Publications by authors named "Shirley K Knauer"

85 Publications

Amyloid precursor protein elevates fusion of promyelocytic leukemia nuclear bodies in human hippocampal areas with high plaque load.

Acta Neuropathol Commun 2021 04 13;9(1):66. Epub 2021 Apr 13.

Department of Molecular Biochemistry, Cell Signalling, Faculty of Chemistry and Biochemistry, Ruhr University Bochum, Bochum, Germany.

The amyloid precursor protein (APP) is a type I transmembrane protein with unknown physiological function but potential impact in neurodegeneration. The current study demonstrates that APP signals to the nucleus causing the generation of aggregates consisting of its adapter protein FE65, the histone acetyltransferase TIP60 and the tumour suppressor proteins p53 and PML. APP C-terminal (APP-CT50) complexes co-localize and co-precipitate with p53 and PML. The PML nuclear body generation is induced and fusion occurs over time depending on APP signalling and STED imaging revealed active gene expression within the complex. We further show that the nuclear aggregates of APP-CT50 fragments together with PML and FE65 are present in the aged human brain but not in cerebral organoids differentiated from iPS cells. Notably, human Alzheimer's disease brains reveal a highly significant reduction of these nuclear aggregates in areas with high plaque load compared to plaque-free areas of the same individual. Based on these results we conclude that APP-CT50 signalling to the nucleus takes place in the aged human brain and is involved in the pathophysiology of AD.
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http://dx.doi.org/10.1186/s40478-021-01174-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8042982PMC
April 2021

Specific inhibition of the Survivin-CRM1 interaction by peptide-modified molecular tweezers.

Nat Commun 2021 03 8;12(1):1505. Epub 2021 Mar 8.

Department of Molecular Biology II, Centre for Medical Biotechnology (ZMB), University of Duisburg-Essen, Essen, Germany.

Survivin's dual function as apoptosis inhibitor and regulator of cell proliferation is mediated via its interaction with the export receptor CRM1. This protein-protein interaction represents an attractive target in cancer research and therapy. Here, we report a sophisticated strategy addressing Survivin's nuclear export signal (NES), the binding site of CRM1, with advanced supramolecular tweezers for lysine and arginine. These were covalently connected to small peptides resembling the natural, self-complementary dimer interface which largely overlaps with the NES. Several biochemical methods demonstrated sequence-selective NES recognition and interference with the critical receptor interaction. These data were strongly supported by molecular dynamics simulations and multiscale computational studies. Rational design of lysine tweezers equipped with a peptidic recognition element thus allowed to address a previously unapproachable protein surface area. As an experimental proof-of-principle for specific transport signal interference, this concept should be transferable to any protein epitope with a flanking well-accessible lysine.
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http://dx.doi.org/10.1038/s41467-021-21753-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7940618PMC
March 2021

PEGylated sequence-controlled macromolecules using supramolecular binding to target the Taspase1/Importin α interaction.

Chem Commun (Camb) 2021 Mar 24;57(25):3091-3094. Epub 2021 Feb 24.

Department for Organic Chemistry and Macromolecular Chemistry, Heinrich Heine University Düsseldorf, Universitätsstraße 1, Düsseldorf 40225, Germany.

A novel strategy to inhibit the oncologically relevant protease Taspase1 is explored by developing PEGylated macromolecular ligands presenting the supramolecular binding motif guanidiniocarbonylpyrrole (GCP). Taspase1 requires interaction of its nuclear localization signal (NLS) with import receptor Importin α. We show the synthesis and effective interference of PEGylated multivalent macromolecular ligands with Taspase1-Importin α-complex formation.
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http://dx.doi.org/10.1039/d0cc07139kDOI Listing
March 2021

TNF-α-Inhibition Improves the Biocompatibility of Porous Polyethylene Implants In Vivo.

Tissue Eng Regen Med 2021 04 30;18(2):297-303. Epub 2021 Jan 30.

Walter Brendel Centre of Experimental Medicine, Ludwig-Maximilians-University Munich, Marchioninistr. 27, 81377, Munich, Germany.

Background: To improve the biocompatibility of porous polyethylene (PPE) implants and expand their application range for reconstructive surgery in poorly vascularized environments, implants were coated with tumor necrosis factor α (TNFα) inhibitor Etanercept. While approved for systemic application, local application of the drug is a novel experimental approach. Microvascular and mechanical integration as well as parameters of inflammation were analyzed in vivo.

Methods: PPE implants were coated with Etanercept and extracellular matrix (ECM) components prior to implantation into dorsal skinfold chambers of C57BL/6 mice. Fluorescence microscopy analyses of angiogenesis and local inflammatory response were thrice performed in vivo over a period of 14 days to assess tissue integration and biocompatibility. Uncoated implants and ECM-coated implants served as controls.

Results: TNFα inhibition with Etanercept led to a reduced local inflammatory response: leukocyte-endothelial cell adherence was significantly lowered compared to both control groups (n = 6/group) on days 3 and 14, where the lowest values were reached: 3573.88 leukocytes/mm-2 ± 880.16 (uncoated implants) vs. 3939.09 mm-2 ± 623.34 (Matrigel only) vs. 637.98 mm-2 + 176.85 (Matrigel and Etanercept). Implant-coating with Matrigel alone and Matrigel and Etanercept led to significantly higher vessel densities 7 and 14 days vs. 3 days after implantation and compared to uncoated implants. Mechanical implant integration as measured by dynamic breaking strength did not differ after 14 days.

Conclusion: Our data show a reduced local inflammatory response to PPE implants after immunomodulatory coating with Etanercept in vivo, suggesting improved biocompatibility. Application of this tissue engineering approach is therefore warranted in models of a compromised host environment.
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http://dx.doi.org/10.1007/s13770-020-00325-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8012447PMC
April 2021

Luminescent Amphiphilic Aminoglycoside Probes to Study Transfection.

Chembiochem 2021 May 11;22(9):1563-1567. Epub 2021 Feb 11.

Faculty of chemistry (Organic Chemistry) and, Centre for Nanointegration Duisburg-Essen (CENIDE), University of Duisburg-Essen, Universitätsstrasse 7, 45117, Essen, Germany.

We report the characterization of amphiphilic aminoglycoside conjugates containing luminophores with aggregation-induced emission properties as transfection reagents. These inherently luminescent transfection vectors are capable of binding plasmid DNA through electrostatic interactions; this binding results in an emission "on" signal due to restriction of intramolecular motion of the luminophore core. The luminescent cationic amphiphiles effectively transferred plasmid DNA into mammalian cells (HeLa, HEK 293T), as proven by expression of a red fluorescent protein marker. The morphologies of the aggregates were investigated by microscopy as well as ζ-potential and dynamic light-scattering measurements. The transfection efficiencies using luminescent cationic amphiphiles were similar to that of the gold-standard transfection reagent Lipofectamine® 2000.
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http://dx.doi.org/10.1002/cbic.202000725DOI Listing
May 2021

New Tools to Probe the Protein Surface: Ultrasmall Gold Nanoparticles Carry Amino Acid Binders.

J Phys Chem B 2021 01 23;125(1):115-127. Epub 2020 Dec 23.

Inorganic Chemistry and Center for Nanointegration Duisburg-Essen (CeNIDE), University of Duisburg-Essen, Universitätsstr. 5-7, 45117 Essen, Germany.

A strategy toward epitope-selective functionalized nanoparticles is introduced in the following: ultrasmall gold nanoparticles (diameter of the metallic core about 2 nm) were functionalized with molecular tweezers that selectively attach lysine and arginine residues on protein surfaces. Between 11 and 30 tweezer molecules were covalently attached to the surface of each nanoparticle by copper-catalyzed azide alkyne cycloaddition (CuAAC), giving multiavid agents to target proteins. The nanoparticles were characterized by high-resolution transmission electron microscopy, differential centrifugal sedimentation, and H NMR spectroscopy (diffusion-ordered spectroscopy, DOSY, and surface composition). The interaction of these nanoparticles with the model proteins hPin1 (WW domain; hPin1-WW) and Survivin was probed by NMR titration and by isothermal titration calorimetry (ITC). The binding to the WW domain of hPin1 occurred with a of 41 ± 2 μM, as shown by ITC. The nanoparticle-conjugated tweezers targeted cationic amino acids on the surface of hPin1-WW in the following order: N-terminus (G) ≈ R17 > R14 ≈ R21 > K13 > R36 > K6, as shown by NMR spectroscopy. Nanoparticle recognition of the larger protein Survivin was even more efficient and occurred with a of 8 ± 1 μM, as shown by ITC. We conclude that ultrasmall nanoparticles can act as versatile carriers for artificial protein ligands and strengthen their interaction with the complementary patches on the protein surface.
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http://dx.doi.org/10.1021/acs.jpcb.0c09846DOI Listing
January 2021

Mechanisms of nanotoxicity - biomolecule coronas protect pathological fungi against nanoparticle-based eradication.

Nanotoxicology 2020 11 24;14(9):1157-1174. Epub 2020 Aug 24.

ENT Department, University Medical Center Mainz, Mainz, Germany.

Whereas nanotoxicity is intensely studied in mammalian systems, our knowledge of desired or unwanted nano-based effects for microbes is still limited. Fungal infections are global socio-economic health and agricultural problems, and current chemical antifungals may induce adverse side-effects in humans and ecosystems. Thus, nanoparticles are discussed as potential novel and sustainable antifungals via the desired nanotoxicity but often fail in practical applications. In our study, we found that nanoparticles' toxicity strongly depends on their binding to fungal spores, including the clinically relevant pathogen as well as common plant pests, such as or . Employing a selection of the model and antimicrobial nanoparticles, we found that nanoparticle-spore complex formation is influenced by the NM's physicochemical properties, such as size, identified as a key determinant for our silica model particles. Biomolecule coronas acquired in pathophysiologically and ecologically relevant environments, protected fungi against nanoparticle-induced toxicity as shown by employing antimicrobial ZnO, Ag, or CuO nanoparticles as well as dissolution-resistant quantum dots. Mechanistically, dose-dependent corona-mediated resistance was conferred via reducing the physical adsorption of nanoparticles to fungi. The inhibitory effect of biomolecules on nano-based toxicity of Ag NPs was further verified , using the invertebrate as an alternative non-mammalian infection model. We provide the first evidence that biomolecule coronas are not only relevant in mammalian systems but also for nanomaterial designs as future antifungals for human health, biotechnology, and agriculture.
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http://dx.doi.org/10.1080/17435390.2020.1808251DOI Listing
November 2020

The other side of the corona: nanoparticles inhibit the protease taspase1 in a size-dependent manner.

Nanoscale 2020 Oct 14;12(37):19093-19103. Epub 2020 Jul 14.

Structural and Medicinal Biochemistry, Department of Biology, University Duisburg-Essen and Zentrum für Molekulare Biotechnologie (ZMB), Universitätsstrasse 5, Essen, 45141 Germany.

When nanoparticles enter a physiological environment, they rapidly adsorb biomolecules, in particular cellular proteins. This biological coating, the so-called nanoparticle protein corona, undoubtedly affects the biological identity and potential cytotoxicity of the nanomaterial. To elucidate a possible impact on the adsorbed biomolecules, we focused on an important group of players in cellular homeostasis, namely proteolytic enzymes. We could demonstrate that amorphous silica nanoparticles are not only able to bind to the oncologically relevant threonine protease Taspase1 as revealed by microscale thermophoresis and fluorescence anisotropy measurements, but moreover inhibit its proteolytic activity in a non-competitive manner. As revealed by temperature-dependent unfolding and CD spectroscopy, binding did not alter the stability of Taspase1 or its secondary structure. Noteworthy, inhibition of protein function seems not a general feature of nanoparticles, as several control enzymes were not affected in their proteolytic activity. Our data suggests that nanoparticles bind Taspase1 as an αβ-dimer in a single layer without conformational change, resulting in noncompetitive inhibition that is either allostery-like or occludes the active site. Nanoparticle-based inhibition of Taspase1 could be also achieved in cell lysates and in live cells as shown by the use of a protease-specific cellular cleavage biosensor. Collectively, we could demonstrate that nanoparticles could not only bind but also selectively inhibit cellular enzymes, which might explain observed cytotoxicity but might serve as a starting point for the development of nanoparticle-based inhibitors as therapeutics.
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http://dx.doi.org/10.1039/d0nr01631dDOI Listing
October 2020

Supramolecular subphthalocyanine complexes-cellular uptake and phototoxicity.

Chem Commun (Camb) 2020 Jul;56(55):7653-7656

Institute of Organic Chemistry, University of Duisburg-Essen, Universitätsstrasse 7, 45117, Essen, Germany.

In this communication we report on the synthesis and application of axially functionalized boron-subphthalocyanines (SubPC) which are able to form host-guest complexes with cyclodextrins. Here, a tert-butylphenyl substituted SubPC was investigated concerning its complexation with β-cyclodextrin (β-CD) and a β-cyclodextrin polymer. NMR-titrations showed the formation of a 1 : 1 complex with β-CD. These assemblies were analyzed for their cellular distribution as well as their phototoxicity towards HeLa cells.
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http://dx.doi.org/10.1039/d0cc03065aDOI Listing
July 2020

Smart Glycopolymeric Nanoparticles for Multivalent Lectin Binding and Stimuli-Controlled Guest Release.

Biomacromolecules 2020 06 10;21(6):2356-2364. Epub 2020 Apr 10.

Organic Chemistry and Cenide, University of Duisburg-Essen, Universitätsstrasse 7, D-45117 Essen, Germany.

The synthesis and self-assembly of a polymer featuring a self-complementary supramolecular binding motif guanidiniocarbonyl pyrrole carboxylate zwitterion (GCP-zwitterion) bearing lactose moieties are reported. The GCP-zwitterion acts as a cross-linker to facilitate self-assembly of the polymeric chain into nanoparticles (NPs) at neutral pH in an aqueous medium. The formation of polymeric NPs can be controlled by addition of external stimuli (acid or base), which disfavors self-assembly of the GCP-zwitterion because of protonation or deprotonation of the GCP units in the polymer chain. The small-sized (<40 nm) NPs have a hydrophobic cavity and accessible lactose units on the outer shell for multivalent lectin binding. The multivalent interaction between NPs and the lectin peanut agglutinin was confirmed by agglutination experiments. In addition, the stimuli-responsive property of NPs was exploited for the uptake and release of a hydrophobic guest Nile red. Furthermore, the selectivity toward different cell lines (HEK 296T, HeLa, and Hep2G) was tested, and a cellular uptake of cargo-loaded NPs was found for Hep2G cells bearing the lactose-specific asialogylcoprotein receptor, whereas all other cells showed no NP interaction.
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http://dx.doi.org/10.1021/acs.biomac.0c00292DOI Listing
June 2020

Functional Disruption of the Cancer-Relevant Interaction between Survivin and Histone H3 with a Guanidiniocarbonyl Pyrrole Ligand.

Angew Chem Int Ed Engl 2020 03 28;59(14):5567-5571. Epub 2020 Jan 28.

Department of Molecular Biology II, University of Duisburg-Essen, Universitätsstraße 5, 45141, Essen, Germany.

The protein Survivin is highly upregulated in most cancers and considered to be a key player in carcinogenesis. We explored a supramolecular approach to address Survivin as a drug target by inhibiting the protein-protein interaction of Survivin and its functionally relevant binding partner Histone H3. Ligand L1 is based on the guanidiniocarbonyl pyrrole cation and serves as a highly specific anion binder in order to target the interaction between Survivin and Histone H3. NMR titration confirmed binding of L1 to Survivin's Histone H3 binding site. The inhibition of the Survivin-Histone H3 interaction and consequently a reduction of cancer cell proliferation were demonstrated by microscopic and cellular assays.
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http://dx.doi.org/10.1002/anie.201915400DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7155087PMC
March 2020

Correction: Nanoparticle binding attenuates the pathobiology of gastric cancer-associated Helicobacter pylori.

Nanoscale 2020 01;12(3):2154-2155

Department of Nanobiomedicine/ENT, University Medical Center of Mainz, Langenbeckstrasse 1, 55101 Mainz, Germany.

Correction for 'Nanoparticle binding attenuates the pathobiology of gastric cancer-associated Helicobacter pylori' by Dana Westmeier et al., Nanoscale, 2018, 10, 1453-1463.
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http://dx.doi.org/10.1039/c9nr90287bDOI Listing
January 2020

Is small smarter? Nanomaterial-based detection and elimination of circulating tumor cells: current knowledge and perspectives.

Int J Nanomedicine 2019 6;14:4187-4209. Epub 2019 Jun 6.

Nanobiomedicine Department/ENT, University Medical Center Mainz, Mainz 55131, Germany,

Circulating tumor cells (CTCs) are disseminated cancer cells. The occurrence and circulation of CTCs seem key for metastasis, still the major cause of cancer-associated deaths. As such, CTCs are investigated as predictive biomarkers. However, due to their rarity and heterogeneous biology, CTCs' practical use has not made it into the clinical routine. Clearly, methods for the effective isolation and reliable detection of CTCs are urgently needed. With the development of nanotechnology, various nanosystems for CTC isolation and enrichment and CTC-targeted cancer therapy have been designed. Here, we summarize the relationship between CTCs and tumor metastasis, and describe CTCs' unique properties hampering their effective enrichment. We comment on nanotechnology-based systems for CTC isolation and recent achievements in microfluidics and lab-on-a-chip technologies. We discuss recent advances in CTC-targeted cancer therapy exploiting the unique properties of nanomaterials. We conclude by introducing developments in CTC-directed nanosystems and other advanced technologies currently in (pre)clinical research.
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http://dx.doi.org/10.2147/IJN.S198319DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6560927PMC
September 2019

A Branched Tripeptide with an Anion-Binding Motif as a New Delivery Carrier for Efficient Gene Transfection.

Chembiochem 2019 06 25;20(11):1410-1416. Epub 2019 Apr 25.

Institute for Organic Chemistry, University of Duisburg-Essen, Universitätsstrasse 7, 45141, Essen, Germany.

Branched and dendrimeric cationic peptides have shown better transfection efficiency than linear peptides, owing to their superior capacity for inducing DNA condensation. We have designed and synthesized two analogously guanidinocarbonylpyrrole-substituted (GCP-substituted) branched cationic tripeptides that provide extremely strong electrostatic attraction towards DNA. Both ligands 1 and 2 can bind to DNA and form condensed complexes, owing to the branched structure and high positive charges, as demonstrated by isothermal titration calorimetry (ITC), ζ potential and atomic force microscopy (AFM). After the replacement of the carboxylate group by an amide group, binding of ligand 2 to DNA shows exothermic enthalpy and positive entropy changes relative to ligand 1. Rational interpretation would suggest that ligand 2 might aid the translocation of plasmid pF143 to HEK 293T cells, showing high gene transfection efficiency. This work therefore provides a facile way, by modifying a branched cationic tripeptide with GCP, to turn a peptide even a tripeptide into an efficient gene transfection vector.
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http://dx.doi.org/10.1002/cbic.201800728DOI Listing
June 2019

Resistance to Nano-Based Antifungals Is Mediated by Biomolecule Coronas.

ACS Appl Mater Interfaces 2019 Jan 18;11(1):104-114. Epub 2018 Dec 18.

Nanobiomedicine Department , University Medical Center Mainz , Langenbeckstrasse 1 , 55131 Mainz , Germany.

Fungal infections are a growing global health and agricultural threat, and current chemical antifungals may induce various side-effects. Thus, nanoparticles are investigated as potential novel antifungals. We report that nanoparticles' antifungal activity strongly depends on their binding to fungal spores, focusing on the clinically important fungal pathogen Aspergillus fumigatus as well as common plant pathogens, such as Botrytis cinerea. We show that nanoparticle-spore complex formation was enhanced by the small nanoparticle size rather than the material, shape or charge, and could not be prevented by steric surface modifications. Fungal resistance to metal-based nanoparticles, such as ZnO-, Ag-, or CuO-nanoparticles as well as dissolution-resistant quantum dots, was mediated by biomolecule coronas acquired in pathophysiological and ecological environments, including the lung surfactant, plasma or complex organic matters. Mechanistically, dose-dependent corona-mediated resistance occurred via reducing physical adsorption of nanoparticles to fungal spores. The inhibitory effect of biomolecules on the antifungal activity of Ag-nanoparticles was further verified in vivo, using the invertebrate Galleria mellonella as an A. fumigatus infection model. Our results explain why current nanoantifungals often show low activity in realistic application environments, and will guide nanomaterial designs that maximize functionality and safe translatability as potent antifungals for human health, biotechnology, and agriculture.
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http://dx.doi.org/10.1021/acsami.8b12175DOI Listing
January 2019

Biomolecule-corona formation confers resistance of bacteria to nanoparticle-induced killing: Implications for the design of improved nanoantibiotics.

Biomaterials 2019 02 21;192:551-559. Epub 2018 Nov 21.

Department of Nanobiomedicine/ENT, University Medical Center of Mainz, Langenbeckstrasse 1, 55101, Mainz, Germany. Electronic address:

Multidrug-resistant bacterial infections are a global health threat. Nanoparticles are thus investigated as novel antibacterial agents for clinical practice, including wound dressings and implants. We report that nanoparticles' bactericidal activity strongly depends on their physical binding to pathogens, including multidrug-resistant primary clinical isolates, such as Staphylococcus aureus, Klebsiella pneumoniae or Enterococcus faecalis. Using controllable nanoparticle models, we found that nanoparticle-pathogen complex formation was enhanced by small nanoparticle size rather than material or charge, and was prevented by 'stealth' modifications. Nanoparticles seem to preferentially bind to Gram-positive pathogens, such as Listeria monocytogenes, S. aureus or Streptococcus pyrogenes, correlating with enhanced antibacterial activity. Bacterial resistance to metal-based nanoparticles was mediated by biomolecule coronas acquired in pathophysiological environments, such as wounds, the lung, or the blood system. Biomolecule corona formation reduced nanoparticles' binding to pathogens, but did not impact nanoparticle dissolution. Our results provide a mechanistic explanation why nano-sized antibiotics may show reduced activity in clinically relevant environments, and may inspire future nanoantibiotic designs with improved and potentially pathogen-specific activity.
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http://dx.doi.org/10.1016/j.biomaterials.2018.11.028DOI Listing
February 2019

From Supramolecular Vesicles to Micelles: Controllable Construction of Tumor-Targeting Nanocarriers Based on Host-Guest Interaction between a Pillar[5]arene-Based Prodrug and a RGD-Sulfonate Guest.

Small 2018 12 19;14(52):e1803952. Epub 2018 Nov 19.

Institute for Organic Chemistry, University of Duisburg-Essen, Essen, 45117, Germany.

The targeting ability, drug-loading capacity, and size of the drug nanocarriers are crucial for enhancing the therapeutic index for cancer therapy. Herein, the morphology and size-controllable fabrication of supramolecular tumor-targeting nanocarriers based on host-guest recognition between a novel pillar[5]arene-based prodrug WP5-DOX and a Arg-Gly-Asp (RGD)-modified sulfonate guest RGD-SG is reported. The amphiphilic WP5-DOX⊃RGD-SG complex with a molar ratio of 5:1 self-assembles into vesicles, whereas smaller-sized micelles can be obtained by changing the molar ratio to 1:3. This represents a novel strategy of controllable construction of supramolecular nanovehicles with different sizes and morphologies based on the same host-guest interactions by using different host-guest ratios. Furthermore, in vitro and in vivo studies reveal that both these prodrug nanocarriers could selectively deliver doxorubicin to RGD receptor-overexpressing cancer cells, leading to longer blood retention time, enhanced antitumor efficacy, and reduced systematic toxicity in murine tumor model, suggesting their potential application for targeted drug delivery.
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http://dx.doi.org/10.1002/smll.201803952DOI Listing
December 2018

Small Meets Smaller: Effects of Nanomaterials on Microbial Biology, Pathology, and Ecology.

ACS Nano 2018 07 16;12(7):6351-6359. Epub 2018 Jul 16.

Department of Molecular Biology II, Centre for Nanointegration (CENIDE) , University Duisburg-Essen , Universitätsstraße 5 , 45117 Essen , Germany.

As functionalities and levels of complexity in nanomaterials have increased, unprecedented control over microbes has been enabled, as well. In addition to being pathogens and relevant to the human microbiome, microbes are key players for sustainable biotechnology. To overcome current constraints, mechanistic understanding of nanomaterials' physicochemical characteristics and parameters at the nano-bio interface affecting nanomaterial-microbe crosstalk is required. In this Perspective, we describe key nanomaterial parameters and biological outputs that enable controllable microbe-nanomaterial interactions while minimizing design complexity. We discuss the role of biomolecule coronas, including the problem of nanoantibiotic resistance, and speculate on the effects of nanomaterial-microbe complex formation on the outcomes and fates of microbial pathogens. We close by summarizing our current knowledge and noting areas that require further exploration to overcome current limitations for next-generation practical applications of nanotechnology in medicine and agriculture.
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http://dx.doi.org/10.1021/acsnano.8b03241DOI Listing
July 2018

Survivin antagonizes chemotherapy-induced cell death of colorectal cancer cells.

Oncotarget 2018 Jun 12;9(45):27835-27850. Epub 2018 Jun 12.

Department of Toxicology, University Medical Center of the Johannes Gutenberg University Mainz, 55131 Mainz, Germany.

Irinotecan (CPT-11) and oxaliplatin (L-OHP) are among the most frequently used drugs against colorectal tumors. Therefore, it is important to define the molecular mechanisms that these agents modulate in colon cancer cells. Here we demonstrate that CPT-11 stalls such cells in the G/M phase of the cell cycle, induces an accumulation of the tumor suppressor p53, the replicative stress/DNA damage marker γH2AX, phosphorylation of the checkpoint kinases ATM and ATR, and an ATR-dependent accumulation of the pro-survival molecule survivin. L-OHP reduces the number of cells in S-phase, stalls cell cycle progression, transiently triggers an accumulation of low levels of γH2AX and phosphorylated checkpoint kinases, and L-OHP suppresses survivin expression at the mRNA and protein levels. Compared to CPT-11, L-OHP is a stronger inducer of caspases and p53-dependent apoptosis. Overexpression and RNAi against survivin reveal that this factor critically antagonizes caspase-dependent apoptosis in cells treated with CPT-11 and L-OHP. We additionally show that L-OHP suppresses survivin through p53 and its downstream target p21, which stalls cell cycle progression as a cyclin-dependent kinase inhibitor (CDKi). These data shed new light on the regulation of survivin by two clinically significant drugs and its biological and predictive relevance in drug-exposed cancer cells.
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http://dx.doi.org/10.18632/oncotarget.25600DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6021236PMC
June 2018

Nanoparticle decoration impacts airborne fungal pathobiology.

Proc Natl Acad Sci U S A 2018 07 20;115(27):7087-7092. Epub 2018 Jun 20.

Department of Nanobiomedicine/ENT, University of Mainz Medical Center, 55101 Mainz, Germany;

Airborne fungal pathogens, predominantly , can cause severe respiratory tract diseases. Here we show that in environments, fungal spores can already be decorated with nanoparticles. Using representative controlled nanoparticle models, we demonstrate that various nanoparticles, but not microparticles, rapidly and stably associate with spores, without specific functionalization. Nanoparticle-spore complex formation was enhanced by small nanoparticle size rather than by material, charge, or "stealth" modifications and was concentration-dependently reduced by the formation of environmental or physiological biomolecule coronas. Assembly of nanoparticle-spore surface hybrid structures affected their pathobiology, including reduced sensitivity against defensins, uptake into phagocytes, lung cell toxicity, and TLR/cytokine-mediated inflammatory responses. Following infection of mice, nanoparticle-spore complexes were detectable in the lung and less efficiently eliminated by the pulmonary immune defense, thereby enhancing infections in immunocompromised animals. Collectively, self-assembly of nanoparticle-fungal complexes affects their (patho)biological identity, which may impact human health and ecology.
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http://dx.doi.org/10.1073/pnas.1804542115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6142250PMC
July 2018

Formation of Twisted β-Sheet Tapes from a Self-Complementary Peptide Based on Novel Pillararene-GCP Host-Guest Interaction with Gene Transfection Properties.

Chemistry 2018 Jul 12;24(39):9754-9759. Epub 2018 Jun 12.

Institute for Organic Chemistry, University of Duisburg-Essen, 45117, Essen, Germany.

Small peptides capable of assembling into well-defined nanostructures have attracted extensive attention due to their interesting applications as biomaterials. This work reports the first example of a pillararene functionalized with a guanidiniocarbonyl pyrrole (GCP)-conjugated short peptide segment. The obtained amphiphilic peptide 1 spontaneously self-assembles into a supramolecular β-sheet in aqueous solution based on host-guest interaction between pillararene and GCP unit as well as hydrogen-bonding between the peptide strands. Interestingly, peptide 1 at low concentration shows transitions from small particles to "pearl necklace" assemblies, and finally to branched fibers in a time-dependent process. At higher concentration, it directly assembles into twisted β-sheet tapes. Notably, without pillararene moiety, the control peptide A forms α-helix structure with morphology changing from particles to bamboo-like assemblies depending on concentration, indicating a significant role of the pillararene-GCP host-guest interaction for the secondary structure formation. Moreover, peptide 1 can serve as an efficient gene transfection vector.
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http://dx.doi.org/10.1002/chem.201801315DOI Listing
July 2018

Expressional analysis of disease-relevant signalling-pathways in primary tumours and metastasis of head and neck cancers.

Sci Rep 2018 05 9;8(1):7326. Epub 2018 May 9.

Molecular and Cellular Oncology, ENT/University Hospital of Mainz, Mainz, 55131, Germany.

Head and neck squamous cell carcinoma (HNSCC) often metastasize to lymph nodes resulting in poor prognosis for patients. Unfortunately, the underlying molecular mechanisms contributing to tumour aggressiveness, recurrences, and metastasis are still not fully understood. However, such knowledge is key to identify biomarkers and drug targets to improve prognosis and treatments. Consequently, we performed genome-wide expression profiling of 15 primary HNSSCs compared to corresponding lymph node metastases and non-malignant tissue of the same patient. Differentially expressed genes were bioinformatically exploited applying stringent filter criteria, allowing the discrimination between normal mucosa, primary tumours, and metastases. Signalling networks involved in invasion contain remodelling of the extracellular matrix, hypoxia-induced transcriptional modulation, and the recruitment of cancer associated fibroblasts, ultimately converging into a broad activation of PI3K/AKT-signalling pathway in lymph node metastasis. Notably, when we compared the diagnostic and prognostic value of sequencing data with our expression analysis significant differences were uncovered concerning the expression of the receptor tyrosine kinases EGFR and ERBB2, as well as other oncogenic regulators. Particularly, upregulated receptor tyrosine kinase combinations for individual patients varied, implying potential compensatory and resistance mechanisms against specific targeted therapies. Collectively, we here provide unique transcriptional profiles for disease predictions and comprehensively analyse involved signalling pathways in advanced HNSCC.
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http://dx.doi.org/10.1038/s41598-018-25512-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5943339PMC
May 2018

Human DHEA sulfation requires direct interaction between PAPS synthase 2 and DHEA sulfotransferase SULT2A1.

J Biol Chem 2018 06 9;293(25):9724-9735. Epub 2018 May 9.

From the Institute of Metabolism and Systems Research (IMSR), University of Birmingham, Birmingham B15 2TT, United Kingdom.

The high-energy sulfate donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS), generated by human PAPS synthase isoforms PAPSS1 and PAPSS2, is required for all human sulfation pathways. Sulfotransferase SULT2A1 uses PAPS for sulfation of the androgen precursor dehydroepiandrosterone (DHEA), thereby reducing downstream activation of DHEA to active androgens. Human mutations manifest with undetectable DHEA sulfate, androgen excess, and metabolic disease, suggesting that ubiquitous PAPSS1 cannot compensate for deficient PAPSS2 in supporting DHEA sulfation. In knockdown studies in human adrenocortical NCI-H295R1 cells, we found that PAPSS2, but not PAPSS1, is required for efficient DHEA sulfation. Specific APS kinase activity, the rate-limiting step in PAPS biosynthesis, did not differ between PAPSS1 and PAPSS2. Co-expression of cytoplasmic SULT2A1 with a cytoplasmic PAPSS2 variant supported DHEA sulfation more efficiently than co-expression with nuclear PAPSS2 or nuclear/cytosolic PAPSS1. Proximity ligation assays revealed protein-protein interactions between SULT2A1 and PAPSS2 and, to a lesser extent, PAPSS1. Molecular docking studies showed a putative binding site for SULT2A1 within the PAPSS2 APS kinase domain. Energy-dependent scoring of docking solutions identified the interaction as specific for the PAPSS2 and SULT2A1 isoforms. These findings elucidate the mechanistic basis for the selective requirement for PAPSS2 in human DHEA sulfation.
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http://dx.doi.org/10.1074/jbc.RA118.002248DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6016456PMC
June 2018

A dipeptide with enhanced anion binding affinity enables cell uptake and protein delivery.

Org Biomol Chem 2018 03;16(13):2312-2317

Institute for Organic Chemistry, University of Duisburg-Essen, 45117 Essen, Germany.

Herein, we report a rather simple strategy to enhance the anion binding ability of a dipeptide to achieve cell uptake and also protein delivery. Peptide 1, composed of only two synthetic amino acids with an artificial anion binding site in the side chains, has an overall molecular weight of only 630 Da and demonstrated strong binding affinity (107 M-1) and clustering ability with heparin as a model for cell surface sugars. Furthermore, peptide 1 is also efficiently taken up by cells most likely via endocytosis. The uptake efficiency is dependent on the amount of glycosaminoglycans on the cell surface. Cells with reduced amounts of surface bound glycosaminoglycans show significantly less uptake of peptide 1. Moreover, 1 induced the uptake of a model protein (avidin, around 67 kDa) into cells, which makes 1 a highly attractive candidate for drug and protein delivery, especially as 1 has negligible cytotoxicity.
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http://dx.doi.org/10.1039/c7ob02721dDOI Listing
March 2018

MTOR inhibitor-based combination therapies for pancreatic cancer.

Br J Cancer 2018 02 2;118(3):366-377. Epub 2018 Jan 2.

Medical Clinic and Polyclinic II, Klinikum rechts der Isar, Technical University Munich, 81675 München, Germany.

Background: Although the mechanistic target of rapamycin (MTOR) kinase, included in the mTORC1 and mTORC2 signalling hubs, has been demonstrated to be active in a significant fraction of patients with pancreatic ductal adenocarcinoma (PDAC), the value of the kinase as a therapeutic target needs further clarification.

Methods: We used Mtor floxed mice to analyse the function of the kinase in context of the pancreas at the genetic level. Using a dual-recombinase system, which is based on the flippase-FRT (Flp-FRT) and Cre-loxP recombination technologies, we generated a novel cellular model, allowing the genetic analysis of MTOR functions in tumour maintenance. Cross-species validation and pharmacological intervention studies were used to recapitulate genetic data in human models, including primary human 3D PDAC cultures.

Results: Genetic deletion of the Mtor gene in the pancreas results in exocrine and endocrine insufficiency. In established murine PDAC cells, MTOR is linked to metabolic pathways and maintains the glucose uptake and growth. Importantly, blocking MTOR genetically as well as pharmacologically results in adaptive rewiring of oncogenic signalling with activation of canonical extracellular signal-regulated kinase and phosphoinositide 3-kinase-AKT pathways. We provide evidence that interfering with such adaptive signalling in murine and human PDAC models is important in a subgroup.

Conclusions: Our data suggest developing dual MTORC1/TORC2 inhibitor-based therapies for subtype-specific intervention.
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http://dx.doi.org/10.1038/bjc.2017.421DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5808033PMC
February 2018

Nanoparticle binding attenuates the pathobiology of gastric cancer-associated Helicobacter pylori.

Nanoscale 2018 Jan;10(3):1453-1463

Department of Nanobiomedicine/ENT, University Medical Center of Mainz, Langenbeckstrasse 1, 55101 Mainz, Germany.

Enteric bacteria may cause severe diseases, including gastric cancer-associated Helicobacter pylori. Their infection paths overlap with the oro-gastrointestinal uptake route for nanoparticles, increasingly occurring during environmental or consumer/medical exposure. By comprehensive independent analytical methods, such as live cell fluorescence, electron as well as atomic force microscopy and elemental analysis, we show that a wide array of nanoparticles (NPs) but not microparticles form complexes with H. pylori and enteric pathogens without the need for specific functionalization. The NP-assembly that occurred rapidly was not influenced by variations in physiological temperature, though affected by the NPs' physico-chemical characteristics. Improved binding was observed for small NPs with a negative surface charge, whereas binding could be reduced by surface 'stealth' modifications. Employing human gastric epithelial cells and 3D-organoid models of the stomach, we show that NP-coating did not inhibit H. pylori's cellular attachment. However, even the assembly of non-bactericidal silica NPs attenuated H. pylori infection by reducing CagA phosphorylation, cytoskeletal rearrangement, and IL-8 secretion. Here we demonstrate that NP binding to enteric bacteria may impact their pathobiology which could be further exploited to rationally modulate the (patho)biology of microbes by nanomaterials.
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http://dx.doi.org/10.1039/c7nr06573fDOI Listing
January 2018

Nanosized food additives impact beneficial and pathogenic bacteria in the human gut: a simulated gastrointestinal study.

NPJ Sci Food 2018 4;2(1):22. Epub 2018 Dec 4.

Department of Nanobiomedicine/ENT, University Medical Center of Mainz, Langenbeckstrasse 1, 55131 Mainz, Germany.

Nanotechnology provides the food industry with new ways to modulate various aspects of food. Hence, engineered nanoparticles (NPs) are increasingly added to food and beverage products as functional ingredients. However, the impact of engineered as well as naturally occurring NPs on both commensal and pathogenic microorganisms within the gastrointestinal tract (GI) is not fully understood. Here, well-defined synthetic NPs and bacterial models were used to probe nanoparticle-bacteria interactions, from analytical to in situ to in vitro. NP-bacteria complexation occurred most efficiently for small NPs, independent of their core material or surface charge, but could be reduced by NPs' steric surface modifications. Adsorption to bacteria could also be demonstrated for naturally occurring carbon NPs isolated from beer. Complex formation affected the (patho)biological behavior of both the NPs and bacteria, including their cellular uptake into epithelial cells and phagocytes, pathogenic signaling pathways, and NP-induced cell toxicity. NP-bacteria complex formation was concentration-dependently reduced when the NPs became coated with biomolecule coronas with sequential simulation of first oral uptake and then the GI. However, efficient NP adsorption was restored when the pH was sufficiently low, such as in simulating the conditions of the stomach. Collectively, NP binding to enteric bacteria may impact their (patho)biology, particularly in the stomach. Nanosized-food additives as well as naturally occurring NPs may be exploited to (rationally) shape the microbiome. The information contained in this article should facilitate a "safe by design" strategy for the development and application of engineered NPs as functional foods ingredients.
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http://dx.doi.org/10.1038/s41538-018-0030-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6420113PMC
December 2018

Translocation Biosensors-Versatile Tools to Probe Protein Functions in Living Cells.

Methods Mol Biol 2018 ;1683:195-210

Institute for Molecular Biology, Centre for Medical Biotechnology (ZMB), Mainz Screening Center UG & Co. KG, University of Duisburg-Essen, Essen, Germany.

In this chapter, you will learn how to use translocation biosensors to investigate protein functions in living cells. We here present three classes of modular protein translocation biosensors tailored to investigate: (1) signal-mediated nucleo-cytoplasmic transport, (2) protease activity, and (3) protein-protein interactions. Besides the mapping of protein function, the biosensors are also applicable to identify chemicals and/or (nano) materials modulating the respective protein activities and can also be exploited for RNAi-mediated genetic screens.
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http://dx.doi.org/10.1007/978-1-4939-7357-6_12DOI Listing
June 2018