Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy

We have investigated the pH dependence of the dynamics of conformational fluctuations of green fluorescent protein mutants EGFP (F64L/S65T) and GFP-S65T in small ensembles of molecules in solution by using fluorescence correlation spectroscopy (FCS). FCS utilizes time-resolved measurements of fluctuations in the molecular fluorescence emission for determination of the intrinsic dynamics and thermodynamics of all processes that affect the fluorescence. Fluorescence excitation of a bulk solution of EGFP decreases to zero at low pH (pKa = 5.8) paralleled by a decrease of the absorption at 488 nm and an increase at 400 nm. Protonation of the hydroxyl group of Tyr-66, which is part of the chromophore, induces these changes. When FCS is used the fluctuations in the protonation state of the chromophore are time resolved. The autocorrelation function of fluorescence emission shows contributions from two chemical relaxation processes as well as diffusional concentration fluctuations. The time constant of the fast, pH-dependent chemical process decreases with pH from 300 μs at pH 7 to 45 μs at pH 5, while the time-average fraction of molecules in a nonfluorescent state increases to 80% in the same range. A second, pH-independent, process with a time constant of 340 μs and an associated fraction of 13% nonfluorescent molecules is observed between pH 8 and 11, possibly representing an internal proton transfer process and associated conformational rearrangements. The FCS data provide direct measures of the dynamics and the equilibrium properties of the protonation processes. Thus FCS is a convenient, intrinsically calibrated method for pH measurements in subfemtoliter volumes with nanomolar concentrations of EGFP.

Intranuclear diffusion and hybridization state of oligonucleotides measured by fluorescence correlation spectroscopy in living cells

Fluorescein-labeled oligodeoxynucleotides (oligos) were introduced into cultured rat myoblasts, and their molecular movements inside the nucleus were studied by fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP). FCS revealed that a large fraction of both intranuclear oligo(dT) (43%) and oligo(dA) (77%) moves rapidly with a diffusion coefficient of 4 × 10−7 cm2/s. Interestingly, this rate of intranuclear oligo movement is similar to their diffusion rates measured in aqueous solution. In addition, we detected a large fraction (45%) of the intranuclear oligo(dT), but not oligo(dA), diffusing at slower rates (≤1 × 10−7 cm2/s). The amount of this slower-moving oligo(dT) was greatly reduced if the oligo(dT) was prehybridized in solution with (unlabeled) oligo(dA) prior to introduction to cells, presumably because the oligo(dT) was then unavailable for subsequent hybridization to endogenous poly(A) RNA. The FCS-measured diffusion rate for much of the slower oligo(dT) population approximated the diffusion rate in aqueous solution of oligo(dT) hybridized to a large polyadenylated RNA (1.0 × 10−7 cm2/s). Moreover, this intranuclear movement rate falls within the range of calculated diffusion rates for an average-sized heterogeneous nuclear ribonucleoprotein particle in aqueous solution. A subfraction of oligo(dT) (15%) moved over 10-fold more slowly, suggesting it was bound to very large macromolecular complexes. Average diffusion coefficients obtained from FRAP experiments were in agreement with the FCS data. These results demonstrate that oligos can move about within the nucleus at rates comparable to those in aqueous solution and further suggest that this is true for large ribonucleoprotein complexes as well.

High-resolution correlation of coastal cliff sections in the Lagos- Portimao Formation (Lower - Middle Miocene, central Algarve, Portugal)

This paper describes a high-resolution stratigraphic correlation scheme for the early to middle Miocene Lagos-Portimão Formation of central Algarve, southern Portugal. The Lagos Portimão-Formation of central Algarve is a 60 m thick package of horizontally bedded siliciclastics and carbonates. The bryozoan and mollusc dominated biofacies is typical of a shallow marine, warm-temperate climatic environment. We define four stratigraphic marker beds based on biofacies, lithology, and gamma-ray signatures. Marker bed 1 is a reddish shell bed composed predominantly of bivalve shells in various stages of fragmentation. Marker bed 2 is a fossiliferous sandstone / sandy rudstone characterized by bryozoan masses. Marker bed 3 is also a fossiliferous sandstone with abundant larger foraminifers and foliate bryozoans. Marker bed 4 is composed of three distinct layers; two fossiliferous sandstones with an intercalated shell bed. The upper sandstone unit displays thickets of the bryozoan Celleporaria palmate associated with the coral Culizia parasitica. This stratigraphic framework allows to correlate isolated outcrops within the stratigraphic context of the Lagos-Portimão Formation and to establish high resolution chronostratigraphic Sr-isotopic dating.

Correlation of Modified Natural Rubber Properties by Artificial Neural Networks

Natural rubber (NR) is a raw material largely used by the modern industry; however, it is common that chemical modifications must be made to NR in order to improve properties such as hydrophobicity or mechanical resistance. This work deals with the correlation of properties of NR modified with dimethylaminoethylmethacrylate or methylmethacrylate as grafting agents. Dynamic-mechanical behavior and stress/strain relations are very important properties because they furnish essential characteristics of the material such as glass transition temperature and rupture point. These properties are concerned with different physical principles; for this reason, normally they are not related to each other. This work showed that they can be correlated by artificial neural networks (ANN). So, from one type of assay, the properties that as a rule only could be obtained from the other can be extracted by ANN correlation. POLYM. ENG. SCI., 49:499-505, 2009. (c) 2009 Society of Plastics Engineers

Produção de hidrogénio através da gasificação da biomassa

A procura de uma forma limpa de combustível, aliada à crescente instabilidade de preços dos combustíveis fósseis verificada nos mercados faz com que o hidrogénio se torne num combustível a considerar devido a não resultar qualquer produto poluente da sua queima e de se poder utilizar, por exemplo, desperdícios florestais cujo valor de mercado não está inflacionado por não pertencer à cadeia alimentar humana. Este trabalho tem como objetivo simular o processo de gasificação de biomassa para produção de hidrogénio utilizando um gasificador de leito fluidizado circulante. O oxigénio e vapor de água funcionam como agentes gasificantes. Para o efeito usou-se o simulador de processos químicos ASPEN Plus. A simulação desenvolvida compreende três etapas que ocorrem no interior do gasificador: pirólise, que foi simulada por um bloco RYIELD, combustão de parte dos compostos voláteis, simulada por um bloco RSTOIC e, por fim, as reações de oxidação e gasificação do carbonizado “char”, simuladas por um bloco RPLUG. Os valores de rendimento dos compostos após a pirólise, obtidos por uma correlação proposta por Gomez-Barea, et al. (2010), foram os seguintes: 20,33% “char”, 22,59% alcatrão, 36,90% monóxido de carbono, 16,05%m/m dióxido de carbono, 3,33% metano e 0,79% hidrogénio (% em massa). Como não foi possível encontrar valores da variação da composição do gás à saída do gasificador com a variação da temperatura, para o caso de vapor de água e oxigénio, optou-se por utilizar apenas vapor na simulação de forma a comparar os seus valores com os da literatura. Às temperaturas de 700, 770 e 820ºC, para um “steam-to-biomass ratio”, (SBR) igual a 0,5, os valores da percentagem molar de monóxido de carbono foram, respetivamente, 56,60%, 55,84% e 53,85%, os valores de hidrogénio foram, respetivamente, 17,83%, 18,25% e 19,31%, os valores de dióxido de carbono foram, respetivamente, 16,40%, 16,85% e 17,93% e os valores de metano foram, respetivamente, 9,00%, 8,95% e 8,83%. Os valores da composição à saída do gasificador, à temperatura de 820ºC, para um SBR de 0,5 foram: 53,85% de monóxido de carbono, 19,31% de hidrogénio, 17,93% de dióxido de carbono e 8,83% de metano (% em moles). Para um SBR de 0,7 a composição à saída foi de 54,45% de monóxido de carbono, 19,01% de hidrogénio, 17,59% de dióxido de carbono e 8,87% de metano. Por fim, quando SBR foi igual a 1 a composição do gás à saída foi de 55,08% de monóxido de carbono, 18,69% de hidrogénio, 17,24% de dióxido de carbono e 8,90% de metano. Os valores da composição obtidos através da simulação, para uma mistura de ar e vapor de água, ER igual a 0,26 e SBR igual a 1, foram: 34,00% de monóxido de carbono, 14,65% de hidrogénio, 45,81% de dióxido de carbono e 5,41% de metano. A simulação permitiu-nos ainda dimensionar o gasificador e determinar alguns parâmetros hidrodinâmicos do gasificador, considerando que a reação “water-gas shift” era a limitante, e que se pretendia obter uma conversão de 95%. A velocidade de operação do gasificador foi de 4,7m/s e a sua altura igual a 0,73m, para um diâmetro de 0,20m.

Deconvolução de perfis de poços através do ajuste de energia

As medidas de resistividade são de fundamental importância para o cálculo da saturação de óleo em reservatórios potencialmente produtores. A combinação das medidas de resistividade rasa e profunda permite a obtenção dos parâmetros R_t, R_xo e d_i. Mas, em reservatórios complexos existem dificuldades em se obter leituras confiáveis de R_t, devido à baixa resolução vertical das ferramentas de investigação profunda. Em reservatórios laminados, por exemplo, as leituras obtidas pela ferramenta de indução profunda (ILD) podem levar a uma interpretação errônea das mesmas, levando a acreditar que as medidas obtidas do perfil referem-se a uma única camada. Este problema pode ser em parte resolvido através de uma metodologia que melhore a resolução vertical dos perfis de investigação profunda, valendo-se do uso de informações obtidas de um perfil de alta resolução vertical, i.e; a curva de resistividade rasa. Uma abordagem neste sentido seria usar um perfil de alta resolução que apresente uma boa correlação com o perfil de investigação profunda. Esta correlação pode ser melhor avaliada se aplicarmos um filtro no perfil de alta resolução, de tal forma que o perfil resultante tenha teoricamente a mesma resolução vertical do perfil de baixa resolução. A obtenção deste filtro, porém, recai na premissa de que as funções respostas verticais para as ferramentas de alta e baixa resolução são disponíveis, o que não ocorre na prática. Este trabalho se propõe mostrar uma nova abordagem onde o filtro pode ser obtido a partir de um tratamento no domínio da freqüência. Tal tratamento visa igualar a energia espectral do perfil de alta resolução à energia espectral do perfil de baixa resolução tendo como base o Teorema de Parseval. Será mostrado que a resolução vertical depende fundamentalmente da energia espectral do perfil em questão. A seguir, uma regressão linear é aplicada sobre os perfis de alta resolução filtrado e de baixa resolução. Para cada ponto amostrado dos perfis, uma rotina de minimização é aplicada visando escolher o melhor intervalo de correlação entre os perfis. Finalmente, um fator de correção é aplicado sobre cada ponto do perfil de baixa resolução. Os resultados obtidos com os perfis de indução são promissores, demonstrando a eficácia da abordagem e mesmo quando aplicada em perfis com diferentes propriedades petrofísicas, a metodologia funcionou satisfatoriamente, sem danificar os perfis originais.

Correlation between volume of apical periodontitis determined by cone-beam computed tomography analysis and endotoxin levels found in primary root canal infection

This clinical study was conducted to correlate the levels of endotoxins and bacterial counts found in primary endodontic infection with the volume of periapical bone destruction determined by cone-beam computed tomography (CBCT) analysis. Moreover, the levels of bacteria and endotoxins were correlated with the development of clinical features. Twenty-four root canals with primary endodontic disease and apical periodontitis were selected. Clinical features such as pain on palpation, pain on percussion, and previous episode of pain were recorded. The volume (cubic millimeters) of periapical bone destruction was determined by CBCT analysis. Endotoxins and bacterial samplings were collected by using sterile/apyrogenic paper points. Endotoxins were quantified by using limulus amebocyte lysate assay (KQCL test), and bacterial count (colony-forming units [CFU]/mL) was determined by using anaerobic culture techniques. Data were analyzed by Pearson correlation and multiple logistic regression (P < .05). Endotoxins and bacteria were detected in 100% of the root canal samples (24 of 24), with median values of 10.92 endotoxin units (EU)/mL (1.75-128 EU/mL) and 7.5 × 10(5) CFU/mL (3.20 × 10(5)-8.16 × 10(6) CFU/mL), respectively. The median volume of bone destruction determined by CBCT analysis was 100 mm(3) (10-450 mm(3)). The multiple regression analysis revealed a positive correlation between higher levels of endotoxins present in root canal infection and larger volume of bone destruction (P < .05). Moreover, higher levels of endotoxins were also correlated with the presence of previous pain (P < .05). Our findings revealed that the levels of endotoxins found in root canal infection are related to the volume of periapical bone destruction determined by CBCT analysis. Moreover, the levels of endotoxin are related to the presence of previous pain.

Fluorescence correlation analysis of probe diffusion simplifies quantitative pathogen detection by PCR

A sensitive, labor-saving, and easily automatable nonradioactive procedure named APEX-FCS (amplified probe extension detected by fluorescence correlation spectroscopy) has been established to detect specific in vitro amplification of pathogen genomic sequences. As an example, Mycobacterium tuberculosis genomic DNA was subjected to PCR amplification with the Stoffel fragment of Thermus aquaticus DNA polymerase in the presence of nanomolar concentrations of a rhodamine-labeled probe (third primer), binding to the target in between the micromolar amplification primers. The probe becomes extended only when specific amplification occurs. Its low concentration avoids false-positives due to unspecific hybridization under PCR conditions. With increasing portion of extended probe molecules, the probe’s average translational diffusion properties gradually change over the course of the reaction, reflecting amplification kinetics. Following PCR, this change from a stage of high to a stage of low mobility can directly be monitored during a 30-s measurement using a fluorescence correlation spectroscopy device. Quantitation down to 10 target molecules in a background of 2.5 μg unspecific DNA without post-PCR probe manipulations could be achieved with different primer/probe combinations. The assay holds the promise to concurrently perform amplification, probe hybridization, and specific detection without opening the reaction chamber, if sealable foils are used.

Sedimentology of a Middle Jurassic Base-of-Slope environment, Cutri Formation, Mallorca

The Cutri Formation’s, type location, exposed in the NW of Mallorca, Spain has previously been described by Álvaro et al., (1989) and further interpreted by Abbots (1989) unpublished PhD thesis as a base-of-slope carbonate apron. Incorporating new field and laboratory analysis this paper enhances this interpretation. From this analysis, it can be shown without reasonable doubt that the Cutri Formation was deposited in a carbonate base-of-slope environment on the palaeowindward side of a Mid-Jurassic Tethyan platform. Key evidence such as laterally extensive exposures, abundant deposits of calciturbidtes and debris flows amongst hemipelagic deposits strongly support this interpretation.

Analysis of Apical Root Transportation Associated with Protaper Universal F3 and F4 Instruments by Using Digital Subtraction Radiography

Introduction: The aim of this study was to assess the occurrence of apical root transportation after the use of Pro Taper Universal rotary files sizes 3 (F3) and 4 (F4). Methods: Instruments were worked to the apex of the original canal, always by the same operator. Digital subtraction radiography images were produced in buccolingual and mesiodistal projections. A total of 25 radiographs were taken from root canals of human maxillary first molars with curvatures varying from 23-31 degrees. Quantitative data were analyzed by intraclass correlation coefficient and Wilcoxon nonparametric test (P = .05). Results: Buccolingual images revealed a significantly higher degree of apical transportation associated with F4 instruments when compared with F3 instruments in relation to the original canal (Wilcoxon test, P = .007). No significant difference was observed in mesiodistal images (P = .492). Conclusions: F3 instruments should be used with care in curved canals, and F4 instruments should be avoided in apical third preparation of curved canals. (J Endod 2010;36:1052-1055)

Viscosity and self-diffusion coefficients of dialkyl adipates: a correlation scheme with predictive capabilities

A correlation and predictive scheme for the viscosity and self-diffusivity of liquid dialkyl adipates is presented. The scheme is based on the kinetic theory for dense hard-sphere fluids, applied to the van der Waals model of a liquid to predict the transport properties. A "universal" curve for a dimensionless viscosity of dialkyl adipates was obtained using recently published experimental viscosity and density data of compressed liquid dimethyl (DMA), dipropyl (DPA), and dibutyl (DBA) adipates. The experimental data are described by the correlation scheme with a root-mean-square deviation of +/- 0.34 %. The parameters describing the temperature dependence of the characteristic volume, V-0, and the roughness parameter, R-eta, for each adipate are well correlated with one single molecular parameter. Recently published experimental self-diffusion coefficients of the same set of liquid dialkyl adipates at atmospheric pressure were correlated using the characteristic volumes obtained from the viscosity data. The roughness factors, R-D, are well correlated with the same single molecular parameter found for viscosity. The root-mean-square deviation of the data from the correlation is less than 1.07 %. Tests are presented in order to assess the capability of the correlation scheme to estimate the viscosity of compressed liquid diethyl adipate (DEA) in a range of temperatures and pressures by comparison with literature data and of its self-diffusivity at atmospheric pressure in a range of temperatures. It is noteworthy that no data for DEA were used to build the correlation scheme. The deviations encountered between predicted and experimental data for the viscosity and self-diffusivity do not exceed 2.0 % and 2.2 %, respectively, which are commensurate with the estimated experimental measurement uncertainty, in both cases.

Prediction of cross-recognition of peptide-HLA A2 by Melan-A-specific cytotoxic T lymphocytes using three-dimensional quantitative structure-activity relationships.

The cross-recognition of peptides by cytotoxic T lymphocytes is a key element in immunology and in particular in peptide based immunotherapy. Here we develop three-dimensional (3D) quantitative structure-activity relationships (QSARs) to predict cross-recognition by Melan-A-specific cytotoxic T lymphocytes of peptides bound to HLA A*0201 (hereafter referred to as HLA A2). First, we predict the structure of a set of self- and pathogen-derived peptides bound to HLA A2 using a previously developed ab initio structure prediction approach [Fagerberg et al., J. Mol. Biol., 521-46 (2006)]. Second, shape and electrostatic energy calculations are performed on a 3D grid to produce similarity matrices which are combined with a genetic neural network method [So et al., J. Med. Chem., 4347-59 (1997)] to generate 3D-QSAR models. The models are extensively validated using several different approaches. During the model generation, the leave-one-out cross-validated correlation coefficient (q (2)) is used as the fitness criterion and all obtained models are evaluated based on their q (2) values. Moreover, the best model obtained for a partitioned data set is evaluated by its correlation coefficient (r = 0.92 for the external test set). The physical relevance of all models is tested using a functional dependence analysis and the robustness of the models obtained for the entire data set is confirmed using y-randomization. Finally, the validated models are tested for their utility in the setting of rational peptide design: their ability to discriminate between peptides that only contain side chain substitutions in a single secondary anchor position is evaluated. In addition, the predicted cross-recognition of the mono-substituted peptides is confirmed experimentally in chromium-release assays. These results underline the utility of 3D-QSARs in peptide mimetic design and suggest that the properties of the unbound epitope are sufficient to capture most of the information to determine the cross-recognition.

Comparison of energy evaluation systems and a mechanistic model for milk production by dairy cattle offered fresh grass-based diets

Grass-based diets are of increasing social-economic importance in dairy cattle farming, but their low supply of glucogenic nutrients may limit the production of milk. Current evaluation systems that assess the energy supply and requirements are based on metabolisable energy (ME) or net energy (NE). These systems do not consider the characteristics of the energy delivering nutrients. In contrast, mechanistic models take into account the site of digestion, the type of nutrient absorbed and the type of nutrient required for production of milk constituents, and may therefore give a better prediction of supply and requirement of nutrients. The objective of the present study is to compare the ability of three energy evaluation systems, viz. the Dutch NE system, the agricultural and food research council (AFRC) ME system, and the feed into milk (FIM) ME system, and of a mechanistic model based on Dijkstra et al. [Simulation of digestion in cattle fed sugar cane: prediction of nutrient supply for milk production with locally available supplements. J. Agric. Sci., Cambridge 127, 247-60] and Mills et al. [A mechanistic model of whole-tract digestion and methanogenesis in the lactating dairy cow: model development, evaluation and application. J. Anim. Sci. 79, 1584-97] to predict the feed value of grass-based diets for milk production. The dataset for evaluation consists of 41 treatments of grass-based diets (at least 0.75 g ryegrass/g diet on DM basis). For each model, the predicted energy or nutrient supply, based on observed intake, was compared with predicted requirement based on observed performance. Assessment of the error of energy or nutrient supply relative to requirement is made by calculation of mean square prediction error (MSPE) and by concordance correlation coefficient (CCC). All energy evaluation systems predicted energy requirement to be lower (6-11%) than energy supply. The root MSPE (expressed as a proportion of the supply) was lowest for the mechanistic model (0.061), followed by the Dutch NE system (0.082), FIM ME system (0.097) and AFRCME system(0.118). For the energy evaluation systems, the error due to overall bias of prediction dominated the MSPE, whereas for the mechanistic model, proportionally 0.76 of MSPE was due to random variation. CCC analysis confirmed the higher accuracy and precision of the mechanistic model compared with energy evaluation systems. The error of prediction was positively related to grass protein content for the Dutch NE system, and was also positively related to grass DMI level for all models. In conclusion, current energy evaluation systems overestimate energy supply relative to energy requirement on grass-based diets for dairy cattle. The mechanistic model predicted glucogenic nutrients to limit performance of dairy cattle on grass-based diets, and proved to be more accurate and precise than the energy systems. The mechanistic model could be improved by allowing glucose maintenance and utilization requirements parameters to be variable. (C) 2007 Elsevier B.V. All rights reserved.

Enhanced solubilization of iron and calcium phosphates by Aspergillus niger by the addition of alcohols

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Structural Properties Induced by the Composition of Biocompatible Phospholipid-Based Microemulsion and Amphotericin B Association

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)