Identifying potential mechanisms enabling acidophily in the ammonia-oxidizing archaeon "Candidatus Nitrosotalea devanaterra"

FUNDING INFORMATION This work, including the efforts of Luis Sayavedra-Soto, was funded by National Science Foundation (NSF) (0541797). This work, including the efforts of Laura E. Lehtovirta-Morley, James I. Prosser, and Graeme W. Nicol, was funded by Natural Environment Research Council (NERC) (NE/I027835/1). Graeme W. Nicol is also supported by the AXA Research Fund.

Reaproveitamento de meio de cultivo de Arthrospira platensis tratado por processos de microfiltração e ultrafiltração

Micro-organismos fotossintetizantes, incluído aqui o gênero Arthrospira, vêm sendo amplamente produzidos em larga escala em vários países, detendo um mercado que gera mais de 1 bilhão de dólares ao ano. A produção industrial utiliza grande volume de água com alta concentração salina para produzir milhares de toneladas de biomassa microalgal. É crescente a utilização de tratamento de águas por processo de separação por membranas, demonstrando ser uma técnica que gera água de ótima qualidade, de instalação compacta e de fácil automação. No presente trabalho, foi avaliada esta tecnologia para o reaproveitamento do meio de cultura em novos cultivos de micro-organismos fotossintetizantes, visando contribuir para a sustentabilidade deste processo produtivo. O efluente do cultivo de Arthrospira platensis oriundo de processo descontínuo em minitanques foi submetido a tratamento por membranas de filtração tangencial, incluindo microfiltração (MF) (porosidades de 0,65 µm e de 0,22 µm) e ultrafiltração (UF) (peso molecular de corte de 5.000 Da), em pressões transmembrana (TMP) de 22,5 a 90 kPa. Os processos de MF levaram a reduções médias de 53,9±1,3 % e 93,1±1,1 % de matéria orgânica natural (NOM) e pigmentos nos meios residuais, respectivamente. Com o uso de processos de UF, cujos meios foram previamente tratados por MF (0,22 µm e 22,5 kPa), as reduções médias de NOM e pigmentos foram de 57,2±0,5 % e 94,0±0,8 %, respectivamente. Os processos de MF com TMP de 22,5 kPa levaram a concentrações celulares máximas (Xm) equivalentes às obtidas com meio novo. O uso de membrana de 0,65 µm e TMP de 22,5 kPa levou a uma perda média de 2,9 %, 22,7 % e 16,4% dos nutrientes carbonato, fosfato e nitrato, respectivamente, mas a correção desses valores aos mesmos do meio padrão levou à obtenção dos mais altos valores de Xm (3586,6±80 mg L-1), produtividade em células (505,0±11,6 mg L-1 d-1) e fator de conversão de nitrogênio em células (29,6±0,7 mg mg-1). O teor protéico da biomassa foi estatisticamente igual ao da biomassa obtida de cultivo com meio padrão novo. Os dados deste trabalho evidenciam que processos de filtração por membrana são promissores para o reuso de meio de micro-organismos fotossintetizantes.

Avaliação de indicadores biológicos na validação de processos de esterilização de isoladores por peróxido de hidrogênio

A resistência de cinco microrganismos presentes na microbiota da área de produção estéril (Cristalização Estéril), frente a ação do gás de peróxido de hidrogênio foi determinada e o valor O obtido para cada microrganismo foi comparado ao valor D do Bacillus stearothermophilus ATCC 12980 exposto ao mesmo agente. Os microrganismos testados foram Bacillus sp, M. luteus, Corynebacterium, Staphylococcus sp e Penicillium sp. Este teste tinha a finalidade de comprovar que a resistência do Bacillus stearothermophilus é maior quando da exposição ao peróxido de hidrogênio se comparada a outros microrganismos presentes na área produtiva. A metodologia consistiu da inoculação de 0,01 mL da suspensão de cada microrganismo na contagem de 102UFC/0,01 mL em cupons de aço inoxidável, previamente esterilizados por calor seco e posterior exposição ao gás de peróxido de hidrogênio. O experimento demonstrou que o valor D obtido para o Bacillus stearothermophilus ésuperior aos obtidos para os outros microrganismos em teste comprovando que a escolha deste microrganismo para o desafio contra o peróxido de hidrogênio é apropriada. Também executou-se o teste que visava garantir que o aço inoxidável é o material de suporte mais recomendado para este fim, utilizando-se suportes de diversos materiais normalmente encontrados no interior dos isoladores (PVC, aço inoxidável, CKC, teflon, polipropileno, látex, silicone, Hypalon, vidro, nylon, saco de alumínio) com 0,01 mL de inóculo de Bacillus stearothermophilus na contagem de 102UFC/O,01 mL, o que foi devidamente comprovado.

Irradiação de drogas vegetais: aspectos microbiológicos e químicos

Apesar da industrialização no setor farmacêutico, o emprego de drogas vegetais constitui desafio atual quando considerado alternativa terapêutica para as populações de baixa renda ou aquelas que apresentam tradição no uso dessas drogas. Além disso, tendências modernas valorizam a variedade de espécies com propriedades curativas, em particular as espécies brasileiras, desafiando os pesquisadores a intensificar investigações nessa área e induzindo à população um crescente consumo. Assim, questões relacionadas à qualidade dessas drogas apresentam fundamental importância. Devido à origem, a carga microbiana detectada nas mesmas é normalmente elevada, oferecendo riscos potenciais ao usuário. Desta forma, a avaliação de sua qualidade sanitária constitui etapa obrigatória no que se refere ao aspecto de segurança ao consumidor. Além disso, a eficácia terapêutica pode igualmente ser comprometida por decomposição de componentes, decorrente da ação de microrganismos. Com o objetivo de eliminar os efeitos decorrentes da biocarga presente nas drogas vegetais, agentes descontaminantes, de natureza física ou química, têm sido empregados. A utilização de tais procedimentos de descontaminação, prevista na legislação vigente, requer estudos relacionados à estabilidade dos princípios ativos após exposição ao agente selecionado. Dentre os agentes destaca-se a irradiação gama, amplamente utilizada em função de sua aplicabilidade na ausência de água e de temperatura elevada, além de apresentar alta penetrabilidade e reduzir, com eficácia, a carga microbiana viável. Os objetivos do presente trabalho foram avaliar os efeitos de diferentes doses de radiação ionizante sobre a carga microbiana de quatro espécies de drogas vegetais: alcachofra (Cynara scolymus L.), camomila (Matricaria recutita L.), ginkgo (Ginkgo biloba L.) e guaraná (Paullinia cupana H.B.K.), bem como detectar possíveis alterações provocadas pela radiação sobre os teores de seus princípios ativos. As análises microbiológicas e químicas foram realizadas antes e após irradiação com doses médias de 5,5 kGy, 11,4 kGy e 17,8 kGy. Os resultados obtidos anteriormente à irradiação revelaram elevados níveis de contaminação: média de 4,1 x106 para microrganismos aeróbicos totais e 3,3x105 para fungos. Após descontaminação, a dose média de 11,4 kGy, reduziu a carga de microrganismos aeróbicos totais a níveis menores ou iguais a 102 em todas as drogas, com exceção da camomila proveniente do fornecedor B (3,2x104). Para os fungos, a menor dose aplicada (5,5 kGy) foi suficiente para reduzir a contagem a níveis da ordem de 10. Com relação à determinação dos marcadores nas drogas vegetais, os resultados obtidos não revelaram alterações significativas nos teores de cafeína no guaraná e de glicosídeos flavonoídicos no ginkgo. Para a camomila, as amostras antes a após irradiação, apresentaram o mesmo teor de óleo volátil bem como ausência de diferenças significativas no teor de α-bisabolol. Em contraste, observou-se redução no teor de 7-glicosil apigenina após submissão à radiação ionizante, indicando degradação decorrente do processo. Com relação à alcachofra, permanece ainda desconhecida a influência da radiação devido à ausência de metodologias adequadas para extração e determinação da cinarina.

Analysis of RNA-protein interactions by flow cytometry

Flow cytometry, in combination with advances in bead coding technologies, is maturing as a powerful high-throughput approach for analyzing molecular interactions. Applications of this technology include antibody assays and single nucleotide polymorphism mapping. This review describes the recent development of a microbead flow cytometric approach to analyze RNA-protein interactions and discusses emerging bead coding strategies that together will allow genome-wide identification of RNA-protein complexes. The microbead flow cytometric approach is flexible and provides new opportunities for functional genomic studies and small-molecule screening.

Mechanisms of Thermal Adaptation Revealed From the Genomes of the Antarctic Archaea Methanogenium frigidum and Methanococcoides burtonii

We generated draft genome sequences for two cold-adapted Archaea, Methanogenium frigidum and Methanococcoides burtonii, to identify genotypic characteristics that distinguish them from Archaea with a higher optimal growth temperature (OGT). Comparative genomics revealed trends in amino acid and tRNA composition, and structural features of proteins. Proteins from the cold-adapted Archaea are characterized by a higher content of noncharged polar amino acids, particularly Gin and Thr and a lower content of hydrophobic amino acids, particularly Leu. Sequence data from nine methanogen genomes (OGT 15degrees-98degreesC) were used to generate IIII modeled protein structures. Analysis of the models from the cold-adapted Archaea showed a strong tendency in the solvent-accessible area for more Gin, Thr, and hydrophobic residues and fewer charged residues. A cold shock domain (CSD) protein (CspA homolog) was identified in M. frigidum, two hypothetical proteins with CSD-folds in M. burtonii, and a unique winged helix DNA-binding domain protein in M. burtonii. This suggests that these types of nucleic acid binding proteins have a critical role in cold-adapted Archaea. Structural analysis of tRNA sequences from the Archaea indicated that GC content is the major factor influencing tRNA stability in hyperthermophiles, but not in the psychrophiles, mesophiles or moderate thermophiles. Below an OGT of 60degreesC, the GC content in tRNA was largely unchanged, indicating that any requirement for flexibility of tRNA in psychrophiles is mediated by other means. This is the first time that comparisons have been performed with genome data from Archaea spanning the growth temperature extremes. from psychrophiles to hyperthermophiles

Effects of leukapheresis protocol, cell processing and cryopreservation on the generation of monocyte-derived DC for immune therapy

Background Many clinical trials of DC-based immunotherapy involve administration of monocyte-derived DCs (Mo-DC) on multiple occasions. We aimed to determine the optimal cell processing procedures and timing (leukapheresis, RBC depletion and cryopreservation) for generation of Mo-DC for clinical purposes. Methods Leukapheresis was undertaken using a COBE Spectra. Two instrument settings were compared - the standard semi-automated software (Version 4.7) (n = 10) and the fully automated software (Version 6.0) (n = 40). Density gradient centrifugation using Ficoll, Percoll, a combination of these methods or neither for RBC depletion were compared. Outcomes (including cell yield and purity) were compared for cryopreserved unmanipulated monocytes and cryopreserved Mo-DC. Results Software Version 6.0 provided significantly better enrichment for monocytes (P

Identification and analysis of chromodomain-containing proteins encoded in the mouse transcriptome

The chromodomain is 40-50 amino acids in length and is conserved in a wide range of chromatic and regulatory proteins involved in chromatin remodeling. Chromodomain-containing proteins can be classified into families based on their broader characteristics, in particular the presence of other types of domains, and which correlate with different subclasses of the chromodomains themselves. Hidden Markov model (HMM)-generated profiles of different subclasses of chromodomains were used here to identify sequences encoding chromodomain-containing proteins in the mouse transcriptome and genome. A total of 36 different loci encoding proteins containing chromodomains, including 17 novel loci, were identified. Six of these loci (including three apparent pseudogenes, a novel HP1 ortholog, and two novel Msl-3 transcription factor-like proteins) are not present in the human genome, whereas the human genome contains four loci (two CDY orthologs and two apparent CDY pseuclogenes) that are not present in mouse. A number of these loci exhibit alternative splicing to produce different isoforms, including 43 novel variants, some of which lack the chromodomain. The likely functions of these proteins are discussed in relation to the known functions of other chromodomain-containing proteins within the same family.

Rhizopus arrhizus - a producer for simultaneous saccharification and fermentation of starch waste materials to L(+)-lactic acid

Rhizopus arrhizus, strain DAR 36017, produced L(+)-lactic acid in a simultaneous saccharification and fermentation process using starch waste effluents. Lactic acid at 19.5 - 44.3 g l(-1) with a yield of 0.85 - 0.96 g g(-1) was produced in 40 h using 20 - 60 g starch l(-1). Supplementation of nitrogen source may be unnecessary if potato or corn starch waste effluent was used as a production medium.

Biotechnological production of lactic acid integrated with potato wastewater treatment by Rhizopus arrhizus

This paper describes a feasibility study of a for lactic acid production integrated with are treatment of wastewater from an industrial starch plant. Rhizopus oryzae two strains, Rhizopus arrhizus and Rhizopus oligosporus were tested with respect to their capability to carry out simultaneous saccharification and fermentation to lactic acid using potato wastewater. Rhizopus arrhizus DAR 36017 was identified as a suitable strain that demonstrated a high capacity for starch saccharification and lactic acid synthesis. The optimal conditions, in terms of pH, temperature and starch concentration, for lactic acid production were determined. The selected fungal strain grew well in a pH range from 3.0 to 7.0. The addition of CaCO(3)10 g dm(-3) maintained the pH at 5.0-6.0 and significantly enhanced lactic acid production. Kinetic study revealed that almost complete starch saccharification and a lactic acid yield of 450g kg(-1) could be achieved in 20 h and 28 h cultivation, respectively. The maximum lactic acid production 21 g dm(-3) and mycelial biomass (1.7 g dm(-3)) were obtained at 30degreesC. Besides the multiple bioproducts, total removal of suspended solids and 90% reduction of COD were achieved in a single no-aseptic operation. (C) 2003 Society of Chemical Industry.

In vivo gene expression: DNA electrotransfer

The use of electric pulses to deliver therapeutic molecules to tissues and organs in vivo is a rapidly growing field of research. Electrotransfer can be used to deliver a wide range of potentially therapeutic agents, including drugs, proteins, oligonucleotides, RNA and DNA. Optimization of this approach depends upon a number of parameters such as target organ accessibility, cell turnover, microelectrode design, electric pulsing protocols and the physiological response to the therapeutic agent. Many organs have been successfully transfected by electroporation, including skin, liver, skeletal and cardiac muscle, male and female germ cells, artery, gut, kidney, retinal ganglion cells, cornea, spinal cord, joint synovium and brain. Electrotransfer technology is relevant in a variety of research and clinical settings including cancer therapy, modulation of pathogenic immune reactions, delivery of therapeutic proteins and drugs, and the identification of drug targets by the modulation of normal gene expression. This, together with the capacity to deliver very large DNA constructs, greatly expands the research and clinical applications of in vivo DNA electrotransfer.

Molecular analysis of the pathway for the synthesis of thiol tripeptides in the model legume Lotus japonicus

The thiol tripeptides, glutathione (GSH) and homoglutathione (hGSH), perform multiple roles in legumes, including protection against toxicity of free radicals and heavy metals. The three genes involved in the synthesis of GSH and hGSH in the model legume, Lotus japonicus, have been fully characterized and appear to be present as single copies in the genome. The gamma-glutamylcysteine synthetase (gammaecs) gene was mapped on the long arm of chromosome 4 (70.0 centimorgans [cM]) and consists of 15 exons, whereas the glutathione synthetase (gshs) and homoglutathione synthetase (hgshs) genes were mapped on the long arm of chromosome 1 (81.3 cM) and found to be arranged in tandem, with a separation of approximately 8 kb. Both genes consist of 12 exons of exactly the same size (except exon 1, which is similar). Two types of transcripts were detected for the gshs gene, which putatively encode proteins localized in the plastids and cytosol. Promoter regions contain cis-acting regulatory elements that may be involved in the plant's response to light, hormones, and stress. Determination of transcript levels, enzyme activities, and thiol contents in nodules, roots, and leaves revealed that gammaecs and hgshs are expressed in all three plant organs, whereas gshs is significantly functional only in nodules. This strongly suggests an important role of GSH in the rhizobia-legume symbiosis.

Gut content analysis to distinguish between seed feeding and mycophagy of a biphyllid beetle larva found on Acacia melanoxylon

In a search for potential biocontrol agents for Acacia melanoxylon R. Br. (Mimosaceae), larvae of the beetle Diplocoelus dilataticollis Lea (Coleoptera; Biphyllidae) were found within damaged seeds of A. melanoxylon. The gut contents of larvae and adults were examined to determine whether their diet included seeds, in apparent contradiction to the known mycophagous diet of members of this family of beetles. Calcofluor M2R White, a plant cell-wall staining optical brightener was used to differentiate between plant cell fragments and fungal tissue in the gut content smears. Gut contents of adults of a known seed predator of A. melanoxylon, a weevil of the genus Melanterius, were examined in the same way to provide a benchmark. The gut contents of D. dilataticollis differed from those of Melanterius sp. Fungal structures and microbes were found in the gut of D. dilataticollis, in contrast to plant cell fragments found in the gut of the weevil and from scrapes made directly from seeds. We conclude that larvae of D. dilataticollis feed primarily on fungi associated with damaged seed and therefore may not be the proximate cause of seed damage.

Identification of a major locus conferring resistance to powdery mildew (Erysiphe polygoni DC) in mungbean (Vigna radiata L. Wilczek) by QTL analysis

A major locus conferring resistance to the causal organism of powdery mildew, Erysiphe polygoni DC,, in mungbean (Vigna radiata L. Wilczek) was identified using QTL analysis with a population of 147 recombinant inbred individuals. The population was derived from a cross between 'Berken', a highly susceptible variety, and ATF 3640, a highly resistant line. To test for response to powdery mildew, F-7 and F-8 lines were inoculated by dispersing decaying mungbean leaves with residual conidia of E. polygoni amongst the young plants to create an artificial epidemic and assayed in a glasshouse facility. To generate a linkage map, 322 RFLP clones were tested against the two parents and 51 of these were selected to screen the mapping population. The 51 probes generated 52 mapped loci, which were used to construct a linkage map spanning 350 cM of the mungbean genome over 10 linkage groups. Using these markers, a single locus was identified that explained up to a maximum of 86% of the total variation in the resistance response to the pathogen.