Synthesis and characterization of delta-atracotoxin-ar1a, the lethal neurotoxin from venom of the Sydney funnel-web spider (Atrax robustus)

delta-Atracotoxin-Ar1a (delta-ACTX-Ar1a) is the major polypeptide neurotoxin isolated from the venom of the male Sydney funnel-web spider, Atrax robustus. This neurotoxin targets both insect and mammalian voltage-gated sodium channels, where it competes with scorpion alpha-toxins for neurotoxin receptor site-3 to slow sodium-channel inactivation. Progress in characterizing the structure and mechanism of action of this toxin has been hampered by the limited supply of pure toxin from natural sources. In this paper, we describe the first successful chemical synthesis and oxidative refolding of the four-disulfide bond containing delta-ACTX-Ar1a. This synthesis involved solid-phase Boc chemistry using double coupling, followed by oxidative folding of purified peptide using a buffer of 2 M GdnHCl and glutathione/glutathiol in a 1:1 mixture of 2-propanol (pH 8.5). Successful oxidation and refolding was confirmed using both chemical and pharmacological characterization. Ion spray mass spectrometry was employed to confirm the molecular weight. H-1 NMR analysis showed identical chemical shifts for native and synthetic toxins, indicating that the synthetic toxin adopts the native fold. Pharmacological studies employing whole-cell patch clamp recordings from rat dorsal root ganglion neurons confirmed that synthetic delta-ACTX-Ar1a produced a slowing of the sodium current inactivation and hyperpolarizing shifts in the voltage-dependence of activation and inactivation similar to native toxin. Under current clamp conditions, we show for the first time that delta-ACTX-Ar1a produces spontaneous repetitive plateau potentials underlying the clinical symptoms seen during envenomation. This successful oxidative refolding of synthetic delta-ACTX-Ar1a paves the way for future structure-activity studies to determine the toxin pharmacophore.

Abordagens metodológicas para avaliar risco de extinção de espécies de Brachyteles (Primates : Atelidae)

A distribuição geográfica de um táxon é limitada por aspectos ecológicos e históricos. Muitas atividades humanas têm causado modificações na cobertura vegetal, o que leva à fragmentação e perda do habitat. Isso tem levado à extinção local de populações de várias espécies, alterando sua distribuição geográfica. Entre elas estão as duas espécies do gênero Brachyteles (os muriquis), que são primatas endêmicos de um dos biomas mais afetados por esses processos, a Mata Atlântica. A União Internacional para a Conservação da Natureza (UICN) é uma organização que busca conservar a biodiversidade. Entre outros critérios, utiliza o conhecimento sobre as distribuições geográficas restritas das espécies para classificá-las em categorias de ameaça de extinção, nas chamadas listas vermelhas. Para isso, utiliza parâmetros espaciais, cujos resultados indicam o risco de extinção de determinado táxon em relação à sua distribuição geográfica. Muitas vezes os cálculos desses parâmetros são realizados de maneira subjetiva, de maneira que é importante a busca de métodos que tornem as classificações mais objetivas, precisas e replicáveis. Nesse contexto, o presente trabalho testou diferentes métodos de cálculos de três parâmetros relacionados à distribuição geográfica de B. hypoxanthus e B. arachnoides. Tratam-se de espécies ameaçadas de extinção, com localidades de ocorrência bem conhecidas, que foram profundamente afetadas pela degradação da Mata Atlântica. Assim, podem ser consideradas bons modelos para essas análises. Foi construído um banco de dados de localidades de ocorrência atuais das duas espécies. Por meio de abordagens de Sistemas de Informação Geográfica (SIG), foram estimadas a Extensão de Ocorrência (EOO) através de Mínimo Polígono Convexo e α-hull e Área de Ocupação (AOO) e Subpopulações por meio de métodos de grids, buffers circulares e α-hull, em diferentes escalas espaciais. Os resultados dos cálculos desses parâmetros foram comparados para identificar as abordagens e escalas mais adequadas para a avaliação de risco de extinção. Esses resultados indicam que as listas de localidades e os mapas de distribuição disponibilizados pela UICN precisam ser atualizados. Além disso, sugerem que α-hull é uma abordagem vantajosa para EOO e o método de buffers é mais adequado para os parâmetros de AOO e Subpopulações, quando utiliza escalas espaciais menores. Também foi utilizada a ferramenta GeoCAT, para as duas espécies. Essa ferramenta, por realizar análises de EOO e AOO instantâneas e por seus resultados serem semelhantes aos de outras análises, serve como uma abordagem preliminar de risco de extinção baseado no critério de distribuição geográfica.

Real-Time Application Mapping for Many-Cores Using a Limited Migrative Model

Many-core platforms are an emerging technology in the real-time embedded domain. These devices offer various options for power savings, cost reductions and contribute to the overall system flexibility, however, issues such as unpredictability, scalability and analysis pessimism are serious challenges to their integration into the aforementioned area. The focus of this work is on many-core platforms using a limited migrative model (LMM). LMM is an approach based on the fundamental concepts of the multi-kernel paradigm, which is a promising step towards scalable and predictable many-cores. In this work, we formulate the problem of real-time application mapping on a many-core platform using LMM, and propose a three-stage method to solve it. An extended version of the existing analysis is used to assure that derived mappings (i) guarantee the fulfilment of timing constraints posed on worst-case communication delays of individual applications, and (ii) provide an environment to perform load balancing for e.g. energy/thermal management, fault tolerance and/or performance reasons.

Smart Plastic Antibody Material for Hemoglobin Tailored by Silica Surface Imprinting and with Charged Binding Sites: Its use as Ionophore in Potentiometric Transduction

JORNADAS DE ELECTROQUÍMICA E INOVAÇÃO 2013

Waste collection routing-limited multiple landfills and heterogeneous fleet

This article deals with a real-life waste collection routing problem. To efficiently plan waste collection, large municipalities may be partitioned into convenient sectors and only then can routing problems be solved in each sector. Three diverse situations are described, resulting in three different new models. In the first situation, there is a single point of waste disposal from where the vehicles depart and to where they return. The vehicle fleet comprises three types of collection vehicles. In the second, the garage does not match any of the points of disposal. The vehicle is unique and the points of disposal (landfills or transfer stations) may have limitations in terms of the number of visits per day. In the third situation, disposal points are multiple (they do not coincide with the garage), they are limited in the number of visits, and the fleet is composed of two types of vehicles. Computational results based not only on instances adapted from the literature but also on real cases are presented and analyzed. In particular, the results also show the effectiveness of combining sectorization and routing to solve waste collection problems.

COMPARISON OF SIX COMMERCIALLY-AVAILABLE DNA POLYMERASES FOR DIRECT PCR

SUMMARYThe use of a “direct PCR” DNA polymerase enables PCR amplification without any prior DNA purification from blood samples due to the enzyme's resistance to inhibitors present in blood components. Such DNA polymerases are now commercially available. We compared the PCR performance of six direct PCR-type DNA polymerases (KOD FX, Mighty Amp, Hemo KlenTaq, Phusion Blood II, KAPA Blood, and BIOTAQ) in dried blood eluted from a filter paper with TE buffer. GoTaq Flexi was used as a standard DNA polymerase. PCR performance was evaluated by a nested PCR technique for detecting Plasmodium falciparum genomic DNA in the presence of the blood components. Although all six DNA polymerases showed resistance to blood components compared to the standard Taq polymerase, the KOD FX and BIOTAQ DNA polymerases were resistant to inhibitory blood components at concentrations of 40%, and their PCR performance was superior to that of other DNA polymerases. When the reaction mixture contained a mild detergent, only KOD FX DNA polymerase retained the original amount of amplified product. These results indicate that KOD FX DNA polymerase is the most resistant to inhibitory blood components and/or detergents. Thus, KOD FX DNA polymerase could be useful in serological studies to simultaneously detect antibodies and DNA in eluents for antibodies. KOD FX DNA polymerase is thus not limited to use in detecting malaria parasites, but could also be employed to detect other blood-borne pathogens.

Econet wireless Zimbawe Limited: Building the future

In February 2015, when Econet Wireless Zimbabwe Limited, EWZL, the market leader of the telecommunications sector in Zimbabwe, published the financial results, the management team was worried about the shareholders’ reaction. With over 60% market share in its core business (the voice and SMS market), it has achieved remarkable results due to the great diversification strategy. However, the share price was continuously falling due to exogenous factors that were pressuring margins and threatening future sustainability of the company. EWZL was highly exposed to Zimbabwe’s specific risks and regulatory policies. This case explores both the business environment surrounding the company and the strategic decisions necessary to fight against the economic and political challenges. Students will step into Econet’s obstacles by analyzing the strategies and diagnosing the multiple risk factors affecting the profitability of the firm. Furthermore, they will have to compute the valuation of the firm, facing several challenges of a developing country.

Multi-agent contracting with countervailing incentives and limited liability

We consider a principal who deals with a privately informed agent protected by limited liability in a correlated information setting. The agent's technology is such that the fixed cost declines with the marginal cost (the type), so that countervailing incentives may arise. We show that, with high liability, the first-best outcome can be effected for any type if (1) the fixed cost is non-concave in type, under the contract that yields the smallest feasible loss to the agent; (2) the fixed cost is not very concave in type, under the contract that yields the maximum sustainable loss to the agent. We further show that, with low liability, the first-best outcome is still implemented for a non-degenerate range of types if the fixed cost is less concave in type than some given threshold, which tightens as the liability reduces. The optimal contract entails pooling otherwise.

Limited liability and mechanism design in procurement

In the presence of cost uncertainty, limited liability introduces the possibility of default in procurement with its associated bank-ruptcy costs. When financial soundness is not perfectly observable, we show that incentive compatibility implies that financially less sound contractors are selected with higher probability in any feasible mechanism. Informational rents are associated with unsound financial situations. By selecting the financially weakest contractor, stronger price competition (auctions) may not only increase the probability of default but also expected rents. Thus, weak conditions are suffcient for auctions to be suboptimal. In particular, we show that pooling firms with higher assets may reduce the cost of procurement even when default is costless for the sponsor.

Altered ligands reveal limited plasticity in the T cell response to a pathogenic epitope.

Experimental leishmaniasis offers a well characterized model of T helper type 1 cell (Th1)-mediated control of infection by an intracellular organism. Susceptible BALB/c mice aberrantly develop Th2 cells in response to infection and are unable to control parasite dissemination. The early CD4(+) T cell response in these mice is oligoclonal and reflects the expansion of Vbeta4/ Valpha8-bearing T cells in response to a single epitope from the parasite Leishmania homologue of mammalian RACK1 (LACK) antigen. Interleukin 4 (IL-4) generated by these cells is believed to direct the subsequent Th2 response. We used T cells from T cell receptor-transgenic mice expressing such a Vbeta4/Valpha8 receptor to characterize altered peptide ligands with similar affinity for I-Ad. Such altered ligands failed to activate IL-4 production from transgenic LACK-specific T cells or following injection into BALB/c mice. Pretreatment of susceptible mice with altered peptide ligands substantially altered the course of subsequent infection. The ability to confer a healer phenotype on otherwise susceptible mice using altered peptides that differed by a single amino acid suggests limited diversity in the endogenous T cell repertoire recognizing this antigen.