Omics Center Graz

Graz, Austria

Omics Center Graz

Graz, Austria
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Triebl A.,Medical University of Graz | Hartler J.,Medical University of Graz | Hartler J.,University of Graz | Hartler J.,Omics Center Graz | And 3 more authors.
Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids | Year: 2017

Over the last two decades, lipidomics has evolved into an 'omics' technology pari passu with benchmarking 'omics' technologies, such as genomics or proteomics. The driving force behind this development was a constant advance in mass spectrometry and related technologies. The aim of this opinion article is to give the interested reader a concise but still comprehensive overview about the technological state of the art in lipidomics, current challenges and perspectives for future development. As such, this article guides through the whole workflow of lipidomics, from sampling to data analysis. This article is part of a Special Issue entitled: BBALIP_Lipidomics Opinion Articles edited by Sepp Kohlwein. © 2017 Elsevier B.V.

Eicher B.,University of Graz | Heberle F.A.,University of Tennessee at Knoxville | Heberle F.A.,Oak Ridge National Laboratory | Marquardt D.,University of Graz | And 4 more authors.
Journal of Applied Crystallography | Year: 2017

Low- and high-resolution models describing the internal transbilayer structure of asymmetric lipid vesicles have been developed. These models can be used for the joint analysis of small-angle neutron and X-ray scattering data. The models describe the underlying scattering length density/electron density profiles either in terms of slabs or through the so-called scattering density profile, previously applied to symmetric lipid vesicles. Both models yield structural details of asymmetric membranes, such as the individual area per lipid, and the hydrocarbon thickness of the inner and outer bilayer leaflets. The scattering density profile model, however, comes at a cost of increased computational effort but results in greater structural resolution, showing a slightly lower packing of lipids in the outer bilayer leaflet of ∼120 nm diameter palmitoyloleoyl phosphatidylcholine (POPC) vesicles, compared to the inner leaflet. Analysis of asymmetric dipalmitoyl phosphatidylcholine/POPC vesicles did not reveal evidence of transbilayer coupling between the inner and outer leaflets at 323 K, i.e. above the melting transition temperature of the two lipids. © 2017 Barbara Eicher et al.

Triebl A.,Medical University of Graz | Trotzmuller M.,Medical University of Graz | Hartler J.,University of Graz | Stojakovic T.,Medical University of Graz | And 2 more authors.
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences | Year: 2017

An improved approach for selective and sensitive identification and quantitation of lipid molecular species using reversed phase chromatography coupled to high resolution mass spectrometry was developed. The method is applicable to a wide variety of biological matrices using a simple liquid-liquid extraction procedure. Together, this approach combines multiple selectivity criteria: Reversed phase chromatography separates lipids according to their acyl chain length and degree of unsaturation and is capable of resolving positional isomers of lysophospholipids, as well as structural isomers of diacyl phospholipids and glycerolipids. Orbitrap mass spectrometry delivers the elemental composition of both positive and negative ions with high mass accuracy. Finally, automatically generated tandem mass spectra provide structural insight into numerous glycerolipids, phospholipids, and sphingolipids within a single run. Calibration showed linearity ranges of more than four orders of magnitude, good values for accuracy and precision at biologically relevant concentration levels, and limits of quantitation of a few femtomoles on column. Hundreds of lipid molecular species were detected and quantified in three different biological matrices, which cover well the wide variety and complexity of various model organisms in lipidomic research. Together with a software package, this method is a prime choice for global lipidomic analysis of even the most complex biological samples. © 2017 Elsevier B.V.

Lebrun-Julien F.,ETH Zurich | Bachmann L.,ETH Zurich | Norrmen C.,ETH Zurich | Trotzmuller M.,Medical University of Graz | And 6 more authors.
Journal of Neuroscience | Year: 2014

The mammalian target of rapamycin (mTOR) pathway integrates multiple signals and regulates crucial cell functions via the molecular complexesmTORC1and mTORC2. These complexes are functionally dependent on their raptor (mTORC1) or rictor (mTORC2) subunits. mTOR has been associated with oligodendrocyte differentiation and myelination downstream of the PI3K/Akt pathway, but the functional contributions of individual complexes are largely unknown.Weshow, by oligodendrocyte-specific genetic deletion of Rptor and/or Rictor in the mouse, that CNS myelination is mainly dependent on mTORC1 function, with minor mTORC2 contributions. Myelinassociated lipogenesis and protein gene regulation are strongly reliant on mTORC1. We found that also oligodendrocyte-specific overactivation of mTORC1, via ablation of tuberous sclerosis complex 1 (TSC1), causes hypomyelination characterized by downregulation of Akt signaling and lipogenic pathways. Our data demonstrate that a delicately balanced regulation of mTORC1 activation and action in oligodendrocytes is essential for CNS myelination, which has practical overtones for understanding CNS myelin disorders. © 2014 the authors.

Triebl A.,Medical University of Graz | Trotzmuller M.,Medical University of Graz | Trotzmuller M.,Omics Center Graz | Eberl A.,Joanneum Research | And 5 more authors.
Journal of Chromatography A | Year: 2014

A method for a highly selective and sensitive identification and quantitation of lysophosphatidic acid (LPA) and phosphatidic acid (PA) molecular species was developed using hydrophilic interaction liquid chromatography (HILIC) followed by negative-ion electrospray ionization high resolution mass spectrometry.Different extraction methods for the polar LPA and PA species were compared and a modified Bligh & Dyer extraction by addition of 0.1. M hydrochloric acid resulted in a ≈1.2-fold increase of recovery for the 7 PA and a more than 15-fold increase for the 6 LPA molecular species of a commercially available natural mix compared to conventional Bligh & Dyer extraction. This modified Bligh & Dyer extraction did not show any artifacts resulting from hydrolysis of natural abundant phospholipids.The developed HILIC method is able to separate all PA and LPA species from major polar membrane lipid classes which might have suppressive effects on the minor abundant lipid classes of interest. The elemental compositions of intact lipid species are provided by the high mass resolution of 100,000 and high mass accuracy below 3. ppm of the Orbitrap instrument. Additionally, tandem mass spectra were generated in a parallel data dependent acquisition mode in the linear ion trap to provide structural information at molecular level. Limits of quantitation were identified at 45. fmol on column and the dynamic range reaches 20. pmol on column, covering the range of natural abundance well.By applying the developed method to mouse brain it can be shown that phosphatidic acid contains less unsaturated fatty acids with PA 34:1 and PA 36:1 as the major species. In contrast, for LPA species a high content of polyunsaturated fatty acids (LPA 20:4 and LPA 22:6) was quantified. © 2014 Elsevier B.V.

Kavscek M.,University of Graz | Bhutada G.,University of Graz | Madl T.,Medical University of Graz | Madl T.,Omics Center Graz | And 3 more authors.
BMC Systems Biology | Year: 2015

Background: Yarrowia lipolytica is a non-conventional yeast that is extensively investigated for its ability to excrete citrate or to accumulate large amounts of storage lipids, which is of great significance for single cell oil production. Both traits are thus of interest for basic research as well as for biotechnological applications but they typically occur simultaneously thus lowering the respective yields. Therefore, engineering of strains with high lipid content relies on novel concepts such as computational simulation to better understand the two competing processes and to eliminate citrate excretion. Results: Using a genome-scale model (GSM) of baker's yeast as a scaffold, we reconstructed the metabolic network of Y. lipolytica and optimized it for use in flux balance analysis (FBA), with the aim to simulate growth and lipid production phases of this yeast. We validated our model and found the predictions of the growth behavior of Y. lipolytica in excellent agreement with experimental data. Based on these data, we successfully designed a fed-batch strategy to avoid citrate excretion during the lipid production phase. Further analysis of the network suggested that the oxygen demand of Y. lipolytica is reduced upon induction of lipid synthesis. According to this finding we hypothesized that a reduced aeration rate might induce lipid accumulation. This prediction was indeed confirmed experimentally. In a fermentation combining these two strategies lipid content of the biomass was increased by 80 %, and lipid yield was improved more than four-fold, compared to standard conditions. Conclusions: Genome scale network reconstructions provide a powerful tool to predict the effects of genetic modifications and the metabolic response to environmental conditions. The high accuracy and the predictive value of a newly reconstructed GSM of Y. lipolytica to optimize growth conditions for lipid accumulation are demonstrated. Based on these findings, further strategies for engineering Y. lipolytica towards higher efficiency in single cell oil production are discussed. © 2015 Kavšček et al.

Knittelfelder O.L.,University of Graz | Weberhofer B.P.,University of Graz | Eichmann T.O.,University of Graz | Kohlwein S.D.,University of Graz | And 2 more authors.
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences | Year: 2014

A new UPLC-based untargeted lipidomic approach using a qTOF hybrid mass spectrometer is introduced. The applied binary gradient enables separations of lipid species including constitutional isomeric compounds and low abundant lipid classes such as phosphatidic acid (PA). Addition of phosphoric acid to the solvents improves peak shapes for acidic phospholipids. MSE scans allow simultaneous acquisition of full scan data and collision induced fragmentation to improve identification of lipid classes and to obtain structural information. The method was used to investigate the lipidome of yeast. © 2014 The Authors.

Deutsch A.J.A.,Medical University of Graz | Rinner B.,Medical University of Graz | Wenzl K.,Medical University of Graz | Pichler M.,Medical University of Graz | And 18 more authors.
Blood | Year: 2014

NR4A1 (Nur77) and NR4A3 (Nor-1) function as tumor suppressor genes as demonstrated by the rapid development of acute myeloid leukemia in the NR4A1 and NR4A3 knockout mouse. The aim of our study was to investigate NR4A1 and NR4A3 expression and function in lymphoid malignancies. We found a vastly reduced expression of NR4A1 and NR4A3 in chronic lymphocytic B-cell leukemia (71%), in follicular lymphoma (FL, 70%), and in diffuse large B-cell lymphoma (DLBCL, 74%). In aggressive lymphomas (DLBCL and FL grade 3), low NR4A1 expression was significantly associated with a non-germinal center B-cell subtype and with poor overall survival. To investigate the function of NR4A1 in lymphomas, we overexpressed NR4A1 in several lymphoma cell lines. Overexpression of NR4A1 led to a higher proportion of lymphoma cells undergoing apoptosis. To test the tumor suppressor function of NR4A1 in vivo, the stable lentiviral-transduced SuDHL4 lymphoma cell line harboring an inducible NR4A1 construct was further investigated in xenografts. Induction of NR4A1 abrogated tumor growth in the NSG mice, in contrast to vector controls, which formed massive tumors. Our data suggest that NR4A1 has proapoptotic functions in aggressive lymphoma cells and define NR4A1 as a novel gene with tumor suppressor properties involved in lymphomagenesis. © 2014 by The American Society of Hematology.

Birner-Gruenberger R.,Medical University of Graz | Birner-Gruenberger R.,Omics Center Graz | Schittmayer M.,Medical University of Graz | Schittmayer M.,Omics Center Graz | And 2 more authors.
Progress in Lipid Research | Year: 2014

Although the epidemiology of high-density lipoprotein (HDL) cholesterol and cardiovascular risk has been consistent, pharmacologic interventions to increase HDL-cholesterol by delaying HDL catabolism did not translate into reduction in cardiovascular risk. HDL particles are small, protein-rich when compared to other plasma lipoprotein classes. Latest progresses in proteomics technology have dramatically increased our understanding of proteins carried by HDL. In addition to proteins with well-established functions in lipid transport, iron transport proteins, members of the complement pathway, and proteins involved in immune function and acute phase response were repeatedly identified on HDL particles. With the unraveling of the complexity of the HDL proteome, different laboratories have started to monitor its changes in various disease states. In addition, dynamic aspects of HDL subgroups are being discovered. These recent studies clearly illustrate the promise of HDL proteomics for deriving new biomarkers for disease diagnosis and to measure the effectiveness of current and future treatment regimens. This review summarizes recent advances in proteomics and lipidomics helping to understand HDL function in health and disease. © 2014 The Authors. Published by Elsevier Ltd.

Tomazic P.V.,Medical University of Graz | Birner-Gruenberger R.,Medical University of Graz | Birner-Gruenberger R.,Austrian Center of Industrial Biotechnology | Birner-Gruenberger R.,Omics Center Graz | And 6 more authors.
Journal of Allergy and Clinical Immunology | Year: 2014

Background Nasal mucus is the first-line defense barrier against (aero-) allergens. However, its proteome and function have not been clearly investigated. Objective The role of nasal mucus in the pathophysiology of allergic rhinitis was investigated by analyzing its proteome in patients with allergic rhinitis (n = 29) and healthy control subjects (n = 29). Methods Nasal mucus was collected with a suction device, tryptically digested, and analyzed by using liquid chromatography-tandem mass spectrometry. Proteins were identified by searching the SwissProt database and annotated by collecting gene ontology data from databases and existing literature. Gene enrichment analysis was performed by using Cytoscape/BINGO software tools. Proteins were quantified with spectral counting, and selected proteins were confirmed by means of Western blotting. Results In total, 267 proteins were identified, with 20 (7.5%) found exclusively in patients with allergic rhinitis and 25 (9.5%) found exclusively in healthy control subjects. Five proteins were found to be significantly upregulated in patients with allergic rhinitis (apolipoprotein A-2 [APOA2], 9.7-fold; α2-macroglobulin [A2M], 4.5-fold; apolipoprotein A-1 [APOA1], 3.2-fold; α1-antitrypsin [SERPINA1], 2.5-fold; and complement C3 [C3], 2.3-fold) and 5 were found to be downregulated (antileukoproteinase [SLPI], 0.6-fold; WAP 4-disulfide core domain protein [WFDC2], 0.5-fold; haptoglobin [HP], 0.7-fold; IgJ chain [IGJ], 0.7-fold; and Ig hc V-III region BRO, 0.8-fold) compared with levels seen in healthy control subjects. Conclusion The allergic rhinitis mucus proteome shows an enhanced immune response in which apolipoproteins might play an important role. Furthermore, an imbalance between cysteine proteases and antiproteases could be seen, which negatively affects epithelial integrity on exposure to pollen protease activity. This reflects the important role of mucus as the first-line defense barrier against allergens. © 2013 American Academy of Allergy, Asthma and Immunology.

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