Heimo Wolinski

Medical University of Graz, Gratz, Styria, Austria

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Publications (55)223.41 Total impact

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    Full-text · Dataset · Dec 2015
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    Full-text · Dataset · Dec 2015
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    ABSTRACT: Peripheral leukocytes aggravate brain damage by releasing cytotoxic mediators that compromise blood-brain barrier function. One of the oxidants released by activated leukocytes is hypochlorous acid (HOCl) that is formed via the myeloperoxidase-H2O2-chloride system. The reaction of HOCl with the endogenous plasmalogen pool of brain endothelial cells results in the generation of 2-chlorohexadecanal (2-ClHDA), a toxic, lipid-derived electrophile that induces blood-brain barrier dysfunction in vivo. Here, we synthesized an alkynyl-analogue of 2-ClHDA, 2-chlorohexadec-15-yn-1-al (2-ClHDyA) to identify potential protein targets in the human brain endothelial cell line hCMEC/D3. Similar to 2-ClHDA, 2-ClHDyA administration reduced cell viability/metabolic activity, induced processing of pro-caspase-3 and PARP, and led to endothelial barrier dysfunction at low micromolar concentrations. Protein-2-ClHDyA adducts were fluorescently labeled with tetramethylrhodamine azide (N3-TAMRA) by 1,3-dipolar cycloaddition in situ, which unveiled a preferential accumulation of 2-ClHDyA adducts in mitochondria, the Golgi, endoplasmic reticulum, and endosomes. Thirty-three proteins that are subject to 2-ClHDyA-modification in hCMEC/D3 cells were identified by mass spectrometry. Identified proteins include cytoskeletal components that are central to tight junction patterning, metabolic enzymes, induction of the oxidative stress response, and electrophile damage to the caveolar/endosomal Rab machinery. A subset of the targets was validated by a combination of N3-TAMRA click chemistry and specific antibodies by fluorescence microscopy. This novel alkyne analogue is a valuable chemical tool to identify cellular organelles and protein targets of 2-ClHDA-mediated damage in settings where myeloperoxidase-derived oxidants may play a disease-propagating role.
    Full-text · Article · Nov 2015 · Free Radical Biology and Medicine
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    ABSTRACT: α/β Hydrolase domain-containing 6 (ABHD6) can act as monoacylglycerol (MG) hydrolase and is believed to play a role in endocannabinoid signaling as well as in the pathogenesis of obesity and liver steatosis. Yet, the mechanistic link between gene function and disease is incompletely understood. Here, we aimed to further characterize the role of ABHD6 in lipid metabolism. We show that mouse and human ABHD6 degrade bis(monoacylglycero)phosphate (BMP) with high specific activity. BMP, also known as lysobisphosphatidic acid (LBPA), is enriched in late endosomes/lysosomes, where it plays a key role in the formation of intraluminal vesicles and in lipid sorting. Up to now, little is known about the catabolism of this lipid. Our data demonstrate that ABHD6 is responsible for ~ 90% of the BMP hydrolase activity detected in liver and that knockdown of ABHD6 increases hepatic BMP levels. Tissue fractionation and live cell imaging experiments revealed that ABHD6 co-localizes with late endosomes/lysosomes. The enzyme is active at cytosolic pH and lacks acid hydrolase activity implicating that it degrades BMP exported from acidic organelles or de novo formed BMP. In conclusion, our data suggest that ABHD6 controls BMP catabolism and is therefore part of the late endosomal/lysosomal lipid sorting machinery.
    Full-text · Article · Oct 2015 · Journal of Biological Chemistry
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    ABSTRACT: Yeast Fld1 and Ldb16 resemble mammalian seipin, implicated in neutral lipid storage. Both proteins form a complex at the endoplasmic reticulum-lipid droplet (LD) interface. Malfunction of this complex either leads to LD clustering or to the generation of supersized LD (SLD) in close vicinity to the nuclear envelope, in response to altered phospholipid (PL) composition. We show that similar to mutants lacking Fld1, deletion of LDB16 leads to abnormal proliferation of a subdomain of the nuclear envelope, which is tightly associated with clustered LD. The human lipin-1 ortholog, the PAH1 encoded phosphatidic acid (PA) phosphatase, and its activator Nem1 are highly enriched at this site. The specific accumulation of PA-binding marker proteins indicates a local enrichment of PA in the fld1 and ldb16 mutants. Furthermore, we demonstrate that clustered LD in fld1 or ldb16 mutants are transformed to SLD if phosphatidylcholine synthesis is compromised by additional deletion of the phosphatidylethanolamine methyltransferase, Cho2. Notably, treatment of wild-type cells with oleate induced a similar LD clustering and nuclear membrane proliferation phenotype as observed in fld1 and ldb16 mutants. These data suggest that the Fld1-Ldb16 complex affects PA homeostasis at an LD-forming subdomain of the nuclear envelope. Lack of Fld1-Ldb16 leads to locally elevated PA levels that induce an abnormal proliferation of nER membrane structures and the clustering of associated LD. We suggest that the formation of SLD is a consequence of locally altered PL metabolism at this site. Copyright © 2015. Published by Elsevier B.V.
    Full-text · Article · Aug 2015 · Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids
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    ABSTRACT: A key component of eukaryotic lipid homeostasis is the esterification of sterols with fatty acids by sterol O-acyltransferases (SOATs). The esterification reactions are allosterically activated by their sterol substrates, the majority of which accumulate at the plasma membrane. We demonstrate that in yeast, sterol transport from the plasma membrane to the site of esterification is associated with the physical interaction of the major SOAT, acyl-coenzyme A:cholesterol acyltransferase (ACAT)-related enzyme (Are)2p, with 2 plasma membrane ATP-binding cassette (ABC) transporters: Aus1p and Pdr11p. Are2p, Aus1p, and Pdr11p, unlike the minor acyltransferase, Are1p, colocalize to sterol and sphingolipid-enriched, detergent-resistant microdomains (DRMs). Deletion of either ABC transporter results in Are2p relocalization to detergent-soluble membrane domains and a significant decrease (53-36%) in esterification of exogenous sterol. Similarly, in murine tissues, the SOAT1/Acat1 enzyme and activity localize to DRMs. This subcellular localization is diminished upon deletion of murine ABC transporters, such as Abcg1, which itself is DRM associated. We propose that the close proximity of sterol esterification and transport proteins to each other combined with their residence in lipid-enriched membrane microdomains facilitates rapid, high-capacity sterol transport and esterification, obviating any requirement for soluble intermediary proteins.-Gulati, S., Balderes, D., Kim, C., Guo, Z. A., Wilcox, L., Area-Gomez, E., Snider, J., Wolinski, H., Stagljar, I., Granato, J. T., Ruggles, K. V., DeGiorgis, J. A., Kohlwein, S. D., Schon, E. A., Sturley, S. L. ATP-binding cassette transporters and sterol O-acyltransferases interact at membrane microdomains to modulate sterol uptake and esterification. © FASEB.
    Full-text · Article · Jul 2015 · The FASEB Journal
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    ABSTRACT: In the multidisciplinary field of heart research it is of utmost importance to identify accurate myocardium material properties for the description of phenomena such as mechano-electric feedback or heart wall thickening. A rationally-based material model is required to understand the highly nonlinear mechanics of complex structures such as the passive myocardium under different loading conditions. Unfortunately, to date there are no experimental data of human heart tissues available to estimate material parameters and to develop adequate material models. This study aimed to determine biaxial extension and triaxial shear properties and the underlying microstructure of the passive human ventricular myocardium. Using new state-of-the-art equipment, planar biaxial extension tests were performed to determine the biaxial extension properties of the passive ventricular human myocardium. Shear properties of the myocardium were examined by triaxial simple shear tests performed on small cubic specimens excised from an adjacent region of the biaxial extension specimens. The three-dimensional microstructure was investigated through second-harmonic generation (SHG) microscopy on optically cleared tissues, which emphasized the 3D orientation and dispersion of the myofibers and adjacent collagen fabrics. The results suggest that the passive human LV myocardium under quasi-static and dynamic multiaxial loadings is a nonlinear, anisotropic (orthotropic), viscoelastic and history-dependent soft biological material undergoing large deformations. Material properties of the tissue components along local microstructural axes drive the nonlinear and orthotropic features of the myocardium. SHG microscopy investigation revealed detailed information about the myocardial microstructure due to its high resolution. It enabled the identification of structural parameters such as the fiber and the sheet orientations and corresponding dispersions. With this complete set of material data, a sophisticated material model and associated material parameters can be defined for a better description of the biomechanical response of the ventricular myocardium in humans. Such a model will lead to more accurate computational simulations to better understand the fundamental underlying ventricular mechanics, a step needed in the improvement of medical treatment of heart diseases. Copyright © 2015. Published by Elsevier Ltd.
    Full-text · Article · Jun 2015 · Acta biomaterialia
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    ABSTRACT: Monoglyceride lipase (MGL) degrades 2-arachidonoyl glycerol (2-AG), an endogenous agonist of cannabinoid receptors (CB1/2 ). Since CB1 receptor is involved in the control of gut function, we investigated the effects of pharmacological inhibition and genetic deletion of MGL on intestinal motility. Furthermore, we were interested whether defective 2-AG degradation affects μ-opioid receptor (μ-receptor) signaling, a parallel pathway regulating gut motility. Gut motility was investigated by monitoring Evan's blue transit and colonic bead propulsion in response to MGL inhibition and CB1 receptor or μ-receptor stimulation. Ileal contractility was investigated by electrical field stimulation. CB1 receptor expression in ileum and colon was studied by immunohistochemical analyses. Pharmacological inhibition of MGL slows down whole gut transit in a CB1 receptor-dependent manner. Conversely, genetic deletion of MGL does not affect gut transit despite increased 2-AG levels. Notably, MGL-deficiency causes complete insensitivity to CB1 receptor agonist-mediated inhibition of whole gut transit and ileal contractility suggesting local desensitization of CB1 receptors. Accordingly, immunohistochemical analyses of myenteric ganglia of MGL-deficient mice revealed that CB1 receptors are trapped in endocytic vesicles. Finally, MGL-deficient mice display accelerated colonic propulsion and respond hypersensitive to μ-opioid receptor agonist-mediated inhibition of colonic motility. This phenotype can be reproduced by chronic pharmalogical inhibition of MGL. Constantly elevated 2-AG levels induce severe desensitization of intestinal CB1 receptors and increased sensitivity to μ-receptor-mediated inhibition of colonic motility. These changes should be considered for the use of cannabinoid-based drugs in the therapy of gastrointestinal diseases. This article is protected by copyright. All rights reserved.
    Full-text · Article · Jun 2015 · British Journal of Pharmacology
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    ABSTRACT: Pex11 is a peroxin that regulates the number of peroxisomes in eukaryotic cells. Recently it was found that a mutation in one of the three mammalian paralogs, PEX11β, results in a neurological disorder. The molecular function of Pex11, however, is not known. Saccharomyces cerevisiae Pex11 has been shown to recruit to peroxisomes the mitochondrial fission machinery, thus enabling proliferation of peroxisomes. This process is essential for efficient fatty acid β-oxidation. In this study, we used high-content microscopy on a genome-wide scale to determine the subcellular localization pattern of yeast Pex11 in all non-essential gene deletion mutants, and in temperature sensitive essential gene mutants. Pex11 localization and morphology of peroxisomes was profoundly affected by mutations in 104 different genes that were functionally classified. A group of genes encompassing MDM10, MDM12 and MDM34 that encode the mitochondrial and cytosolic components of the ERMES complex was analyzed in greater detail. Deletion of these genes caused a specifically altered Pex11 localization pattern, whereas deletion of MMM1, the gene encoding the fourth, endoplasmic reticulum-associated component of the complex, did not result in an altered Pex11 localization or peroxisome morphology phenotype. Moreover, we found that Pex11 and Mdm34 physically interact and that Pex11 plays a role in establishing the contact sites between peroxisomes and mitochondria through the ERMES complex. Based on these results we propose that the mitochondrial/cytosolic components of the ERMES complex establish a direct interaction between mitochondria and peroxisomes through Pex11. Copyright © 2015. Published by Elsevier Ltd.
    Full-text · Article · Mar 2015 · Journal of Molecular Biology
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    ABSTRACT: Hepatic stellate cells (HSCs) store triglycerides (TGs) and retinyl esters (REs) in cytosolic lipid droplets. RE stores are degraded following retinoid starvation or in response to pathogenic stimuli resulting in HSC activation. At present, the major enzymes catalyzing lipid degradation in HSCs are unknown. In this study, we investigated whether adipose triglyceride lipase (ATGL) is involved in RE catabolism of HSCs. Additionally, we compared the effects of ATGL-deficiency and hormone-sensitive lipase (HSL)-deficiency, a known RE hydrolase (REH), on RE stores in liver and adipose tissue. We show that ATGL degrades REs even in the presence of TGs implicating that these substrates compete for ATGL binding. REH activity was stimulated and inhibited by comparative gene identification-58 and G0/G1 switch gene-2, respectively, the physiological regulators of ATGL activity. In cultured primary murine HSCs, pharmacological inhibition of ATGL, but not HSL, increased RE accumulation. In mice globally lacking ATGL or HSL, RE contents in white adipose tissue were decreased or increased, respectively, while plasma retinol and liver RE levels remained unchanged. In conclusion, our study shows that ATGL acts as REH in HSCs promoting the degradation of RE stores in addition to its established function as TG lipase. HSL is the predominant REH in adipocytes but does not affect lipid mobilization in HSCs. Copyright © 2015. Published by Elsevier B.V.
    Full-text · Article · Feb 2015 · Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids
  • Heimo Wolinski · Sepp D Kohlwein
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    ABSTRACT: In spite of some progress in understanding the molecular basis of lipid-associated disorders, major questions about the regulation of synthesis and degradation of lipids and the interaction of these processes with other aspects of cellular physiology are still unresolved. Studies in reference organisms such as various yeast species, the fruit fly Drosophila melanogaster, or the nematode Caenorhabditis elegans complement efforts in mouse models as well as clinical studies in humans to address these questions. Imaging techniques play a pivotal role in understanding lipid droplet biology, and the implementation of imaging-based high-content screens of mutant collections has led to the identification of novel molecular players. This study focuses on novel fluorescent probes as well as spectroscopic imaging techniques to investigate lipid droplet formation and turnover in yeast. The application and limitations of such techniques in understanding lipid storage and turnover are discussed.
    No preview · Article · Feb 2015 · Methods in molecular biology (Clifton, N.J.)
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    ABSTRACT: To obtain mechanistic insights into the cross talk between lipolysis and autophagy, two key metabolic responses to starvation, we screened the autophagy-inducing potential of a panel of fatty acids in human cancer cells. Both saturated and unsaturated fatty acids such as palmitate and oleate, respectively, triggered autophagy, but the underlying molecular mechanisms differed. Oleate, but not palmitate, stimulated an autophagic response that required an intact Golgi apparatus. Conversely, autophagy triggered by palmitate, but not oleate, required AMPK, PKR and JNK1 and involved the activation of the BECN1/PIK3C3 lipid kinase complex. Accordingly, the downregulation of BECN1 and PIK3C3 abolished palmitate-induced, but not oleate-induced, autophagy in human cancer cells. Moreover, Becn1+/− mice as well as yeast cells and nematodes lacking the ortholog of human BECN1 mounted an autophagic response to oleate, but not palmitate. Thus, unsaturated fatty acids induce a non-canonical, phylogenetically conserved, autophagic response that in mammalian cells relies on the Golgi apparatus.
    No preview · Article · Jan 2015 · The EMBO Journal
  • A. Kuijper · B. Heise · Y. Zhou · L. He · H. Wolinski · S. Kohlwein
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    ABSTRACT: With the huge amount of cell images produced in bio-imaging, automatic methods for segmentation are needed in order to evaluate the content of the images with respect to types of cells and their sizes. Traditional PDE-based methods using level-sets can perform automatic segmentation, but do not perform well on images with clustered cells containing sub-structures. We present two modifications for popular methods and show the improved results.
    No preview · Article · Jan 2015
  • Heimo Wolinski · Sepp D Kohlwein
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    ABSTRACT: Microscopic imaging techniques play a pivotal role in the life sciences. Here we describe labeling and imaging methods for live yeast cell imaging. Yeast is an excellent reference organism for biomedical research to investigate fundamental cellular processes, and has gained great popularity also for large-scale imaging-based screens. Methods are described to label live yeast cells with organelle-specific fluorescent dyes or GFP-tagged proteins, and how cells are maintained viable over extended periods of time during microscopy. We point out common pitfalls and potential microscopy artifacts arising from inhomogeneous labeling and depending on cellular physiology. Application and limitation of bleaching techniques to address dynamic processes in the yeast cell are described.
    No preview · Article · Sep 2014 · Methods in Molecular Biology
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    ABSTRACT: In the multidisciplinary field of heart research it is of utmost importance, for the description of phenomena like mechanoelectric feedback or heart wall thickening, to identify accurate myocardium material properties. Therefore this study aims to determine biaxial tensile and triaxial shear properties of the passive human ventricular myocardium. This novel combination of biaxial and shear testing, together with the investigation of the myocardial microstructure, will yield new innovative and essential information about material properties to fulfil the short term goals of constructing realistic myocardial models capable of capturing the mechanics of the heart, as well as aiding the long term goals of improving methods of medical treatment and quality of life for people suffering from heart diseases. For the biaxial tests, squared specimens (25x25x2mm) were prepared with their sides aligned with the fiber and cross-fiber axis. During the experiments, the specimens were submerged in a cardioplegic solution at physiological conditions and different stretch ratios were applied consecutively. For the triaxial shear testing, three adjoining cubic specimens (4x4x4mm) were prepared with their sides aligned according to the fiber axis, sheet axis and sheet-normal axis. Three cycles of sinusoidal simple shear (0.1-0.5 in 0.1 steps of specimen thickness) were applied to each cubic specimens in two orthogonal directions. A novel combination of optical clearing and multiphoton microscopy was utilized to explore the 3D microstructure of the tissue emphasizing the 3-D orientation and dispersion of the muscle fibers and adjacent collagen fabrics. The tissue showed pronounced nonlinear and highly orientation dependent behavior. The donor's age was greatly influencing the mechanical behavior of the myocardial tissue. Microstructural investigations affirmed an orthotropic composition of the investigated tissue and showed highly aligned myofibers with small dispersion in the healthy human myocardium. An invariant-based constitutive model showed the ability to give a good representation of both the biaxial tensile and the triaxial shear responses. The material data from this study is intended to be used in numerical (Finite Element) simulations for better understanding of fundamental underlying ventricular mechanics, a step needed in the improvement of medical treatment of heart diseases.
    Preview · Article · Jul 2014 · Cardiovascular Research
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    ABSTRACT: Membrane phospholipids typically contain fatty acids (FAs) of 16 and 18 carbon atoms. This particular chain length is evolutionarily highly conserved and presumably provides maximum stability and dynamic properties to biological membranes in response to nutritional or environmental cues. Here, we show that the relative proportion of C16 versus C18 FAs is regulated by the activity of acetyl-CoA carboxylase (Acc1), the first and rate-limiting enzyme of FA de novo synthesis. Acc1 activity is attenuated by AMPK/Snf1-dependent phosphorylation, which is required to maintain an appropriate acyl-chain length distribution. Moreover, we find that the transcriptional repressor Opi1 preferentially binds to C16 over C18 phosphatidic acid (PA) species: thus, C16-chain containing PA sequesters Opi1 more effectively to the ER, enabling AMPK/Snf1 control of PA acyl-chain length to determine the degree of derepression of Opi1 target genes. These findings reveal an unexpected regulatory link between the major energy-sensing kinase, membrane lipid composition, and transcription.
    Full-text · Article · Jun 2014 · Developmental Cell
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    ABSTRACT: Cytosolic lipid droplets (LD) are ubiquitous organelles in pro- and eukaryotes that play a key role in cellular and organismal lipid homeostasis. Triacylglycerols (TAG) and steryl esters which are stored in LDs, are typically mobilized in growing cells or upon hormonal stimulation by LD-associated lipases and steryl ester hydrolases. Here we show that in the yeast Saccharomyces cerevisiae, LDs can also be turned over in vacuoles/lysosomes by a process that morphologically resembles microautophagy. A distinct set of proteins involved in LD autophagy was identified, which includes the core autophagic machinery, but not Atg11 or Atg20. Thus, LD autophagy is distinct from ER-phagy, pexophagy or mitophagy, despite the close association between these organelles. Atg15 is responsible for TAG breakdown in vacuoles, and is required to support growth when de novo fatty acid synthesis is compromised. Furthermore, none of the core autophagy proteins, including Atg1 and Atg8, are required for LD formation, in yeast.
    Full-text · Article · Nov 2013 · Molecular biology of the cell
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    ABSTRACT: Nanostructured substrates have been recognized to initiate transcriptional programs promoting cell proliferation. Specifically β-catenin has been identified as transcriptional regulator, activated by adhesion to nanostructures. We set out to identify processes responsible for nanostructure-induced endothelial β-catenin signaling. Transmission electron microscopy (TEM) of cell contacts to differently sized polyethylene terephthalate (PET) surface structures (ripples with 250 to 300 nm and walls with 1.5 μm periodicity) revealed different patterns of cell-substrate interactions. Cell adhesion to ripples occurred exclusively on ripple peaks, while cells were attached to walls continuously. The Src kinase inhibitor PP2 was active only in cells grown on ripples, while the Abl inhibitors dasatinib and imatinib suppressed β-catenin translocation on both structures. Moreover, Gd(3+) sensitive Ca(2+) entry was observed in response to mechanical stimulation or Ca(2+) store depletion exclusively in cells grown on ripples. Both PP2 and Gd(3+) suppressed β-catenin nuclear translocation along with proliferation in cells grown on ripples but not on walls. Our results suggest that adhesion of endothelial cells to ripple structured PET induces highly specific, interface topology-dependent changes in cellular signalling, characterized by promotion of Gd(3+) -sensitive Ca(2+) entry and Src/Abl activation. We propose that these signaling events are crucially involved in nanostructure-induced promotion of cell proliferation.
    Full-text · Article · Oct 2013 · Journal of Nanomaterials
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    ABSTRACT: The 'discovery' of lipid droplets as a metabolically highly active subcellular organelle has sparked great scientific interest in its research in recent years. The previous view of a rather inert storage pool of neutral lipids-triacylglycerol and sterols or steryl esters-has markedly changed. Driven by the endemic dimensions of lipid-associated disorders on the one hand, and the promising biotechnological application to generate oils ('biodiesel') from single-celled organisms on the other, multiple model organisms are exploited in basic and applied research to develop a better understanding of biogenesis and metabolism of this organelle. This article summarizes the current status of LD research in yeast and experimental approaches to obtain insight into the regulatory and structural components driving lipid droplet formation and their physiological and pathophysiological roles in lipid homeostasis.
    Full-text · Article · Sep 2013 · Current Genetics
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    Full-text · Article · Sep 2013 · Biomedizinische Technik/Biomedical Engineering