Protein digestion and phosphopeptide enrichment on a glass microchip

This work describes an integrated glass microdevice for proteomics, which directly couples proteolysis with affinity selection. Initial results with standard phosphopeptide fragments from β-casein in peptide mixtures showed selective capture of the phosphorylated fragments using immobilized metal affinity chromatography (IMAC) beads packed into a microchannel. Complete selectivity was seen with angiotensin, with capture of only the phosphorylated form. On-chip proteolysis, using immobilized trypsin beads packed into a separate channel, was directly coupled to the phosphopeptide capture and the integrated devices evaluated using β-casein. Captured and eluted fragments were analyzed using both capillary electrophoresis (CE) and capillary liquid chromatography/mass spectrometry (cLC/MS). The results show selective capture of only phosphopeptide fragments, but incomplete digestion of the protein was apparent from multiple peaks in the CE separations. The MS analysis indicated a capture bias on the IMAC column for the tetraphosphorylated peptide fragment over the monophosphorylated fragment. Application to digestion and capture of a serum fraction showed capture of material; however, non-specific binding was evident. Additional work will be required to fully optimize this system, but this work represents a novel sample preparation method, incorporating protein digestion on-line with affinity capture for proteomic applications.     

Microfluidic multi-analyte gradient generator

A microfluidic device was developed to produce temporal concentration gradients of multiple analytes. Four on-chip pumps delivered pulses of three analytes and buffer to a 14-cm channel where the pulses were mixed to homogeneity. The final concentration of each analyte was dependent on the temporal density of the pulses from each pump. The concentration of each analyte was varied by changing the number of pump cycles from each reservoir while maintaining the total number of pump cycles per unit time to ensure a constant total flow rate in the device. To gauge the independent nature of each pump, sinusoidal waves of fluorescein concentration were produced from each pump with independent frequencies and amplitudes. The resulting fluorescence intensity was compared with a theoretical summation of the waves and the experimental data matched the theoretical waves within 1%, indicating that the pumps were operating independently and outputting the correct frequency and amplitude. The device was used to demonstrate the role of adenosine triphosphate-sensitive K⁺ channels in glucose-stimulated increases in intracellular [Ca²⁺] in islets of Langerhans. Perfusion of single islets of Langerhans with combinations of glucose, diazoxide, and K⁺ resulted in intracellular Ca²⁺ patterns similar to what has been observed using conventional perfusion devices. The system will be useful in other studies with islets of Langerhans, as well as other assays that require the modulation of multiple analytes in time.     

Polyethylene glycol matrix reduces the rates of photochemical and thermal release of nitric oxide from S-nitroso-N-acetylcysteine

S -nitrosothiols have many biological activities and may act as nitric oxide (NO) carriers and donors, prolonging NO half-life in vivo. In spite of their great potential as therapeutic agents, most S -nitrosothiols are too unstable to isolate. We have shown that the S -nitroso adduct of N -acetylcysteine (SNAC) can be synthesized directly in aqueous and polyethylene glycol (PEG) 400 matrix by using a reactive gaseous (NO/O₂) mixture. Spectral monitoring of the S–N bond cleavage showed that SNAC, synthesized by this method, is relatively stable in nonbuf-fered aqueous solution at 25°C in the dark and that its stability is greatly increased in PEG matrix, resulting in a 28-fold decrease in its initial rate of thermal decomposition. Irradiation with UV light (λ= 333 nm) accelerated the rate of decomposition of SNAC to NO in both matrices, indicating that SNAC may find use for the photogeneration of NO. The quantum yield for SNAC decomposition decreased from 0.65 ± 0.15 in aqueous solution to 0.047 ± 0.005 in PEG 400 matrix. This increased stability in PEG matrix was assigned to a cage effect promoted by the PEG microenvironment that increases the rate of geminated radical pair recombination in the homolytic S–N bond cleavage process. This effect allowed for the storage of SNAC in PEG at −20°C in the dark for more than 10 weeks with negligible decomposition. Such stabilization may represent a viable option for the synthesis, storage and handling of S -nitrosothiol solutions for biomedical applications.     

Novel fluorogenic calix[4]arene-bis(crown-6-ether) for selective recognition of thallium(I)

A new fluorogenic, dansyl group-containing derivative of 1,3-alternate calix[4]arene-bis(crown-6-ether) provides optical recognition of Tl+ with selectivity over many other metal cations, including Na+, K+, Ca2+, Ag+, Hg2+ and Pb2+, and embodies the first example of a calixarene-based fluorescent Tl+-chemosensor.     

Inherent Ethyl Acetate Selectivity in a Trianglimine Molecular Solid

Ethyl acetate is an important chemical raw material and solvent. It is also a key volatile organic compound in the brewing industry and a marker for lung cancer. Materials that are highly selective toward ethyl acetate are needed for its separation and detection. Here, we report a trianglimine macrocycle ( TAMC ) that selectively adsorbs ethyl acetate by forming a solvate. Crystal structure prediction showed this to be the lowest energy solvate structure available. This solvate leaves a metastable, “templated” cavity after solvent removal. Adsorption and breakthrough experiments confirmed that TAMC has adequate adsorption kinetics to separate ethyl acetate from azeotropic mixtures with ethanol, which is a challenging and energy-intensive industrial separation.     

Scheduling UET task systems with concurrency on two parallel identical processors

Problems with unit execution time tasks and two identical parallel processors have received a great deal of attention in scheduling theory. In contrast to the conventional models, where each task requires only one processor, we consider a situation when a task may require both processors simultaneously. For problems without precedence constraints we present several polynomial time algorithms which complement recent results of Lee and Cai. We also show that the introduction of precedence constraints leads to NP-hardness results for maximum lateness and mean flow time objective functions. For the maximum lateness problem, a family of algorithms, based upon the idea of modified due dates, is considered. The worst case behaviour of these algorithms is analysed, and it is shown that the same upper bound is tight for each algorithm of this family.     

Unsteady axisymmetric stagnation flow on a circular cylinder

Unsteady, axisymmetric stagnation flow about a circular cylinderis examined when the far-field flow is a periodic function oftime with a fixed time average and an oscillatory part of prescribedamplitude and frequency. Solutions are computed for arbitraryvalues of the Reynolds number, quantifying the effects of surfacecurvature, and a frequency parameter based on the period ofthe far-field flow. It is found that solutions remain regularand periodic provided that the far-field amplitude lies belowa critical value. Above this value, solutions terminate in afinite-time singularity. The blow-up time is delayed by increasingthe curvature of the surface. These results are corroboratedby asymptotic predictions valid in the limits of small and largeamplitude and frequency. For large Reynolds number, the problemreduces to the two-dimensional stagnation-point flow againsta plane wall studied by previous authors.