Single-molecule fluorescence studies of nucleosome dynamics.

Single-molecule techniques have transformed dramatically the way we think about biophysics, making it possible to address questions about the dynamics of systems in equilibrium that were practically unthinkable just a decade ago. This review focuses on how single-molecule fluorescence and fluorescence correlation techniques have allowed the investigation of the mechanisms by which nucleosomes allow enzymes and other proteins to access DNA regions that are buried within the nucleosome structure. It has been established that DNA-protein and protein-protein interactions in nucleosomes are very dynamic. The dynamics of the interactions between the DNA and the histone proteins have been investigated by single-molecule FRET and fluorescence correlation spectroscopy. Results are consistent with the so-called site exposure model, in which DNA transiently and spontaneously unwraps from the histone core. DNA accessibility is greatest for sites close to the DNA termini, and decreases towards the nucleosome dyad. Evidence also suggests that DNA sequence plays an important role. The dynamics of the H2A-H2B dimers within the nucleosome has also been addressed by several groups in terms of their implications in determining nucleosome stability and DNA dynamics.

Is the Alifax Test-1TH useful to determine the Disease Activity Score (DAS28) in rheumatoid arthritis patients?

Treatment of rheumatoid arthritis (RA) is monitored with the disease activity score (DAS28), for which the erythrocyte sedimentation rate (ESR) is needed. Apart from the original gold standard method, other methods like the Alifax Test-1TH apparatus are widely used in laboratory worldwide. We compared ESR values obtained by the Alifax Test-1Th apparatus and the gold standard method for 218 RA patients. We found a good correlation (r=0.87) between the Alifax Test-1TH results and the gold standard method. A good correlation (r=0.96) was also found for the DAS28 results obtained with both methods. The number of patients that were misclassified when the Alifax Test-1TH is used is reasonable for both the ESR (14.7%) and the DAS28 (10.6%). These results suggest that it may be useful to determine the ESR by the Alifax Test-1TH, with a DAS28 misclassification in less than 11% of the patients.

Steps to demarcate the effects of chromophore aggregation and planarization in poly(phenyleneethynylene)s. 1. Rotationally interrupted conjugation in the excited states of 1,4-bis(phenylethynyl)benzene.

A series of photophysical measurements and semiempirical calculations were carried out with 1,4-bis(phenylethynyl)benzene in search of evidence on the effects of phenyl group rotation and chromophore aggregation of oligo- and poly(phenyleneethynylene)s. It is suggested that planarization gives rise to relatively modest shifts of ca. 20-30 nm, which preserve the vibronic structure of the monomer and retain a high emission quantum yield. In contrast, it is proposed that aggregation gives rise to larger shifts and loss of vibronic structure.

Steps to demarcate the effects of chromophore aggregation and planarization in poly(phenyleneethynylene)s. 2. The photophysics of 1,4-diethynyl-2-fluorobenzene in solution and in crystals.

Crystals of 1,4-bis(2-hydroxy-2-methyl-3-butynyl)-2-fluorobenzene 4 have a rich packing structure with four distinct molecules in the unit cell. A complex hydrogen bonding network results in the formation of cofacial trimers, cofacial dimers, and monomers within the same unit cell. Given a remarkable opportunity to investigate the effect of aggregation on the photophysics of 1,4-diethynylbenzenes, we analyzed the absorption, diffuse reflectance, and emission spectra of compound 4 in solutions and in crystals. Diffuse reflectance and fluorescence excitation revealed a red-shifted absorption that is absent in dilute solution but becomes observable at high concentrations and low temperatures. The fluorescence emission in the solid state is dual with components assigned to monomers and aggregates. The excitation and emission assigned to the monomer are nearly identical in crystals and dilute solutions. The absorption and emission bands assigned to aggregates are broad and red-shifted by 60--80 nm. As expected for a sample with absorbers and emitters with different energies and incomplete equilibration, efficient monomer-to-aggregate energy transfer was observed by a proper selection of excitation wavelengths. The fluorescence quantum yield of 4 in solution is relatively low (Phi(F) = 0.15) and the singlet lifetime short (tau(F) = 3.8 ns). A lower limit for the triplet yield of Phi(T) = 0.64 was determined indirectly in solution by (1)O(2) sensitization, and a relatively strong and long-lived phosphorescence was observed in low-temperature glasses and in crystals at 77 K.

Complementation analysis in Fanconi anemia: assignment of the reference FA-H patient to group A.

Fanconi anemia (FA) is an autosomal recessive disorder with diverse clinical symptoms and extensive genetic heterogeneity. Of eight FA genes that have been implicated on the basis of complementation studies, four have been identified and two have been mapped to different loci; the status of the genes supposed to be defective in groups B and H is uncertain. Here we present evidence indicating that the patient who has been the sole representative of the eighth complementation group (FA-H) in fact belongs to group FA-A. Previous exclusion from group A was apparently based on phenotypic reversion to wild-type rather than on genuine complementation in fusion hybrids. To avoid the pitfall of reversion, future assignment of patients with FA to new complementation groups should conform with more-stringent criteria. A new group should be based on at least two patients with FA whose cell lines are excluded from all known groups and that fail to complement each other in fusion hybrids, or, if only one such cell line were available, on a new complementing gene that carries pathogenic mutations in this cell line. On the basis of these criteria, the current number of complementation groups in FA is seven.