Effect of hydrogen bonding interaction on the photophysics of α-amino-orcein.

This paper reports for the first time a detailed spectroscopic investigation into the ground- and excited-state properties of α-amino-orcein (α-AO), one of the main components of the orcein dye, in solvents of different proticity and water at different pHs. In order to gain insight into the nature of the involved transitions and excited state deactivation pathways, the study was carried out by means of UV-Visible steady state and ultrafast spectroscopic techniques with the support of quantum mechanical calculations (DFT and TDDFT). The results highlight that the photophysical and photodynamic behaviour of α-AO are highly sensitive to the solvent proticity and pH. In particular, protic environment induces a red shift (55 nm) of the absorption spectrum together with a relevant decrease of the fluorescence quantum yield (from 0.19 in acetonitrile to 6.6 × 10-3 in methanol) and radiative rate constant (two orders of magnitude). A notable red shift is also caused by increasing the pH leading the molecule from monocationic to neutral and then monoanionic form through two deprotonation steps (pKa = 3.539 ±â€¯0.006 and 11.180 ±â€¯0.006). Following deprotonation, the molecule assumes spectral and photophysical properties very similar to those retrieved in protic media. The observed behaviour has been rationalized through the occurrence of hydrogen bonding, likely involving to a greater extent the carbonyl oxygen of α-AO and the protic solvent, that favours the charge delocalization on the whole chromophore as well as fast non-radiative excited state deactivation. The ultrafast spectroscopic investigation revealed in fact the presence, in protic solvent, of a short living component (tens of picoseconds), assignable to solvent complexed S1 state, alongside the long living component (few nanoseconds) observed in aprotic media and attributed to the solvent free S1 state. The results achieved in this study for α-AO provides an important contribution to the interpretation of absorption and fluorescence features of orcein dye mixture in more complex systems (protein based substrates within the many aspects of the cultural heritage and biomedical field) where hydrogen bonds are expected to play a crucial role in mediating the interaction with the environment.

Doxycycline and oxytetracycline loading of a zwitterionic amphoteric surfactant-gel and their controlled release.

Oxytetracycline (OX) and doxycycline (DX) are antibiotics belonging to the family of tetracyclines. We present a UV-Visible steady state and time-resolved experimental study of OX and DX and their biologically active Mg(2+) complexes loaded within a hydrogel matrix. Hydrogels are a three dimensional network of worm-like micelles, mutually intertwined, forming a pattern of hydrophobic domains and water pools. We resorted to a hydrogel, made of a zwitterionic N-oxide surfactant (p-dodecyloxybenzyldimethylamine N-oxide, pDoAO), which showed promising features as a drug vehicle. The spectral and photophysical properties of the drugs are significantly altered by the inclusion in the hydrophobic domains of the gel and these variations are indicators of the permeation ratio of the drug in between the micelles forming the gel network. We thus get a clear picture of the distribution of the drug molecules and metal chelates into the two different kinds of environment, where the hydrophobic domains are also able to cause a gel-induced deprotonation of these two drugs. Furthermore, the amphoteric nature of the surfactant is responsible for its peculiar acid-base behaviour: under acidic pH conditions, the surfactant gets protonated and the stability of the gel network is damaged. This feature can be thus exploited for the pH controlled release of the tetracycline drugs.

The Book of Kells: a non-invasive MOLAB investigation by complementary spectroscopic techniques.

This paper highlights the efficacy of non-invasive portable spectroscopy for assessing the execution technique and constituent materials in one of the most important medieval manuscripts, the Book of Kells. An aimed campaign of in situ measurements by the MObile LABoratory (MOLAB) has analyzed its elemental composition and vibrational and electronic molecular properties. The ample analytical toolbox has afforded complementary diagnostic information of the pigment palette permitting the characterization of both inorganic and organic materials as pigments and dyes in the white, purple, blue, red, orange, green and black areas. In particular, the novel widespread use of calcinated gypsum (anhydrite) as both a white pigment and in correlation to the organic dyes in this manuscript has been noted. The non-invasive identification of the organic dye orchil is significant considering its rare non invasive detection in medieval manuscripts. Finally the occurrence of particular alterations of the organic black areas giving rise to calcium carboxylate and calcium oxalate has been specifically highlighted. Importantly, this work elaborates complex aspects of the employed painting materials which have given rise to numerous significant points of interest for a more elaborate understanding of this Irish treasure.

In-situ fluorimetry: a powerful non-invasive diagnostic technique for natural dyes used in artefacts. Part II. Identification of orcein and indigo in Renaissance tapestries.

In this paper, three Renaissance tapestries depicting scenes painted by Raffaello Sanzio, conserved at the Vatican Museum, were investigated using in-situ UV-Visible fluorimetric measurements. The results show that this technique is suitable for the detection of natural organic colorants used for dyeing the threads woven in these tapestries. The emission signals detected on red-purple colours were assigned to the colorant orcein and those on different nuances of blue and green colours to indigo by comparison with data from reference laboratory samples. The assignments were supported by chromatographic experiments carried out on threads taken from the back side of the tapestry in the same points analysed by spectrofluorimentry.

Portable equipment for luminescence lifetime measurements on surfaces.

The prototype of a portable instrument, based on the time-correlated single-photon counting method, purposely assembled for in situ measurements of luminescence lifetimes on artwork surfaces, is here presented. Preliminary tests have been carried out using the portable instrument and the results have been compared with those obtained using a bench instrument. In this way we have proven that the prototype provides lifetime measurements with good precision. It is also shown that fluorescence lifetime determinations, coupled with steady-state fluorescence spectra, allow the distinguishing, on laboratory samples, of different red organic lakes having similar fluorescence spectra, achieving new boundaries in the nondestructive diagnosis of artwork materials. The first in situ application of the technique on an original work of art, The Book of Kells, held at Trinity College Library, Dublin, highlighted the diagnostic potential of coupled steady-state and time-resolved luminescence spectroscopy in the identification of organic colorants.

Acidichromism and ionochromism of luteolin and apigenin, the main components of the naturally occurring yellow weld: a spectrophotometric and fluorimetric study.

Luteolin and apigenin, extracted from Reseda luteola L., were spectrophotometrically and fluorimetrically studied. The spectra were investigated as a function of pH in methanol/water solutions (1/2, v/v) in the 2-12 pH range. The absorption spectra markedly shifted to the red by increasing the pH. Three acid-base dissociation steps were detected for luteolin (pK (a) = 6.9; 8.6; 10.3) and two for apigenin (pK (a) = 6.6; 9.3). Fluorescence emission was very weak or undetectable (Phi (F) < 10(-4)) in acidic solution, but increased in intensity with increasing the pH. Both molecules exhibited a great propensity towards complex formation with metal ions, with association constants on the order of 10(5)-10(7) for the first complexation step; in the presence of excess Al(3+) ions, multiple equilibria were detected. A marked fluorescence enhancement was observed upon complexation with Al(3+) ions (Phi (F) approximately 1 for luteolin and approximately 10(-2) for apigenin).

A spectrometric and chromatographic approach to the study of ageing of madder (Rubia tinctorum L.) dyestuff on wool.

In this work, the lightfastness of wool textile samples, dyed with madder and its principal components alizarin and purpurin, was investigated using two complementary experimental techniques: absorption and emission UV-vis spectroscopy and chromatography (HPLC-PDA). Spectroscopic techniques were used to follow the time course of ageing, whereas chromatography was applied to determine relative compositional changes that occurred after exposure of wool dyed samples to natural and artificial ageing. The results from the two techniques integrate well each other and provide complementary and useful indications about the sensitivity of the dyed textiles to ageing, showing that purpurin is the principal component responsible for the spectral and chromatic properties of madder as well as for its degradation. The fading of both the fibre and dye is reduced in the presence of alum and in the absence of oxygen. The multi-analytical approach used highlights the potential of the UV-vis spectroscopy for the investigation of dyes on textiles. The great sensitivity of the spectrofluorimetry makes this technique particularly promising for a non-destructive study of dyes on works of art.

In situ fluorimetry: a powerful non-invasive diagnostic technique for natural dyes used in artefacts Part I. Spectral characterization of orcein in solution, on silk and wool laboratory-standards and a fragment of Renaissance tapestry.

In this paper the potentialities of spectrophotometric and fluorimetric techniques for identifying the materials used in artistic textiles are investigated. A portable non-destructive instrument suitable to record fluorescence spectra on surfaces was set up and successfully experienced. A naturally occurring dye, orcein, which was widely used in antiquity for textile dyeing, has been spectrally characterized in both solution and powder. Laboratory samples of wool and silk orcein-dyed threads were analysed before and after ageing. An original fragment of Renaissance tapestry was also analysed. The textile (wool) and the colourant (orcein) were recognised by comparison with the data from the laboratory samples.

Prediction of folding mechanism for circular-permuted proteins.

Recent theoretical and experimental studies have suggested that real proteins have sequences with sufficiently small energetic frustration that topological effects are central in determining the folding mechanism. A particularly interesting and challenging framework for exploring and testing the viability of these energetically unfrustrated models is the study of circular-permuted proteins. Here we present the results of the application of a topology-based model to the study of circular permuted SH3 and CI2, in comparison with the available experimental results. The folding mechanism of the permuted proteins emerging from our simulations is in very good agreement with the experimental observations. The differences between the folding mechanisms of the permuted and wild-type proteins seem then to be strongly related to the change in the native state topology.

Hamiltonian dynamics and geometry of phase transitions in classical XY models

The Hamiltonian dynamics associated with classical, planar, Heisenberg XY models is investigated for two- and three-dimensional lattices. In addition to the conventional signatures of phase transitions, here obtained through time averages of thermodynamical observables in place of ensemble averages, qualitatively different information is derived from the temperature dependence of Lyapunov exponents. A Riemannian geometrization of Newtonian dynamics suggests consideration of other observables of geometric meaning tightly related to the largest Lyapunov exponent. The numerical computation of these observables--unusual in the study of phase transitions--sheds light on the microscopic dynamical counterpart of thermodynamics, also pointing to the existence of some major change in the geometry of the mechanical manifolds at the thermodynamical transition. Through the microcanonical definition of the entropy, a relationship between thermodynamics and the extrinsic geometry of the constant energy surfaces sigma E of phase space can be naturally established. In this framework, an approximate formula is worked out determining a highly nontrivial relationship between temperature and topology of sigma E. From this it can be understood that the appearance of a phase transition must be tightly related to a suitable major topology change of sigma E. This contributes to the understanding of the origin of phase transitions in the microcanonical ensemble.

Topological and energetic factors: what determines the structural details of the transition state ensemble and "en-route" intermediates for protein folding? An investigation for small globular proteins.

Recent experimental results suggest that the native fold, or topology, plays a primary role in determining the structure of the transition state ensemble, at least for small, fast-folding proteins. To investigate the extent of the topological control of the folding process, we studied the folding of simplified models of five small globular proteins constructed using a Go-like potential to retain the information about the native structures but drastically reduce the energetic frustration and energetic heterogeneity among residue-residue native interactions. By comparing the structure of the transition state ensemble (experimentally determined by Phi-values) and of the intermediates with those obtained using our models, we show that these energetically unfrustrated models can reproduce the global experimentally known features of the transition state ensembles and "en-route" intermediates, at least for the analyzed proteins. This result clearly indicates that, as long as the protein sequence is sufficiently minimally frustrated, topology plays a central role in determining the folding mechanism.

How native-state topology affects the folding of dihydrofolate reductase and interleukin-1beta.

The overall structure of the transition-state and intermediate ensembles observed experimentally for dihydrofolate reductase and interleukin-1beta can be obtained by using simplified models that have almost no energetic frustration. The predictive power of these models suggests that, even for these very large proteins with completely different folding mechanisms and functions, real protein sequences are sufficiently well designed, and much of the structural heterogeneity observed in the intermediates and the transition-state ensembles is determined by topological effects.

Protein design is a key factor for subunit-subunit association.

Fundamental questions about role of the quaternary structures are addressed by using a statistical mechanics off-lattice model of a dimer protein. The model, in spite of its simplicity, captures key features of the monomer-monomer interactions revealed by atomic force experiments. Force curves during association and dissociation processes are characterized by sudden jumps followed by smooth behavior and form hysteresis loops. Furthermore, the process is reversible in a finite range of temperature stabilizing the dimer, and the width of the hysteresis loop increases as the design procedure improves: i.e., stabilizes the dimer more. It is shown that, in the interface between the two monomeric subunits, the design procedure naturally favors those amino acids whose mutual interaction is stronger.

Folding Lennard-Jones proteins by a contact potential.

We studied the possibility to approximate a Lennard-Jones interaction by a pairwise contact potential. First we used a Lennard-Jones potential to design off-lattice, protein-like heteropolymer sequences, whose lowest energy (native) conformations were then identified by molecular dynamics. Then we turned to investigate whether one can find a pairwise contact potential, whose ground states are the contact maps associated with these native conformations. We show that such a requirement cannot be satisfied exactly, i.e., no such contact parameters exist. Nevertheless, we found that one can find contact energy parameters for which an energy minimization procedure, acting in the space of contact maps, yields maps whose corresponding structures are close to the native ones. Finally, we show that when these structures are used as the initial point of a molecular dynamics energy minimization process, the correct native folds are recovered with high probability.

Liver cirrhosis: epidemiological aspects in Italy.

In order to improve our knowledge of the incidence of liver cirrhosis in Italy, we conducted two epidemiological studies. The first study showed that about 15% of asymptomatic subjects with persistent increase in alanine aminotransferase had histological evidence of cirrhosis. In this setting, cirrhosis was associated with viral aetiology in 91.4% of cases. In the second study, which enrolled cirrhosis patients from 13 centres from all regions of the country, viral infections were detected in 82.6% of patients, the large majority of whom, 71.2%, were positive for hepatitis C virus (HCV). Alcohol abuse was present in 8.7% of cases as exclusive aetiological factor. All the patients were classified according to Child-Pugh and were scored as class A in 62.4%, as class B in 23.8% and as class C in 13.8% of cases. The age distribution showed that about 55% of cirrhosis patients were under 60 years of age; 34.3% of them had a Child-Pugh score of class B or C. These data show that HCV infection represents the predominant aetiological factor of cirrhosis in Italy and that cirrhosis can be found frequently in asymptomatic subjects.

Hepatitis C virus RNA in peripheral blood mononuclear cells: relation with response to interferon treatment.

The polymerase chain reaction (PCR) was used to investigate the presence of positive and negative hepatitis C virus (HCV) RNA strands in serum and peripheral blood mononuclear cells (PBMC) of 20 patients with histologically proven HCV-related chronic liver disease. All patients completed a course of interferon (IFN) treatment (6 MU of IFN-alpha 2b three times a week for 24 weeks) and were followed-up for 12 months after treatment was discontinued. Pre-treatment, end-treatment and 6-month follow-up serum and PBMC samples were examined. At enrollment, the positive strand of HCV-RNA was detected in serum of 18 patients (90%), the negative strand in none. Positive-stranded HCV-RNA was detected in PBMC of 15 patients (75%), 13 of whom also had detectable levels of negative-stranded HCV-RNA in PBMC. By the end of the treatment, 12 patients (60%) were responders. The pre-treatment HCV infection of PBMC, indicated by the presence of both RNA strands, was found in 8 (66.7%) responders compared to 5 (62.5%) non-responders (P = n.s.). End-treatment loss of PBMC HCV-RNA correlated significantly with the response since it occurred in all responders compared to 2 non-responders (P = 0.02). However, end-treatment-negative serum and PBMC HCV-RNA did not predict the occurrence of a sustained response, which was observed at month 12 in 5 of 12 responders (P = n.s.). On the other hand, the persistent absence of HCV RNA in serum and PBMC at the end of the 6-month follow-up was significantly associated with the occurrence of a sustained response (P < 0.0001).

High prevalence of hepatitis C virus infection in a small central Italian town: lack of evidence of parenteral exposure.

In the spring of 1994, anti-HCV prevalence and associated risk factors were evaluated in 681 subjects representing all age-groups in the general population of a small central Italian town. The overall anti-HCV prevalence was 8.4%, ranging from 3.7% in the 30-39 age-group to 18.2% (p < 0.01) in the 60-70 age-group; no subject below 30 years of age was positive. Multiple logistic regression analysis showed that the only variables independently associated with anti-HCV positivity were awareness of unspecified liver disease (O.R. 3.58), age > 45 years (O.R. 2.72), and lowest number of years of schooling (O.R. 11.0) while no association was found with any parenteral exposure such as blood transfusion, intravenous drug use, major or minor surgical intervention, use of glass syringes or dental therapy. The HBsAg prevalence in this population was 1.3%, which corresponds to the rate reported in central Italy. These findings show a high level of HCV endemicity, with no evidence of parenteral exposure.