Assay principle for modulators of protein-protein interactions and its application to non-ATP-competitive ligands targeting protein kinase A

Targeting sites that modulate protein-protein interactions represents an ongoing challenge for drug discovery. We have devised an assay principle, named ligand-regulated competition (LiReC), in an effort to find non-ATP competitive small-molecule regulators for type Ialpha cAMP-dependent Protein kinase (PKA-Ialpha), a protein complex that is implicated in disease. Our assay based on the LiReC principle utilizes a competitive fluorescent peptide probe to assess the integrity of the PKA-Ialpha complex upon introduction of an allosteric ligand. The developed fluorescence polarization method screens for small molecules that specifically protect (antagonists) or conversely activate (agonists) this protein complex. In high-throughput format, various cyclic nucleotide-derived agonists and antagonists are successfully detected with high precision. Furthermore, assay performance (Z'-factors above 0.7) far exceeds the minimum requirement for small-molecule screening. To identify compounds that operate through novel modes of action, our method shields the ATP-binding site and purposely excludes ATP-competitive ligands. These proof-of-principle experiments highlight the potential of the LiReC technique and suggest its application to other protein complexes, thereby providing a novel approach to identify and characterize modulators (small molecules, proteins, peptides, or nucleic acids) of protein-protein systems.     

Evaluation of cortical bone by peripheral quantitative computed tomography in continuous ambulatory peritoneal dialysis patients

Several studies have suggested an increased prevalence of osteopenia in dialysis. Peripheral quantitative computed tomography (pQCT) is a new technique that allows the noninvasive evaluation of trabecular and cortical bone separately. The aim of the study was: (1) to evaluate cortical bone by pQCT in continuous ambulatory peritoneal dialysis (CAPD) patients and compare the data with that obtained in healthy controls; and (2) to correlate cortical bone parameters with bone mineral density (BMD) of the lumbar spine and femoral neck and total bone mineral content (TBMC). Cortical bone parameters were obtained in 22 CAPD patients and 27 healthy individuals at the distal radius using a Stratec XCT 960 pQCT machine. In the dialysis patients, we also determined BMD and TBMC by bone densitometry. Dialysis patients, compared with controls, showed a significant reduction in volumetric cortical BMD (VcBMD) (p = 0.04) and cortical thickness (cThk) (p < 0.0001) with a significant increase in radial total cross-sectional area (TA) (p = 0.006), endosteal circumference (p < 0.0001), and buckling ratio (p < 0.0001). In CAPD patients, total time on dialysis correlated negatively with radial total BMD (p < 0.01) and VcBMD (p < 0.01). Age correlated positively with TA (p < 0.01), endosteal (p < 0.01), and periosteal circumferences (p < 0.01). Serum intact parathyroid hormone (PTH) levels correlated positively with endosteal (p = 0.04) and periosteal perimeter (p = 0.01). Total alkaline phosphatase correlated negatively with VcBMD (p < 0.01), and positively with endosteal perimeter (p = 0.02). Total bone mineral content correlated significantly with radial cortical content (p < 0.001), cross-sectional cortical area (cA; p < 0.001), and cThk (p < 0.01) but not with total radial BMD, VcBMD, or buckling ratio. No correlations were found between radial cortical parameters and BMD measured at the lumbar spine or femoral neck. We conclude that dialysis patients show cortical osteopenia with marked cortical thinning partially mediated by PTH action on bone. Total bone mineral content correlated with various radial cortical parameters (content, area, and thickness) but not with others. No correlations were found between cortical bone parameters measured at the peripheral skeleton with areal bone density measured at the axial skeleton. These findings suggest that pQCT may be a new tool in the assessment of bone fragility in dialysis patients.     

An Unbiased Method for Probabilistic Fire Safety Engineering,Requiring a Limited Number of Model Evaluations

The rise of Performance Based Design methodologies for fire safety engineering has increased the interest of the fire safety community in the concepts of risk and reliability. Practical applications have however been severely hampered by the lack of an efficient unbiased calculation methodology. This is because on the one hand, the distribution types of model output variables in fire safety engineering are not known and traditional distribution types as for example the normal and lognormal distribution may result in unsafe approximations. Therefore unbiased methods must be applied which make no (implicit) assumptions on the PDF type. Traditionally these unbiased methods are based on Monte Carlo simulations. On the other hand, Monte Carlo simulations require a large number of model evaluations and are therefore too computationally expensive when large and nonlinear calculation models are applied, as is common in fire safety engineering. The methodology presented in this paper avoids this deadlock by making an unbiased estimate of the PDF based on only a very limited number of model evaluations. The methodology is known as the Maximum Entropy Multiplicative Dimensional Reduction Method (ME-MDRM) and results in a mathematical formula for the probability density function (PDF) describing the uncertain output variable. The method can be applied with existing models and calculation tools and allows for a parallelization of model evaluations. The example applications given in the paper stem from the field of structural fire safety and illustrate the excellent performance of the method for probabilistic structural fire safety engineering. The ME-MDRM can however be considered applicable to other types of engineering models as well.     

Improving access to biodiversity data for,and from,EIAs – a data publishing framework built to global standards

Biodiversity information obtained during environmental impact assessments (EIAs) is rarely accessible for other uses following the completion of the EIA. Such data need to be made readily accessible; adding them to publicly accessible national datasets is important if biodiversity science, conservation and future decisions based on environmental assessment are to benefit from new biodiversity data and improved biodiversity data coverage. An ‘EIA Biodiversity Data Publishing Framework’, based on the Global Biodiversity Information Facility (GBIF) global standards, is thus proposed to meet this need. This paper outlines the GBIF-catalysed initiative to establish such an operational framework for uptake by the EIA community, as well as options that are available for data publishing in the absence of such a framework. It reviews the current state of accessibility and management of the primary biodiversity data associated with EIA studies, and highlights the urgent need for uptake of a range of data-publishing tools and best practices for making EIA biodiversity data exchangeable using globally accepted standards. Lessons learnt from pilot projects in India and South Africa underline the call for the rapid uptake of a national-to-global scale EIA Biodiversity Data Publishing Framework.     

Diffusion of NaOH into a protein gel

The diffusivity of NaOH in a layer of protein gel on an inert surface is measured by combining experimental study of the reactive dissolution of the gel with simulation of transport and reaction within the gel. Pure β-lactoglobulin gels, formed under three different gelation conditions, were used as a model system. The alkaline solutions cause the gel to swell, and destroy the interprotein interactions in the gel matrix. The swelling and cleavage reactions depend on the local concentration of hydroxide and so are sensitive to the rate of transport of hydroxide through the gel. Experiments were performed to determine the effect of the NaOH concentration on the hydroxide penetration thickness and on the velocity of the penetration front marked by phenolthalein indicator. Simple simulations and a more advanced semi-theoretical analysis were performed, with proteins being treated as ideal polyelectrolyte polymers. Both yielded good agreement with the experimental results. The effective diffusivity of NaOH in the protein gels was found to be similar to that in water. The analytical procedure can be extended in principle to any protein gel.     

Supramolecular spin valves

Magnetic molecules are potential building blocks for the design of spintronic devices. Moreover, molecular materials enable the combination of bottom-up processing techniques, for example with conventional top-down nanofabrication. The development of solid-state spintronic devices based on the giant magnetoresistance, tunnel magnetoresistance and spin-valve effects has revolutionized magnetic memory applications. Recently, a significant improvement of the spin-relaxation time has been observed in organic semiconductor tunnel junctions, single non-magnetic molecules coupled to magnetic electrodes have shown giant magnetoresistance and hybrid devices exploiting the quantum tunnelling properties of single-molecule magnets have been proposed. Herein, we present an original spin-valve device in which a non-magnetic molecular quantum dot, made of a single-walled carbon nanotube contacted with non-magnetic electrodes, is laterally coupled through supramolecular interactions to TbPc(2) single-molecule magnets (Pc=phthalocyanine). Their localized magnetic moments lead to a magnetic field dependence of the electrical transport through the single-walled carbon nanotube, resulting in magnetoresistance ratios up to 300% at temperatures less than 1 K. We thus demonstrate the functionality of a supramolecular spin valve without magnetic leads. Our results open up prospects of new spintronic devices with quantum properties.     

C-1s NEXAFS spectroscopy reveals chemical fractionation of humic acid by cation-induced coagulation

The influence of cation-induced coagulation on the chemical composition of dissolved and coagulated fractions of humic acid was investigated in batch coagulation experiments for additions of aluminum at pH 4 and 5, iron at pH 4, and calcium and lead at pH 6. The partitioning of organic carbon and metals was determined by analyzing total organic carbon and total metal contents of the dissolved phase. Both the dissolved and the coagulated humic acid fractions were characterized using synchrotron scanning transmission X-ray microscopy (STXM) and C-1s near-edge X-ray absorption fine structure (NEXAFS) spectroscopy. Intensities of pi* transitions of carboxyl carbon and sigma* transitions of alkyl, O-alkyl, and carboxyl carbon decreased with increasing metal concentration for the dissolved humic acid fractions. This decrease was accompanied by an increase of the respective intensities in the coagulated fraction as shown for lead. Intensities of aromatic and phenolic carbon were affected to a larger extent only by aluminum and iron additions. The changes observed in the C-1s NEXAFS spectra coincided with an increasing removal of organic carbon from the dissolved phase with increasing total metal concentrations. We conclude that humic acid was chemically fractionated by cation-induced coagulation, which preferentially removed functional groups involved in metal-cation binding from solution.     

Molecular quantum spintronics: supramolecular spin valves based on single-molecule magnets and carbon nanotubes

We built new hybrid devices consisting of chemical vapor deposition (CVD) grown carbon nanotube (CNT) transistors, decorated with TbPc(2) (Pc = phthalocyanine) rare-earth based single-molecule magnets (SMMs). The drafting was achieved by tailoring supramolecular π-π interactions between CNTs and SMMs. The magnetoresistance hysteresis loop measurements revealed steep steps, which we can relate to the magnetization reversal of individual SMMs. Indeed, we established that the electronic transport properties of these devices depend strongly on the relative magnetization orientations of the grafted SMMs. The SMMs are playing the role of localized spin polarizer and analyzer on the CNT electronic conducting channel. As a result, we measured magneto-resistance ratios up to several hundred percent. We used this spin valve effect to confirm the strong uniaxial anisotropy and the superparamagnetic blocking temperature (T(B) ~ 1 K) of isolated TbPc(2) SMMs. For the first time, the strength of exchange interaction between the different SMMs of the molecular spin valve geometry could be determined. Our results introduce a new design for operable molecular spintronic devices using the quantum effects of individual SMMs.