In vitro evaluation of hydroxyapatite, chitosan, and carbon nanotube composite biomaterial to support bone healing / Avaliação in vitro do biomaterial compósito à base de hidroxiapatita, quitosana e nanotubo de carbono como adjuvante no reparo ósseo

Hydroxyapatite, chitosan, and carbon nanotube composite biomaterial were developed to improve bone healing. Previous studies suggested that a combination of biomaterials and mesenchymal stem cells (MSCs) can potentially help promote bone regeneration. In the present study, we first developed hydroxyapatite, chitosan, and carbon nanotube composite biomaterial. Then, the effect of different concentrations of the extract on the viability of Vero cells (ATCC CCL-81) and MSCs obtained from sheep bone marrow using methylthiazol tetrazolium (MTT) and propidium iodide (PI) assays were evaluated. The biomaterial group demonstrated an absence of cytotoxicity, similar to the control group. Samples with 50% and 10% biomaterial extract concentrations showed higher cell viability compared to samples from the control group (MTT assay). These results suggest that the presence of this composite biomaterial can be used with MSCs. This study also concluded that hydroxyapatite, chitosan, and carbon nanotube composite biomaterial were not cytotoxic. Therefore, these could be used for performing in vivo tests.(AU)

O compósito à base de hidroxiapatita, quitosana e nanotubo de carbono foi desenvolvido com o intuito de auxiliar na consolidação óssea. Estudos anteriores sugerem que a combinação de substitutos ósseos e células-tronco mesenquimais (CTM) podem auxiliar a potencializar e promover a regeneração óssea. No presente estudo, o biomaterial foi desenvolvido e a viabilidade e a citotoxicidade de células Vero (ATCC CCL-81) e CTM obtidas de medula óssea provenientes de ovinos utilizando ensaios metil-tiazol-tetrazólio, MTT e iodeto de propídeo (PI) foram avaliadas em diferentes concentrações de extrato desse compósito. O compósito demonstrou ausência de citotoxicidade com comportamento semelhante ao grupo controle. Amostras com 50% e 10% de concentração de extrato do compósito mostraram resultados maiores comparados ao grupo controle (ensaio MTT). Esses resultados também sugerem que a presença do biomaterial pode ser utilizada em associação a CTM. Assim, esse estudo conclui que o compósito apresentado de hidroxiapatita, quitosana e nanotubo de cabono não foi considerado citotóxico e pode ser utilizado em teste in vivo.(AU)

Luz, giberelinas y profundidad de siembra inciden sobre la germinación de semillas de Amaranthus hybridus L / Light, gibberellins and burial depth affect seed germination of Amaranthus hybridus L

RESUMEN Amaranthus hybridus L., conocido como bledo, es una planta maleza en una gran variedad de cultivos en el mundo, que se caracteriza por producir alto número de semillas latentes. Para generar estrategias de manejo sostenibles en esta especie es necesario comprender mejor la fisiología de sus semillas. Por tanto, el objetivo fue evaluar el efecto de la luz, de las giberelinas y la profundidad de la siembra sobre la germinación de semillas de bledo. En un primer experimento, se evaluaron cuatro tratamientos de luz (rojo, azul, sin luz y luz natural «testigo¼); en el segundo, se imbibieron semillas en diferentes concentraciones de ácido giberélico (0, 200, 400 y 800mg L-1 de GA3) y, en un último experimento, se evaluaron ocho profundidades de siembra (10, 20, 30, 40, 50, 60, 70 y 80mm), utilizando turba como sustrato. Se encontró mayor porcentaje de germinación en oscuridad, indicando que son semillas fotoblásticas negativas. Para el experimento de giberelinas, el mejor resultado se obtuvo con 400 y 800mg L-1 de ácido giberélico. En el tercer experimento, se observó alto porcentaje de emergencia de plántulas en las primeras profundidades (10mm a 30mm); a mayor profundidad hubo menor emergencia. La respuesta diferencial a la luz, a la aplicación de las giberelinas y el alto porcentaje de semillas viables que no germinaron permite concluir que semillas de bledo poseen latencia fisiológica.

ABSTRACT Amaranthus hybridus L., known as bledo, is a weed in a wide variety of crops worldwide, characterized by producing high number of seeds with dormancy. To generate sustainable management strategies of this species, it is necessary to better understand the physiology of its seeds. Therefore, the objective was to evaluate the effect of light, gibberellins, and seed burying depth on seed germination of bledo. In the first experiment, four light conditions were evaluated (red, blue, without light and natural light (control)), in the second experiment the seeds were imbibed in different concentrations of gibberellic acid (0, 200, 400 and 800mg L-1 of GA3), and in the last experiment, 8 planting depths were evaluated (10, 20, 30, 40, 50, 60, 70 or 80mm) using peat as a substrate. A higher percentage of seed germination in darkness was found, indicating that this species had negative photoblastic seeds. For the gibberellin experiment, the best result was obtained with treatment with 400 and 800mg L-1 of gibberellic acid. In the third experiment, a high percentage of seedling emergence was observed from the first depths (10mm to 30mm); the greater was the depth, the less emergence was found. The differential responses to light, the application of gibberellins, and the high percentage of non-germinated viable seeds allowed to conclude that bledo seeds possess physiological dormancy.

Morphological characteristics and seed viability of Schizolobium parahyba (Vell.) S.F. Blake / Características morfológicas y viabilidad de semillas de Schizolobium parahyba (Vell.) S.F. Blake

ABSTRACT Schizolobium parahyba is a fast-growing tropical tree, with various uses: forestry, agroforestry, landscaping, boat building, cabinetmaking and light packaging. Studies of morphometry and viability of forest species seeds are important for their identification, conservation and sustainable use. The objective of the study was to describe the morphometric and viability characteristics of the seeds of S. parahyba. Seeds of free pollination of plantations from three locations in Tierralta (Córdoba - Colombia) were used. In each plantation, five trees were randomly taken and, of each, 100 seeds to estimate the morphometric characteristics and seed weight. The morphological description was made with a sample of 10 seeds. The viability was determined by the tetrazolium test in an experiment under a completely randomized design, with six treatments and four replicates of 25 seeds each. The six treatments corresponded to the combinations of three concentrations of tetrazolium (0.5; 1.0 and 1.5%) and two staining times (2 and 3 hours). The width, length, and ratio width / length of the seed showed little variation, compared to the freshweight of a seed, freshweight of 100 seeds and number of seeds per kilogram. Tetrazolium and germination tests showed similar and reliable results. The concentration of 0.5% tetrazolium, with immersion of 2 hours, was sufficient to determine the viability of seeds of S. parahyba.

RESUMEN Schizolobium parahyba es un árbol tropical de crecimiento rápido, con diversos usos: forestería, agroforestería, paisajismo, construcción de embarcaciones, ebanistería y embalajes livianos. Los estudios de morfometría y viabilidad de semillas de especies forestales son importantes para la identificación, la conservación y el uso sostenible de éstas. El objetivo del estudio fue describir las características morfométricas y viabilidad de la semilla de S. parahyba. Se utilizó semilla sexual de polinizaciónlibre de plantaciones de tres localidades de Tierralta (Córdoba, Colombia). En cada plantación, se tomaron al azar cinco árboles y de cada uno 100 semillas, para la estimación de las características morfométricas y el peso de la semilla. La descripción morfológica, se hizo con una muestra de 10 semillas. La viabilidad, se determinó mediante la prueba de tetrazolio, en un experimento bajo un diseño completamente al azar, con seis tratamientos y cuatro repeticiones de 25 semillas cada una; los seis tratamientos correspondieron a las combinaciones de tres concentraciones de tetrazolio (0,5; 1,0 y 1,5%) y dos tiempos de tinción (2 y 3 horas). Las características de ancho, longitud y relación ancho/longitud de la semilla, acusaron poca variación, en comparación con el peso de una semilla, peso frescode 100 semillas y número de semilla por kilogramo. Las pruebas de tetrazolio y de germinaciónmostraron resultados similares y confiables. La concentración de 0,5% de tetrazolio, con inmersión de 2 horas, es suficiente para determinar la viabilidad de la semilla de S. parahyba.

Viability of Cyperus rotundus L. tubers after application of herbicide in pre- and postemergence / Viabilidade de tubérculos de Cyperus rotundus L. após a aplicação de herbicidas em pré e pós-emergência

Chemical management is the most widely adopted technique to control weeds in sugarcane crops. Purple nutsedge (Cyperus rotundus L.) is among the most difficult species to control. Herbicides need to be absorbed and translocated in a sufficient amount for the tuber chain to be effective. Thus, this study aimed to evaluate the effectiveness of the herbicides sulfentrazone, diclosulam, imazapic, imazapyr, halosulfuron, ethoxysulfuron, monosodium methyl arsenate (MSMA), and 2,4-dichlorophenoxyacetic acid (2,4-D) in controlling C. rotundus and reducing the viability of its tubers. The experiment was carried out in a greenhouse in a completely randomized design, with nine treatments and four replications. The herbicides sulfentrazone (800 g active ingredient ­ ai·ha?1), diclosulam (193.17 g ai·ha?1), imazapic (133 g ai·ha?1), and imazapyr (500 g acid equivalent ­ ae·ha?1) were applied in preemergence at five days after planting the tubers, while halosulfuron (112.5 g ai·ha?1), ethoxysulfuron (135 g ai·ha?1), MSMA (1,975 g ai·ha?1), and 2,4-D (1,340 g ae·ha?1) were applied in postemergence (4 to 5 leaves). The parameters visual control, shoot dry matter, number of tubers and bulbs, weight of tubers + bulbs, number of epigeal manifestations, and tuber viability were analyzed. The herbicides diclosulam, halosulfuron, and ethoxysulfuron provided 100% control of the shoot at 90 days after application (DAA). All herbicides reduced the number of bulbs, weight of tubers + bulbs, and shoot dry matter. The herbicides sulfentrazone, imazapic, halosulfuron, ethoxysulfuron, and MSMA provided the highest reduction in tuber viability.(AU)

O manejo químico é o mais adotado para o controle de plantas daninhas na cultura da cana-de-açúcar. Entre as espécies de difícil controle está a tiririca (Cyperus rotundus L.). Os herbicidas utilizados precisam ser absorvidos e transportados para a cadeia de tubérculos em quantidades suficientes para serem eficazes. Assim, objetivou-se com este estudo avaliar a eficácia dos herbicidas sulfentrazona, diclosulam, imazapique, imazapir, halossulfurom, etoxissulfurom, metano arseniato ácido monossodico (MSMA) e ácido diclorofenoxiacético (2,4-D) no controle de C. rotundus e na redução da viabilidade de seus tubérculos. O experimento foi realizado em casa de vegetação, em delineamento inteiramente randomizado, com nove tratamentos e quatro repetições. Os herbicidas sulfentrazone (800 g ingrediente ativo ­ i.a.·ha-1), diclosulam (193,17 g i.a.·ha-1), imazapique (133 g i.a.·ha-1) e imazapir (500 g equivalente ­ e.a.·ha-1) foram aplicados cinco dias após o plantio dos tubérculos em pré-emergência, enquanto que o halossulfurom (112,5 g i.a.·ha-1), etoxissulfurom (135 g i.a.·ha-1), MSMA (1.975 g i.a.·ha-1) e 2,4-D (1.340 g e.a.·ha-1) foram aplicados em pós-emergência (4 a 5 folhas). Foram feitas avaliações visuais de controle, massa seca da parte aérea, número de tubérculos e bulbos, peso de tubérculos + bulbos, número de manifestações epígeas e viabilidade de tubérculos. Aos 90 dias após a aplicação (DAA) os herbicidas diclosulam, halossulfurom e etoxissulfurom propiciaram 100% de controle da parte aérea. Todos os herbicidas reduziram o número de bulbos, peso de tubérculos + bulbos e massa seca da parte aérea. Sulfentrazona, imazapic, halossulfurom, etoxissulfurom e MSMA foram os herbicidas que mais reduziram a viabilidade dos tubérculos.(AU)

Vigor tests in seeds creole of Phaseolus lunatus L / Testes de vigor em sementes crioulas de Phaseolus lunatus L

Seeds with a good physiological quality are essential to high productivity. However, for some seeds, Phaseolus lunatus L., the viability and vigor analyze show deficiency in quality. This work aimed to evaluate the physiological quality of Phaseolus lunatus L. seeds using several vigor tests. The study was realized at the Laboratory of Seed Analysis of the Agricultural Sciences Center of the Federal University of Paraíba, in Areia ­ PB, Brazil. We used seeds from five cultivars of P. lunatus (Branca, Orelha de Vó, Cearense, Rosinha, and Roxinha). To determine the physiological quality, we measured the water content, the viability and vigor test (germination, emergence, electrical conductivity, tetrazolium, first germination count and emergence, seedling length and dry mass). We used a completely randomized design for seeds evaluation, and the field test was performed in randomized blocks. The water content of P. lunatus did not differ among the cultivars, indicating a uniformity in the percentage of moisture. Also, the percentage and speed of emergency and the dry mass of seedlings did not differ among the varieties. The tetrazolium and electrical conductivity tests indicated that Rosinha cultivar had the worst viability concerning the other cultivars, showing a low percentage of living tissue and lower electrical conductivity. The seeds of Roxinha cultivar had the better physiological quality, and the Cearense cultivar had the worst quality evaluation. We recommend the tetrazolium test but not the electrical conductivity test to evaluate the vigor of P. lunatus.

Em qualquer cultura, para se ter uma elevada produtividade é indispensável à aquisição de sementes com alta qualidade fisiológica, mas para sementes de Phaseolus lunatus L., ainda ocorre deficiência em análises de viabilidade e vigor. Dessa forma, o objetivo neste trabalho foi avaliar a qualidade fisiológica das sementes de cultivares de Phaseolus lunatus por diferentes testes de vigor. A pesquisa foi realizada no Laboratório de Análise de Sementes do Centro de Ciências Agrárias da Universidade Federal da Paraíba, em Areia - PB, com sementes de cinco cultivares de Phaseolus lunatus (Branca, Orelha de Vó, Cearense, Rosinha e Roxinha). Para avaliação da qualidade fisiológica das sementes determinou-se o teor de água e realizou-se testes de viabilidade e vigor (germinação, emergência, condutividade elétrica, tetrazólio, primeira contagem de germinação e emergência, comprimento e massa seca de plântulas). O delineamento utilizado foi o inteiramente casualizado, com exceção dos testes realizados em campo, que foi em blocos ao acaso. O teor de água das sementes dos cultivares de feijão-fava não diferiu estatisticamente entre si, indicando que houve uniformidade na porcentagem de umidade. Não houve diferença significativa entre as sementes dos cultivares de feijão-fava quando avaliou-se a porcentagem e velocidade de emergência, bem como a massa seca de plântulas. Os resultados dos testes de tetrazólio e condutividade elétrica constatou-se maior viabilidade para as sementes do cultivar Roxinha, Branca, Cearense e Orelha de Vó quando comparadas com as da Rosinha, uma vez que houve um maior percentual de tecidos vivos e uma menor condutividade elétrica, respectivamente. As sementes do cultivar Roxinha são de melhor qualidade fisiológica e, o cultivar Cearense de pior qualidade; o teste de tetrazólio é recomendado para avaliação do vigor de sementes de cultivares de Phaseolus lunatus, enquanto, o teste de condutividade elétrica não é recomendado para avaliar o vigor das sementes dos cultivares de P. lunatus.

Efeitos citotóxicos do aripiprazol nas linhagens celulares MKN45 e NIH3T3 e efeitos genotóxicos nos linfócitos sanguíneos periféricos humanos / Cytotoxic effects of aripiprazole on mkn45 and nih3t3 cell lines and genotoxic effects on human peripheral blood lymphocytes

ABSTRACT BACKGROUND: Gastric cancer is known as the fourth most common cancer. Current treatments for cancer have damaged the sensitive tissues of the healthy body, and in many cases, cancer will be recurrent. Therefore, need for treatments that are more effective is well felt. Researchers have recently shifted their attention towards antipsychotic dopamine antagonists to treat cancer. The anticancer activities of aripiprazole remain unknown. OBJECTIVE: This study aimed to evaluate the efficacy and safety of aripiprazole on gastric cancer and normal cell lines. METHODS: In this regard, the cytotoxicity and genotoxicity of aripiprazole were investigated in MKN45 and NIH3T3 cell lines by methyl tetrazolium assay and on peripheral blood lymphocytes by micronucleus assay. For this purpose, cells were cultured in 96 wells plate. Stock solutions of aripiprazole and cisplatin were prepared. After cell incubation with different concentrations of aripiprazole (1, 10, 25, 50, 100 and 200 μL), methyl tetrazolium solution was added. For micronucleus assay fresh blood was added to RPMI culture medium 1640 supplemented, and different concentrations of aripiprazole (50, 100 and 200 μL) were added. RESULTS: The finding of present study showed that the IC50 of aripiprazole in the cancer cell line (21.36 μg/mL) was lower than that in the normal cell line (54.17 μg/mL). Moreover, the micronucleus assay showed that the frequency of micronuclei of aripiprazole at concentrations below 200 μM was much less than cisplatin. CONCLUSION: Aripiprazole can be a good cytotoxic compound and good candidate for further studies of cancer therapy.

RESUMO CONTEXTO: O câncer gástrico é conhecido como o quarto câncer mais comum. Os tratamentos atuais para o câncer danificaram os tecidos sensíveis do corpo saudável e, em muitos casos, o cancro será recorrente. Portanto, a necessidade de tratamentos que são mais eficazes é desejada. Recentemente, os pesquisadores mudaram sua atenção para os antagonistas antipsicóticos da dopamina para tratar o câncer. As atividades anticâncer de aripiprazol permanecem desconhecidas. OBJETIVO: Este estudo objetivou avaliar a eficácia e a segurança do aripiprazol no câncer gástrico e nas linhagens celulares normais. MÉTODOS: A este respeito, a citotoxicidade e a genotoxicidade do aripiprazol foram investigadas em linhas celulares MKN45 e NIH3T3 por ensaio de metil tetrazólio e em linfócitos periféricos de sangue por ensaio de micronúcleos. Para este efeito, as células foram cultivadas em 96 placas. As soluções de estoque de aripiprazol e cisplatina foram preparadas. Após incubação celular com diferentes concentrações de aripiprazol (1, 10, 25, 50, 100 e 200 μL), a solução de metil tetrazólio foi adicionada. Para o ensaio do micronúcleo o sangue fresco foi adicionado ao meio de cultura RPMI 1640 suplementado, e as concentrações diferentes de aripiprazole (50, 100 e 200 μL) foram adicionadas. RESULTADOS: O presente estudo mostrou que o IC50 de aripiprazol na linhagem celular cancerosa (21,36 μg/mL) foi menor do que na linha celular normal (54,17 μg/ mL). Além disso, o ensaio de micronúcleos demonstrou que a frequência de micronúcleos de aripiprazol em concentrações inferiores a 200 μM foi muito inferior à cisplatina. CONCLUSÃO: O aripiprazol pode ser um bom composto citotóxico e bom candidato para estudos adicionais da terapia do câncer.

Viabilidad y germinación de semillas de Hechtiaperotensis (Bromeliaceae) / Viability and germination of Hechtia perotensis (Bromeliaceae) seed

ResumenPoblaciones endémicas de Hechtiaperotensis se han descrito en Puebla y Veracruz, México. La recolección de semillas de buena calidad permite su uso en conservación, investigación y restauración ecológica. Algunos compuestos para evaluar la calidad de las semillas silvestres y endémicas, como nitrato de potasio (KNO3) y ácido giberílico (AG3), se utilizan para incrementar la germinación de las semillas y disminuir la latencia. La prueba de cloruro de trifeniltetrazolio (tetrazolio) se correlaciona con la viabilidad de las semillas porque se basa en la actividad de las deshidrogenasas de tejidos vivos que catalizan la respiración mitocondrial. El objetivo de este estudio fue conocer el tamaño y el peso de las cápsulas y las semillas y la germinación y viabilidad de las semillas de H. perotensis recolectadas en Veracruz en 2012 y 2015. Las hipótesis fueron (1) que la germinación y la viabilidad de las semillas son independientes del año de recolecta, (2) que hay una concentración adecuada de tetrazolio para identificar la viabilidad de las semillas y (3) que el pretratamiento con KNO3 o AG3 incrementa la germinación de las semillas. La germinación se evaluó con un diseño completamente al azar con tres tratamientos (testigo y promotores de la germinación KNO3 0.2 % y AG3 500 mg/L), cuatro tratamientos para la prueba de viabilidad (testigo, 0.2, 0.5 y 1.0 % de tetrazolio) y seis repeticiones de cada tratamiento. La germinación se evaluó en 100 semillas y la viabilidad en 25. Los resultados entre y dentro de años se analizaron con ANDEVA y prueba de comparación múltiple de medias de Tukey. La proporción de semillas no germinadas se cuantificó junto con el número de plántulas normales y anormales, semillas con embrión viable o sin él, y con viabilidad baja o sin viabilidad. En promedio la muestra recolectada en 2012 tuvo 36 % de semillas con embrión viable, 7 con viabilidad baja, 24 % no viables y 33 % sin embrión. Este resultado fue significativamente diferente al de 2015, que presentó 87 % de embriones viables, 10 % con viabilidad baja, 0 % no viables y 3 % sin embrión. La germinación también fue significativamente diferente entre los años (22 y 92 %). Los tratamientos pregerminativos no modificaron la germinación. La germinación y la viabilidad de las semillas de H. perotensis varían significativamente entre los años de recolecta.

Abstract:Endemic populations of Hechtiaperotensis have been described in Puebla and Veracruz, Mexico. Good quality seed collections can be used in conservation, research and ecological restoration. To evaluate seed quality of wild and endemic species, some compounds are used as effective promoters of germination, such as potassium nitrate (KNO3) and gibberellic acid (AG3), because they increase seed germination capacity and reduce latency. The triphenyl tetrazolium chloride (tetrazolium) test correlates seed viability because it is based on the activity of dehydrogenases in live tissues that catalyze mitochondrial respiration. The objective of this study was to obtain information on size and weight of capsules and seeds and seed germination and viability of H.perotensis, collected in Veracruz in the year 2012 and 2015. The hypotheses were 1) that seed germination and viability are independent of the year of collection, 2) that there is a tetrazolium concentration that can identify seed viability better than others, and 3) that pretreatment with KNO3 or AG3 improves seed germination. Seed germination was assessed using a completely randomized design with three treatments (control and the germination promoters 0.2 % KNO3 and 500 mg/L AG3), four treatments for the viability test (control, 0.2, 0.5 and 1.0 % of tetrazolium) and six replicates for each treatment. A total of one hundred seeds for germination experiments, and 25 seeds for the viability test were used. The results between and within years were analyzed with ANOVA and multiple comparison with the Tukey test. The proportion of non-germinated seeds was quantified along with the number of normal and abnormal seedlings, seeds with viable embryo, seeds without embryo, and seeds with low or no viability. On average, for the 2012 collected sample, 36 % had viable embryos, 7 % had low viability, 24 % were not viable and 33 % had no embryo. This result was significantly different from the 2015 sample, for which 87 % of seed showed viable embryos, 10 % had low viability, 0 % was not viable and 3 % had no embryo. Seed germination was also significantly different between years (22 and 92 %) Pregerminative treatments did not improve germination. Seed germination and viability of H. perotensis significantly varied between years of seed collection. Rev. Biol. Trop. 65 (1): 153-165. Epub 2017 March 01.

Histochemical Evaluation of the Vessel Wall Destruction and Selectivity After Treatment with Intense Pulsed Light in Capillary Malformations.

BACKGROUND: Among the different approaches for improving the effectiveness in the treatment of Capillary Malformations type Port Wine Stain (CM type PWS) are the intense pulsed light sources. There are few clinical studies prove useful in the treatment of CM. Furthermore, no studies have been published yet demonstrating the histological effects of IPL in CM. OBJECTIVES: To assess the histological effects of pulsed light in capillary malformations type port wine stain. We wanted to compare epidermal, dermal and vessel wall damage after treatment with different combinations of IPL parameters. MATERIAL AND METHODS: Fifty-five post-treatment biopsies were performed in 15 consenting patients with CM and stained with nitroblue-tetrazolium chloride (NBTC). Patients had not been treated previously. RESULTS: Fifteen patients with CM, with a median age of 39 years-old were enrolled in this study. In this series, the patients with the most severe epidermal damage were those with a darker phototype. Pink CM were especially resistant to treatment, even using high fluences, short pulse durations and stacking pulses. Longer intra- and interpulse delays were effective in purple CM, achieving adequate vessel destruction. CONCLUSIONS: IPL devices provide a vast amount of treatment possibilities and further studies are necessary to optimize therapeutic approaches to CM. In this study we have observed the histological effects of different pulses on the MC type PWS.

Comparação entre duas metodologias para a realização do teste de redução do tetrazólio nitroazul (NBT) em caprinos / Comparison among two methodologys at realization nitroblue tetrazolium reduction test (NBT) in caprines

O teste de redução do tetrazólio nitroazul (NBT) é um dos métodos mais utilizados para a avaliação do metabolismo oxidativo dos neutrófilos. Porém, o custo por amostra e o tempo de utilização do corante são desvantagens do método. O objetivo deste trabalho foi comparar duas metodologias para realização do teste do NBT a fim de maximizar a sua utilização e diminuir o custo por amostra processada. Foram utilizadas 10 cabras, adultas clinicamente sadias e colhidas amostras de sangue por meio de venipunção jugular em tubos de 10mL sendo retirada uma alíquota de 500µL em tubo "eppendorff" contendo 2µL de heparina. Através desta alíquota foram realizadas três metodologias para o teste do NBT: A) técnica original Park & Good (1970); B) redução de 50% do volume do corante; C) redução de 50% e armazenamento do corante a -20°C por 180 dias. Foram confeccionados e corados esfregaços sanguíneos (May Grünwald-Giemsa) das amostras com as metodologias propostas e realizada contagem de 100 neutrófilos em microscopia óptica para determinar o percentual de neutrófilos reativos ao NBT. A análise estatística pelo método de Análise de Medidas Repetida (ANOVA) demonstrou diferenças significativas (P<0,05) entre os dois métodos e o original (A=14,4±4,6; B=1,9±1,4; C=1,1±0,9), porém apresentou alta correlação pelo teste de Spearman entre os métodos (rs=0,82 (AxB), rs=0,75 (BxC) e rs=0,93 (AxC)). Conclui-se que o teste de Redução do Tetrazólio Nitroazul (NBT) segundo a metodologia descrita por Park & Good (1970) e a redução do corante em 50% do seu volume e a sua redução associada ao congelamento por 180 dias a -20ºC podem ser realizadas em caprinos, porém os valores devem ser comparados com as respectivas metodologias utilizadas.(AU)

The nitroblue tetrazolium reduction test (NBT) is one of the most used methods to evaluate the oxidative metabolism of neutrophils. However, the cost for each sample and dye's life-time are disadvantages of the method. This paper aim was to compare two NBT test methodologies in order to maximize its use and decrease the cost for each sample. It was made using 10 adult and healthy goats and blood samples were taken through venipuncion in jugular vein, using 10mL tubes, and then a 500µL sample was taken in eppendoff tubes with 2µL of heparin. Using this sample were made three methodologies for the NBT test: A) Park & Good (1970) original technique; B) dye reduction in 50% of the volume; C) 50% reduction and dye storage at -20°C for 180 days. It was made and dyed the blood smear (May Grünwald-Giemsa) from the samples, using the proposed methodologies and then made the counting of 100 neutrophils in optic microscopy, to determinate the percentage of NBT reactive neutrophils. The statistic analysis by the method of Repeated Measuring Analysis (ANOVA) evinced significant differences (P<0,05) between the two methods and the original (A=14.4±4.6; B=1.9±1.4; C=1.1±0.9), however it showed high correlation by the Spearman test between the methods (rs=0,82 (AxB), rs=0,75 (BxC) e rs=0.93 (AxC)). It was concluded that the original Park & Good (1970) method and two methodologies for dye reduction in 50% and it's reduction associated with NBT dye freezing for 180 days can be used in caprines, but the values should be compared with their respective methodologies.(AU)

Comparação entre duas metodologias para a realização do teste de redução do tetrazólio nitroazul (NBT) em caprinos

Abstract: The nitroblue tetrazolium reduction test (NBT) is one of the most used methods to evaluate the oxidative metabolism of neutrophils. However, the cost for each sample and dye's life-time are disadvantages of the method. This paper aim was to compare two NBT test methodologies in order to maximize its use and decrease the cost for each sample. It was made using 10 adult and healthy goats and blood samples were taken through venipuncion in jugular vein, using 10mL tubes, and then a 500L sample was taken in eppendoff tubes with 2L of heparin. Using this sample were made three methodologies for the NBT test: A) Park & Good (1970) original technique; B) dye reduction in 50% of the volume; C) 50% reduction and dye storage at -20°C for 180 days. It was made and dyed the blood smear (May Grünwald-Giemsa) from the samples, using the proposed methodologies and then made the counting of 100 neutrophils in optic microscopy, to determinate the percentage of NBT reactive neutrophils. The statistic analysis by the method of Repeated Measuring Analysis (ANOVA) evinced significant differences (P 0,05) between the two methods and the original (A=14.4±4.6; B=1.9±1.4; C=1.1±0.9), however it showed high correlation by the Spearman test between the methods (rs=0,82 (AxB), rs=0,75 (BxC) e rs=0.93 (AxC)). It was concluded that the original Park & Good (1970) method and two methodologies for dye reduction in 50% and it's reduction associated with NBT dye freezing for 180 days can be used in caprines, but the values should be compared with their respective methodologies.

Resumo: O teste de redução do tetrazólio nitroazul (NBT) é um dos métodos mais utilizados para a avaliação do metabolismo oxidativo dos neutrófilos. Porém, o custo por amostra e o tempo de utilização do corante são desvantagens do método. O objetivo deste trabalho foi comparar duas metodologias para realização do teste do NBT a fim de maximizar a sua utilização e diminuir o custo por amostra processada. Foram utilizadas 10 cabras, adultas clinicamente sadias e colhidas amostras de sangue por meio de venipunção jugular em tubos de 10mL sendo retirada uma alíquota de 500L em tubo "eppendorff" contendo 2L de heparina. Através desta alíquota foram realizadas três metodologias para o teste do NBT: A) técnica original Park & Good (1970); B) redução de 50% do volume do corante; C) redução de 50% e armazenamento do corante a -20°C por 180 dias. Foram confeccionados e corados esfregaços sanguíneos (May Grünwald-Giemsa) das amostras com as metodologias propostas e realizada contagem de 100 neutrófilos em microscopia óptica para determinar o percentual de neutrófilos reativos ao NBT. A análise estatística pelo método de Análise de Medidas Repetida (ANOVA) demonstrou diferenças significativas (P 0,05) entre os dois métodos e o original (A=14,4±4,6; B=1,9±1,4; C=1,1±0,9), porém apresentou alta correlação pelo teste de Spearman entre os métodos (rs=0,82 (AxB), rs=0,75 (BxC) e rs=0,93 (AxC)). Conclui-se que o teste de Redução do Tetrazólio Nitroazul (NBT) segundo a metodologia descrita por Park & Good (1970) e a redução do corante em 50% do seu volume e a sua redução associada ao congelamento por 180 dias a -20ºC podem ser realizadas em caprinos, porém os valores devem ser comparados com as respectivas metodologias utilizadas.

Morphological characteristics and cell viability of coffee plants calli / Características morfológicas e viabilidade celular de calos de cafeeiro

The aim of this research was to characterize and compare two types of calli from leaf explants of Coffea arabica (cultivar Catiguá). Cells of different types of callus were successfully characterized regarding viability and internal and external morphological characteristics. It was obtained two morphologically distinct types of callus: (i) yellow friable and (ii) transparent watery. The yellow friable calli showed higher cell viability and embryogenic characteristics. Scanning and transmission electron microscopy showed embryogenic characteristics in cells of the yellow friable calli evidenced by the presence of small and isodiametric cells, while transparent watery calli showed elongated cells and large cytoplasm vacuolization.

O objetivo deste trabalho foi caracterizar e comparar dois tipos de calos de explantes foliares de Coffea arabica (cultivar Catiguá). Células de diferentes tipos de calos foram caracterizadas quanto a viabilidade e características morfológicas externas e internas. Foram obtidos dois tipos de calos morfologicamente distintos: (a) amarelo friável e (b) transparente aquoso. Os calos amarelos friáveis apresentaram maior viabilidade celular e características embriogênicas. Microscopia eletrônica de varredura e transmissão mostraram características embriogênicas em calos amarelos friáveis evidenciadas pela presença de células pequenas e isodiamétricas. Os calos transparentes aquosos apresentaram células alongadas e vacuolizadas.

Nanoencapsulação de um peptídeo isolado de anuros: citotoxicidade in vitro em células tumorais humanas / Nanoencapsulation of a peptide isolated from anurans: in vitro cytotoxicity in human tumor cells

A terapia antineoplásica tradicional apresenta algumas limitações que podem ser superadas através da utilização dos lipossomas. Estes nanossistemas de carreamento possibilitam o direcionamento de fármacos e reduzem efeitos secundários. Alguns peptídeos catiônicos sintetizados na pele de anuros apresentam atividade citotóxica seletiva (microorganismos e/ou tumores). Neste sentido, espécies do gênero Phyllomedusa secretam as dermaseptinas. Objetivou-se neste estudo, avaliar a citotoxicidade in vitro da dermaseptina 01 (DS 01) livre e encapsulada em lipossomas unilamelares pequenos (SUVs) em células tumorais humanas. Os lipossomas foram preparados pelo método de hidratação do filme lipídico seguido de sonicação. Foram produzidas formulações neutras e catiônicas, convencionais e furtivas. A citotoxicidade foi analisada em células tumorais de pulmão (NCI-H292), cólon (HT-29) e laringe (HEp-2), pelo ensaio de redução do sal tetrazólio (MTT) em placas de 96 poços. Os lipossomas foram submetidos a testes de estabilidade acelerada e em longo prazo. Em NCI-H292, a DS 01 livre apresentou efeito citostático médio de 35,6%. A encapsulação do peptídeo em lipossomas convencionais neutros aumentou o efeito, ao contrário dos furtivos. Para HT-29 e HEp-2, a DS 01 livre inibiu o crescimento celular em aproximadamente 50%, em média. A encapsulação em lipossomas catiônicos potencializou o efeito; os lipossomas convencionais inibiram na faixa de 80% e os furtivos, mais que 95% para as duas linhagens celulares. A DS 01, um peptídeo catiônico antimicrobiano, apresentou efeito citotóxico in vitro para células tumorais humanas que foi potencializado com a nanoencapsulação...

The conventional anticancer therapies show some limitations that can be overcome by using liposomes. This type of nanocarriers allows preferential targeting of drugs and reduces undesirable secondary effects. Some cationic peptides synthesized by the skin of anurans exhibit selective cytotoxicity (to pathogens and/or tumors). Species of Phyllomedusa secrete dermaseptins (DSs). The aim of this study was to assess the in vitro cytotoxicity of free and small unilamellar vesicle (SUV)-encapsulated dermaseptin 01 (DS 01) in various human tumor cells. Liposomes were prepared by lipid film hydration followed by sonication. Neutral and cationic, conventional and stealth liposomes were produced. Cytotoxicity was analyzed in lung (NCI-H292), colon (HT-29) and larynx (HEp-2) cancer cells, by the tetrazolium reduction method (MTT) carried out in 96-well microplates. Liposomes were tested for accelerated and long-term stability. In NCI-H292, free DS 01 showed a medium cytostatic effect of 36.5%. The conventional neutral liposome encapsulation of peptide increased this effect, whereas the stealth did not. In HT-29 and HEp-2, free DS 01 inhibited the cell growth by approximately 50% on average. The cationic liposome encapsulation was synergic, inhibition being about 80% in conventional and higher than 95% in stealth liposomes for both celllines. Thus, DS 01, an antimicrobial cationic peptide, showed in vitro cytotoxicity to human tumor cells that was potentiated by nanoencapsulation...

Increase of mesenchymal stem cell migration by cannabidiol via activation of p42/44 MAPK.

Migration and differentiation of mesenchymal stem cells (MSCs) are known to be involved in various regenerative processes such as bone healing. However, little is known about the pharmacotherapeutical options aiming at the mobilization and differentiation of MSCs. The present study therefore focussed on cannabinoids which have been demonstrated to exhibit tissue healing properties. Using Boyden chamber assays, the non-psychoactive phytocannabinoid cannabidiol (CBD) was found to increase the migration of adipose-derived MSCs in a time- and concentration-dependent manner. CBD-induced migration was inhibited by AM-630 (CB2 receptor antagonist) and O-1602 (G protein-coupled receptor 55 [GRP55] agonist). Moreover, the promigratory effect of CBD was antagonized by inhibition of the p42/44 mitogen-activated protein kinase (MAPK) pathway which became activated upon CBD treatment. In line with this data, AM-630 and O-1602 attenuated CBD-induced p42/44 MAPK phosphorylation. A p42/44 MAPK-dependent promigratory effect was likewise demonstrated for the GPR55 antagonist O-1918 and the selective CB2 receptor agonist JWH-133. Additional evidence for a functional effect of CBD on MSCs was provided by experiments demonstrating long-term stimulation with CBD to induce differentiation of MSCs into the osteoblastic lineage as evidenced by increased mineralization assessed by cresolphthalein complexone assay and enhanced activity of alkaline phosphatase. Collectively, this study demonstrates CBD to promote the migration of MSCs via activation of the CB2 receptor and inhibition of GPR55 and to induce osteoblastic differentiation. CBD may therefore recruit MSCs to sites of calcifying tissue regeneration and subsequently support bone regeneration via an osteoanabolic action on MSCs.

Leucograma e metabolismo oxidativo de neutrófilos em cabras da raça Saanen nos períodos de gestação, parto e pós-parto / Leukocyte count and neutrophil oxidative burst in Saanen goats in the pregnancy, parturition and postpartum periods

O presente estudo teve como objetivo a avaliação do leucograma e do metabolismo oxidativo de neutrófilos em fêmeas caprinas da raça Saanen, nos períodos de gestação, parto e pós-parto. Amostras de sangue foram colhidas de 20 fêmeas nulíparas da raça Saanen, clinicamente sadias por venipunção jugular aos 49 (M1), 42 (M2), 35 (M3), 28 (M4), 21 (M5), 14 (M6), sete (M7), três (M8) dias antes do parto, no dia do parto (M9), três (M10) e sete (M11) dias após o parto, para a realização do leucograma e dosagens séricas de cortisol, estradiol e progesterona. A partir de 28 dias (M4) antes do parto até sete dias do pós-parto (M11) foram colhidas amostras de sangue para a avaliação do metabolismo oxidativo de neutrófilos por meio do teste de redução do tetrazólio nitroazul (NBT). Os resultados demonstraram que no dia do parto houve aumento nas concentrações séricas de cortisol e estradiol, e diminuição da progesterona, leucocitose por neutrofilia e desvio à esquerda leve, diminuição dos linfócitos, aumento da relação neutrófilo:linfócito, eosinopenia, monocitose e basofilia. No sétimo dia do pós-parto houve leucocitose por neutrofilia e aumento da relação neutrófilo:linfócito. Não houve nos períodos de gestação, parto e pós-parto alterações significativas no metabolismo oxidativo dos neutrófilos. Conclui-se que o parto determina elevação da concentração sérica de cortisol e estradiol, e diminuição da progesterona determinando quadro de leucocitose por neutrofilia e desvio à esquerda leve, com diminuição dos linfócitos, aumento da relação neutróflo:linfócito, eosinopenia, monocitose e basofilia. Ao sétimo dia do pós-parto há leucocitose por neutrofilia, aumento da relação neutrófilo:linfócito e do fibrinogênio. A gestação, o parto e o período do pós-parto não alteram o metabolismo oxidativo de neutrófilos avaliado por meio do teste de redução do NBT.

The purpose of this study was to evaluate the leukocyte count and the oxidative metabolism of neutrophil in Saanen goats during periods of pregnancy, parturition and postpartum. Were used 20 Saanen goats, clinically healthy and serologically negative for caprine arthritis encephalitis virus (CAEV). Blood samples were collected by jugular venipuncture 49 (M1), 42 (M2), 35 (M3), 28 (M4), 21 (M5), 14 (M6), seven (M7), three (M8) days before the parturition, on the day of birth (M9), three (M10) and seven (M11) days postpartum, for the leukocyte count, and serum for cortisol, estradiol and progesterone determination. From 28 days (M4) before parturition until seven days postpartum (M11) blood samples were collected for evaluation of oxidative metabolism of neutrophils by the nitroblue tetrazolium reduction test (NBT). The results showed that at parturition day there were an increase in cortisol and estradiol levels and a decrease in progesterone serum, neutrophilic leukocytosis and left shift slight, decrease of lymphocytes, increase in the neutrophil: lymphocyte, eosinopenia, monocytosis and basophilia. There was a neutrophilic leukocytosis and an increase in the neutrophil: lymphocyte on the seventh day postpartum. There were not significant alterations in oxidative metabolism of neutrophils during pregnancy, parturition and postpartum. It was concluded that parturition causes an elevation in cortisol and estradiol levels and a decrease in progesterone serum determining a neutrophilic leukocytosis and left shift slight, with a reduction of lymphocytes, increase in the neutrophil:lymphocyte, eosinopenia, monocytosis and basophilia. Neutrophilic leukocytosis, increase in the neutrophil: lymphocyte and fibrinogen are detected on the seventh day postpartum. Pregnancy, parturition and the postpartum do not change the oxidative metabolism of neutrophils evaluated by NBT reduction test.

Mechanisms of heparanase inhibition by the heparan sulfate mimetic PG545 and three structural analogues.

The tetrasaccharide heparan sulfate (HS) mimetic PG545, a clinical anti-cancer candidate, is an inhibitor of the HS-degrading enzyme heparanase. The kinetics of heparanase inhibition by PG545 and three structural analogues were investigated to understand their modes of inhibition. The cholestanol aglycon of PG545 significantly increased affinity for heparanase and also modified the inhibition mode. For the tetrasaccharides, competitive inhibition was modified to parabolic competition by the addition of the cholestanol aglycon. For the trisaccharides, partial competitive inhibition was modified to parabolic competition. A schematic model to explain these findings is presented.

Prevention of Reg I-induced ß-cell apoptosis by IL-6/dexamethasone through activation of HGF gene regulation.

Reg (regenerating gene) product, Reg protein, is induced in pancreatic ß-cells and acts as autocrine/paracrine growth factor for regeneration via the cell surface Reg receptor. However, high concentrations of Reg I protein induced ß-cell apoptosis. In the present study, we found that hepatocyte growth factor (HGF) attenuated the ß-cell apoptosis induced by the high concentrations of Reg I protein and that the combined stimulation of interleukin-6 (IL-6) and dexamethasone (Dx) induced the accumulation of HGF mRNA as well as Reg I mRNA in ß-cells. The accumulation of the HGF mRNA was caused by the activation of the HGF promoter. Deletion analysis revealed that the region of -96 to -92 of the HGF gene was responsible for the promoter activation by IL-6+Dx. The promoters contain a consensus transcription factor binding sequence for signal transducer and activator of transcription (STAT). Site-directed mutations of STAT-binding motif in the region markedly attenuated the HGF promoter activity. Chromatin immunoprecipitation assay showed that STAT3 is located at the active HGF promoter in response to IL-6+Dx stimulation. These results strongly suggest that the combined stimulation of IL-6 and glucocorticoids induces the activation of both Reg and HGF genes and that the anti-apoptotic effects of HGF against the Reg I-induced apoptosis may help ß-cell regeneration by Reg I protein.

Protective effect of polypeptides from larva of housefly (Musca domestica) on hydrogen peroxide-induced oxidative damage in HepG2 cells.

Housefly (Musca domestica) is an important medical insect and its larva is an ideal high protein food source. We isolated from housefly larvae the polypeptides hydrolyzed by neutral protease (PHNP), and investigated the protective effect of PHNP on hydrogen peroxide (H2O2)-induced oxidative damage in HepG2 cells. Cells exposed to H2O2 showed a marked decrease in proliferation and intracellular superoxide dismutase (SOD) activity, and a significant increase in reactive oxygen species (ROS) level and malondialdehyde (MDA) content. H2O2 also caused apoptosis and mitochondrial dysfunction including mitochondrial fragmentation and the loss of mitochondrial membrane potential. Pretreatment with PHNP at concentrations of 2.5, 5, 10 µg/mL blocked these H2O2-induced cellular events in a dose-dependent manner. The effect of PHNP at 10 µg/mL is equal to that of ascorbic acid at 10 µM. In summary, PHNP has a protective effect against H2O2-induced oxidative injury in cells due to its ability to decrease intracellular ROS and elevate antioxidant enzyme activities.

Characterization of a novel proinflammatory effect mediated by BK and the kinin B2 receptor in human preadipocytes.

Obesity and adipose tissue contribute to local and systemic inflammation. However the role of the inflammatory mediator bradykinin (BK) in this context is not known. We therefore evaluated the effect of BK on adipokines secretion in human preadipocytes during the course of differentiation and characterized the receptors involved. Results obtained from antibody array and ELISA experiments showed that several adipokines are released by human preadipocytes under basal conditions while BK specifically stimulated the production of interleukin(IL)-6 and IL-8. The effect of BK diminished with the progression of differentiation, being almost inactive on adipocytes. In preadipocytes, BK also induced a rapid and transient [Ca²âº](i) mobilization, a rapid and sustained increase in ERK1/2 activation and enhanced forskolin-stimulated cAMP accumulation. BK was without effect on cell proliferation and viability as assessed by bromodeoxyuridine incorporation, WST-1 conversion, or lactate dehydrogenase leakage and was without effect on adipogenesis as measured by triglyceride accumulation, GPDH activity and leptin release. The B1 receptor agonist, Lys-[des-Arg9]-BK, displayed poor activity or was without effect while overall BK effects were prevented by the selective B2 receptor antagonist, fasitibant chloride, but not by the B1 selective antagonist, Lys-[Leu8][des-Arg9]-BK. Immunoblot analysis and immunofluorescence studies showed that the kinin B2 receptor was essentially expressed at the beginning of the differentiation program. In conclusion, human preadipocytes expressed kinin B2 receptors linked to multiple signaling pathways, IL-6 and IL-8 production, and BK proinflammatory response in adipose tissue could be prevented by fasitibant chloride.

Metodologia alternativa para condução do teste de envelhecimento acelerado em sementes de milho / Alternative methodology for the accelerated aging test for corn seeds

Os testes de vigor são rotineiramente empregados em programas internos de controle de qualidade por empresas sementeiras. Para tanto, é necessária a escolha de métodos eficientes que possibilitem a obtenção de respostas rápidas para a tomada de decisões relacionadas ao manuseio, descarte e comercialização dos lotes de sementes. A pesquisa objetivou verificar a redução do período de execução do teste de envelhecimento acelerado (EA) em sementes de milho, empregando-se, para a avaliação do desempenho das sementes após o envelhecimento, o teste de tetrazólio - TZ (viabilidade e vigor) em substituição ao de germinação (TG) em 10 lotes de sementes de sete genótipos de milho, com e sem tratamento fungicida. Os dados obtidos com a metodologia proposta (EA+TZ) foram comparados com os valores determinados pelo teste de envelhecimento acelerado realizado com a metodologia tradicional (EA+TG). O uso do teste de tetrazólio (vigor), associado ao teste de envelhecimento acelerado, possibilitou a obtenção de informações semelhantes às fornecidas pelo teste de germinação empregado para o mesmo fim, reduzindo de oito para três dias o tempo necessário para a obtenção dos resultados em sementes de milho.

Some vigor tests are routinely used by seed industry for internal programs of seed quality control. Then, it is requested the use of efficient methods to obtain quick answers to take right decisions related to the management, discard and trade of seed lots. This research was carried out in order to study the possibility to short the period to get the accelerated aging test (AA) results, using the tetrazolium test (TZ) instead of germination test (GT) to evaluate the seed performance after the seed aging. Tem corn seed lots were used, with and without fungicide treatment. The data obtained using the alternative method (AA+TZ) were compared with those determined by the traditional one (AA+GT). There was discrimination among seed lots using the traditional AA test and aging combined with TZ test. The corn seed vigor can be evaluate using the TZ test after seed aging instead of germination test. It allows similar results with the advantage of reducing the test period from eight to three days.

Evaluación del efecto de dos suplementos orgánicos en la germinación in vitro de orquídeas nativas de la provincia de Pamplona, Colombia / Evaluation of the effects of two organics supplements on in vitro germination of native orchids in the province of Pamplona, Colombia

La continua pérdida de hábitat de las orquídeas nativas en Colombia, y las limitaciones de la germinación en estado silvestre, ha dado lugar a un mayor énfasis en la conservación de las orquídeas. Por consiguiente el cultivo in vitro es una herramienta alternativa para la conservación de especies en peligro de extinción. En esta investigación se evaluó la germinación asimbiótica y la formación de plántulas de semillas de orquídeas, de las especies Prosthechea vespa Vell. y Sobralia klotzscheana Rchb. f. en el medio de cultivo Murashige y Skoog (MS) con dos suplementos orgánicos (jugo de piña y agua de coco). Se colectaron cápsulas maduras de las especies P. vespa y S. klotzscheana, en la región nororiental de Colombia (Pamplona, Norte de Santander) y se determinó la viabilidad de las semillas con la prueba de Tetrazolio. Las semillas se desinfectaron y se sembraron con el método de la jeringuilla. La viabilidad de las semillas fue del 87,2% en P. vespa y 80,6% en S. klotzscheana. El porcentaje de viabilidad corregido con respecto a la germinación fue mayor, entre 2,8% (P. vespa) y 0,5% (S. klotzscheana). Este estudio demostró que el medio MS suplementado con jugo de piña tiene una mayor respuesta a la geminación asimbiótica y formación de plántulas en las orquídeas P. vespa (22%) y S. klotzscheana (43%) con diferencias estadísticamente significativas (P≤0,05: Tukey HSD).

The continued habitat destruction of native orchids in Colombia and its germination limitations in the wild has led to greater emphasis of the species conservation, therefore to preserve endangered orchids in vitro culture is an alternative tool. This research evaluated orchid seeds asymbiotic germination and seedling formation of Prosthechea vespa Vell. and Sobralia klotzscheana Rchb. f. species in Murashige and Skoog (MS) culture medium with two organic supplements (pineapple juice and coconut water). Mature capsules from P. vespa and S. klotzscheana species were collected in the Province of Pamplona, northeastern region of Colombia and their viability with tetrazolium staining was determined. The seeds were disinfected and sown by the syringe method. The seeds viability were 87,2% in P. vespa and 80,6% in S. klotzscheana. The corrected percentage of viability compared to the germination was higher, between 2,8% (P. vespa) and 0,5% (S. klotzscheana). This study showed that MS medium supplemented with pineapple juice has a greater response to asymbiotic germination and seedling formation in P. vespa (22%) and S. klotzscheana (43%) with statistically significant differences (P≤0,05: Tukey HSD).