Technical tip: high-resolution isolation of nanoparticle–protein corona complexes from physiological fluids

Nanoparticles (NPs) in contact with biological fluids are generally coated with environmental proteins, forming a stronger layer of proteins around the NP surface called the hard corona. Protein corona complexes provide the biological identity of the NPs and their isolation and characterization are essential to understand their in vitro and in vivo behaviour. Here we present a one-step methodology to recover NPs from complex biological media in a stable non-aggregated form without affecting the structure or composition of the corona. This method allows NPs to be separated from complex fluids containing biological particulates and in a form suitable for use in further experiments. The study has been performed systematically comparing the new proposed methodology to standard approaches for a wide panel of NPs. NPs were first incubated in the biological fluid and successively recovered by sucrose gradient ultracentrifugation in order to separate the NPs and their protein corona from the loosely bound proteins. The isolated NP–protein complexes were characterized by size and protein composition through Dynamic Light Scattering, Nanoparticle Tracking Analysis, SDS-PAGE and LC-MS. The protocol described is versatile and can be applied to diverse nanomaterials and complex fluids. It is shown to have higher resolution in separating the multiple protein corona complexes from a biological environment with a much lower impact on their in situ structure compared to conventional centrifugal approaches

Stephen Murray and Andrew Tallon, 2012-. Mapping Gothic France. http://mappinggothic.org

The 'Mapping Gothic' website (http://mappinggothic.org/) was originally conceived by Stephen Murray, Professor of Art and Archaeology at Columbia University, and Andrew Tallon, Assistant Professor of Art at Vassar College, as a space to represent Gothic architecture digitally. Aware of the insufficiencies of a two-dimensional screen for rendering these structures, the site compiles panoramic, gigapan images; exact architectural elevations; timelines; and historical narratives to show these buildings in both time and space. The site derives its guiding Hrinciple from Henri Lefèbvre in seeking connections between what it describes as "the architectural space of individual buildings, geo-political space, and the social space resulting from the interaction (collaboration and conflict) between multiple agents – builders and users." Murray and Tallon hope that in addition to providing digital access to these churches, cathedrals, and abbeys, the manipulable platform will allow users to draw their own connections between these Gothic buildings

Biologischer Lichtsammler (LHCII) für Halbleiternanokristalle (Quantum Dots)

Der light harvesting complex II (LHCII) ist ein pflanzliches Membranprotein, das in seiner trimeren Form über 40 Chlorophylle bindet. In der Pflanze kann er besonders effizient Licht sammeln und die Anregungsenergie anschließend fast verlustfrei über andere chlorophyll-bindende Proteine an die Reaktionszentren weiterleiten. Aufgrund dieser besonderen Eigenschaften war es ein Ziel dieser Arbeit, rekombinanten LHCII mit synthetischen Komponenten zu kombinieren, die zur Ladungstrennung befähigt sind. Zu diesem Zweck wurden unter anderem Halbleiternanokristalle (Quantum Dots, QDs) ausgewählt, die je nach Zusammensetzung sowohl als Energieakzeptoren als auch als Energiedonoren in Frage kamen. Durch Optimierung des Puffers gelang es, die Fluoreszenzquantenausbeute der QDs in wässriger Lösung zu erhöhen und zu stabilisieren, so dass die Grundvoraussetzungen für die spektroskopische Untersuchung verschiedener LHCII-QD-Hybridkomplexe erfüllt waren.rnUnter Verwendung bereits etablierter Affinitätssequenzen zur Bindung des LHCII an die QDs konnte gezeigt werden, dass die in dieser Arbeit verwendeten Typ-I QDs aus CdSe und ZnS sich kaum als Energie-Donoren für den LHCII eignen. Ein Hauptgrund lag im vergleichsweise kleinen Försterradius R0 von 4,1 nm. Im Gegensatz dazu wurde ein R0 von 6,4 nm für den LHCII als Donor und Typ-II QDs aus CdTe, CdSe und ZnS als Akzeptor errechnet, wodurch in diesem System eine höhere Effizienz des Energietransfers zu erwarten war. Fluoreszenzspektroskopische Untersuchungen von Hybridkomplexen aus LHCII und Typ-II QDs ergaben eine hohe Plausibilität für einen Fluoreszenz Resonanz Energietransfer (FRET) vom Lichtsammler auf die QDs. Weitere QD-Affinitätssequenzen für den LHCII wurden identifiziert und deren Bindekonstanten ermittelt. Versuche mit dem Elektronenakzeptor Methylviologen lieferten gute Hinweise auf eine LHCII-sensibilisierte Ladungstrennung der Typ-II QDs, auch wenn dies noch anhand alternativer Messmethoden wie z.B. durch transiente Absorptionsspektroskopie bestätigt werden muss. rnEin weiteres Ziel war die Verwendung von LHCII als Lichtsammler in dye-sensitized solar cells (DSSC). Geeignete dotierte TiO2-Platten wurden ermittelt, das Verfahren zur Belegung der Platten optimiert und daher mit wenig Aufwand eine hohe LHCII-Belegungsdichte erzielt. Erste Messungen von Aktionsspektren mit LHCII und einem zur Ladungstrennung fähigen Rylenfarbstoff zeigen eine, wenn auch geringe, LHCII sensibilisierte Ladungstrennung. rnDie Verwendung von Lanthanide-Binding-Tags (LBTs) ist ein potentielles Verfahren zur in vivo-Markierung von Proteinen mit Lanthanoiden wie Europium und Terbium. Diese Metalle besitzen eine überdurchschnittlich lange Lumineszenzlebensdauer, so dass sie leicht von anderen fluoreszierenden Molekülen unterschieden werden können. Im Rahmen der vorliegenden Arbeit gelang es, eine LBT in rekombinanten LHCII einzubauen und einen Lumineszenz Resonanz Energietransfer (LRET) vom Europium auf den LHCII nachzuweisen.rnThe light harvesting complex II (LHCII) is a membrane protein and consists of more than 40 chlorophylls in its trimeric version. In plants it performs efficient light harvesting and transfers the excitation energy nearly quantitatively via other pigment-binding proteins to the reaction center. Due to these LHCII properties it was of interest to combine recombinant LHCII with synthetic compounds that are capable of charge separation. To this end semiconductor nanocrystals, so-called Quantum dots (QDs), where chosen as energy acceptors. Depending on their composition, QDs can also serve as energy donors. By optimizing the buffer system, the QDs fluorescence quantum yield in aqueous solution has been enhanced and stabilized, fulfilling the prerequisites for spectroscopic investigations of different LHCII-QD hybrid complexes.rnBy using established affinity tags to bind LHCII to QDs it was shown that type-I nanocrystals from CdSe and ZnS were no efficient energy donors for LHCII, presumably due to the small Förster radius (R0) of 4.1 nm. By contrast, a larger R0 of 6.4 nm was estimated for hybrid complexes of LHCII as donors and type-II QDs (CdTe, CdSe, ZnS), thus a higher efficiency of energy transfer was expected. Complexes of LHCII and type-II QDs exhibited fluorescence properties that were indicative of Foerster-type energy transfer from LHCII to QD. Additional QD-affinity tags have been established for the LHCII and their binding constants were estimated. Experiments with the electron acceptor methyl viologen indicated an LHCII sensitized charge separation in QDs. This preliminary result still needs to be confirmed, for example transient absorption spectroscopy.rnAnother objective was to integrate LHCII-hybrid complexes into dye-sensitized solar cells (DSSCs). Suitably doted TiO2 plates were loaded by an optimized procedure, enhancing the LHCII density on the plates. Preliminary recordings of action spectra with LHCII and a rylen dye as a sensitizer showed a small but significant LHCII-sensitized charge separation. rnThe use of lanthanide binding tags (LBTs) is a possibility for in vivo labeling of proteins with lanthanides like terbium and europium. These metals have an extraordinary long luminescence lifetime making them easily distinguishable from other fluorescent molecules. In this work an LBT was introduced into recombinant LHCII and luminescence resonance energy transfer was shown to take place from europium to LHCII.r

Remote monitoring of vigilance behavior in large herbivores using acceleration data

Abstract Background Biotelemetry offers an increasing set of tools to monitor animals. Acceleration sensors in particular can provide remote observations of animal behavior at high temporal resolution. While recent studies have demonstrated the capability of this technique for a wide range of species and behaviors, a coherent methodology is still missing (1) for behavior monitoring of large herbivores that are usually tagged with neck collars and frequently switch between diverse behaviors and (2) for monitoring of vigilance behavior. Here, we present an approach that aims at remotely monitoring different types of large herbivore behavior including vigilance with acceleration data. Methods We pioneered this approach with field observations of eight collared roe deer (Capreolus capreolus). First, we trained a classification model for distinguishing seven structural behavior categories: lying, standing, browsing, walking, trotting, galloping and ‘others’. Second, we developed a model that predicted the internal states, active and resting, based on the predicted sequence of structural behaviors and expert-based rules. Further, we applied both models to automatically monitor vigilance behavior and compared model predictions with expert judgment of vigilance behavior. To exemplify the practical application of this approach, we predicted behavior, internal state and vigilance continuously for a collared roe deer. Results The structural behaviors were predicted with high accuracy (overall cross-validated accuracy 71%). Only behaviors that are similar in terms of posture and dynamic body movements were prone to misclassification. Active and resting states showed clear distinction and could be utilized as behavioral context for the detection of vigilance behavior. Here, model predictions were characterized by excellent consistency with expert judgment of vigilance behavior (mean accuracy 96%). Conclusion In this study, we demonstrated the strong potential and practical applicability of acceleration data for continuous, high-resolution behavior monitoring of large herbivores and showed that vigilance behavior is well detectable. In particular, when combined with spatial data, automated behavior recognition will enrich many fields in behavioral ecology by providing extensive access to behaviors of animals in the wild

Integração energética de plantas petroquímicas utilizando a abordagem de Análise Pinch

Devido à crescente conscientização ambiental e à busca pela redução de custos de processos químicos, a integração energética tem sido amplamente utilizada pelas indústrias. A análise Pinch foi desenvolvida com o objetivo de encontrar as condições operacionais e, consequentemente, a integração energética mais otimizada, seguindo a termodinâmica, para plantas de processos. O presente trabalho lida com a simulação de produção de tolueno e de ciclohexano em plantas integradas energeticamente, com e sem análise Pinch. As simulações dos processos foram realizadas no software UNISIM e os resultados das análises Pinch obtidos, realizados no software Microsoft Excel, foram comparados aos valores dos processos integrados energeticamente reportados por Melo (2016) e Cardoso et al. (2014). Porém, assim como qualquer método, a análise Pinch possui limitações, tal qual a brusca alteração na capacidade calorífica devido à mudança de fase dentro do trocador de calor, que foram evidenciadas neste trabalho. No processo de tolueno, a alteração na capacidade calorífica resultou em resultados não compatíveis com a termodinâmica, observados através da temperatura cruzada no UNISIM. Apesar disso, foi possível encontrar a temperatura onde há uma redução, quando comparada com Melo (2016), de 4,45% nas emissões de CO2 devido à combustão e de 4,28% no consumo de água. Foi observado no processo de ciclohexano que a proposta realizada por Cardoso et al. (2014) viola a teoria da análise Pinch, ademais de verificar, através da simulação, que a alteração brusca na capacidade calorífica resultou na necessidade de um aquecedor, além do refrigerador e trocador de calor, já previstos pela abordagem de análise PinchDue to increasing environmental awareness and efforts to reduce costs of chemical processes, energy integration has been widely used by industries. The pinch analysis was created in order to find the operating conditions and therefore the most optimized energy integration, respecting the thermodynamics, for plant processes. This work deals with the simulation of the production of toluene and cyclohexane in energetic integrated plants, with and without Pinch analysis. The simulations of the processes were carried out in UNISIM software and the results obtained for the Pinch analysis, performed in Microsoft Excel software, were compared to the values of energetic integrated processes reported by Melo (2016) and Cardoso et al. (2014). However, like any method, the Pinch analysis has limitations, such as the sudden change in heat capacity due to the phase change in the heat exchanger, which were evidenced in this work. In the toluene process, the change in heat capacity has resulted in inconsistent results with thermodynamics, observed through temperature cross at UNISIM. Nevertheless, it was possible to find the temperature where there is a reduction, when compared to Melo (2016), of 4.45% of CO2 emissions due to combustion and 4.28% in water consumption. It was observed in the cyclohexane process that the proposal made by Cardoso et al. (2014) violates the theory of Pinch analysis, in addition to verify, through simulation, that the abrupt change in heat capacity resulted in the need for a heater, in addition to the refrigerator and heat exchanger, as already predicted by Pinch analysis approac

A psicologia na corda bamba do circo social: construindo redes

Este trabalho surgiu através das práticas do Projeto de Extensão “Psicologia social, alteridade e intervenção em instituições do terceiro setor na cidade do Rio de Janeiro”, financiado pelo programa de bolsas PIBEX. Ele partiu do desejo das alunas em articular a psicologia às atividades de uma ONG que trabalha com circo social localizada no bairro de Laranjeiras, Rio de Janeiro. A iniciativa foi acolhida pela orientadora, dando início a um percurso de presenças semanais das bolsistas na instituição, em vigor há 10 meses. A horizontalidade de caráter autogestionário que deu origem ao projeto, é a mesma premissa que sustenta nossas formas de atuação no campo. Ao longo do processo de habitação neste território, de acordo com a proposta metodológica da cartografia psicossocial de Suely Rolnik, foi possível perceber um distanciamento entre o espaço universitário e a realidade das práticas que acontecem fora dele. A extensão se apresentou como uma possibilidade de fazer essa costura mais claramente, ao nos depararmos com os problemas cotidianos do fazer da psicologia e buscarmos reflexões teóricas que possibilitassem a compreensão desses processos não como dicotômicos, mas com limites e separações que se complementam em gradações. O convite para a entrada na ONG se deu a partir da identificação de problemas por seus coordenadores, e a psicologia surgiu como uma resposta possível a estas demandas. Nossa presença mapeou dificuldades nos profissionais de fazer conversar a dimensão do ensino da técnica circense e a construção de cidadania que também norteia o trabalho da institução. O resultados deste acompanhamento demonstram a mudança na construção da demanda de nossa presença, considerando mais importante o cuidado da saúde mental dos educadores em relação aos educandos, ao percebermos traços de sofrimentos éticos e políticos neste fazer, além da necessidade de trabalhar temas como racismo e preconceitos em geral. Deste modo, a partir desses resultados, buscamos promover reflexões sobre assuntos que emergiram como questão no grupo: saúde mental, homofobia, racismo, formas de intervenção pedagógica e outros temas. Os trabalhos de Sawaia, que discutem o sofrimento ético político, e Yamamoto, que problematiza o terceiro setor, a psicologia e seu compromisso social foram importantes referenciais que permitiram promover reflexões a partir deste espaço, mas que também podem se estender a instituições similares. Os problemas aqui identificados não são exclusivos e remetem à compreensão dos modos de funcionamento deste tipo de empresa. Neste sentido, a afetação surge como uma estratégia para nortear a sensibilidade no campo e tornar possíveis as negociações para encaminhamentos de problemas. Acreditamos ser também de responsabilidade da psicologia a criação de alternativas a realidades já naturalizadas, buscando colocar perguntas onde não tinham sido formuladas

Stability of water-soluble chlorophyll protein (WSCP) depends on phytyl conformation

Water-soluble chlorophyll proteins (WSCP) from Brassicaceae form homotetrameric chlorophyll (Chl)–protein complexes binding one Chl per apoprotein and no carotenoids. Despite the lack of photoprotecting photoprotecting pigments, the complex-bound Chls displays a remarkable stability toward photodynamic photodynamic damage. On the basis of a mutational study, we show that not only the presence of the phytyls phytyls is necessary for photoprotection in WSCPs, as we previously demonstrated, but also is their correct correct conformation and localization. The extreme heat stability of WSCP also depends on the presence presence of the phytyl chains, confirming their relevance for the unusual stability of WSCP

Water-Soluble Chlorophyll Protein (WSCP) Stably Binds Two or Four Chlorophylls

Water-soluble chlorophyll proteins (WSCPs) of class IIa from Brassicaceae form tetrameric complexes containing one chlorophyll (Chl) per apoprotein but no carotenoids. The complexes are remarkably stable toward dissociation and protein denaturation even at 100 °C and extreme pH values, and the Chls are partially protected against photooxidation. There are several hypotheses that explain the biological role of WSCPs, one of them proposing that they function as a scavenger of Chls set free upon plant senescence or pathogen attack. The biochemical properties of WSCP described in this paper are consistent with the protein acting as an efficient and flexible Chl scavenger. At limiting Chl concentrations, the recombinant WSCP apoprotein binds substoichiometric amounts of Chl (two Chls per tetramer) to form complexes that are as stable toward thermal dissociation, denaturation, and photodamage as the fully pigmented ones. If more Chl is added, these two-Chl complexes can bind another two Chls to reach the fully pigmented state. The protection of WSCP Chls against photodamage has been attributed to the apoprotein serving as a diffusion barrier for oxygen, preventing its access to triplet excited Chls and, thus, the formation of singlet oxygen. By contrast, the sequential binding of Chls by WSCP suggests a partially open or at least flexible structure, raising the question of how WSCP photoprotects its Chls without the help of carotenoids