Monitoring cytoplasmic protein complexes with blue native gel electrophoresis and stable isotope labelling with amino acids in cell culture: analysis of changes in the 20S proteasome

Analysis of protein complexes is of increasing interest in the field of proteomics. A challenge is to develop methods for monitoring changes in the quantity and subunit composition of protein complexes on a proteome-wide scale. Here, we describe the combination of 1-D blue native polyacrylamide gel electrophoresis (BN-PAGE) with stable isotope labelling of amino acids in cell culture (SILAC) and tandem mass spectrometry (MS/MS). Cleared lysates from normal and perturbed samples, one incorporating heavy stable isotopes and the other light isotopes, are co-separated by blue native PAGE and then analysed and quantitated with MS/MS and appropriate software. This permits the analysis of cytoplasmic complexes. To demonstrate this technique, we explored how the 20S proteasome changes when the Pre9/α3 subunit, the only non-essential subunit of this complex, was deleted. Our results showed that ΔPre9/α3 cells can form the 20S proteasome complex, although with reduced efficiency. This involves an increase in expression of the α4 subunit. Our findings suggest this technique as an approach for the study of quantitative and qualitative differences in protein complexes, from cleared cell lysates.     

Physicochemical characterization of nanotemplate engineered solid lipid nanoparticles

As the physicochemical characteristics of solid lipid nanoparticles (SLNs) play a critical role in their success, it is important to understand how the materials and process used in their preparation affect these properties. In this study, two stearyl alcohol-based formulations were prepared using nanotemplate engineering technology and characterized. Both formulations were of a small particle size (<100 nm), ellipsoidal shape, and low polydispersity. (1)H NMR spectroscopy confirmed that the SLNs have the expected solid core structure and PEGylated surface. Analysis of the bulk materials indicated that a number of complex interactions are present among the SLN components, including a eutectic between stearyl alcohol and Brij 78. The decreased crystallinity resulting from these interactions may allow for enhanced drug loading. Physiological stability was identified and confirmed as a potential problem due to the low melting point of the eutectic. However, it is expected that with appropriate formulation modifications nanotemplate engineered SLNs will possess the properties necessary for a successful drug delivery system.     

Dynamics of colloidal particles in ice

We use x-ray photon correlation spectroscopy (XPCS) to probe the dynamics of colloidal particles in polycrystalline ice. During freezing, the dendritic ice morphology and rejection of particles from the ice created regions of high particle density, where some of the colloids were forced into contact and formed disordered aggregates. The particles in these high density regions underwent ballistic motion, with a characteristic velocity that increased with temperature. This ballistic motion is coupled with both stretched and compressed exponential decays of the intensity autocorrelation function. We suggest that this behavior could result from ice grain boundary migration.     

Pseudoceramines A-D, new antibacterial bromotyrosine alkaloids from the marine sponge Pseudoceratina sp

Bioassay-guided fractionation of the CH(2)Cl(2)/MeOH extract of the Australian marine sponge Pseudoceratina sp. resulted in the purification of four new bromotyrosine alkaloids, pseudoceramines A-D (1-4), along with a known natural product, spermatinamine (5). The structures of 1-5 were determined by spectroscopic methods. Pseudoceramines A (1) and B (2) feature a rare bromotyrosyl-spermine-bromotyrosyl sequence, and pseudoceramine C (3) is the first example of bromotyrosine coupled with an N-methyl derivative of spermidine. Compounds 1-5 were screened for inhibition of toxin secretion by the type III secretion (T3S) pathway in Yersinia pseudotuberculosis. Compounds 2 and 5 inhibited secretion of the Yersinia outer protein YopE (IC(50) = 19 and 6 μM, respectively) and the enzyme activity of YopH (IC(50) = 33 and 6 μM, respectively).     

Fast nuclear spin conversion in water clusters and ices: a matrix isolation study

Single water molecules have been isolated in solid Ar matrices at 4 K and studied by rovibrational spectroscopy using FTIR in the regions of the ν(1), ν(2), and ν(3) modes. Upon nuclear spin conversion at 4 K, essentially pure para-H(2)O was prepared, followed by subsequent fast annealing generating ice particles. FTIR studies of the vapor above the condensed water upon annealing to T ≥ 250 K indicate fast reconversion of nuclear spin to equilibrium conditions. Our results indicate that nuclear spin conversion is fast in water dimers and larger clusters, which preclude preparation of concentrated samples of para-H(2)O, such as in ice or vapor.     

Mutually orthogonal equitable Latin rectangles

Let ab=n². We define an equitable Latin rectangle as an a×b matrix on a set of n symbols where each symbol appears either or times in each row of the matrix and either or times in each column of the matrix. Two equitable Latin rectangles are orthogonal in the usual way. Denote a set of ka×b mutually orthogonal equitable Latin rectangles as a k– MOELR (a,b;n). When a≠9,18,36, or 100, then we show that the maximum number of k– MOELR (a,b;n)≥3 for all possible values of (a,b).     

The coherent-constructible correspondence and Fourier-Mukai transforms

As evidence for his conjecture in birational log geometry, Kawamata constructed a family of derived equivalences between toric orbifolds. In a previous paper, the authors showed that the derived category of a toric orbifold is naturally identified with a category of polyhedrally-constructible sheaves on ℝ ⁿ . In this paper we investigate and reprove some of Kawamata’s results from this perspective.     

5-(Indol-2-yl)pyrazolo[3,4-b]pyridines as a New Family of TASK-3 Channel Blockers: A Pharmacophore-Based Regioselective Synthesis

TASK channels belong to the two-pore-domain potassium (K_2P) channels subfamily. These channels modulate cellular excitability, input resistance, and response to synaptic stimulation. TASK-channel inhibition led to membrane depolarization. TASK-3 is expressed in different cancer cell types and neurons. Thus, the discovery of novel TASK-3 inhibitors makes these bioactive compounds very appealing to explore new cancer and neurological therapies. TASK-3 channel blockers are very limited to date, and only a few heterofused compounds have been reported in the literature. In this article, we combined a pharmacophore hypothesis with molecular docking to address for the first time the rational design, synthesis, and evaluation of 5-(indol-2-yl)pyrazolo[3,4-b]pyridines as a novel family of human TASK-3 channel blockers. Representative compounds of the synthesized library were assessed against TASK-3 using Fluorometric imaging plate reader—Membrane Potential assay (FMP). Inhibitory properties were validated using two-electrode voltage-clamp (TEVC) methods. We identified one active hit compound (MM-3b) with our systematic pipeline, exhibiting an IC₅₀ ≈ 30 μM. Molecular docking models suggest that compound MM-3b binds to TASK-3 at the bottom of the selectivity filter in the central cavity, similar to other described TASK-3 blockers such as A1899 and PK-THPP. Our in silico and experimental studies provide a new tool to predict and design novel TASK-3 channel blockers.     

Extension of the 2-p-opt and 1-shift algorithms to the heterogeneous probabilistic traveling salesman problem

The probabilistic traveling salesman problem is a well known problem that is quite challenging to solve. It involves finding the tour with the lowest expected cost for customers that will require a visit with a given probability. There are several proposed algorithms for the homogeneous version of the problem, where all customers have identical probability of being realized. From the literature, the most successful approaches involve local search procedures, with the most famous being the 2-p-opt and 1-shift procedures proposed by Bertsimas [D.J. Bertsimas, L. Howell, Further results on the probabilistic traveling salesman problem, European Journal of Operational Research 65 (1) (1993) 68–95]. Recently, however, evidence has emerged that indicates the equations offered for these procedures are not correct, and even when corrected, the translation to the heterogeneous version of the problem is not simple. In this paper we extend the analysis and correction to the heterogeneous case. We derive new expressions for computing the cost of 2-p-opt and 1-shift local search moves, and we show that the neighborhood of a solution may be explored in O(n²) time, the same as for the homogeneous case, instead of O(n³) as first reported in the literature.     

Searching for the Center on the Mathematics-Science Continuum

The history of mathematics and science integration in American schools can be illustrated through the use of a continuum which runs from math for math's sake at one end to science for science's sake at the other. True integration occurs at the center point. While published examples of integration focusing on process skills are common, those featuring integration of content are less often found. Two such lessons, developed around radioactive decay and efficiency in nature, are presented as examples of science and mathematics concepts taught in concert. Changes in preservice and in-service teacher training must occur if the potential for this type of integration is to be realized.