Exploring Bacterial Diversity of Endodontic Microbiota by Cloning and Sequencing 16S rRNA

Introduction: The characterization of microbial communities infecting the endodontic system in each clinical condition may help on the establishment of a correct prognosis and distinct strategies of treatment. The purpose of this study was to determine the bacterial diversity in primary endodontic infections by 16S ribosomal-RNA (rRNA) sequence analysis. Methods: Samples from root canals of untreated asymptomatic teeth (n = 12) exhibiting periapical lesions were obtained, 165 rRNA bacterial genomic libraries were constructed and sequenced, and bacterial diversity was estimated. Results: A total of 489 clones were analyzed (mean, 40.7 +/- 8.0 clones per sample). Seventy phylotypes were identified of which six were novel phylotypes belonging to the family Ruminococcaceae. The mean number of taxa per canal was 10.0, ranging from 3 to 21 per sample; 65.7% of the cloned sequences represented phylotypes for which no cultivated isolates have been reported. The most prevalent taxa were Atopobium rimae (50.0%), Dialister invisus, Pre-votella oris, Pseudoramibacter alactolyticus, and Tannerella forsythia (33.3%). Conclusions: Although several key species predominate in endodontic samples of asymptomatic cases with periapical lesions, the primary endodontic infection is characterized by a wide bacterial diversity, which is mostly represented by members of the phylum Firmicutes belonging to the class Clostridia followed by the phylum Bacteroidetes. (J Ended 2011;37:922-926)

Cloning and overexpression of the xylose isomerase gene from Burkholderia sacchari and production of polyhydroxybutyrate from xylose

A different organization for the xyl operon was found in different genomes of Burkholderia and Pseudomomas species. Degenerated primers were designed based on Burkholderia genomes and used to amplify the xylose isomerase gene (xylA) from Burkholderia sacchari IPT101 The gene encoded a protein of 329 amino acids, which showed the highest similarity (90%) to the homologous gene of Burkholderia dolosa. It was cloned in the broad host range plasmid pBBR1MCS-2, which partially restored growth and polyhydroxybutyrate production capability in xylose to a B. sacchari xyl(-) mutant. When xylA was overexpressed in the wild-type strain, it was not able to increase growth and polyhydroxybutyrate production, suggesting that XylA activity is not limiting for xylose utilization in B. sacchari.

Cloning, expression, purification and characterization of recombinant glutathione-S-transferase from Xylella fastidiosa

Xylella fastidiosa is an important pathogen bacterium transmitted by xylem-feedings leafhoppers that colonizes the xylem of plants and causes diseases on several important crops including citrus variegated chlorosis (CVC) in orange and lime trees. Glutathione-S-transferases (GST) form a group of multifunctional isoenzymes that catalyzes both glutathione (GSH)-dependent conjugation and reduction reactions involved in the cellular detoxification of xenobiotic and endobiotic compounds. GSTs are the major detoxification enzymes found in the intracellular space and mainly in the cytosol from prokaryotes to mammals, and may be involved in the regulation of stress-activated signals by suppressing apoptosis signal-regulating kinase 1. In this study, we describe the cloning of the glutathione-S-transferase from X. fastidiosa into pET-28a(+) vector, its expression in Escherichia coli, purification and initial structural characterization. The purification of recombinant xfGST (rxfGST) to near homogeneity was achieved using affinity chromatography and size-exclusion chromatography (SEC). SEC demonstrated that rxfGST is a homodimer in solution. The secondary and tertiary structures of recombinant protein were analyzed by circular dichroism and fluorescence spectroscopy, respectively. The enzyme was assayed for activity and the results taken together indicated that rxfGST is a stable molecule, correctly folded, and highly active. Several members of the GST family have been extensively studied. However, xfGST is part of a less-studied subfamily which yet has not been structurally and biochemically characterized. In addition, these studies should provide a useful basis for future studies and biotechnological approaches of rxfGST. (C) 2008 Elsevier Inc. All rights reserved.

Aspectos epidemiológicos da fluorose dentária no Brasil: pesquisas no período 1993-2006

A epidemiologia da fluorose dentária resulta de inquéritos realizados recentemente, em decorrência da melhor compreensão de aspectos metabólicos dos fluoretos no organismo humano e de preocupações no âmbito da saúde coletiva. Objetiva-se apresentar os estudos realizados sobre a fluorose entre 1993 e 2006. O período 1993-2004 demarca o intervalo entre a 2ªe a 3ª Conferência Nacional de Saúde Bucal. Em 2005-2006, a busca de dados primários apresentados em reuniões científicas confirmou os achados existentes na literatura, mostrando que a agenda científica brasileira não foi substancialmente influenciada pelas discussões travadas durante a 3ª Conferência. A maioria dos estudos concentra-se em áreas urbanas e a predominância no Brasil dos graus "muito leve" e "leve" de fluorose mostra não haver comprometimentos de ordem funcional. A baixa percepção da fluorose pela população, aliada à sua baixa prevalência, evoca o necessário debate sobre as questões de saúde coletiva no país. Visto que a produção científica nacional constitui uma importante fonte de conhecimentos para subsidiar a elaboração de políticas públicas para o setor da saúde, os sucessivos e pontuais estudos analisados mostram que, no que diz respeito à fluorose, o diagnóstico epidemiológico reafirma a necessidade, a importância e a segurança da fluoretação das águas de abastecimento público como medida de saúde coletiva.

Distribution of biotypes and leukotoxic activity of Aggregatibacter actinomycetemcomitans isolated from Brazilian patients with chronic periodontitis

Aggregatibacter actinomycetemcomitans is an important etiologic agent of the periodontitis and is associated with extra-oral infections. In this study, the detection of the ltxA gene as well as the ltx promoter region from leukotoxic A. actinomycetemcomitans isolated from 50 Brazilian patients with periodontitis and 50 healthy subjects was performed. The leukotoxic activity on HL-60 cells was also evaluated. Leukotoxic activity was determined using a trypan blue exclusion method. The 530 bp deletion in the promoter region was evaluated by PCR using a PRO primer pair. A. actinomycetemcomitans was detected by culture and directly from crude subgingival biofilm by PCR using specific primers. By culture, A. actinomycetemcomitans was detected in nine (18%) of the periodontal patients and one (2%) healthy subject. However, by PCR, this organism was detected in 44% of the periodontal patients and in 16% of the healthy subjects. It was verified a great discrepancy between PCR detection of the ltx operon promoter directly from crude subgingival biofilm and from bacterial DNA. Only one periodontal sample harbored highly leukotoxic A. actinomycetemcomitans. Moreover, biotype II was the most prevalent and no correlation between biotypes and leukotoxic activity was observed. The diversity of leukotoxin expression by A. actinomycetemcomitans suggests a role of this toxin in the pathogenesis of periodontal disease and other infectious diseases.

A fototerapia com diodo emissor de luz (LEDT) aplicada pré-exercício inibe a peroxidação lipídica em atletas após exercício de alta intensidade: um estudo preliminar

Estresse oxidativo é o termo geralmente utilizado para descrever os danos causados pelo desequilíbrio entre pró-oxidantes e antioxidantes no organismo. O aumento no consumo de O2 induzido pelo exercício físico está associado ao aumento das espécies reativas de oxigênio (EROs) sendo estas indutoras do estresse oxidativo. Embora as evidências indiquem um provável efeito inibitório da fototerapia com diodos emissores de luz (LEDT) sobre a produção das EROs, não existem estudos observando tal efeito em atletas. Este estudo preliminar destina-se a verificar os efeitos da aplicação de LEDT previamente ao exercício de alta intensidade sobre a peroxidação lipídica, mensurada através dos níveis sanguíneos de substâncias reativas ao ácido tiobarbitúrico (TBARS). Todos os seis atletas de voleibol do sexo masculino foram submetidos às duas situações: aplicação de LEDT efetiva e aplicação de LEDT placebo. O desempenho no protocolo de exercício adotado não revelou diferença (p > 0,05) entre as duas situações nas variáveis potência pico, potência média e índice de fadiga. Os resultados relacionados com a peroxidação lipídica foram: na situação LEDT efetiva, não foi possível observar diferença estatisticamente significante (p > 0,05) entre os níveis pré e pós-exercício (6,98 ± 0,81 e 7,02 ± 0,47nmol/mL); na situação LEDT (LBP) placebo, houve diferença estatisticamente significante (p = 0,05) entre os valores pré e pós-exercício (7,09 ± 1,28 e 8,43 ± 0,71nmol/mL). Tais resultados demonstram que a aplicação efetiva de LEDT parece ser eficaz no controle da peroxidação lipídica em atletas submetidos a exercício intenso

TiO2-Photocatalyzed degradation of phenol in saline media in an annular reactor: hydrodynamics, lumped kinetics, intermediates, and acute toxicity

The photocatalytic degradation of phenol in aqueous suspensions of TiO2 under different salt concentrations in an annular reactor has been investigated. In all cases, complete removal of phenol and mineralization degrees above 90% were achieved. The reactor operational parameters were optimized and its hydrodynamics characterized in order to couple mass balance equations with kinetic ones. The photodegradation of the organics followed a Langmuir-Hinshelwood-Hougen-Watson lumped kinetics. From GC/MS analyses, several intermediates formed during oxidation have been identified. The main ones were catechol, hydroquinone, and 3-phenyl-2-propenal, in this order. The formation of negligible concentrations of 4-chlorophenol was observed only in high salinity medium. Acute toxicity was determined by using Artemia sp. as the test organism, which indicated that intermediate products were all less toxic than phenol and a significant abatement of the overall toxicity was accomplished, regardless of the salt concentration.

Nutraceuticals in reproduction of bulls and stallions

The industry has made available in the market a series of substances (nutraceuticals) which intent would be to optimize the use of nutrients in some metabolic paths, influencing positively reproductive performance in animals. However, the response to the use of nutraceuticals varies according to the animal. As the organism is highly complex and in order to achieve a perfect activity of the hypothalamic-pituitary-gonadal axis, an ideal interaction in molecular basis is needed, where the nutraceuticals can have their direct action. The aim of this study was to review the function and research results using the main nutraceuticals (β carotene, vitamin A, L-carnitine, omegas 3, 6 and 9 and Gamma-oryzanol) on reproductive characteristics of bulls and stallions.

Produção in vitro de embriões de ovinos: uma visão crítica do método e de seu resultado a campo

A metodologia de produção in vitro de embriões de ovinos implica no desenvolvimento de meios de maturação, fertilização e cultivo que permitam aumentar a taxa de clivagem e desenvolvimento, tanto para o investimento biotecnológico em programas comerciais, quanto para sua utilização em clonagem e transgenia dessa espécie animal. Do ponto de vista da pesquisa, os ovócitos podem ser obtidos pelas técnicas de punção e slicing a partir de ovários oriundos de matadouros, ou através de aspiração folicular por laparoscopia. Como vantagem, este método permite o uso de uma mesma doadora estimulada hormonialmente em intervalos periódicos, mantida sob rigoroso controle sanitário, o que é de vital importância para a produção de biofármacos em programas que utilisem os ovinos como modelo biológico. Por outro lado, em nosso país a demanda pela multiplicação de animais de alto valor genético, seja pela produtividade ou pelo elevado valor comercial dos mesmos, impõe o desenvolvimento, adaptação e otimização das diferentes metodologias desenvolvidas ao longo dos ultimos anos em laboratórios de referência mundiais. Nesse contexto, cresce de importância o perfeito conhecimento da fisiologia dessa espécie e das raças criadas em nosso país, e da problemática da produção in vitro de seus embriões. Respeitando essas premissas, gerar o desenvolvimento de protocolos que permitam não apenas aumentar a população de ovócitos passíveis de maturação in vitro, mas de sua competência ao desenvolvimento ao estágio de blastocisto, ou, alternativamente, sua transferência a receptoras em estágios precoces do desenvolvimento, evitando assim as conhecidas perdas durante o desenvolvimento in vitro, e o baixo percentual de gestações que chegam a termo, com cordeiro saudáveis. Trata-se de um desafio, que já apresenta os primeiros resultados em nosso país, tanto na produção comercial de embriões produzidos in vitro, quanto em programas de clonagem e transgenia.

Emergence and disappearance of a virulent clone of Haemophilus influenzae biogroup aegyptius, cause of Brazilian Purpuric Fever

Summary: In 1984, children presented to the emergency department of a hospital in the small town of Promissão, São Paulo State, Brazil, with an acute febrile illness that rapidly progressed to death. Local clinicians and public health officials recognized that these children had an unusual illness, which led to outbreak investigations conducted by Brazilian health officials in collaboration with the U.S. Centers for Disease Control and Prevention. The studies that followed are an excellent example of the coordinated and parallel studies that are used to investigate outbreaks of a new disease, which became known as Brazilian purpuric fever (BPF). In the first outbreak investigation, a case-control study confirmed an association between BPF and antecedent conjunctivitis but the etiology of the disease could not be determined. In a subsequent outbreak, children with BPF were found to have bacteremia caused by Haemophilus influenzae biogroup aegyptius (H. aegyptius), an organism previously known mainly to cause self-limited purulent conjunctivitis. Molecular characterization of blood and other isolates demonstrated the clonal nature of the H. aegyptius strains that caused BPF, which were genetically distant from the diverse strains that cause only conjunctivitis. This led to an intense effort to identify the factors causing the unusual invasiveness of the BPF clone, which has yet to definitively identify the virulence factor or factors involved. After a series of outbreaks and sporadic cases through 1993, no additional cases of BPF have been reported

Elementos protetores do leite materno na prevenção de doenças gastrintestinais e respiratórias

A Epistemologia Genética defende que o indivíduo passa por várias etapas de desenvolvimento ao longo da sua vida. O desenvolvimento é observado pela sobreposição do equilíbrio entre a assimilação e a acomodação, resultando em adaptação. Assim, nesta formulação, o ser humano assimila os dados que obtém do exterior, mas uma vez que já tem uma estrutura mental que não está vazia, precisa adaptar esses dados à estrutura mental já existe. O processo de modificação de si próprio é chamado de acomodação. Este esquema revela que nenhum conhecimento chega do exterior sem que sofra alguma alteração pelo indivíduo, sendo que tudo o que se aprende é influenciado por aquilo que já havia sido aprendido. A assimilação ocorre quando a informação é incorporada às estruturas já pré-existentes nessa dinâmica estrutura cognitiva, enquanto que a adaptação ocorre quando o organismo se modifica de alguma maneira de modo a incorporar dinamicamente a nova informação. Por fim, de um pensamento moderno que, buscando a síntese inusitada entre o biológico e o lógico-matemático, parece encontrar seus limites na desconstrução ainda mais inusitada a que tende sistematicamente todo o pensamento na atualidade: a de si mesmo se construindo de modo essencialmente esclarecido

A epistemologia genética de Piaget e o construtivismo

A Epistemologia Genética defende que o indivíduo passa por várias etapas de desenvolvimento ao longo da sua vida. O desenvolvimento é observado pela sobreposição do equilíbrio entre a assimilação e a acomodação, resultando em adaptação. Assim, nesta formulação, o ser humano assimila os dados que obtém do exterior, mas uma vez que já tem uma estrutura mental que não está vazia, precisa adaptar esses dados à estrutura mental já existe. O processo de modificação de si próprio é chamado de acomodação. Este esquema revela que nenhum conhecimento chega do exterior sem que sofra alguma alteração pelo indivíduo, sendo que tudo o que se aprende é influenciado por aquilo que já havia sido aprendido. A assimilação ocorre quando a informação é incorporada às estruturas já pré-existentes nessa dinâmica estrutura cognitiva, enquanto que a adaptação ocorre quando o organismo se modifica de alguma maneira de modo a incorporar dinamicamente a nova informação. Por fim, de um pensamento moderno que, buscando a síntese inusitada entre o biológico e o lógico-matemático, parece encontrar seus limites na desconstrução ainda mais inusitada a que tende sistematicamente todo o pensamento na atualidade: a de si mesmo se construindo de modo essencialmente esclarecido

Ticks produce highly selective chemokine binding proteins with antiinflammatory activity

Bloodsucking parasites such as ticks have evolved a wide variety of immunomodulatory proteins that are secreted in their saliva, allowing them to feed for long periods of time without being detected by the host immune system. One possible strategy used by ticks to evade the host immune response is to produce proteins that selectively bind and neutralize the chemokines that normally recruit cells of the innate immune system that protect the host from parasites. We have identified distinct cDNAs encoding novel chemokine binding proteins (CHPBs), which we have termed Evasins, using an expression cloning approach. These CHBPs have unusually stringent chemokine selectivity, differentiating them from broader spectrum viral CHBPs. Evasin-1 binds to CCL3, CCL4, and CCL18; Evasin-3 binds to CXCL8 and CXCL1; and Evasin-4 binds to CCL5 and CCL11. We report the characterization of Evasin-1 and -3, which are unrelated in primary sequence and tertiary structure, and reveal novel folds. Administration of recombinant Evasin-1 and - 3 in animal models of disease demonstrates that they have potent antiinflammatory properties. These novel CHBPs designed by nature are even smaller than the recently described single-domain antibodies (Hollinger, P., and P. J. Hudson. 2005. Nat. Biotechnol. 23: 1126-1136), and may be therapeutically useful as novel antiinflammatory agents in the future.

Convergence of developmental mutants into a single tomato model system: 'Micro-Tom' as an effective toolkit for plant development research

Background: The tomato (Solanum lycopersicum L.) plant is both an economically important food crop and an ideal dicot model to investigate various physiological phenomena not possible in Arabidopsis thaliana. Due to the great diversity of tomato cultivars used by the research community, it is often difficult to reliably compare phenotypes. The lack of tomato developmental mutants in a single genetic background prevents the stacking of mutations to facilitate analysis of double and multiple mutants, often required for elucidating developmental pathways. Results: We took advantage of the small size and rapid life cycle of the tomato cultivar Micro-Tom (MT) to create near-isogenic lines (NILs) by introgressing a suite of hormonal and photomorphogenetic mutations (altered sensitivity or endogenous levels of auxin, ethylene, abscisic acid, gibberellin, brassinosteroid, and light response) into this genetic background. To demonstrate the usefulness of this collection, we compared developmental traits between the produced NILs. All expected mutant phenotypes were expressed in the NILs. We also created NILs harboring the wild type alleles for dwarf, self-pruning and uniform fruit, which are mutations characteristic of MT. This amplified both the applications of the mutant collection presented here and of MT as a genetic model system. Conclusions: The community resource presented here is a useful toolkit for plant research, particularly for future studies in plant development, which will require the simultaneous observation of the effect of various hormones, signaling pathways and crosstalk.

DEVELOPMENT OF A NOVEL SET OF MICROSATELLITE MARKERS FOR CASTOR BEAN, RICINUS COMMUNIS (EUPHORBIACEAE)

Premise of study: Microsatellite primers were developed for castor bean (Ricinus communis L.) to investigate genetic diversity and population structure, and to provide support to germplasm management. Methods and Results: Eleven microsatellite loci were isolated using an enrichment cloning protocol and used to characterize castor bean germplasm from the collection at the Instituto Agronomico de Campinas (IAC). In a survey of 76 castor bean accessions, the investigated loci displayed polymorphism ranging from two to five alleles. Conclusions: The information derived from microsatellite markers led to significant gains in conserved allelic richness and provides support to the implementation of several molecular breeding strategies for castor bean.