The Difficult Case of Crystallization and Structure Solution for the ParC55 Breakage-Reunion Domain of Topoisomerase IV from Streptococcus pneumoniae

BACKGROUND: Streptococcus pneumoniae is the major cause of community-acquired pneumonia and is also associated with bronchitis, meningitis, otitis and sinusitis. The emergence and increasing prevalence of resistance to penicillin and other antibiotics has led to interest in other anti-pneumonococcal drugs such as quinolones that target the enzymes DNA gyrase and topoisomerase IV. During crystallization and in the avenues to finding a method to determine phases for the structure of the ParC55 breakage-reunion domain of topoisomerase IV from Streptococcus pneumoniae, obstacles were faced at each stage of the process. These problems included: majority of the crystals being twinned, either non-diffracting or exhibiting a high mosaic spread. The crystals, which were grown under conditions that favoured diffraction, were difficult to flash-freeze without loosing diffraction. The initial structure solution by molecular replacement failed and the approach proved to be unviable due to the complexity of the problem. In the end the successful structure solution required an in-depth data analysis and a very detailed molecular replacement search. METHODOLOGY/PRINCIPAL FINDINGS: Crystal anti-twinning agents have been tested and two different methods of flash freezing have been compared. The fragility of the crystals did not allow the usual method of transferring the crystals into the heavy atom solution. Consequently, it was necessary to co-crystallize in the presence of the heavy atom compound. The multiple isomorphous replacement approach was unsuccessful because the 7 cysteine mutants which were engineered could not be successfully derivatized. Ultimately, molecular replacement was used to solve the structure by sorting through a large number of solutions in space group P1 using CNS. CONCLUSIONS/SIGNIFICANCE: The main objective of this paper is to describe the obstacles which were faced and overcome in order to acquire data sets on such difficult crystals and determine phases for successful structure solution

Effluent Organic Nitrogen (EON): Bioavailability and Photochemical and Salinity-Mediated Release

The goal of this study was to investigate three potential ways that the soluble organic nitrogen (N) fraction of wastewater treatment plant (WWTP) effluents, termed effluent organic N (EON), could contribute to coastal eutrophication - direct biological removal, photochemical release of labile compounds, and salinity-mediated release of ammonium (NH4+). Effluents from two WWTPs were used in the experiments. For the bioassays, EON was added to water from four salinities (similar to 0 to 30) collected from the James River (VA) in August 2008, and then concentrations of N and phosphorus compounds were measured periodically over 48 h. Bioassay results, based on changes in DON concentrations, indicate that some fraction of the EON was removed and that the degree of EON removal varied between effluents and with salinity. Further, we caution that bioassay results should be interpreted within a broad context of detailed information on chemical characterization. EON from both WWTPs was also photoreactive, with labile NH4+ and dissolved primary amines released during exposure to sunlight. We also present the first data that demonstrate that when EON is exposed to higher salinities, increasing amounts of NH4+ are released, further facilitating EON use as effluent transits from freshwater through estuaries to the coast

Improper program management-induced system archetypes

Projects and programs: those two concepts are not one same side of a coin or even two different sides of a same coin. Despite the growing consensus among the project management community around the differences between the two topics, many organizations still fail to adopt a governance framework that considers such differences. The consequence is a domino effect: programs are treated as large projects, short-term focus values only the delivery of capabilities by component projects, an unhealthy internal competition between said projects is established, the organization's resources are burdened, and ultimately, organizations fail to realize benefits to their full potential. In this paper, we show that such situation fits into two well-known system archetypes, namely "Success to the Successful" and "Tragedy of the Commons". As case-study, we present a large, multi-year, multi-million Brazilian Government initiative, as well as the results achieved by said initiative, before and after a proper governance framework was in place. We also use System Dynamics (SD) to simulate and demonstrate said results, but also to forecast the expected results for the years yet to come, now that the programs and projects are given the proper treatment

Trapping of the transport-segment DNA by the ATPase domains of a type II topoisomerase

Type II topoisomerases alter DNA topology to control DNA supercoiling and chromosome segregation and are targets of clinically important anti-infective and anticancer therapeutics. They act as ATP-operated clamps to trap a DNA helix and transport it through a transient break in a second DNA. Here, we present the first X-ray crystal structure solved at 2.83 Å of a closed clamp complete with trapped T-segment DNA obtained by co-crystallizing the ATPase domain of S. pneumoniae topoisomerase IV with a nonhydrolyzable ATP analogue and 14-mer duplex DNA. The ATPase dimer forms a 22 Å protein hole occupied by the kinked DNA bound asymmetrically through positively charged residues lining the hole, and whose mutagenesis impacts the DNA decatenation, DNA relaxation and DNA-dependent ATPase activities of topo IV. These results and a side-bound DNA-ParE structure help explain how the T-segment DNA is captured and transported by a type II topoisomerase, and reveal a new enzyme–DNA interface for drug discovery

Preliminary estimate of contribution of Arctic nitrogen fixation to the global nitrogen budget

Dinitrogen (N-2) fixation is the source of all biologically available nitrogen on earth, and its presence or absence impacts net primary production and global biogeochemical cycles. Here, we report rates of 3.5-17.2 nmol N L-1 d(-1) in the ice-free coastal Alaskan Arctic to show that N-2 fixation in the Arctic Ocean may be an important source of nitrogen to a seasonally nitrogen-limited system. If widespread in surface waters over ice-free shelves throughout the Arctic, N-2 fixation could contribute up to 3.5 Tg N yr(-1) to the Arctic nitrogen budget. At these rates, N-2 fixation occurring in ice-free summer waters would offset up to 27.1% of the Arctic denitrification deficit and contribute an additional 2.7% to N-2 fixation globally, making it an important consideration in the current debate of whether nitrogen in the global ocean is in steady state. Additional investigations of high-latitude marine diazotrophic physiology are required to refine these N-2 fixation estimates

Clusters of Galaxies: New Results from the CLEF Hydrodynamics Simulation

Preliminary results are presented from the CLEF hydrodynamics simulation, a large (N=2(428)^3 particles within a 200 Mpc/h comoving box) simulation of the LCDM cosmology that includes both radiative cooling and a simple model for galactic feedback. Specifically, we focus on the X-ray properties of the simulated clusters at z=0 and demonstrate a reasonable level of agreement between simulated and observed cluster scaling relations.Comment: 7 pages, 4 figures, accepted for publication in Advances in Space Research (proceedings of the COSPAR 2004 Assembly, Paris

A Estratigrafia do Abandono: dois museus públicos potiguares

As coleções arqueológicas são pouco articuladas com outros conjuntos patrimoniais brasileiros, o que revela camadas de relações estabelecidas para com estes artefatos e evidencia um processo de isolamento e esquecimento destes enquanto constituintes das memórias regionais ou nacionais. Esta "estratigrafia do abandono" é observada no Rio Grande do Norte a partir das relações estabelecidas para com os acervos arqueológicos nos museus Câmara Cascudo e Lauro da Escóssia. Estudando realidades institucionais distintas, se busca contribuir para a percepção não só do potencial informativo e comunicativo das coleções arqueológicas potiguares, mas também para evidenciar a maneira como este campo do patrimônio cultural brasileiro tem sido abordado em terras norte-rio-grandenses

Education, curriculum and differences: an analysis of indigenous people into the school education from Mato Grosso do Sul state, Brazil

This paper work is based on a research carried out about the school education curriculum from Mato Grosso do Sul State and it has as aim to analyze how the contents related to cultures and histories of indigenous peoples, instituted by Law no 11.645/2008, are presented inside the Curriculum Referential for the High School in the human sciences area. With the implementation of the Law of Directives and Basis for National Education and the creation of the Law no 11.645/2008 new challenges have emerged for basic education professionals. The failure to comply with these laws may hinder the construction of a critical attitude of teachers and students about the knowledge that they deal with indigenous people who struggle for the survival of their histories, cultures and rights beyond the historical interethnic contact of prejudice and violence by the non-indigenous people.Este artigo é fundamentado em pesquisa realizada sobre o currículo da educação escolar do estado de Mato Grosso do Sul (MS) e tem como objetivo analisar como os conteúdos relacionados às culturas e histórias dos povos indígenas, instituídos pela Lei n. 11.645/08, são apresentados no Referencial Curricular para o Ensino Médio (RCEM) na área de ciências humanas. Com a implantação da Lei de Diretrizes e Bases da Educação Nacional (LDB) e a criação da Lei n. 11.645, de 2008, novos desafios surgiram aos profissionais da educação básica. O não cumprimento dessas leis pode prejudicar a construção de uma postura crítica dos professores e estudantes sobre os conhecimentos que tratam dos povos indígenas que lutam pela sobrevivência de suas histórias, culturas e direitos diante do histórico contato interétnico de preconceito e de violência por parte dos não indígena

Exploring the active site of the Streptococcus pneumoniae topoisomerase IV-DNA cleavage complex with novel 7,8-bridged fluoroquinolones.

As part of a programme of synthesizing and investigating the biological properties of new fluoroquinolone antibacterials and their targeting of topoisomerase IV from Streptococcus pneumoniae, we have solved the X-ray structure of the complexes of two new 7,8-bridged fluoroquinolones (with restricted C7 group rotation favouring tight binding) in complex with the topoisomerase IV from S. pneumoniae and an 18-base-pair DNA binding site-the E-site-found by our DNA mapping studies to bind drug strongly in the presence of topoisomerase IV (Leo et al. 2005 J. Biol. Chem. 280, 14 252-14 263, doi:10.1074/jbc.M500156200). Although the degree of antibiotic resistance towards fluoroquinolones is much lower than that of β-lactams and a range of ribosome-bound antibiotics, there is a pressing need to increase the diversity of members of this successful clinically used class of drugs. The quinolone moiety of the new 7,8-bridged agents ACHN-245 and ACHN-454 binds similarly to that of clinafloxocin, levofloxacin, moxifloxacin and trovofloxacin but the cyclic scaffold offers the possibility of chemical modification to produce interactions with other topoisomerase residues at the active site

O RECONHECIMENTO DA MULTIPARENTALIDADE SOB A ÓTICA DO PRINCÍPIO DA DIGNIDADE HUMANA

O presente artigo tem por propósito estudar os recentes regramentos a respeito do Direito de Família no ordenamento jurídico brasileiro. Apesar de a Legislação brasileira não ter sofrido alterações, os entendimentos doutrinários e jurisprudenciais, voltados aos novos modelos de institutos familiares, se encarregaram de tentar resolver os problemas enfrentados pelas novas configurações familiares. Com o passar dos anos, o instituto da família vem sofrendo diversas modificações. Como se sabe é nato do homem o desejo de conviver em grupos. A família vem de um fato natural, sua formação se dá através de constituição social espontânea (DIAS, 2010)