Elucidation of Nucleic Acid–Drug Interactions by Tandem Mass Spectrometry

In continuation of the long tradition of mass spectrometric research at the University of Bern, our group focuses on the characterization of nucleic acids as therapeutic agents and as drug targets. This article provides a short overview of our recent work on platinated single-stranded and higher-order nucleic acids. Nearly three decades ago the development of soft ionization techniques opened a whole new chapter in the mass spectrometric analysis of not only nucleic acids themselves, but also their interactions with potential drug candidates. In contrast to modern next generation sequencing approaches, though, the goal of the tandem mass spectrometric investigation of nucleic acids is by no means the complete sequencing of genetic DNA, but rather the characterization of short therapeutic and regulatory oligonucleotides and the elucidation of nucleic acid–drug interactions. The influence of cisplatin binding on the gas-phase dissociation of nucleic acids was studied by the means of electrospray ionization tandem mass spectrometry. Experiments on native and modified DNA and RNA oligomers confirmed guanine base pairs as the preferred platination site and laid the basis for the formulation of a gas-phase fragmentation mechanism of platinated oligonucleotides. The study was extended to double stranded DNA and DNA quadruplexes. While duplexes are believed to be the main target of cisplatin in vivo, the recently discovered DNA quadruplexes constitute another promising target for anti-tumor drugs owing to their regulatory functions in the cell cycle.

Electrospray tandem mass spectrometry of mixed-sequence RNA/DNA oligonucleotides

The fragmentation of electrospray-generated multiply deprotonated RNA and mixed-sequence RNA/DNA pentanucleotides upon low-energy collision-induced dissociation (CID) in a hybrid quadrupole time-of-flight mass spectrometer was investigated. The goal of unambiguous sequence identification of mixed-sequence RNA/DNA oligonucleotides requires detailed understanding of the gas-phase dissociation of this class of compounds. The two major dissociation events, base loss and backbone fragmentation, are discussed and the unique fragmentation behavior of oligoribonucleotides is demonstrated. Backbone fragmentation of the all-RNA pentanucleotides is characterized by abundant c-ions and their complementary y-ions as the major sequence-defining fragment ion series. In contrast to the dissociation of oligodeoxyribonucleotides, where backbone fragmentation is initiated by the loss of a nucleobase which subsequently leads to the formation of the w- and [a-base]-ions, backbone dissociation of oligoribonucleotides is essentially decoupled from base loss. The different behavior of RNA and DNA oligonucleotides is related to the presence of the 2'-hydroxyl substituent, which is the only structural alteration between the DNA and RNA pentanucleotides studied. CID of mixed-sequence RNA/DNA pentanucleotides results in a combination of the nucleotide-typical backbone fragmentation products, with abundant w-fragment ions generated by cleavage of the phosphodiester backbone adjacent to the deoxy building blocks, whereas backbone cleavage adjacent to ribonucleotides induces the formation of c- and y-ions. (C) 2002 American Society for Mass Spectrometry.

Tandem Mass Spectrometric Analysis of Metallocene Adducts with Short Oligonucleotides

Metallocene dichlorides constitute a remarkable class of antineoplastic agents that are highly effective against several cancer cell lines. They were shown to accumulate in the DNA-rich region, which suggests DNA as the primary target. These compounds exhibit two cyclopentadienyl ligands and two labile halide ligands, resulting in a bent sandwich structure. The cis-dihalide motif is structurally related to the cis-chloro configuration of cisplatin and similar modes of action can thus be assumed. Cisplatin binds to two neighboring guanine nucleobases in DNA and consequently, distorts the double-helix, thereby inhibiting DNA replication and transcription. Platinum is classified as a soft Lewis acid and binds preferentially to the nitrogen atoms within the nucleobases. The metallocene dichlorides investigated in this study comprise the metal centers Ti, V, Nb, Mo, Hf, and W, which are classified as hard or intermediate Lewis acids, and thus, favor binding to the phosphate oxygen. Although several studies reported adduct formation of metallocene dichlorides with nucleic acids, substantial information about the adduct composition, the binding pattern, and the nucleobase selectivity has not been provided yet. ESI-MS analyses gave evidence for the formation of metallocene adducts (M = Ti, V, Mo, and W) with single-stranded DNA homologues at pH 7. No adducts were formed with Nb and Hf at neutral pH, albeit adducts with Nb were observed at a low pH. MS2 data revealed considerable differences of the adduct compositions. The product ion spectra of DNA adducts with hard Lewis acids (Ti, V) gave evidence for the loss of metallocene ligands and only moderate backbone fragmentation was observed. By contrast, adducts with intermediate Lewis acids (Mo, W) retained the hydroxy ligands. Preliminary results are in good agreement with the Pearson concept and DFT calculations. Since the metallodrugs were not lost upon CID, the nucleobase selectivity, stoichiometry, and binding patterns can be elucidated by means of tandem mass spectrometry.

Increased spread and replication efficiency of Listeria monocytogenes in organotypic brain-slices is related to multilocus variable number of tandem repeat analysis (MLVA) complex.

BACKGROUND Listeria (L.) monocytogenes causes fatal infections in many species including ruminants and humans. In ruminants, rhombencephalitis is the most prevalent form of listeriosis. Using multilocus variable number tandem repeat analysis (MLVA) we recently showed that L. monocytogenes isolates from ruminant rhombencephalitis cases are distributed over three genetic complexes (designated A, B and C). However, the majority of rhombencephalitis strains and virtually all those isolated from cattle cluster in MLVA complex A, indicating that strains of this complex may have increased neurotropism and neurovirulence. The aim of this study was to investigate whether ruminant rhombencephalitis strains have an increased ability to propagate in the bovine hippocampal brain-slice model and can be discriminated from strains of other sources. For this study, forty-seven strains were selected and assayed on brain-slice cultures, a bovine macrophage cell line (BoMac) and a human colorectal adenocarcinoma cell line (Caco-2). They were isolated from ruminant rhombencephalitis cases (n = 21) and other sources including the environment, food, human neurolisteriosis cases and ruminant/human non-encephalitic infection cases (n = 26). RESULTS All but one L. monocytogenes strain replicated in brain slices, irrespectively of the source of the isolate or MLVA complex. The replication of strains from MLVA complex A was increased in hippocampal brain-slice cultures compared to complex C. Immunofluorescence revealed that microglia are the main target cells for L. monocytogenes and that strains from MLVA complex A caused larger infection foci than strains from MLVA complex C. Additionally, they caused larger plaques in BoMac cells, but not CaCo-2 cells. CONCLUSIONS Our brain slice model data shows that all L. monocytogenes strains should be considered potentially neurovirulent. Secondly, encephalitis strains cannot be conclusively discriminated from non-encephalitis strains with the bovine organotypic brain slice model. The data indicates that MLVA complex A strains are particularly adept at establishing encephalitis possibly by virtue of their higher resistance to antibacterial defense mechanisms in microglia cells, the main target of L. monocytogenes.

Adam vetus tandem laetus novum promat canticum : Adán de San Víctor y la novedad del canto victorino en la lírica mariana medieval

La poesía latina del maestro Adán de San Víctor (ca.11121192), oriundo de Bretaña y canónigo agustino de la abadía parisina de SaintVictor, representa, en su ámbito y en su época, una novedad trascendente dentro de la lírica litúrgica y particularmente de la lírica mariana. La ductilidad rítmica y retórica del subgénero de la sequentia lograda en sus piezas hace de este autor un paradigma de la creación poética litúrgica en la Europa de los siglos XII y XIII. En este sentido, tanto la mención a su obra como la inclusión de sus sequentiae (junto con otras del mismo período y "estilo" gótico) en diversos manuscritos hispánicos, desde las Cantigas de Santa María hasta el ms. Alcobacense 149, permiten considerar la relevante influencia de esta refinada lírica del siglo XII en la confección de poemas y compilaciones marianas del siglo siguiente en la Península Ibérica.

Adam vetus tandem laetus novum promat canticum : Adán de San Víctor y la novedad del canto victorino en la lírica mariana medieval

La poesía latina del maestro Adán de San Víctor (ca.11121192), oriundo de Bretaña y canónigo agustino de la abadía parisina de SaintVictor, representa, en su ámbito y en su época, una novedad trascendente dentro de la lírica litúrgica y particularmente de la lírica mariana. La ductilidad rítmica y retórica del subgénero de la sequentia lograda en sus piezas hace de este autor un paradigma de la creación poética litúrgica en la Europa de los siglos XII y XIII. En este sentido, tanto la mención a su obra como la inclusión de sus sequentiae (junto con otras del mismo período y "estilo" gótico) en diversos manuscritos hispánicos, desde las Cantigas de Santa María hasta el ms. Alcobacense 149, permiten considerar la relevante influencia de esta refinada lírica del siglo XII en la confección de poemas y compilaciones marianas del siglo siguiente en la Península Ibérica.

Adam vetus tandem laetus novum promat canticum : Adán de San Víctor y la novedad del canto victorino en la lírica mariana medieval

La poesía latina del maestro Adán de San Víctor (ca.11121192), oriundo de Bretaña y canónigo agustino de la abadía parisina de SaintVictor, representa, en su ámbito y en su época, una novedad trascendente dentro de la lírica litúrgica y particularmente de la lírica mariana. La ductilidad rítmica y retórica del subgénero de la sequentia lograda en sus piezas hace de este autor un paradigma de la creación poética litúrgica en la Europa de los siglos XII y XIII. En este sentido, tanto la mención a su obra como la inclusión de sus sequentiae (junto con otras del mismo período y "estilo" gótico) en diversos manuscritos hispánicos, desde las Cantigas de Santa María hasta el ms. Alcobacense 149, permiten considerar la relevante influencia de esta refinada lírica del siglo XII en la confección de poemas y compilaciones marianas del siglo siguiente en la Península Ibérica.

TanDEM-X ice thickness dataset for Wilkins Ice Shelf, Antarctic Peninsula, with link to GeoTIFF

The dataset shows the ice thickness over Wilkins Ice Shelf, Antarctic Peninsula derived from TanDEM-X Interferometry. The data has been acquired between June and August 2012. The TanDEM-X heights have been linked to CryoSAT-2 heights (V. Helm) from the respective time stamp. Elevations have been transformed from WGS84 ellipsoidal heights to the EGM2008 geoid. The ice shelf thickness was estimated assuming hydrostatic equilibrium and a mean ice density of 915 kg/m³.

Development of Robotized Laser Welding Applications for Joining Thin Sheets

Laser Welding (LW) is more often used in manufacturing due to its advantages, such as accurate control, good repeatability, less heat input, opportunities for joining of special materials, high speed, capability to join small dimension parts etc. LW is dedicated to robotized manufacturing, and the fabrication cells are using various level of flexibility, from specialized robots to very flexible setups. This paper features several LW applications using two industrially-scaled manufacturing cells at UPM Laser Centre (CLUPM) of Polytechnical University of Madrid (Universidad Politécnica de Madrid). The one dedicated to Remote Laser Welding (RLW) of thin sheets for automotive and other sectors uses a CO2 laser of 3500 W. The second has a high flexibility, is based on a 6-axis ABB robot and a Nd:YAG laser of 3300 W, and is meant for various laser processing methods, including welding. After a short description of each cell, several LW applications experimented at CLUPM and recently implemented in industry are briefly presented: RLW of automotive coated sheets, LW of high strength automotive sheets, LW vs. laser hybrid welding (LHW) of Double Phase steel thin sheets, and LHW of thin sheets of stainless steel and carbon steel (dissimilar joints). The main technological issues overcame and the critical process parameters are pointed out. Conclusions about achievements and trends are provided.

Preceptorium Nider, hoc est, opus preclarissimu[m] ... Iohannis Nider ... in expositionem preceptorum decalogi : diligentissime nunc tandem cum originalibus collatum et recognitum ac multis locis tersum et emendatum.

Hojas imp. por ambas caras

Residual stress distribution in ferritic-austenitic steel joints made by laser welding

The present investigation addresse the influence of laser welding process-ing parameters used for joining dis-similar metals (ferritic to austenitic steel), on the induced residual stress field. Welding was performed on a Nd:YAG laser DY033 (3300 W) in a continuous wave (CW), keyhole mode. The base metals (BM) employed in this study are AISI 1010 carbon steel (CS) and AISI 304L austenitic stainless steel (SS). Pairs of dissimilar plates of 200 mm x 45 mm x 3 mm were butt joined by laser welding. Different sets of parameters were used to engineer the base metals apportionment at joint formation, namely distinct dilution rates. Residual strain scanning, carried out by neutron diffraction was used to assess the joints. Through-thickness residual stress maps were determined for the laser welded samples of dis-similar steels using high spatial reso-lution. As a result, an appropriate set of processing parameters, able to mi-nimize the local tensile residual stress associated to the welding process, was found.