BACKGROUND:Huntington's disease (HD) is a monogenic disorder caused by an aberrant expansion of CAG repeats in the huntingtin gene (HTT). Pathogenesis is associated with expression of the mutant (mHTT) protein in the CNS, with its levels most likely related to disease progression and symptom severity. Since non-invasive methods to quantify HTT in the CNS do not exist, measuring amount of soluble HTT in peripheral cells represents an important step in development of disease-modifying interventions in HD.RESULTS:An ELISA assay using commercially available antibodies was developed to quantify HTT levels in complex matrices like mammalian cell cultures lysates and human samples. The immunoassay was optimized using a recombinant full-length HTT protein, and validated both on wild-type and mutant HTT species. The ability of the assay to detect significant variations of soluble HTT levels was evaluated using an HSP90 inhibitor that is known to enhance HTT degradation. Once optimized, the bioassay was applied to peripheral blood mononuclear cells (PBMCs) from HD patients, demonstrating good potential in tracking the disease course.CONCLUSIONS:The method described here represents a validated, simple and rapid bio-molecular assay to evaluate soluble HTT levels in blood cells as useful tool in disease and pharmacodynamic marker identification for observational and clinical trials.

The TWEAK-Fn14 pathway is upregulated in models of inflammation, autoimmune diseases, and cancer. Both TWEAK and Fn14 show increased expression also in the CNS in response to different stimuli, particularly astrocytes, microglia, and neurons, leading to activation of NF-κB and release of proinflammatory cytokines. Although neutralizing antibodies against these proteins have been shown to have therapeutic efficacy in animal models of inflammation, no small-molecule therapeutics are yet available. Here, we describe the development of a novel homogeneous time-resolved fluorescence (HTRF)–based screening assay together with several counterassays for the identification of small-molecule inhibitors of this protein-protein interaction. Recombinant HIS-TWEAK and Fn14-Fc proteins as well as FLAG-TWEAK and Fn14-FLAG proteins and an anti-Fn14 antibody were used to establish and validate these assays and to screen a library of 60 000 compounds. Two HTRF counterassays with unrelated proteins in the same assay format, an antiaggregation assay and a redox assay, were applied to filter out potential false-positive compounds. The novel assay and associated screening cascade should be useful for the discovery of small-molecule inhibitors of the TWEAK-Fn14 protein interaction.

Background. DKK1 antagonizes canonical Wnt signalling through high-affinity binding to LRP5/6, an essential component of the Wnt receptor complex responsible for mediating downstream canonical Wnt signalling. DKK1 overexpression is known for its pathological implications in osteoporosis, cancer, and neurodegeneration, suggesting the interaction with LRP5/6 as a potential therapeutic target. Results. We show that the small-molecule NCI8642 can efficiently displace DKK1 from LRP6 and block DKK1 inhibitory activity on canonical Wnt signalling, as shown in binding and cellular assays, respectively. We further characterize NCI8642 binding activity on LRP6 by Surface Plasmon Resonance (SPR) technology. Conclusions. This study demonstrates that the DKK1-LRP6 interaction can be the target of small molecules and unlocks the possibility of new therapeutic tools for diseases associated with DKK1 dysregulation.

Proteomics studies employing primary neurons are difficult due to the neurons' characteristics. We have developed a detergent‐based fractionation method which reduces complexity of the protein extracts, is sufficiently fast to allow differential proteomics analysis after treatments of neurons for short time periods, can be applied to small numbers of cells directly in culture plates, and allows differential extraction of proteins in a compartment‐specific manner. The sequential use of detergent‐containing buffers on neurons in culture plates yields four extracts enriched in cytosolic, membrane‐bound or enclosed, nuclear, and cytoskeletal proteins. Fractionation of neurons was validated by comparison of the distribution of known subcellular marker proteins in the four extracts using Western blotting. Comparison of extracts by DIGE showed a clear difference in protein composition demonstrating significant variations with a fold change (FC) of at least 1.20 for 82% of the detected spots. Using proteins identified in these spots that could be assigned a subcellular localization based on descriptions in the Uniprot database, an extraction efficiency of 85% was calculated for cytosolic proteins in extract 1, 90% for membrane‐bound and membrane‐enclosed proteins in extract 2, 82% for nuclear proteins in extract 3 and 38% for cytoskeletal and RAFT proteins in extract 4.

The early events in the thrombin-induced formation of fibrin have been studied by the use of stopped-flow multiangle laser light scattering (SF-MALLS). This technological advancement has allowed the recovering, as a function of time with a resolution of about 0.5 sec, of the mean square radius of gyration (Rg2)z and of the molecular weight Mw, and to place an upper bound to the values of the mass/unit length ML. The ionic strength, pH and salt type conditions investigated were all close to physiological, starting with a 50 mM Tris, 104 mM NaCl, pH 7.4 buffer (TBS), to which either 1 mM EDTA-Na2 or 2.5 mM CaCl2 were also added. Fibrinogen was 0.2-0.3 mg/ml and rate-limiting concentrations of thrombin were used (0.05-0.25 NIH units/mg fibrinogen). By plotting (Rg2)z and ML versus Mw on log-log scales, runs proceeding at different velocities and under different solvent conditions could be compared and confronted with model curves. It was found that: (1) within this thrombin range, the mechanism of association does not depend on its concentration, nor on the buffers employed; (2) the (Rg2)z versus Mw curves could all be reasonably fitted with a bifunctional polycondensation scheme involving semiflexible worm-like, double-stranded, half-staggered polymers with persistence length between 200-600 nm, provided that a ratio Q = 16 between the rate of release of the two fibrinopeptides A was employed; (3) the ML versus Mw data seemed more compatible with lower Q values (4 < Q < 8), but their uncertainty prevented a better assessment of this issue; the formation of fibrinogen-fibrin monomer complexes may also play a role in the polymer distributions; (4) in the very early stages (e.g., when Mw < 7 x 10(5)), the (Rg2)z versus Mw data were fitted well only in TBS and at the lowest thrombin concentration, suggesting that a transient, either sequential or concurrent fast second mechanism, involving longer and thinner polymers, may be at work.

The solution properties of a novel soluble polydiacetylene, poly[1,6-bis(3,6-dihexadecyl-9H-carbazol-9-yl)hexa-2,4-diyne] (polyDCHD-HS), have been investigated by using UV-Vis absorption, fluorescence, and FT-Raman spectroscopies. The polymer gives rise to stable, brilliant red solutions in aromatic solvents and in chloroform at room temperature with no evidence of the colour transition to yellow that usually takes place by increasing the temperature with other soluble polydiacetylenes. A particular role appears to be played by benzene-polyDCHD-HS interactions, because in this solvent a very narrow and intense excitonic peak is observed at room temperature in the electronic spectrum. Interestingly, these solutions show also a well-resolved fluorescence spectrum, with a very small Stokes shift and a rather high quantum yield (congruent to 10(-2)). Furthermore, a very efficient transfer of the excitation energy from the carbazolyl substituents to the conjugated backbone is found to take place. On account of the observation that by increasing the temperature of the benzene solutions up to 75 degreesC the excitonic feature in both the absorption and the emission spectra undergoes fully reversible broadening and intensity reduction accompanied by the increase of the Stokes shift, without any dramatic colour change, it is likely that the rates of torsional motions in the polymer chains are thermally activated without modifying the average conjugation length of the polymer chains. FT-Raman measurements in benzene solutions in the same temperature range have confirmed this conclusion. The photophysical properties of polyDCHD-HS in benzene around 60 degreesC are quite similar to those observed in other aromatic solvents at room temperature. By heating up the red solutions in toluene or in chlorobenzene, no spectral shift is observed but only a limited increase of the absorption band broadening. All these findings strongly suggest that by the appropriate choice of the substituents it is possible to obtain stable solutions of the red form of polydiacetylenes.

Many biological supramolecular structures are formed by polymerization of macromolecular monomers. Light scattering techniques can provide structural information from such systems, if suitable procedures are used to collect the data and then to extract the relevant parameters. We present an experimental set-up in which a commercial multiangle laser light scattering photometer is linked to a stopped-flow mixer, allowing, in principle, the time-resolved extrapolation of the weight-average molecular weight M-w and of the z-average square radius of gyration [R-g(2)](z) of the polymers from Zimm-like plots. However, if elongated structures are formed as the polymerization proceeds, curved plots rapidly arise, from which M-w and [R-g(2)](z) cannot be recovered by linear fitting. To verify the correctness of a polynomial fitting procedure, polydisperse collections of rod-like or worm-like particles of different lengths, generated at various stages during bifunctional polycondensations of rod-like macromolecular monomers, were considered. Then, the angular dependence of their time-averaged scattered intensity was calculated in the Rayleigh-Gans-Debye approximation, with random and systematic noise also added to the data. For relatively narrow size distributions, a third-degree polynomial fitting gave satisfactory results across a broad range of conversion degrees, yielding M-w and [R-g(2)](z) values within 2% and no greater than 10-20%, respectively, of the calculated values. When more broad size distributions were analyzed, the procedure still performed well for semiflexible polymers, but started to seriously underestimate both M-w and [R-g(2)](z) when rigid rod-like particles were analyzed, even at relatively low conversion degrees. The data were also analyzed in the framework of the Casassa approximation, from which the mass per unit length of the polymers can be derived. These procedures were applied to a set of data taken on the early stages of the thrombin-catalyzed polymerization of fibrinogen, a rod-like macromolecule similar to 50 nm long. The polymers, grown in the absence of Ca2+ by rate-limiting amounts of thrombin, appeared to be characterized by a much broader size distribution than the one expected for a classical Flory bifunctional polycondensation, and they seem to behave as relatively flexible worm-like double-stranded chains. Evidence for the formation of fibrinogen-fibrin monomer complexes is also inferred from the time dependence of the mass/length ratio. However, our data are also compatible with the presence of limited amounts of single-stranded structures in the very early stages, either as a secondary, less populated pathway, or as transient intermediates to the classical double-stranded fibrils.