INMdok (Leibniz Institute for New Materials)
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Effect of fluoride mouthrinses and stannous ions on the erosion protective properties of the in situ pellicle
Get PDFThe particular feature of this study is the investigation of effects of pure fluoride- or stannous ions based mouthrinses on the erosion protective properties and the ultrastructure of the in situ pellicle (12 volunteers). Experimental solutions were prepared either from 500 ppm NaF, SMFP, AmF or SnF2 or 1563 ppm SnCl2, respectively. After 1 min of in situ pellicle formation on bovine enamel slabs, rinses with one of the preparations were performed for 1 min and intraoral specimens’ exposure was continued for 28 min. Native enamel slabs and rinses with bidestilled water served as controls. After oral exposure, slabs were incubated in HCl (pH 2; 2.3; 3) for 120 s and kinetics of calcium- and phosphate release were measured photometrically; representative samples were analysed by TEM and EDX. All mouthrinses reduced mineral loss compared to the native 30-min pellicle. The effect was pH-dependent and significant at all pH values only for the tin-containing mouthrinses. No significant differences were observed between the SnF2- and the SnCl2-containing solutions. TEM/EDX confirmed ultrastructural pellicle modifications. SnF2 appears to be the most effective type of fluoride to prevent erosive enamel demineralisation. The observed effects primarily have to be attributed to the stannous ions’ content
Photoactivatable Hsp47: A Tool to Regulate Collagen Secretion and Assembly
Get PDFAbstract Collagen 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
Nanopillar Diffraction Gratings by Two-Photon Lithography
Get PDFTwo-dimensional photonic structures such as nanostructured pillar gratings are useful for various applications including wave coupling, diffractive optics, and security features. Two-photon lithography facilitates the generation of such nanostructured surfaces with high precision and reproducibility. In this work, we report on nanopillar diffraction gratings fabricated by two-photon lithography with various laser powers close to the polymerization threshold of the photoresist. As a result, defect-free arrays of pillars with diameters down to 184 nm were fabricated. The structure sizes were analyzed by scanning electron microscopy and compared to theoretical predictions obtained from Monte Carlo simulations. The optical reflectivities of the nanopillar gratings were analyzed by optical microscopy and verified by rigorous coupled-wave simulations
Vimentin Intermediate Filament Rings Deform the Nucleus During the First Steps of Adhesion
Get PDFDuring cell spreading, cells undergo many changes to their architecture and their mechanical properties. Vimentin, as an integral part of the cell architecture, and its mechanical stability must adapt to the new state of the cell. This study focuses on the structures formed by vimentin during the first steps of cell adhesion. Very early, ball-like structures, or ‘knots’, are seen and often vimentin filaments emerge in the shape of rings around the nucleus. Although intermediate filaments are not known to be associated to motor proteins to form contractile systems, these rings can nonetheless strongly deform the cell nucleus. In the first 6 h to 12 h of adhesion, these vimentin knots and rings disappear, and the intermediate filament network returns to the state seen before detachment of the cells. As these vimentin structures are very transient in the early steps of cell spreading, they have rarely been described in the literature. However, they can also be seen during mitosis, which is an event that involves partial detachment and re-spreading of the cells. Interestingly, the turnover dynamics of vimentin are reduced in both the knots and rings, compared to vimentin in the lamelipodia. It remains to de defined how the force is transmitted from the ball-like structures to the rings, and to measure the impact of such strong nuclear deformation on gene expression during cell re-spreading and the rearrangement of the vimentin network
Enantiomer-selective magnetization of conglomerates for quantitative chiral separation
Get PDFSelective crystallization represents one of the most economical and convenient methods to provide large-scale optically pure chiral compounds. Although significant development has been achieved since Pasteur’s separation of sodium ammonium tartrate in 1848, this method is still fundamentally low efficient (low transformation ratio or high labor). Herein, we describe an enantiomer-selective-magnetization strategy for quantitatively separating the crystals of conglomerates by using a kind of magnetic nano-splitters. These nano-splitters would be selectively wrapped into the S-crystals, leading to the formation of the crystals with different physical properties from that of R-crystals. As a result of efficient separation under magnetic field, high purity chiral compounds (99.2 ee% for R-crystals, 95.0 ee% for S-crystals) can be obtained in a simple one-step crystallization process with a high separation yield (95.1%). Moreover, the nano-splitters show expandability and excellent recyclability. We foresee their great potential in developing chiral separation methods used on different scales
Electroencephalographic Responses to Frictional Stimuli: Measurement Setup and Processing Pipeline
Get PDFTactility is a key sense in the human interaction with the environment. The understanding of tactile perception has become an exciting area in industrial, medical and scienti c research with an emphasis on the development of new haptic technologies. Surprisingly, the quanti cation of tactile perception has, compared to other senses, only recently become a eld of scienti c investigation. The overall goal of this emerging scienti c discipline is an understanding of the causal chain from the contact of the skin with materials to the brain dynamics representing recognition of and emotional reaction to the materials. Each link in this chain depends on individual and environmental factors ranging from the in uence of humidity on contact formation to the role of attention for the perception of touch. This thesis reports on the research of neural correlates to the frictional stimulation of the human ngertip. Event-related electroencephalographic potentials (ERPs) upon the change in ngertip friction are measured and studied, when pins of a programmable Braille-display were brought into skin contact. In order to contribute to the understanding of the causal chain mentioned above, this work combines two research areas which are usually not connected to each other, namely tribology and neuroscience. The goal of the study is to evaluate contributions of friction to the process of haptic perception. Key contributions of this thesis are: 1) Development of a setup to simultaneously record physical forces and ERPs upon tactile stimulation. 2) Implementation of a dedicated signal processing pipeline for the statistical analysis of ERP -amplitudes, -latencies and -instantaneous phases. 3) Interpretation of skin friction data and extraction of neural correlates with respect to varying friction intensities. The tactile stimulation of the ngertip upon raising and lowering of di erent lines of Braille-pins (one, three and ve) caused pronounced N50 and P100 components in the event-related ERPsequences, which is in line with the current literature. Friction between the ngertip and the Braille-system exhibited a characteristic temporal development which is attributed to viscoelastic skin relaxation. Although the force stimuli varied by a factor of two between the di erent Braillepatterns, no signi cant di erences were observed between the amplitudes and latencies of ERPs after standard across-trial averaging. Thus, for the rst time a phase measure for estimating singletrial interactions of somatosensory potentials is proposed. Results show that instantaneous phase coherency is evoked by friction, and that higher friction induces stronger and more time-localized phase coherencyDie Taktilität ist ein zentraler Sinn in der Interaktion mit unserer Umwelt. Das Bestreben, fundierte Erkenntnisse hinsichtlich der taktilenWahrnehmung zu gewinnen erhält groÿen Zuspruch in der industriellen, medizinischen und wissenschaftlichen Forschung, meist mit einem Fokus auf der Entwicklung von haptischen Technologien. Erstaunlicherweise ist jedoch die wissenschaftliche Quanti zierung der taktilen Wahrnehmung, verglichen mit anderen Sinnesmodalitäten, erst seit kurzem ein sich entwickelnder Forschungsbereich. Fokus dieser Disziplin ist es, die kognitive und emotionale Reaktion nach physischem Kontakt mit Materialien zu beschreiben, und die kausale Wirkungskette von der Berührung bis zur Reaktion zu verstehen. Dabei unterliegen die einzelnen Faktoren dieser Kette sowohl individuellen als auch externen Ein üssen, welche von der Luftfeuchtigkeit während des Kontaktes bis hin zur Rolle der Aufmerksamkeit für die Wahrnehmung reichen. Die vorliegende Arbeit beschäftigt sich mit der Untersuchung von neuronalen Korrelaten nach Reibungsstimulation des menschlichen Fingers. Dazu wurden Reibungsänderungen, welche durch den Kontakt der menschlichen Fingerspitze mit schaltbaren Stiften eines Braille-Display erzeugt wurden, untersucht und die entsprechenden neuronalen Korrelate aufgezeichnet. Um zu dem Verst ändnis der oben erwähnten Wirkungskette beizutragen, werden Ansätze aus zwei für gewöhnlich nicht zusammenhängenden Forschungsbereichen, nämlich der Tribologie und der Neurowissenschaft, kombiniert. Folgende Beiträge sind Hauptbestandteile dieser Arbeit: 1) Realisierung einer Messumgebung zur simultanen Ableitung von Kräften und ereigniskorrelierten Potentialen nach taktiler Stimulation der Fingerspitze. 2) Aufbau einer speziellen Signalverarbeitungskette zur statistischen Analyse von stimulationsabh ängigen EEG -Amplituden, -Latenzen und -instantanen Phasen. 3) Interpretation der erhobenen Reibungsdaten und Extraktion neuronaler Korrelate hinsichtlich variierender Stimulationsintensitäten. Unsere Resultate zeigen, dass die taktile Stimulation der Fingerspitze nach Anheben und Senken von Braille-Stiften zu signi kanten N50 und P100 Komponenten in den ereigniskorrelierten Potentialen führt, im Einklang mit der aktuellen Literatur. Die Reibung zwischen der Fingerspitze und dem Braille-System zeigte einen charakteristischen Signalverlauf, welcher auf viskoelastische Hautrelaxation zurückzuführen ist. Trotz der um einen Faktor zwei verschiedenen Intensit ätsunterschiede zwischen den Stimulationsmustern zeigten sich keine signi kanten Unterschiede zwischen den einfach gemittelten Amplituden der evozierten Potentialen. Erstmalig wurde ein Phasen-Maÿ zur Identi zierung von Unterschieden zwischen somatosensorischen "single-trial" Interaktionen angewandt. Diese Phasenanalyse zeigte, im Gegensatz zur Amplituden- und Latenzanalyse, deutlichere und signi kantere Unterschiede zwischen den Stimulationsparadigmen. Es wird gefolgert, dass Kohärenz zwischen den Momentanphasen durch Reibungsereignisse herbeigef ührt wird und dass durch stärkere Reibung diese Kohärenz, im zeitlichen Verlauf, stärker und lokalisierter wird
Cellular Deformations Induced by Conical Silicon Nanowire Arrays Facilitate Gene Delivery
Get PDFAbstract Engineered cell–nanostructured interfaces generated by vertically aligned silicon nanowire (SiNW) arrays have become a promising platform for orchestrating cell behavior, function, and fate. However, the underlying mechanism in SiNW-mediated intracellular access and delivery is still poorly understood. This study demonstrates the development of a gene delivery platform based on conical SiNW arrays for mechanical cell transfection, assisted by centrifugal force, for both adherent and nonadherent cells in vitro. Cells form focal adhesions on SiNWs within 6 h, and maintain high viability and motility. Such a functional and dynamic cell–SiNW interface features conformational changes in the plasma membrane and in some cases the nucleus, promoting both direct penetration and endocytosis; this synergistically facilitates SiNW-mediated delivery of nucleic acids into immortalized cell lines, and into difficult-to-transfect primary immune T cells without pre-activation. Moreover, transfected cells retrieved from SiNWs retain the capacity to proliferate—crucial to future biomedical applications. The results indicate that SiNW-mediated intracellular delivery holds great promise for developing increasingly sophisticated investigative and therapeutic tools
Mathematical modeling of drug-induced receptor internalization in the HER2-positive SKBR3 breast cancer cell-line
Get PDFAbout 20% of breast cancer tumors over-express the HER2 receptor. Trastuzumab, an approved drug to treat this type of breast cancer, is a monoclonal antibody directly binding at the HER2 receptor and ultimately inhibiting cancer cell growth. The goal of our study was to understand the early impact of trastuzumab on HER2 internalization and recycling in the HER2-overexpressing breast cancer cell line SKBR3. To this end, fluorescence microscopy, monitoring the amount of HER2 expression in the plasma membrane, was combined with mathematical modeling to derive the flux of HER2 receptors from and to the membrane. We constructed a dynamic multi-compartment model based on ordinary differential equations. To account for cancer cell heterogeneity, a first, dynamic model was expanded to a second model including two distinct cell phenotypes, with implications for different conformational states of HER2, i.e. monomeric or homodimeric. Our mathematical model shows that the hypothesis of fast constitutive HER2 recycling back to the plasma membrane does not match the experimental data. It conclusively describes the experimental observation that trastuzumab induces sustained receptor internalization in cells with membrane ruffles. It is also concluded that for rare, non-ruffled (flat) cells, HER2 internalization occurs three orders of magnitude slower than for the bulk, ruffled cell population
Adhesion of a rigid punch to a confined elastic layer revisited
Get PDFThe adhesion of a punch to a linear elastic, confined layer is investigated. Numerical analysis is performed to determine the equivalent elastic modulus in terms of layer confinement. The size of the layer relative to the punch radius and its Poisson?s ratio are found to affect the layer stiffness. The results reveal that the equivalent modulus of a highly confined layer depends on its Poisson?s ratio, whereas, in contrast, an unconfined layer is only sensitive to the extent of the elastic film. The solutions of the equivalent modulus obtained from the simulations are fitted by an analytical function that, subsequently, is utilized to deduce the energy release rate for detachment of the punch via linear elastic fracture mechanics. The energy release rate strongly varies with layer confinement. Regimes for stable and unstable crack growth can be identified that, in turn, are correlated to interfacial stress distributions to distinguish between different detachment mechanisms
Toll-Like Receptor 2 Release by Macrophages: An Anti-inflammatory Program Induced by Glucocorticoids and Lipopolysaccharide
Get PDFGlucocorticoids (GCs) are widely prescribed therapeutics for the treatment of inflammatory diseases, and endogenous GCs play a key role in immune regulation. Toll-like receptors (TLRs) enable innate immune cells, such as macrophages, to recognize a wide variety of microbial ligands, thereby promoting inflammation. The interaction of GCs with macrophages in the immunosuppressive resolution phase upon prolonged TLR activation is widely unknown. Treatment of human alveolar macrophages (AMs) with the synthetic GC dexamethasone (Dex) did not alter the expression of TLRs -1, -4, and -6. In contrast, TLR2 was upregulated in a GC receptor-dependent manner, as shown by Western blot and qPCR. Furthermore, long-term lipopolysaccharide (LPS) exposure mimicking immunosuppression in the resolution phase of inflammation synergistically increased Dex-mediated TLR2 upregulation. Analyses of publicly available data sets suggested that TLR2 is induced during the resolution phase of inflammatory diseases, i.e., under conditions associated with high endogenous GC production. TLR2 induction did not enhance TLR2 signaling, as indicated by reduced cytokine production after treatment with TLR2 ligands in Dex- and/or LPS-primed AMs. Thus, we hypothesized that the upregulated membrane-bound TLR2 might serve as a precursor for soluble TLR2 (sTLR2), known to antagonize TLR2-dependent cell actions. Supernatants of LPS/Dex-primed macrophages contained sTLR2, as demonstrated by Western blot analysis. Activation of metalloproteinases resulted in enhanced sTLR2 shedding. Additionally, we detected full-length TLR2 and assumed that this might be due to the production of TLR2-containing extracellular vesicles (EVs). EVs from macrophage supernatants were isolated by sequential centrifugation. Both untreated and LPS/Dex-treated cells produced vesicles of various sizes and shapes, as shown by cryo-transmission electron microscopy. These vesicles were identified as the source of full-length TLR2 in macrophage supernatants by Western blot and mass spectrometry. Flow cytometric analysis indicated that TLR2-containing EVs were able to bind the TLR2 ligand Pam3CSK4. In addition, the presence of EVs reduced inflammatory responses in Pam3CSK4-treated endothelial cells and HEK Dual reporter cells, demonstrating that TLR2-EVs can act as decoy receptors. In summary, our data show that sTLR2 and full-length TLR2 are released by macrophages under anti-inflammatory conditions, which may contribute to GC-induced immunosuppression