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- 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.
- ItemHow to minimize dye-induced perturbations while studying biomembrane structure and dynamics: PEG linkers as a rational alternative(Amsterdam : Elsevier, 2018) Mobarak, Edouard; Javanainen, Matti; Kulig, Waldemar; Honigmann, Alf; Sezgin, Erdinc; Aho, Noora; Eggeling, Christian; Rog, Tomasz; Vattulainen, IlpoOrganic dye-tagged lipid analogs are essential for many fluorescence-based investigations of complex membrane structures, especially when using advanced microscopy approaches. However, lipid analogs may interfere with membrane structure and dynamics, and it is not obvious that the properties of lipid analogs would match those of non-labeled host lipids. In this work, we bridged atomistic simulations with super-resolution imaging experiments and biomimetic membranes to assess the performance of commonly used sphingomyelin-based lipid analogs. The objective was to compare, on equal footing, the relative strengths and weaknesses of acyl chain labeling, headgroup labeling, and labeling based on poly-ethyl-glycol (PEG) linkers in determining biomembrane properties. We observed that the most appropriate strategy to minimize dye-induced membrane perturbations and to allow consideration of Brownian-like diffusion in liquid-ordered membrane environments is to decouple the dye from a membrane by a PEG linker attached to a lipid headgroup. Yet, while the use of PEG linkers may sound a rational and even an obvious approach to explore membrane dynamics, the results also suggest that the dyes exploiting PEG linkers interfere with molecular interactions and their dynamics. Overall, the results highlight the great care needed when using fluorescent lipid analogs, in particular accurate controls.
- ItemUncertainty in the measurement of indoor temperature and humidity in naturally ventilated dairy buildings as influenced by measurement technique and data variability(Amsterdam : Elsevier, 2017) Hempel, Sabrina; König, Marcel; Menz, Christoph; Janke, David; Amon, Barbara; Banhazi, Thomas M.; Estellés, Fernando; Amon, ThomasThe microclimatic conditions in dairy buildings affect animal welfare and gaseous emissions. Measurements are highly variable due to the inhomogeneous distribution of heat and humidity sources (related to farm management) and the turbulent inflow (associated with meteorologic boundary conditions). The selection of the measurement strategy (number and position of the sensors) and the analysis methodology adds to the uncertainty of the applied measurement technique. To assess the suitability of different sensor positions, in situations where monitoring in the direct vicinity of the animals is not possible, we collected long-term data in two naturally ventilated dairy barns in Germany between March 2015 and April 2016 (horizontal and vertical profiles with 10 to 5 min temporal resolution). Uncertainties related to the measurement setup were assessed by comparing the device outputs under lab conditions after the on-farm experiments. We found out that the uncertainty in measurements of relative humidity is of particular importance when assessing heat stress risk and resulting economic losses in terms of temperature-humidity index. Measurements at a height of approximately 3 m–3.5 m turned out to be a good approximation for the microclimatic conditions in the animal occupied zone (including the air volume close to the emission active zone). However, further investigation along this cross-section is required to reduce uncertainties related to the inhomogeneous distribution of humidity. In addition, a regular sound cleaning (and if possible recalibration after few months) of the measurement devices is crucial to reduce the instrumentation uncertainty in long-term monitoring of relative humidity in dairy barns. © 2017 The Authors
- ItemEncapsulation of bacteria in bilayer Pluronic thin film hydrogels: A safe format for engineered living materials(Amsterdam : Elsevier, 2023) Bhusari, Shardul; Kim, Juhyun; Polizzi, Karen; Sankaran, Shrikrishnan; del Campo, AránzazuIn engineered living materials (ELMs) non-living matrices encapsulate microorganisms to acquire capabilities like sensing or biosynthesis. The confinement of the organisms to the matrix and the prevention of overgrowth and escape during the lifetime of the material is necessary for the application of ELMs into real devices. In this study, a bilayer thin film hydrogel of Pluronic F127 and Pluronic F127 acrylate polymers supported on a solid substrate is introduced. The inner hydrogel layer contains genetically engineered bacteria and supports their growth, while the outer layer acts as an envelope and does not allow leakage of the living organisms outside of the film for at least 15 days. Due to the flat and transparent nature of the construct, the thin layer is suited for microscopy and spectroscopy-based analyses. The composition and properties of the inner and outer layer are adjusted independently to fulfil viability and confinement requirements. We demonstrate that bacterial growth and light-induced protein production are possible in the inner layer and their extent is influenced by the crosslinking degree of the used hydrogel. Bacteria inside the hydrogel are viable long term, they can act as lactate-sensors and remain active after storage in phosphate buffer at room temperature for at least 3 weeks. The versatility of bilayer bacteria thin-films is attractive for fundamental studies and for the development of application-oriented ELMs.
- 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.
- ItemMembrane Tension Orchestrates Rear Retraction in Matrix-Directed Cell Migration(Amsterdam : Elsevier, 2019) Hetmanski, J.H.R.; de, Belly, H.; Busnelli, I.; Waring, T.; Nair, R.V.; Sokleva, V.; Dobre, O.; Cameron, A.; Gauthier, N.; Lamaze, C.; Swift, J.; del, Campo, A.; Starborg, T.; Zech, T.; Goetz, J.G.; Paluch, E.K.; Schwartz, J.-M.; Caswell, P.T.In development, wound healing, and cancer metastasis, vertebrate cells move through 3D interstitial matrix, responding to chemical and physical guidance cues. Protrusion at the cell front has been extensively studied, but the retraction phase of the migration cycle is not well understood. Here, we show that fast-moving cells guided by matrix cues establish positive feedback control of rear retraction by sensing membrane tension. We reveal a mechanism of rear retraction in 3D matrix and durotaxis controlled by caveolae, which form in response to low membrane tension at the cell rear. Caveolae activate RhoA-ROCK1/PKN2 signaling via the RhoA guanidine nucleotide exchange factor (GEF) Ect2 to control local F-actin organization and contractility in this subcellular region and promote translocation of the cell rear. A positive feedback loop between cytoskeletal signaling and membrane tension leads to rapid retraction to complete the migration cycle in fast-moving cells, providing directional memory to drive persistent cell migration in complex matrices. © 2019 The AuthorsCell migration through 3D matrix is critical to developmental and disease processes, but the mechanisms that control rear retraction are poorly understood. Hetmanski et al. show that differential membrane tension allows caveolae to form at the rear of migrating cells and activate the contractile actin cytoskeleton to promote rapid retraction. © 2019 The Authors
- ItemDatasets from a vapor diffusion mineral precipitation protocol for Dictyostelium stalks(Amsterdam : Elsevier, 2016) Eder, Magdalena; Muth, Christina; Weiss, Ingrid M.Datasets from a slow carbonate vapor diffusion and mineral precipitation protocol for Dictyostelium ECM and cellulose stalks show examples for composite materials obtained by an in vitro approach, which differs substantially from the in vivo approach reported in The Journal of Structural Biology, doi: 10.1016/j.jsb.2016.03.015 [1]. Methods for obtaining the datasets include bright field transmitted light microscopy, fluorescence microscopy, LC-PolScope birefringence microscopy, variable pressure scanning electron microscopy (VP-SEM/ESEM), and Raman imaging spectroscopy.
- ItemGli protein activity is controlled by multisite phosphorylation in vertebrate hedgehog signaling(Amsterdam : Elsevier, 2013) Niewiadomski, Pawel; Kong, Jennifer H.; Ahrends, Robert; Ma, Yan; Humke, Eric W.; Khan, Sohini; Teruel, Mary N.; Novitch, Bennett G.; Rohatgi, RajatGli proteins are transcriptional effectors of the Hedgehog (Hh) pathway in both normal development and cancer. We describe a program of multisite phosphorylation that regulates the conversion of Gli proteins into transcriptional activators. In the absence of Hh ligands, Gli activity is restrained by the direct phosphorylation of six conserved serine residues by protein kinase A (PKA), a master negative regulator of the Hh pathway. Activation of signaling leads to a global remodeling of the Gli phosphorylation landscape: the PKA target sites become dephosphorylated, while a second cluster of sites undergoes phosphorylation. The pattern of Gli phosphorylation can regulate Gli transcriptional activity in a graded fashion, suggesting a phosphorylation-based mechanism for how a gradient of Hh signaling in a morphogenetic field can be converted into a gradient of transcriptional activity.
- ItemLink to glow - iEDDA conjugation of a Ruthenium(II) tetrazine complex leading to dihydropyrazine and pyrazine complexes with improved 1O2 formation ability(Amsterdam : Elsevier, 2022) Müller, Carolin; Wintergerst, Pascal; Nair, Shruthi Santhosh; Meitinger, Nicolas; Rau, Sven; Dietzek-Ivanšić, BenjaminThe synthesis and photophysical properties of the Ru-polypyridyl type complex [(tbbpy)2Ru(bptz)]2+ (Ru-bptz, tbbpy: 4,4’-di-tert-butyl-2,2’-bipyridine, bptz: 2,6-dipyrido-1,2,4,5-tetrazine), and the complexes [(tbbpy)2Ru(L)]2+ formed by inverse electron demand Diels Alder reaction (iEDDA) of Ru-bptz with with alkenes and alkynes, where L is 3,6-dipyrido-2,5-dihydropyridazine (bpdhpn) or 3,6-dipyrido-pyridazine (bppn) are described. A combination of steady-state and time-resolved spectroscopy complemented by the computation of state-specific absorption properties by means of time-dependent density functional theory reveals that the intense visible absorption band stems from Ru → tbbpy and Ru → L metal-to-ligand charge-transfer (MLCT) excitations. The studies show that lowest-lying L-centered MLCT states (3MLCTL) show comparably low emission quantum yields (3–9%) and lifetimes (90–150 ns). This correlates with the singlet oxygen generation ability, following the trend: Ru-bppn > Ru-bpdhpn > Ru-bptz.
- ItemStability of hepatitis E virus at high hydrostatic pressure processing(Amsterdam : Elsevier, 2021) Johne, R.; Wolff, A.; Gadicherla, A.K.; Filter, M.; Schlüter, O.Hepatitis E virus (HEV) is the causative agent of acute and chronic hepatitis in humans. The zoonotic HEV genotype 3 is the main genotype in Europe. The foodborne transmission via consumption of meat and meat products prepared from infected pigs or wild boars is considered the major transmission route of this genotype. High hydrostatic pressure processing (HPP) is a technique, which can be used for inactivation of pathogens in food. Here, preparations of a cell culture-adapted HEV genotype 3 strain in phosphate-buffered saline (PBS) were subjected to HPP and the remaining infectivity was titrated in cell culture by counting fluorescent foci of replicating virus. A gradual decrease in infectivity was found by application of 100 to 600 MPa for 2 min. At 20 °C, infectivity reduction of 0.5 log10 at 200 MPa and 1 log10 at 400 MPa were observed. Slightly higher infectivity reduction of 1 log10 at 200 MPa and 2 log10 at 400 MPa were found by application of the pressure at 4 °C. At both temperatures, the virus was nearly completely inactivated (>3.5 log10 infectivity decrease) at 600 MPa; however, low amounts of remaining infectious virus were observed in one of three replicates in both cases. Transmission electron microscopy showed disassembled and distorted particles in the preparations treated with 600 MPa. Time-course experiments at 400 MPa showed a continuous decline of infectivity from 30 s to 10 min, leading to a 2 log10 infectivity decrease at 20 °C and to a 2.5 log10 infectivity decrease at 4 °C for a 10 min pressure application each. Predictive models for inactivation of HEV by HPP were generated on the basis of the generated data. The results show that HPP treatment can reduce HEV infectivity, which is mainly dependent on pressure height and duration of the HPP treatment. Compared to other viruses, HEV appears to be relatively stable against HPP and high pressure/long time combinations have to be applied for significant reduction of infectivity.