri, Penicillium y Mucor estuvieron presentes en las muestras analizadas, donde la avena en hojuelas, presentó el mayor número de aislamientos para Fusarium (6 UFC/g). La muestra de Avena Instantánea con nueces, pasas y dátiles, presentó el mayor número de aislamientos con el género Aspergillus sección nigri (14 UFC/g). En contraste, las muestras con menos contaminación fueron de Avena Natural en Hojuela; que presentó el género Rizhopus (1 UFC/g) y la muestra de Cereales de Avena fortificado sabor Fresas con Crema con el género Penicillium (1 UFC/g). Además en todos los productos se presentaron levaduras. Las fumonisinas se presentaron en todos los productos, pero las muestras con mayor concentración de la micotoxina fueron Avena Instantánea Integral con Arándanos (3033 µg/mL) y Cereal de Avena Fortificado Sabor Fresas con Crema (3727 µg/mL) que conforme a la regulación vigente de la Unión Europea, exceden el límite que establece para Cereales de desayuno a base de maíz y aperitivos de maíz, que es de 800 µg/mL. Todas las muestras analizadas presentaron contaminación con hongos de diferentes géneros y fumonisinas lo que representa un riesgo para la salud en humanos y animales. En países del continente americano no hay regulaciones para el control de fumonisinas ni para muchas otras toxinas presentes, por lo que se sugiere hacer hincapié en este estudio de investigación para que se propongan normativas, se establezcan límites y principalmente prevenir su desarrollo.

N-O 031

DESIGN AND VALIDATION OF PCR ASSAYS FOR RAPID DETECTION OF SHIGA TOXIN-PRODUCING E. coli IN ENVIRONMENTAL SAMPLES Ahumada-Cora R1., Esquivel-Hernandez Y1., Gerardo M. Nava1 1Universidad Autonoma de Queretaro, Queretaro, Mexico. [email protected] Shiga toxin-producing E. coli (STEC) is a well-known foodborne pathogen capable of colonizing the gastrointestinal tract of food-producing animals and producing serious infection in humans. Two types of Shiga toxin (Stx), Stx1 and Stx2; are the main virulence factors linked to the development of hemorrhagic colitis and hemolytic–uremic syndrome in humans. Thus, stx genes are an important molecular marker for detection of STEC in food and environmental samples. However, identification of this marker has been challenging because Stx1 and Stx2 are encoded by 10 different genes (stx1a, stx1c, stx1d, stx2a, stx2b, stx2c, stx2d, stx2e, stx2f and stx2g). In the present study, bioinformatics analyses were applied to identify PCR primers capable of detecting the broad diversity of stx alleles. Then, these set of primers were used to design and validate PCR assays for detection of stx1 and stx2 STEC in fecal samples from swine, beef and dairy farms. The studies revealed that two pairs of primers were able to identify 100% and 94.9% of the stx gene known alleles archived in the GenBank. Additionally, the PCR assay designed with these primers detected the presence of stx1 and stx2 in 81.17% of the swine, beef and dairy farms evaluated. In conclusion, the new PCR assay represents a rapid and lowcost tool for the detection of STEC in environmental samples.

V Symposium of Biochemistry and Molecular Biology

MARTES/TUESDAY OCT. 9th

N-P 001 EFFECT OF BRASSINOSTEROIDS TREATMENT IN GROWTH PROMOTION OF STRAWBERRY PLANTS Furio R.N., Coll Y., Hael Conrad V., Martínez Zamora G.M., Sergio Miguel Salazar S.M.3,4, Díaz Ricci J.C. 3 Instituto Nacional de Tecnología Agropecuaria. EEA Famaillá. Argentina 4 Facultad de Agronomía y Zootecnia. UNT. Argentina [email protected] Brassinosteroids (BRs) are steroidal compounds involved in plant growth and development. BRs are considered a class of hormone with great potential to boost crop yield andit was shown that the applications of BRs enhance plant tolerance toward different pathogens. The aim of this work was to evaluate the effect of the treatment with 24-epibrasinolide (EP24) and a brassinosteroidspirostanic analogue DI-31 (BB16) in the growth promotion of strawberry plants (Fragariaananassa).The parameters analyzed were number of leaves, leaf area, number of stolons, verdure index, root length and dry weight.For the determinations of the green index, the relative chlorophyll content was measured by using a Minolta SPAD-502 chlorophyll-meter. To determine the foliar area, photographs of all the leaflets were taken and the measurement was carried out using the ImageJ program. The plants treated with BB16 and EP24 showed a significant increase in the greenness index, number of leaves and number of stolons, with respect to the control plants. Another important observation was the increase in leaf area in response to these treatments. When analyzing the biomass, a higher root dry weight was observed in the plants treated with BB16, while the plants treated with EP24 did not show significant differences with the control plants. When evaluating the dry weight of the aerial part of the plants, it was possible to observe an increase in the plants treated with BB16 and EP24, but this increase was not significant. In this way, we can think that the increase in the total dry weight observed in response to treatment with BB16 can be attributed mainly to the increase observed in the dry weight of the root. These results suggest that BRs could be used as a new crop management strategy, achieving to increase the quality of the crops and their yields.

N-P 002 EFECTO DEL ANÁLOGO DE BRASINOESTERIODE DI-31 SOBRE LA ARQUITECTURA DE LA RAÍZ EN CULTIVARES DE INTERÉS COMERCIAL INCA LP-5 Y PERLA DE CUBA DE Oryza sativa L. Alena Vazquez-Glaría, Duvergel A., Coll Y., Ortega-Rodés P., Naidoo S., Loiret FG. Lab. de Fisiología Vegetal, Fac. Biología, Universidad de La Habana, Cuba [email protected] El sistema radical de las plantas es esencial para su crecimiento y tiene numerosas funciones como la toma de agua y nutrientes del sustrato. Se ha demostrado que en el desarrollo del mismo influyen diferentes hormonas como son los brasinoesteroides; estos pueden potenciar el alargamiento de la raíz y la formación de raíces laterales. El objetivo de este trabajo es la evaluación de un análogo de brasinoesteroide DI-31 en la arquitectura de la raíz de arroz (Oryza sativa L.) cv. Inca Lp-5 y Perla de Cuba. Las semillas de arroz fueron germinadas en rolos de papel de germinación durante 7 días. Dichas semillas fueron tratadas previamente con tres concentraciones de DI-31 (1 ppm, 10-1ppm y 10-2ppm) durante 30 min. Transcurrido este tiempo las plántulas fueron fotografiadas y se procedió a medir la longitud de la raíz principal, número de raíces y densidad de raíces laterales mediante el programa SmartRoot de Image J. Se observaron diferencias en todas las variables analizadas para los tratamientos y el control, siendo la concentración de 1 ppm la que mayores valores presenta en cuanto a longitud de la raíz y número de raíces laterales. La densidad de raíces laterales fue influenciada por la presencia de brasisnoesteriodes en comparación con el control no tratado, sin embargo pero no se encontraron diferencias entre las concentraciones probadas.

N-P 003 ACTIVATION OF GENES RELATED WITH PLANT INNATE IMMUNITY BY APPLICATION OF BRASSINOSTEROIDS. Yamilet Coll, Hernández I, Niebla V, Rodríguez M, Canales E, Hernández G, Coll F Chemistry Faculty, Havana, University, Havana, Cuba [email protected] Brassinosteroids (BRs) is a group of phytohormones that regulates many common developmental processes throughout the plant life cycle. It was shown that the exogenous applications of BRs induce protection against different pathogens and can provide plants tolerance/resistance to different abiotic stresses. Similar to other growth regulators, BRs play ambiguous roles in molding pathological outcomes, the effects of which may depend not only on the pathogen’s lifestyle and infection strategy, but also on specialized features of each interaction. In this work we show the effect of exogenous treatment with a natural BR (24- epibrasinolide (eBL)) and two synthetic spirostanic analogues, on expression of different genes associated with the defence of Arabidopsis thaliana plants. These results suggest that BRs plays a role in the activation of innate immunity in Arabidopsis plants, which envisages the potential for a new safer strategy, alternative to agrochemicals, for crop health management.

N-P 004 FORMULATED SYNTHETIC COMPOUNDS IMPROVE INNATE IMMUNITY IN PLANTS. Ingrid Hernández,, Canales, E, Guirola O, Portieles, R, Rodríguez, M, López Y, González L.J, BorrásHidalgo, O, Rodríguez, M Center for Genetic Engineering and Biotechnology (CIGB), Havana, Cuba. [email protected] Protection of plants against infectious microorganisms depends on both constitutive and induced defense mechanisms. Induction of disease resistance is a method of great importance and interest at present, which allows the use of biochemical and molecular mechanisms that already exist in the plant for use in disease control. The defense of plants to disease comprises a series of events related to the recognition, signaling and response defined as innate immunity in plants. In this regard, formulation technologies are used to improve delivery, shelf life and efficacy of products for biological control, bio-pesticides, among others. Previously, we demonstrate the potential of different synthetic compounds to stimulate natural defense and induce resistance to diseases in plants. However, the formulation of the same for its application is a subject of research today. In our work, we first studied the formulation of these molecules to improve the activity and persistence of the active ingredient in Arabidopsis plant´s leaves. In this regard, we have found that combination of DMF, Silwet L-77 and PEG (1500) was the best variant which allowed an enhance response of relative expression in some defense related genes. Additionally, citrus plants were foliar sprayed with these synthetic compounds and tested the competence of these molecules for stimulating the natural defense. In conclusion, there is a need to explore new strategies based on activating the plant's own immune system and defense mechanism to control plant diseases. Keywords: innate immunity in plants, formulation, plant defense

N-P 005 EFECTO DE DOS ESTEROIDES SEMI-SINTÉTICOS SOBRE LA INDUCCIÓN DE RESPUESTA DE DEFENSA EN PLANTAS ARABIDOPSIS THALIANA Elaine Puentes, Hernández I, Canales E, Rodríguez M, Espinosa G, Coll Y Departamento de Bioquímica, Facultad de Biología, Universidad de la Habana, Cuba [email protected] Los brasinoesteroides son fitohormonas esenciales en el mantenimiento de la homeostasia vegetal, ya que regulan procesos fisiológicos fundamentales relacionados con el crecimiento, desarrollo, reproducción e inducción de tolerancia a estrés ambiental. A pesar de ser las rutas del jasmónico/etileno y la del ácido salicílico, las más conocidas en la respuesta de defensa vegetal, cada vez hay más evidencias que demuestran que otras hormonas, como las auxinas, ácido abscísico y los brasinoesteroides, juegan un papel primordial en la inducción de tolerancia a estrés. En este trabajo se evaluó el efecto del brasinoesteroide natural 24 epibrasinólida (eBL) y dos análogos funcionales obtenidos por vía semi sintética en el Centro de Estudio de Productos Naturales de la Facultad de Química, Universidad de la Habana, sobre la disminución de la concentración de la bacteria Pseudomonas syringae pv Tomato DC3000 en su interacción con Arabidopsis thaliana.

N-P 006 A SINTHETIC STEROID AS A NOVEL ABA-INDEPENDET DROUGTH RESPONSE INDUCTOR Lucia S. Pérez-Borroto1-2, Pardo M., Castagnaro A. , González-Olmedo J., Coll F., Coll Y. 1 Centro Bioplantas, Universidad de Ciego de Ávila “Máximo Gómez Báez”, Ciego de Ávila, Cuba. 2 Instituto de Tecnología Agroindustrial del Noreste Argentino, San Miguel de Tucumán, Argentina. [email protected] Water deficit is the greatest abiotic stress in worldwide agriculture, therefore research aimed at reducing its effects, constituted a priority. Among them is the application of synthetic compounds like DI-31, a spirostanic brassinosteroid analog with greater persistence in the field. During the investigation, DI-31 (2,23 µM) was analyzed as a regulator of the response to water and osmotic stress, by evaluating parameters such as oxidative burst, reduction of lipoperoxidation products, antioxidant activity of Superoxide dismutase (SOD), Catalase (CAT), Ascorbate peroxidase (APX) and Phenol peroxidase (POX) enzymes in Arabidopsis thaliana wild-type and ABA-mutant plants, besides the relative expression by qRT-PCR of abiotic abscisic acid (ABA)-related responsive genes. The oxidative burst induced by the DI-31 was registered six hours after the application of the compound, reaching the highest levels after 48 hours; time at which the antioxidant enzyme SOD showed the greatest increases with concomitant decreases of lipid peroxidation products. At the same time, DI-31 significantly decreased the activity of APX and POX peroxidases in both wildtype and ABA-hypersensitive pp2ca mutant plants subjected to stress, unlike all the control plants treated with the compound and the ABA-insensitive SnRK2 mutant under stress. These peroxidases are regulated by ABA-dependent transcriptional factors, so their decrease in both wild-type and pp2ca plants, such as the increase in SnRK2 mutant could be associated with an antagonist interaction between brassinosteroid and ABA stress-response transcriptional regulation. Also the gene expression patterns in DI-31 treated wild-type plants showed similar trends, by an increase in the expression of ABA-independent P5CS gene and the significant repression of Rab18, RD22 and RD29A ABA-responsive genes. These results suggest that DI-31 may masks ABA effects in stress responses, although acts as an effective antioxidant-activity regulator in A. thaliana. However, it´s possible to consider that most of its action will be developed as ABA-independent water-stress responsive-inductor.

N-P 007 FOSFOMANOSA ISOMERASA, PMI, COMO MARCADOR DE SELECCIÓN POSITIVA PARA LA TRANSFORMACIÓN DE EXPLANTES MERISTEMATICOS DE SOYA Alejandro E. Morales-Basulto, Soto N., Carrillo L., Delgado C. , Enriquez G.A. Centro de Ingeniería Genética y Biotecnología, La Habana, Cuba [email protected] Los marcadores de selección son necesarios para el desarrollo de la tecnología de transformación de plantas, debido a su capacidad de proteger a las células transformadas en presencia de un sustrato tóxico. Pocas células son transformadas en la mayoría de los experimentos y la probabilidad de distinguir las células transgénicas sin selección es, normalmente, muy baja. Dentro de estos genes, uno de los más utilizados y en soya casi en exclusiva, es el gen cp4epsps que confiere resistencia frente al herbicida glifosato. Este uso casi exclusivo del gen cp4epsps en soya dificulta la selección de nuevas plantas transgénicas que ya presenten este gen. Por tanto en la búsqueda de nuevos marcadores de selección para la producción de plantas transgénicas, hemos adaptado el sistema pmi/manosa para la transformación de soya (Glycine max [L.] Merrill). Primeramente se realizó una evaluación de la sensibilidad de la manosa en explantes meristematicos de soya genotipo DT84. Se probaron concentraciones crecientes de manosa desde 5g L-1 a 20g L-1 así como diferentes combinaciones de manosa-sacarosa. Posteriormente el gen pmi de Escherichia coli (E.C. 5.1.3.8) que codifica una fosfomanosa isomerasa que convierte manosa-6-fosfato, un inhibidor de la glucólisis, en fructosa-6-fosfato, intermediario de la glucólisis, fue aislado del evento MIR162. Este gen fue clonado en el plásmido comercial pCAMBIA3300 bajo el promotor constitutivo 35S del virus de mosaico de la coliflor. El gen de interés fue introducido en las planta mediante transformación por Agrobacterium cepa LBA4404, generando una eficiencia de transformación superior al 10%. Las plantas transgénicas capaces de enraizar en un medio de selección con manosa 5g L-1 fueron confirmadas molecularmente mediante PCR. Esta es la primera vez que se transforman plantas de soya con pmi lo que permitirá la utilización de este sistema de selección en la discriminación de plantas transgénicas que porten genes de interés.

N-P 008 EFFECT OF DIOSGENIN-DERIVED SYNTHETIC COMPOUNDS ON BIOFILM FORMATION IN BACTERIA WITH POTENTIAL AGRICULTURAL USE. Leonardo D. Martín-Ríos, Mora-González N, Rojas-Badía M., Coll-García Y Centro de Estudio de Productos Naturales, Facultad de Química, Universidad de la Habana, Cuba [email protected] The study of plant-microorganism relationships continuously reveals important tools for the development of sustainable agriculture, aimed to the exploitation of natural mechanisms to increase plant yield. The field of study of bacterial biofilms shows a wide range of potential research related to agriculture, among which is the search for compounds able to stimulate or inhibit the formation of these biological microstructures. Taking into account the biological activity of diosgenin (free sapogenin or as glycosidic glycosides in nature), we evaluated the effect of several synthetic derivatives obtained at the Center for the Study of Natural Products (CEPN) on the formation of biofilms of different bacterial strains of agricultural interest. The data obtained were processed by the GraphPad program and there were significant differences between the treatments, with a specific dose / compound ratio for each strain studied. These results constitute preliminary evidences to carry out further studies focused to the use of this type of steroidal compounds to increase the efficiency of these microorganisms in their interaction with host plants.

N-P 009 LATINOAMÉRICA APUESTA POR LA BIOTECNOLOGÍA AGROALIMENTARIA. PANORAMA GLOBAL DE LA PRODUCCIÓN DE CONOCIMIENTO DE LA BIOTECNOLOGÍA AGROALIMENTARIA EN LATINOAMÉRICA. Dante I. León-de la O, Thorsteinsdóttir H., Calderón-Salinas J V. CINVESTAV-IPN, UPIBI-IPN, Universidad Simón Bolívar, CDMX, México [email protected] La biotecnología como una sub-categoría de las ciencias químicas-biológicas se ha convertido en una herramienta que ha traído cambios radicales en el proceso de innovación. Por otro lado, la biotecnología tiene considerables impactos (económico, social, ambiental, etc.) en las sociedades que desarrollan innovación en cualquiera de sus campos. La biotecnología agroalimentaria es un campo de la biotecnología que tiene gran impacto en la producción, y conservación de los alimentos, y puede brindar soluciones a problemas alimentarios, agrícolas, y sociales. Diversas innovaciones en biotecnología agroalimentaria han sido desarrolladas en Latinoamérica, por ejemplo: papas resistentes a virus y mejoramiento de vides (Argentina); mejoramiento de la caña de azúcar y maíz (Brasil); yuca y café modificado (Colombia); vacunas veterinarias de nueva generación y enzimas utilizadas en la industria alimentaria (Cuba); bioinsecticidas y mejoramiento de cultivos acuícolas (Chile); y, frijol y algodón resistente a sequía (México). El concepto de sistema de innovación destaca al aprendizaje como la clave principal para que se desarrolle innovación en una región o país, y por ello es importante analizar la producción de conocimiento que tienen los países Latinoamericanos en biotecnología agroalimentaria. Para hacerse una idea de las potencialidades y características de la biotecnología agroalimentaria en los países de Latinoamérica, se realizó el análisis bibliométrico de las publicaciones en la biotecnología agroalimentaria en los países de Latinoamérica que han tenido éxito en este campo. Se analizaron los patrones de publicaciones de biotecnología agroalimentaria de seis países de Latinoamérica (Argentina, Brasil, Colombia, Cuba, Chile, y México) entre 2004 - 2018. El análisis bibliométrico incluye el nivel de publicaciones, la colaboración entre naciones, los sectores de la biotecnología agroalimentaria en los que más publican y la distribución de los actores que realizan las investigaciones en cada país. Nuestros resultados mostraron diferentes tendencias de crecimiento en las publicaciones de biotecnología agroalimentaria en Latinoamérica.

N-P 010 RELACIÓN ENTRE ÁREA FOLIAR Y BIOMASA EN POBLACIONES SILVESTRES Y CULTIVADAS DE “TOTORA” (Schoenoplectus californicus - Cyperaceae) DE LOS LAGOS YAHUARCOCHA E IMBACOCHA, IMBABURA-ECUADOR Pabón Galo, Ortega E, Rodés R, Vásquez L Universidad Técnica del Norte, Ibarra, Ecuador [email protected] La especie Schoenoplectus californicus (Cyperaceae), conocida vulgarmente como “totora”, es una planta acuática cuya Biomasa (en su mayoría tallos), posee gran valor socio-económico. Con ellos se fabrican variedad de artesanías, que representan ingresos monetarios para las comunidades locales. Los lagos Yahuarcocha e Imbacocha (Imbabuara-Ecuador), son las principales fuentes de materia prima y sus productos son comercializados en mercados nacionales e internacionales. El presente estudio comparó la relación entre Área Foliar (AF) y la Biomasa en cinco poblaciones silvestres del norte de los antes ecuatorianos: tres de Yahurcocha y dos deI mbacocha, y plantas cultivadas en el Laboratorio de Fisiología Vegetal (Universidad de la Habana, Cuba). El trabajo se realizó entre febrero de 2017 y febrero de 2018. En los tallos fértiles se midió: altura (cm), diámetro base (cm), peso fresco (g) “in situ”, y peso seco (g) luego de ser sometidos a 100oC por 24 horas. El cálculo de AF se realizó mediante la ecuación Al=π.radio.generatriz (Al: área lateral cono). El nivel de significancia fue establecido mediante un Diseño Completamente Aleatorizado (DCA), una vez comprobado el cumplimiento de las premisas de normalidad y homogeneidad de varianzas. Se detectó diferencias significativas entre lagos y plantas desarrolladas en laboratorio, para todas las variables. Las poblaciones de un mismo lago, no presentan diferencias. Mayores valores corresponden a Yahuarcocha, tanto en altura del tallo (350cm), diámetro de la base (2.49cm), Área Foliar (1362.92cm2) y Biomasa (35.5g); los menores valores corresponden al Laboratorio, excepto para contenido de humedad (87.6%), y podría deberse a que fueron cultivadas en suelos no inundados, produciendo menor cantidad de aerénquima. En todos los casos existe una correlación positiva entre AF y Biomasa (r=0.9848). En conclusión, las tres poblaciones de Yahuarcocha, posee plantas más grandes (más Biomasa) que aportan mayor cantidad de materia prima, a los artesanos de la zona.

N-P 011 EVIDENCIA BIOQUÍMICA DE LA ACTIVACIÓN DE LA RUTA QUE INVOLUCRA MESA EN SIMPLASTO DE CLAVEL (DIANTHUSCARYOPHYLLUS L.) DURANTE SU INTERACCIÓN CON Fusarium oxysporum f. sp. dianthi Leidy Vanegas-Cano, Orduz-Díaz L. L., Coy-Barrera E. A, Martínez-Peralta S., Ardila-Barrantes H. D Laboratorio de Investigación en Actividades metabólicas vegetales, Departamento de Química, Universidad Nacional de Colombia Bogotá, Colombia [email protected] El clavel (Dianthus caryophyllus L.) es un producto de exportación importante para Colombia. Su producción ha disminuido debido al marchitamiento vascular causado por Fusarium oxysporum f. sp. dianthi(Fod). El desarrollo de nuevas alternativas de control que sean eficientes con los sistemas de cultivos actuales requiere conocer los mecanismos asociados a la resistencia de la planta a esta enfermedad. Es por ello, que la presente investigación se constituye como una primera aproximación al estudio de los mecanismos de defensa que se activan a nivel desimplasto de la planta, durante su interacción con el patógeno. Para tal fin, en extractos simplasticos de cultivares resistentes (Golem) y susceptibles (Solex) inoculadas con el patógeno, se evaluó la acumulación de la hormona de estrés vegetal Metil-salicilato (MeSA), e inducción de enzimas asociadas, como son la Fenilalanina amonio liasa (PAL) y Fosfolipasa D (PLD). Después de un ensayo in vivo, el material vegetal fue sometido a la remoción previa de apoplasto[1], con el fin de obtener la fracción simplastica que fue usada para la obtención de extractos de metabolitos y enzimas. Mediante análisis cromatográfico por HPLC se cuantificó en los extractos obtenidos, el MeSA utilizando patrones estándar. La actividad dePAL y PLD se evaluó por métodos espectrofotométricos reportados para cada enzima[2].Se determinó que en simplasto de tallos de la variedad resistente a las 24h post-inoculación, se presentó una acumulación de MeSA y aumento en la actividad PAL y PLD. Dichos cambios fueron inexistentes en la variedad susceptible. En general se confirman la participación de metabolitos y enzimas objeto de estudio, en los mecanismos de defensa del clavel ante este patógeno. Agradecimientos a la Universidad Nacional de Colombia, a Colciencias por la financiación del proyecto (110174558226) y a Florval SAS sede QFC por el material vegetal suministrado [1] A. P. Martinez Gonzalez, S. T. Martínez Peralta, H. D. Ardila Barrantes, A. P. Martínez, S. T. Martínez, and H. D. Ardila, “Condiciones para el análisis electrofóretico de proteínas apoplásticas de tallos y raíces de clavel (Dianthus caryophyllus L) para estudios proteómicos,” Rev. Colomb. Química, vol. 46, no. 2, p. 5, 2017. [2] H. D. Ardila, B. Baquero, and S. T. Martínez, “Inducción de la actividad de la enzima fenilalanina amonio liasa en clavel (Dianthus caryophyllus L) por elicitores del hongo Fusarium oxysporum f. sp. Dianthi raza 2,” Rev. Colomb. Química, vol. 36, no. 2, pp. 151–167, 2007.

N-P 012 IDENTIFICACIÓN Y ANÁLISIS DE LA EXPRESIÓN DE GENES CODIFICANTES DE POLIFENOL OXIDASAS EN Musa paradisiaca DE ECUADOR Victor Hugo Huebla-Concha Escuela Superior Politécnica de Chimborazo-Extensión Morona Santiago-Macas-Ecuador. [email protected] Las polifenol oxidasas (PPOs), son enzimas ubicuas que catalizan la reacción dependiente de oxígeno y transforman o-difenoles en o-quinonas. Estas quinonas son reactivas y capaces de modificar covalentemente un amplio abanico de especies nucleófilas del interior de las células que conduce a la formación de polímeros marrones, conocido como pardeamiento enzimático (Morante y col., 2014). Hasta el momento se ha descrito la existencia de familias multigénicas que codifican PPOs diferentes en tomate (Newman y col., 1993) patata (Thygesen y col., 1995), plátano (Gooding y col., 2001), un híbrido de álamo (Wang y Constabel, 2004) y algunas rosáceas (Haruta y col., 1999). Aunque la función biológica de las PPOs no se ha aclarado todavía en detalle, la bibliografía sugiere como función la participación de estas enzimas en la defensa de las plantas frente a patógenos y herbívoros (Dongfeng et al., 2004). Por tanto, este trabajo pretende demostrar el origen mono/multigénico de las polifenol oxidasas en plantas de banano. La identificación, clonaje y caracterización molecular de las PPOs, servirá de base para estudios de expresión génica y proponer investigaciones futuras orientadas a la mejora génica de plantas de banano, con especial atención en la defensa endógena de las plantas frente a patógenos y herbívoros.

N-P 013 BUFADIENOLIDES FROM PAROTOID GLAND SECRETIONS OF CUBAN TOAD Peltophryne fustiger (BUFONIDAE): INHIBITION OF HUMAN KIDNEY NA+/K+-ATPASE ACTIVITY Perera Cordova WH, Guimaraes Leitao S, Cunha-Filho G, Roberto Alonso Bosch, Pascual Alonso I, Pereda-Miranda R, Gervou R, Araújo Touza N, Quintas LEM, François Noel Facultad de Biología, Universidad de La Habana, Cuba [email protected] Parotoid gland secretions of toad species are a vast reservoir of bioactive molecules with a wide range of biological properties. Herein, for the first time, it is described the isolation by preparative reversed-phase HPLC and the structure elucidation by NMR spectroscopy and/or mass spectrometry of nine major bufadienolides from parotoid gland secretions of the Cuban endemic toad Peltophryne fustiger: ѱ-bufarenogin, gamabufotalin, bufarenogin, arenobufagin, 3-(Nsuberoylargininyl) marinobufagin, bufotalinin, telocinobufagin, marinobufagin and bufalin. In addition, the secretion was analyzed by UPLC-MS/MS which also allowed the identification of azelayl arginine. The effect of arenobufagin, bufalin and ѱ-bufarenogin on Na+/K+-ATPase activity in a human kidney preparation was evaluated. These bufadienolides fully inhibited the Na+/K+ATPase in a concentration-dependent manner, although arenobufagin (IC50 28.3 nM) and bufalin (IC50 28.7 nM) were 100 times more potent than ѱ-bufarenogin (IC50 3020 nM). These results provided evidence about the importance of the hydroxylation at position C-14 in the bufadienolide skeleton for the inhibitory activity on the Na+/K+-ATPase. Acknowledgments: The authors are grateful to CAPES and FAPERJ for the postdoctoral support to WHPC and GCF. We also thank CNRMN National Center of Nuclear Magnetic Resonance Jiri Jonas from Federal University of Rio de Janeiro for kindly allowing us to record the NMR spectra as well as Dr. Ari Miranda da Silva and Marina Amaral for HRESI and ESI mass spectrometric measurements. FN and LEMQ are research fellows from CNPq and RG is a student fellow from PIBIC-EM/CNPq. R.P.-M. was a visiting research scientist at Faculda de de Farmacia, Universida de Federal do Rio de Janeiro with partial financial support from Direccion General de Asuntos del Personal Academico, UNAM. We also thanks Ms. Vanessa Dias for helping us with some experiments. We wish to thank L.Y. Garcia for his assistance in the fieldwork and Dr. James D. McChesney for checking the English grammar.

N-P 014 BUFADIENOLIDES ISOLATED FROM Peltophryne fustiger (AMPHIBIA: BUFONIDAE) ARE POTENT AND SELECTIVE INHIBITORS OF THE MEMBRANE-BOUND NEUTRAL AMINOPEPTIDASE N. Laura Rivera, Alonso Bosch R,, Valdes-Tresanco ME, Perera-Córdova W, Sánchez L, Sánchez B, Guimaraes Leitao, S, Charli JL, Pascual I Center for Protein Studies, Faculty of Biology, University of Havana, Cuba [email protected] Neutral aminopeptidase (APN) is a M1 family membrane ectopeptidase that plays pivotal roles in many physiological processes, such as pain sensation, tumor angiogenesis and metastasis, immune cell chemotaxis, cell-cell adhesion, among others. Accordingly, APN is a major target for the treatment of diseases related to those physiological processes. APN inhibitors from natural sources are scarce being bestatin and betulinic acid, the most used molecules with IC50 values in the range of µmol/L and very low specificity. Recently, we identified compounds, belonging to bufadienolides family, characterized by a high hydrophobicity and voluminous groups in their structures; both properties agreed with the substrate preference of APN. Taking into account the biomedical relevance of the development of new APN inhibitors, we validated (using biochemical, bioinformatics and in vitro cellular approaches) various bufadienolides previously isolated from parotoid gland secretions of Peltophryne fustiger, as a new class of APN inhibitors. We used microsomal preparations of porcine APN (80% of identity with the human enzyme) to test the enzyme in a native structure and natural context. We studied the Enzyme-Inhibitor interaction using a kinetic approach with a specific substrate. Additionally, we determined for the first time the presence of APN on the surface of the HTB – 65 (MEWO) melanoma cell line using kinetic assays, and evaluated the effect of the new inhibitors on cell viability, cell cycle and mechanism of death. The new molecules inhibited APN in the 10-6 - 10-7 mol/L range, with kinetic mechanisms that differed according to inhibitor structure. The strongest effect on the MEWO cell line was detected for bufalin, with IC50 values in the sub-micromolar range, directly correlating with the inhibition of APN activity at cell surface. Acknowledgment: Laboratorio Conjunto UH-CIM, IUBMB Mid Career fellowship to I. Pascual.

N-P 015 INHIBITION OF THREE MEMBRANE ECTOPEPTIDASES DIPEPTIDIL-PEPTIDASE-IV, NEUTRAL METALLO-AMINOPEPTIDASE, AND ACID AMINOPEPTIDESE (ANTICANCER TARGETS) BY THE PEPTIDIC INHIBITOR BACITRACIN. Yarini Arrebola, Valdés M, Díaz L., Mc. Whire R, Rivera L ,Sánchez B, Pedroso A, Pascual I. Centro de Estudios de Proteínas, Facultad de Biología, Universidad de La Habana, Cuba. [email protected] Dipeptidilpeptidase-IV (DPP-IV), Neutralmetallo-aminopeptidase (APN), and Acid aminopeptidese (APA) are well known membrane ectopeptidases that catalyze the cleavage of specific residues from the N-terminus of peptide substrates. These enzymes are often simultaneously up-regulated in pathologies such as some cancers, inflammation and skin diseases. Although their simultaneous inhibition could be promissory as a therapeutic target, strategies into that way haven’t been developed yet. The aim of this work was to study the inhibition of DPP-IV, APN, and APA by the inhibitor bacitracin, a natural deca-peptide. Considering the high identity of sequence between these three human enzymes and their porcine homologous, we prepared microsomes of membrane purified from porcine kidney to have the enzymes in native structure as closer to nature. We studied the interaction Enzyme-Inhibitor using kinetic approach with a specific substrates for each enzyme. We in depth in the structure-function relation of the EnzymeInhibitor complexes using Docking simulations conducted with the Autodock4Zn force field implemented in the Autodock4 software. The inhibition mechanism determinations showed a competitive inhibition for DPP-IV, as well as noncompetitive inhibitions (with α ˃ 1 and α ˂ 1respectively) for APN and APA. The Ki values were in the micromolar order in all cases. New insights regarding the structure-function relationship for the three enzymes were established based on in silico studies. Since DPP-IV and APN are up-regulated in several skin pathologies, we demonstrated their susceptibility to be inhibit by bacitracin on the cellular surface of de melanoma line MEWO. Acknowledgment: Laboratorio Conjunto CIM-UH, IUBMB Mid career fellowship to I. Pascual.

N-P 016 PREDICTING A NON-CANONICAL INHIBITION MECHANISM OF CMPI-II, A NON-CLASSICAL KAZAL INHIBITOR, WITH THE HUMAN NEUTROPHIL ELASTASE Mario Valdés-Tresanco, Valiente, P. A. Computational Biology and Biomolecular Dynamics Laboratory, Center for Proteins Studies, Faculty of Biology, University of Havana, Havana, Cuba [email protected] CmPI-II is a ‘non-classical’ Kazal type inhibitor isolated from the marine mollusk Cenchritis muricatus. CmPI-II, with arginine at P1 position, shows a tight-binding inhibition profile not only against typical serine proteases, i.e. trypsin (Ki = 1.1nM), but others such as subtilisin A (Ki = 30.8 nM) and human neutrophil elastase (HNE) (Ki = 2.6 nM). This is one of the most striking features of this inhibitor because the presence of a basic residue at P1 site has been widely related with the trypsin inhibition, but not with subtilisin A and HNE inhibition. We focused our study in predicting the binding mode of both, the wild-type (CmPI-II/WT) and R12A mutant (CmPI-II/R12A) with HNE, through molecular dynamics (MD) simulations and binding free energy calculations with MMPBSA method. MD simulations showed a non-canonical binding mode of CmPI-II/WT vs HNE through formation of a salt-bridge between R12 (Inhibitor) and E90 (Enzyme). The computational alanine scanning of the P1 position (assuming similar binding modes for both systems) suggested a significant loss of the mutant affinity (∆GCmPI-II/R12A - ∆GCmPI-II/WT = 6 kcal/mol) by HNE. However, the enzymatic assays showed a slightly increased of the mutant affinity (∆∆Gexp = -0.98 kcal/mol) compared to CmPI-II/WT, thus suggesting that the assumption of similar binding modes may be at fault. Notably, MD simulation of HNE+CmPI-II/R12A complex revealed a non-canonical binding mode as well, although CmPI-II/R12A adopted a different binding mode compared to CmPI-II/WT. Free energy calculations with the MM-PBSA method reproduced accurately the affinity differences of CmPI-II/WT and CmPI-II/R12A with HNE (∆∆Gcalc = -1.72 kcal/mol). This model provides a structural framework for the interpretation of its experimentally determined Ki value and understanding the inhibition mechanism. Nevertheless, future mutagenesis experiments will be necessary to confirm the proposed binding mode.

N-P 017 UNDERSTANDING THE CONFORMATIONAL DYNAMICS AND SUBTILISIN A INHIBITION OF CMPIII, A NON- CLASSICAL KAZAL PROTEASE INHIBITOR Aymara Cabrera-Muñoz, Valiente P, Rojas L, Pires J.R., Alonso-del-Rivero M Center for Protein Studies, Faculty of Biology, University of Havana, Havana, Cuba. [email protected] Kazal protease inhibitors are widely distributed in all groups of organisms and are able to inhibit S1 and S8 serine proteases. CmPI-II is a Kazal proteinase inhibitor isolated from Cenchritis muricatus that inhibits trypsin, elastases and subtilisin A. The analysis of its amino acids sequence allows to define a new group in non-classical Kazal inhibitors (also composed by Kazal-type proteinase inhibitor (Litopenaeus vannamei) and hemocyte Kazal-type proteinase inhibitor (Penaeus monodon). However, there are not described tridimensional structures for these inhibitors. Here, we solved for first time the 3D structure of CmPI-II using NMR techniques. CmPIII adopts the typical fold described for Kazal-type inhibitors but has significant structural differences in the N-terminal moiety, the disposition of CysI-CysV disulfide bond and in the reactive site loop (RSL) conformation. We observed by NMR dynamics, high flexibility of the Nterminal region, the RSL, and the α-helix, in agreement with the results of molecular dynamics simulations, which suggest coupled motion of these regions. To understand the energetic basis of the subtilisin A inhibition, the 3D structure of the CmPI-II/subtilisin A complex was modeled. Notably, we predicted that residues at the P2 (Thr11) and P2’ (Trp14) positions of the RSL displayed a higher contribution to the binding free energy of the complex than the P1 (Arg12) position of the inhibitor. The whole results obtained in this work support structurally the existence of a new group in non-classical Kazal inhibitors and allow explaining the broad specificity observed for CmPI-II.

N-P 018 ISOLATION OF A NEW SUBTILISIN INHIBITOR FROM THE MOLLUSK Nerita peloronta Jesus D. Mojarena, Rojas L, Cabrera-Muñoz A, Chávez M A, Alonso del Rivero M, Betzel C Center for Protein Studies, Faculty of Biology, University of Havana, Havana [email protected] Subtilisins are serine proteases of the S8 family, essential during the life cycle of human pathogens such as Plasmodium, Trypanosoma and Leishmania and therefore of great interest from the biomedical point of view. Recently, González et al. performed a screening to detect subtilisin inhibitory activity in 20 extracts of marine invertebrates from Cuban coast. As a result of this study it was obtained that the extract of the mollusk Nerita peloronta is a promising source of subtilisin inhibitors. The objective of this work was to isolate the subtilisin inhibitor present in the aqueous extract of the mollusk. The extract was clarified using TCA treatment and then applied to ion exchange chromatography, followed by a molecular exclusion chromatography on Superdex 75 matrix. During the purification the subtilisin inhibitory activity and the concentration of proteins were evaluated at each step. Samples obtained were analyzed by SDS-PAGE. The major subtilisin inhibitor was purified and characterized in terms of kinetics and specificity against serine proteases. The purification protocol established in this work will be useful for obtaining the major subtilisin inhibitor from N. peloronta in sufficient quantities to perform structural and functional characterization.

N-P 019 EXPRESSION, PURIFICATION AND KINETIC CHARACTERIZATION OF LAPTC, AN M17 AMINOPEPTIDASE OF Trypanosoma cruzi Izquierdo M., Mirtha E. Aguado-Casas, Zoltner M., Field M.C., González-Bacerio J. Centro de Estudio de Proteínas, Facultad de Biología, Universidad de La Habana, La Habana, Cuba [email protected] A novel target in Chagas disease is the leucil-aminopeptidase M17 of Trypanosoma cruzi (LAPTc). The objective of this work was to obtain a recombinant variant of LAPTc in Escherichia coli. A LAPTc gene was designed, was optimized for its expression in E. coli, was synthesized and cloned in the vector pET-19b, contracting the services of the company Eurofins Genomics (Germany). The Codon Adaptation Index and the average content of G/C results adequate for a high efficiency of expression. The genetic construction pET-19b rLAPTc allows an soluble and active production of rLAPTc in E. coli BL21(DE3)pLysS, by induction during 20 h at 25ºC with IPTG 1 mM, added in the late exponential phase of the bacterial growth. A level of expression of 12.53 % of the total cellular protein was obtained. The rLAPTc enzyme was purified in a single step by IMAC, exploiting the Nterminal tag of 10 His encoded by the vector. The recombinant protein was obtained with a 90 % of purity and a yield of 90 mg per liter of culture. The enzyme has an optimal pH of 9.0, and preference for Leu p-nitroanilide among nine tested substrates (KMapp = 74 M, kcatap = 4.4 s1, kcat/KM (ap) = 59,459 L mol-1 s-1). The optimal temperature is 50ºC, and the cations Mg2+, Cd2+, Ba2+, Ca2+ and Zn2+ at 4 mM inhibited the activity in a 60 % or more, but Mn2+ inhibited only a 15 % and Co2+ activated in a 40 %. The recombinant enzyme is insensitive toward the protease inhibitors PMSF, TLCK, E-64 and pepstatin A, but is inhibited by EDTA and bestatin. Therefore, the enzyme is a good target for the inhibitors identification.

N-P 020 IDENTIFICATION OF PEPTIDOMIMETICS AND TETRAZOLES INHIBITORS OF THE NEUTRAL M1 AMINOPEPTIDASE FROM Plasmodium falciparum Ana C. Varela, Izquierdo M., Méndez Y., Rivera D.G., González-Bacerio J. Centro de Estudio de Proteínas, Facultad de Biología, Universidad de La Habana, La Habana, Cuba [email protected] Plasmodium falciparum neutral M1-aminopeptidase (PfA-M1) is a novel target identified for the development of antimalarials. A well-known PfA-M1 inhibitor is the non-selective compound bestatin, which combines a Zn2+-binding group in the active site with substituents that recognize the subsites S1 and S1’. From these antecedents, the aim of this work was the identification of potent and selective inhibitors of the recombinant PfA-M1 (rPfA-M1) in two series of synthetic compounds: 10 bestatin-derived peptidomimetics and 22 tetrazoles. Firstly, it was checked that 15 min of preincubation enzyme bestatin is enough to reach the inhibition equilibrium. The IC50 value of bestatin toward recombinant PfA-M1 is 1.79 µM. The competitive inhibition observed for this compound confirms the reports of other authors and suggests that it is possible to achieve the potent rPfA-M1 inhibition in this manner. The evaluation of the bestatin-derived peptidomimetic library toward the enzyme does not allowed to identify neither interesting inhibitor, but made possible to do the analysis of the structure-activity relationship. By doseresponse studies, the synthetic tetrazoles YTE003 (IC50 = 1.35 µM) and YTE008 (IC50 = 1.59 µM) were identified as potent and selective rPfA-M1 inhibitors, regarding the porcine orthologue (selectivity indexes of 1,167 and 3,832, respectively), what supports their potentialities as leader compounds for the development of antimalarial agents. For the tetrazoles YTE003 and YTE008 also is enough a preincubation time of 15 min with the enzyme to reach the inhibition equilibrium. The type of inhibition of these compounds is competitive and noncompetitive toward rPfA-M1, respectively. This knowledge can facilitate the study of the mode of binding of the inhibitors to the enzyme in silico, guides the future optimization of these structures and contributes to the design of novel PfA-M1 inhibitors.

N-P 021 IDENTIFICATION OF PEPTIDOMIMETICS AND TETRAZOLES INHIBITORS OF THE NEUTRAL M1 AMINOPEPTIDASE FROM Escherichia coli Pérez-Leyva I., De Armas-Guitart G., Izquierdo M., Méndez Y., Rivera D.G., Jorge González-Bacerio Centro de Estudio de Proteínas, Facultad de Biología, Universidad de La Habana, La Habana, Cuba [email protected] Escherichia coli neutral M1-aminopeptidase (ePepN) is a novel target identified for the development of antimicrobials. A well-known ePepN inhibitor is the non-selective compound bestatin, which combines a Zn2+-binding group in the active site with substituents that recognize the subsites S1 and S1’. From these antecedents, the aim of this work was the identification of potent and selective inhibitors of the recombinant ePepN in two series of synthetic compounds: 10 bestatin-derived peptidomimetics and 22 tetrazoles. The kinetic characteristics determined for this enzyme (activity at pH 7.0 and 8.0 and KM value) indicate that this is a good model of its natural counterpart for the inhibitors identification. On the other hand, the noncompetitive or mixed inhibition observed with bestatin suggested that is possible to achieve the potent ePepN inhibition in this manner. By dose-response studies, the synthetic tetrazoles YTE003 (IC50 = 1.39 µM), YTE007 (IC50 = 2.21 µM) and YTE008 (IC50 = 0.26 µM) were identified as potent and selective ePepN inhibitors, regarding the porcine orthologue (selectivity indexes of 1,133, 924 and 23,434, respectively). In addition, relevant data about the structural determinants of the ePepN inhibition by the tetrazoles and bestatin-derived peptidomimetics were obtained. On the contrary to the expected, the type of inhibition of the bacterial enzyme by the representing of the compound series (KBE053 and YTE008) is uncompetitive and noncompetitive (α < 1), respectively. This knowledge can facilitate the study of the mode of binding of the inhibitors to the enzyme in silico, guides the future optimization of these structures and contributes to the design of novel ePepN inhibitors. Interestingly, the tetrazole YTE003 shows in vitro antibacterial activity, toward the bacterium E. coli, what reinforces its potentialities as a leader compound for the development of antibacterial agents.

N-P 022 BINDING OF THE PORE-FORMING TOXIN STICHOLYSIN II TO MEMBRANE SPHINGOID-DERIVED LIPIDS AND THE INTRACELLULAR PATHWAYS RESPONSE. Carmen Soto, A. Valle, P. Valiente, G. Bergado, R. Blanco ,J. C. Rodríguez, F. Pazos, M. E. Lanio, A. M. Hernández, Carlos Alvarez Center for Protein Studies, Havana University, Cuba. [email protected], [email protected] SticholysinII (StII) is an actinoporin produced by the anemone Stichodactyla helianthus. Despite their well-documented affinity for sphingomyelin, the molecular determinants that define this affinity have not been clarified. In order to deepen into the understanding of the lipidic determinants governing StII-membrane interaction, we examined the association of StII with ceramide-derived lipids and their combination with phosphatidylcholine. Binding of StII to lipids was assessed by thin layer immunostaining. We demonstrated that StII recognizes ceramide, gangliosides but neither phosphatidic acid nor sphingosine. The StII interaction with ceramidederived lipids that might also contribute to pore formation was evaluated in lipidic monolayers and liposomes. The relevance of these findings for pore formation was confirmed through carboxyfluorescein leakage assays by fluorescence spectroscopy. In order to understand the forces involved in the StII differential association with phosphatidylcholine, sphingomyelin or ceramide, molecular dynamics simulation and energetic approaches allowed postulating the molecular determinants that distinguish binding to these lipids. In addition, StII binding to cellular membrane causes cytotoxic effect. By Flow Cytometry, we showed that StII action on human B lymphoma and murine P3X63Ag8.653 tumor cells led to cellular swelling. In Raji cells, StII at sublytic concentrations did not induce caspases activation. StII provoked calcium release from endoplasmic reticulum. Moreover, StII induced ERK1/2 activation and PD98059, an ERK1/2 inhibitor, reduced their cytotoxic activity. Necrostatin-1, a necroptosis inhibitor, reduced StII toxicity. Additionally, StII induced an increase of RIP1. Anti-tumor effect in vivo was study in a P3X63Ag8.653 tumor model in Balb/c mice. The intra-tumoral administration of StII reduced tumor volume without systemic toxicity. In summary, StII-membrane interaction, may respond to changes in lipid head properties and the access to sphingomyelin interfacial motif. StII induces cell death by a regulated necrosis. StII showed an anti-tumor effect in a murine model suggesting the antitumor properties of this toxin.

N-P 023 STICHOLYSIN II, A PORE FORMING TOXIN FROM SEA ANEMONE STICHODATYLA HELIANTHUS, INDUCES A STRONG IMMUNOMODULATORY EFFECTS AND COMPARABLE TO THOSE ELICITED BY THE EVOLUTIVELY DISTANT LISTERIOLYSIN O Anaixis del Valle, Laborde R.J. , Cruz Leal Y., Acosta-Rivero N., Labrada M., Nogueira V., Grubaugha D., Higgins D., Fernandez L.E. , Lanio M.E. Center for Protein Studies, Faculty of Biology, Havana University, Cuba. [email protected] Sticholysin II (StII) is a pore-forming protein (PFP) from the sea anemone Stichodactyla helianthus, whilst listeriolysin O (LLO) is a cholesterol-depending PFP produced by the bacterium Lysteria monocitogenes with widely demonstrated immunomodulatory properties. We have previously shown that liposomes comprised of dipalmitoyl phosphatidylcholine and cholesterol (DPPC:Chol,1:1 molar ratio) (Lp1:1) co-encapsulating ovalbumin (OVA) and StII (Lp1:1/OVA/StII), induce robust cytotoxic T lymphocytes (CTL) responses and anti-tumoral activity in mice. In this work, we aimed to compare the abilities of StII and LLO to induce bone marrow-derived dendritic cells (BMDCs) maturation, to stimulate cross-presentation of OVA to CD8+ T cells and to promote CTL responses. Interestingly, activation assays of BMDCs in vitro revealed that both StII and LLO induced BMDCs maturation. Besides, LLO stimulated the cross-presentation of OVA in both BMDCs and bone-marrow-derived macrophages (BMMs); while StII increased OVA crosspresentation just in BMDCs. However, StII increased OVA cross-presentation in BMMs when coencapsulated into Lp1:1/OVA/StII. To compare the capacity of StII and LLO to induce in vivo CTL responses, they were encapsulated into DPPC:Chol (molar ratio 2:1) liposomes to diminish the effect of Chol in LLO encapsulation. Importantly, liposomal formulations carrying either StII or LLO with OVA, showed similar CTL responses in vivo. Interestingly, mice immunized with Lp1:1/OVA/StII exhibited a higher antigen-specific CTL response in vivo as compared to that observed in mice treated with Lp2:1/OVA/LLO. Our results demonstrate for the first time that StII induce strong immunomodulatory effects and comparable to those elicited by the evolutively distant LLO.

N-P 024 PHYSICAL-CHEMICAL ASPECTS OF LIPOSOMES ENCAPSULATING THE HREGF-RP64K CONJUGATE Pedro L. Echevarria-Torres, Hechavarría Y., Santo JF. , Hernández YS., Cruz M, González G., Lanio ME., Luzardo MC. Departamento de Bioquimica y Centro de Estudio de Proteínas (CEP), Facultad de Biología, Universidad de la Habana(UH), La Habana, Cuba [email protected] CIMAvax-EGF® therapeutic vaccine is constituted by the hrEGF-rP64k conjugate and Montanide ISA 51VG. This adjuvant induces granulomas in injection sites and is not metabolized, hence the need to explore other vehicles for vaccine antigen. Liposomes are bio-compatible and not immunogenic particles without toxic effects. They could constitute an alternative adjuvant to obtain efficient anti-EGF immune response. Dehydration-rehydration technology allows the obtaining of liposomes (DRVs) with advantages for the encapsulation of labile molecules such as proteins. The use of sugars (e.g. sucrose) as additives in the liposome preparations increases their stability. This paper characterized physical-chemical aspects of DRVs liposomes that encapsulated hrEGF-rP64k in the presence or not of sucrose: particle size, polydispersity index and encapsulation efficiency (EE) by fluorescence and EGF-UMELISA. DRVs composed by dipalmitoyl phosphatidylcholine and cholesterol (DPPC:Cho; 1:1 molar ratio), were moderately polydisperses and about 1 μm size and encapsulated approximately 27% of hrEGF-rP64k. The addition of 14 mM sucrose previous to lyophilization of the vesicles, in sugar : phospholipid 3:1 mass ratio, reduced three times the size of liposomes and twice the EE of proteins. However, the presence of 40 mM sucrose did not modify the EE of the conjugate in DRVs compared to those that do not present the sugar. The addition of 14 mM sucrose favored the stability of liposomes stored in lyophilized form, by reducing the aggregation of vesicles and release of the encapsulated hrEGF-rP64k.

N-P 025 STICHOLYSIN II AS A POSSIBLE ENDOSOMOLYTIC COMPONENT FOR A NON-VIRAL GENE DELIVERY SYSTEM Felipe A. Escalona-Rodríguez, Cruz-Leal, Y., Lopetegui, I., Rivero-Hernández, A.L., Campos, G.S.M., Barbosa, L.R.S, Itri R., Sánchez B., Pérez, R., Lanio M.E. Center for Protein Studies (CEP), Biology Faculty, University of Havana, Havana, Cuba [email protected], [email protected] Non-viral delivery systems hold great promise for gene therapy. Despite synthetic vectors such as cationic polymers/lipids offer versatility, safety and relatively low cost for large-scale manufacturing compared to their viral counterparts, they are hampered by their low delivery efficiencies. Endocytosis is the major route of cellular entry for non-viral nucleic acid delivery. Efficient vectors should release their contents from endosome compartments at an early stage to prevent the fate of lysosomal destruction. A non-viral gene delivery system should carry functional components that can facilitate overcoming this barrier. Sticholysin II (StII) is a basic pore-forming toxin purified from the sea anemone Stichodactyla helianthus. This protein is positively charged at physiologic pH and shows permeabilizing activity on lipid membranes containing sphingomyelin by forming oligomeric pores with a diameter around 2 nm. In this study, we evaluated the capability of StII of interaction with model plasmids as a first step for design a vector based on this protein with enhanced endosomal escape. The ability of StII to bind DNA with high electrostatic stability was demonstrated by using electrophoretic mobility shift assay (EMSA). The structure and size of StII/DNA complexes depended on charge ratio used to produce them as was shown by atomic force microscopy (AFM) measurement in air. Nanometric particles were observed at a positive/negative charges ratio of 3.2. However, dynamic light scattering (DLS) revealed colloidal suspension of the complexes to be unstable resulting in formation of micrometric particles. Interestingly, complex formation did not affect pore-forming capacity of StII. This study suggests it is necessary to avoid the StII-plasmid direct interaction maybe by adding a condensing agent to achieve smaller complexes. StII could be an attractive endosomolytic agent in a gene delivery system although the protein activity outside the cell should be control. An approach using a StII mutant will be explored.

N-P 026 IN VIVO AND IN VITRO EFFECTS ON ANTIGEN-PRESENTING CELLS OF STICHOLYSIN II AND LIPOSOMES ENCAPSULATING THIS PORE-FORMING PROTEIN Rady J. Laborde, Fernández A., Del Valle A., Cruz-Leal Y., Luzardo M. C., Acosta N., Mesa C., Pazos F., Álvarez C., Alonso M. E., Abreu L., Longo I. M., Viana C. S., Grubaugha D., Higgins D., Fernández L. E., Lanio M. E. Center for Protein Studies and Dpt. Biochemistry, Faculty of Biology, University of Havana, Cuba [email protected] Cytotoxic CD8+ T lymphocytes (CTL) are crucial against diseases such as cancer. These cells need to be elicited by recognizing antigenic peptides in the context of the main histocompatibility complex class-I (MHC-I) molecules on antigen-presenting cells (APCs). Bacterial pore-forming proteins (PFPs) and liposomes encapsulating them, have been employed for favoring exogenous antigens access to MHC-I presentation on APCs and therefore to improve CTL responses. Sticholysin II (StII) is a PFP from the sea anemone Stichodactyla helianthus. Due to the functional homology of StII with bacterial PFPs, we studied its ability in solution or encapsulated into liposomes for enhancing the MHC-I presentation of the model antigen ovalbumin (OVA) on APCs such as dendritic cells and macrophages. Mouse bone marrow derived-dendritic cells (BM-DCs) pulsed with OVA simply mixed with StII activated CD8+ T lymphocytes from OT-1 mice and B3Z CD8+ T-cell line, suggesting StII potentialities to mediate the antigen access to MHC-I presentation. OVA-containing liposomes and co-encapsulating (Lp/OVA/StII) or not StII (Lp/OVA) were prepared. Interestingly, Lp/OVA/StII significantly enhanced the B3Z CD8+ T-cells activation as a consequence of antigen MHC-I presentation by BM-macrophages but not by BM-DCs. This process occurred even at antigen doses lower than those used with the simple mixture of StII and OVA. The ability of Lp/OVA/StII of mediating antigen MHC-I presentation was also demonstrated by in vivo cytotoxic assays. In this scenery, Lp/OVA/StII enhanced significantly an OVA-specific CTL response in relation to Lp/OVA and induced a potent antitumoral activity in a murine model. Besides, StII was able to activate BM-DCs and it was dependent on toll like receptor 4, as well as the stimulation of CTL response by Lp/OVA/StII. Our results suggest the potentialities of StII and StII-liposomes for the design of new MHC-I delivery system addressed to enhance a potent CTL immune response.

N-P 027 DEGRADATION OF ANTHRAQUINONIC AND AZOIC ACID DYES BY LACCASE OF GANODERMA WEBERIANUM B-18: IN VITRO AND IN SILICO STUDY Yosberto Cárdenas-Moreno, González-Durruthy Michael , Guerra-Rivera Gilda1, Sánchez-López M. I. Laboratory of Biotechnology, Department of Microbiology, Faculty of Biology, University of Havana, Cuba. [email protected], [email protected] Laccase catalyzes oxidation of certain aromatic compounds and similar structures reducing molecular oxygen to H20. Their low substrate specificity results on degradation of similar phenolics compounds. These characteristics make laccase an attractive solution to xenobiotic contaminants degradation, including antaquinonic and azoic acids. Kinetics of laccase enzyme production from Ganoderma weberianum B-18 strain, was carried out in Solid State Fermentation in cane bagasse, to determine the maximum days of laccase production, using as Intracid navy TR (INTR) and Remazol azul brillante (RBBR) substrates. In addition, 2,6 DMP was used to reveal this activity in native electrophoresis of the total of proteins in the enzymatic extracts. Molecular Docking was performed with INTR and RBBR ligands suggesting potential degradation by these enzymes. Binding active-sites prediction of fungal laccase (access number uniprotkb: A0A166P2X0), from Ganoderma weberianum was performed using machine learning algorithm based on Deep Convolutional Neural Networks (DeepSite-CNNs chemoinformatic tool).The eighth day was determined as the maximum production of laccase enzymes by both methods. From Free Energy of Gibbs, affinity values were obtained in the molecular docking studies and interacting aminoacid residues were described as well . Affinity values for INTR and RBBR ( kcal/mol) were 5.9 Kcal/mol and -6.8 Kcal/mol. These in silico and in vitro analyses suggest potential degradation of these compounds and possible application of these enzymes in degradation of xenobiotics compounds present in effluents of the textile industry such as anthraquinone and azo acids dyes.

N-P 028 REDISEÑO DE LA ESTRATEGIA DRDI PARA LA INMOVILIZACIÓN DE CÉLULAS EN SOLIDOS POROSOS: OPTIMIZACIÓN DE LA CARGA CELULAR EN EL BIOCATALIZADOR Castillo-Alfonso F, Bello MA , Palomino M, Salgado I, Rojas M, González Bacerio J, Guisán JM, Alberto del Monte-Martínez Centro de Estudio de Proteínas, Facultad de Biología, Universidad de La Habana, La Habana, Cuba. [email protected] Las técnicas de inmovilización no covalente como la adsorción iónica o el intercambio iónico con un soporte pueden representar una buena opción, pues este es un método simple, emplea poco tiempo y el soporte puede reutilizarse después de la desorción del derivado inmovilizado; de esta manera se reduce el precio final de la tecnología y se generan menos residuos. El Diseño Racional de Derivados Inmovilizados (DRDI) es una estrategia que combina herramientas matemáticas y bioinformáticas para diseñar procesos de inmovilización óptimos. En este trabajo se describen nuevos algoritmos matemáticos para perfeccionar la inmovilización de células a través de las interacciones electrostáticas en los intercambiadores iónicos; estos algoritmos pertenecen a la estrategia de DRDI. Específicamente se estimó la capacidad máxima de carga en el soporte para la inmovilización de células bacterianas con características de bacilos Gram positivos. Palabras Claves: inmovilización de células, diseño racional, diámetro celular, algoritmos matematicos.

N-P 029 ESTRATEGIA DE DISEÑO RACIONAL DE DERIVADOS INMOVILIZADOS APLICADA A LA INMOVILIZACIÓN DE LA LISOZIMA DE CLARA DE HUEVO EN EL SOPORTE SP-SEPHAROSE® FF P. L. Arruebo-Rivera, A. Troya Pérez, . Cárdenas-Moreno, P. Pérez Ramos, J. González-Bacerio, J. M. Guisán, Alberto del Monte-Martínez Centro de Estudio de Proteínas, Facultad de Biología, Universidad de La Habana, La Habana, Cuba. [email protected] Las técnicas de inmovilización por adsorción electrostáticas o de intercambio iónico con un soporte pueden representar una buena opción para la inmovilización de proteínas. Este es un método simple en el cual se emplea poco tiempo y el soporte puede reutilizarse después de la desorción del derivado inmovilizado. Como consecuencia se reduce el costo final del proceso. La inmovilización de proteínas se puede definir como aquel proceso mediante el cual se restringen completa o parcialmente los grados de libertad de movimiento de las proteínas, mediante su unión o confinamiento en el espacio de una superficie sólida. La aplicación de la metodología del DRDI a la inmovilización por adsorción electrostática de Lisozima de clara de huevo en el soporte SP-Sepharose® Fast Flow y los cálculos de predicción realizados mediante el programa RDID1.0 resultaron en una correspondencia y acercamiento entre los valores del parámetro eMQ predicho y el parámetro pMQ obtenido de manera experimental en el soporte SP-Sepharose® FF. El ensayo de actividad funcional con el sustrato “no convencional” Quitosana permite avalar la aplicación de estos derivados inmovilizados como biocatalizadores en procesos de bioconversión enzimática con aplicación en campos como la Industria Alimentaria, la Medico-Farmacéutica y la Cosmecéutica.

N-P 030 CHARACTERIZATION OF ENZYMATIC EXTRACTS FROM CUBAN GANODERMA STRAINS: APPLICABLE FOR THE DEGRADATION OF ENVIRONMENTAL POLLUTANTS? Lucía L. Ledo Alonso, Torres-Farradá G, Manzano León AM, Sánchez López MI, Guerra G , Vangronsveld J Laboratory of Biotechnology, Department of Microbiology and Virology, Faculty of Biology, University of Havana, Havana, Cuba. [email protected] Environmental pollution is one of the big problems in the world. The industrial development has enhanced the discharge to the environment of large quantities of chemical compounds with high toxicity and limited possibility of biodegradation. The use of enzymatic methods for remediation of polluted areas is a cost-effective and promising strategy. White rot fungi are the most efficient producers of oxidative extracellular enzymes with capacity to transform hazardous pollutants. The biotechnological potential of strains of genus Ganoderma remains underexplored. TorresFarradá et al., (2017) determined the diversity of the genes coding ligninolytic enzymes and their application for biodegradation of xenobiotic compounds of native strains of genus Ganoderma isolated in Cuba. However for a future biotechnological application, it is essential the determination of the stability of the ligninolytic enzymes to tolerate a wide range of abiotic conditions. The objective of this work was to characterize the crude enzyme extract produced by three Ganoderma strains with capacity to degrade xenobiotic compounds. The strains were cultured in SB-U medium and the crude enzymatic extracts were obtained after four days of incubation. The stability of the laccase enzymes and non-specific peroxidases from the enzymatic extracts was determined at different temperatures (30-60 0C), pH (3-9) and salinity (NaCl, Na2SO4, NaCO3). Moreover the decolorization capacity against four synthetic dyes was evaluated in the range of abiotic factors evaluated. The results showed that laccase enzymes produced by the strains Ganoderma weberianum B-18, Ganoderma sp. UH-L and Ganoderma sp. UH-M were stable for one week at basic and slightly acidic pH, temperature of 35 °C, and a range of salt concentrations which makes possible the enzymatic treatment of industrial effluents and the bioremediation of ecosystems with these characteristics. The catalytic versatility detected can give these strains advantages in their habitat and potential for the degradation of environmental pollutants.

N-P 031 LACCASE GENES AND THEIR ISOZYMES OF TWO GANODERMA STRAINS ASSOCIATED TO THE DEGRADATION OF XENOBIOTIC COMPOUNDS. Giselle Torres-Farradá, J-P. Noben, Francois Rineau, Erick Royackers, Ana M. Manzano, Miguel Ramos-Leal, Sofie Thijs, Lucía L. Ledo, M.I Sánchez, Gilda Guerra and Jaco Vangronsveld. Laboratory of Biotechnology, Department of Microbiology and Virology, Faculty of Biology, University of Havana. Cuba. [email protected] Laccases representing the largest subgroup of blue multi-copper oxidases; laccases use the redox ability of copper ions to catalyze the oxidation of aromatic substrates concomitantly with the fourelectron reduction of molecular oxygen to water. The broad range of substrate makes laccases excellent candidates for the bioremediation of toxic effluents and polluted ecosystems. Whiterot Fungi (WRF) are the most efficient- laccase producers. The ligninolytic machinery and biotechnological potential of well-adapted autochthonous strains belonging to genus Ganoderma remains underexplored. The objective of this study was: To analyze the laccase genes and their isozymes of Ganoderma weberianum B-18 and Ganoderma sp. UH-M associated to the degradation of xenobiotic compounds such as RBBR (synthetic dye) and naphthalene by laccase transcripts analysis and comparative secretome approach. The laccase transcripts were amplified with specific primers and the secretome analysis was performed by SDS-PAGE and LC-MS/MS. The combination of these methodologies allowed us to detect that laccase genes are differentially regulated in these strains. The genes lac 2, lac 4 and lac 6 from strain B-18 and genes lac II and lac VII from strain UH-M were constitutively expressed; however, the presence of pollutants induced the synthesis of the laccase isoforms encoded by genes lac 1, lac 3 from B-18 and lac V from UHM. Interesting, some of these genes, such as lac 4, lac 6 and lac II encode for laccase isoforms with different molecular weights, suggesting the presence of monomeric and dimeric structures of laccase isoforms. This study demonstrated the presence of some constitutive laccase genes while others are specifically induced by the presence of pollutants such as dyes and PAHs. With both approaches we could correlate the isoforms synthetized in different conditions with the laccase genes expressed. The laccase isoforms produced by Ganoderma strains are suitable for the degradation of a range of pollutants.

N-P 032 GENERATION

OF HIGH EXPRESSING CHO-DG44 CELL LINESOF A RITUXIMAB MABBIOSIMILARUSING IRES-MEDIATED TRICISTRONIC VECTORS Adriana Castillo-Caballero, Lao-González T., Sosa-Aguiar K., Hernández-García T., de la LuzHernández K., Boggiano-AyoT. Process Development Direction, INIM, Center of Molecular Immunology, Havana, Cuba [email protected] In order to obtain a high producer cell line of a Rituximab biosimilar, and at the same time, to establish an optimized protein expression platform, a system of tricistronic vectors based on IRES elements for the expression of mAb was developed in the CIM. AnIRES-mediated tricistronicvector expressing CHO cell lines to express the LC,HC, and a DHFR selection marker from one transcript isdesigned for generation of mAb. As compared to the commonly used vectors in CIM (lentiviral system), benefits of this design include: minimized non-expressing clones, enhanced stable mAb productivity, control of LC:HC expression ratios and consistent product quality. Two versions of tricistronic vectors expressing LC, HC, and DHFRto compare wild type (wt) and attenuate (att) IRES for their influence on mAb expression level and quality in CHODG44 cells were designed. Techniques of molecular biology to design the family of tricistronic vector, and clone the LC and HC of Rituximab Mab in this vectors and in the lentiviral vectors were employed. We characterize the pool obtained from stable transfection with tricistronic vectors in CHO-DG44 and lentiviral transduction in CHO-K1 by ELISA, SDS-PAGE, Western Blotting and FACS; and to obtain high producer clone by limiting dilution. The results showed that the transfection with attIRES-mediated tricistronic vector exhibits an increasein volumetric productivity and mAb expression level with respect towtIRES vector in the DG44 cell line. Bothtricistronic vector were more efficient in terms of better growth, minimized product aggregation, stability, quality and development timelines that co-transduction methodology with the lentiviral system in the CHOK1 cell line. The IRES-mediated tricistronic vector provides an attractive alternative to commonly used vectors for fast generation of mAb CHO cell lines with high productivity.

N-P 033 IN VITRO ASSESSMENT OF HER2 RECEPTOR EXPRESSION IN TUMOR CELL LINES BY RELAXOMETRY Yamirka Alonso, Harteman O, Reyes K, Lores MA, Gutierrez E, Toledano M, Álvarez ED Centro de Biofísica Médica (CBM), Patricio Lumumba s/n, Santiago de Cuba, Cuba [email protected] HER2 is a tyrosine kinase receptor, member of Epithermal Growth Factor Receptors family; it is over expressed in several types of tumours like breast, gastric and ovarian cancer. HER2 is used for diagnostic proposes in gene amplification techniques by Fluorescent in situ Hybridization (FISH) and in detection of receptor expression by immunohistochemistry (IH). Both procedures are very complex and take a lot of time to achieve the diagnosis. Recently advances in nanotechnology have been developed the immunodiagnostic techniques using magnetic nanoparticles. We propose assess the feasibility to use this technique for HER2 semiquantification in different tumoral cell lines. We conjugated the antibody anti-HER2 with carbon coated cobalt nanoparticles and we determined the expression of HER2 in cellular suspension of SKBr3, A431, A549 and Ramos cell lines (5x106 cells) by relaxometry using pulse series CarrPurcell-Meiboon-Gill (CPMG) at 4 MHz. The results show that SKBr3 cells have the lowest T2 value (51 ms), followed by A431 and A549 with 78 ms and 154 ms respectively. Additionally, we explored the behaviour of Ramos cells and it show the highest T2 value (457 ms). Comparing the T2 values of the cell lines targeted with antibody-nanoparticle system, we proved the statistically significant differences between the expression of HER2 receptor in all cell lines. T2 varies inversely HER2 expression level, therefore, the relative HER2 expression in studied cell lines is the follow order: SKBr3>A549>A431>>Ramos. According to our results, it is feasible the differential detection of HER2 expression level in the studied cell line by relaxometry.

N-P 034 CASEIN KINASE 2 IS REGULATED BY THE EIF4E RNA REGULON IN HUMAN OSTEOSARCOMA CELLS Perera Y., Culjkovic B., Ailyn de la Caridad Ramón, Perea SE., Borden KLB Laboratory of Molecular Oncology, Division of Pharmaceuticals, Center for Genetic Engineering and Biotechnology (CIGB), Havana, Cuba. [email protected] Casein Kinase K2 (CK2) is a serine-threonine protein kinase which phosphorylates more than 300 substrates including proteins related to cell survival and proliferation. The holoenzyme is a tetramer composed by two catalytic (α and/or α`) and two regulatory β subunits. Although CK2 over-expression is common in cancer, the molecular mechanism that leads to such aberrant expression is hardly known. Interestingly, an RNA Immunoprecipitation Assay (RIP) followed by high-throughput sequencing (RIP-seq) evidenced that eIF4E binds to CK2 mRNA in the nuclear fraction. eIF4E is a central node of an RNA regulon that coordinates the expression of several mRNAs connected to cell proliferation and malignant transformation. In the nucleus, eIF4E binds to 5`cap structure of mRNAs and the 4E-Sensitive Element (4ESE) to enhance their export and translation in the cytoplasm. Here, we conducted RIP experiments in eIF4E-over-expressed U2OS osteosarcoma cells followed by qPCR to detect eIF4E interaction with CK2α/α´/β mRNAs in nuclear fractions. mRNA export assays and Western blot experiments were done to associated such binding with enhanced export and protein levels of CK2 subunits. eIF4E and CK2 levels were donwregulated by siRNAs and their functional consecuences were evaluated in clonogenic and wound-healing assays. Our results indicated that eIF4E binds the mRNA of the three CK2 subunits in the nuclear fraction and enhance their export to cytoplasm, producing an slight increase of CK2α/α´/β functional protein levels. Accordingly, eIF4E-over-expression significantly reduced the antiproliferative potency of the CK2 inhibitor CX-4945. Of note, knockdown or pharmacologic inhibition of eIF4E decreased CK2α´/ β protein levels both in plain U2OS as well as eIF4E overexpressed U2OS cells. Finally, CK2α` knockdown decreased wound-healing and clonogenic potential of eIF4E-over-expressing cells. Altogether, these evidences suggest a mechanistic link between eIF4E and CK2, where CK2α´ but not CK2α might be instrumental to the eIF4E-mediated oncogenic program.

N-P 035 SÍNTESIS Y CARACTERIZACIÓN DE MUTANTES DE UN PÉPTIDO TIPO Β-DEFENSINA DE INVERTEBRADO Roberto Bello Madruga, Roque Diaz Y, Vázquez González A, Ortiz Castro D, Diago Abreu D., Perdomo Morales R, Montero Alejo V. Centro de Investigación y Desarrollo de Medicamentos, Cuba [email protected] Panusín es el primer representante peptídico de una nueva familia de péptidos antimicrobianos de amplio espectro, clasificado como defensina e identificado recientemente en crustáceos. Las características estructurales de este péptido, que incluye la presencia de un motivo α/β estabilizado por enlaces disulfuro, lo relacionan a las defensinas de vertebrados. La conservación estructural de aminoácidos básicos e hidrofóbicos en la familia de péptidos de tipo panusín sugiere la importancia de éstos residuos para ejercer su función biológica. La obtención y evaluación de las variantes mutadas en los residuos de tirosina presentes en panusín es el objetivo de estudio de nuestro trabajo. Las variantes de panusín con mutaciones puntuales Tyr/Ala se obtuvieron mediante síntesis en fase sólida y se evaluaron sus purezas mediante cromatografía líquida de alta resolución en fase reversa. La identificación de secuencias se determinó por espectrometría de masas. La actividad antimicrobiana se evaluó mediante el método de microdilución en caldo. Los resultados de este experimento reflejaron la importancia de los residuos de tirosina en la conservación de la actividad antibacteriana. La potencia microbicida de los péptidos se afectó significativamente en dependencia del número de residuos mutados, sugiriendo una participación de los mismos en su mecanismo de acción sobre la membrana bacteriana. La importancia e influencia de las tirosinas en la unión a modelos de membrana se realizó por la combinación de la fluorescencia intrínseca y apagamiento con acrilamida. De esta manera se confirmó que estos residuos determinan el grado de interacción y comprometimiento de panusín con la membrana bacteriana. La estructura secundaria de las variantes lineales se evaluó por dicroísmo circular y además se analizó en presencia de micelas de dodecilsulfato de sodio. Los péptidos se reorganizaron estructuralmente al interaccionar con sistemas micelares, lo que indica que los residuos de tirosina no influyen en la estructura secundaria adoptada.

N-P 036 SÍNTESIS Y CARACTERIZACIÓN DE ANÁLOGOS LINEALES DE UN PÉPTIDO TIPO B-DEFENSINA DE INVERTEBRADOS Yessica Roque Diaz, Bello Madruga R, Perdomo Morales R, Vásquez González A, Ortiz Castro D, Martínez Rodríguez JC, Montero Alejo V. Centro de Investigación y Desarrollo de Medicamentos, Cuba [email protected] Recientemente se ha reportado la presencia de una nueva familia de péptidos antimicrobianos tipo-defensina en invertebrados marinos. El miembro representativo de esta familia, panusín, es un péptido antimicrobiano de amplio espectro estructuralmente relacionado con las defensinas de mamíferos, mostrando un dominio α/β estabilizado por enlaces disulfuro. A partir de este molde natural se ha comenzado el estudio de su relación estructura actividad, siguiendo estrategias del diseño racional de péptidos. La influencia de los enlaces disulfuro de panusín sobre la actividad antibacteriana del mismo, es nuestro objeto de estudio. La obtención de variantes lineales de panusín se logró mediante la síntesis en fase sólida. La cromatografía líquida de alta resolución en fase reversa preparativa y analítica permitió la purificación y evaluación de pureza de los análogos sintetizados, respectivamente. La identificación de secuencia se realizó por espectrometría de masas. Los péptidos fueron evaluados mediante el método de microdilución en caldo para determinar su actividad antibacteriana. A partir de la construcción de vesículas liposomales y la fluorescencia intrínseca de las variantes se determinó la capacidad de unión de éstas a modelos de membrana. Mediante la espectropolarimetría de dicroísmo circular, se realizó el análisis de la estructura secundaria de las variantes y la molécula molde, empleando como disolventes agua, 2,2,2-trifluoroetanol y micelas de dodecilsulfato de sodio. Los resultados mostraron que se lograron obtener dos variantes lineales de panusín con buen rendimiento y pureza. Los péptidos conservaron la actividad antibacteriana, sugiriendo que los enlaces de disulfuro no son determinantes para ejercer la actividad de panusín, aunque influyen en la misma. La capacidad de unión a vesículas de estos péptidos mostró mayor afinidad con membranas negativas que mimetizan la composición de células bacterianas. El estudio de estructura secundaria mostró que a medida que el ambiente se hacía más hidrófobo, las variantes adoptaban una estructura secundaria definida.

N-P 037 ESPECIFICIDAD Y POSIBLE MECANISMO DE ACCIÓN DEL PÉPTIDO ANTIMICROBIANO GAM019 Y SUS ANÁLOGOS GAM020, GAM022 Y GAM026 CONTRA BACTERIAS GRAM POSITIVAS Y GRAM NEGATIVAS Stefania Correa, Bautista Y., Ortiz C., Méndez-Sánchez S., Urquiza M. Universidad Industrial de Santander, Bucaramanga, Colombia [email protected], [email protected] En los últimos años, el diseño de fármacos con potencial antibiótico ha surgido como una alternativa contra bacterias resistentes a los antibióticos convencionales. Los Péptidos Antimicrobianos (PAMs), son parte de esta alternativa debido a sus múltiples mecanismos de acción, amplio espectro antibacteriano y baja generación de resistencia. Algunos de estos PAMs son hélices anfipáticas y su principal blanco de acción es la membrana bacteriana, debido a interacciones electrostáticas entre los péptidos catiónicos y lípidos aniónicos expuestos en la superficie de las bacterias. El péptido GIBIM-P5 recientemente reportado, presenta potente actividad antibacteriana contra Staphylococcus aureus resistente a la meticilina (SARM), Escherichia coli O157:H7 (E.coli) y Pseudomona aeruginosa (P.aeruginosa); partiendo de él, se diseñó el péptido GAM019 y análogos a este con el fin de determinar el efecto de la estructura en la actividad biológica. La cinética de crecimiento de 12 cepas Gramnegativas y Grampositivas en presencia del péptido GAM019 y sus análogos a 50 μM fue determinada mediante el método de microdilución en caldo. Aquellos péptidos que presentaron inhibición del crecimiento bacteriano a esta concentración fueron probados a nuevas diferentes concentraciones de péptido contra cepas seleccionadas anteriormente. El efecto de los péptidos sobre membrana eucariota se determinó por actividad hemolítica sobre eritrocitos. Se encontró que estos péptidos, tienen amplio espectro de actividad independiente de la composición de membrana bacteriana por ejemplo, la Concentración Mínima Inhibitoria (CMI) en cepas como SARM y P.aeruginosa fue de CMI90 16 μM. Interesantemente, GAM019 y GAM026 presentan solo un 26% y 10% de actividad hemolítica a 128 μM. Además, los péptidos GAM019 y GAM022 a 20μM modifican drásticamente el potencial de membrana de P. aeruginosa, pasando de -62.4mV a -13.16mV y -28.3mV respectivamente. En conclusión, estos péptidos poseen amplio espectro de actividad antibacterial y muestran un efecto desestabilizador en las membranas favoreciendo su actividad antibacterial.

N-P 038 DIFERENCIAS EN LA CONCENTRACIÓN DE LA PROTEÍNA Β-AMILOIDE EN EL PLASMA DE SUJETOS SANOS Y PACIENTES CON ENFERMEDAD DE ALZHEIMER DETECTADOS POR ESPECTROSCOPIA RAMAN Nadia Tzayaka Castillo Mendieta, Soriano M. Segura J. Campos V. Guerra C. Instituto Mexicano del Seguro Social, Ciudad de México, México [email protected] La enfermedad de Alzheimer (EA) es una enfermedad neurodegenerativa, es la más común de las demencias. Actualmente, no hay investigación clínica, de laboratorio, genética o de neuroimagen que pueda dar un diagnóstico oportuno. La confirmación del diagnóstico de la EA solo se puede realizar con un examen post mortem del cerebro, por esto es necesario buscar un método no invasivo de diagnóstico temprano. En el presente trabajo se analizó el uso de la Espectroscopia Raman (ER) como una herramienta diagnóstica para la EA. Se analizó plasma de pacientes Mexicanos >60 años de edad diagnosticados clínicamente con EA (n=10) y sujetos control sin enfermedades neurológicas asociadas (n=10), las muestras de plasma fueron procesadas por ER. Para la búsqueda de biomarcadores en plasma se analizó un estándar puro de beta amiloide (Aβ) por ER, el cual fue comparado con las muestras de plasma recolectadas. Se observó que los espectros por ER son diferentes entre los pacientes con EA y los sujetos control. Al analizar el plasma de pacientes con EA por ER, se observaron bandas en un rango de 1000-1200 cm1, éstas coinciden con las bandas obtenidas al analizar el estándar de Aβ por ER. Las muestras de sujetos control muestran bandas en el rango para la proteína Aβ con una menor intensidad en relación a los pacientes de EA, lo que indica una menor concentración de esta proteína. En los espectros de pacientes con EA se observaron bandas en las amidas III, éstas se asocian al paso conformacional de α-hélice a β-plegada, lo cual indica un aumento en la concentración de Aβ. En conclusión, la ER nos permite detectar bandas asociadas a la proteína Aβ y diferencias en su concentración, lo cual sugiere que la ER puede ser utilizada como herramienta no invasiva para la detección de marcadores de la EA.

N-P 039 CHARACTERIZATION OF LOW‐ABUNDANCE SPECIES IN THE ACTIVE PHARMACEUTICAL INGREDIENT OF CIGB‐300: A CLINICAL‐GRADE ANTICANCER SYNTHETIC PEPTIDE Luis A. Espinosa, Garay H., Perera Y., Sánchez A, Diago D., Perea S., Besada V., Reyes O., González L. J. Mass Spectrometry Laboratory, Department of Proteomics, Center for Genetic Engineering and Biotechnology, Cuba [email protected] CIGB-300 is a first-in-class synthetic peptide-based drug of 25 amino acids currently undergoing clinical trials in cancer patients. It contains an amidated disulfide cyclic undecapeptide fused to the TAT cell-penetrating peptide through a beta-alanine spacer. CIGB-300 inhibits the CK2mediated phosphorylation leading to apoptosis of tumor cells in vitro, and in vivo in cancer patients. Despite the clinical development of CIGB-300, the characterization of peptide-related impurities present in the active pharmaceutical ingredient has not been reported earlier. In the decision tree of ICHQ3A (R2) guidelines, the daily doses intake, the abundance, and the identity of the peptide-related species are pivotal nodes that define actions to be taken (reporting, identification, and qualification). For this, purity was first assessed by reverse-phase chromatography (>97%) and low-abundance impurities (≤0.27%) were collected and identified by mass spectrometry. Most of the impurities were generated during peptide synthesis, the spontaneous air oxidation of the reduced peptide, and the lyophilization step. The most abundant impurity, with no biological activity, was the full-length peptide containing Met17 transformed into a sulfoxide residue. Interestingly, parallel and antiparallel dimers of CIGB-300 linked by 2 intermolecular disulfide bonds exhibited a higher antiproliferative activity than the CIGB-300 monomer. Likewise, very low abundance trimers and tetramers of CIGB-300 linked by disulfide bonds (≤0.01%) were also detected. Here we describe for the first time the presence of active dimeric species whose feasibility as novel CIGB-300 derived entities merits further investigation.

N-P 040 RAZÓN PSAT/E2 EN EL DIAGNÓSTICO DEL CÁNCER DE PRÓSTATA EN EL INOR. Isbel García Figueredo, Pereda Meira CM, Alonso Hernández C, Bouzó A, Chappé A, Quintero Cayola S. Instituto de Oncología y Radiobiología, Cuba [email protected] La elevada sensibilidad (S) y la baja especificidad (E) del PSA total, ocasiona un número elevado de biopsias innecesarias. El objetivo fue determinar el valor diagnóstico para el cáncer de próstata de los niveles de la razón PSAT/E, en el suero de pacientes sometidos a biopsia prostática, en el INOR. Se realizó un estudio de casos y controles en pacientes que con criterios para realizarles una biopsia prostática. Se incluyeron 86 pacientes, de éstos 35 pacientes con cáncer y 51 con hiperplasia (HPB) como control. Se observaron diferencias significativas entre los valores de los pacientes con cáncer respecto al grupo control (HPB), para las variables: PSAT, PSA/E2, (p 256,2. La razón PSAT/E2 mostró una elevada sensibilidad comparable con la obtenida para el PSAT y una mayor especificidad (72,50%), superior a los determinados para el PSAT, generando solamente un 30% de falsos positivos y un 9% de falsos negativos. El riesgo para valores de la razón PSAT/E2 superiores a 256,2 fue de OR=19,55 (IC=5,2572.87; p≤0.00001), lo que indicó que los pacientes tienen 19,55 veces más riesgo de tener un cáncer de próstata que aquellos que no. La razón PSAT/E2, permitió identificar los pacientes con cáncer respecto a los pacientes con hiperplasia prostática, mediante la implementación del valor de corte. Por lo que resultó ser un biomarcador más específico que el PSAT para el diagnóstico del cáncer de próstata. Además, se asoció con el riesgo al cáncer de próstata por lo que su empleo permitirá reducir el número de biopsias en los pacientes sometidos a ésta.

N-P 041 TESTOSTERONA, PSA Y VOLUMEN PROSTÁTICO, COMO VARIABLES DIAGNÓSTICAS PARA EL CÁNCER DE PRÓSTATA. ESTUDIO PRELIMINAR. Isbel García Figueredo, Candia MN, Pereda Meira CM, Alonso Hernández C, Bouzó A, Chappé A, Picans F, Quintero Cayola S. Instituto de Oncología y Radiobiología, Cuba [email protected]; [email protected] El tumor de próstata es la primera causa de muerte por cáncer en el hombre cubano. La baja especificidad de las herramientas diagnósticas pudiera influir en ello. El objetivo fue estimar el valor diagnóstico de la razón T/PSAT y T/PSAD en pacientes con diagnóstico presuntivo de cáncer de próstata. Se incluyeron 71 pacientes que se les realizó la biopsia prostática, 25 pacientes con diagnóstico de cáncer, 25 con lesiones intraepiteliales prostáticas (PIN) y 21 con hiperplasia (HPB).Se observaron diferencias significativas de los pacientes con cáncer respecto al resto de los grupos, para las variables: PSAT, PSAL, PSAD (p