Butyrylcholinesterase (BChE) is regarded as a promising drug target as its levels and activity significantly increase in the late stages of Alzheimer's disease. To discover novel BChE inhibitors, we used a hierarchical virtual screening protocol followed by biochemical evaluation of 40 highest scoring hit compounds. Three of the compounds identified showed significant inhibitory activities against BChE. The most potent, compound 1 (IC50 = 21.3 nM), was resynthesized and resolved into its pure enantiomers. A high degree of stereoselective activity was revealed, and a dissociation constant of 2.7 nM was determined for the most potent stereoisomer (+)-1. The crystal structure of human BChE in complex with compound (+)-1 was solved, revealing the binding mode and providing clues for potential optimization. Additionally,compound 1 inhibited amyloid Ž1-42 peptide self-induced aggregation into fibrils (by 61.7% at 10 ŽM), and protected cultured SH-SY5Y cells against amyloid-Ž-induced toxicity. These data suggest that compound 1 represents a promising candidate for hit-to-lead follow-up in the drug-discovery process against Alzheimer's disease.
COBISS.SI-ID: 3713393
Paraoxonase-1 has been widely investigated as potential biomarker in cardiovascular and neurodegenerative diseases in the last ten years. However, phenylacetate and paraoxonases enzame activities and ELISA assay only are currently in use for its quantity-determination. The development of other diagnostic assays demands the study of catalytic mechanism this calcium-dependent enzyme and binding/inhibition of adequate ligands (e.g. transition-state analogs) into its active site. The latter could be good starting point for the development of lanthanide-based luminescence time-resolved assays which would enable the enzyme-concentration quantification in samples. Our study present the basis for further developments of methodology and translational research.
COBISS.SI-ID: 31520729
The standard Michaelis-Menten model equation must be extended to describe the kinetics of enzyme-catalyzed reactions that exhibit also spontaneous substrate depletion. Hence, its use is limited because the integrated form cannot be expressed in an explicit closed-form reformulation of the time-dependent solution. Thus, we construct explicit approximations to the solution of the extended Michaelis-Menten equation using the Adomian decomposition method, and this report presents its use to an illustrative problem that has been considered solvable only by numerical methods. We demonstrate here that the algebraic nature of these approximations to the solution of the extended Michaelis-Menten equation makes progress-curve analysis an attractive and useful alternative that can be simply performed using a standard nonlinear regression curve-fitting computer program.
COBISS.SI-ID: 32324825
Wallemia comprises air- and food-borne, mycotoxigenic contaminants including the halophilic W. ichthyophaga, xerotolerant W. sebi and xerophilic W. muriae. Wallemia isolates are easily overlooked and only a comparably small number of strains have been deposited in culture collections so far. In order to better understand the natural distribution of Wallemia spp. and to encounter their natural habitats, we tested more than 300 low-water-activity substrates and 30 air samples from a wide geographical coverage. We isolated more than 150 new Wallemia strains. Wallemia sebi and W. muriae were isolated mostly from hypersaline water, low-water-activity foods, plant materials and indoor. Wallemia muriae is the dominant Wallemia species in the air of natural and human influenced environments in Europe. New isolates of W. ichthyophaga were obtained from hypersaline environments such as brine, salt crystals, salty foods and MgCl2-rich bitterns, and from the air of hay barns in Denmark. Five halotolerant strains were recognised as a hitherto undescribed species Wallemia hederae, the phylogenetic sister of the halophilic W. ichthyophaga. Wallemia spp. show in-vitro growth on media that contain the chaotropic salt MgCl2. Wallemia ichthyophaga can grow in liquid medium enriched with 2 M MgCl2. Never before has a microorganism been grown on comparably high MgCl2 concentrations. Tests of the activity of a wide range of extracellular enzymes in the presence of NaCl also suggested that Wallemia is well-adapted to substrates with a reduced water activity.
COBISS.SI-ID: 3436111
Discovery of the extremely halotolerant and adaptable fungus Hortaea werneckii and the obligate halophile Wallemia ichthyophaga introduced two new model organisms into studies on the mechanisms of salt tolerance in eukaryotes. In collaboration with BGI Genomics Institute Shenzhen, China and Canada's Michael Smith Genome Sciences Centre, Vancouver, Canada, the whole genomes of H.werneckii and W.ichthyophaga were sequences recently and published in 2013 in PlosOne and BMC Genomics respectively. The present review article in Frontiers in Microbiology is the first example of comparative analysis of genomes of halotolerant/halophilic fungi. We addressed differences that have been revealed from analysis of sequenced genomes of halotolerant H.werneckii and halophilic W.ichthyophaga. The most striking characteristics associated with H. werneckii are the large genetic redundancy, the expansion of genes encoding metal cation transporters, and a relatively recent whole genome duplication. In contrast, the genome of W. ichthyophaga is very compact, as less than 5000 protein-coding genes are predicted, which cover almost three quarters of the sequence. Importantly, there has been a significant increase in their hydrophobins, cell-wall proteins that have multiple cellular functions. Our previous studies and analysis of genomes have revealed the novel and intricate molecular mechanisms used to combat high salt concentrations, which differ in many aspects between the extremely halotolerant H. werneckii and the halophilic W. ichthyophaga. Specifically, the high osmolarity glycerol signaling pathway that is important for sensing and responding to increased salt concentrations was compared between H. werneckii and W. ichthyophaga. In both of these fungi, the key signaling components are conserved, but there are structural and regulation differences between these pathways in H. werneckii and W. ichthyophaga.
COBISS.SI-ID: 3130191
In the past few years extracellular vesicles called exosomes have gained huge interest of scientific community since they show a great potential for human diagnostic and therapeutic applications. However, an ongoing challenge is accurate size characterization and quantification of exosomes because of the lack of reliable characterization techniques. In this work, the emphasis was focused on a method development to size-separate, characterize, and quantify small amounts of exosomes by asymmetricalflow field-flow fractionation (AF4) technique coupled to a multidetection system (UV and MALS). Batch DLS (dynamic light-scattering) and NTA (nanoparticle tracking analysis) analyses of unfractionated exosomes were also conducted to evaluate their shape and internal structure, as well as their number density. The results show significant influence of cross-flow conditions and channel thickness on fractionation quality of exosomes, whereas the focusing time has less impact. The AF4/UV-MALS and DLS results display the presence of two particles subpopulations, that is, the larger exosomes and the smaller vesicle-like particles, which coeluted in AF4 together with impurities in early eluting peak. Compared to DLS and AF4-MALS results, NTA somewhat overestimates the size and the number density for larger exosome population, but it discriminates the smaller particle population.
COBISS.SI-ID: 5754138
Background: Current non-invasive diagnostic methods for endometriosis lack sensitivity and specificity. In search for new diagnostic biomarkers for ovarian endometriosis, we used a hypothesis-generating targeted metabolomics approach. Methods: In a case-control study, we collected plasma of study participants and analysed their metabolic profiles. We selected a group of 40 patients with ovarian endometriosis who underwent laparoscopic surgery and a control group of 52 healthy women who underwent sterilization at the University Clinical Centre Ljubljana, Slovenia. Over 140 targeted analytes included glycerophospholipids, sphingolipids and acylcarnitines. The analytes were quantified by electrospray ionization tandem mass spectrometry. For assessing the strength of association between the metabolite or metabolite ratios and the disease, we used crude and adjusted odds ratios. A stepwise logistic regression procedure was used for selecting the best combination of biomarkers. Results: Eight lipid metabolites were identified as endometriosis-associated biomarkers due to elevated levels in patients compared with controls. A model containing hydroxysphingomyelin SMOH C16:1 and the ratio between phosphatidylcholine PCaa C36:2 to etherphospholipid PCae C34:2, adjusted for the effect of age and the BMI, resulted in a sensitivity of 90.0%, a specificity of 84.3% and a ratio of the positive likelihood ratio to the negative likelihood ratio of 48.3. Conclusions: Our results suggest that endometriosis is associated with elevated levels of sphingomyelins and phosphatidylcholines, which might contribute to the suppression of apoptosis and affect lipid-associated signalling pathways. Our findings suggest novel potential routes for therapy by specifically blocking highly up-regulated isoforms of phosphpolipase A2 and lysophosphatidylcholine acyltransferase 4.
COBISS.SI-ID: 30025945
The occurrence of adverse events and resistance to treatment is one of the major clinical problems in cancer treatment. It has been well established that genetic variability in drug pathways or DNA repair mechanisms might influence treatment outcome. In addition, genetic polymorphisms in DNA repair mechanisms may influence the risk of malignant disease. The aim of our study was to evaluate theinfluence of platinum pathway polymorphisms on treatment outcome in patients with MM. GST polymorphisms were not associated with treatment outcome in patients with MM. In the group of platinum-treated patients with MM, ERCC1 8092C/C genotype significantly influenced progression-free survival (PFS). XPD 312Asp/Asp and ERCC1 8092C/C wild-type genotypes also increased the odds of treatment-related toxic effects and of better PFS. Our results suggest that polymorphisms in RNA repair pathway influence platinum-treatment efficacy and toxicity. If confirmed as markers of clinical outcome in further pharmacogenetics studies, the analysis of these polymorphisms may contribute to the individualisation of cancer treatment. Award: The paper was selected as an outstanding scientific contribution to Slovenian science in the field of medicine in 2010 (Slovenian Researh Agency - ARRS)
COBISS.SI-ID: 28755417
We describe a novel approach to investigate and evaluate combined effect of a large number of clinical and pharmacogenetic factors on treatment outcome. We have used this approach to investigate predictors of methotrexate (MTX)-induced adverse events (AEs) leading to treatment discontinuation in rheumatoid arthritis (RA) patients. In total, 333 RA patients were genotyped for 34 polymorphisms in MTX transporters, folate and adenosine pathways. The effect of clinical and pharmacogenetic factors was assessed with penalized regression in the cause-specific Cox proportional hazards model. The predictive capacity was evaluated with the area under time-dependent receiver operating characteristic curve where cross-validation was applied. SLC19A1, ABCG2, ADORA3 and TYMS were associated with discontinuation because of AEs in clinical-pharmacogenetic model. Cross-validation showed that both clinical-pharmacogenetic model and nongenetic model had worthless predictive ability for MTX discontinuation because of AEs. These models could be further improved, either with additional polymorphisms or with epigenetic predictors.
COBISS.SI-ID: 32687833
Despite scientific and clinical advances in the field of pharmacogenomics (PGx), application into routine care remains limited. Opportunely, several implementation studies and programmes have been initiated over recent years. This article presents an overview of these studies and identifies current research gaps. Importantly, one such gap is the undetermined collective clinical utility of implementing a panel of PGx-markers into routine care, because the evidence base is currently limited to specific, individual drug-gene pairs. The Ubiquitous Pharmacogenomics Consortium (U-PGx), which has been funded by the European Commission's Horizon-2020 programme, aims to address this unmet need. In a prospective, block-randomized, controlled clinical study (PREPARE), pre-emptive genotyping of a panel of clinically relevant PGx-markers, for which guidelines are available, will be implemented across healthcare institutions in seven European countries. The impact on patient outcomes and cost-effectiveness will be investigated. The program is unique in its multi-center, multi-gene, multi-drug, multi-ethnic, and multi-healthcare system approach.
COBISS.SI-ID: 33082329