Fluorescence lifetime imaging microscopy (FLIM) to demonstrate the nuclear binding of flavanols and (--epigallocatechin gallate

The use of light microscopy and DMACA staining strongly suggested that plant and animal cell nuclei act as sinks for flavanols [1, 2]. Detailed uv-vis spectroscopic titration experiments indicated that histone proteins are the likely binding sites in the nucleus [2]. Here we report the development of a multi-photon excitation microscopy technique combined with fluorescent lifetime measurements of flavanols. Using this technique, (+) catechin, (-) epicatechin and (-) epigallocatechin gallate (EGCG) showed strikingly different excited state lifetimes in solution. Interaction of histone proteins with flavanols was indicated by the appearance of a significant τ2-component of 1.7 to 4.0ns. Tryptophan interference could be circumvented in the in vivo fluorescence lifetime imaging microscopy (FLIM) experiments with 2-photon excitation at 630nm. This enabled visualisation and semi-quantitative measurements that demonstrated unequivocally the absorption of (+)catechin, (-)epicatechin and EGCG by nuclei of onion cells. 3D FLIM revealed for the first time that externally added EGCG penetrated the whole nucleus in onion cells. The relative proportions of EGCG in cytoplasm: nucleus: nucleoli were ca. 1:10:100. FLIM experiments may therefore facilitate probing the health effects of EGCG, which is the major constituent of green tea.

Simple solution for a complex problem: Proanthocyanidins, galloyl glucoses and ellagitannins fit on a single calibration curve in high performance-gel permeation chromatography

This study was undertaken to explore gel permeation chromatography (GPC) for estimating molecular weights of proanthocyanidin fractions isolated from sainfoin (Onobrychis viciifolia). The results were compared with data obtained by thiolytic degradation of the same fractions. Polystyrene, polyethylene glycol and polymethyl methacrylate standards were not suitable for estimating the molecular weights of underivatized proanthocyanidins. Therefore, a novel HPLC-GPC method was developed based on two serially connected PolarGel-L columns using DMF that contained 5% water, 1% acetic acid and 0.15 M LiBr at 0.7 ml/min and 50 degrees C. This yielded a single calibration curve for galloyl glucoses (trigalloyl glucose, pentagalloyl glucose), ellagitannins (pedunculagin, vescalagin, punicalagin, oenothein B, gemin A), proanthocyanidins (procyanidin B2, cinnamtannin B1), and several other polyphenols (catechin, epicatechin gallate, epicallocatechin gallate, amentoflavone). These GPC predicted molecular weights represented a considerable advance over previously reported HPLC-GPC methods for underivatized proanthocyanidins. (C) 2011 Elsevier B.V. All rights reserved.

Two-photon excitation with pico-second fluorescence lifetime imaging to detect nuclear association of flavanols

Two-photon excitation enabled for the first time the observation and measurement of excited state fluorescence lifetimes from three flavanols in solution, which were ∼1.0 ns for catechin and epicatechin, but <45 ps for epigallocatechin gallate (EGCG). The shorter lifetime for EGCG is in line with a lower fluorescence quantum yield of 0.003 compared to catechin (0.015) and epicatechin (0.018). In vivo experiments with onion cells demonstrated that tryptophan and quercetin, which tend to be major contributors of background fluorescence in plant cells, have sufficiently low cross sections for two-photon excitation at 630 nm and therefore do not interfere with detection of externally added or endogenous flavanols in Allium cepa or Taxus baccata cells. Applying two-photon excitation to flavanols enabled 3-D fluorescence lifetime imaging microscopy and showed that added EGCG penetrated the whole nucleus of onion cells. Interestingly, EGCG and catechin showed different lifetime behaviour when bound to the nucleus: EGCG lifetime increased from <45 to 200 ps, whilst catechin lifetime decreased from 1.0 ns to 500 ps. Semi-quantitative measurements revealed that the relative ratios of EGCG concentrations in nucleoli associated vesicles: nucleus: cytoplasm were ca. 100:10:1. Solution experiments with catechin, epicatechin and histone proteins provided preliminary evidence, via the appearance of a second lifetime (τ2 = 1.9–3.1 ns), that both flavanols may be interacting with histone proteins. We conclude that there is significant nuclear absorption of flavanols. This advanced imaging using two-photon excitation and biophysical techniques described here will prove valuable for probing the intracellular trafficking and functions of flavanols, such as EGCG, which is the major flavanol of green tea.

Initial distribution spread: A density forecasting approach

Ensemble forecasting of nonlinear systems involves the use of a model to run forward a discrete ensemble (or set) of initial states. Data assimilation techniques tend to focus on estimating the true state of the system, even though model error limits the value of such efforts. This paper argues for choosing the initial ensemble in order to optimise forecasting performance rather than estimate the true state of the system. Density forecasting and choosing the initial ensemble are treated as one problem. Forecasting performance can be quantified by some scoring rule. In the case of the logarithmic scoring rule, theoretical arguments and empirical results are presented. It turns out that, if the underlying noise dominates model error, we can diagnose the noise spread.

Proanthocyanidin diversity in the EU 'HealthyHay' sainfoin (onobrychis viciifolia) germplasm collection

This study investigated 37 diverse sainfoin (Onobrychis viciifolia Scop.) accessions from the EU ‘HealthyHay’ germplasm collection for proanthocyanidin (PA) content and composition. Accessions displayed a wide range of differences: PA contents varied from 0.57 to 2.80 g/100 g sainfoin; the mean degree of polymerisation from 12 to 84; the proportion of prodelphinidin tannins from 53% to 95%, and the proportion of trans-flavanol units from 12% to 34%. A positive correlation was found between PA contents (thiolytic versus acid–butanol degradation; P < 0.001; R2 = 0.49). A negative correlation existed between PA content (thiolysis) and mDP (P < 0.05; R2 = −0.30), which suggested that accessions with high PA contents had smaller PA polymers. Cluster analysis revealed that European accessions clustered into two main groups: Western Europe and Eastern Europe/Asia. In addition, accessions from USA, Canada and Armenia tended to cluster together. Overall, there was broad agreement between tannin clusters and clusters that were based on morphological and agronomic characteristics.

Dare voce agli invisibili: Il teatro di Beppe Rosso

The article – which is co-authored with Irene Zagrebelsky – focuses on the work of the Italian playwright and actor Beppe Rosso, and it is the most detailed critical account on Rosso’s production so far. Based on original material and interviews, it not only analyses Rosso’s widely acclaimed “Trilogy of invisibility” in the light of recent political events in Italy, but it also – in its first two sections – introduces the so called “teatro di narrazione” to Italian scholars in the UK, and in its two last sections opens up new perspectives on the relationship between contemporary theatre, citizenship and political participation.

Standardised ileal digestibility of crude protein and amino acids of UK-grown peas and faba beans by broilers

In view of the increasing interest in home-grown legumes as components of diets for non-ruminant livestock and in an attempt to reduce the reliance on imported soya bean meal (SBM), two experiments were conducted to evaluate samples of peas and faba beans for their standardised ileal digestibility (SID) of amino acids determined with young broiler chicks. Experiment 1 evaluated six faba bean and seven pea cultivars and Experiment 2 evaluated two faba bean and three pea cultivars as well as a sample of soya bean meal provided as a reference material. Peas and beans were added at 750g/kg as the only source of protein/amino acids in a semi-synthetic diet containing the inert marker titanium dioxide; SBM was added, in a control diet, at 500g/kg. Each diet was fed to six replicates of a cage containing two Ross-type broilers for 96h at which point birds were culled allowing removal of ileal digesta. Chemical analyses allowed the calculation of the coefficient of SID of amino acids. There were no differences between samples of the same pulse species (P>0.05) but peas had higher values (P<0.05), similar to SBM, than beans. Trypsin inhibitor content (expressed as g trypsin inhibitor units/mg sample) of all pea samples was low and in the range 0.83–1.77mg/kg. There was relatively little variation in bean tannin content and composition amongst the coloured-flowered varieties; however, the white-flowered cultivar had no tannins. There was no correlation between tannin content and coefficient of SID. The content of SID of amino acids (g/kg legume) was higher in SBM when compared with peas and beans by virtue of having higher total concentrations.

Les variations formelles d'un moule stylistique: rubrique, texte et image à la charnière du Merlin et de sa Suite Vulgate

Le passage du Merlin propre à sa Suite Vulgate, espace de suture entre le texte et sa continuation est dans la tradition manuscrite l'objet de variances et de déplacements. A la paix annoncée à la fin du Merlin succède en effet le conflit qui oppose le jeune roi Arthur à ses barons. Les passages de rubrication et d'entrelacement sont le lieu privilégié de variations formelles gommées par l'adoption et la reprise de moules stylistiques attendus. Ceux-ci exhibent tout en les masquant de subtiles divergences relevant de partis pris à la fois narratifs, interprétatifs et idéologiques.

Diviser, choisir et condenser: rubriques et illustrations du règne d'Uter et Pandragon dans le Merlin de Robert de Boron

La sélection opérée par les rubriques et les enluminures, formes contraintes qui échappent dans une certaine mesure à la linéarité du développement textuel, détermine la réception de l'œuvre. Les manuscrits comprenant des miniatures synthétiques (comme celles qui représentent l'enfance de Merlin) ou des séries iconographiques (comme celles qui rendent compte des amours d'Uterpandragon et Ygerne et de leurs conséquences) présentent donc un caractère exceptionnel, orientant la lecture du texte et son interprétation.

'Conment Merlin se mua en guise de cerf' : écrire et représenter la métamorphose animale dans les manuscrits enluminés de la Suite Vulgate du Merlin

Les manuscrits enluminés du Merlin illustrent l’arrivée du personnage transformé en cerf à la table de Jules César, mais sa transformation elle-même n’est jamais représentée. Cette intervention théâtralisée permet ultimement de dénoncer les faux semblants et le désordre qui règnent autour de l’empereur. Elle a une dimension purgative, visant la restauration de l’ordre moral, politique et sexuel, tout en promouvant une démarche heuristique placée au cœur du processus de lecture et d’interprétation.