Simultaneous two-photon excitation of distinct labels for dual-color fluorescence crosscorrelation analysis

Confocal fluorescence correlation spectroscopy as a time-averaging fluctuation analysis combining maximum sensitivity with high statistical confidence has proved to be a very versatile and powerful tool for detection and temporal investigation of biomolecules at ultralow concentrations on surfaces, in solutions, and in living cells. To probe the interaction of different molecular species for a detailed understanding of biologically relevant mechanisms, crosscorrelation studies on dual or multiple fluorophore assays with spectrally distinct excitation and emission are particularly promising. Despite the considerable improvement of detection specificity provided by fluorescence crosscorrelation analysis, few applications have so far been reported, presumably because of the practical challenges of properly aligning and controlling the stability of the experimental setup. In this work, we demonstrate that two-photon excitation combined with dual-color fluorescence correlation spectroscopy can be the key to simplifying simultaneous investigations of multiple fluorescent species significantly on a single-molecule scale. Two-photon excitation allows accession of common fluorophores of largely distinct emission by the same excitation wavelength, because differences in selection rules and vibronic coupling can induce considerable shifts between the one-photon and two-photon excitation spectra. The concept of dual-color two-photon fluorescence crosscorrelation analysis is introduced and experimentally demonstrated with an established assay probing the selective cleavage of dual-labeled DNA substrates by restriction endonuclease EcoRI.

Constraints on nebular dynamics and chemistry based on observations of annealed magnesium silicate grains in comets and in disks surrounding Herbig Ae/Be stars

Understanding dynamic conditions in the Solar Nebula is the key to prediction of the material to be found in comets. We suggest that a dynamic, large-scale circulation pattern brings processed dust and gas from the inner nebula back out into the region of cometesimal formation—extending possibly hundreds of astronomical units (AU) from the sun—and that the composition of comets is determined by a chemical reaction network closely coupled to the dynamic transport of dust and gas in the system. This scenario is supported by laboratory studies of Mg silicates and the astronomical data for comets and for protoplanetary disks associated with young stars, which demonstrate that annealing of nebular silicates must occur in conjunction with a large-scale circulation. Mass recycling of dust should have a significant effect on the chemical kinetics of the outer nebula by introducing reduced, gas-phase species produced in the higher temperature and pressure environment of the inner nebula, along with freshly processed grains with “clean” catalytic surfaces to the region of cometesimal formation. Because comets probably form throughout the lifetime of the Solar Nebula and processed (crystalline) grains are not immediately available for incorporation into the first generation of comets, an increasing fraction of dust incorporated into a growing comet should be crystalline olivine and this fraction can serve as a crude chronometer of the relative ages of comets. The formation and evolution of key organic and biogenic molecules in comets are potentially of great consequence to astrobiology.

Generalized transduction in Streptomyces coelicolor

We report the isolation of generalized transducing phages for Streptomyces species able to transduce chromosomal markers or plasmids between derivatives of Streptomyces coelicolor, the principal genetic model system for this important bacterial genus. We describe four apparently distinct phages (DAH2, DAH4, DAH5, and DAH6) that are capable of transducing multiple chromosomal markers at frequencies ranging from 10−5 to 10−9 per plaque-forming unit. The phages contain DNA ranging in size from 93 to 121 kb and mediate linked transfer of genetic loci at neighboring chromosomal sites sufficiently close to be packaged within the same phage particle. The key to our ability to demonstrate transduction by these phages was the establishment of conditions expected to severely reduce superinfection killing during the selection of transductants. The host range of these phages, as measured by the ability to form plaques, extends to species as distantly related as Streptomyces avermitilis and Streptomyces verticillus, which are among the most commercially important species of this genus. Transduction of plasmid DNA between S. coelicolor and S. verticillus was observed at frequencies of ≈10−4 transductants per colony-forming unit.

Comparative genetics in the grasses

Genetic mapping of wheat, maize, and rice and other grass species with common DNA probes has revealed remarkable conservation of gene content and gene order over the 60 million years of radiation of Poaceae. The linear organization of genes in some nine different genomes differing in basic chromosome number from 5 to 12 and nuclear DNA amount from 400 to 6,000 Mb, can be described in terms of only 25 “rice linkage blocks.” The extent to which this intergenomic colinearity is confounded at the micro level by gene duplication and micro-rearrangements is still an open question. Nevertheless, it is clear that the elucidation of the organization of the economically important grasses with larger genomes, such as maize (2n = 10, 4,500 Mb DNA), will, to a greater or lesser extent, be predicted from sequence analysis of smaller genomes such as rice, with only 400 Mb, which in turn may be greatly aided by knowledge of the entire sequence of Arabidopsis, which may be available as soon as the turn of the century. Comparative genetics will provide the key to unlock the genomic secrets of crop plants with bigger genomes than Homo sapiens.

Differential toxicity of mitomycin C and porfiromycin to aerobic and hypoxic Chinese hamster ovary cells overexpressing human NADPH:cytochrome c (P-450) reductase.

Purified NADPH:cytochrome c (P-450) reductase (FpT; NADPH-ferrihemoprotein oxidoreductase, EC 1.6.2.4) can reductively activate mitomycin antibiotics through a one-electron reduction to species that alkylate DNA. To assess the involvement of FpT in the intracellular activation of the mitomycins, transfectants overexpressing a human FpT cDNA were established from a Chinese hamster ovary cell line deficient in dihydrofolate reductase (CHO-K1/dhfr-). The parental cell line was equisensitive to the cytotoxic action of mitomycin C under oxygenated and hypoxic conditions. In contrast, porfiromycin was considerably less cytotoxic to wild-type parental cells than was mitomycin C in air and markedly more cytotoxic under hypoxia. Two FpT-transfected clones were selected that expressed 19- and 27-fold more FpT activity than the parental line. Levels of other oxidoreductases implicated in the activation of the mitomycins were unchanged. Significant increases in sensitivity to mitomycin C and porfiromycin in the two FpT-transfected clones were seen under both oxygenated and hypoxic conditions, with the increases in toxicity being greater under hypoxia than in air. These findings demonstrate that FpT can bioreductively activate the mitomycins in living cells and implicate FpT in the differential aerobic/hypoxic toxicity of the mitomycins.

Revisão taxonômica de Octostruma Forel, 1912 (Formicidae, Myrmicinae)

O gênero de formigas Octostruma Forel 1912 (Formicidae: Myrmicinae) está restrito à região Neotropical; suas espécies ocorrem do México até a Argentina, são predadoras de pequenos artrópodes e podem nidificar no solo, serapilheira e em epífitas. No presente trabalho, a taxonomia do gênero é revista com base no exame de cerca de 4.000 exemplares. Reconheço 43 espécies de Octostruma, 34 espécies previamente conhecidas são consideradas válidas e redescritas e, portanto, nove são reconhecidas como novas. Além disso, indivíduos de castas e sexos ainda não registrados (machos, gines e ergatóides) são aqui descritos pela primeira vez para diferentes espécies. Novos registros de localidades expandem a distribuição geográfica conhecida para parte das espécies e informações sobre a biologia das espécies a partir de dados de rótulo e observações de campo, somados às poucas informações encontradas na literatura são compiladas neste trabalho. Uma chave de identificação para as operárias de todas as espécies do gênero é fornecida.

Estudo de catalisadores de Ni/ZnO promovidos com CeO2 aplicados em reações de reforma a vapor do etanol e acetona

A produção de energia é um dos grandes desafios deste século, principalmente com a necessidade no desenvolvimento de processos que utilizem preferencialmente fontes renováveis. Nesse contexto, é claro o interesse por pesquisas baseada no hidrogênio. Porém, os ganhos ambientais efetivos estão associados também à matéria-prima usada no processo de geração do hidrogênio, senso assim mais significativo quando do uso de fontes renováveis. No presente trabalho foi estudado o efeito da adição de diferentes teores de CeO2 em catalisadores de NiZnO, preparados pelo método da co-precipitação. Os catalisadores foram estudados frente à reação de reforma a vapor do etanol (RVE) para produção de H2, e no decorrer do trabalho foi pertinente incluir o estudo da reforma a vapor da acetona (RVA), como complemento dos estudos da RVE. Além disso, esta é uma molécula modelo para reforma a vapor do bio-óleo. Nos sistemas catalíticos sintetizados houve a formação da liga NiZn3, o que aparenta proporcionar um efeito sinérgico entre esses elementos. Observou-se um efeito altamente promotor do CeO2 com relação a diminuição do coque formado, devido a uma menor da formação da acetona, durante as etapas de reforma do etanol. O que consequentemente interferiu na produção de H2. O catalisador contendo 20% de CeO2 denominado NiZn20Ce apresentou um desempenho altamente promissor, pois de acordo com as análises de DRIFT, a presença do CeO2 levou a formação de espécies de formiato, o que consequentemente interferiu em uma menor formação de coque e maior produção de H2. Foi comprovado também que a natureza dos depósitos carbonáceos depende tanto do substrato utilizado quanto das etapas precursoras inicias que levarão a formação desse coque, sendo o ponto chave para um melhor desempenho do catalisador.

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.

Access Barriers to Services Markets: Mapping, tracing, understanding and measuring. CEPS Special Report No. 77, June 2013

EU and national policy-makers argue that the single services market is a key to EU growth, but that many barriers to services market access remain. Grasping the scope, nature and economic meaning of these barriers, however, has proven rather difficult. This is exactly what the present CEPS Special Report helps the reader to do. We trace all market access barriers in services, as far as the data allow, and attempt to understand their nature and economic meaning (given that they are usually forms of domestic regulation) and discuss aspects of the measurement of restrictiveness. We make a sharp distinction between market access barriers restrictions in a non-EU WTO/GATS environment and intra-EU ones, and demonstrate the significant difference in ambition between the two. The paper specifies in detail the progress made by the EU's horizontal reform in services markets, documenting the removal of many cross-border obstacles to trade in services and establishment. Finally, following these conceptual and descriptive analyses, a brief assessment of access restrictiveness indices is provided for both the non-EU WTO environment and for intra-EU services access barriers.

La disoccupazione giovanile in tempo di crisi: un monito all’Europa (continentale) per rifondare il diritto del lavoro? = Youth Unemployment in Recessionary Times: a Caveat to (Continental) Europe to Reconstruct its Labour Law. WP C.S.D.L.E. "Massimo D'Antona" .INT - 91/2012

The current debate taking place in continental Europe on the need to reform labour law to reduce the duality between labour market insiders and outsiders, thus giving new employment opportunities to young people seems to be, at its best, a consequence of the crisis, or at its worst, an excuse. The considerable emphasis placed on the power of legislation to reduce youth unemployment prevents real labour market problems from being clearly identified, thus reducing the scope to adopt more effective measures. Action is certainly required to help young people during the current crisis, yet interventions should not be exclusively directed towards increased flexibility and deregulation. This paper questions the “thaumaturgic power” wrongly attributed to legislative interventions and put forward a more holistic approach to solve the problem of youth employment, by focusing on the education systems, school-to-work transition and industrial relations. As a comparative analysis demonstrates, in order to effectively tackle the issue of youth employment, it is not enough to reform labour law. High quality education systems, apprenticeship schemes, efficient placement and employment services, cooperative industrial relations and flexible wage determination mechanisms are the key to success when it comes to youth employment, not only in times of recession.

Why trying to keep the Germans close on EU competences is risky for Britain. EPIN Commentary No. 13, 3 December 2013

A new Commentary published by the European Policy Institutes Network (EPIN) offers an interesting piece of advice to the UK if it is to succeed in winning over Germany on EU reform. The author, Almut Möller, asserts that the UK needs to understand how Germany’s federal system, with its intricate balance of competences between the various levels, is an integral part of modern Germany and key to the country’s thinking on Europe.

Ukraine – Trying to build a future beyond the past. EPC Commentary, 2 June 2014

On 25 May Ukrainian businessman Petro Poroshenko became Ukraine’s fifth President, winning in the first round with some 54% of the vote, far ahead of Yulia Tymoshenko. While Poroshenko has been involved in Ukrainian politics for several years, including a short stint in the government of disposed President Viktor Yanukovych, his support and involvement in the EuroMaiden anti-government protests, along with the decision of Vitali Klitschko to drop out of the presidential race and support Poroshenko’s candidacy, were key to his success.

Insight into the evolution of the Solanaceae from the parental genomes of Petunia hybrida

Petunia hybrida is a popular bedding plant that has a long history as a genetic model system. We report the whole-genome sequencing and assembly of inbred derivatives of its two wild parents, P. axillaris N and P. inflata S6. The assemblies include 91.3% and 90.2% coverage of their diploid genomes (1.4 Gb; 2n = 14) containing 32,928 and 36,697 protein-coding genes, respectively. The genomes reveal that the Petunia lineage has experienced at least two rounds of hexaploidization: the older gamma event, which is shared with most Eudicots, and a more recent Solanaceae event that is shared with tomato and other solanaceous species. Transcription factors involved in the shift from bee to moth pollination reside in particularly dynamic regions of the genome, which may have been key to the remarkable diversity of floral colour patterns and pollination systems. The high-quality genome sequences will enhance the value of Petunia as a model system for research on unique biological phenomena such as small RNAs, symbiosis, self-incompatibility and circadian rhythms.

(Table 2) Radiocarbon ages of lake sediments from Boresø, East Greenland

This chapter provides a review of proxy data from a variety of natural archives sampled in the Wollaston Forland region, central Northeast Greenland. The data are used to describe long-term environmental and climatic changes. The focus is on reconstructing the Holocene conditions particularly in the Zackenberg area. In addition, this chapter provides an overview of the archaeological evidence for prehistoric occupation of the region. The Zackenberg area has been covered by the Greenland Ice Sheet several times during the Quaternary. At the Last Glacial Maximum (LGM, about 22,000 years BP), temperatures were much lower than at present, and only very hardy organisms may have survived in the region, even if ice-free areas existed. Marked warming at around 11,700 years BP led to ice recession, and the Zackenberg area was deglaciated in the early Holocene, prior to 10,100 years BP. Rapid early Holocene land emergence was replaced by a slight transgression in the late Holocene. During the Holocene, summer solar insolation decreased in the north. Following deglaciation of the region, summer temperatures probably peaked in the early to mid-Holocene, as indicated by the occurrence of a southern beetle species. However, the timing for the onset of the Holocene thermal maximum is rather poorly constrained because of delayed immigration of key plant species. During the thermal maximum, the mean July temperature was at least 2-3°C higher than at present. Evidence for declining summer temperatures is seen at around 5500, 4500 and 3500 years BP. The cooling culminated during the Little Ice Age that peaked about 100-200 years ago. The first plants that immigrated to the region were herbs and mosses. The first dwarf shrubs arrived in Northeast Greenland prior to 10,400 years BP, and dwarf birch arrived around 8800 years BP. The first people arrived about 4500 years BP, but the region was depopulated several times before the last people disappeared some time after 1823 AD, perhaps as a consequence of poor hunting conditions during the peak of the Little Ice Age.