Shirley K Knauer

University of Duisburg-Essen, Essen, North Rhine-Westphalia, Germany

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Publications (73)463.09 Total impact

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    ABSTRACT: Functionalization of the tetra-cationic cyclic peptide (Ka)4 with a single guanidiniocarbonyl pyrrole (GCP) moiety, a weakly basic but highly efficient arginine analogue, completely alters the self-assembly properties of the peptide. In contrast to the non-functionalized peptide 2, which does not self-assemble, the GCP-containing peptide 1 forms cationic nanotubes of micrometer length. These nanotubes are efficient gene transfection vectors. DNA binds to their cationic surface and is efficiently delivered into cells.
    No preview · Article · Jan 2016 · Angewandte Chemie International Edition
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    ABSTRACT: Proteolysis is not only a critical requirement for life, but the executing enzymes also play important roles in numerous pathological conditions, including cancer. Therefore, targeting proteases is clearly relevant for improving cancer patient care. However, to effectively control proteases, a profound knowledge of their mechanistic function as well as their regulation and downstream signalling in health and disease is required. The highly conserved protease Threonine Aspartase1 (Taspase1) is overexpressed in numerous liquid and solid malignancies and was characterized as a ‘non-oncogene addiction’ protease. Although Taspase1 was shown to cleave various regulatory proteins in humans as well as leukaemia provoking mixed lineage leukaemia fusions, our knowledge on its detailed functions and the underlying mechanisms contributing to cancer is still incomplete. Despite superficial similarity to type 2 asparaginases as well as Ntn proteases, such as the proteasome, Taspase1-related research so far gives us the picture of a unique protease exhibiting special features. Moreover, neither effective genetic nor chemical inhibitors for this enzyme are available so far, thus hampering not only to further dissect Taspase1’s pathobiological functions but also precluding the assessment of its clinical impact. Based on recent insights, we here critically review the current knowledge of Taspase1’s structure–function relationship and its mechanistic relevance for tumorigenesis obtained from in vitro and in vivo cancer models. We provide a comprehensive overview of tumour entities for which Taspase1 might be of predictive and therapeutic value, and present the respective experimental evidence. To stimulate progress in the field, a comprehensive overview of Taspase1 targeting approaches is presented, including coverage of Taspase1-related patents. We conclude by discussing future inhibition strategies and relevant challenges, which need to be resolved by the field.
    No preview · Article · Dec 2015 · Oncogene
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    ABSTRACT: Functionalization of the tetracationic cyclic peptide (Ka)4 with a single guanidiniocarbonyl pyrrole (GCP) moiety, a weakly basic but highly efficient arginine analogue, completely alters the self-assembly properties of the peptide. In contrast to the nonfunctionalized peptide 2, which does not self-assemble, GCP-containing peptide 1 forms cationic nanofibers of micrometer length. These aggregates are efficient gene transfection vectors. DNA binds to their cationic surface and is efficiently delivered into cells.
    No preview · Article · Nov 2015 · Angewandte Chemie International Edition
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    ABSTRACT: From the beginning of life, proteases are key to organismal development comprising morphogenesis, cellular differentiation, and cell growth. Regulated proteolytic activity is essential for the orchestration of multiple developmental pathways, and defects in protease activity can account for multiple disease patterns. The highly conserved protease threonine aspartase 1 is a member of such developmental proteases and critically involved in the regulation of complex processes, including segmental identity, head morphogenesis, spermatogenesis, and proliferation. Additionally, threonine aspartase 1 is overexpressed in numerous liquid as well as in solid malignancies. Although threonine aspartase 1 is able to cleave the master regulator mixed lineage leukemia protein as well as other regulatory proteins in humans, our knowledge of its detailed (patho)biological function and the underlying molecular mechanisms contributing to development and disease is still incomplete. Moreover, neither effective genetic nor chemical inhibitors for this enzyme are available so far precluding the detailed dissection of the (patho)biological functions of threonine aspartase 1. Here, we review the current knowledge of the structure-function relationship of threonine aspartase 1 and its mechanistic impact on substrate-mediated coordination of the cell cycle and development. We discuss threonine aspartase 1-mediated effects on cellular transformation and conclude by presenting a short overview of recent interference strategies.-Stauber, R. H., Hahlbrock, A., Knauer, S. K., Wünsch, D. Cleaving for growth: threonine aspartase 1-a protease relevant for development and disease.
    No preview · Article · Nov 2015 · The FASEB Journal
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    ABSTRACT: Survivin (BIRC5) is highly expressed in the vast majority of human cancers and is associated with chemotherapy resistance, increased tumor recurrence and shortened patient survival, making it an attractive therapeutic target. Initially identified as an inhibitor of apoptosis protein, it also plays a major role in the regulation of cell division. As such, it acts as a subunit of the chromosomal passenger complex, composed of the mitotic kinase aurora B, borealin and inner centromere protein, and is essential for proper chromosome segregation and cytokinesis. For both biological functions, interaction of survivin's nuclear export signal with the nuclear export receptor chromosome region maintenance 1 is absolutely essential. The timely orchestration of survivin's wide protein interaction repertoire is further modulated by different posttranslational modifications occurring in a cell-cycle-dependent manner. Recent data furthermore indicate additional roles of survivin in the DNA damage response, contributing to therapy resistance, yet the underlying molecular details are still not completely resolved. This also holds true for a potential involvement of survivin in senescence regulation. An age-related accumulation of survivin probably contributes to the apoptosis resistance observed in aged as well as in senescent cells, while it might promote escape from therapy-induced senescence. This review seeks to integrate the current knowledge on survivin's diverse and complex biological functions. By linking the 'old' facts about survivin with recent findings in research areas such as DNA damage response and aging, we want to highlight survivin's crucial role in a variety of cellular processes. © 2015 S. Karger AG, Basel.
    No preview · Article · Jul 2015 · Gerontology
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    ABSTRACT: Besides the wide use of engineered nanomaterials (NMs) in technical products, their applications are not only increasing in biotechnology and biomedicine, but also in the environmental field. While the physico-chemical properties and behaviour of NMs can be characterized accurately under idealized conditions, this is no longer the case in complex physiological or natural environments. Herein, proteins and other biomolecules rapidly bind to NMs, forming a protein/biomolecule corona that critically affects the NMs' (patho)biological and technical identities. As the corona impacts the in vitro and/or in vivo NM applications in humans and ecosystems, a mechanistic understanding of its relevance and of the biophysical forces regulating corona formation is mandatory. Based on recent insights, we here critically review and present an updated concept of corona formation and evolution. We comment on how corona signatures may be linked to effects at the nano-bio interface in physiological and environmental systems. In order to comprehensively analyse corona profiles and to mechanistically understand the coronas' biological/ecological impact, we present a tiered multidisciplinary approach. To stimulate progress in this field, we introduce the potential impact of the corona for NM-microbiome-(human)host interactions and the novel concept of 'nanologicals', i.e., the nanomaterial-specific targeting of molecular machines. We conclude by discussing the relevant challenges that still need to be resolved in this field.
    Full-text · Article · Jun 2015 · Chemical Society Reviews
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    ABSTRACT: The ubiquitin-dependent proteasomal degradation of proteins controls signaling and cellular survival. An increasing body of evidence suggests that the E3 ubiquitin ligases SIAH1 and SIAH2 are able to dictate the growth, development, and chemo-/radiosensitivity of breast and prostate cancer cells. Here we review the current knowledge on the impact of SIAHs on breast and prostate tumorigenesis. Furthermore, we summarize how stress, hormones, and cytokines regulate SIAH1 and SIAH2 in transformed mammalian cells. Copyright © 2015 Elsevier Ltd. All rights reserved.
    No preview · Article · May 2015 · Cytokine & Growth Factor Reviews

  • No preview · Article · May 2015 · Oral Oncology
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    ABSTRACT: MicroRNAs (miRNAs) are deregulated in a variety of human cancers, including neuroblastoma, the most common extracranial tumor of childhood. We previously reported a signature of 42 miRNAs to be highly predictive of neuroblastoma outcome. One miRNA in this signature, miR-542, was downregulated in tumors from patients with adverse outcome. Reanalysis of quantitative PCR and next-generation sequencing transcript data revealed that miR-542-5p as well as miR-542-3p expression is inversely correlated with poor prognosis in neuroblastoma patients. We, therefore, analyzed the function of miR-542 in neuroblastoma tumor biology. Ectopic expression of miR-542-3p in neuroblastoma cell lines reduced cell viability and proliferation, induced apoptosis and downregulated Survivin. Survivin expression was also inversely correlated with miR-542-3p expression in primary neuroblastomas. Reporter assays confirmed that miR-542-3p directly targeted Survivin. Downregulating Survivin using siRNA copied the phenotype of miR-542-3p expression in neuroblastoma cell lines, while cDNA-mediated ectopic expression of Survivin partially rescued the phenotype induced by miR-542-3p expression. Treating nude mice bearing neuroblastoma xenografts with miR-542-3p-loaded nanoparticles repressed Survivin expression, decreased cell proliferation and induced apoptosis in the respective xenograft tumors. We conclude that miR-542-3p exerts its tumor suppressive function in neuroblastoma, at least in part, by targeting Survivin. Expression of miR-542-3p could be a promising therapeutic strategy for treating aggressive neuroblastoma. What's new? The profile of microRNAs in a tumor can help predict the severity of the disease. Neuroblastomas with poor outcome have been shown to contain less of miR-542-3p. In this paper, the authors investigated this microRNA's function and found that it downregulates the protein Survivin, an apoptosis inhibitor. When the authors delivered nanoparticles bearing miR-542-3p to human neuroblastomas in mice, the treatment slowed Survivin production, induced apoptosis, and stopped tumor growth. Thus, miR-542-3p appears to hold promise for treating this often deadly childhood tumor.
    Full-text · Article · Mar 2015 · International Journal of Cancer
  • Mao Li · Stefanie Schlesiger · Shirley K. Knauer · Carsten Schmuck
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    ABSTRACT: Arginine-rich cell-penetrating peptides are widely utilized as vectors for gene delivery. However, their transfection efficacy still needs to be optimized. To accomplish this, guanidinocarbonylpyrrole groups, which are tailor-made anion binding sites, were introduced into the side chains of tetralysine to obtain the peptide analogue 1. In contrast to the common strategy of adding a lipophilic tail to peptide vectors, this novel method most likely enhances transfection efficacy through more specific interactions between the binding motifs and DNA or the cell membrane. Tetrapeptide analogue 1 is thus the smallest peptidic transfection vector that has been reported to date. The transfection efficacy of 1, which on average has less than two positive charges under physiological conditions, is even better than that of polyethylenimine (PEI). Furthermore, 1 exhibits only negligible cytotoxicity, which makes it an interesting candidate for further development.
    No preview · Article · Mar 2015 · Angewandte Chemie International Edition
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    ABSTRACT: Besides the wide use of nanomaterials in technical products, their application spectrum in biotechnology and biomedicine is steadily increasing. Whereas the physico-chemical properties and behavior of nanomaterials can be engineered and characterized accurately under idealized conditions, this is no longer the case in complex physiological environments. In biological fluids, proteins rapidly bind to nanomaterials forming the protein corona, critically affecting the nanomaterials' biological identity. As the corona impacts in vitro and/or in vivo nanomaterial applications, we here review the concept of the protein corona and its analytical dissection. We comment on how corona signatures may be linked to effects at the nano-bio interface and conclude how such knowledge is offering novel opportunities for improved nanomedicine.
    No preview · Article · Feb 2015 · Nanomedicine
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    ABSTRACT: Human Taspase1 is essential for development and cancer by processing critical regulators, such as the mixed-lineage leukemia protein. Likewise, its ortholog, trithorax, is cleaved by Drosophila Taspase1 (dTaspase1), implementing a functional coevolution. To uncover novel mechanism regulating protease function, we performed a functional analysis of dTaspase1 and its comparison to the human ortholog. dTaspase1 contains an essential nucleophile threonine(195), catalyzing cis cleavage into its α- and β-subunits. A cell-based assay combined with alanine scanning mutagenesis demonstrated that the target cleavage motif for dTaspase1 (Q(3)[F/I/L/M](2)D(1)↓G(1')X(2')X(3')) differs significantly from the human ortholog (Q(3)[F,I,L,V](2)D(1)↓G(1')x(2')D(3')D(4')), predicting an enlarged degradome containing 70 substrates for Drosophila. In contrast to human Taspase1, dTaspase1 shows no discrete localization to the nucleus/nucleolus due to the lack of the importin-α/nucleophosmin1 interaction domain (NoLS) conserved in all vertebrates. Consequently, dTaspase1 neither interacts with the Drosophila nucleoplasmin-like protein nor human nucleophosmin1. The impact of localization on the protease's degradome was confirmed by demonstrating that dTaspase1 did not efficiently process nuclear substrates, such as upstream stimulatory factor 2. However, genetic introduction of the NoLS into dTaspase1 restored its nucleolar localization, nucleophosmin1 interaction, and efficient cleavage of nuclear substrates. We report that evolutionary functional divergence separating vertebrates from invertebrates can be achieved for proteases by a transport/localization-regulated mechanism.-Wünsch, D., Hahlbrock, A., Heiselmayer, C., Bäcker, S., Heun, P., Goesswein, D., Stöcker, W., Schirmeister, T., Schneider, G., Krämer, O. H., Knauer, S. K., Stauber, R. H. Fly versus man: Evolutionary impairment of nucleolar targeting affects the degradome of Drosophila's Taspase1. © FASEB.
    No preview · Article · Jan 2015 · The FASEB Journal
  • Mao Li · Stefanie Schlesiger · Shirley K. Knauer · Carsten Schmuck
    [Show abstract] [Hide abstract]
    ABSTRACT: Arginine-rich cell-penetrating peptides are widely utilized as vectors for gene delivery. However, their transfection efficacy still needs to be optimized. To accomplish this, guanidinocarbonylpyrrole groups, which are tailor-made anion binding sites, were introduced into the side chains of tetralysine to obtain the peptide analogue 1. In contrast to the common strategy of adding a lipophilic tail to peptide vectors, this novel method most likely enhances transfection efficacy through more specific interactions between the binding motifs and DNA or the cell membrane. Tetrapeptide analogue 1 is thus the smallest peptidic transfection vector that has been reported to date. The transfection efficacy of 1, which on average has less than two positive charges under physiological conditions, is even better than that of polyethylenimine (PEI). Furthermore, 1 exhibits only negligible cytotoxicity, which makes it an interesting candidate for further development.
    No preview · Article · Jan 2015 · Angewandte Chemie International Edition
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    ABSTRACT: The transcription factor nuclear factor-κB (NF-κB) is crucial for the maintenance of homeostasis. It is incompletely understood how nuclear NF-κB and the crosstalk of NF-κB with other transcription factors are controlled. Here, we demonstrate that the epigenetic regulator histone deacetylase 2 (HDAC2) activates NF-κB in transformed and primary cells. This function depends on both, the catalytic activity and an intact HDAC2 sumoylation motif. Several mechanisms account for the induction of NF-κB through HDAC2. The expression of wild-type HDAC2 can increase the nuclear presence of NF-κB. In addition, the ribosomal S6 kinase 1 (RSK1) and the tumor suppressor p53 contribute to the regulation of NF-κB by HDAC2. Moreover, TP53 mRNA expression is positively regulated by wild-type HDAC2 but not by sumoylation-deficient HDAC2. Thus, sumoylation of HDAC2 integrates NF-κB signaling involving p53 and RSK1. Since HDAC2-dependent NF-κB activity protects colon cancer cells from genotoxic stress, our data also suggest that high HDAC2 levels, which are frequently found in tumors, are linked to chemoresistance. Accordingly, inhibitors of NF-κB and of the NF-κB/p53-regulated anti-apoptotic protein survivin significantly sensitize colon carcinoma cells expressing wild-type HDAC2 to apoptosis induced by the genotoxin doxorubicin. Hence, the HDAC2-dependent signaling node we describe here may offer an interesting therapeutic option.
    Full-text · Article · Jan 2015 · Oncotarget
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    ABSTRACT: Abstract Proteases are key regulators of life. Human Threonine Aspartase1 processes substrates, such as the mixed-lineage leukemia (MLL) protein, containing two cleavage sites, CS1 and CS2. Likewise, MLL's Drosophila ortholog trithorax is cleaved by D̲rosophila T̲hreonine A̲s̲p̲artase1 (dTasp), suggesting a mechanistic co-evolution. However, a detailed analysis of dTasp's function was missing so far. Here, active and inactive dTasp mutants allowed to compare substrate recognition and cleavage-site selectivity of human and Drosophila enzymes. In contrast to the human protease, our cell-based assay revealed a preferential processing of CS2-like (QLD↓Gx[xD/Dx]) targets for dTasp, whereas cleavage of CS1-like targets (QVD↓Gx[xD/Dx]) was significantly impaired. Systematic mutagenesis of the CS2-sequence defined the motif x[FILMW]D↓Gx[xD/Dx] as the consensus cleavage sequence for dTasp. Substrate species-selectivity of the enzymes was uncovered by demonstrating that dTasp cleaves Drosophila TFIIA, but not the human ortholog, suggesting evolutionary divergence of TFIIA downstream networks. Also, Drosophila USF2 was neither predicted, nor cleaved by dTasp. Moreover, we found that dTasp cleavage-site selectivity is independent of heterocomplex formation, as dTasp exists predominantly as an αβ-monomer. Collectively, we provide novel insights into evolutionary similarities and divergence concerning Threonine Aspartase1 function in different species, which may aid to dissect and better target human Threonine Aspartase1 in malignancies.
    No preview · Article · Jan 2015 · Biological Chemistry
  • Shirley K. Knauer · Wolf Mann · Roland H. Stauber
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    ABSTRACT: Survivin is proposed to function as a mitotic regulator and an apoptosis inhibitor duringdevelopment and pathogenesis. As such, survivin has aroused keen interest in disparateareas of basic and translational research. Survivin acts as a subunit of the chromosomalpassenger complex (CPC), composed of the mitotic kinase Aurora-B, Borealin 5 and INCENP,and is essential for proper chromosome segregation and cytokinesis. Our recent findingsindicate that the nuclear export receptor Crm1 is critically involved in tethering the CPC to thecentromere by interacting with a leucine-rich nuclear export signal (NES), evolutionaryconserved in all mammalian survivin proteins. In addition, the survivin/Crm1 interaction10 seems to be required for the cytoprotective activity of survivin, because export deficientsurvivin fails to protect tumor cells against cancer therapy-induced apoptosis. These findingsappear of clinical relevance since preferential nuclear localization of survivin turned out to bea favorable prognostic factor in cancer patients. Besides emphasizing the functionalsignificance of the Crm1/survivin interface, we suggest to exploit the pharmacogenetic15 interference with survivin’s export as a novel strategy to antagonize survivin’s activity.
    No preview · Article · Oct 2014 · Cell cycle (Georgetown, Tex.)
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    ABSTRACT: The cover picture shows the bright-field microscopy image of HeLa tumor cells overlaid with the respective fluorescence microscopy image of overexpressed exogenous human Taspase1 (colored in dark blue), located in nucleoli within the nucleus (stained in light blue). The colors of the bright-field image were altered to brown/gray. The right circular inset depicts the crystal structure surface of active Taspase1 (PDB ID: 2a8j) with its two subunits colored in blue and green. The active site (red patch) is targeted by a peptidyl-succinimidyl peptide (left circular insert), displayed as sticks with a semitransparent surface (orange). The picture thus illustrates the efforts of M. Kaiser et al. (see the full paper on p. 2233 ff.) to design a substrate analogue inhibitor of the putative cancer target Taspase1, a threonine protease that is frequently overexpressed in many different leukemias or Taspase1-dependent solid tumors.
    No preview · Article · Oct 2014 · ChemBioChem
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    ABSTRACT: Taspase 1 is an N-terminal threonine protease implicated in leukemia and other cancers. Despite intensive efforts in recent years, only a limited number of Taspase 1 inhibitors are currently available, and they lack general applicability. Here we present a novel class of Taspase 1 inhibitors based on a peptidyl succinimidyl peptide motif. These inhibitors were obtained from the substrate cleavage sequence and mechanistic considerations involving the previously proposed asparaginase-type cleavage mechanism. We anticipate that this class of Taspase 1 inhibitor will find wide application in further biochemical and structural studies, for example for better investigating the molecular details of the unusual enzymatic cleavage mechanism of Taspase 1.
    Full-text · Article · Oct 2014 · ChemBioChem
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    ABSTRACT: Calcium phosphate/poly(D,L-lactide-co-glycolide acid) (PLGA) nanoparticles with a diameter below 200 nm, loaded with either nucleic acids or proteins, were synthesized by a water-in-oil-in-water (W1/O/W2) emulsion solvent evaporation technique. The particles were stabilized by polyvinyl alcohol (PVA) and had a negative charge (zeta potential -26 mV). By the addition of calcium phosphate into the inner aqueous phase of the W1/O/W2-emulsion, the encapsulation efficiency of siRNA was increased to 37%, of DNA to 52%, and of bovine serum albumin to 78%, i.e. by a factor of 3 to 10 compared to PLGA nanoparticles without calcium phosphate. Total loadings of 8 µg siRNA, 5 µg DNA and 280 µg fluorescein isothiocyanate-labelled bovine serum albumin (FITC-BSA) per mg of PLGA were achieved by this method. The addition of an outer layer of either chitosan or polyethyleneimine (PEI) reversed the charge of the particles (zeta potential > +30 mV) and improved the cellular uptake as well as the endosomal escape of these particles as demonstrated by confocal laser scanning microscopy. Calcium phosphate-PLGA nanoparticles loaded with DNA encoding for green fluorescent protein (eGFP-DNA) showed a good transfection efficiency for epithelial cells (HeLa) without any toxic effects. Gene silencing with HeLa cells expressing eGFP gave knockdown efficiencies of 53% for anionic nanoparticles, of 68% for chitosan-coated cationic nanoparticles, and of 89% for polyethyleneimine-coated cationic nanoparticles.
    Preview · Article · Sep 2014
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    ABSTRACT: Nanoparticle applications in biotechnology and biomedicine are steadily increasing. In biological fluids, proteins bind to nanoparticles that form the protein corona, crucially affecting the nanoparticles' biological identity. As the corona affects in vitro and/or in vivo nanoparticle applications, we developed a method to obtain time-resolved protein corona profiles formed on various nanoparticles. After incubation in plasma or a similar biofluid, or after injection into a mouse, the first analytical step is sedimentation of the nanoparticle-protein complexes through a sucrose cushion, thereby allowing analysis of early corona formation time points. Next, corona profiles are visualized by gel electrophoresis and quantitatively analyzed after tryptic digestion using label-free liquid chromatography-high-resolution mass spectrometry. In contrast to other approaches, our established methodology allows the researcher to obtain qualitative and quantitative high-resolution corona signatures. The protocol can be readily extended to the investigation of protein coronas from various nanomaterials (as an example, we applied this protocol to different silica nanoparticles (SiNPs) and polystyrene nanoparticles (PSNPs)). Depending on the number of samples, the protocol from nanoparticle-protein complex recovery to data evaluation takes ∼8-12 d to complete.
    No preview · Article · Sep 2014 · Nature Protocols

Publication Stats

2k Citations
463.09 Total Impact Points

Institutions

  • 2010-2015
    • University of Duisburg-Essen
      • • Center for Medical Biotechnology
      • • Arbeitsgruppe Molekularbiologie I
      Essen, North Rhine-Westphalia, Germany
  • 2007-2009
    • Johannes Gutenberg-Universität Mainz
      Mayence, Rheinland-Pfalz, Germany
  • 2007-2008
    • University Hospital Frankfurt
      Frankfurt, Hesse, Germany
  • 2005-2007
    • Georg-Speyer-Haus
      Frankfurt, Hesse, Germany