21 results
Search Results
Now showing 1 - 10 of 21
- ItemQuantifying ligand-cell interactions and determination of the surface concentrations of ligands on hydrogel films: The measurement challenge(Melville, NY : AIP Publishing, 2015) Beer, Meike V.; Hahn, Kathrin; Diederichs, Sylvia; Fabry, Marlies; Singh, Smriti; Spencer, Steve J.; Salber, Jochen; Möller, Martin; Shard, Alexander G.; Groll, JürgenHydrogels are extensively studied for biomaterials application as they provide water swollen noninteracting matrices in which specific binding motifs and enzyme-sensitive degradation sites can be incorporated to tailor cell adhesion, proliferation, and migration. Hydrogels also serve as excellent basis for surface modification of biomaterials where interfacial characteristics are decisive for implant success or failure. However, the three-dimensional nature of hydrogels makes it hard to distinguish between the bioactive ligand density at the hydrogel-cell interface that is able to interact with cells and the ligands that are immobilized inside the hydrogel and not accessible for cells. Here, the authors compare x-ray photoelectron spectrometry (XPS), time-of-flight secondary ion mass spectroscopy (ToF-SIMS), enzyme linked immunosorbent assay (ELISA), and the correlation with quantitative cell adhesion using primary human dermal fibroblasts (HDF) to gain insight into ligand distribution. The authors show that although XPS provides the most useful quantitative analysis, it lacks the sensitivity to measure biologically meaningful concentrations of ligands. However, ToF-SIMS is able to access this range provided that there are clearly distinguishable secondary ions and a calibration method is found. Detection by ELISA appears to be sensitive to the ligand density on the surface that is necessary to mediate cell adhesion, but the upper limit of detection coincides closely with the minimal ligand spacing required to support cell proliferation. Radioactive measurements and ELISAs were performed on amine reactive well plates as true 2D surfaces to estimate the ligand density necessary to allow cell adhesion onto hydrogel films. Optimal ligand spacing for HDF adhesion and proliferation on ultrathin hydrogel films was determined as 6.5 ± 1.5 nm.
- ItemPolymer Brush-Functionalized Chitosan Hydrogels as Antifouling Implant Coatings(Columbus, Ohio : American Chemical Society, 2017) Buzzacchera, Irene; Vorobii, Mariia; Kostina, Nina Yu; de Los Santos Pereira, Andres; Riedel, Tomáš; Bruns, Michael; Ogieglo, Wojciech; Möller, Martin; Wilson, Christopher J.; Rodriguez-Emmenegger, CesarImplantable sensor devices require coatings that efficiently interface with the tissue environment to mediate biochemical analysis. In this regard, bioinspired polymer hydrogels offer an attractive and abundant source of coating materials. However, upon implantation these materials generally elicit inflammation and the foreign body reaction as a consequence of protein fouling on their surface and concomitant poor hemocompatibility. In this report we investigate a strategy to endow chitosan hydrogel coatings with antifouling properties by the grafting of polymer brushes in a "grafting-from" approach. Chitosan coatings were functionalized with polymer brushes of oligo(ethylene glycol) methyl ether methacrylate and 2-hydroxyethyl methacrylate using photoinduced single electron transfer living radical polymerization and the surfaces were thoroughly characterized by XPS, AFM, water contact angle goniometry, and in situ ellipsometry. The antifouling properties of these new bioinspired hydrogel-brush coatings were investigated by surface plasmon resonance. The influence of the modifications to the chitosan on hemocompatibility was assessed by contacting the surfaces with platelets and leukocytes. The coatings were hydrophilic and reached a thickness of up to 180 nm within 30 min of polymerization. The functionalization of the surface with polymer brushes significantly reduced the protein fouling and eliminated platelet activation and leukocyte adhesion. This methodology offers a facile route to functionalizing implantable sensor systems with antifouling coatings that improve hemocompatibility and pave the way for enhanced device integration in tissue.
- ItemMulti-scale processes of beech wood disintegration and pretreatment with 1-ethyl-3-methylimidazolium acetate/water mixtures(London : BioMed Central, 2016) Viell, Jörn; Inouye, Hideyo; Szekely, Noemi K.; Frielinghaus, Henrich; Marks, Caroline; Wang, Yumei; Anders, Nico; Spiess, Antje C.; Makowski, LeeBackground: The valorization of biomass for chemicals and fuels requires efficient pretreatment. One effective strategy involves the pretreatment with ionic liquids which enables enzymatic saccharification of wood within a few hours under mild conditions. This pretreatment strategy is, however, limited by water and the ionic liquids are rather expensive. The scarce understanding of the involved effects, however, challenges the design of alternative pretreatment concepts. This work investigates the multi length-scale effects of pretreatment of wood in 1-ethyl-3-methylimidazolium acetate (EMIMAc) in mixtures with water using spectroscopy, X-ray and neutron scattering. Results: The structure of beech wood is disintegrated in EMIMAc/water mixtures with a water content up to 8.6 wt%. Above 10.7 wt%, the pretreated wood is not disintegrated, but still much better digested enzymatically compared to native wood. In both regimes, component analysis of the solid after pretreatment shows an extraction of few percent of lignin and hemicellulose. In concentrated EMIMAc, xylan is extracted more efficiently and lignin is defunctionalized. Corresponding to the disintegration at macroscopic scale, SANS and XRD show isotropy and a loss of crystallinity in the pretreated wood, but without distinct reflections of type II cellulose. Hence, the microfibril assembly is decrystallized into rather amorphous cellulose within the cell wall. Conclusions: The molecular and structural changes elucidate the processes of wood pretreatment in EMIMAc/water mixtures. In the aqueous regime with >10.7 wt% water in EMIMAc, xyloglucan and lignin moieties are extracted, which leads to coalescence of fibrillary cellulose structures. Dilute EMIMAc/water mixtures thus resemble established aqueous pretreatment concepts. In concentrated EMIMAc, the swelling due to decrystallinization of cellulose, dissolution of cross-linking xylan, and defunctionalization of lignin releases the mechanical stress to result in macroscopic disintegration of cells. The remaining cell wall constituents of lignin and hemicellulose, however, limit a recrystallization of the solvated cellulose. These pretreatment mechanisms are beyond common pretreatment concepts and pave the way for a formulation of mechanistic requirements of pretreatment with simpler pretreatment liquors. © 2016 Viell et al.
- ItemBiadhesive Peptides for Assembling Stainless Steel and Compound Loaded Micro-Containers(Weinheim : Wiley-VCH, 2019) Apitius, Lina; Buschmann, Sven; Bergs, Christian; Schönauer, David; Jakob, Felix; Pich, Andrij; Schwaneberg, UlrichBiadhesive peptides (peptesives) are an attractive tool for assembling two chemically different materials—for example, stainless steel and polycaprolactone (PCL). Stainless steel is used in medical stents and PCL is used as a biodegradable polymer for fabrication of tissue growth scaffolds and drug delivering micro-containers. Biadhesive peptides are composed of two domains (e.g., dermaseptin S1 and LCI) with different material-binding properties that are separated through a stiff peptide-spacer. The peptesive dermaseptin S1-domain Z-LCI immobilizes antibiotic-loaded PCL micro-containers on stainless steel surfaces. Immobilization is visualized by microscopy and field emission scanning electron microscopy analysis and released antibiotic from the micro-containers is confirmed through growth inhibition of Escherichia coli cells.
- ItemDecreased Effective Macromolecular Crowding in Escherichia coli Adapted to Hyperosmotic Stress(Washington, DC : Soc., 2019) Liu, Boqun; Hasrat, Zarief; Poolman, Bert; Boersma, Arnold J.; Mullineaux, Conrad W.Escherichia coli adapts to changing environmental osmolality to survive and maintain growth. It has been shown that the diffusion of green fluorescent protein (GFP) in cells adapted to osmotic upshifts is higher than expected from the increase in biopolymer volume fraction. To better understand the physicochemical state of the cytoplasm in adapted cells, we now follow the macromolecular crowding during adaptation with fluorescence resonance energy transfer (FRET)-based sensors. We apply an osmotic upshift and find that after an initial increase, the apparent crowding decreases over the course of hours to arrive at a value lower than that before the osmotic upshift. Crowding relates to cell volume until cell division ensues, after which a transition in the biochemical organization occurs. Analysis of single cells by microfluidics shows that changes in cell volume, elongation, and division are most likely not the cause for the transition in organization. We further show that the decrease in apparent crowding upon adaptation is similar to the apparent crowding in energy-depleted cells. Based on our findings in combination with literature data, we suggest that adapted cells have indeed an altered biochemical organization of the cytoplasm, possibly due to different effective particle size distributions and concomitant nanoscale heterogeneity. This could potentially be a general response to accommodate higher biopolymer fractions yet retaining crowding homeostasis, and it could apply to other species or conditions as well.IMPORTANCE Bacteria adapt to ever-changing environmental conditions such as osmotic stress and energy limitation. It is not well understood how biomolecules reorganize themselves inside Escherichia coli under these conditions. An altered biochemical organization would affect macromolecular crowding, which could influence reaction rates and diffusion of macromolecules. In cells adapted to osmotic upshift, protein diffusion is indeed faster than expected on the basis of the biopolymer volume fraction. We now probe the effects of macromolecular crowding in cells adapted to osmotic stress or depleted in metabolic energy with a genetically encoded fluorescence-based probe. We find that the effective macromolecular crowding in adapted and energy-depleted cells is lower than in unstressed cells, indicating major alterations in the biochemical organization of the cytoplasm.
- ItemPolymer-based controlled-release fed-batch microtiter plate - diminishing the gap between early process development and production conditions(Berlin ; Heidelberg : Springer, 2019) Keil, T.; Dittrich, B.; Lattermann, C.; Habicher, T.; Büchs, J.Background: Fed-batch conditions are advantageous for industrial cultivations as they avoid unfavorable phenomena appearing in batch cultivations. Those are for example the formation of overflow metabolites, catabolite repression, oxygen limitation or inhibition due to elevated osmotic concentrations. For both, the early bioprocess development and the optimization of existing bioprocesses, small-scale reaction vessels are applied to ensure high throughput, low costs and prompt results. However, most conventional small-scale procedures work in batch operation mode, which stands in contrast to fed-batch conditions in large-scale bioprocesses. Extensive expenditure for installations and operation accompany almost all cultivation systems in the market allowing fed-batch conditions in small-scale. An alternative, more cost efficient enzymatic glucose release system is strongly influenced by environmental conditions. To overcome these issues, this study investigates a polymer-based fed-batch system for controlled substrate release in microtiter plates. Results: Immobilizing a solid silicone matrix with embedded glucose crystals at the bottom of each well of a microtiter plate is a suitable technique for implementing fed-batch conditions in microtiter plates. The results showed that the glucose release rate depends on the osmotic concentration, the pH and the temperature of the medium. Moreover, the applied nitrogen source proved to influence the glucose release rate. A new developed mathematical tool predicts the glucose release for various media conditions. The two model organisms E. coli and H. polymorpha were cultivated in the fed-batch microtiter plate to investigate the general applicability for microbial systems. Online monitoring of the oxygen transfer rate and offline analysis of substrate, product, biomass and pH confirmed that fed-batch conditions are comparable to large-scale cultivations. Furthermore, due to fed-batch conditions in microtiter plates, product formation could be enhanced by the factor 245 compared to batch cultivations. Conclusions: The polymer-based fed-batch microtiter plate represents a sophisticated and cost efficient system to mimic typical industrial fed-batch conditions in small-scale. Thus, a more reliable strain screening and early process development can be performed. A systematical scale-down with low expenditure of work, time and money is possible. © 2019 The Author(s).
- ItemDirected Evolution of P450 BM3 towards Functionalization of Aromatic O-Heterocycles(Basel : Molecular Diversity Preservation International (MDPI), 2019) Santos, Gustavo de Almeida; Dhoke, Gaurao V.; Davari, Mehdi D.; Ruff, Anna Joëlle; Schwaneberg, UlrichThe O-heterocycles, benzo-1,4-dioxane, phthalan, isochroman, 2,3-dihydrobenzofuran, benzofuran, and dibenzofuran are important building blocks with considerable medical application for the production of pharmaceuticals. Cytochrome P450 monooxygenase (P450) Bacillus megaterium 3 (BM3) wild type (WT) from Bacillus megaterium has low to no conversion of the six O-heterocycles. Screening of in-house libraries for active variants yielded P450 BM3 CM1 (R255P/P329H), which was subjected to directed evolution and site saturation mutagenesis of four positions. The latter led to the identification of position R255, which when introduced in the P450 BM3 WT, outperformed all other variants. The initial oxidation rate of nicotinamide adenine dinucleotide phosphate (NADPH) consumption increased ≈140-fold (WT: 8.3 ± 1.3 min−1; R255L: 1168 ± 163 min−1), total turnover number (TTN) increased ≈21-fold (WT: 40 ± 3; R255L: 860 ± 15), and coupling efficiency, ≈2.9-fold (WT: 8.8 ± 0.1%; R255L: 25.7 ± 1.0%). Computational analysis showed that substitution R255L (distant from the heme-cofactor) does not have the salt bridge formed with D217 in WT, which introduces flexibility into the I-helix and leads to a heme rearrangement allowing for efficient hydroxylation.
- ItemSynthesis, Characterization, and Antimicrobial Properties of Peptides Mimicking Copolymers of Maleic Anhydride and 4-Methyl-1-pentene(Basel : Molecular Diversity Preservation International (MDPI), 2018) Szkudlarek, Marian; Heine, Elisabeth; Keul, Helmut; Beginn, Uwe; Möller, MartinSynthetic amphiphilic copolymers with strong antimicrobial properties mimicking natural antimicrobial peptides were obtained via synthesis of an alternating copolymer of maleic anhydride and 4-methyl-1-pentene. The obtained copolymer was modified by grafting with 3-(dimethylamino)-1-propylamine (DMAPA) and imidized in a one-pot synthesis. The obtained copolymer was modified further to yield polycationic copolymers by means of quaternization with methyl iodide and dodecyl iodide, as well as by being sequentially quaternized with both of them. The antimicrobial properties of obtained copolymers were tested against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, and Staphylococcus aureus. Both tested quaternized copolymers were more active against the Gram-negative E. coli than against the Gram-positive S. aureus. The copolymer modified with both iodides was best when tested against E. coli and, comparing all three copolymers, also exhibited the best effect against S. aureus. Moreover, it shows (limited) selectivity to differentiate between mammalian cells and bacterial cell walls. Comparing the minimum inhibitory concentration (MIC) of Nisin against the Gram-positive bacteria on the molar basis instead on the weight basis, the difference between the effect of Nisin and the copolymer is significantly lower.
- ItemDisulfide Bond Engineering of an Endoglucanase from Penicillium verruculosum to Improve Its Thermostability(Basel : Molecular Diversity Preservation International (MDPI), 2019) Bashirova, Anna; Pramanik, Subrata; Volkov, Pavel; Rozhkova, Aleksandra; Nemashkalov, Vitaly; Zorov, Ivan; Gusakov, Alexander; Sinitsyn, Arkady; Schwaneberg, Ulrich; Davari, Mehdi D.Endoglucanases (EGLs) are important components of multienzyme cocktails used in the production of a wide variety of fine and bulk chemicals from lignocellulosic feedstocks. However, a low thermostability and the loss of catalytic performance of EGLs at industrially required temperatures limit their commercial applications. A structure-based disulfide bond (DSB) engineering was carried out in order to improve the thermostability of EGLII from Penicillium verruculosum. Based on in silico prediction, two improved enzyme variants, S127C-A165C (DSB2) and Y171C-L201C (DSB3), were obtained. Both engineered enzymes displayed a 15–21% increase in specific activity against carboxymethylcellulose and β-glucan compared to the wild-type EGLII (EGLII-wt). After incubation at 70 °C for 2 h, they retained 52–58% of their activity, while EGLII-wt retained only 38% of its activity. At 80 °C, the enzyme-engineered forms retained 15–22% of their activity after 2 h, whereas EGLII-wt was completely inactivated after the same incubation time. Molecular dynamics simulations revealed that the introduced DSB rigidified a global structure of DSB2 and DSB3 variants, thus enhancing their thermostability. In conclusion, this work provides an insight into DSB protein engineering as a potential rational design strategy that might be applicable for improving the stability of other enzymes for industrial applications.
- ItemCellular responses to beating hydrogels to investigate mechanotransduction([London] : Nature Publishing Group UK, 2019) Chandorkar, Yashoda; Castro Nava, Arturo; Schweizerhof, Sjören; Van Dongen, Marcel; Haraszti, Tamás; Köhler, Jens; Zhang, Hang; Windoffer, Reinhard; Mourran, Ahmed; Möller, Martin; De Laporte, LauraCells feel the forces exerted on them by the surrounding extracellular matrix (ECM) environment and respond to them. While many cell fate processes are dictated by these forces, which are highly synchronized in space and time, abnormal force transduction is implicated in the progression of many diseases (muscular dystrophy, cancer). However, material platforms that enable transient, cyclic forces in vitro to recreate an in vivo-like scenario remain a challenge. Here, we report a hydrogel system that rapidly beats (actuates) with spatio-temporal control using a near infra-red light trigger. Small, user-defined mechanical forces (~nN) are exerted on cells growing on the hydrogel surface at frequencies up to 10 Hz, revealing insights into the effect of actuation on cell migration and the kinetics of reversible nuclear translocation of the mechanosensor protein myocardin related transcription factor A, depending on the actuation amplitude, duration and frequency.
- «
- 1 (current)
- 2
- 3
- »