Background: The colonial ascidian Didemnum vexillum, sea carpet squirt, is not only a key marine organism to study morphological ancestral patterns of chordates evolution but it is also of great ecological importance due to its status as a major invasive species. Non-coding RNAs, in particular microRNAs (miRNAs), are important regulatory genes that impact development and environmental adaptation. Beyond miRNAs, not much in known about tunicate ncRNAs. Results: We provide here a comprehensive homology-based annotation of non-coding RNAs in the recently sequenced genome of D. vexillum. To this end we employed a combination of several computational approaches, including blast searches with a wide range of parameters, and secondary structured centered survey with infernal. The resulting candidate set was curated extensively to produce a high-quality ncRNA annotation of the first draft of the D. vexillum genome. It comprises 57 miRNA families, 4 families of ribosomal RNAs, 22 isoacceptor classes of tRNAs (of which more than 72% of loci are pseudogenes), 13 snRNAs, 12 snoRNAs, and 1 other RNA family. Additionally, 21 families of mitochondrial tRNAs and 2 of mitochondrial ribosomal RNAs and 1 long non-coding RNA. Conclusions: The comprehensive annotation of the D. vexillum non-coding RNAs provides a starting point towards a better understanding of the restructuring of the small RNA system in ascidians. Furthermore it provides a valuable research for efforts to establish detailed non-coding RNA annotations for other recently published and recently sequences in tunicate genomes.

Amostras de Didemnum psammatodes foram coletadas em Icapuí (CE) e Trairi (CE). O extrato metanólico de D. psammatodes, proveniente de Icapuí, foi submetido a processo de fracionamento e purificação por diversas técnicas cromatográficas, obtendo-se quatro nucleosídeos (2-deoxiuridina, 2-deoxiinosina, timidina e 2-deoxiguanosina), três esteróides em mistura (colestanol, colestanona e estigmasterol) e três derivados do glicerol éter em mistura (2,3-propanodiol, 1- heptadeciloxi; álcool batílico e 2,3-propanodiol, 1-nonadeciloxi). O extrato metanólico de D. psammatodes, proveniente de Trairi, foi inicialmente particionado, originando as fases aquosa, hexânica, clorofórmica, butanólica e hidroalcoólica. A fase hexânica inibiu o ciclo celular de ovos de ouriço do mar e exibiu toxicidade em células tumorais. Ela foi fracionada por técnicas cromatográficas e obtiveram-se três ácidos graxos metilados em mistura (miristato de metila, palmitato de metila e estearato de metila), dois esteróides em mistura (colestanol e colestanona), dois ácidos graxos em mistura (ácido palmítico e ácido esteárico) e três derivados do glicerol éter em mistura (2,3-propanodiol, 1-heptadeciloxi; álcool batílico e 2,3- propanodiol, 1-nonadeciloxi). A fase clorofórmica também inibiu o ciclo celular de ovos de ouriço do mar e exibiu toxicidade em células tumorais. Ela foi fracionada por técnicas cromatográficas e obtiveram-se seis ácidos graxos em mistura (ácido láurico, ácido mirístico, ácido pentadecanóico, ácido palmítico, ácido margárico e ácido esteárico). A fase aquosa foi submetida ao processo de fracionamento e purificação por diversas técnicas cromatográficas, obtendo-se dois nucleosídeos (uridina e timidina) e um núcleo purínico (hipoxantina). A fase butanólica resultou no isolamento e identificação de dois nucleosídeos (2-deoxiguanosina e timidina). Amostras de Eudistoma vannamei foram coletadas em São Gonçalo do Amarante (CE). O extrato metanólico de E. vannamei foi particionado, originando as fases hexânica e hidroalcoólica. A fase hexânica inibiu o ciclo celular de ovos de ouriço do xi mar e exibiu toxicidade em células de linhagem B-16. Ela foi fracionada por técnicas cromatográficas e obtiveram-se cinco ácidos graxos metilados (miristato de metila, palmitato de metila, estearato de metila, palmitoleato de metila e oleato de metila), dois esteróides (colestanona e colesterol) e cinco ácidos graxos (ácido mirístico, ácido pentadecanóico, ácido palmítico, ácido margárico e ácido esteárico). A fase hidroalcoólica resultou no isolamento e identificação de dois nucleosídeos (guanosina e adenosina). As substâncias foram identificadas por métodos espectroscópicos e cromatográficos, comparando-se os valores obtidos com aqueles da literatura e padrões. Os nucleosídeos 2-deoxiuridina e 2-deoxiguanosina inibiram o ciclo celular de ovos de ouriço do mar em ambas fases examinadas. Os ácidos graxos metilados de D. psammatodes, o ácido palmítico junto com ácido esteárico e os derivados do glicerol éter inibiram o ciclo celular de ovos de ouriço do mar durante a primeira divisão. Os ácidos graxos metilados de D. psammatodes apresentaram maior toxicidade nas linhagens tumorais analisadas, sendo que a mistura de ácidos graxos metilados de E. vannamei também mostrou toxicidade em células tumorais leucêmicas. Ambas amostras de ácidos graxos metilados mostraram efeitos antiproliferativos e citotóxicos e estas atividades envolveram a inibição da síntese de DNA e indução de apoptose e necrose. / Samples of Didemnum psammatodes were collected in Icapui (Ceara State) and Trairi (Ceara State). The methanolic extract from D. psammatodes, acquired from Icapui, was submitted to fractionation and purification through several chromatographic techniques and were obtained four nucleosides (2?-deoxyuridine, 2- deoxyinosine, thyimidine, and 2?-deoxyguanosine), three steroids in mixture (cholestanol, cholestanone, and stigmasterol) and three glyceryl ethers in mixture (2,3-propanediol, 1-(heptadecyloxy), batyl alcohol, and 2,3-propanediol, 1- (nonadecyloxy)). The methanolic extract from D. psammatodes, acquired from Trairi, was initially partitionated, giving rise to aqueous, hexane, chloroform, and butanol phases. The hexane phase inhibited the sea urchin egg cell cycle and exhibited toxicity in tumor cell lines. It was fractionated by several chromatographic techniques and obtained three fatty acid methyl esters in mixture (methyl myristate, methyl palmitate, and methyl stearate), four steroids in mixture (cholestanol and cholestanone), two fatty acids in mixture (palmitic acid and stearic acid) and three glyceryl ethers in mixture (2,3-propanediol, 1-(heptadecyloxy), batyl alcohol, and 2,3- propanediol, 3-(nonadecyloxy)). The chloroform phase also inhibited the sea urchin egg cell cycle and exhibited toxicity in tumor cell lines. It was fractionated through several chromatographic techniques and obtained six fatty acids in mixture (lauric acid, myristic acid, pentadecanoic acid, palmitic acid, margaric acid, and stearic acid). The aqueous phase was submitted to fractionation and purification by several chromatographic techniques and obtained two nucleosides (uridine and thymidine) and one purinic nucleus (hypoxanthine). The butanol phase resulted in the isolation and identification of two nucleosides (2?-deoxyguanosine and thymidine). Samples of Eudistoma vannamei were collected in São Gonçalo do Amarante (Ceara State). The methanolic extract from E. vannamei was partitionated, giving rise to hexane and hydroalcoholic phases. The hexane phase inhibited the sea urchin egg cell cycle and xiii exhibited toxicity in cell line B-16. It was fractionated by chromatographic techniques and obtained five fatty acid methyl esters in mixture (methyl myristate, methyl palmitate, methyl stearate, methyl palmitoleate, and methyl oleate), two steroids (cholestanol and cholesterol), and five fatty acids (myristic acid, pentadecanoic acid, palmitic acid, margaric acid, and stearic acid). The hydroalcoholic phase resulted in the isolation and identification of two nucleosides (guanosine and adenosine). The substances were identified by spectroscopic and chromatographic methods comparing the obtained values with those of the literature and standards. The nucleosides 2?-deoxyuridine and 2?-deoxyguanosine inhibited the sea urchin egg cell cycle in both stages. The fatty acid methyl esters from D. psammatodes, the palmitic acid plus stearic acid and the glyceryl ethers inhibited the sea urchin egg cell during first cleavage. The fatty acid methyl esters from D. psammatodes presented the highest toxicity in the cell lines tested and the mixture of fatty acid methyl esters from E. vannamei showed toxicity to leukemia cell lines too. Both samples of fatty acid methyl esters showed antiproliferative and cytotoxic effects and these activities involved the inhibition of DNA synthesis and induction of both necrosis and apoptosis.

Amostras de Didemnum psammatodes foram coletadas em Icapuí (CE) e Trairi (CE). O extrato metanólico de D. psammatodes, proveniente de Icapuí, foi submetido a processo de fracionamento e purificação por diversas técnicas cromatográficas, obtendo-se quatro nucleosídeos (2-deoxiuridina, 2-deoxiinosina, timidina e 2-deoxiguanosina), três esteróides em mistura (colestanol, colestanona e estigmasterol) e três derivados do glicerol éter em mistura (2,3-propanodiol, 1- heptadeciloxi; álcool batílico e 2,3-propanodiol, 1-nonadeciloxi). O extrato metanólico de D. psammatodes, proveniente de Trairi, foi inicialmente particionado, originando as fases aquosa, hexânica, clorofórmica, butanólica e hidroalcoólica. A fase hexânica inibiu o ciclo celular de ovos de ouriço do mar e exibiu toxicidade em células tumorais. Ela foi fracionada por técnicas cromatográficas e obtiveram-se três ácidos graxos metilados em mistura (miristato de metila, palmitato de metila e estearato de metila), dois esteróides em mistura (colestanol e colestanona), dois ácidos graxos em mistura (ácido palmítico e ácido esteárico) e três derivados do glicerol éter em mistura (2,3-propanodiol, 1-heptadeciloxi; álcool batílico e 2,3- propanodiol, 1-nonadeciloxi). A fase clorofórmica também inibiu o ciclo celular de ovos de ouriço do mar e exibiu toxicidade em células tumorais. Ela foi fracionada por técnicas cromatográficas e obtiveram-se seis ácidos graxos em mistura (ácido láurico, ácido mirístico, ácido pentadecanóico, ácido palmítico, ácido margárico e ácido esteárico). A fase aquosa foi submetida ao processo de fracionamento e purificação por diversas técnicas cromatográficas, obtendo-se dois nucleosídeos (uridina e timidina) e um núcleo purínico (hipoxantina). A fase butanólica resultou no isolamento e identificação de dois nucleosídeos (2-deoxiguanosina e timidina). Amostras de Eudistoma vannamei foram coletadas em São Gonçalo do Amarante (CE). O extrato metanólico de E. vannamei foi particionado, originando as fases hexânica e hidroalcoólica. A fase hexânica inibiu o ciclo celular de ovos de ouriço do xi mar e exibiu toxicidade em células de linhagem B-16. Ela foi fracionada por técnicas cromatográficas e obtiveram-se cinco ácidos graxos metilados (miristato de metila, palmitato de metila, estearato de metila, palmitoleato de metila e oleato de metila), dois esteróides (colestanona e colesterol) e cinco ácidos graxos (ácido mirístico, ácido pentadecanóico, ácido palmítico, ácido margárico e ácido esteárico). A fase hidroalcoólica resultou no isolamento e identificação de dois nucleosídeos (guanosina e adenosina). As substâncias foram identificadas por métodos espectroscópicos e cromatográficos, comparando-se os valores obtidos com aqueles da literatura e padrões. Os nucleosídeos 2-deoxiuridina e 2-deoxiguanosina inibiram o ciclo celular de ovos de ouriço do mar em ambas fases examinadas. Os ácidos graxos metilados de D. psammatodes, o ácido palmítico junto com ácido esteárico e os derivados do glicerol éter inibiram o ciclo celular de ovos de ouriço do mar durante a primeira divisão. Os ácidos graxos metilados de D. psammatodes apresentaram maior toxicidade nas linhagens tumorais analisadas, sendo que a mistura de ácidos graxos metilados de E. vannamei também mostrou toxicidade em células tumorais leucêmicas. Ambas amostras de ácidos graxos metilados mostraram efeitos antiproliferativos e citotóxicos e estas atividades envolveram a inibição da síntese de DNA e indução de apoptose e necrose. / Samples of Didemnum psammatodes were collected in Icapui (Ceara State) and Trairi (Ceara State). The methanolic extract from D. psammatodes, acquired from Icapui, was submitted to fractionation and purification through several chromatographic techniques and were obtained four nucleosides (2?-deoxyuridine, 2- deoxyinosine, thyimidine, and 2?-deoxyguanosine), three steroids in mixture (cholestanol, cholestanone, and stigmasterol) and three glyceryl ethers in mixture (2,3-propanediol, 1-(heptadecyloxy), batyl alcohol, and 2,3-propanediol, 1- (nonadecyloxy)). The methanolic extract from D. psammatodes, acquired from Trairi, was initially partitionated, giving rise to aqueous, hexane, chloroform, and butanol phases. The hexane phase inhibited the sea urchin egg cell cycle and exhibited toxicity in tumor cell lines. It was fractionated by several chromatographic techniques and obtained three fatty acid methyl esters in mixture (methyl myristate, methyl palmitate, and methyl stearate), four steroids in mixture (cholestanol and cholestanone), two fatty acids in mixture (palmitic acid and stearic acid) and three glyceryl ethers in mixture (2,3-propanediol, 1-(heptadecyloxy), batyl alcohol, and 2,3- propanediol, 3-(nonadecyloxy)). The chloroform phase also inhibited the sea urchin egg cell cycle and exhibited toxicity in tumor cell lines. It was fractionated through several chromatographic techniques and obtained six fatty acids in mixture (lauric acid, myristic acid, pentadecanoic acid, palmitic acid, margaric acid, and stearic acid). The aqueous phase was submitted to fractionation and purification by several chromatographic techniques and obtained two nucleosides (uridine and thymidine) and one purinic nucleus (hypoxanthine). The butanol phase resulted in the isolation and identification of two nucleosides (2?-deoxyguanosine and thymidine). Samples of Eudistoma vannamei were collected in São Gonçalo do Amarante (Ceara State). The methanolic extract from E. vannamei was partitionated, giving rise to hexane and hydroalcoholic phases. The hexane phase inhibited the sea urchin egg cell cycle and xiii exhibited toxicity in cell line B-16. It was fractionated by chromatographic techniques and obtained five fatty acid methyl esters in mixture (methyl myristate, methyl palmitate, methyl stearate, methyl palmitoleate, and methyl oleate), two steroids (cholestanol and cholesterol), and five fatty acids (myristic acid, pentadecanoic acid, palmitic acid, margaric acid, and stearic acid). The hydroalcoholic phase resulted in the isolation and identification of two nucleosides (guanosine and adenosine). The substances were identified by spectroscopic and chromatographic methods comparing the obtained values with those of the literature and standards. The nucleosides 2?-deoxyuridine and 2?-deoxyguanosine inhibited the sea urchin egg cell cycle in both stages. The fatty acid methyl esters from D. psammatodes, the palmitic acid plus stearic acid and the glyceryl ethers inhibited the sea urchin egg cell during first cleavage. The fatty acid methyl esters from D. psammatodes presented the highest toxicity in the cell lines tested and the mixture of fatty acid methyl esters from E. vannamei showed toxicity to leukemia cell lines too. Both samples of fatty acid methyl esters showed antiproliferative and cytotoxic effects and these activities involved the inhibition of DNA synthesis and induction of both necrosis and apoptosis.

Made available in DSpace on 2016-08-17T18:39:35Z (GMT). No. of bitstreams: 1 3286.pdf: 1985838 bytes, checksum: e5ef56af190be032fb21c75bb7198227 (MD5) Previous issue date: 2010-06-01 / Universidade Federal de Sao Carlos / Studies of marine natural products in 20 years of research have revealed a wide variety of molecules with complex structures and hitherto unknown, and potent biological activities. The aims of this study were to isolate microbial strains associated with Didemnum granulatum, to fermentate such strains for the production of crude extracts and the screening of these extracts for bioactive secondary metabolites. For the isolation of the strains, six different culture media were prepared with artificial sea water: malt extract 2% and 3%, carrot/potato, corn, oat and GPY. The strains were cultivated for 30 days in 250mL of the same culture media. The crude extracts were obtained by partition with ethyl acetate. The extracts were sent for the cytotoxic activity bioassay and analyzed by thin layer chromatography (TLC) on silica gel plate (with absorption in UV-vis). Among the 26 extracts, samples DG (M3) 6'C and DG (M3) 5'C showed potent cytotoxic activity, samples DG (B) 13, DG (M3) 1 and DG (G) 2B a moderate activity. The analysis by TLC indicated the presence of bioactive secondary metabolites in the extracts. The strains were identified as bioactive three fungal: DG (M 3) 6'C (Penicillium sp.) DG (M3) 5'C (Cladosporium sp.) DG (M3) 1 (Aspergillus sp.) And two bacterial: DG (B) 13 (Aurantimonas sp.) DG (G) 2B (Nocardiopsis sp.). These were grown on a larger scale of broth (500 mL) and extracted after 7, 14, 21 and 30 days of incubation. We carried out a cleaning process of these extracts by solid phase extraction (SPE) column of silica gel derivatized with C18 (35 mL of eluent: 100% H2O, MeOH/H2O 1:1 and MeOH 100%). The fractions generated were analyzed by HPLC-UV-MS trying to find characteristic profiles of secondary metabolites. From the DG sample (M3) 6'C (A + B), was isolated and identified the compound 13- deoxy-fomenona previously obtained by Tirilly et al. in 1983. Although this compound is not unheard of, no literature reports of studies involving bioassays cytotoxic against tumor cells and even this compound has been isolated from other species of microorganisms other than the fungus Dicyma pulvinata. / Os estudos sobre produtos naturais marinhos, durante os ultimos 20 anos de pesquisa, revelaram uma grande variedade de moleculas com estruturas complexas e ate entao desconhecidas, alem de potentes atividades biologicas. Assim, os objetivos deste trabalho foram isolar linhagens de micro-organismos associadas a ascidia Didemnum granulatum, sua posterior fermentacao para a producao de extratos brutos e a analise destes extratos na busca por metabolitos secundarios bioativos. Para o isolamento das linhagens foram utilizados os seguintes meios de cultura preparados com agua do mar artificial: extrato de malte 2% e 3%, cenoura/batata, fuba, aveia e GPY. A fermentacao das linhagens foi feita em 250 mL dos mesmos meios de cultura sem agar, durante 30 dias. Os extratos brutos foram obtidos por particao com acetato de etila. Estes extratos foram enviados para bioensaio de atividade citotoxica contra celulas tumorais e analisados por cromatografia em camada delgada (CCD) em placa de silica gel (com diferentes reveladores). Dentre os 26 extratos, as amostras DG(M3) 6 C e DG(M3) 5 C apresentaram potente atividade citotoxica, as amostras DG(B)13, DG(M3)1 e DG(G)2B uma atividade moderada. As analises por CCD indicaram a presenca de metabolitos secundarios nos extratos bioativos. As linhagens bioativas foram identificadas como sendo tres fungicas: DG(M3)6 C (Penicilium sp.); DG(M3)5 C (Cladosporium sp.); DG(M3)1 (Aspergillus sp.) e duas bacterianas: DG(B)13 (Aurantimonas sp.); DG(G)2B (Nocardiopsis sp.). Estas foram cultivadas em escala maior de meio liquido (500 mL) e extraidas apos 7, 14, 21 e 30 dias de incubacao. Realizou-se um processo de limpeza destes extratos por extracao em fase solida (SPE) em coluna de silica-gel derivatizada com C18 (35 mL dos eluentes: H2O 100%, MeOH/H2O 1:1 e MeOH 100%). As fracoes geradas foram analisadas por CLAE-UV-EM buscando-se encontrar perfis caracteristicos de metabolitos secundarios. A partir da amostra DG(M3) 6 C (A+B), foi isolado e identificado o composto 13-desoxi-fomenona, previamente obtido por Tirilly et al. em 1983. Apesar deste composto nao ser inedito, nao ha relatos na literatura de estudos envolvendo bioensaios citotoxicos contra celulas tumorais e nem mesmo deste composto ter sido isolado de outras especies de microrganismos que nao seja do fungo Dicyma pulvinata.

Background: The colonial ascidian Didemnum vexillum, sea carpet squirt, is not only a key marine organism to study morphological ancestral patterns of chordates evolution but it is also of great ecological importance due to its status as a major invasive species. Non-coding RNAs, in particular microRNAs (miRNAs), are important regulatory genes that impact development and environmental adaptation. Beyond miRNAs, not much in known about tunicate ncRNAs. Results: We provide here a comprehensive homology-based annotation of non-coding RNAs in the recently sequenced genome of D. vexillum. To this end we employed a combination of several computational approaches, including blast searches with a wide range of parameters, and secondary structured centered survey with infernal. The resulting candidate set was curated extensively to produce a high-quality ncRNA annotation of the first draft of the D. vexillum genome. It comprises 57 miRNA families, 4 families of ribosomal RNAs, 22 isoacceptor classes of tRNAs (of which more than 72% of loci are pseudogenes), 13 snRNAs, 12 snoRNAs, and 1 other RNA family. Additionally, 21 families of mitochondrial tRNAs and 2 of mitochondrial ribosomal RNAs and 1 long non-coding RNA. Conclusions: The comprehensive annotation of the D. vexillum non-coding RNAs provides a starting point towards a better understanding of the restructuring of the small RNA system in ascidians. Furthermore it provides a valuable research for efforts to establish detailed non-coding RNA annotations for other recently published and recently sequences in tunicate genomes.