Publications by authors named "Martin Svoboda"

50 Publications

Ring-closing metathesis of prochiral oxaenediynes to racemic 4-alkenyl-2-alkynyl-3,6-dihydro-2-pyrans.

Beilstein J Org Chem 2020 13;16:2757-2768. Epub 2020 Nov 13.

Department of Organic Chemistry, University of Chemistry and Technology, Prague, Technická 5, 166 28 Prague 6, Czech Republic.

The prochiral 4-(allyloxy)hepta-1,6-diynes, optionally modified in the positions 1 and 7 with an alkyl or ester group, undergo a chemoselective ring-closing enyne metathesis yielding racemic 4-alkenyl-2-alkynyl-3,6-dihydro-2-pyrans. Among the catalysts tested, Grubbs 1st generation precatalyst in the presence of ethene (Mori conditions) gave superior results compared to the more stable Grubbs or Hoveyda-Grubbs 2nd generation precatalysts. This is probably caused by a suppression of the subsequent side-reactions of the enyne metathesis product with ethene. On the other hand, the 2nd generation precatalysts gave better yields in the absence of ethene. The metathesis products, containing both a triple bond and a conjugated system, can be successfully orthogonally modified. For example, the metathesis product of 5-(allyloxy)nona-2,7-diyne reacted chemo- and stereoselectively in a Diels-Alder reaction with -phenylmaleimide affording the tricyclic products as a mixture of two separable diastereoisomers, the configuration of which was estimated by DFT computations. The reported enediyne metathesis paves the way to the enantioselective enyne metathesis yielding chiral building blocks for compounds with potential biological activity, e.g., norsalvinorin or cacospongionolide B.
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http://dx.doi.org/10.3762/bjoc.16.226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7670115PMC
November 2020

Interrelations of Sphingolipid and Lysophosphatidate Signaling with Immune System in Ovarian Cancer.

Comput Struct Biotechnol J 2019 10;17:537-560. Epub 2019 Apr 10.

Molecular Systems Biology and Pathophysiology Research Group, Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.

The sphingolipid and lysophosphatidate regulatory networks impact diverse mechanisms attributed to cancer cells and the tumor immune microenvironment. Deciphering the complexity demands implementation of a holistic approach combined with higher-resolution techniques. We implemented a multi-modular integrative approach consolidating the latest accomplishments in gene expression profiling, prognostic/predictive modeling, next generation digital pathology, and systems biology for epithelial ovarian cancer. We assessed patient-specific transcriptional profiles using the sphingolipid/lysophosphatidate/immune-associated signature. This revealed novel sphingolipid/lysophosphatidate-immune gene-gene associations and distinguished tumor subtypes with immune high/low context. These were characterized by robust differences in sphingolipid-/lysophosphatidate-related checkpoints and the drug response. The analysis also nominates novel survival models for stratification of patients with , , , , and emerging as the most prognostically important genes. Alignment of proprietary data with curated transcriptomic data from public databases across a variety of malignancies (over 600 categories; over 21,000 arrays) showed specificity for ovarian carcinoma. Our systems approach identified novel sphingolipid-lysophosphatidate-immune checkpoints and networks underlying tumor immune heterogeneity and disease outcomes. This holds great promise for delivering novel stratifying and targeting strategies.
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http://dx.doi.org/10.1016/j.csbj.2019.04.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6479272PMC
April 2019

Controlled Transport of Flexible Polymers in Slit and Cylindrical Pores Coated with Polymer Brushes: Insight from Dissipative Particle Dynamics.

J Nanosci Nanotechnol 2019 May;19(5):2943-2949

Laboratory of Aerosols Chemistry and Physics, Institute of Chemical Process Fundamentals of the Czech Academy of Science, v. v. i., Rozvojová 135/1, 165 02-Prague, Czech Republic.

We use dissipative particle dynamics to simulate the controlled transport of flexible polymers through coated slit and cylindrical pores. Pores are coated inside with solvent-sensitive polymer brushes. Stretch-to-collapse transition then controls the permeability of the coated pores. We change the solvent quality with respect to the polymer brushes and study the flow of flexible polymers through the pores. We show that stretched brush chains close the pores and compress the polymers in the centre of the pores. The collapsed brush chains relieve compression and rapid change in permeability is observed. In open pore state, polymers partially accommodate on the brush layer and partially migrate towards the center of the pores, where the flux is maximal. Finally, we observe that polymers tend to align in the direction of the flow.
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http://dx.doi.org/10.1166/jnn.2019.15865DOI Listing
May 2019

Nonhydroxylated 1--acylceramides in vernix caseosa.

J Lipid Res 2018 11 25;59(11):2164-2173. Epub 2018 Sep 25.

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, CZ-166 10 Praha 6, Czech Republic

Vernix caseosa, the waxy substance that coats the skin of newborn babies, has an extremely complex lipid composition. We have explored these lipids and identified nonhydroxylated 1--acylceramides (1--ENSs) as a new class of lipids in vernix caseosa. These ceramides mostly contain saturated C11-C38 ester-linked (1-) acyls, saturated C12-C39 amide-linked acyls, and C16-C24 sphingoid bases. Because their fatty acyl chains are frequently branched, numerous molecular species were separable and detectable by HPLC/MS: we found more than 2,300 molecular species, 972 of which were structurally characterized. The most abundant 1--ENSs contained straight-chain and branched fatty acyls with 20, 22, 24, or 26 carbons in the 1- position, 24 or 26 carbons in the position, and sphingosine. The 1--ENSs were isolated using multistep TLC and HPLC and they accounted for 1% of the total lipid extract. The molecular species of 1--ENSs were separated on a C18 HPLC column using an acetonitrile/propan-2-ol gradient and detected by APCI-MS, and the structures were elucidated by high-resolution and tandem MS. Medium-polarity 1--ENSs likely contribute to the cohesiveness and to the waterproofing and moisturizing properties of vernix caseosa.
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http://dx.doi.org/10.1194/jlr.M088864DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6210899PMC
November 2018

Clinical Significance of Organic Anion Transporting Polypeptide Gene Expression in High-Grade Serous Ovarian Cancer.

Front Pharmacol 2018 7;9:842. Epub 2018 Aug 7.

Institute of Clinical Biometrics, Center for Medical Statistics, Informatics, and Intelligent Systems, Medical University of Vienna, Vienna, Austria.

High-grade serous ovarian cancer (HGSOC) is considered the most deadly and frequently occurring type of ovarian cancer and is associated with various molecular compositions and growth patterns. Evaluating the mRNA expression pattern of the organic anion transporters (OATPs) encoded by genes may allow for improved stratification of HGSOC patients for targeted invention. The expression of mRNA and genes coding for putative functionally related ABC-efflux pumps, enzymes, pregnane-X-receptor, and (coding for estrogen receptors ERα and ERß) and HER-2 were assessed using RT-qPCR. The expression levels were assessed in a cohort of 135 HGSOC patients to elucidate the independent impact of the expression pattern on the overall survival (OS). For identification of putative regulatory networks, Graphical Gaussian Models were constructed from the expression data with a tuning parameter K varying between meaningful borders (Pils et al., 2012; Auer et al., 2015, 2017; Kurman and Shih Ie, 2016; Karam et al., 2017; Labidi-Galy et al., 2017; Salomon-Perzynski et al., 2017; Sukhbaatar et al., 2017). The final value used ( = 4) was determined by maximizing the proportion of explained variation of the corresponding LASSO Cox regression model for OS. The following two networks of directly correlated genes were identified: (i) with implicated in estrogen homeostasis; and (ii) two ABC-efflux pumps in the immune regulation () with and . Combining LASSO Cox regression and univariate Cox regression analyses, coding for OATP5A1, an estrogen metabolite transporter located in the cytoplasm and plasma membranes of ovarian cancer cells, was identified as significant and independent prognostic factor for OS (HR = 0.68, CI 0.49-0.93; = 0.031). Furthermore, results indicated the benefits of patients with high expression by adding 5.1% to the 12.8% of the proportion of explained variation (PEV) for clinicopathological parameters known for prognostic significance (FIGO stage, age and residual tumor after debulking). Additionally, overlap with previously described signatures that indicated a more favorable prognosis for ovarian cancer patients was shown for , the network as well as . Furthermore, expression of and , which are important for PGE degradation, was associated with the non-miliary peritoneal tumor spreading. In conclusion, the present findings suggested that and the related molecules identified as potential biomarkers in HGSOC may be useful for the development of novel therapeutic strategies.
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http://dx.doi.org/10.3389/fphar.2018.00842DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6090214PMC
August 2018

Concentrated aqueous sodium chloride solution in clays at thermodynamic conditions of hydraulic fracturing: Insight from molecular dynamics simulations.

J Chem Phys 2018 Jun;148(22):222806

Department of Molecular and Mesoscopic Modelling, Institute of Chemical Process Fundamentals of the CAS, v. v. i., Prague, Czech Republic.

To address a high salinity of flow-back water during hydraulic fracturing, we use molecular dynamics (MD) simulations and study the thermodynamics, structure, and diffusion of concentrated aqueous salt solution in clay nanopores. The concentrated solution results from the dissolution of a cubic NaCl nanocrystal, immersed in an aqueous NaCl solution of varying salt concentration and confined in clay pores of a width comparable to the crystal size. The size of the nanocrystal equals to about 18 Å which is above a critical nucleus size. We consider a typical shale gas reservoir condition of 365 K and 275 bar, and we represent the clay pores as pyrophyllite and Na-montmorillonite (Na-MMT) slits. We employ the Extended Simple Point Charge (SPC/E) model for water, Joung-Cheatham model for ions, and CLAYFF for the slit walls. We impose the pressure in the normal direction and the resulting slit width varies from about 20 to 25 Å when the salt concentration in the surrounding solution increased from zero to an oversaturated value. By varying the salt concentration, we observe two scenarios. First, the crystal dissolves and its dissolution time increases with increasing salt concentration. We describe the dissolution process in terms of the number of ions in the crystal, and the crystal size and shape. Second, when the salt concentration reaches a system solubility limit, the crystal grows and attains a new equilibrium size; the crystal comes into equilibrium with the surrounding saturated solution. After crystal dissolution, we carry out canonical MD simulations for the concentrated solution. We evaluate the hydration energy, density profiles, orientation distributions, hydrogen-bond network, radial distribution functions, and in-plane diffusion of water and ions to provide insight into the microscopic behaviour of the concentrated aqueous sodium chloride solution in interlayer galleries of the slightly hydrophobic pyrophyllite and hydrophilic Na-MMT pores.
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http://dx.doi.org/10.1063/1.5017166DOI Listing
June 2018

Adsorption of amphiphilic graft copolymers in solvents selective for the grafts on a lyophobic surface: a coarse-grained simulation study.

Phys Chem Chem Phys 2018 Feb;20(9):6533-6547

Department of Informatics, Faculty of Science, J. E. Purkinje University, České mládeŽe 8, 400 96 Ústí n. Lab., Czech Republic.

The sorption of graft copolymers on surfaces attractive only for the backbone and its effect on the conformational behavior of adsorbed/desorbed chains in solvents good for the grafts and poor for the backbone was studied by coarse-grained computer simulations. It was found that the sorption and conformational behavior are very complex and are results of an intricate interplay of solvent quality (polymer-solvent interactions) and solvent strength (polymer-surface vs. solvent-surface interactions). Increasing grafting density and length of grafts protect the backbone against adsorption, but the behavior is non-trivial. A decrease in solvent quality promotes the adsorption, because it lowers the overall solubility, but the backbone collapses and the probability of backbone-surface contacts decreases, which simultaneously hinders the adsorption. The results of simulations are presented in the form of phase diagrams depicting the decisive features of the conformational and sorption behavior.
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http://dx.doi.org/10.1039/c7cp08327kDOI Listing
February 2018

Modelling aqueous solubility of sodium chloride in clays at thermodynamic conditions of hydraulic fracturing by molecular simulations.

Phys Chem Chem Phys 2017 Jun;19(25):16586-16599

Laboratory of Aerosols Chemistry and Physics, Institute of Chemical Process Fundamentals of the CAS, v. v. i., Rozvojová 135/1, 165 02 Prague 6-Suchdol, Czech Republic.

To address the high salinity of flow-back water during hydraulic fracturing, we have studied the equilibrium partitioning of NaCl and water between the bulk phase and clay pores. In shale rocks, such a partitioning can occur between fractures with a bulk-like phase and clay pores. We use an advanced Grand Canonical Monte Carlo (GCMC) technique based on fractional exchanges of dissolved ions and water molecules. We consider a typical shale gas reservoir condition of a temperature of 365 K and pressure of 275 bar, and we represent clay pores by pyrophyllite and Na-montmorillonite slits of a width ranging from about 7 to 28 Å, covering clay pores from dry clay to clay pores with a bulk-like layer in the middle of the pore. We employ the Joung-Cheatham model for ions, SPC/E model for water and CLAYFF for the clay pores. We first determine the chemical potentials for NaCl and water in the bulk phase using Osmotic Ensemble Monte Carlo simulations. The chemical potentials are then used in GCMC to simulate the adsorption of ions and water molecules in the clay pores, and in turn to predict the salt solubility in confined solutions. Besides the thermodynamic properties, we evaluate the structure and in-plane diffusion of the adsorbed fluids, and ion conductivities.
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http://dx.doi.org/10.1039/c7cp02121fDOI Listing
June 2017

Flow and aggregation of rod-like proteins in slit and cylindrical pores coated with polymer brushes: an insight from dissipative particle dynamics.

Soft Matter 2017 Feb;13(8):1634-1645

Laboratory of Aerosols Chemistry and Physics, Institute of Chemical Process Fundamentals of the CAS, v. v. i., Prague, Czech Republic and Department of Physics, Faculty of Science, J. E. Purkinje University, Ústí n. Lab., Czech Republic.

We use a meso-scale dissipative particle dynamics method to simulate the flow and aggregation of rod-like protein solutions through pores grafted with a solvent-sensitive polymer brush. The coated pores can control protein permeability and aggregation by a stretch-to-collapse conformational transition of the brush polymers in response to changes in the solvent quality. The protein solutions mimic aqueous glycoprotein solutions and proteins are represented as rod-like objects formed by coarse-grain beads. The model further employs two types of beads to represent the existence of cystein-like terminal groups in real glycoproteins and mimic the aggregation of real glycoproteins in aqueous solutions. We vary the solvent quality with respect to the brush chains and study the flow and aggregation of rod-like proteins in the slit and cylindrical pores as the brush polymers undergo the stretch-to-collapse transition. The results show that stretched brush chains close the pore, hamper proteins' flow and promote proteins' aggregation. The collapsed brush chains open the pores for proteins' flow and suppress their aggregation. Therefore, we observe more than a ten-fold reduction in the permeation rate of proteins in both pore geometries. Finally, due to pore confinement, larger proteins' aggregates are formed in the slit pore than in the cylindrical pore, while more pronounced orientation of proteins in the flow direction is seen in the cylindrical pore than in the slit pore.
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http://dx.doi.org/10.1039/c6sm02751bDOI Listing
February 2017

Complementary Glycomic Analyses of Sera Derived from Colorectal Cancer Patients by MALDI-TOF-MS and Microchip Electrophoresis.

Anal Chem 2016 10 13;88(19):9597-9605. Epub 2016 Sep 13.

Department of Chemistry, Indiana University , Bloomington, Indiana 47405, United States.

Colorectal cancer is the fourth most prevalent cancer in the United States, yet there are no reliable noninvasive early screening methods available. Serum-based glycomic profiling has the necessary sensitivity and specificity to distinguish disease states and provide diagnostic potential for this deadly form of cancer. We applied microchip electrophoresis and MALDI-TOF-MS-based glycomic procedures to 20 control serum samples and 42 samples provided by patients diagnosed with colorectal cancer. Within the identified glycans, the position of fucose units was located to quantitate possible changes of fucosyl isomeric species associated with the pathological condition. MALDI-MS data revealed several fucosylated tri- and tetra-antennary glycans which were significantly elevated in their abundance levels in the cancer samples and distinguished the control samples from the colorectal cancer cohort in the comprehensive profiles. When compared to other cancers studied previously, some unique changes appear to be associated with colorectal cancer, being primarily associated with fucosyl isomers. Through MS and microchip electrophoresis-based glycomic methods, several potential biomarkers were identified to aid in the diagnosis and differentiation of colorectal cancer. With its unique capability to resolve isomers, microchip electrophoresis can yield complementary analytical information to MS-based profiling.
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http://dx.doi.org/10.1021/acs.analchem.6b02310DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5097869PMC
October 2016

Physicochemical and biological properties of novel amide-based steroidal inhibitors of NMDA receptors.

Steroids 2017 01 17;117:52-61. Epub 2016 Aug 17.

Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i., Flemingovo nam. 2, Prague 6 - Dejvice, 166 10, Czech Republic. Electronic address:

Herein, we report a new class of amide-based inhibitors (1-4) of N-methyl-d-aspartate receptors (NMDARs) that were prepared as analogues of pregnanolone sulfate (PAS) and pregnanolone glutamate (PAG) - the steroidal neuroprotective NMDAR inhibitors. A series of experiments were conducted to evaluate their physicochemical and biological properties: (i) the inhibitory effect of compounds 3 and 4 on NMDARs was significantly improved (IC=1.0 and 1.4μM, respectively) as compared with endogenous inhibitor - pregnanolone sulfate (IC=24.6μM) and pregnanolone glutamate (IC=51.7μM); (ii) physicochemical properties (logP and logD) were calculated; (iii) Caco-2 assay revealed that the permeability properties of compounds 2 and 4 are comparable with pregnanolone glutamate; (iv) compounds 1-4 have minimal or no adverse hepatic effect; (v) compounds 1-4 cross blood-brain-barrier.
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http://dx.doi.org/10.1016/j.steroids.2016.08.010DOI Listing
January 2017

AID/APOBEC-network reconstruction identifies pathways associated with survival in ovarian cancer.

BMC Genomics 2016 08 16;17(1):643. Epub 2016 Aug 16.

Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.

Background: Building up of pathway-/disease-relevant signatures provides a persuasive tool for understanding the functional relevance of gene alterations and gene network associations in multifactorial human diseases. Ovarian cancer is a highly complex heterogeneous malignancy in respect of tumor anatomy, tumor microenvironment including pro-/antitumor immunity and inflammation; still, it is generally treated as single disease. Thus, further approaches to investigate novel aspects of ovarian cancer pathogenesis aiming to provide a personalized strategy to clinical decision making are of high priority. Herein we assessed the contribution of the AID/APOBEC family and their associated genes given the remarkable ability of AID and APOBECs to edit DNA/RNA, and as such, providing tools for genetic and epigenetic alterations potentially leading to reprogramming of tumor cells, stroma and immune cells.

Results: We structured the study by three consecutive analytical modules, which include the multigene-based expression profiling in a cohort of patients with primary serous ovarian cancer using a self-created AID/APOBEC-associated gene signature, building up of multivariable survival models with high predictive accuracy and nomination of top-ranked candidate/target genes according to their prognostic impact, and systems biology-based reconstruction of the AID/APOBEC-driven disease-relevant mechanisms using transcriptomics data from ovarian cancer samples. We demonstrated that inclusion of the AID/APOBEC signature-based variables significantly improves the clinicopathological variables-based survival prognostication allowing significant patient stratification. Furthermore, several of the profiling-derived variables such as ID3, PTPRC/CD45, AID, APOBEC3G, and ID2 exceed the prognostic impact of some clinicopathological variables. We next extended the signature-/modeling-based knowledge by extracting top genes co-regulated with target molecules in ovarian cancer tissues and dissected potential networks/pathways/regulators contributing to pathomechanisms. We thereby revealed that the AID/APOBEC-related network in ovarian cancer is particularly associated with remodeling/fibrotic pathways, altered immune response, and autoimmune disorders with inflammatory background.

Conclusions: The herein study is, to our knowledge, the first one linking expression of entire AID/APOBECs and interacting genes with clinical outcome with respect to survival of cancer patients. Overall, data propose a novel AID/APOBEC-derived survival model for patient risk assessment and reconstitute mapping to molecular pathways. The established study algorithm can be applied further for any biologically relevant signature and any type of diseased tissue.
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http://dx.doi.org/10.1186/s12864-016-3001-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4986275PMC
August 2016

Exploring the role of sphingolipid machinery during the epithelial to mesenchymal transition program using an integrative approach.

Oncotarget 2016 Apr;7(16):22295-323

Department of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria.

The epithelial to mesenchymal transition (EMT) program is activated in epithelial cancer cells and facilitates their ability to metastasize based on enhanced migratory, proliferative, anti-apoptotic, and pluripotent capacities. Given the fundamental impact of sphingolipid machinery to each individual process, the sphingolipid-related mechanisms might be considered among the most prominent drivers/players of EMT; yet, there is still limited knowledge. Given the complexity of the interconnected sphingolipid system, which includes distinct sphingolipid mediators, their synthesizing enzymes, receptors and transporters, we herein apply an integrative approach for assessment of the sphingolipid-associated mechanisms underlying EMT program. We created the sphingolipid-/EMT-relevant 41-gene/23-gene signatures which were applied to denote transcriptional events in a lung cancer cell-based EMT model. Based on defined 35-gene sphingolipid/EMT-attributed signature of regulated genes, we show close associations between EMT markers, genes comprising the sphingolipid network at multiple levels and encoding sphingosine 1-phosphate (S1P)-/ceramide-metabolizing enzymes, S1P and lysophosphatidic acid (LPA) receptors and S1P transporters, pluripotency genes and inflammation-related molecules, and demonstrate the underlying biological pathways and regulators. Mass spectrometry-based sphingolipid analysis revealed an EMT-attributed shift towards increased S1P and LPA accompanied by reduced ceramide levels. Notably, using transcriptomics data across various cell-based perturbations and neoplastic tissues (24193 arrays), we identified the sphingolipid/EMT signature primarily in lung adenocarcinoma tissues; besides, bladder, colorectal and prostate cancers were among the top-ranked. The findings also highlight novel regulatory associations between influenza virus and the sphingolipid/EMT-associated mechanisms. In sum, data propose the multidimensional contribution of sphingolipid machinery to pathological EMT and may yield new biomarkers and therapeutic targets.
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http://dx.doi.org/10.18632/oncotarget.7947DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5008362PMC
April 2016

Wetting properties of molecularly rough surfaces.

J Chem Phys 2015 Sep;143(10):104701

Laboratory of Aerosols Chemistry and Physics, Institute of Chemical Process Fundamentals of the Czech Academy of Sciences, v. v. i., 165 02 Prague 6-Suchdol, Czech Republic.

We employ molecular dynamics simulations to study the wettability of nanoscale rough surfaces in systems governed by Lennard-Jones (LJ) interactions. We consider both smooth and molecularly rough planar surfaces. Solid substrates are modeled as a static collection of LJ particles arranged in a face-centered cubic lattice with the (100) surface exposed to the LJ fluid. Molecularly rough solid surfaces are prepared by removing several strips of LJ atoms from the external layers of the substrate, i.e., forming parallel nanogrooves on the surface. We vary the solid-fluid interactions to investigate strongly and weakly wettable surfaces. We determine the wetting properties by measuring the equilibrium droplet profiles that are in turn used to evaluate the contact angles. Macroscopic arguments, such as those leading to Wenzel's law, suggest that surface roughness always amplifies the wetting properties of a lyophilic surface. However, our results indicate the opposite effect from roughness for microscopically corrugated surfaces, i.e., surface roughness deteriorates the substrate wettability. Adding the roughness to a strongly wettable surface shrinks the surface area wet with the liquid, and it either increases or only marginally affects the contact angle, depending on the degree of liquid adsorption into the nanogrooves. For a weakly wettable surface, the roughness changes the surface character from lyophilic to lyophobic due to a weakening of the solid-fluid interactions by the presence of the nanogrooves and the weaker adsorption of the liquid into the nanogrooves.
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http://dx.doi.org/10.1063/1.4930084DOI Listing
September 2015

The major birch pollen allergen Bet v 1 induces different responses in dendritic cells of birch pollen allergic and healthy individuals.

PLoS One 2015 30;10(1):e0117904. Epub 2015 Jan 30.

Department of Pathophysiology and Allergy Research; Center for Pathophysiology, Infectiology, and Immunology, Medical University of Vienna, Vienna, Austria.

Dendritic cells play a fundamental role in shaping the immune response to allergens. The events that lead to allergic sensitization or tolerance induction during the interaction of the major birch pollen allergen Bet v 1 and dendritic cells are not very well studied. Here, we analyzed the uptake of Bet v 1 and the cross-reactive celery allergen Api g 1 by immature monocyte-derived dendritic cells (iMoDCs) of allergic and normal donors. In addition, we characterized the allergen-triggered intracellular signaling and transcriptional events. Uptake kinetics, competitive binding, and internalization pathways of labeled allergens by iMoDCs were visualized by live-cell imaging. Surface-bound IgE was detected by immunofluorescence microscopy and flow cytometry. Allergen- and IgE-induced gene expression of early growth response genes and Th1 and Th2 related cytokines and chemokines were analyzed by real-time PCR. Phosporylation of signaling kinases was analyzed by Western blot. Internalization of Bet v 1 by iMoDCs of both donor groups, likely by receptor-mediated caveolar endocytosis, followed similar kinetics. Bet v 1 outcompeted Api g 1 in cell surface binding and uptake. MoDCs of allergic and healthy donors displayed surface-bound IgE and showed a pronounced upregulation of Th2 cytokine- and NFκB-dependent genes upon non-specific Fcε receptor cross-linking. In contrast to these IgE-mediated responses, Bet v 1-stimulation increased transcript levels of the Th2 cytokines IL-4 and IL-13 but not of NFκB-related genes in MoDCs of BP allergic donors. Cells of healthy donors were either unresponsive or showed elevated mRNA levels of Th1-promoting chemokines. Moreover, Bet v 1 was able to induce Erk1/2 and p38 MAPK activation in BP allergics but only a slight p38 activation in normal donors. In conclusion, our data indicate that Bet v 1 favors the activation of a Th2 program only in DCs of BP allergic individuals.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0117904PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4311984PMC
January 2016

Resveratrol enhances the chemopreventive effect of celecoxib in chemically induced breast cancer in rats.

Eur J Cancer Prev 2014 Nov;23(6):506-13

Departments of aAnimal Physiology bCell Biology, Institute of Biology and Ecology, Faculty of Science, P.J. Šafárik University in Košice, Košice cDepartment of Pathology, Jessenius Faculty of Medicine, Commenius University, Martin, Slovak Republic dDepartment of Pathophysiology and Allergy Research, Center for Pathophysiology, Medical University of Vienna eDepartment of Clinical Pharmacy and Diagnostics, University of Vienna, Vienna fInstitute of Environmental Health, Centre for Public Health, Medical University of Vienna, 1090 Vienna, Austria.

Resveratrol and celecoxib were used as chemopreventive agents in animal models of carcinogenesis, and exert antiproliferative and proapoptotic effects on cancer cells. Therefore, the aim of this study was to evaluate whether combining resveratrol with celecoxib may exert more potent anticarcinogenic effects than the single agents. Mammary carcinogenesis was initiated in 70 female Sprague-Dawley rats with N-methyl-N-nitrosourea (NMU). The chemoprevention with resveratrol, celecoxib, and their combination started 2 weeks before the first carcinogen dose and lasted until the end of the experiment. Tumor incidence and frequency, latency period, tumor volume, the expression of cyclooxygenase 2 (COX2) and growth differentiation factor 15 (GDF15), and also the formation of reactive oxygen species were analyzed using different methods. In addition, the levels of resveratrol and its metabolites in blood and selected tumor tissues were determined by high-performance liquid chromatography. Finally, the anticancer effects of the reagents were studied in the human breast cancer cell line MCF-7. Celecoxib as a single agent significantly decreased tumor frequency, prolonged tumor latency, and decreased the total number of malignant tumors compared with the NMU conditions. Tumor volume was nonsignificantly reduced (0.68±0.25 vs. 0.93±0.28 cm3). Importantly, the addition of resveratrol to celecoxib reduced tumor volume by 60% compared with celecoxib alone (from 0.68±0.25 to 0.27±0.07 cm3, P<0.05). Furthermore, the combination of resveratrol and celecoxib reduced tumor frequency by 29% compared with celecoxib alone (P=0.53). Tumor latency was not influenced by this combination compared with celecoxib alone (126.56±3.45 vs. 120.71±4.08 days). In addition, COX2 mRNA and immunoreactive protein stained on tumor sections were reduced and GDF15 protein increased significantly by the combination studied compared with the NMU conditions. In agreement with these data, a significant reduction in reactive oxygen species in blood lymphocytes of the combination was detected, which may have contributed toward the cancer-preventive effects of this application. This study showed that in NMU-induced mammary cancer in rats, the combination of resveratrol and celecoxib led to a significant reduction in all tumor parameters. In addition, in terms of tumor volume, the combination was more efficient than celecoxib as a single agent.
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http://dx.doi.org/10.1097/CEJ.0000000000000083DOI Listing
November 2014

B cells and ectopic follicular structures: novel players in anti-tumor programming with prognostic power for patients with metastatic colorectal cancer.

PLoS One 2014 6;9(6):e99008. Epub 2014 Jun 6.

Department of Pathophysiology and Allergy Research, Center of Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.

Remarkably limited information is available about biological mechanisms that determine the disease entity of metastatic colorectal cancer in the liver (CRCLM) with no good clinical parameters to estimate prognosis. For the last few years, understanding the relationship between tumor characteristics and local immune response has gained increasing attention. Given the multifaceted roles of B-cell-driven responses, we aimed to elucidate the immunological imprint of B lymphocytes at the metastatic site, the interrelation with macrophages, and their prognostic relevance. Here we present novel algorithm allowing to assess a link between the local patient-specific immunological capacity and clinical outcome. The microscopy-based imaging platform was used for automated scanning of large-scale tissue sections and subsequent qualitative and quantitative analyses of immune cell subtypes using lineage markers and single-cell recognition strategy. Results indicate massive infiltration of CD45-positive leukocytes confined to the metastatic border. We report for the first time the accumulation of CD20-positive B lymphocytes at the tumor-liver interface comprising the major population within the large CD45-positive aggregates. Strikingly, functionally active, activation-induced cytidine deaminase (AID)-positive ectopic lymphoid structures were found to be assembled within the metastatic margin. Furthermore, the CD20-based data set revealed a strong prognostic power: patients with high CD20 content and/or ectopic follicles had significantly lower risk for disease recurrence as revealed by univariate analysis (p<0.001 for both) and in models adjusted for clinicopathological variables (p<0.001 and p = 0.01, respectively), and showed prolonged overall survival. In contrast, CD68 staining-derived data set did not show an association with clinical outcome. Taken together, we nominate the magnitude of B lymphocytes, including those organized in ectopic follicles, as novel prognostic marker which is superior to clinicopathological parameters. Findings emphasize anti-tumoral role of B cell-driven mechanism(s) and thus indicate a new way of thinking about potential treatment strategies for CRCLM patients.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0099008PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4048213PMC
January 2015

The effects of mycotoxin deoxynivalenol (DON) on haematological and biochemical parameters and selected parameters of oxidative stress in piglets.

Neuro Endocrinol Lett 2013 ;34 Suppl 2:84-9

Ruminant and Swine Clinic, University of Veterinary and Pharmaceutical Sciences Brno, Czech Republic.

Objectives: Deoxynivalenol (DON) - trichothecene mycotoxin, is frequently detected in high concentrations in cereals in the temperate region of Europe. The aim of this study was to determine the effect of DON in feed on haematological and biochemical parameters and on oxidative stress in piglets.

Methods: Two concentrations of DON in feedstuff for pigs were chosen: 0.6 mg/kg (group C) and 2.0 mg/kg (group M). Twelve weaned pigs were used in each group. Pigs were fed with naturally contaminated feed for 4 weeks. On days 14, 21 and at the end of the experiment (day 28) samples of blood were taken to determine haematological parameters, plasma biochemical parameters, ceruloplasmin activity and FRAP (ferric reducing ability of plasma).

Results: The haematological variables did not show changes in response to contaminated diet with exception of the mean corpuscular volume, which was significantly decreased at the end of the experiment in the group M. A significant increase of alkaline phosphatase activity (140%, p<0.01) was found in the group M compared to the group C at the end of the experiment. A significant decrease was found on the day 21 in FRAP (85%, p<0.001) and on the day 28 in ceruloplasmin (75%, p<0.01) in the group M compared to the group C.

Conclusions: The decrease of FRAP and ceruloplasmin indicate a lowered ability of organism to scavenge reactive oxygen species. The higher concentration of DON in feedstuffs had a negative influence on the antioxidant ability of piglet's plasma.
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May 2014

Cyclin E1 (CCNE1) as independent positive prognostic factor in advanced stage serous ovarian cancer patients - a study of the OVCAD consortium.

Eur J Cancer 2014 Jan 28;50(1):99-110. Epub 2013 Oct 28.

Department of Obstetrics and Gynecology, Molecular Oncology Group, Medical University of Vienna, Vienna, Austria; Ludwig Boltzmann Cluster "Translational Oncology", General Hospital Vienna, Vienna, Austria.

Cyclin E, coded by the genes CCNE1 and CCNE2, is the main regulator for transition from G1 to S phase determining cell division. CCNE1 and CCNE2 are known oncogenes in many cancer entities. Especially CCNE1 has frequently been associated with gene amplifications in various malignancies, emphasising its role as a putative oncogene. We determined gene expression and copy number of CCNE1 and CCNE2 by quantitative polymerase chain reaction (PCR) from 172 International Federation of Obstetrics and Gynecology (FIGO) II/III/IV stage serous epithelial ovarian cancer (EOC) tissues and analysed its impact on outcome. Furthermore, whole transcriptome gene expression changes correlating with CCNE1 expression were determined by microarray technology, interpreted by Signalling Pathway Impact Analysis (SPIA), Tool for Inferring Network of Genes (TINGe), and illustrated by hive plots. Protein-protein interaction (PPI) networks were also used for the interpretation. Interestingly, and contradictory to most reports and intuitive expectations, high CCNE1 expression correlated with better overall survival (p=0.005) if corrected for usual clinicopathologic parameters and a molecular subclassification. Using different grading systems or only high graded tumours had no impact on this correlation. Copy number of CCNE1 was increased in 25% of cases which correlated highly significantly with expression but showed no impact on outcome. CCNE2 had no impact on outcomes at all. Whole genome transcriptome analysis revealed 1872 differentially expressed genes correlated to CCNE1 expression, which were significantly enriched with genes from five pathways (e.g. cell cycle and viral carcinogenesis pathway were up-regulated and the Fanconi anaemia pathway was down-regulated). High CCNE1 gene expression is a significant and independent predictor for prolonged overall survival in FIGO III/IV EOC patients. This upside down impact of CCNE1 on survival probably reflects the special characteristic of EOC with tumour dissemination in the near anaerobic peritoneal cavity as the predominant cause of death, compared to other cancer entities where distant metastasis are predominantly lethal.
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http://dx.doi.org/10.1016/j.ejca.2013.09.011DOI Listing
January 2014

The sulfatase pathway for estrogen formation: targets for the treatment and diagnosis of hormone-associated tumors.

J Drug Deliv 2013 13;2013:957605. Epub 2013 Feb 13.

Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria.

The extragonadal synthesis of biological active steroid hormones from their inactive precursors in target tissues is named "intracrinology." Of particular importance for the progression of estrogen-dependent cancers is the in situ formation of the biological most active estrogen, 17beta-estradiol (E2). In cancer cells, conversion of inactive steroid hormone precursors to E2 is accomplished from inactive, sulfated estrogens in the "sulfatase pathway" and from androgens in the "aromatase pathway." Here, we provide an overview about expression and function of enzymes of the "sulfatase pathway," particularly steroid sulfatase (STS) that activates estrogens and estrogen sulfotransferase (SULT1E1) that converts active estrone (E1) and other estrogens to their inactive sulfates. High expression of STS and low expression of SULT1E1 will increase levels of active estrogens in malignant tumor cells leading to the stimulation of cell proliferation and cancer progression. Therefore, blocking the "sulfatase pathway" by STS inhibitors may offer an attractive strategy to reduce levels of active estrogens. STS inhibitors either applied in combination with aromatase inhibitors or as novel, dual aromatase-steroid sulfatase inhibiting drugs are currently under investigation. Furthermore, STS inhibitors are also suitable as enzyme-based cancer imaging agents applied in the biomedical imaging technique positron emission tomography (PET) for cancer diagnosis.
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http://dx.doi.org/10.1155/2013/957605DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3586502PMC
March 2013

Tumor-specific expression of organic anion-transporting polypeptides: transporters as novel targets for cancer therapy.

J Drug Deliv 2013 3;2013:863539. Epub 2013 Feb 3.

Department of Medicine, Donauspital, Ludwig Boltzmann, Cluster for Translational Oncology, Vienna, Austria.

Members of the organic anion transporter family (OATP) mediate the transmembrane uptake of clinical important drugs and hormones thereby affecting drug disposition and tissue penetration. Particularly OATP subfamily 1 is known to mediate the cellular uptake of anticancer drugs (e.g., methotrexate, derivatives of taxol and camptothecin, flavopiridol, and imatinib). Tissue-specific expression was shown for OATP1B1/OATP1B3 in liver, OATP4C1 in kidney, and OATP6A1 in testis, while other OATPs, for example, OATP4A1, are expressed in multiple cells and organs. Many different tumor entities show an altered expression of OATPs. OATP1B1/OATP1B3 are downregulated in liver tumors, but highly expressed in cancers in the gastrointestinal tract, breast, prostate, and lung. Similarly, testis-specific OATP6A1 is expressed in cancers in the lung, brain, and bladder. Due to their presence in various cancer tissues and their limited expression in normal tissues, OATP1B1, OATP1B3, and OATP6A1 could be a target for tumor immunotherapy. Otherwise, high levels of ubiquitous expressed OATP4A1 are found in colorectal cancers and their metastases. Therefore, this OATP might serve as biomarkers for these tumors. Expression of OATP is regulated by nuclear receptors, inflammatory cytokines, tissue factors, and also posttranslational modifications of the proteins. Through these processes, the distribution of the transporter in the tissue will be altered, and a shift from the plasma membrane to cytoplasmic compartments is possible. It will modify OATP uptake properties and, subsequently, change intracellular concentrations of drugs, hormones, and various other OATP substrates. Therefore, screening tumors for OATP expression before therapy should lead to an OATP-targeted therapy with higher efficacy and decreased side effects.
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http://dx.doi.org/10.1155/2013/863539DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3574750PMC
February 2013

The impact of high iodine intake on thyroid function in ewes and lambs.

Neuro Endocrinol Lett 2012 ;33(5):517-24

Department of Veterinary Disciplines and Quality of Products, Faculty of Agriculture, University of South Bohemia in České Budějovice, Czech Republic.

Objectives: The objective of the study was to assess the metabolic risk of excessive dietary iodine intake in ewes and neonatal lambs.

Design: Pregnant Šumava ewes received an experimental diet containing 3.1 mg iodine per kg of dietary dry matter in Group A (control, n=13, 6 ewes and 7 lambs) and 5.1 mg iodine per kg of dietary dry matter in Group B (experimental, n=12, 6 ewes, 6 lambs) for eight months. Iodine was administered to ewes as calcium iodate. TSH in blood serum; TT3, TT4, fT3, and fT4 in blood plasma were examined in both groups of ewes and lambs to assess the risks of iodine intake above the permitted limit, as it applies to thyroid gland activity.

Results: Group B ewes showed a significant increase in TSH and TT4 only on day 1 after parturition. The highest values of TT4, TT3, and fT3 in lambs were recorded on day 1 after birth. The lowest values of fT3 and fT4 in lambs were measured on day 60 after birth with no differences observed between the groups. In lambs of Group B the lower concentration of TSH until day 3 after birth was followed by a significant increase from day 10 after birth.

Conclusion: Our results indicate a risk of postnatal hypothyroidism among lambs from pregnant and lactating ewes having a high iodine intake.
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January 2013

Activation-induced cytidine deaminase (AID) linking immunity, chronic inflammation, and cancer.

Cancer Immunol Immunother 2012 Sep 19;61(9):1591-8. Epub 2012 Apr 19.

Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria.

Activation-induced cytidine deaminase (AID) is critically involved in class switch recombination and somatic hypermutation of Ig loci resulting in diversification of antibodies repertoire and production of high-affinity antibodies and as such represents a physiological tool to introduce DNA alterations. These processes take place within germinal centers of secondary lymphoid organs. Under physiological conditions, AID is expressed predominantly in activated B lymphocytes. Because of the mutagenic and recombinogenic potential of AID, its expression and activity is tightly regulated on different levels to minimize the risk of unwanted DNA damage. However, chronic inflammation and, probably, combination of other not-yet-identified factors are able to create a microenvironment sufficient for triggering an aberrant AID expression in B cells and, importantly, in non-B-cell background. Under these circumstances, AID may target also non-Ig genes, including cancer-related genes as oncogenes, tumor suppressor genes, and genomic stability genes, and modulate both genetic and epigenetic information. Despite ongoing progress, the complete understanding of fundamental aspects is still lacking as (1) what are the crucial factors triggering an aberrant AID expression/activity including the impact of Th2-driven inflammation and (2) to what extent may aberrant AID in human non-B cells lead to abnormal cell state associated with an increased rate of genomic alterations as point mutations, small insertions or deletions, and/or recurrent chromosomal translocations during solid tumor development and progression.
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http://dx.doi.org/10.1007/s00262-012-1255-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3427704PMC
September 2012

Examination of glycan profiles from IgG-depleted human immunoglobulins facilitated by microscale affinity chromatography.

Anal Chem 2012 Apr 13;84(7):3269-77. Epub 2012 Mar 13.

Department of Chemistry and National Center for Glycomics and Glycoproteomics, Indiana University, Bloomington, Indiana 47405, USA.

Among the most important proteins involved in disease and healing processes are the immunoglobulins (Igs). Although many of the Igs have been studied through proteomics, aside from IgG, immunoglobulin carbohydrates have not been extensively characterized in different states of health. It seems valuable to develop techniques that permit an understanding of changes in the structures and abundances of Ig glycans in the context of disease onset and progression. We have devised a strategy for characterization of the glycans for the Ig classes other than IgG (i.e., A, D, E, and M) that contain kappa light chains that requires only a few microliters of biological material. First, we designed a microcolumn containing recombinant Protein L that was immobilized on macroporous silica particles. A similarly designed Protein G microcolumn was utilized to first perform an online depletion of the IgG from the sample, human blood serum, and thereby facilitate enrichment of the other Igs. Even though only 3 μL of serum was used in these analyses, we were able to recover a significantly enriched fraction of non-IgG immunoglobulins. The enrichment properties of the Protein L column were characterized using a highly sensitive label-free quantitative proteomics LC-MS/MS approach, and the glycomic profiles of enriched immunoglobulins were measured by MALDI-TOF MS. As a proof of principle, a comparative study was conducted using blood serum from a small group of lung cancer patients and a group of age-matched cancer-free individuals to demonstrate that the method is suitable for investigation of glycosylation changes in disease. The results were in agreement with a glycomic investigation of whole blood serum from a much larger lung cancer cohort.
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http://dx.doi.org/10.1021/ac203336uDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3320794PMC
April 2012

N-linked glycan structures and their expressions change in the blood sera of ovarian cancer patients.

J Proteome Res 2012 Apr 7;11(4):2282-300. Epub 2012 Mar 7.

Department of Chemistry, Indiana University, Bloomington, Indiana, United States.

Glycosylated proteins play important roles in a broad spectrum of biochemical and biological processes, and prior reports have suggested that changes in protein glycosylation occur during cancer initiation and progression. Ovarian cancer (OC) is a fatal malignancy, most commonly diagnosed after the development of metastases. Therefore, early detection of OC is key to improving survival. To this end, specific changes of the serum glycome have been proposed as possible biomarkers for different types of cancers. In this study, we extend this concept to OC. To characterize differences in total N-glycan levels, serum samples provided by 20 healthy control women were compared to those acquired from patients diagnosed with late-stage recurrent OC who were enrolled in an experimental treatment trial prior to receiving therapy (N=19) and one month later, prior to the second treatment cycle (N=11). Additionally, analyses of the N-glycans associated with IgG and characterization of the relative abundance levels of core vs outer-arm fucosylation were also performed. The N-linked glycomic profiles revealed increased abundances of tri- and tetra-branched structures with varying degrees of sialylation and fucosylation and an apparent decrease in the levels of "bisecting" glycans in OC samples compared to controls. Increased levels of a-galactosylation structures were observed on N-linked glycans derived from IgG, which were independent of the presence of fucose residues. Elevated levels of outer-arm fucosylation were also identified in the OC samples. These results allowed the control samples to be distinguished from the baseline ovarian cancer patients prior to receiving the experimental treatment. In some cases, the pre-treatment samples could be distinguished from the post-experimental treatment samples, as many of those patients showed a further progression of the disease.
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http://dx.doi.org/10.1021/pr201070kDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3321095PMC
April 2012

Activation-induced cytidine deaminase (AID)-associated multigene signature to assess impact of AID in etiology of diseases with inflammatory component.

PLoS One 2011 3;6(10):e25611. Epub 2011 Oct 3.

Department of Pathophysiology and Allergy Research, Center of Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.

Activation-induced cytidine deaminase (AID) is expressed in B cells within germinal centers and is critically involved in class switch recombination and somatic hypermutation of immunoglobulin loci. Functionally active AID can additionally be detected within ectopic follicular structures developed at sites of chronic inflammation. Furthermore, AID may target non-Ig genes in B- and non-B-cell background. Therefore, AID-associated effects are of increasing interest in disease areas such as allergy, inflammation, autoimmunity, and cancer.Pathway- or disease-relevant multigene signatures have attracted substantial attention for therapeutic target proposal, diagnostic tools, and monitoring of therapy response. To delineate the impact of AID in etiology of multifactorial diseases, we designed the AID-associated 25-gene signature. Chronic rhinosinusitis with nasal polyps was used as an inflammation-driven airway disease model; high levels of IgE have been previously shown to be present within polyp tissue. Expression levels of 16 genes were found to be modulated in polyps including AID, IgG and IgE mature transcripts which reflect AID activity; clustering algorithm revealed an AID-specific gene signature for the disease state with nasal polyp. Complementary, AID-positive ectopic lymphoid structures were detected within polyp tissues by in situ immunostaining. Our data demonstrate the class switch recombination and somatic hypermutation events likely taking place locally in the airways and in addition to the previously highlighted markers and/or targets as IL5 and IgE suggest novel candidate genes to be considered for treatment of nasal polyposis including among others IL13 and CD23. Thus, the algorithm presented herein including the multigene signature approach, analysis of co-regularities and creation of AID-associated functional network gives an integrated view of biological processes and might be further applied to assess role of altered AID expression in etiology of other diseases, in particular, aberrant immunity and cancer.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0025611PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3184987PMC
January 2012

Glibenclamide reduces proinflammatory cytokines in an ex vivo model of human endotoxinaemia under hypoxaemic conditions.

Life Sci 2011 Nov 10;89(19-20):725-34. Epub 2011 Sep 10.

Institute of Physiology, Center for Physiology and Pharmacology, Medical University of Vienna, Austria.

Aims: In vivo application of the K(ATP)-channel blocker glibenclamide can reverse endotoxin-induced hypotension, vascular hyporeactivity and shock in experimental animals. The hypothesis of the present study is, that the drug effects might not only be based on direct inhibition of K(ATP)-channels of vascular smooth muscle cells, but might also reflect reduction of shock-induced excess proinflammatory cytokines and procoagulatory molecules produced in the blood monocytes.

Main Methods: Human whole blood (normoxaemic or hypoxaemic) supplemented ex vivo with 100 ng/ml LPS was used to assess glibenclamide (3-100 μM) effects on IL-1 beta, IL-6, TNF-alpha, tissue factor, and plasminogen-activator-inhibitor-2 (PAI-2). Co-incubations with monocytes and erythrocytes and cytosolic calcium measurements were performed to reveal their purinergic intercellular interaction.

Key Findings: In heparinized blood, glibenclamide reduced LPS-induced release of IL-1 beta and TNF-alpha, tissue factor and PAI-2 mRNA in a concentration-dependent manner. When samples were subjected to strong hypoxemia using 95% N(2)/5% CO(2), these parameters became even more sensitive to the drug. No drug effect was observable in citrated blood or in isolated monocytes. IL-1 beta mRNA inhibition by glibenclamide appeared to be dependent on P2X7-receptor activation of monocytes by ATP-releasing erythrocytes during hypoxia. Cytosolic calcium values as well as the duration of calcium transients elicited by P2X7-receptor stimulation in isolated monocytes were strongly increased during hypoxia, both of which could be abolished by glibenclamide.

Significance: We conclude that the anti-inflammatory effect of glibenclamide is mainly based on the reduction of calcium entry by drug-induced depolarization of hypoxic monocytes. Thus, glibenclamide possesses a potentially beneficial shock-specific anti-inflammatory action.
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http://dx.doi.org/10.1016/j.lfs.2011.08.017DOI Listing
November 2011

Utilisation of iodine from different sources by sows and their progeny.

Neuro Endocrinol Lett 2011 ;32(4):510-7

Clinic of Ruminant and Swine Diseases, University of Veterinary and Pharmaceutical Sciences Brno, Czech Republic.

Objectives: The aim of the study was to compare iodine utilization from different sources by sows and their progeny and the levels of T3 and T4 in their serum.

Design: Pregnant Czech Large White × Landrace sows were fed with an experimental KPK diet (a diet for lactating sows) 14 days before parturition until weaning (at a piglet age of 28 days). In group A (n=50, 10 sows, 40 piglets) the feed was supplemented with KI (0.6 mg of iodine per kg of feed). Iodine enriched alga Chlorella spp. (0.6 mg of iodine per kg of feed) was used as a supplement in group B (n=50, 10 sows, 40 piglets). In group C (n=50, 10 sows, 40 piglets) the sows were injected i.m. with IFAE at a dose of 100 mg of iodine per sow. Iodine, T3 and T4 were measured in each group for comparison of iodine utilization.

Results: The use of IFAE resulted in higher serum concentrations in sows compared to KI and alga. In contrast, iodine concentrations in milk and piglets were lower when IFAE were used. We found a wide variation in the concentrations of T3 and T4 in the serum of piglets in all groups.

Conclusion: Our results indicate a good utilization of iodized oil by sows. However, its transfer into milk is lower compared to the other iodine sources.
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October 2011

Expression of organic anion-transporting polypeptides 1B1 and 1B3 in ovarian cancer cells: relevance for paclitaxel transport.

Biomed Pharmacother 2011 Sep 12;65(6):417-26. Epub 2011 Jun 12.

Department of Pathophysiology, Medical University of Vienna, 1090 Vienna, Austria.

Purpose: Ovarian cancer remains a deadly malignancy because most patients develop recurrent disease that is resistant to chemotherapy. Organic anion-transporting polypeptides (OATPs) mediate the uptake of clinically important drugs thereby effecting intracellular drug accumulation. In this study, we investigated whether OATPs may also contribute to paclitaxel transport in estrogen-responsive and estrogen-independent ovarian carcinoma cell lines and tumor tissue.

Methods: Expression of all 11 human OATPs in human ovarian cancer tissue samples and in the ovarian carcinoma cell lines OVCAR-3 and SK-OV-3 was investigated using real-time RT-PCR. Kinetic analysis of paclitaxel uptake was characterized in both cell lines and in OATP-transfected Xenopus laevis oocytes. Cytotoxicity of paclitaxel in OVCAR-3, SK-OV-3 and OATP1B1- and OATP1B3-transfected SK-OV-3 cells was performed using the CellTiter-Glo assay.

Results: OATP1B1 and OATP1B3 are active paclitaxel transporters in transfected X. laevis oocytes. Real-time RT-PCR analysis revealed expression of both OATPs in human ovarian cancer tissue specimens and in cancer cell lines. The higher mRNA levels for OATP1B1 and OATP1B3 found in SK-OV-3 cells correlated with higher initial uptake rates for paclitaxel. In addition, cytotoxicity studies with OATP1B1- and OATP1B3-transfected SK-OV-3 cells demonstrated lower IC(50) values compared to cells transfected with the empty vector.

Conclusions: Our results revealed OATP1B1 and OATP1B3 as high-affinity paclitaxel transporters expressed in ovarian cancer cell lines and tumor tissues, suggesting a role for these polypeptides in the disposition of paclitaxel during therapy.
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http://dx.doi.org/10.1016/j.biopha.2011.04.031DOI Listing
September 2011

Expression of organic anion-transporting polypeptides 1B3, 1B1, and 1A2 in human pancreatic cancer reveals a new class of potential therapeutic targets.

Onco Targets Ther 2011 Apr 8;4:27-32. Epub 2011 Apr 8.

Cancer Biobank Center of the University of Ioannina, Greece;

Background: Organic anion-transporting polypeptides (OATPs) are influx transporters that mediate intracellular uptake of selective endogenous and xenobiotic compounds. Identification of new molecular targets and discovery of novel targeted therapies is top priority for pancreatic cancer, which lacks any effective therapy.

Materials And Methods: We studied expression of OATP 1A2, 1B1, and 1B3 in pancreatic cancer tissue and in cell lines. Formalin-fixed paraffin-embedded biopsy material of 12 human pancreatic cancers was immunohistochemically assessed for protein expression of the three studied influx transporters. Immunohistochemistry was evaluated by experienced pathologists and quantified by use of an automated image analysis system. BxPC-3 and MIA PaCa-2 pancreatic cancer cell lines were used to quantify transcripts of OATP 1B1 and 1B3.

Results: OATP 1A2, 1B1, and 1B3 proteins were found ubiquitously expressed in all studied cases. Quantification performed by HistoQuest system revealed that mean intensity was 53 for 1A2, 45 for 1B1, and 167 for OATP 1B1/1B3 on a range scale 0-250 units. At mRNA level, 1B1 and 1B3 were overexpressed in both studied cancer cell lines but not in normal pancreatic tissue.

Conclusion: OATPs 1A2, 1B1, and 1B3 are highly expressed in pancreatic adenocarcinoma. We suggest that expression of these transporters in pancreatic cancer justify research efforts towards discovery of novel therapeutics targeting OATPs.
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http://dx.doi.org/10.2147/OTT.S16706DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3084305PMC
April 2011