41. RAPID UPLC-ESI-MS/MS BASED ASSAY FOR DISCOVERY OF UDP-N-ACETYLMURAMOYL-L-ALANINE:D-GLUTAMATE (MurD) LIGASE INHIBITORSVjekoslava Car, 2016, master's thesis Abstract: A rapid, selective, robust and sensitive analytical assay method, operating in a short time frame with acceptable levels of precision, linear range and the accuracy necessary for successful Mur ligases inhibitors discovery, was developed.
An LC-MS/MS analytical procedure was designed for the determination of a MurD ligase reaction product (UMAG). The special focus of this work was on UDP-N-acetylmuramyl-L-alanine:D-glutamate ligase (MurD) activity. The assay method is especially valuable as an orthogonal (secondary) assay for the primary high throughput fluorescent-based assay screening of potential Mur ligase inhibitors. The LC-MS/MS assay is fully compatible with the components from the primary fluorescent-based assay and enables the analysis of the same samples by both methodologies. The presented LC-MS/MS assay procedure is used for the evaluation of the false positive hits (molecules) from the primary, fluorescence based, high throughput screening assay experiments. This is important for the elimination of false positive hits from the prohibitively expensive and time-consuming investigation process.
Method development describes the evaluation and optimization of the various stages of sample preparation, chromatographic separation, MS/MS determination and quantification. An enzyme reaction is performed in a 96-well plate. The quenched reaction mixture samples were spiked with an internal standard (phenacetin). The permeate was injected onto the U(H)PLC-MS/MS triple quadrupole system after sample ultrafiltration. Chromatographic separation was achieved on the ACQUITY UPLCTM HSS T3 column (100 x 2.1 mm i.d., 1.8 µm particle size) using an ammonium format buffer at pH 2.8 and acetonitrile as eluent. Elution initiated with an isocratic-hold for 1.1 min, followed by a two-step linear gradient of up to 3 min, giving a total run time of 5 min including equilibration. The flow rate was kept at a constant 0.4 mL/min.
UMAG quantitative analysis was performed by positive electrospray ionization, followed by tandem mass spectrometry (ESI-MS/MS). The analytical assay quantifies UMAG in a linear range from 0.25 to 20 µM using 70 µL of samples. Validation results demonstrated that UMAG concentrations can be accurately and precisely determined in samples from the primary assay.
Evaluation of inhibitory activities of compounds measured by both the fluorescence and the LC-MS/MS method demonstrated that the values were in a very good agreement. This analytical method can be used to screen a compound library at a defined concentration of each compound to obtain the percentage of inhibition, or with a series of compound concentrations to obtain inhibition potency of a compound (IC50). The selected Lek compounds no. 1 and 2 from the virtual screening campaign were presented, tested and further investigated due to the expression of significant MurD ligase inhibitory action acquired by primary high throughput tests.
This assay has been developed for MurD, but during development, chromatographic and MS/MS conditions for UM and UMA were studied and defined as well. Therefore, this analytical assay method can easily be applied to other Mur ligases (i.e. MurC, MurE) enzyme activity monitoring in the process of bacteria cell wall peptidoglycan formation. This method enables the identification of many different Mur ligase inhibitors in a continued search for new Gram positive and Gram negative bacteria antibiotics. Keywords: Mur ligases, UDP-N-acetylmuramyl-L-alanine:D-glutamate (MurD) inhibitors, UNAM-Ala-Glu, LC-MS/MS, liquid chromatography, tandem mass spectrometry, antibiotics, drug discovery Published in RUNG: 23.09.2016; Views: 6946; Downloads: 273 Full text (2,62 MB) |
42. Azimuthal asymmetry in the risetime of the surface detector signals of the Pierre Auger ObservatoryA. Aab, Andrej Filipčič, Gašper Kukec Mezek, Ahmed Saleh, Samo Stanič, Marta Trini, Darko Veberič, Serguei Vorobiov, Lili Yang, Danilo Zavrtanik, Marko Zavrtanik, 2016, original scientific article Abstract: The azimuthal asymmetry in the risetime of signals in Auger surface detector stations is a source of information on shower development. The azimuthal asymmetry is due to a combination of the longitudinal evolution of the shower and geometrical effects related to the angles of incidence of the particles into the detectors. The magnitude of the effect depends upon the zenith angle and state of development of the shower and thus provides a novel observable, (secθ)max, sensitive to the mass composition of cosmic rays above 3×1018 eV. By comparing measurements with predictions from shower simulations, we find for both of our adopted models of hadronic physics (QGSJETII-04 and EPOS-LHC) an indication that the mean cosmic-ray mass increases slowly with energy, as has been inferred from other studies. However, the mass estimates are dependent on the shower model and on the range of distance from the shower core selected. Thus the method has uncovered further deficiencies in our understanding of shower modeling that must be resolved before the mass composition can be inferred from (secθ)max. Keywords: ultra-high energy cosmic rays (UHECR), UHECR mass composition, Pierre Auger Observatory, extensive air showers, Auger Surface Detector signals risetime, azimuthal symmetry Published in RUNG: 15.04.2016; Views: 5474; Downloads: 0 This document has many files! More... |
43. Measurement of the muon content in air showers at the Pierre Auger ObservatoryLaura Collica, Andrej Filipčič, Gašper Kukec Mezek, Ahmed Saleh, Samo Stanič, Marta Trini, Darko Veberič, Serguei Vorobiov, Lili Yang, Danilo Zavrtanik, Marko Zavrtanik, 2015, published scientific conference contribution Abstract: The muon content of extensive air showers is an
observable sensitive to the primary composition and to
the hadronic interaction properties. We present here
different methods which allow us to estimate the muon number
at the ground level and the muon production depth by
exploiting the measurement of the longitudinal, lateral and
temporal distribution of particles in air showers recorded at
the Pierre Auger Observatory. The results, obtained at about
10[sup]19 eV (E[inf]CM ∼ 140 TeV center-of-mass energy for
proton primaries), are compared to the predictions
of LHC-tuned hadronic interaction models with different primary
masses and suggest a deficit in the muon content at the
ground predicted by simulations. The Pierre Auger Observatory
uses water-Cherenkov detectors to measure particle
densities at the ground and therefore has a good
sensitivity to the muon content of air showers. Moreover, due
to its hybrid design, the combination of muon measurements with
other independent mass composition analyses such as Xmax
provides additional constraints on hadronic interaction models. Keywords: Pierre Auger Observatory, ultra-high energy cosmic rays, muons, mass composition, hadronic interactions Published in RUNG: 03.03.2016; Views: 4787; Downloads: 218 Full text (298,46 KB) |
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