Quantitative proteomic analysis and functional studies reveal that nucleophosmin is involved in cell death in glioblastoma cell line transfected with siRNA

Previously, we reported that nucleophosmin (NPM) was increased in glioblastoma multiforme (GBM). NPM is a phosphoprotein related to apoptosis, ribosome biogenesis, mitosis, and DNA repair, but details about its function remain unclear. We treated U87MG and A172 cells with small interference RNA (siRNA) and obtained a reduction of 80% in NPM1 expression. Knockdown at the protein level was evident after the 4th day and was maintained until the 7th day of transfection that was investigated by quantitative proteomic analysis using isobaric tags. The comparison of proteomic analysis of NPM1-siRNA against controls allowed the identification of 14 proteins, two proteins showed increase and 12 presented a reduction of expression levels. Gene ontology assigned most of the hypoexpressed proteins to apoptosis regulation, including GRP78. NPM1 silencing did not impair cell proliferation until the 7th day after transfection, but sensitized U87MG cells to temozolomide (TMZ), culminating with an increase in cell death and provoking at a later period a reduction of colony formation. In a large data set of GBM patients, both GRP78 and NPM1 genes were upregulated and presented a tendency to shorter overall survival time. In conclusion, NPM proved to participate in the apoptotic process, sensitizing TMZ-treated U87MG and A172 cells to cell death, and in association with upregulation of GRP78 may be helpful as a predictive factor of poor prognosis in GBM patients.

Natural intracellular peptides can modulate the interactions of mouse brain proteins and thimet oligopeptidase with 14-3-3e and calmodulin

Protein interactions are crucial for most cellular process. Thus, rationally designed peptides that act as competitive assembly inhibitors of protein interactions by mimicking specific, determined structural elements have been extensively used in clinical and basic research. Recently, mammalian cells have been shown to contain a large number of intracellular peptides of unknown function. Here, we investigate the role of several of these natural intracellular peptides as putative modulators of protein interactions that are related to Ca2+-calmodulin (CaM) and 14-3-3 epsilon, which are proteins that are related to the spatial organization of signal transduction within cells. At concentrations of 1-50 mu M, most of the peptides that are investigated in this study modulate the interactions of CaM and 14-3-3 epsilon with proteins from the mouse brain cytoplasm or recombinant thimet oligopeptidase (EP24.15) in vitro, as measured by surface plasmon resonance. One of these peptides (VFDVELL; VFD-7) increases the cytosolic Ca2+ concentration in a dose-dependent manner but only if introduced into HEK293 cells, which suggests a wide biological function of this peptide. Therefore, it is exciting to suggest that natural intracellular peptides are novel modulators of protein interactions and have biological functions within cells.

Automatic design of decision-tree induction algorithms tailored to flexible-receptor docking data

Background: This paper addresses the prediction of the free energy of binding of a drug candidate with enzyme InhA associated with Mycobacterium tuberculosis. This problem is found within rational drug design, where interactions between drug candidates and target proteins are verified through molecular docking simulations. In this application, it is important not only to correctly predict the free energy of binding, but also to provide a comprehensible model that could be validated by a domain specialist. Decision-tree induction algorithms have been successfully used in drug-design related applications, specially considering that decision trees are simple to understand, interpret, and validate. There are several decision-tree induction algorithms available for general-use, but each one has a bias that makes it more suitable for a particular data distribution. In this article, we propose and investigate the automatic design of decision-tree induction algorithms tailored to particular drug-enzyme binding data sets. We investigate the performance of our new method for evaluating binding conformations of different drug candidates to InhA, and we analyze our findings with respect to decision tree accuracy, comprehensibility, and biological relevance. Results: The empirical analysis indicates that our method is capable of automatically generating decision-tree induction algorithms that significantly outperform the traditional C4.5 algorithm with respect to both accuracy and comprehensibility. In addition, we provide the biological interpretation of the rules generated by our approach, reinforcing the importance of comprehensible predictive models in this particular bioinformatics application. Conclusions: We conclude that automatically designing a decision-tree algorithm tailored to molecular docking data is a promising alternative for the prediction of the free energy from the binding of a drug candidate with a flexible-receptor.

Unmanned platform for long-range remote analysis of volatile compounds in air samples

This paper describes a long-range remotely controlled CE system built on an all-terrain vehicle. A four-stroke engine and a set of 12-V batteries were used to provide power to a series of subsystems that include drivers, communication, computers, and a capillary electrophoresis module. This dedicated instrument allows air sampling using a polypropylene porous tube, coupled to a flow system that transports the sample to the inlet of a fused-silica capillary. A hybrid approach was used for the construction of the analytical subsystem combining a conventional fused-silica capillary (used for separation) and a laser machined microfluidic block, made of PMMA. A solid-state cooling approach was also integrated in the CE module to enable controlling the temperature and therefore increasing the useful range of the robot. Although ultimately intended for detection of chemical warfare agents, the proposed system was used to analyze a series of volatile organic acids. As such, the system allowed the separation and detection of formic, acetic, and propionic acids with signal-to-noise ratios of 414, 150, and 115, respectively, after sampling by only 30 s and performing an electrokinetic injection during 2.0 s at 1.0 kV.

SIS: a program to generate draft genome sequence scaffolds for prokaryotes

Background: Decreasing costs of DNA sequencing have made prokaryotic draft genome sequences increasingly common. A contig scaffold is an ordering of contigs in the correct orientation. A scaffold can help genome comparisons and guide gap closure efforts. One popular technique for obtaining contig scaffolds is to map contigs onto a reference genome. However, rearrangements that may exist between the query and reference genomes may result in incorrect scaffolds, if these rearrangements are not taken into account. Large-scale inversions are common rearrangement events in prokaryotic genomes. Even in draft genomes it is possible to detect the presence of inversions given sufficient sequencing coverage and a sufficiently close reference genome. Results: We present a linear-time algorithm that can generate a set of contig scaffolds for a draft genome sequence represented in contigs given a reference genome. The algorithm is aimed at prokaryotic genomes and relies on the presence of matching sequence patterns between the query and reference genomes that can be interpreted as the result of large-scale inversions; we call these patterns inversion signatures. Our algorithm is capable of correctly generating a scaffold if at least one member of every inversion signature pair is present in contigs and no inversion signatures have been overwritten in evolution. The algorithm is also capable of generating scaffolds in the presence of any kind of inversion, even though in this general case there is no guarantee that all scaffolds in the scaffold set will be correct. We compare the performance of SIS, the program that implements the algorithm, to seven other scaffold-generating programs. The results of our tests show that SIS has overall better performance. Conclusions: SIS is a new easy-to-use tool to generate contig scaffolds, available both as stand-alone and as a web server. The good performance of SIS in our tests adds evidence that large-scale inversions are widespread in prokaryotic genomes.

On the formation of carbonate adducts of fatty alcohols, sterols, and sugars in biological conditions

The formation and properties of carbonate adducts of some organic hydroxy compounds in aqueous medium were investigated. Fatty alcohols and sugars were chosen as representative classes of biological interest, and the medium was carbonated aqueous solution with pH ranging from 3.0 to 8.3. Capillary electrophoresis with two capacitively coupled contactless conductivity detectors (C4Ds) was used for quantitation and to obtain the mobility of the monoalkyl carbonates (MACs), which were used to determine the equilibrium and kinetic constants of the reaction as well as the diffusion coefficients. For increasing chain length of the alcohols, the equilibrium constant tends to the unit, which suggests that fatty alcohols can form the corresponding MACs. The formation of MACs for cyclohexanol and cyclopentanol also suggest the existence of similar species for sterols. Carbonate adducts of fructose, glucose, and sucrose were also detected, which suggests that these counterparts of the well-known phosphates can also occur in the cytosol. Our calculations suggest that one in 1000 to one in 10 000 molecules of these hydroxy compounds would be available as the corresponding MAC in such a medium. Experiments carried out at pH values less than 3.0 showed that there is a catalytic effect of hydronium on the interconversion of bicarbonate and a MAC. Taking into account the great number of hydroxy compounds similar to the ones investigated and that bicarbonate is ubiquitous in living cells, one can anticipate the existence of a whole new class of carbonate adducts of these metabolites.

Deconvolution of the EPR spectra of vanadium oxide nanotubes

In this work we report results of continuous wave (CW) electron paramagnetic resonance (EPR) spectroscopy of vanadium oxide nanotubes. The observed EPR spectra are composed of a weak well-resolved spectrum of isolated V4+ ions on top of an intense and broad structure-less line shape, attributed to spin-spin exchanged V4+ clusters. With the purpose to deconvolute the structured weak spectrum from the composed broad line, a new approach based on the Krylov basis diagonalization method (KBDM) is introduced. It is based on the discrimination between broad and sharp components with respect to a selectable threshold and can be executed with few adjustable parameters, without the need of a priori information on the shape and structure of the lines. This makes the method advantageous with respect to other procedures and suitable for fast and routine spectral analysis, which, in conjunction with simulation techniques based on the spin Hamiltonian parameters, can provide a full characterization of the EPR spectrum. Results demonstrate and characterize the coexistence of two V4+ species in the nanotubes and show good progress toward the goal of obtaining high fidelity deconvoluted spectra from complex signals with overlapping broader line shapes. (C) 2012 Elsevier Inc. All rights reserved.

Crystallization and preliminary X-ray diffraction analysis of endoglucanase III from Trichoderma harzianum

Endoglucanases are enzymes that hydrolyze cellulose and are important components of the cellulolytic complex. In contrast to other members of the complex, they cleave internal beta-1,4-glycosidic bonds in the cellulose polymer, allowing cellulose to be used as an energy source. Since biomass is an important renewable source of energy, the structural and functional characterization of these enzymes is of interest. In this study, endoglucanase III from Trichoderma harzianum was produced in Pichia pastoris and purified. Crystals belonging to the orthorhombic space group P212121, with unit-cell parameters a = 47.54, b = 55.57, c = 157.3 angstrom, were obtained by the sitting-drop vapour-diffusion method and an X-ray diffraction data set was collected to 2.07 angstrom resolution.

The contribution of a murine CNS-TB model for the understanding of the host-pathogen interactions in the formation of granulomas

Central nervous system (CNS) tuberculosis (TB) is the most severe form of TB, characterized morphologically by brain granulomas and tuberculous meningitis (TBM). Experimental strategies for the study of the host-pathogen interaction through the analysis of granulomas and its intrinsic molecular mechanisms could provide new insights into the neuropathology of TB. To verify whether cerebellar mycobacterial infection induces the main features of the disease in human CNS and better understand the physiological mechanisms underlying the disease, we injected bacillus Calmette-Guerin (BCG) into the mouse cerebellum. BCG-induced CNS-TB is characterized by the formation of granulomas and TBM, a build up of bacterial loads in these lesions, and microglial recruitment into the lesion sites. In addition, there is an enhanced expression of signaling molecules such as nuclear factor-kappa B (NF-kappa B) and there is a presence of inducible nitric oxide synthase (iNOS) in the lesions and surrounding areas. This murine model of cerebellar CNS-TB was characterized by cellular and biochemical immune responses typically found in the human disease. This model could expand our knowledge about granulomas in TB infection of the cerebellum, and help characterize the physiological mechanisms involved with the progression of this serious illness that is responsible for killing millions people every year. (C) 2012 Elsevier B.V. All rights reserved.

CE-ESI-MS metabolic fingerprinting of Leishmania resistance to antimony treatment

Metabolomics has become an invaluable tool to unveil biology of pathogens, with immediate application to chemotherapy. It is currently accepted that there is not one single technique capable of obtaining the whole metabolic fingerprint of a biological system either due to their different physical-chemical properties or concentrations. In this work, we have explored the capability of capillary electrophoresis mass spectrometry with a sheathless interface with electrospray ionization (CE-ESI-TOF-MS) to separate metabolites in order to be used as a complementary technique to LC. As proof of concept, we have compared the metabolome of Leishmania infantum promastigotes BCN 150 (Sb (III) IC50 = 20.9 mu M) and its variation when treated with 120 mu M of Sb(III) potassium tartrate for 12 h, as well as with its Sb(III) resistant counterpart obtained by growth of the parasites under increasing Sb(III) in a step-wise manner up to 180 mu M. The number of metabolites compared were of 264 for BCN150 Sb(III) treated versus nontreated and of 195 for Sb(III) resistant versus susceptible parasites. After successive data filtering, differences in seven metabolites identified in databases for Leishmania pathways, showed the highest significant differences, corresponding mainly to amino acids or their metabolite surrogates. Most of them were assigned to sulfur containing amino acids and polyamine biosynthetic pathways, of special relevance considering the deterioration of the thiol-dependent redox metabolism in Leishmania by Sb(III). Given the low concentrations typical for most of these metabolites, the assay can be considered a success that should be explored for new biological questions.

Venomics Profiling of Thamnodynastes strigatus Unveils Matrix Metalloproteinases and Other Novel Proteins Recruited to the Toxin Arsenal of Rear-Fanged Snakes

Rear-fanged and aglyphous snakes are usually considered not dangerous to humans because of their limited capacity of injecting venom. Therefore, only a few studies have been dedicated to characterizing the venom of the largest parcel of snake fauna. Here, we investigated the venom proteome of the rear-fanged snake Thamnodynastes strigatus, in combination with a transcriptomic evaluation of the venom gland. About 60% of all transcripts code for putative venom components. A striking finding is that the most abundant type of transcript (similar to 47%) and also the major protein type in the venom correspond to a new kind of matrix metalloproteinase (MMP) that is unrelated to the classical snake venom metalloproteinases found in all snake families. These enzymes were recently suggested as possible venom components, and we show here that they are proteolytically active and probably recruited to venom from a MMP-9 ancestor. Other unusual proteins were suggested to be venom components: a protein related to lactadherin and an EGF repeat-containing transcript. Despite these unusual molecules, seven toxin classes commonly found in typical venomous snakes are also present in the venom. These results support the evidence that the arsenals of these snakes are very diverse and harbor new types of biologically important molecules.

Capillary bioreactors based on human purine nucleoside phosphorylase: A new approach for ligands identification and characterization

The enzyme purine nucleoside phosphorylase (PNP) is a target for the discovery of new lead compounds employed on the treatment severe T-cell mediated disorders. Within this context, the development of new, direct, and reliable methods for ligands screening is an important task. This paper describes the preparation of fused silica capillaries human PNP (HsPNP) immobilized enzyme reactor (IMER). The activity of the obtained IMER is monitored on line in a multidimensional liquid chromatography system, by the quantification of the product formed throughout the enzymatic reaction. The Km value for the immobilized enzyme was about twofold higher than that measured for the enzyme in solution (255 +/- 29.2 mu M and 133 +/- 114.9 mu M, respectively). A new fourth-generation immucillin derivative (DI4G: IC50 = 40.6 +/- 0.36 nM), previously identified and characterized in HsPNP free enzyme assays, was used to validate the IMER as a screening method for HsPNP ligands. The validated method was also used for mechanistic studies with this inhibitor. This new approach is a valuable tool to PNP ligand screening, since it directly measures the hypoxanthine released by inosine phosphorolysis, thus furnishing more reliable results than those one used in a coupled enzymatic spectrophotometric assay. (C) 2011 Elsevier B.V. All rights reserved.

Simultaneous determination of dextromethorphan, dextrorphan and doxylamine in human plasma by HPLC coupled to electrospray ionization tandem mass spectrometry: Application to a pharmacokinetic study

In the present study, a fast, sensitive and robust method to quantify dextromethorphan, dextrorphan and doxylamine in human plasma using deuterated internal standards (IS) is described. The analytes and the IS were extracted from plasma by a liquid-liquid extraction (LLE) using diethyl-ether/hexane (80/20, v/v). Extracted samples were analyzed by high performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Chromatographic separation was performed by pumping the mobile phase (acetonitrile/water/formic acid (90/9/1, v/v/v) during 4.0 min at a flow-rate of 1.5 mL min(-1) into a Phenomenex Gemini (R) C18, 5 mu m analytical column (150 x 4.6 mm id.). The calibration curve was linear over the range from 0.2 to 200 ng mL(-1) for dextromethorphan and doxylamine and 0.05 to 10 ng mL(-1) for dextrorphan. The intra-batch precision and accuracy (%CV) of the method ranged from 2.5 to 9.5%, and 88.9 to 105.1%, respectively. Method inter-batch precision (%CV) and accuracy ranged from 6.7 to 10.3%, and 92.2 to 107.1%, respectively. The run-time was for 4 min. The analytical procedure herein described was used to assess the pharmacokinetics of dextromethorphan, dextrorphan and doxylamine in healthy volunteers after a single oral dose of a formulation containing 30 mg of dextromethorphan hydrobromide and 12.5 mg of doxylamine succinate. The method has high sensitivity, specificity and allows high throughput analysis required for a pharmacokinetic study. (C) 2012 Elsevier B.V. All rights reserved.

End-to-end Distance Distribution in Fluorescent Derivatives of Bradykinin in Interaction with Lipid Vesicles

Cellular membranes have relevant roles in processes related to proteases like human kallikreins and cathepsins. As enzyme and substrate may interact with cell membranes and associated co-factors, it is important to take into account the behavior of peptide substrates in the lipid environment. In this paper we report an study based on energy transfer in two bradykinin derived peptides labeled with the donor-acceptor pair Abz/Eddnp (ortho-aminobenzoic acid/N-[2,4-dinitrophenyl]-ethylenediamine). Time-resolved fluorescence experiments were performed in phosphate buffer and in the presence of large unilamelar vesicles of phospholipids, and of micelles of sodium dodecyl sulphate (SDS). The decay kinetics were analyzed using the program CONTIN to obtain end-to-end distance distribution functions f(r). Despite of the large difference in the number of residues the end-to-end distance of the longer peptide (9 amino acid residues) is only 20 % larger than the values obtained for the shorter peptide (5 amino acid residues). The proline residue, in position 4 of the bradykinin sequence promotes a turn in the longer peptide chain, shortening its end-to-end distance. The surfactant SDS has a strong disorganizing effect, substantially broadening the distance distributions, while temperature increase has mild effects in the flexibility of the chains, causing small increase in the distribution width. The interaction with phospholipid vesicles stabilizes more compact conformations, decreasing end-to-end distances in the peptides. Anisotropy experiments showed that rotational diffusion was not severely affected by the interaction with the vesicles, suggesting a location for the peptides in the surface region of the bilayer, a result consistent with small effect of lipid phase transition on the peptides conformations.

Differential expression of CD90 and CD14 stem cell markers in malignant breast cancer cell lines

The recently emerged concept of cancer stem cell (CSC) has led to a new hypothesis on the basis for tumor progression. Basically, the CSC theory hypothesizes the presence of a hierarchically organized and relatively rare cell population, which is responsible for tumor initiation, self-renewal, and maintenance, in addition to accumulation of mutation and resistance to chemotherapy. CSCs have recently been described in breast cancer. Different genetic markers have been used to isolate breast CSCs, none of which have been correlated with the tumorigenicity or metastatic potential of the cells, limiting their precise characterization and clinical application in the development of therapeutic protocols. Here, we sought for subpopulations of CSCs by analyzing 10 judiciously chosen stem cell markers in a normal breast cell line (MCF10-A) and in four human breast cancer cell lines (MCF-7, MDA-MB-231, MDA-MB-435, and Hs578-T) displaying different degrees of metastatic and invasiveness potential. We were able to identify two markers, which are differentially expressed in nontumorigenic versus tumor cells. The CD90 marker was highly expressed in the malignant cell lines. Interestingly, the CD14 molecule displayed higher expression levels in the nontumorigenic cell line. Therefore, we demonstrated that these two markers, which are more commonly used to isolate and characterize stem cells, are differentially expressed in breast tumor cells, when compared with nontumorigenic breast cells. (C) 2012 International Society for Advancement of Cytometry