TRAP, the trp RNA-binding attenuation protein of Bacillus subtilis, is a toroid-shaped molecule that binds transcripts containing GAG or UAG repeats separated by two nucleotides.

The trp RNA-binding attenuation protein of Bacillus subtilis, TRAP, regulates both transcription and translation by binding to specific transcript sequences. The optimal transcript sequences required for TRAP binding were determined by measuring complex formation between purified TRAP protein and synthetic RNAs. RNAs were tested that contained repeats of different trinucleotide sequences, with differing spacing between the repeats. A transcript containing GAG repeats separated by two-nucleotide spacers was bound most tightly. In addition, transmission electron microscopy was used to examine the structure of TRAP and the TRAP-transcript complex. TRAP was observed to be a toroid-shaped oligomer when free or when bound to either a natural or a synthetic RNA.

Cell-free activation of neutrophil NADPH oxidase by a phosphatidic acid-regulated protein kinase.

The phosphorylation-dependent mechanisms regulating activation of the human neutrophil respiratory-burst enzyme, NADPH oxidase, have not been elucidated. We have shown that phosphatidic acid (PA) and diacylglycerol (DG), products of phospholipase activation, synergize to activate NADPH oxidase in a cell-free system. We now report that activation by PA plus DG involves protein kinase activity, unlike other cell-free system activators. NADPH oxidase activation by PA plus DG is reduced approximately 70% by several protein kinase inhibitors [1-(5-isoquinolinesulfonyl)piperazine, staurosporine, GF-109203X]. Similarly, depletion of ATP by dialysis reduces PA plus DG-mediated NADPH oxidase activation by approximately 70%. Addition of ATP, but not a nonhydrolyzable ATP analog, to the dialyzed system restores activation levels to normal. In contrast, these treatments have little effect on NADPH oxidase activation by arachidonic acid or SDS plus DG. PA plus DG induces the phosphorylation of a number of endogenous proteins. Phosphorylation is largely mediated by PA, not DG. A predominant substrate is p47-phox, a phosphoprotein component of NADPH oxidase. Phosphorylation of p47-phox precedes activation of NADPH oxidase and is markedly reduced by the protein kinase inhibitors. In contrast, arachidonic acid alone or SDS plus DG is a poor activator of protein phosphorylation in the cell-free system. Thus, PA induces activation of one or more protein kinases that regulate NADPH oxidase activation in a cell-free system. This cell-free system will be useful for identifying a functionally important PA-activated protein kinase(s) and for dissecting the phosphorylation-dependent mechanisms responsible for NADPH oxidase activation.

Long-term in vivo expression of the human glucocerebrosidase gene in nonhuman primates after CD34+ hematopoietic cell transduction with cell-free retroviral vector preparations.

Successful gene transfer into stem cells would provide a potentially useful therapeutic modality for treatment of inherited and acquired disorders affecting hematopoietic tissues. Coculture of primate bone marrow cells with retroviral producer cells, autologous stroma, or an engineered stromal cell line expressing human stem cell factor has resulted in a low efficiency of gene transfer as reflected by the presence of 0.1-5% of genetically modified cells in the blood of reconstituted animals. Our experiments in a nonhuman primate model were designed to explore various transduction protocols that did not involve coculture in an effort to define clinically useful conditions and to enhance transduction efficiency of repopulating cells. We report the presence of genetically modified cells at levels ranging from 0.1% (granulocytes) to 14% (B lymphocytes) more than 1 year following reconstitution of myeloablated animals with CD34+ immunoselected cells transduced in suspension culture with cytokines for 4 days with a retrovirus containing the glucocerebrosidase gene. A period of prestimulation for 7 days in the presence of autologous stroma separated from the CD34+ cells by a porous membrane did not appear to enhance transduction efficiency. Infusion of transduced CD34+ cells into animals without myeloablation resulted in only transient appearance of genetically modified cells in peripheral blood. Our results document that retroviral transduction of primate repopulating cells can be achieved without coculture with stroma or producer cells and that the proportion of genetically modified cells may be highest in the B-lymphoid lineage under the given transduction conditions.

Free living astigmatid mites (Astigmatina): new taxa, rearing and use for mesostigmatid (Mesostigmata) predatory mite production

The cohort Astigmatina is divided in two major groups: Psoroptidia, composed mainly by feather and fur mites, and Non-psoroptidia, a dominant component of the acarofauna in ephemeral habitats. In these environments Astigmatina usually are saprophages or feed on fungi or bacteria. Astigmatina protonymphs undergo a complete reorganization of the body structure leading to the production of heteromorphic deutonymphs, generally specialized for dispersion through phoresy using arthropods and vertebrates as phoronts. Although most Astigmatina occur in natural environments, some species live in anthropic environments, such as food deposits, where some of them became pests; some Astigmatina infest subterraneous plant organs. Despite their economic and ecological importance, studies on the diversity and taxonomy of Astigmatina in Brazil have been rare over the last decades. The general objective of this thesis was to collaborate to the knowledge of the diversity and to evaluate the potential practical uses of these mites in Brazil. For this, new genera and species were described, method for rearing dust mites was studied and the efficiency of Astigmatina as prey for edaphic predators was evaluated. A new species of Thyreophagus (Astigmatina: Acaridae) was described based on specimens collected in Brazil, the association of three other species of this genus with stored food was reviewed and a key to all species of this genus was prepared. The genus Neotropacarus (Astigmatina: Acaridae), commonly found on plant leaves, was reviewed with the redescription of two species and description of new species collected in Brazil and from the Philippines. Two new genera and seven new species of Acaridae associated with the bee family Apidae was described and a key to Acaridae genera in subfamily Horstiinae was prepared. Several species of Astigmatina were evaluated as prey for predatory mites Stratiolaelaps scimitus (Womersley) (Mesostigmata: Laelapidae) and Protogamasellopsis zaheri Abo-Shnaf, Castilho and Moraes (Mesostigmata: Rhodacaridae), which oviposited on all evaluated astigmatids, with Tyrophagus putrescentiae (Schrank) and Aleuroglyphus ovatus (Tropeau) (Acaridae) being the most suitable prey. Seven foods and two development period, 30 and 60 days, after the introduction of 400 females of two important dust mite species, Blomia tropicalis van Bronswijk, de Cock e Oshima and Dermatophagoides pteronyssinus (Trouessart) were evaluate. With the most suitable foods, the population growth were higher than 20.2 and 15.3 for B. tropicalis and D. pteronyssinus, respectively.

A Highly Efficient Solvent-Free Asymmetric Direct Aldol Reaction Organocatalyzed by Recoverable (s)-Binam-L-Prolinamides. ESI-MS Evidence of the Enamine-Iminium Formation

Recoverable (Sa)-binam-l-prolinamide in combination with benzoic acid is used as catalysts in the direct aldol reaction between cycloalkyl, alkyl, and α-functionalized ketones and aldehydes under solvent-free reaction conditions. Three different methods are assayed: simple conventional magnetic stirring, magnetic stirring after previous dissolution in THF and evaporation, and ball mill technique. These procedures allow one to reduce not only the amount of required ketone to 2 equiv but also the reaction time to give the aldol products with regio-, diastereo-, and enantioselectivities comparable to those in organic or aqueous solvents. Generally anti-isomers are mainly obtained with enantioselectivities up to 97%. The reaction can be carried out under these conditions also using aldehydes as nucleophiles, yielding after in situ reduction of the aldol products the corresponding chiral 1,3-diols with moderate to high enantioselectivities mainly as anti-isomers. The aldol reaction has been studied by the use of positive ESI-MS technique, providing the evidence of the formation of the corresponding enamine−iminium intermediates.

Solvent-Free Enantioselective Friedländer Condensation with Wet 1,1′-Binaphthalene-2,2′-diamine-Derived Prolinamides as Organocatalysts

Wet unsupported and supported 1,1′-binaphthalene-2,2′-diamine (BINAM) derived prolinamides are efficient organocatalysts under solvent-free conditions at room temperature to perform the synthesis of chiral tacrine analogues in good yields (up to 93%) and excellent enantioselectivies (up to 96%). The Friedländer reaction involved in this process takes place with several cyclohexanone derivatives and 2-aminoaromatic aldehydes, and it is compatible with the presence of either electron-withdrawing or electron-donating groups at the aromatic ring of the 2-aminoaryl aldehyde derivatives used as electrophiles. The reaction can be extended to cyclopentanone derivatives, affording a regioisomeric but separable mixture of products. The use of the wet silica gel supported organocatalyst, under solvent-free conditions, for this process led to the expected product (up to 87% enantiomeric excess), with its reuse being possible at least up to five times.

Solvent-Free Enantioselective Organocatalyzed Aldol Reactions

The use of proline as catalyst for the aldol process has given a boost to the development of organocatalysis as a research area. Since then, a plethora of organocatalysts of diverse structures have been developed for this and other organic transformations under different reaction conditions. The use of an organic molecule as catalyst to promote a reaction meets several principles of Green Chemistry. The implementation of solvent-free methodologies to carry out the aldol reaction was soon envisaged. These solvent-free processes can be performed using conventional magnetic stirring or applying ball milling techniques and are even compatible with the use of supported organocatalysts as promoters, which allows the recovery and reuse of the organocatalysts. In addition, other advantages such as the reduction of the required amount of nucleophile and the acceleration of the reaction are accomplished by using solvent-free conditions leading to a “greener” and more sustainable process.

Mapping Binary Liquid-Vapor or Liquid-Liquid-Vapor Equilibria Regions, including the Different Azeotropic Behaviours, as a Function of the NRTL Binary Parameters

13th Mediterranean Congress of Chemical Engineering (Sociedad Española de Química Industrial e Ingeniería Química, Fira Barcelona, Expoquimia), Barcelona, September 30-October 3, 2014

Transvaal & Orange Free State Regions, South Africa, ca. 1899 (Raster Image)

This layer is a georeferenced raster image of the historic paper map entitled: Bacon's large-print map of the Transvaal and Orange Free State. It was published by G.W. Bacon & Co. ca. 1899. Scale [ca. 1:1,900,000]. Covers also Swaziland, Lesotho, and portions of Botswana, Zimbabwe, and Mozambique.The image inside the map neatline is georeferenced to the surface of the earth and fit to the Africa Sinusoidal projected coordinate system. All map collar and inset information is also available as part of the raster image, including any inset maps, profiles, statistical tables, directories, text, illustrations, index maps, legends, or other information associated with the principal map. This map shows features such as drainage, cities and other human settlements, territorial and administrative boundaries, roads, railroads, shoreline features, and more. Relief shown by shading and spot heights. Includes also insets: "Map showing the routes from England and India to South Africa", "Environs of Cape Town", "Lorenço Marquez [and vicinity]", 'South Africa" and "Durban and Port Natal".This layer is part of a selection of digitally scanned and georeferenced historic maps from the Harvard Map Collection. These maps typically portray both natural and manmade features. The selection represents a range of originators, ground condition dates, scales, and map purposes.

Free State Region, South Africa, 1899 (Raster Image)

This layer is a georeferenced raster image of the historic paper map entitled: Stanford's new map of the Orange Free State, the southern part of the South African Republic, the northern frontier of Cape Colony, Natal, Basutoland and Delagoa Bay. It was published by E. Stanford in 1899. Scale 1:1,000,000 The image inside the map neatline is georeferenced to the surface of the earth and fit to the Africa Lambert Conformal Conic projected coordinate system. All map collar and inset information is also available as part of the raster image, including any inset maps, profiles, statistical tables, directories, text, illustrations, index maps, legends, or other information associated with the principal map. This map shows features such as drainage, cities and other human settlements, roads, railroads and stations, administrative and territorial boundaries, shoreline features, and more. Relief shown by shading and spot heights.This layer is part of a selection of digitally scanned and georeferenced historic maps from the Harvard Map Collection. These maps typically portray both natural and manmade features. The selection represents a range of originators, ground condition dates, scales, and map purposes.

Electricity infrastructure: more border crossings or a borderless Europe? Bruegel Policy Contribution 2013/05, February 2013

Being able to transport electricity seamlessly across borders is essential for achieving three major European Union energy policy goals: (1) enabling competition between national energy companies, (2) cost-effective roll-out of renewables,and (3) security of supply. However, neither the market design nor the framework for infrastructure investment proposed by the European Commission is adequate for enabling free flows of electricity within the EU.

In Search of a Feasible EU-Mercosul Free Trade Agreement. CEPS Working Document No. 378, 25 February 2013

This paper aims at identifying ways to pursue the EU–Mercosul negotiations leading to a free trade agreement (FTA). After reviewing their already long history, it outlines a basic framework, in goods, services and other themes, judged possible. The main point is that, given the prevailing conditions on both sides, an agreement to be signed within a reasonable time must be modest, i.e. along the described lines. It then clearly sets up the decisions confronting the negotiators: either to pursue the modest, feasible option or to terminate negotiations under the FTA heading. The latter, however, does not imply an end to the dialogue. Many actions and measures may be taken – which are easier to discuss and fix – that could pave the way for, in due time, a closer-to-ideal FTA to be considered again. These are the subjects of a last section.

Free movement of workers in the EU. Legal aspects of the transitional arrangements. ZEI Discussion Paper No. 217, 2013

From the Introduction. There are four fundamental freedoms which lay the foundation of the European Union. Those are the free movement of goods, free movement of capital, free movement of services and free movement of persons. They guarantee the existence and effective functioning of an area without internal borders within which goods, capital, services and people move freely. Despite the pivotal importance of these freedoms, there are cases where some freedoms can be partially or fully restricted within the territory of some member states or the Union as a whole. This thesis is going to analyze the restrictions of one of these freedoms: the free movement of persons, resulting from the arrangements applying to new member states. The focus will be the free movement of workers from new to old member states for a transitional period following the date of accession.

British Balance of Competence Reviews, Part III: More reform than renegotiation or repatriation. EPIN [Working] Paper No. 42/December 2014. [see Abstract for I and II]

This paper, the third in a series for a CEPS project on the ‘The British Question’, is pegged on an ambitious exercise by the British government to review all the competences of the European Union on the basis of evidence submitted by independent stakeholders. The reviews considered in this paper cover the following EU policies: the single market for services, financial markets, the free movement of people, cohesion, energy, agriculture, fisheries, competition, social and employment policies, and fundamental rights. The declared objective of Prime Minister Cameron is to secure a ‘new settlement’ between the UK and the EU. From political speeches in the UK one can identify three different types of possible demand: reform of EU policies, renegotiation of the UK’s specific terms of membership, and repatriation of competences from the EU back to the member states. As most of the reviews are now complete, three points are becoming increasingly clear: i) The reform agenda – past, present or future - concerns virtually every branch of EU policy, including several cases reviewed here that are central to stated UK economic interests. The argument that the EU is ‘unreformable’ is shown to be a myth. ii) The highly sensitive cases of immigration from the EU and social policies may translate into requests for renegotiation of specific conditions for the UK, but further large-scale opt-outs, as in the case of the euro and justice and home affairs, are implausible. iii) While demands for repatriation of EU competences are voiced in general terms in public debate in the UK, no specific proposals emerge from the evidence as regards competences at the level at which they are identified in the treaties, and there is no chance of achieving consensus for such ideas among member states. Michael Emerson and Steven Blockmans, “British Balance of Competence Reviews, Part I: ‘Competences about right, so far’”, CEPS/EPIN Working Paper No. 35, October 2013 (www.ceps.eu/book/british-balance-competence-reviews-part-i-%E2%80%98competences-about-right-so-far%E2%80%99)(http://aei.pitt.edu/45599/); Michael Emerson, Steven Blockmans, Steve Peers and Michael Wriglesworth, “British Balance of Competence Reviews, Part II: Again, a huge contradiction between the evidence and Eurosceptic populism”, CEPS/EPIN Working Paper No. 40, June 2014 (www.ceps.eu/book/british-balance-competence-reviews-part-ii-again-huge-contradiction-between-evidence-and-eurosc)(http://aei.pitt.edu/52452/).

Enhancing the Prospects of the EU’s Deep and Comprehensive Free Trade Areas in the Mediterranean: Lessons from the Eastern Partnership. CEPS Commentary, 24 June 2015

In their March 2015 Consultation paper on the review of the European Neighbourhood Policy (ENP), the European Commission and the High Representative raised the question whether the Deep and Comprehensive Free Trade Areas (DCFTAs) “are the right objective for all or should more tailor-made alternatives be developed, to reflect differing interests and ambitions of some partners?” Such ambitious but complex trade agreements have now finally been signed with Ukraine, Moldova and Georgia, but they are still on the table for several Mediterranean ENP countries. Although these Mediterranean partners have a completely different political, economic and legal relationship with the EU, some important lessons can be drawn from the ‘Eastern DCFTA experience’. In particular, the DCFTA negotiators should avoid overly ambitious and ill-defined legislative approximation commitments and develop a comprehensive implementation strategy.