Biochemical suppression of small-molecule inhibitors: a strategy to identify inhibitor targets and signaling pathway components

Identification of small-molecule targets remains an important challenge for chemical genetics. We report an approach for target identification and protein discovery based on functional suppression of chemical inhibition in vitro. We discovered pirl1, an inhibitor of actin assembly, in a screen conducted with cytoplasmic extracts. Pirl1 was used to partially inhibit actin assembly in the same assay, and concentrated biochemical fractions of cytoplasmic extracts were added to find activities that suppressed pirl1 inhibition. Two activities were detected, separately purified, and identified as Arp2/3 complex and Cdc42/RhoGDI complex, both known regulators of actin assembly. We show that pirl1 directly inhibits activation of Cdc42/RhoGDI, but that Arp2/3 complex represents a downstream suppressor. This work introduces a general method for using low-micromolar chemical inhibitors to identify both inhibitor targets and other components of a signaling pathway.     

Preferential sites for InAsP/InP quantum wire nucleation using molecular dynamics

In this paper, stress fields at the surface of the capping layer of self-assembled InAsP quantum wires grown on an InP (001) substrate have been determined from atomistic models using molecular dynamics and Stillinger-Weber potentials. To carry out these calculations, the quantum wire compositional distribution was extracted from previous works, where the As and P distributions were determined by electron energy loss spectroscopy and high-resolution aberration-corrected Z-contrast imaging. Preferential sites for the nucleation of wires on the surface of the capping layer were studied and compared with (i) previous simulations using finite element analysis to solve anisotropic elastic theory equations and (ii) experimentally measured locations of stacked wires. Preferential nucleation sites of stacked wires were determined by the maximum stress location at the MD model surface in good agreement with experimental results and those derived from finite element analysis. This indicates that MD simulations based on empirical potentials provide a suitable and flexible tool to study strain dependent atom processes.     

Multi-way partial least-squares and residual bi-linearization for the direct determination of monohydroxy-polycyclic aromatic hydrocarbons on octadecyl membranes via room-temperature fluorescence excitation emission matrices

Multi-way partial least-squares (N-PLS) is combined to the residual bi-linearization procedure (RBL) for the direct analysis of metabolites of polycyclic aromatic hydrocarbons in urine samples. Metabolite analysis is carried out via a two-step experimental procedure based on solid-phase extraction and room temperature fluorescence spectroscopy. Excitation-emission matrices are recorded from octadecyl (C18) membranes that serve as solid substrates for sample extraction and spectroscopic measurements. Excellent metabolite recoveries were obtained in all cases, which varied from 96.2 ± 1.35% (9-hydroxyphenanthrene) to 99.7 ± 0.49% (3-hydroxybenzo[a]pyrene). Background correction of extraction membranes is carried out with a new alternating least-squares (ALS) procedure adapted to second order data. The performance of N-PLS/RBL is compared to the well-established multivariate curve resolution-alternating least-squares (MCR-ALS) algorithm. Both algorithms provided similar analytical figures of merit, including their ability to handle unknown interference in urine samples. With only 10 mL of sample, the limits of detection varied between 0.06–0.08 ng mL⁻¹ (1-hydroxypyrene) and 0.016–0.018 ng mL⁻¹ (2-hydroxyfluorene). When compared to previously reported univariate calibration data, the limits of detection via N-PLS/RBL and MCR-ALS are approximately one order of magnitude higher. This was somehow expected due to the effect of unexpected components in multivariate figures of merit, i.e. a more realistic approach to the analysis of metabolites in human urine samples.     

Development of a ReaxFF reactive force field for ettringite and study of its mechanical failure modes from reactive dynamics simulations

Ettringite is a hexacalcium aluminate trisulfate hydrate mineral that forms during Portland cement hydration. Its presence plays an important role in controlling the setting rate of the highly reactive aluminate phases in cement paste and has also been associated with severe cracking in cured hardened cement. To understand how it forms and how its properties influence those of hardened cement and concrete, we have developed a first-principles-based ReaxFF reactive force field for Ca/Al/H/O/S. Here, we report on the development of this ReaxFF force field and on its validation and application using reactive molecular dynamics (RMD) simulations to characterize and understand the elastic, plastic, and failure response of ettringite at the atomic scale. The ReaxFF force field was validated by comparing the lattice parameters, pairwise distribution functions, and elastic constants of an ettringite crystal model obtained from RMD simulations with those from experiments. The predicted results are in close agreement with published experimental data. To characterize the atomistic failure modes of ettringite, we performed stress-strain simulations to find that Ca-O bonds are responsible for failure of the calcium sulfate and tricalcium aluminate (C3A) column in ettringite during uniaxial compression and tension and that hydrogen bond re-formation during compression induces an increase in plastic strain beyond the material's stress-strain proportionality limit. These results provide essential insight into understanding the mechanistic role of this mineral in cement and concrete degradation, and the ReaxFF potential developed in this work serves as a fundamental tool to further study the kinetics of hydration in cement and concrete.     

Game-perfect digraphs

In the A-coloring game, two players, Alice and Bob, color uncolored vertices of a given uncolored digraph D with colors from a given color set C, so that, at any time a vertex is colored, its color has to be different from the colors of its previously colored in-neighbors. Alice begins. The players move alternately, where a move of Bob consists in coloring a vertex, and a move of Alice in coloring a vertex or missing the turn. The game ends when Bob is unable to move. Alice wins if every vertex is colored at the end, otherwise Bob wins. This game is a variant of a graph coloring game proposed by Bodlaender (Int J Found Comput Sci 2:133?C147, 1991). The A-game chromatic number of D is the smallest cardinality of a color set C, so that Alice has a winning strategy for the game played on D with C. A digraph is A-perfect if, for any induced subdigraph H of D, the A-game chromatic number of H equals the size of the largest symmetric clique of H. We characterize some basic classes of A-perfect digraphs, in particular all A-perfect semiorientations of paths and cycles. This gives us, as corollaries, similar results for other games, in particular concerning the digraph version of the usual game chromatic number.     

Reductive Ring Opening of Ephedrine and Pseudo-Ephedrine-Derived Oxazolidinium Methiodides by Sodium Borohydride. A Direct Access to New Chiral Amine-Borane Adducts

Sodiun borohydride reacts with oxazolidinium methiodides derived from ephedrine and pseudoephedrine, in ethereal solvents, cleaving regioselectively the C-N bond of the ring and leading, in good yields, to chiral amine-borane adducts of know absolute configuration. The ¹HNMR data allow to predict the most stable conformation.     

Spin fluctuations and magnetic order in the heavy fermion compound CePt2Sn2

We present zero and longitudinal field μ SR measurements of single crystal and polycrystalline specimens of the heavy fermion compound CePt₂Sn₂. Above 1 K the behaviour of the two samples is indistinguishable; the muon 1/T_1 increases with decreasing temperature until 25 K when it plateaus. The 1/T_1 relaxation rate differs strongly for the two cases below \sim\,0.8\ K. At 0.1 K a rate of about 20 μ s^-1 is seen in the polycrystal while in the single crystal it is only about 5 μ s^-1. Even more revealing is the fact that longitudinal field decoupling spectra at very low temperatures demonstrate an essentially static spin system to be present in the polycrystalline material while the single crystal shows definite dynamic spin properties. We conclude that, in the presence of the distortion, long range magnetic order occurs below 0.9 K while in tetragonal symmetry long range order is suppressed (probably due to frustration) and spin fluctuations remain for T\rightarrow0. This revised version was published online in July 2006 with corrections to the Cover Date.     

Anisotropy of the lower critical field and meissner effect in (TMTSF)2ClO4 in the basal plane

The concept of a macroscopically large London-penetration depth due to weak interstack interaction in the c¹-direction is well suited to explain the different low field magnetization behavior for magnetic fields along the b¹ and c¹ directions. In a Ginzburg Landau model with anisotropic effective masses the angular dependence of the lower critical field H_c1 can be accounted for.