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- ItemFormation mechanism for stable hybrid clusters of proteins and nanoparticles(Washington D.C. : American Chemical Society, 2015) Moerz, Sebastian T.; Kraegeloh, Annette; Chanana, Munish; Kraus, TobiasCitrate-stabilized gold nanoparticles (AuNP) agglomerate in the presence of hemoglobin (Hb) at acidic pH. The extent of agglomeration strongly depends on the concentration ratio [Hb]/[AuNP]. Negligible agglomeration occurs at very low and very high [Hb]/[AuNP]. Full agglomeration and precipitation occur at [Hb]/[AuNP] corresponding to an Hb monolayer on the AuNP. Ratios above and below this value lead to the formation of an unexpected phase: stable, microscopic AuNP–Hb agglomerates. We investigated the kinetics of agglomeration with dynamic light scattering and the adsorption kinetics of Hb on planar gold with surface-acoustic wave-phase measurements. Comparing agglomeration and adsorption kinetics leads to an explanation of the complex behavior of this nanoparticle–protein mixture. Agglomeration is initiated either when Hb bridges AuNP or when the electrostatic repulsion between AuNP is neutralized by Hb. It is terminated when Hb has been depleted or when Hb forms multilayers on the agglomerates that stabilize microscopic clusters indefinitely.
- ItemSurviving the surf: The tribomechanical properties of the periostracum of Mytilus sp(Amsterdam : Elsevier, 2014) Wählisch, Felix C.; Peter, Nicolas J.; Torrents Abad, Oscar; Oliveira, Mariana V.G.; Schneider, Andreas S.; Schmahl, Wolfgang; Griesshaber, Erika; Bennewitz, RolandWe investigated the friction and wear behavior as well as the mechanical properties of the periostracum of Mytilus sp. Tribological properties were determined with a reciprocal sliding microtribometer, while mechanical characterization was performed using a nanoindenter. Measurements were performed in dry and wet conditions. On the dry periostracum we found a low friction coefficient of 0.078 ± 0.007 on the young parts and a higher one of 0.63 ± 0.02 on the old parts of the shell. Under wet, saline, conditions we only observed one average coefficient of friction of 0.37 ± 0.01. Microscopic ex situ analysis indicated that dry periostracum wore rather rapidly by plowing and fatigue, while it exhibited a high wear resistance when immersed in salt water. The Young’s modulus and hardness of the periostracum were also investigated in both dry and wet conditions. Under dry conditions the Young’s modulus of the periostracum was 8 ± 3 GPa, while under wet conditions it was 0.21 ± 0.05 GPa. The hardness of dry periostracum samples was 353 ± 127 MPa, whereas the hardness of wet samples was 5 ± 2 MPa. It was found that, in the wet state, viscous behavior plays a significant role in the mechanical response of the periostracum. Our results strongly indicate that the periostracum can provide an important contribution to the overall wear resistance of Mytilus sp. shell.
- ItemBifunctional hydrogels containing the laminin motif IKVAV promote neurogenesis(Amsterdam : Elsevier, 2017) Farrukh, Aleeza; Ortega, Felipe; Fan, Wenqiang; Marichal, Nicolás; Paez, Julieta I.; Berninger, Benedikt; del Campo, Aránzazu; Salierno, Marcelo J.Engineering of biomaterials with specific biological properties has gained momentum as a means to control stem cell behavior. Here, we address the effect of bifunctionalized hydrogels comprising polylysine (PL) and a 19-mer peptide containing the laminin motif IKVAV (IKVAV) on embryonic and adult neuronal progenitor cells under different stiffness regimes. Neuronal differentiation of embryonic and adult neural progenitors was accelerated by adjusting the gel stiffness to 2 kPa and 20 kPa, respectively.While gels containing IKVAV or PL alone failed to support long-term cell adhesion, in bifunctional gels, IKVAV synergized with PL to promote differentiation and formation of focal adhesions containing b1-integrin in embryonic cortical neurons. Furthermore, in adult neural stem cell culture, bifunctionalized gels promoted neurogenesis via the expansion of neurogenic clones. These data highlight the potential of synthetic matrices to steer stem and progenitor cell behavior via defined mechano-adhesive properties.
- ItemLipid droplets as a novel cargo of tunnelling nanotubes in endothelial cells(London : Nature Publishing Group, 2015) Astanina, Ksenia; Koch, Marcus; Jüngst, Christian; Zumbusch, Andreas; Kiemer, Alexandra K.Intercellular communication is a fundamental process in the development and functioning of multicellular organisms. Recently, an essentially new type of intercellular communication, based on thin membrane channels between cells, has been reported. These structures, termed intercellular or tunnelling nanotubes (TNTs), permit the direct exchange of various components or signals (e.g., ions, proteins, or organelles) between non-adjacent cells at distances over 100 μm. Our studies revealed the presence of tunnelling nanotubes in microvascular endothelial cells (HMEC-1). The TNTs were studied with live cell imaging, environmental scanning electron microscopy (ESEM), and coherent anti-Stokes Raman scattering spectroscopy (CARS). Tunneling nanotubes showed marked persistence: the TNTs could connect cells over long distances (up to 150 μm) for several hours. Several cellular organelles were present in TNTs, such as lysosomes and mitochondria. Moreover, we could identify lipid droplets as a novel type of cargo in the TNTs. Under angiogenic conditions (VEGF treatment) the number of lipid droplets increased significantly. Arachidonic acid application not only increased the number of lipid droplets but also tripled the extent of TNT formation. Taken together, our results provide the first demonstration of lipid droplets as a cargo of TNTs and thereby open a new field in intercellular communication research.
- ItemLeben und Werk des Karl Hahn(Berlin : Logos Verlag, 2017) Mensing, PetraVon 1905 bis zu seinem Lebensende 1946 stellte der Musiklehrer und Botaniker Karl Hahn eine umfangreiche Sammlung der Mecklenburger Flora insbesondere der Moose in der Umgebung von Neukloster und Grabow zusammen. Neben den noch vorhandenen Belegen hat er diverse Veröffentlichungen im Archiv der Freunde der Naturgeschichte in Mecklenburg hinterlassen, die neben der Beschreibung der einzelnen Funde auch Wanderbeschreibungen und Naturbeobachtungen thematisierten. In diesem Beitrag werden alle von ihm als „Neu für Mecklenburg“ bezeichneten Moosarten erstmals in einer Veröffentlichung zusammengetragen sowie Anregungen für zukünftige Arbeiten gegeben.
- ItemReal-time monitoring of calcium carbonate and cationic peptide deposition on carboxylate-SAM using a microfluidic SAW biosensor(Frankfurt am Main : Beilstein-Institut, 2014) Pohl, Anna; Weiss, Ingrid M.A microfluidic biosensor with surface acoustic wave technology was used in this study to monitor the interaction of calcium carbonate with standard carboxylate self-assembled monolayer sensor chips. Different fluids, with and without biomolecular components, were investigated. The pH-dependent surface interactions of two bio-inspired cationic peptides, AS8 and ES9, which are similar to an extracellular domain of the chitin synthase involved in mollusc shell formation, were also investigated in a biological buffer system. A range of experimental conditions are described that are suitable to study non-covalent molecular interactions in the presence of ionic substances, such as, mineral precursors below the solubility equilibrium. The peptide ES9, equal to the mollusc chitin synthase epitope, is less sensitive to changes in pH than its counterpart AS8 with a penta-lysine core, which lacks the flanking acidic residues. This study demonstrates the extraordinary potential of microfluidic surface acoustic wave biosensors to significantly expand our experimental capabilities for studying the principles underlying biomineralization in vitro.
- ItemQuantification of internalized silica nanoparticles via STED microscopy(London : Hindawi, 2015) Peuschel, Henrike; Ruckelshausen, Thomas; Cavelius, Christian; Kraegeloh, AnnetteThe development of safe engineered nanoparticles (NPs) requires a detailed understanding of their interaction mechanisms on a cellular level. Therefore, quantification of NP internalization is crucial to predict the potential impact of intracellular NP doses, providing essential information for risk assessment as well as for drug delivery applications. In this study, the internalization of 25 nm and 85 nm silica nanoparticles (SNPs) in alveolar type II cells (A549) was quantified by application of super-resolution STED (stimulated emission depletion) microscopy. Cells were exposed to equal particle number concentrations (9.2 x 10^10 particles mL^-1) of each particle size and the sedimentation of particles during exposure was taken into account. Microscopy images revealed that particles of both sizes entered the cells after 5 h incubation in serum supplemented and serum-free medium. According to the in vitro sedimentation, diffusion, and dosimetry (ISDD) model 20–27 of the particles sedimented. In comparison, 102-103 NPs per cell were detected intracellularly serum-containing medium. Furthermore, in the presence of serum, no cytotoxicity was induced by the SNPs. In serum-free medium, large agglomerates of both particle sizes covered the cells whereas only high concentrations (≥ 3.8 × 10^12 particles mL^-1) of the smaller particles induced cytotoxicity.
- ItemProtein identity and environmental parameters determine the final physico-chemical properties of protein-coated metal nanoparticles(Washington D.C. : American Chemical Society, 2015) Dewald, Inna; Isakin, Olga; Schubert, Jonas; Kraus, Tobias; Chanana, MunishWhen a nanomaterial enters a biological system, proteins adsorb onto the particle surface and alter the surface properties of nanoparticles, causing drastic changes in physico-chemical properties such as hydrodynamic size, surface charge and aggregation state, thus giving a completely new and undefined physico-chemical identity to the nanoparticles. In the present work, we study the impact of the protein identity (molecular weight and isoelectric point) and the environmental conditions (pH and ionic strength) on the final physico-chemical properties of a model nanoparticle system, i.e. gold nanoparticles. Gold nanoparticles either form stable dispersions or agglomerate spontaneously when mixed with protein solutions, depending on the protein and the experimental conditions. Strikingly, the agglomerates redisperse to individually dispersed and colloidally stable nanoparticles, depending on the purification pH. The final protein coated nanoparticles exhibit specific stabilities and surface charges that depend on protein type and the conditions during its adsorption. By understanding the interactions of nanoparticles with proteins under controlled conditions, we can define the protein corona of the NPs and thus their physico-chemical properties in various media.
- ItemPhotoactivatable Hsp47: A tool to control and regulate collagen secretion & assembly(Hoboken, NJ : Wiley, 2018) Khan, Essak; Sankaran, Shrikrishnan; Paez, Julieta; Muth, Christina; Han, Mitchell; Del Campo, AránzazuCollagen is the most abundant structural protein in mammals and is crucial for the mechanical integrity of tissues. Hsp47, an endoplasmic reticulum resident collagen-specific chaperone, is involved in collagen biosynthesis and plays a fundamental role in the folding, stability, and intracellular transport of procollagen triple helices. This work reports on a photoactivatable derivative of Hsp47 that allows regulation of collagen biosynthesis within mammalian cells using light. Photoactivatable Hsp47 contains a non-natural light-responsive tyrosine (o-nitro benzyl tyrosine (ONBY)) at Tyr383 position of the protein sequence. This mutation renders Hsp47 inactive toward collagen binding. The inactive, photoactivatable protein is easily uptaken by cells within a few minutes of incubation, and accumulated at the endoplasmic reticulum via retrograde KDEL receptor-mediated uptake. Upon light exposure, the photoactivatable Hsp47 turns into functional Hsp47 in situ. The increased intracellular concentration of Hsp47 results in stimulated secretion of collagen. The ability to promote collagen synthesis on demand, with spatiotemporal resolution, and in diseased state cells is demonstrated in vitro. It is envisioned that photoactivatable Hsp47 allows unprecedented fundamental studies of collagen biosynthesis, matrix biology, and inspires new therapeutic concepts in biomedicine and tissue regeneration.
- ItemThe mTOR and PP2A pathways regulate PHD2 phosphorylation to Fine-Tune HIF1α levels and colorectal cancer cell survival under hypoxia(Amsterdam : Elsevier, 2017) Di Conza, Giusy; Cafarello, Sarah Trusso; Loroch, Stefan; Mennerich, Daniela; Deschoemaeker, Sofie; Di Matteo, Mario; Ehling, Manuel; Gevaert, Kris; Prenen, Hans; Zahedi, Rene Peiman; Sickmann, Albert; Kietzmann, Thomas; Moretti, Fabiola; Mazzone, MassimilianoOxygen-dependent HIF1α hydroxylation and degradation are strictly controlled by PHD2. In hypoxia, HIF1α partly escapes degradation because of low oxygen availability. Here, we show that PHD2 is phosphorylated on serine 125 (S125) by the mechanistic target of rapamycin (mTOR) downstream kinase P70S6K and that this phosphorylation increases its ability to degrade HIF1α. mTOR blockade in hypoxia by REDD1 restrains P70S6K and unleashes PP2A phosphatase activity. Through its regulatory subunit B55α, PP2A directly dephosphorylates PHD2 on S125, resulting in a further reduction of PHD2 activity that ultimately boosts HIF1α accumulation. These events promote autophagy-mediated cell survival in colorectal cancer (CRC) cells. B55α knockdown blocks neoplastic growth of CRC cells in vitro and in vivo in a PHD2-dependent manner. In patients, CRC tissue expresses higher levels of REDD1, B55α, and HIF1α but has lower phospho-S125 PHD2 compared with a healthy colon. Our data disclose a mechanism of PHD2 regulation that involves the mTOR and PP2A pathways and controls tumor growth.