Open tubular immobilized metal ion affinity chromatography combined with MALDI MS and MS/MS for identification of protein phosphorylation sites

Protein phosphorylation is one of the most important known posttranslational modifications. Tandem mass spectrometry has become an important tool for mapping out the phosphorylation sites. However, when a peptide generated from the enzymatic or chemical digestion of a phosphoprotein is highly phosphorylated or contains many potential phosphorylation residues, phosphorylation site assignment becomes difficult. Separation and enrichment of phosphopeptides from a digest mixture is desirable and often a critical step for MS/MS-based site determination. In this work, we present a novel open tubular immobilized metal ion affinity chromatography (OT-IMAC) method, which is found to be more effective and reproducible for phosphopeptide enrichment, compared to a commonly used commercial product, Ziptip from Millipore. A strategy based on a combination of OT-IMAC, sequential dual-enzyme digestion, and matrix-assisted laser desorption/ionization (MALDI) quadrupole time-of-flight tandem mass spectrometry for phosphoprotein characterization is presented. It is shown that MALDI MS/MS with collision-induced dissociation can be very effective in generating fragment ion spectra containing rich structural information, which enables the identification of phosphorylation sites even from highly phosphorylated peptides. The applicability of this method for real world applications is demonstrated in the characterization and identification of phosphorylation sites of a Na(+)/H(+) exchanger fusion protein, His182, which was phosphorylated in vitro using the kinase Erk2.     

Estimation of scattering error in spectrophotometric measurements of light absorption by aquatic particles from three-dimensional radiative transfer simulations

We present an approach based on three-dimensional Monte Carlo radiative transfer simulations for estimating scattering error in measurements of light absorption by aquatic particles with a typical laboratory double-beam spectrophotometer. The scattering error is calculated by combining the weighting function describing the angular distribution of photon losses that are due to scattering on suspended particles with the volume scattering function of particles. We applied this method to absorption measurements made on marine phytoplankton, a diatom Thalassiosira pseudonana and a cyanobacterium Synechococcus. Assuming that the scattering phase function is described by the Henyey-Greenstein formula, we determined the backscatter probability of phytoplankton, which yields the best correction for scattering error at a light wavelength of 750 nm, where true absorption is null. The backscattering ratio estimated for both phytoplankton species is significantly higher than previously reported data based on Mie-scattering calculations for homogeneous spheres. Depending on the type of particles, the corrected absorption spectra obtained with our method may be similar or significantly different from spectra obtained with the null-point correction based on wavelength-independent scattering error.     

Using the primal-dual interior point algorithm within the branch-price-and-cut method

Branch-price-and-cut has proven to be a powerful method for solving integer programming problems. It combines decomposition techniques with the generation of both columns and valid inequalities and relies on strong bounds to guide the search in the branch-and-bound tree. In this paper, we present how to improve the performance of a branch-price-and-cut method by using the primal-dual interior point algorithm. We discuss in detail how to deal with the challenges of using the interior point algorithm with the core components of the branch-price-and-cut method. The effort to overcome the difficulties pays off in a number of advantageous features offered by the new approach. We present the computational results of solving well-known instances of the vehicle routing problem with time windows, a challenging integer programming problem. The results indicate that the proposed approach delivers the best overall performance when compared with a similar branch-price-and-cut method which is based on the simplex algorithm.     

The Role of Neurotrophins in Multiple Sclerosis��Pathological and Clinical Implications

Multiple sclerosis (MS) is a chronic inflammatory demyelinating and neurodegenerative disease of the central nervous system (CNS) with unknown etiology. It was recently suggested that autoimmunity, which had long been considered to be destructive in MS, might also play a protective role in the CNS of MS patients. Neurotrophins are polypeptides belonging to the neurotrophic factor family. While neurotrophins mediate cell survival and proliferation in the nervous system, they are also expressed within peripheral blood mononuclear cells fraction (PBMCs) of immunological system. In MS additional neurotrophic support from PBMCs might compensate relative neurotrophins deficiency in the damaged CNS tissue that needs to be repaired. Failure to produce the adequate neurotrophins concentrations might result in decreased protection of the CNS, consequently leading to increased atrophy, which is the main determinant of MS patients’ end-point disability. There are several lines of evidence, both from clinical research and animal models, suggesting that neurotrophins play a pivotal role in neuroprotective and neuroregenerative processes that are often defective in the course of MS. It seems that neuroprotective strategies might be used as potentially valuable add-on therapies, alongside traditional immunomodulatory treatment in multiple sclerosis.     

STRESSES IN VISCOELASTIC PLATE DRIED CONVECTIVELY

The deformations and drying-induced stresses in a saturated porous plate insulated on one side and dried convectively on the others were analysedPlate material is assumed to be elastic and viscoelastic. A rigorous solution of the problem is presented using Laplace transformations and the method of variable separation. The results are presented in the form of a series resulting from the solution of a Sturm-Liouville problem. The shrinkage stresses in the plate are caused by non-uniform distribution of the moisture content. The thermal stresses are not included as the consideration were confined to only constant rate drying period.     

Diminazene Aceturate Stabilizes Atherosclerotic Plaque and Attenuates Hepatic Steatosis in apoE-Knockout Mice by Influencing Macrophages Polarization and Taurine Biosynthesis

Atherosclerosis and nonalcoholic fatty liver disease are leading causes of morbidity and mortality in the Western countries. The renin–angiotensin system (RAS) with its two main opposing effectors, i.e., angiotensin II (Ang II) and Ang-(1–7), is widely recognized as a major regulator of cardiovascular function and body metabolic processes. Angiotensin-converting enzyme 2 (ACE2) by breaking-down Ang II forms Ang-(1–7) and thus favors Ang-(1–7) actions. Therefore, the aim of our study was to comprehensively evaluate the influence of prolonged treatment with ACE2 activator, diminazene aceturate (DIZE) on the development of atherosclerotic lesions and hepatic steatosis in apoE^−/− mice fed a high-fat diet (HFD). We have shown that DIZE stabilized atherosclerotic lesions and attenuated hepatic steatosis in apoE^−/− mice fed an HFD. Such effects were associated with decreased total macrophages content and increased α-smooth muscle actin levels in atherosclerotic plaques. Moreover, DIZE changed polarization of macrophages towards increased amount of anti-inflammatory M2 macrophages in the atherosclerotic lesions. Interestingly, the anti-steatotic action of DIZE in the liver was related to the elevated levels of HDL in the plasma, decreased levels of triglycerides, and increased biosynthesis and concentration of taurine in the liver of apoE^−/− mice. However, exact molecular mechanisms of both anti-atherosclerotic and anti-steatotic actions of DIZE require further investigations.     

Hydroxyalkylation of barbituric acid. III. Product analysis and reaction pathway

Polyetherols containing a thermally stable pyrimidine ring were obtained upon the reaction of hydroxymethyl derivatives of barbituric acid with an excess of ethylene or propylene oxide. The reaction was monitored by ¹H‐NMR and IR spectroscopy for the systems with variable starting molar ratios of reagents. We found that formaldehyde rearranged from N‐hydroxymethyl and oxymethylene bridges into the end of the polyetherol chain during the reaction. Simultaneously, the O‐hydroxymethyl groups underwent blocking by oxirane. The structures of the polyetherols was deduced on the basis of the course of the reaction and the analytical data. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

Large area synthesis of conical carbon nanotube arrays on graphite and tungsten foil substrates

Conical carbon nanotube (CCNT) arrays were synthesized over a large area of approximately 1 cm² or more on graphite and tungsten foil substrates. Experimental observations reveal that nucleation is caused by catalyst metal cluster in the initial stages, but the tapered morphology occurs due to the difference in the rates of vertical growth by attachment carbon atoms at edges of growing graphene sheets and radial growth with epitaxial nucleation of new graphene layers near bottom at the substrate. The above mechanism is supported through re-growth experiments on straight multi-walled nanotubes and growth kinetics data, which suggest a linear relationship between the growth rate and ratio of diameter to length (d/l) of CCNT.     

A parallel algorithm evaluating the reliability of a system with known minimal cuts (paths)

A system with known minimal cuts is considered. In order to compute its reliability a binary tree is constructed with nodes being assigned numerical values. The value of each non-leaf node is a linear combination of its child nodes' values. The values of leaf nodes can be computed in a very simple way. The value of the root node is the system's reliability. The presented method is a memory saving one. Moreover, it is possible to apply parallel computation to the nodes located on the same level, which may significantly reduce the computing time.