Parallel Fragments Research Articles

Desorption electrospray ionization mass spectrometric analysis of organophosphorus chemical warfare agents using ion mobility and tandem mass spectrometry

Desorption electrospray ionization mass spectrometry (DESI-MS) has been applied to the direct analysis of sample media for target chemicals, including chemical warfare agents (CWA), without the need for additional sample handling. During the present study, solid-phase microextraction (SPME) fibers were used to sample the headspace above five organophosphorus CWA, O-isopropyl methylphosphonofluoridate (sarin, GB), O-pinacolyl methylphosphonofluoridate (soman, GD), O-ethyl N,N-dimethyl phosphoramidocyanidate (tabun, GA), O-cyclohexyl methylphosphonofluoridate (cyclohexyl sarin, GF) and O-ethyl S-2-diisopropylaminoethyl methyl phosphonothiolate (VX) spiked into glass headspace sampling vials. Following sampling, the SPME fibers were introduced directly into a modified ESI source, enabling rapid and safe DESI of the toxic compounds. A SYNAPT HDMS instrument was used to acquire time-aligned parallel (TAP) fragmentation data, which provided both ion mobility and MS(n) (n = 2 or 3) data useful for the confirmation of CWA. Unique ion mobility profiles were acquired for each compound and characteristic product ions of the ion mobility separated ions were produced in the Triwave transfer collision region. Up to six full scanning MS(n) spectra, containing the [M + H](+) ion and up to seven diagnostic product ions, were acquired for each CWA during SPME fiber analysis. A rapid screening approach, based on the developed methodology, was applied to several typical forensic media, including Dacron sampling swabs spiked with 5 microg of CWA. Background interference was minimal and the spiked CWA were readily identified within one minute on the basis of the acquired ion mobility and mass spectrometric data.

Metal-Assisted Oxazolidine/Oxazine Ring Formation in Dinuclear Zinc(II) Complexes: Synthesis, Structural Aspects, and Bioactivity

Three novel dinuclear Zn(II) complexes of phenol-based compartmental macrocyclic ligands have been synthesized and characterized by routine physicochemical techniques as well as by X-ray single-crystal structure analysis. The dinuclear macrocyclic complexes 1, 2, and 3 were obtained through a 1:2 condensation reaction of 2,6-diformyl-4-methylphenol and N-(hydroxyalkyl) ethylenediamine (L(1), L(2), and L(3), respectively) in the presence of zinc(II) acetate, followed by the addition of thiocyanate anion [L(1) = N-(2-hydroxyethyl)ethylenediamine, L(2) = N-(3-hydroxypropyl)ethylenediamine, and L(3) = N-(2-hydroxypropyl)ethylenediamine]. The synthesized 18-membered macrocycles are noted to be structurally unique, and their formation proceeds with the generation of two oxazolidine side rings in complexes 1 and 3 and two oxazine side rings in 2, along with the creation of four new chiral centers in each case. Complexes 1 and 2 are characterized by a butterfly-like arrangement with the SCN ligands situated on the same side with respect to the Zn(2)O(2) moiety, whereas the centrosymmetric complex 3 exhibits a stepped arrangement with parallel methyl-phenoxy fragments (spaced at ca. 1.5 A) and trans located SCN ligands with respect to the Zn(2)O(2) core. The formation of these unusual macrocycles is considered to be zinc-mediated. Preliminary studies with the complexes show that all of them exhibit an inhibitory effect, on the cell proliferation of human stomach cancer cell line AGS, though with different degrees, where complex 3 shows the highest efficiency.

ParTopS: compact topological framework for parallel fragmentation simulations

Cohesive models are used for simulation of fracture, branching and fragmentation phenomena at various scales. Those models require high levels of mesh refinement at the crack tip region so that nonlinear behavior can be captured and physical results obtained. This imposes the use of large meshes that usually result in computational and memory costs prohibitively expensive for a single traditional workstation. If an extrinsic cohesive model is to be used, support for dynamic insertion of cohesive elements is also required. This paper proposes a topological framework for supporting parallel adaptive fragmentation simulations that provides operations for dynamic insertion of cohesive elements, in a uniform way, for both two- and three-dimensional unstructured meshes. Cohesive elements are truly represented and are treated like any other regular element. The framework is built as an extension of a compact adjacency-based serial topological data structure, which can natively handle the representation of cohesive elements. Symmetrical modifications of duplicated entities are used to reduce the communication of topological changes among mesh partitions and also to avoid the use of locks. The correctness and efficiency of the proposed framework are demonstrated by a series of arbitrary insertions of cohesive elements into some sample meshes.

Novel MMP-9 Substrates in Cancer Cells Revealed by a Label-free Quantitative Proteomics Approach

Matrix metalloproteinase-9 (MMP-9) is implicated in tumor metastasis as well as a variety of inflammatory and pathological processes. Although many substrates for MMP-9, including components of the extracellular matrix, soluble mediators such as chemokines, and cell surface molecules have been identified, we undertook a more comprehensive proteomics-based approach to identify new substrates to further understand how MMP-9 might contribute to tumor metastasis. Previous proteomics approaches to identify protease substrates have depended upon differential labeling of each sample. Instead we used a label-free quantitative proteomics approach based on ultraperformance LC-ESI-high/low collision energy MS. Conditioned medium from a human metastatic prostate cancer cell line, PC-3ML, in which MMP-9 had been down-regulated by RNA interference was compared with that from the parental cells. From more than 200 proteins identified, 69 showed significant alteration in levels after depletion of the protease (>+/-2-fold), suggesting that they might be candidate substrates. Levels of six of these (amyloid-beta precursor protein, collagen VI, leukemia inhibitory factor, neuropilin-1, prostate cancer cell-derived growth factor (PCDGF), and protease nexin-1 (PN-1)) were tested in the conditioned media by immunoblotting. There was a strong correlation between results by ultraperformance LC-ESI-high/low collision energy MS and by immunoblotting giving credence to the label-free approach. Further information about MMP-9 cleavage was obtained by comparison of the peptide coverage of collagen VI in the presence and absence of MMP-9 showing increased sensitivity of the C- and N-terminal globular regions over the helical regions. Susceptibility of PN-1 and leukemia inhibitory factor to MMP-9 degradation was confirmed by in vitro incubation of the recombinant proteins with recombinant MMP-9. The MMP-9 cleavage sites in PN-1 were sequenced. This study provides a new label-free method for degradomics cell-based screening leading to the identification of a series of proteins whose levels are affected by MMP-9, some of which are clearly direct substrates for MMP-9 and become candidates for involvement in metastasis.

On Reading Maupassant's Le Horla Problematologically

My intention in this article is to interpret Guy de Maupassant's Le Horla through the prism of problematology. I do so because I would like to show how and why the thought of Michel Meyer urgently matters to literary criticism, how and why problematology could possibly offer a way out of the current impasse in which criticism vacillates between hermeneutical indeterminacy and ideological over-determinacy, when it is not simply the glib performance of juxtaposing various parallel fragments woven together with a vague argument. Since Meyer has himself written extensively about the application of problematology to literary analysis and the place of problematology vis-a-vis hermeneutics, recep tion theory, and deconstruction, it seems to me futile to simply paraphrase his writings. Yet, I am aware that this article may be read in itself as perhaps a first step toward further readings in problematology. I will therefore first take the liberty of rapidly foreshadowing the concepts gleaned from Meyer's problema tology which are essential to my own reading of Le Horla.

Oxygen Isotopic Substitution of Peptidyl Phosphates for Modification-Specific Mass Spectrometry

The first method of isotopic substitution of a nonbridging oxygen atom in pre-existing phosphates on peptides is reported, solving a long-standing, challenging issue in the sample preparation of phosphopeptides. Peptidyl phosphates, phosphate groups on phosphopeptides, are converted to phosphoramidates with carbodiimide assistance. Acid-catalyzed hydrolysis of the newly formed phosphoramidates incorporates one oxygen atom from H2(16)O or H2(18)O, producing peptidyl phosphates-16O1 or -18O1, respectively. The oxygen labels are stable under common separation and analysis conditions. This labeling method causes minimal structural alteration to peptidyl phosphates and allows the direct application of established phosphate-specific marker ions to the mass spectrometric analysis of differentially labeled phosphopeptide pairs. Using phosphotyrosinyl peptides as model analytes, the characteristic 16O1- and 18O1-labeled phosphotyrosine immonium ions at m/z 216.043 and 218.047 are used for developing a method of phosphopeptide quantitation that is independent of the amino acid sequence of the peptides. From analysis by tandem parallel fragmentation mass spectrometry, it is clear that the phosphate-specific marker ions authentically inherit the quantitative information from precursor phosphopeptides. The dynamic range for relative quantitation of differentially labeled phosphopeptides is at least 2 orders of magnitude for experiments run on a quadrupole time-of-flight mass spectrometer. The use of 16O1 and 18O1 labeling for counting the number of phosphate groups on peptides is also demonstrated.

An efficient plasmid vector for expression cloning of large numbers of PCR fragments in Escherichia coli

The described plasmid pEamTA was designed for parallel polymerase chain reaction (PCR) cloning of open reading frames (ORFs) in Escherichia coli. It relies on the well-known TA-cloning principle, and the "T-vector" can be generated from a plasmid preparation by digestion with the restriction enzyme Eam1105I. The single 3'-T-overhangs of the vector fragment are positioned in a way that A-tailed PCR-products beginning with the start-ATG of an ORF end up in optimal position for expression from a strong tac-promoter when ligated in correct orientation. The orientation of the insert can be checked via a reconstituted NdeI site (catATG) present in correct clones. The protocol works regardless of internal restriction sites of the PCR fragment, a major advantage when cloning a number of fragments in parallel. It also does not require 5'-primer extensions and finally delivers an expression clone for the preparation of untagged protein in less than a week.

Tandem Parallel Fragmentation of Peptides for Mass Spectrometry

Parallel fragmentations of peptides in the source region and in the collision cell of tandem mass spectrometers are sequentially combined to develop parallel collision-induced-dissociation mass spectrometry (p2CID MS). Compared to MS/MS spectra, the p2CID mass spectra show increased signal intensities (2-400-fold) and number of sequence ions. This improvement is attributed to the fact that p2CID MS virtually samples all the ions generated by electrospray ionization, including intact and fragment ions of different charge states from a peptide. We implement the method using a quadrupole time-of-flight tandem mass spectrometer. The instrument is operated in TOF-MS mode that allows the ions from source region broadband-passing the first mass analyzer to enter the collision cell. Cone voltage and collision energy are investigated to optimize the outcome of the two parallel CID processes. In the in-source parallel CID, elevated cone voltage produces singly charged intact peptide ions and large fragment ions, as well as decreases the charge-state distribution of peptide ions mainly to double and single charges. The in-collision-cell parallel CID is optimized to dissociate the ions from the source region to produce small and medium fragment ions. The method of p2CID MS is especially useful for sequencing of large peptides with labile amide bonds and peptides with C-terminal arginine. It has unique potential for de novo sequencing of peptides and proteome analysis, especially for affinity-enriched subproteomes.

Seizure detection algorithm for neonates based on wave-sequence analysis

Objective The description and evaluation of the performance of a new real-time seizure detection algorithm in the newborn infant. Methods The algorithm includes parallel fragmentation of EEG signal into waves; wave-feature extraction and averaging; elementary, preliminary and final detection. The algorithm detects EEG waves with heightened regularity, using wave intervals, amplitudes and shapes. The performance of the algorithm was assessed with the use of event-based and liberal and conservative time-based approaches and compared with the performance of Gotman's and Liu's algorithms. Results The algorithm was assessed on multi-channel EEG records of 55 neonates including 17 with seizures. The algorithm showed sensitivities ranging 83–95% with positive predictive values (PPV) 48–77%. There were 2.0 false positive detections per hour. In comparison, Gotman's algorithm (with 30 s gap-closing procedure) displayed sensitivities of 45–88% and PPV 29–56%; with 7.4 false positives per hour and Liu's algorithm displayed sensitivities of 96–99%, and PPV 10–25%; with 15.7 false positives per hour. Conclusions The wave-sequence analysis based algorithm displayed higher sensitivity, higher PPV and a substantially lower level of false positives than two previously published algorithms. Significance The proposed algorithm provides a basis for major improvements in neonatal seizure detection and monitoring.

The hydrogen-bonding network in (E)-1,2-bis(1-ethylbenzimidazol-2-yl)ethene dihydrate

The title compound, C20H20N4·2H2O, contains centrosymmetric (E)-1,2-bis­(1-ethyl­benzimidazol-2-yl)ethene mol­ecules with parallel benzimidazole fragments separated by 0.131 (4) Å. The structure consists of layers parallel to (100). In each layer, alternating chains of water mol­ecules, linked by O—H⋯O hydrogen bonds, and columns of alkene mol­ecules are linked to each other by O—H⋯Nimine hydrogen bonds to form a two-dimensional polymeric network. An inter­planar separation of 3.413 (3) Å between alkene mol­ecules in a given column is consistent with substantial π–π inter­actions.

Unraveling the possible isomers of CH4CN+ and CH2CN+ through FT-IR matrix-isolation spectroscopy of mass-selected ions and DFT theory

The positive molecular ions of astrophysical interest--protonated and fragment ions of acetonitrile are investigated by means of mass-selective deposition in Ne matrices and FT-IR spectroscopy in the region of 500-4000 cm(-1). Ionic species are generated by electron impact ionization with parallel fragmentation and) ion-molecule reactions of an acetonitrile precursor, CH3CN (CD3CN). The ions are deposited in Ne matrices together with an excess of electrons to prevent a buildup of space charge. Spectral assignments are supported by deuterium isotopic measurements and by density functional (DFT/B3LYP) computations. This work resulted in the spectral characterization of protonated acetonitrile CH3CNH+, as well as the identification of three cationic cyanomethyl C2H2N+ isomers: H2CCN+, H2CNC+ and HCCNH+. Although the spectral assignments for two ionic isomers, H2CCN+ and H2CNC+, are somewhat tentative, experimental results give rather strong evidence for the occurrence of the linear HCCNH+ ion.

Fast fragments: the development of a parallel effective fragment potential method.

The Effective Fragment Potential (EFP) method for solvation decreases the cost of a fully quantum mechanical calculation by dividing a chemical system into an ab initio region that contains the solute plus some number of solvent molecules, if desired, and an "effective fragment" region that contains the remaining solvent molecules. Interactions introduced with this fragment region (for example, Coulomb and polarization interactions) are added as one-electron terms to the total system Hamiltonian. As larger systems and dynamics are just starting to be studied with the EFP method, more needs to be done to decrease the calculation time of the method. This article considers parallelization of both the EFP fragment-fragment and mixed quantum mechanics (QM)-EFP interaction energy and gradient computation within the GAMESS suite of programs. The iteratively self-consistent polarization term is treated with a new algorithm that makes use of nonblocking communication to obtain better scalability. Results show that reasonable speedup is achieved with a variety of sizes of water clusters and number of processors.

Parallel fragment assembly strategy towards multiple-ether mimicry of anticancer annonaceous acetogenins.

A new class of multiple-ether-modifiedanalogues (4–13) of the annonaceous acetogenins was designed based on the structures of mimetics 1 and 2, andthe analogues were synthesized enantioselectively by using aparallel fragment-assembly strategy that involved the combination of prefunctionalized units. This methodology may potentially be applicable to the synthesis of further analoguesof this family, as well as other complex, focused libraries. Directional chemo- or regioselective C- and O-alkylations served as key reactions in the synthesis of the target linear assemblies. An aldol-condensation-centered three-step procedurewas utilized to form terminal g-butenolides. Thestarting materials were all readily availabile and the reagents inexpensive. Biological evaluation of the products showed that introduction of hydroxy groups to the ether-bond regionof the compounds results in different selectivities and potencies for different tumor cells. Most importantly, the parallel synthetic strategy presented herein may accelerate discovery of potent and selective antitumor agents.

Parallel Fragment Assembly Strategy Towards Multiple‐Ether Mimicry of Anticancer Annonaceous Acetogenins

Eine Strategie zur Synthese einer kleinen Bibliothek von Annonaceen-Acetogenin-Mimetika aus Parallel-Fragmenten wurde entwickelt. Bei In-vivo-Studien zeigten einige diastereomere Analoga gute Selektivität zwischen KB- und Bel-7402-Zellen.

Acute ischemia/hypoxia in rat hippocampal neurons activates nuclear ubiquitin and alters both chromatin and DNA.

We investigated early alterations in rat neurons after experimental ischemic stress. Transient ischemia was generated by bilateral occlusion of the carotids after hypoxia. Data show a relevant increase of the nuclear level of ubiquitin 2 h post-stress as evaluated by immuno-cytolocalization. Ubiquitin returns to normal levels after 6 h. The increase in ischemic/hypoxic rats was localized preferentially in nuclei of hippocampal neurons, although some augmentation was also shown essentially in dendrites. The activation of ubiquitin system is related to a defective homeostasis and might trigger different degenerative processes. With respect to this, we observed chromatin alterations by densitometric analysis. The shown extensive DNA degeneration is consistent with the occurrence of necrotic phenomena at an early stage. However the parallel internucleosomal specific DNA fragmentation, strongly suggests that apoptotic events also occur. In any case both necrosis and apoptosis are likely to occur at same time, although apoptosis is less extensive, and the two phenomena take place in different neural cells.

Digital analysis of cDNA abundance; expression profiling by means of restriction fragment fingerprinting.

BackgroundGene expression profiling among different tissues is of paramount interest in various areas of biomedical research. We have developed a novel method (DADA, Digital Analysis of cDNA Abundance), that calculates the relative abundance of genes in cDNA libraries.ResultsDADA is based upon multiple restriction fragment length analysis of pools of clones from cDNA libraries and the identification of gene-specific restriction fingerprints in the resulting complex fragment mixtures. A specific cDNA cloning vector had to be constructed that governed missing or incomplete cDNA inserts which would generate misleading fingerprints in standard cloning vectors. Double stranded cDNA was synthesized using an anchored oligo dT primer, uni-directionally inserted into the DADA vector and cDNA libraries were constructed in E. coli. The cDNA fingerprints were generated in a PCR-free procedure that allows for parallel plasmid preparation, labeling, restriction digest and fragment separation of pools of 96 colonies each. This multiplexing significantly enhanced the throughput in comparison to sequence-based methods (e.g. EST approach). The data of the fragment mixtures were integrated into a relational database system and queried with fingerprints experimentally produced by analyzing single colonies. Due to limited predictability of the position of DNA fragments on the polyacrylamid gels of a given size, fingerprints derived solely from cDNA sequences were not accurate enough to be used for the analysis. We applied DADA to the analysis of gene expression profiles in a model for impaired wound healing (treatment of mice with dexamethasone).ConclusionsThe method proved to be capable of identifying pharmacologically relevant target genes that had not been identified by other standard methods routinely used to find differentially expressed genes. Due to the above mentioned limited predictability of the fingerprints, the method was yet tested only with a limited number of experimentally determined fingerprints and was able to detect differences in gene expression of transcripts representing 0.05% of the total mRNA population (e.g. medium abundant gene transcripts).

High-Speed, Multicolor Fluorescent Two-Dimensional Gene Scanning

Two-dimensional gene scanning (TDGS) is a method for analyzing multiple DNA fragments in parallel for all possible sequence variations, using extensive multiplex PCR and two-dimensional electrophoretic separation on the basis of size and melting temperature. Highthroughput application of TDGS is limited by the prolonged time periods necessary to complete the second-dimension electrophoretic separation step–denaturing gradient gel electrophoresis–and the current need for gel staining. To address these problems, we constructed a high-voltage, automatic, two-dimensional electrophoresis system and used this in combination with thinner gels to reduce two-dimensional electrophoresis time about 80%. Instead of gel staining, we used three different fluorophores to simultaneously analyze three samples in the same gel. These improvements greatly increase TDGS speed and throughput and make the method highly suitable for large-scale single-nucleotide polymorphism discovery and genetic testing.

In situ observation of a rolling interface and modeling of the surface texturing of rolled sheets

To observe the tool–workpiece interface in sheet rolling, a visual rolling system is constructed on a laboratory scale. In situ observation is by a small CCD camera with a macro-lens embedded into the upper roll so that the optical axis coincides with the roll axis. A strip specimen of half-hard pure aluminium 2.0 mm thick was rolled at 167 mm s −1 at a thickness reduction of 5–13%. Paraffinic base oils with differing viscosities were used as lubricants. The original ridges and valleys of the sheet specimen were set to be transverse to the experimental rolling direction. From observation of the rolling interface with image processing together with scanning laser microscopy after rolling, a model of surface texture formation on the sheet is proposed as follows. At a low thickness reduction of 5% the initial ridge-and-valley pattern of the sheet survives, but the lubricant introduced into the rolling interface is hydrodynamically compressed and is emitted across the outlet. At an increased reduction of 10%, after contact of the top of the ridges of the sheet surface and the tool, and pressurization of lubricant, the saddle points of the ridges provide new channels for the pressurized lubricant. As a result, the original valleys and the new “valleys” parallel to rolling direction are connected. This causes local and fragmented emission of lubricant at the outlet of the roll. Under tighter contact at 13% reduction, the lubricant present in the initial valleys play a moderating role in the contact, enabling partial free surface deformation and consequent roughening. Parallel ridges and valleys fragment but asperities are finally flattened to leave minor isolated micropools.

Vibrational modes of graphitic fragments and the nucleation of carbon nanotubes

We study the nucleation of carbon nanotubes based on the hypothesis that a cylindrical seed can be formed by the spontaneous rolling of a small graphite fragment under thermal fluctuations. We calculate the vibrational spectra of fragments of different sizes by means of a tight-binding model, and compare the spectra to that of an infinite graphite sheet, as a function of temperature. The spectra of finite-size fragments contain an increasing density of low-frequency, large amplitude modes with increasing temperature; it is shown that such modes tend to fold over the fragment into a cylindrical shape. Finally, direct tight-binding molecular-dynamics simulations of the high-temperature fluctuation of parallel graphite fragments demonstrate that nanotube seeds closed at one end can spontaneously form.

Modulation of urokinase-type plasminogen activator and metalloproteinase activities in cultured mouse mammary-carcinoma cells: enhancement by paclitaxel and inhibition by nocodazole.

Paclitaxel is a potent anti-tumor drug used in the treatment of breast cancer. It induces de-centralization of the microtubular system in tumor cells, blocking cell division. In the search for dissemination to a secondary site, cancer cells are capable of degrading most components of the extracellular matrix via an extracellular proteolytic cascade, including urokinase-type plasminogen activator (uPA) and the matrix metalloproteinases (MMPs). In the present study, the effects of paclitaxel and nocodazole, 2 drugs known to affect microtubules with opposite mechanisms of action, have been tested for their effect on the secretion of uPA and MMPs in cultures of F3II mouse mammary-tumor cells. Tumor-derived uPA activity significantly increased after pre-treatment of tumor cells for 24 hr with micromolar concentrations of paclitaxel (4 microM), while decreasing after pre-treatment with nocodazole (1 microM). A similar modulation was found for MMP-9 by zymographic analysis. Immunofluorescence and Western-blot analysis confirmed the formation of parallel microtubule fragments in paclitaxel-treated cells and almost complete de-polymerization of microtubules in nocodazole-treated ones. Our data suggest that, through opposite actions on microtubule organization and dynamics, paclitaxel and nocodazole exert inverse modulation of tumor-derived proteolytic activity in mammary tumor cells.