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1.
RAPID UPLC-ESI-MS/MS BASED ASSAY FOR DISCOVERY OF UDP-N-ACETYLMURAMOYL-L-ALANINE:D-GLUTAMATE (MurD) LIGASE INHIBITORS
Vjekoslava Car, 2016, magistrsko delo

Opis: 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.
Najdeno v: ključnih besedah
Ključne besede: 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
Objavljeno: 23.09.2016; Ogledov: 2803; Prenosov: 157
.pdf Polno besedilo (2,62 MB)

2.
Outlier based literature exploration for cross-domain linking of Alzheimer's disease and gut microbiota
Tanja Urbančič, Nada Lavrač, Bojan Cestnik, Elsa Fabbretti, Donatella Gubiani, 2017, izvirni znanstveni članek

Opis: In knowledge discovery, experts frequently need to combine knowledge from different domains to get new insights and derive new conclusions. Intelligent systems should support the experts in the search for relationships between concepts from different domains, where huge amounts of possible combinations require the systems to be efficient but also sufficiently general, open and interactive to enable the experts to creatively guide the discovery process. The paper proposes a cross-domain literature mining methodology that achieves this functionality by combining the functionality of two complementary text mining tools: clustering and topic ontology creation tool OntoGen and cross-domain bridging terms exploration tool CrossBee. Focusing on outlier documents identified by OntoGen contributes to the efficiency, while CrossBee allows for flexible and user-friendly bridging concepts exploration and identification. The proposed approach, which is domain independent and can support cross-domain knowledge discovery in any field of science, is illustrated on a biomedical case study dealing with Alzheimer’s dis- ease, one of the most threatening age-related diseases, deteriorating lives of numerous individuals and challenging the ageing society as a whole. By applying the proposed methodology to Alzheimer’s disease and gut microbiota PubMed articles, we have identified Nitric oxide synthase (NOS) as a potentially valuable link between these two domains. The results support the hypothesis of neuroinflammatory nature of Alzheimer’s disease, and is indicative for the quest for identifying strategies to control nitric oxide- associated pathways in the periphery and in the brain. By addressing common mediators of inflammation using literature-based discovery, we have succeeded to uncover previously unidentified molecular links between Alzheimer’s disease and gut microbiota with a multi-target therapeutic potential.
Najdeno v: ključnih besedah
Ključne besede: Literature-based discovery, Outlier detection, Alzheimer's disease, Gut microbiome
Objavljeno: 26.05.2017; Ogledov: 1709; Prenosov: 0
.pdf Polno besedilo (3,13 MB)

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