Multiple genetic modifications of the erythromycin polyketide synthase to produce a library of novel “unnatural” natural products

The structures of complex polyketide natural products, such as erythromycin, are programmed by multifunctional polyketide synthases (PKSs) that contain modular arrangements of functional domains. The colinearity between the activities of modular PKS domains and structure of the polyketide product portends the generation of novel organic compounds—“unnatural” natural products—by genetic manipulation. We have engineered the erythromycin polyketide synthase genes to effect combinatorial alterations of catalytic activities in the biosynthetic pathway, generating a library of >50 macrolides that would be impractical to produce by chemical methods. The library includes examples of analogs with one, two, and three altered carbon centers of the polyketide products. The manipulation of multiple biosynthetic steps in a PKS is an important milestone toward the goal of producing large libraries of unnatural natural products for biological and pharmaceutical applications.

Rapid generation of nested chromosomal deletions on mouse chromosome 2

Nested chromosomal deletions are powerful genetic tools. They are particularly suited for identifying essential genes in development either directly or by screening induced mutations against a deletion. To apply this approach to the functional analysis of mouse chromosome 2, a strategy for the rapid generation of nested deletions with Cre recombinase was developed and tested. A loxP site was targeted to the Notch1 gene on chromosome 2. A targeted line was cotransfected with a second loxP site and a plasmid for transient expression of Cre. Independent random integrations of the second loxP site onto the targeted chromosome in direct repeat orientation created multiple nested deletions. By virtue of targeting in an F1 hybrid embryonic stem cell line, F1(129S1×Cast/Ei), the deletions could be verified and rapidly mapped. Ten deletions fell into seven size classes, with the largest extending six or seven centiMorgans. The cytology of the deletion chromosomes were determined by fluorescent in situ hybridization. Eight deletions were cytologically normal, but the two largest deletions had additional rearrangements. Three deletions, including the largest unrearranged deletion, have been transmitted through the germ line. Several endpoints also have been cloned by plasmid rescue. These experiments illustrate the means to rapidly create and map deletions anywhere in the mouse genome. They also demonstrate an improved method for generating nested deletions in embryonic stem cells.

A human myeloma cell line suitable for the generation of human monoclonal antibodies

Ever since monoclonal antibodies were produced in 1975 with mouse myeloma cells there has been interest in developing human myeloma cultures for the production of monoclonal antibodies. However, despite multiple attempts, no human myeloma line suitable for hybridoma production has been described. Here we report the derivation of a hypoxanthine–aminopterin–thymidine-sensitive and ouabain-resistant human myeloma cell line (Karpas 707H) that contains unique genetic markers. We show that this line is useful for the generation of stable human hybridomas. It can easily be fused with ouabain-sensitive Epstein–Barr virus-transformed cells as well as with fresh tonsil and blood lymphocytes, giving rise to stable hybrids that continuously secrete very large quantities of human immunoglobulins. The derived hybrids do not lose immunoglobulin secretion over many months of continuous growth. The availability of this cell line should enable the in vitro immortalization of human antibody-producing B cells that are formed in vivo. The monoclonal antibodies produced may have advantages in immunotherapy.

Rapid generation of incremental truncation libraries for protein engineering using α-phosphothioate nucleotides

Incremental truncation for the creation of hybrid enzymes (ITCHY) is a novel tool for the generation of combinatorial libraries of hybrid proteins independent of DNA sequence homology. We herein report a fundamentally different methodology for creating incremental truncation libraries using nucleotide triphosphate analogs. Central to the method is the polymerase catalyzed, low frequency, random incorporation of α-phosphothioate dNTPs into the region of DNA targeted for truncation. The resulting phosphothioate internucleotide linkages are resistant to 3′→5′ exonuclease hydrolysis, rendering the target DNA resistant to degradation in a subsequent exonuclease III treatment. From an experimental perspective the protocol reported here to create incremental truncation libraries is simpler and less time consuming than previous approaches by combining the two gene fragments in a single vector and eliminating additional purification steps. As proof of principle, an incremental truncation library of fusions between the N-terminal fragment of Escherichia coli glycinamide ribonucleotide formyltransferase (PurN) and the C-terminal fragment of human glycinamide ribonucleotide formyltransferase (hGART) was prepared and successfully tested for functional hybrids in an auxotrophic E.coli host strain. Multiple active hybrid enzymes were identified, including ones fused in regions of low sequence homology.

Stimulation of phosphatidylinositol 3-kinase by fibroblast growth factor receptors is mediated by coordinated recruitment of multiple docking proteins

The docking protein FRS2 is a major downstream effector that links fibroblast growth factor (FGF) and nerve growth factor receptors with the Ras/mitogen-activated protein kinase signaling cascade. In this report, we demonstrate that FRS2 also plays a pivotal role in FGF-induced recruitment and activation of phosphatidylinositol 3-kinase (PI3-kinase). We demonstrate that tyrosine phosphorylation of FRS2α leads to Grb2-mediated complex formation with the docking protein Gab1 and its tyrosine phosphorylation, resulting in the recruitment and activation of PI3-kinase. Furthermore, Grb2 bound to tyrosine-phosphorylated FRS2 through its SH2 domain interacts primarily via its carboxyl-terminal SH3 domain with a proline-rich region in Gab1 and via its amino-terminal SH3 domain with the nucleotide exchange factor Sos1. Assembly of FRS2α:Grb2:Gab1 complex induced by FGF stimulation results in activation of PI3-kinase and downstream effector proteins such as the S/T kinase Akt, whose cellular localization and activity are regulated by products of PI3-kinase. These experiments reveal a unique mechanism for generation of signal diversity by growth factor-induced coordinated assembly of a multidocking protein complex that can activate the Ras/mitogen-activated protein kinase cascade to induce cell proliferation and differentiation, and PI3-kinase to activate a mediator of a cell survival pathway.

The generation of oscillations in networks of electrically coupled cells

In several biological systems, the electrical coupling of nonoscillating cells generates synchronized membrane potential oscillations. Because the isolated cell is nonoscillating and electrical coupling tends to equalize the membrane potentials of the coupled cells, the mechanism underlying these oscillations is unclear. Here we present a dynamic mechanism by which the electrical coupling of identical nonoscillating cells can generate synchronous membrane potential oscillations. We demonstrate this mechanism by constructing a biologically feasible model of electrically coupled cells, characterized by an excitable membrane and calcium dynamics. We show that strong electrical coupling in this network generates multiple oscillatory states with different spatio-temporal patterns and discuss their possible role in the cooperative computations performed by the system.

Adaptive mutations in Escherichia coli as a model for the multiple mutational origins of tumors.

The cells in most tumors are found to carry multiple mutations; however, based upon mutation rates determined by fluctuation tests, the frequency of such multiple mutations should be so low that tumors are never detected within human populations. Fluctuation tests, which determine the cell-division-dependent mutation rate per cell generation in growing cells, may not be appropriate for estimating mutation rates in nondividing or very slowly dividing cells. Recent studies of time-dependent, "adaptive" mutations in nondividing populations of microorganisms suggest that similar measurements may be more appropriate to understanding the mutation origins of tumors. Here I use the ebgR and ebgA genes of Escherichia coli to measure adaptive mutation rates where multiple mutations are required for rapid growth. Mutations in either ebgA or ebgR allow very slow growth on lactulose (4-O-beta-D-galactosyl-D-fructose), with doubling times of 3.2 and 17.3 days, respectively. However, when both mutations are present, cells can grow rapidly with doubling times of 2.7 hr. I show that during prolonged (28-day) selection for growth on lactulose, the number of lactulose-utilizing mutants that accumulate is 40,000 times greater than can be accounted for on the basis of mutation rates measured by fluctuation tests, but is entirely consistent with the time-dependent adaptive mutation rates measured under the same conditions of prolonged selection.

Efetividade das áreas protegidas para a conservação da natureza sob a abordagem dos serviços ecossistêmicos na bacia do rio Corumbataí

A sustentabilidade da paisagem trata-se de um tema central no que se refere às questões de conservação e desenvolvimento de paisagens altamente antropizadas. Está embasada nos conceitos tradicionais de desenvolvimento sustentável, que visam balancear o desenvolvimento humano com a proteção ambiental, pautada na equidade intra e extra-geração. Considera, ainda, os conceitos de bem-estar humano nesta esfera. Paralelamente, os conceitos de serviços ecossistêmicos reconhecem a dependência das atividades antrópicas e seu bem-estar à qualidade dos ecossistemas, reacendendo os debates sobre capital natural e desenvolvimento sustentável. Neste contexto, este trabalho aborda a sustentabilidade da paisagem questionando a potencialidade dos remanescentes florestais em fornecer, de maneira equilibrada, diferentes serviços ecossistêmicos na bacia do rio Corumbataí. Para isso, o trabalho foi dividido em duas etapas. A primeira buscou analisar questões de demanda e oferta por múltiplos serviços ecossistêmicos (controle de erosão, regulação hídrica, regulação microclimática, informação estética e qualidade de habitat). Isso baseado em indicadores da paisagem, como a dinâmica do uso do solo e padrões do meio físico e antrópico. A segunda investigou a viabilidade de integrar o sinergismo entre os serviços de controle de erosão e qualidade de habitat às prioridades de conservação e restauração florestal. Isso baseado no cenário atual da paisagem e por meio de diferentes simulações de incremento em 10% da cobertura florestal na paisagem, inserindo florestas nos locais de alta erodibilidade (situação criteriosa) ou de forma randômica. Os resultados demonstraram que, apesar do aumento de 60% na cobertura florestal durante os últimos 30 anos, apenas 37% das florestas possuem alto potencial para ofertar serviços ecossistêmicos e que, quando ponderadas perante as demandas da paisagem, apenas 20% das florestas encontram-se em equilíbrio. Além disso, foi verificado sinergismo entre os serviços de controle de erosão e qualidade de habitat em aproximadamente 80% da cobertura florestal. No entanto, nos cenários de restauração florestal, o sinergismo foi alcançado em todas as situações, sejam elas criteriosas ou randômicas. Deste modo, ficou evidente o limite das florestas e áreas protegidas como potenciais prestadoras de serviços ecossistêmicos na paisagem. Também ficou evidente que, em paisagens com alto grau de fragmentação e baixa proporção florestal, os processos de restauração não necessariamente devem almejar o sinergismo entre serviços ecossistêmicos. Por fim, atribui-se grande importância ao papel das áreas agrícolas e pastagens para compensar demandas, restaurar serviços ecossistêmicos, almejando, portanto, a sustentabilidade da paisagem.

The first extrasolar planet discovered with a new-generation high-throughput Doppler instrument

We report the detection of the first extrasolar planet, ET-1 (HD 102195b), using the Exoplanet Tracker (ET), a new-generation Doppler instrument. The planet orbits HD 102195, a young star with solar metallicity that may be part of the local association. The planet imparts radial velocity variability to the star with a semiamplitude of 63.4 ± 2.0 m s^-1 and a period of 4.11 days. The planetary minimum mass (m sin i) is 0.488MJ ± 0.015M_J. The planet was initially detected in the spring of 2005 with the Kitt Peak National Observatory (KPNO) 0.9 m coudé feed telescope. The detection was confirmed by radial velocity observations with the ET at the KPNO 2.1 m telescope and also at the 9 m Hobby-Eberly Telescope (HET) with its High Resolution Spectrograph. This planetary discovery with a 0.9 m telescope around a V = 8.05 magnitude star was made possible by the high throughput of the instrument: 49% measured from the fiber output to the detector. The ET's interferometer-based approach is an effective method for planet detection. In addition, the ET concept is adaptable to multiple-object Doppler observations or very high precision observations with a cross-dispersed echelle spectrograph to separate stellar fringes over a broad wavelength band. In addition to spectroscopic observations of HD 102195, we obtained brightness measurements with one of the automated photometric telescopes at Fairborn Observatory. Those observations reveal that HD 102195 is a spotted variable star with an amplitude of ~0.015 mag and a 12.3 ± 0.3 day period. This is consistent with spectroscopically observed Ca II H and K emission levels and line-broadening measurements but inconsistent with rotational modulation of surface activity as the cause of the radial velocity variability. Our photometric observations rule out transits of the planetary companion.

Avaliação de conflitos de usos múltiplos de água na bacia hidrográfica do Rio Jaguari - SP, através de simulação computacional

No gerenciamento de recursos hídricos, a bacia hidrográfica é considerada a unidade natural de gestão. Suas características físicas definem um espaço onde a água converge para uma saída comum. O desenvolvimento integrado de uma bacia, bem como os possíveis conflitos ocasionados por demandas concorrentes do mesmo recurso, devem ser administrados respeitando esse conceito de unidade. No caso específico da bacia hidrográfica do Rio Jaguari, existe uma crescente preocupação relacionada aos usos múltiplos da água superficial. O rio Jaguari, importante afluente do Paraíba do Sul no estado de São Paulo, tem suas águas represadas para geração de energia elétrica e regularização de vazões. Nos últimos anos, tem-se constatado um crescente rebaixamento dos níveis operacionais da represa do Jaguari. Segundo estudos da sociedade civil organizada local, a tendência é de esgotamento do reservatório em poucos anos. Este trabalho aborda a questão do rebaixamento dos níveis de Jaguari através da aplicação do software DHI Mike Basin 2000. Trata-se de um simulador genérico para sistemas de recursos hídricos, de ampla aplicação. A bacia do Jaguari é representada em um modelo matemático e são simulados quatro cenários distintos de usos de água na bacia. Verifica-se que o problema é real e requer uma intervenção multi-institucional para ser solucionado.

Renewable Energy Generation at Copper Mountain Resort

Copper Mountain, a Colorado ski area, evaluated onsite renewable energy generation to save on energy costs and reduce carbon emissions. Multiple resort locations were analyzed to determine suitable sites for implementation of solar electricity generation, wind electricity generation and biomass heat production. Potential project sites were assessed based on four criteria: costs and financial returns, environmental impacts, implementation and maintenance, and public relations/marketing opportunities. Solar projects had the lowest capital cost of the three types of renewable energy, and wind projects had high capital costs and low financial returns. Biomass projects had high capital costs, solid financial projections and good marketing value compared to wind and solar technologies. Project implementation recommendations were given based upon the evaluation.

COMPENDIUM: a text summarisation tool for generating summaries of multiple purposes, domains, and genres

In this paper, we present a Text Summarisation tool, compendium, capable of generating the most common types of summaries. Regarding the input, single- and multi-document summaries can be produced; as the output, the summaries can be extractive or abstractive-oriented; and finally, concerning their purpose, the summaries can be generic, query-focused, or sentiment-based. The proposed architecture for compendium is divided in various stages, making a distinction between core and additional stages. The former constitute the backbone of the tool and are common for the generation of any type of summary, whereas the latter are used for enhancing the capabilities of the tool. The main contributions of compendium with respect to the state-of-the-art summarisation systems are that (i) it specifically deals with the problem of redundancy, by means of textual entailment; (ii) it combines statistical and cognitive-based techniques for determining relevant content; and (iii) it proposes an abstractive-oriented approach for facing the challenge of abstractive summarisation. The evaluation performed in different domains and textual genres, comprising traditional texts, as well as texts extracted from the Web 2.0, shows that compendium is very competitive and appropriate to be used as a tool for generating summaries.

Prospects for Energy Supply and Demand in the Southern Mediterranean: Scenarios for 2010-30. MEDPRO Technical Report No. 22/December 2012

The aim of this technical report is to quantify alternative energy demand and supply scenarios for ten southern and eastern Mediterranean countries up to 2030. The report presents the model-based results of four alternative scenarios that are broadly in line with the MEDPRO scenario specifications on regional integration and cooperation with the EU. The report analyses the main implications of the scenarios in the following areas: • final energy demand by sector (industry, households, services, agriculture and transport); • the evolution of the power generation mix, the development of renewable energy sources and electricity exports to the EU; • primary energy production and the balance of trade for hydrocarbons; • energy-related CO2 emissions; and • power generation costs.

(Table 1) Chemical composition of interstitial waters from the area of gas hydrate generation, Sea of Okhotsk

Distributions of halogens (Cl, Br and I) in interstitial waters from sediments containing methane hydrate and in water of the hydrate itself are presented. High concentrations of halogens do not occur in interstitial waters from sediments that contain gas hydrates. The main reason for their low concentrations is the poverty of organic matter in sediments.

Stickleback F2 generation transgenerational plasticity experiment: egg and body size data

Nongenetic inheritance mechanisms such as transgenerational plasticity (TGP) can buffer populations against rapid environmental change such as ocean warming. Yet, little is known about how long these effects persist and whether they are cumulative over generations. Here, we tested for adaptive TGP in response to simulated ocean warming across parental and grandparental generations of marine sticklebacks. Grandparents were acclimated for two months during reproductive conditioning, whereas parents experienced developmental acclimation, allowing us to compare the fitness consequences of short-term vs. prolonged exposure to elevated temperature across multiple generations. We found that reproductive output of F1 adults was primarily determined by maternal developmental temperature, but carry-over effects from grandparental acclimation environments resulted in cumulative negative effects of elevated temperature on hatching success. In very early stages of growth, F2 offspring reached larger sizes in their respective paternal and grandparental environment down the paternal line, suggesting that other factors than just the paternal genome may be transferred between generations. In later growth stages, maternal and maternal granddam environments strongly influenced offspring body size, but in opposing directions, indicating that the mechanism(s) underlying the transfer of environmental information may have differed between acute and developmental acclimation experienced by the two generations. Taken together, our results suggest that the fitness consequences of parental and grandparental TGP are highly context dependent, but will play an important role in mediating some of the impacts of rapid climate change in this system.