Resumo A presente revisão discute o impacto das iniciativas de bioprospecção desenvolvidas pelos projetos associados ao BIOprospecTA, subprograma do programa BIOTA, apoiado pela FAPESP. Esta revisão traz um resumo dos principais resultados produzidos pelos projetos de investigação de produtos naturais (PNs) de organismos não vegetais, como exemplos do sucesso desta iniciativa, com foco nos avanços alcançados pelos projetos relacionados a PNs de macroalgas, invertebrados marinhos, artrópodes e microrganismos associados. As macroalgas são um dos grupos mais estudados no Brasil com o isolamento de muitas substâncias bioativas, incluindo lipídios, carotenóides, ficocolóides, lectinas, aminoácidos do tipo micosporina e substâncias halogenadas. Invertebrados marinhos e microrganismos associados têm sido estudados de forma mais sistemática nos últimos trinta anos, revelando substâncias únicas, com potentes atividades biológicas. Os venenos de insetos himenópteros também foram amplamente estudados, resultando na identificação de centenas de peptídeos, que foram utilizados para criar uma biblioteca química que contribuiu para a identificação de modelos para o desenvolvimento de substâncias antifúngicas, antiparasitárias e anticancerígenas. O conhecimento construído dos venenos de himenópteros permitiu o desenvolvimento de um soro equino anti-peçonha de abelha. Dentre os microrganismos associados a insetos, a estratégia de bioprospecção foi compreender as bases moleculares das interações intra e interespécies (Ecologia Química), traduzindo esse conhecimento para possíveis aplicações biotecnológicas. Os resultados aqui discutidos reforçam a importância do programa BIOprospecTA no desenvolvimento de pesquisas com alto potencial inovador no Brasil.

Abstract The present review aims the discussion of the impact of the bioprospection initiative developed by the projects associated to BIOprospecTA, a subprogram of the program BIOTA, supported by FAPESP. This review brings a summary of the main results produced by the projects investigating natural products (NPs) from non-plants organisms, as examples of the success of this initiative, focusing on the progresses achieved by the projects related to NPs from macroalgae, marine invertebrates, arthropods and associated microorganisms. Macroalgae are one of the most studied groups in Brazil with the isolation of many bioactive compounds including lipids, carotenoids, phycocolloids, lectins, mycosporine-like amino acids and halogenated compounds. Marine invertebrates and associated microorganisms have been more systematically studied in the last thirty years, revealing unique compounds, with potent biological activities. The venoms of Hymenopteran insects were also extensively studied, resulting in the identification of hundreds of peptides, which were used to create a chemical library that contributed for the identification of leader models for the development of antifungal, antiparasitic, and anticancer compounds. The built knowledge of Hymenopteran venoms permitted the development of an equine hyperimmune serum anti honeybee venom. Amongst the microorganisms associated with insects the bioprospecting strategy was to understand the molecular basis of intra- and interspecies interactions (Chemical Ecology), translating this knowledge to possible biotechnological applications. The results discussed here reinforce the importance of BIOprospecTA program on the development of research with highly innovative potential in Brazil.

Resumo Diante da toxicidade do mercúrio para o ser humano e para o meio ambiente, o objetivo deste trabalho foi avaliar a sua bioacumulação em 13 espécies de peixes com diferentes hábitos alimentares, capturados no reservatório de Três Marias, bacia do rio São Francisco, no período de março de 2012 a abril de 2013. Os resultados obtidos revelam que as espécies com hábito alimentar carnívoro apresentaram as maiores concentrações médias de mercúrio em músculo, com destaque para Serrasalmus brandtii (pirambeba) e Pygocentrus piraya (piranha), com concentrações de 0,4147 ± 0,2744 μg g-1 e 0,1774 ± 0,2980 μg g-1, respectivamente. Considerando as concentrações médias, todas as espécies estudadas apresentaram teores de mercúrio abaixo dos limites máximos de 0,5 μg g-1 em peixes não predadores e de 1,0 μg g-1 em peixes predadores, estabelecidos pela Agência Nacional de Vigilância Sanitária. Levando em conta cada espécie, os valores mínimos e máximos de mercúrio nos tecidos muscular e hepático demonstraram uma ampla dispersão. Em 4% dos espécimes de pirambeba e 7% de piranha, os teores de mercúrio em músculo ultrapassaram o limite estabelecido pela ANVISA para peixes predadores, e, portanto, sendo impróprios para o consumo humano. As demais espécies não apresentaram amostras de músculo com teor de Hg acima do estabelecido em lei. Devido ao seu hábito alimentar carnívoro e sua ampla distribuição na bacia do rio São Francisco, Serrasalmus brandtii pode ser considerada como biomonitor, capaz de bioacumular mercúrio, indicando, assim, o nível de contaminação desse metal no ambiente aquático em que vive.

Abstract The objective of this work was to evaluate the bioaccumulation of mercury in 13 species of fish with different eating habits, captured in the Três Marias Reservoir, São Francisco Basin, from March 2012 to April 2013. The results obtained showed that the species with carnivorous eating habit presented the highest average concentrations of mercury in muscle, especially Serrasalmus brandtii (pirambeba) and Pygocentrus piraya (piranha), with concentrations of 0.4147 ± 0.2744 μg g-1 and 0.17774 ± 0.2980 μg g-1, respectively. Considering the average concentrations, all the species studied showed levels of mercury below the maximum limit of 0.5 μg g-1 in non-predatory fish and 1.0 μg g-1 in predatory fish, established by the Agência Nacional de Vigilância Sanitária. Taking each species into account, the minimum and maximum values of mercury in the muscular and hepatic tissues showed a wide dispersion. In 4% of the specimens of pirambeba and 7% of piranha, the contents of mercury in muscle exceeded the limit established by ANVISA for predatory fish, and therefore, were unfit for human consumption. The other species did not present muscle samples with Hg content above that established by law. Due to its carnivorous eating habit and its wide distribution in the São Francisco Basin, Serrasalmus brandtii can be considered a biomonitor, capable of bioaccumulating mercury, thus indicating the level of contamination in the aquatic environment in which it lives.

RESUMO Nos últimos anos há um interesse crescente no uso de diversos resíduos agroindustriais. No Brasil, as indústrias de alimentos acumulam uma enorme quantidade de resíduos, principalmente as de processamento de frutos cítricos.Entre as possibilidades de valorização destes resíduos, a digestão anaeróbia é uma alternativa promissora na geração de energia renovável e controle da poluição. Nesse sentido, esse estudo avaliou a digestão anaeróbia de água residuária citrícola, visando à produção biológica de hidrogênio a partir de três inóculos: (1) Clostridium Acetobutylicum ATCC 824,(2) Clostridium Beijerinckii ATCC 10132 e (3) consórcio anaeróbio obtido da água residuária citrícola. O experimento foi realizado em 51h, em triplicatas de reatores anaeróbios em batelada, alimentados com 2 fontes distintas de carbono: glicose (10,7 g DQO L-1) e água residuária citrícola (10,0 g DQO L-1), headspace preenchido com N2 (99,99%), pH inicial 7,0 e mantidos a 37°C, sob modo estático. Nos reatores alimentados com glicose foram verificados geração de hidrogênio apenas com os inóculos 1 e 2, que foram, respectivamente, de 36,8 mmol L-1 e 64,1 mmol L-1. Nos reatores operados com água residuária citrícola foram obtidos 21,2; 15,7 e; 37,6 mmol H2 L-1 com os inóculos 1; 2 e 3, respectivamente. Além disso, foram verificadas gerações expressivas de etanol para todos os inóculos testados nos reatores alimentados com glicose. Na caracterização do consórcio anaeróbio citrícola foi verificado predomínio de bacilos Gram + e quantificados os gêneros de bactérias anaeróbias geradoras de H2 (UFC mL-1): Clostridium sp. (3x105), Bacteroides sp. (4x105), Lactobacillus sp. (4x105), Streptococcus sp. (5x104), e Veillonella sp. (3x105), revelando diversidade elevada de bactérias anaeróbias produtoras de hidrogênio presentes na água residuária agroindustrial. Tais resultados ressaltam que a geração de hidrogênio pode ocorrer em vias metabólicas distintas com consequente favorecimento na geração de biocombustíveis como etanol ou butanol.

ABSTRACT In recent years there has been a growing interest in the use of various agro-industrial wastes. In Brazil, the food industries accumulate an enormous amount of waste, especially those that process citrus fruits. Among the possibilities for valuing these residues, anaerobic digestion is a promising alternative in the generation of renewable energy and pollution control. In this sense, this study evaluated the anaerobic digestion of citrus wastewater, aiming at the biological production of hydrogen from three inocula: (1) Clostridium Acetobutylicum ATCC 824, (2) Clostridium Beijerinckii ATCC 10132 and (3) anaerobic consortium obtained from citrus wastewater. The experiment was carried out in 51h, in triplicates of anaerobic batch reactors, fed with 2 different carbon sources: glucose (10.7 g COD L-1) and citrus wastewater (10.0 g COD L-1), headspace filled with N2 (99.99%), initial pH 7.0 and maintained at 37 ° C, under static mode. In the reactors fed with glucose, hydrogen generation was verified only with the inocula 1 and 2, which were, respectively, 36.8 mmol L-1 and 64.1 mmol L-1. In the reactors operated with citrus wastewater, were obtained 21.2; 15.7 e; 37.6 mmol H2 L-1 with inocula 1; 2 and 3, respectively. In addition, expressive generations of ethanol were verified for all inocula tested in the reactors fed with glucose. In the characterization of the citrus anaerobic consortium, a predominance of Gram + bacilli was verified and the genera of anaerobic H2-generating bacteria (UFC mL-1) were quantified: Clostridium sp. (3x105), Bacteroides sp. (4x105), Lactobacillus sp. (4x105), Streptococcus sp. (5x104), and Veillonella sp. (3x105), revealing a high diversity of anaerobic hydrogen-producing bacteria present in agro-industrial wastewater. Such results emphasize that the generation of hydrogen can occur in different metabolic pathways with consequent favor in the generation of biofuels such as ethanol or butanol.

RESUMO O esgotamento das reservas de petróleo e a crescente preocupação com as mudanças climáticas têm acelerado novas pesquisas para obtenção de energias renováveis. A produção de biogás através da digestão anaeróbia vem se mostrando uma ótima opção, não só para produção de energia, como também para manejo adequado de resíduos orgânicos. O biodiesel teve sua produção aumentada em diversos países por ser um combustível renovável e biodegradável. Entretanto, é largamente obtido da transesterificação de óleos vegetais e animais, onde seu rendimento é 9 litros de biodiesel para 1 litro de glicerol. O glicerol bruto possui grau reduzido de pureza, se tornando uma matéria-prima barata com potencial para produção de energia renovável, principalmente gás hidrogênio (H2) e metano (CH4). Nesse sentido, os objetivos deste estudo foram: operar um reator horizontal de leito fixo (RAHLF), com alimentação contínua e determinar a partida do mesmo na produção de H2 a partir de glicerol bruto residual de biodiesel, co-digerido em esgoto sanitário nas fases de: partida, ensaio 1 e ensaio 2. Durante a fase de partida e Ensaio 1, o afluente foi constituído de 99% esgoto 1% glicerol bruto (v/v) e 1,5% de glicerol bruto no Ensaio 2. Na partida e ensaio 1 foram verificadas médias de produção de 1 mol H2 m3 d-1 e 3,2 mols H2 m3 d-1, respectivamente. A maior produção diária atingida foi 11,34 mols H2 m3 d-1 no Ensaio 2 com geração média de ácidos graxos voláteis (AGV) de 3414,0 mg L-1. Tais resultados foram promissores e novos avanços com proporções mais elevadas do glicerol bruto e agentes tamponantes poderão evitar grandes variações de pH, viabilizando assim gerações mais elevadas e mais estáveis de H2, em reatores contínuos.

ABSTRACT The depletion of oil reserves and the growing concern about climate change have accelerated new research to obtain renewable energy. The production of biogas through anaerobic digestion has proved to be a great option, not only for energy production, but also for the proper management of organic waste. Biodiesel had its production increased in several countries as it is a renewable and biodegradable fuel; moreover, it does not require major modifications for its use in diesel engines. However, it is largely obtained from the transesterification of vegetable oils and animal fat, where its yield is 9 volumes of biodiesel to 1 volume of glycerol. The crude glycerol has a low degree of purity, becoming an inexpensive raw material with the potential to produce renewable energy, mainly hydrogen gas (H2) and methane (CH4). In this sense, the objectives of this study were: to operate a horizontal anaerobic reactor with fixed bed (HARFB), with continuous feeding and to determine its start in the production of H2 from residual biodiesel glycerol co-digested in sanitary sewage in the phases from: Startup, Test 1 and Test 2. During the Startup and Test 1 assay, the affluent was constituted by 99% sewage 1% crude glycerol (v/v) and 1.5% crude glycerol in Test 2. At Startup and Test 1, production averages of 1 mol H2 m3 d-1 and 3.2 moles H2 m3 d-1 were verified, respectively. The highest daily production achieved was 11.34 moles H2 m3 d-1 in Test 2 with an average generation of volatile fatty acids (VFA) of 3414.0 mg L-1. Such results were promising and new advances with higher proportions of crude glycerol and buffering agents could avoid large pH variations, thus enabling higher and more stable generations of H2 in continuous reactors.

The inhibition effect of limonene on the organisms involved in anaerobic reactors may affect obtaining H2 in this process. Rapid and precise limonene quantification is desirable to characterize wastewaters. The objective of this research was to develop a Gas Chromatography Coupled to Mass Spectrometry (GC-MS) method to determine limonene concentration in liquid samples from batch reactors applied to H2 production at 30 ºC. Method validation was carried out through calibration curves (3 to 20 mg L-1of limonene), in addition to evaluating its precision (CV =0.1%), linearity (R2=0.979), limits of detection (1.7 mg L-1) and quantification (4.2 mg L-1). Its inhibitory effect was evaluated with increasing concentrations of limonene (without addition to 2000 mg L-1). Limonene affected the starting time of H2 production (λ), since λ was 8.7 h in control assays and 15 h with 2000 mg L-1 of limonene. The same was observed for obtaining H2, since 19.5 mmol H2 L-1 was obtained in control assays and 8.7 mmol H2 L-1 was obtained in assays with 2000 mg L-1 of limonene. This method was effective for limonene determination in liquid samples from batch reactors and could be used for characterization and control of anaerobic reactors.

ABSTRACT Aspergillus spp. cause economic impacts due to aflatoxins production. Although the toxicity of aflatoxins is already known, little information about their ecological roles is available. Here we investigated the compounds produced by Aspergillus nomius ASR3 directly from a dead leaf-cutter queen ant Atta sexdens rubropilosa and the fungal axenic culture. Chemical analyses were carried out by high-resolution mass spectrometry and tandem mass spectrometry techniques. Aflatoxins B1 and G1 were detected in both the axenic culture and in the dead leaf-cutter queen ant. The presence of these mycotoxins in the dead leaf-cutter queen ant suggests that these compounds can be related to the insect pathogenicity of A. nomius against A. sexdens rubropilosa.

Mortality and adverse neurologic sequelae from HIV-associated cryptococcal meningitis (HIV-CM) remains high due to raised intracranial pressure (ICP) complications. Cerebrospinal fluid (CSF) high opening pressure occurs in more than 50% of HIV-CM patients. Repeated lumbar puncture with CSF drainage and external lumbar drainage might be required in the management of these patients. Usually, there is a high grade of uncertainty and the basis for clinical decisions regarding ICP hypertension tends to be from clinical findings (headache, nausea and vomiting), a low Glasgow coma scale score, and/or fundoscopic papilledema. Significant neurological decline can occur if elevated CSF pressures are inadequately managed. Various treatment strategies to address intracranial hypertension in this setting have been described, including: medical management, serial lumbar punctures, external lumbar and ventricular drain placement, and either ventricular or lumbar shunting. This study aims to evaluate the role of a non-invasive intracranial pressure (ICP-NI) monitoring in a critically ill HIV-CM patient.

The best time of production of the cyclohexadepsipeptide beauvericin by the endophytic fungus Fusarium oxysporum SS46 in Czapek medium was evaluated. The highest level of beauvericin production was found on day 21 of fermentative culture, as assessed by quantitative analysis by high performance liquid chromatography coupled with a photodiode array detector. Precursor-directed biosynthesis experiments were carried out to produce new analogues of beauvericin by feeding F. oxysporum with ten analogues of L-phenylalanine. In order to evaluate which precursor analogues were incorporated by the microorganism, the obtained extracts were analyzed using matrix-assisted laser desorption ionization - time-of-flight mass spectrometry (MALDI-TOF/TOF). The precursor-directed biosynthesis studies led to the biosynthesis of novel beauvericin derivatives by replacement of one, two, or all three L-phenylalanine residues in beauvericin with DL-3-fluorophenylalanine, L-3-fluorophenylalanine, L-4-fluorophenylalanine, or L-tyrosine. Beyond these precursor analogues, one unit of L-4-aminophenylalanine, L-4-chlorophenylalanine, DL-4-bromophenylalanine, or L-4-bromophenylalanine was also incorporated by the endophyte F. oxysporum SS46. Units of L-4-nitrophenylalanine and L-histidine were not incorporated by the microorganism to produce unnatural beauvericins.

The chemistry of natural products has been remarkably growing in the past few decades in Brazil. Aspects related to the isolation and identification of new natural products, as well as their biological activities, have been achieved in different laboratories working on this subject in the country. More recently, the introduction of new molecular biology tools has strongly influenced the research on natural products, mainly those produced by microorganisms, creating new possibilities to assess the chemical diversity of secondary metabolites. This paper describes some ideas on how the research on natural products can have a considerable input from molecular biology in the generation of chemical diversity. We also explore the role of microbial natural products in mediating interspecific interactions and their relevance to ecological studies. Examples of the generation of chemical diversity are highlighted by using genome mining, mutasynthesis, combinatorial biosynthesis, metagenomics, and synthetic biology, while some aspects of microbial ecology are also discussed. The idea to bring up this topic is linked to the remarkable development of molecular biology techniques to generate useful chemicals from different organisms. Here, we focus mainly on microorganisms, even though similar approaches have also been applied to the study of plants and other organisms. Investigations in the frontier of chemistry and biology require interactions between different areas, characterizing the interdisciplinarity of this research field. The necessity of a real integration of chemistry and biology is pivotal to finding correct answers to a number of biological phenomena. The use of molecular biology tools to generate chemical diversity and control biosynthetic pathways is largely explored in the production of important biologically active compounds. Finally, we briefly comment on the Brazilian organization of research in this area, the necessity of new strategies for the graduation programs, and the establishment of networks as a way of organization to overcome some of the problems faced in the area of natural products.

The aim of this work was to identify groups of microorganisms that are capable of degrading organic matter utilizing sulfate as an electron acceptor. The assay applied for this purpose consisted of running batch reactors and monitoring lactate consumption, sulfate reduction and sulfide production. A portion of the lactate added to the batch reactors was consumed, and the remainder was converted into acetic, propionic and butyric acid after 111 hours of operation These results indicate the presence of sulfate-reducing bacteria (SRB) catalyzing both complete and incomplete oxidation of organic substrates. The sulfate removal efficiency was 49.5% after 1335 hours of operation under an initial sulfate concentration of 1123 mg/L. The SRB concentrations determined by the most probable number (MPN) method were 9.0x10(7) cells/mL at the beginning of the assay and 8.0x10(5) cells/mL after 738 hours of operation.

The enantioselective biotransformation of propranolol (Prop) by the endophytic fungi Phomopsis sp., Glomerella cingulata, Penicillium crustosum, Chaetomium globosum and Aspergillus fumigatus was investigated by studying the kinetics of the aromatic hydroxylation reaction with the formation of 4-hydroxypropranolol (4-OH-Prop). Both Prop enantiomers were consumed by the fungi in the biotransformation process, but the 4-hydroxylation reaction yielded preferentially (-)-(S)-4-OH-Prop. The quantity of metabolites biosynthesized varied slightly among the evaluated endophytic fungi. These results show that all investigated endophytic fungi could be used as biosynthetic tools in biotransformation processes to obtain the enantiomers of 4-OH-Prop.

Mesmo havendo grandes investimentos, a resistência a fármacos continua sendo um grave problema de saúde pública. Produtos naturais têm prevalecido como a maior fonte de novas drogas e muitos deles são isolados de sistemas simbióticos: microorganismos-plantas. Os fungos endofíticos Papulaspora immersa e Arthrinium state of Apiospora montagnei Sacc. foram isolados de raízes de Smallanthus sonchifolius (yacón). Extratos de culturas destes fungos apresentaram atividade antibacteriana frente Staphylococcus aureus, Kocuria rhizophila, Pseudomonas aeruginosa e Escherichia coli, sendo as mais relevantes observadas nas frações acetato de etila provenientes dos fungos P. immersa e Arthrinium state of Apiospora montagnei Sacc. frente a P. aeruginosa, apresentando CIM de 90µg/mL e 160µg/mL, respectivamente.

Papulaspora immersa and Arthrinium state of Apiospora montagnei Sacc. were isolated from the roots of Smallanthus sonchifolius (yacón). The crude extracts from their cultures inhibited the growth of Staphylococcus aureus, Kocuria rhizophila, Pseudomonas aeruginosa and Escherichia coli. The more relevant results were observed in the ethyl acetate extract from P immersa against P aeruginosa (90 µg/mL) and ethyl acetate extract from Arthrinium state of A montagnei Sacc. against P aeruginosa (160 µg/mL). The two endophytic fungi isolated from yacón roots as well as their antimicrobial activity detected in the crude extracts cultures were being reported for the first time.

There is a continuous need for antibiotics, mainly with new mechanisms of action, since infectious diseases represent the second major cause of death in the world and bacteria resistance levels are high. This review describes the contribution of microbial natural products for the development of the major antibiotic classes, the mechanisms of action of current antibiotics, some modern approaches involving genetic tools for the discovery and development of new antibiotics from microbial products and antibiotics in clinical trials.

This paper presents an overview of the Natural Products Research in Brazil in the last five years (2002-2006), and also discusses how some relevant aspects of the Chemical Biology area could create new research opportunities and challenges for the natural product chemists. In addition, some aspects of the scientific policies and their impact on current projects are discussed.

Três antraquinonas conhecidas (1,7-diidroxi-3-metil-9,10-antraquinona, 1,6-diidroxi-3-metil-9,10-antraquinona e 1-hidroxi-3-metil-9,10-antraquinona), uma nova antraquinona (1,7-diidroxi-3-hidroximetil-9,10-antraquinona), e dois novos derivados hexaidroantraquinônicos, dendrióis E e F, foram isolados da cultura do fungo endofítico Phoma sorghina, associado a Tithonia diversifolia (Asteraceae). Suas estruturas foram identificadas com base em dados espectroscópicos, principalmente RMN 1D e 2D.

Three known anthraquinones (1,7-dihydroxy-3-methyl-9,10-anthraquinone, 1,6-dihydroxy-3-methyl-9,10-anthraquinone and 1-hydroxy-3-methyl-9,10-anthraquinone), one new anthraquinone (1,7-dihydroxy-3-hydroxymethyl-9,10-anthraquinone), and two new hexahydroanthraquinone derivatives, dendryols E and F, were isolated from the culture of the endophytic fungus Phoma sorghina, found in association with Tithonia diversifolia (Asteraceae). Their structures were identified on the basis of spectroscopic data, mainly 1D and 2D NMR.