Universität Stuttgart
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Item Open Access Sudden elevation of carbon dioxide concentration causes perturbation of the electron transport chain and triggers defense responses in Arabidopsis thaliana(2026) Shokouhi, Danial; Hernandez, Jakob Sebastian; Walther, Dirk; Kepp, Gabriele; Schwenkert, Serena; Leister, Dario; Gremmels, Jürgen; Zuther, Ellen; Alpers, Jessica; Nägele, Thomas; Heyer, Arnd G.Main conclusionArabidopsis wildtype plants suffer symptoms of stress at a sudden increase in CO2 concentration, resulting from perturbation of photosynthetic electron transport. Defense-related gene induction includes increased methionine cycle and glucosinolates metabolism.AbstractElevated CO2 (eCO2) increases photosynthetic performance of plants, but also leads to decreased nitrogen-to-carbon ratio and a long-term decline in photosynthetic activity, known as photosynthetic acclimation. It is unclear whether initially increased CO2 assimilation or perturbation of the physiological homeostasis triggers acclimation. Here, we used a combination of omics analysis to investigate immediate (1 day) and delayed (7 days) responses of plants to rising atmospheric CO2, thus allowing us to discriminate regulatory from metabolic effects. Responses of wildtype Arabidopsis plants, Columbia-0, were compared to those of the hpr1-1 mutant of peroxisomal hydroxy-pyruvate reductase that has reduced photorespiratory turnover at ambient CO2. Comparisons enabled separating the impact of eCO2 (1000 ppm) on increased carbon assimilation from that of reduced photorespiration. While both genotypes had elevated sugar levels at eCO2, the wildtype displayed symptoms of stress that were accompanied by perturbation of the photosynthetic electron transport chain. These were consistent with physiological parameters, including non-photochemical quenching and chlorophyll fluorescence. The induction of defense-related mechanisms was tightly associated with increased sulfate assimilation, methionine cycle activity and glucosinolates metabolism, all being early responses of the wildtype to eCO2. Transcriptome data pointed to hexokinase1 as a central regulatory hub in orchestrating these responses. In contrast, eCO2 enabled the hpr1-1 mutant to metabolically align with the wildtype. Results offer new interpretations of how the impairment of carbon and nitrogen recycling is compensated in the hpr1-1 mutant.Item Open Access Anti-adhesive surfaces inspired by bee mandible surfaces(2023) Saccardi, Leonie; Schiebl, Jonas; Balluff, Franz; Christ, Ulrich; Gorb, Stanislav N.; Kovalev, Alexander; Schwarz, OliverPropolis, a naturally sticky substance used by bees to secure their hives and protect the colony from pathogens, presents a fascinating challenge. Despite its adhesive nature, honeybees adeptly handle propolis with their mandibles. Previous research has shown a combination of an anti-adhesive fluid layer and scale-like microstructures on the inner surface of bee mandibles. Our aim was to deepen our understanding of how surface energy and microstructure influence the reduction in adhesion for challenging substances like propolis. To achieve this, we devised surfaces inspired by the intricate microstructure of bee mandibles, employing diverse techniques including roughening steel surfaces, creating lacquer structures using Bénard cells, and moulding resin surfaces with hexagonal patterns. These approaches generated patterns that mimicked the bee mandible structure to varying degrees. Subsequently, we assessed the adhesion of propolis on these bioinspired structured substrates. Our findings revealed that on rough steel and resin surfaces structured with hexagonal dimples, propolis adhesion was significantly reduced by over 40% compared to unstructured control surfaces. However, in the case of the lacquer surface patterned with Bénard cells, we did not observe a significant reduction in adhesion.Item Open Access Precision 3D‐printed cell scaffolds mimicking native tissue composition and mechanics(2020) Erben, Amelie; Hörning, Marcel; Hartmann, Bastian; Becke, Tanja; Eisler, Stephan A.; Southan, Alexander; Cranz, Séverine; Hayden, Oliver; Kneidinger, Nikolaus; Königshoff, Melanie; Lindner, Michael; Tovar, Günter E. M.; Burgstaller, Gerald; Clausen‐Schaumann, Hauke; Sudhop, Stefanie; Heymann, MichaelCellular dynamics are modeled by the 3D architecture and mechanics of the extracellular matrix (ECM) and vice versa. These bidirectional cell‐ECM interactions are the basis for all vital tissues, many of which have been investigated in 2D environments over the last decades. Experimental approaches to mimic in vivo cell niches in 3D with the highest biological conformity and resolution can enable new insights into these cell‐ECM interactions including proliferation, differentiation, migration, and invasion assays. Here, two‐photon stereolithography is adopted to print up to mm‐sized high‐precision 3D cell scaffolds at micrometer resolution with defined mechanical properties from protein‐based resins, such as bovine serum albumin or gelatin methacryloyl. By modifying the manufacturing process including two‐pass printing or post‐print crosslinking, high precision scaffolds with varying Young's moduli ranging from 7‐300 kPa are printed and quantified through atomic force microscopy. The impact of varying scaffold topographies on the dynamics of colonizing cells is observed using mouse myoblast cells and a 3D‐lung microtissue replica colonized with primary human lung fibroblast. This approach will allow for a systematic investigation of single‐cell and tissue dynamics in response to defined mechanical and bio‐molecular cues and is ultimately scalable to full organs.Item Open Access Flux calculation for primary metabolism reveals changes in allocation of nitrogen to different amino acid families when photorespiratory activity changes(2024) Friedrichs, Nils; Shokouhi, Danial; Heyer, Arnd G.Photorespiration, caused by oxygenation of the enzyme Rubisco, is considered a wasteful process, because it reduces photosynthetic carbon gain, but it also supplies amino acids and is involved in amelioration of stress. Here, we show that a sudden increase in photorespiratory activity not only reduced carbon acquisition and production of sugars and starch, but also affected diurnal dynamics of amino acids not obviously involved in the process. Flux calculations based on diurnal metabolite profiles suggest that export of proline from leaves increases, while aspartate family members accumulate. An immense increase is observed for turnover in the cyclic reaction of glutamine synthetase/glutamine-oxoglutarate aminotransferase (GS/GOGAT), probably because of increased production of ammonium in photorespiration. The hpr1-1 mutant, defective in peroxisomal hydroxypyruvate reductase, shows substantial alterations in flux, leading to a shift from the oxoglutarate to the aspartate family of amino acids. This is coupled to a massive export of asparagine, which may serve in exchange for serine between shoot and root.Item Open Access Insights into the structural and functional properties of the eukaryotic porin Tom40(2012) Gessmann, Dennis; Nußberger, Stephan (Prof. Dr.)Tom40 forms the preprotein conducting channel in the outer membrane of mitochondria enabling transport of up to 1500 different preproteins through an optimized pore environment. Moreover, Tom40 exhibits a voltage-dependent gating mechanism in terms of an ‘electrical switch’ making this eukaryotic beta-barrel a promising target for nanopore based applications. In this work, new bioinformatics methods were developed and verified through practical approaches to shed light on the structural elements of Tom40 facilitating its particular function in mitochondria. Based on these results, Tom40 proteins were designed with modified and optimized structural properties. TmSIP, a physical interaction model developed for TM beta-barrel proteins, was used to identify weakly stable regions in the TM domain of Tom40 from mammals and fungi. Three unfavorable beta-strands were determined for human Tom40A. Via CD and Trp-fluorescence spectroscopy it was shown that substitution of key amino acid residues in theses strands resulted in an improved resistance of the protein to chemical and thermal perturbations. Further, the mutated form of hTom40A was strictly found in its monomeric state. Equal improvements were gained for the apparent stability of Tom40 from Aspergillus fumigatus. Tom40 was isolated and purified in its native state from Neurospora crassa mitochondria. Time-limited proteolysis of native NcTom40 coupled to mass spectrometry revealed comparable protease-accessibility to VDAC isoform 1 from mammals suggesting a similar fold. Thus, a homology model of NcTom40 was developed on the basis of the solved mouse VDAC-1 crystal structure. It was found that Tom40 forms a 19-stranded beta-barrel with an N-terminal alpha-helix inside the pore. Further, a conserved ‘polar slide’ in the pore interior is possibly involved in preprotein translocation and a second conserved domain, termed ‘helix anchor region’, in arresting the helix inside the Tom40 pore. Based on the homology model of NcTom40, the structure and function of the N-terminal domain of Tom40 was addressed. Examination of the model structure revealed two different domains for the N-terminus, the inner-barrel and outer-barrel N-terminus. In vivo investigations showed that both parts prevent a heat-induced dysfunction of Tom40 in N. crassa mitochondria independently. By applying CD spectroscopy the predicted N-terminal alpha-helix could be allocated to the inner-barrel N-terminus. Further, in combination with Trp-fluorescence spectroscopy it was found that the N-terminal alpha-helix unfolds independently from the Tom40 beta-barrel, but is not necessary for pore stability or integrity. However, a conserved amino acid residue, Ile47 of NcTom40, in the inner-barrel N-terminus is essential for the structural integrity of the N-terminal alpha-helix. In conclusion, these results may offer a basis for future works on TM beta-barrel proteins with the aim to alter the structural properties in the absence of a high atomic resolution structure or an established knowledge of the biochemical and biophysical properties.Item Open Access A systems biology approach to analyse leaf carbohydrate metabolism in Arabidopsis thaliana(2011) Henkel, Sebastian; Nägele, Thomas; Hörmiller, Imke; Sauter, Thomas; Sawodny, Oliver; Ederer, Michael; Heyer, Arnd G.Plant carbohydrate metabolism comprises numerous metabolite interconversions, some of which form cycles of metabolite degradation and re-synthesis and are thus referred to as futile cycles. In this study, we present a systems biology approach to analyse any possible regulatory principle that operates such futile cycles based on experimental data for sucrose (Scr) cycling in photosynthetically active leaves of the model plant Arabidopsis thaliana. Kinetic parameters of enzymatic steps in Scr cycling were identified by fitting model simulations to experimental data. A statistical analysis of the kinetic parameters and calculated flux rates allowed for estimation of the variability and supported the predictability of the model. A principal component analysis of the parameter results revealed the identifiability of the model parameters. We investigated the stability properties of Scr cycling and found that feedback inhibition of enzymes catalysing metabolite interconversions at different steps of the cycle have differential influence on stability. Applying this observation to futile cycling of Scr in leaf cells points to the enzyme hexokinase as an important regulator, while the step of Scr degradation by invertases appears subordinate.Item Open Access Mechanical properties of architected polymer lattice materials : a comparative study of additive manufacturing and CAD using FEM and µ‐CT(2025) Walker, Oliver; Roland, Thierry; Favier, Damien; Egele, Antoine; Heymann, Michael; Stubenrauch, CosimaMechanical properties of porous lattice materials can be tuned by the structure. Thus, the effect of the unit cell design on the mechanical properties is of special interest. However, studies often lack an in‐depth investigation of the underlying mechanisms. Additionally, inhomogeneous bulk material properties and structural deviations introduced during manufacturing are often neglected. Here, the impact of the pore shape on Young's modulus, initial peak stress, and energy absorbance of lattice materials is investigated. Structural deviations of 3D‐printed lattice materials from the computer‐aided design (CAD) are examined, and analyze how they affect the mechanical properties. For comparability, the porosity, overall dimensions, and unit cell size of the lattice material are kept constant. Flexible lattice materials assembled of 3 × 3 × 3 unit cells are 3D‐printed (stereolithography). Mechanical properties are determined through uniaxial compression tests and finite element modeling (FEM). Deviations between CAD and 3D‐printed lattice materials are analyzed using micro‐computed tomography (µ‐CT). Combining µ‐CT with FEM revealed the impact of these deviations on the mechanical properties. It is found that the mechanical properties depend indeed on the pore shape. Furthermore, a pyramidal shape along the printing direction and displaced or deformed pores are observed in the 3D‐printed lattice materials, all of which result in reduced mechanical properties.Item Open Access Acoustic streaming‐induced multimodal locomotion of bubble‐based microrobots(2023) Mahkam, Nima; Aghakhani, Amirreza; Sheehan, Devin; Gardi, Gaurav; Katzschmann, Robert; Sitti, MetinAcoustically‐driven bubbles at the micron scale can generate strong microstreaming flows in its surrounding fluidic medium. The tunable acoustic streaming strength of oscillating microbubbles and the diversity of the generated flow patterns enable the design of fast‐moving microrobots with multimodal locomotion suitable for biomedical applications. The acoustic microrobots holding two coupled microbubbles inside a rigid body are presented; trapped bubbles inside the L‐shaped structure with different orifices generate various streaming flows, thus allowing multiple degrees of freedom in locomotion. The streaming pattern and mean streaming speed depend on the intensity and frequency of the acoustic wave, which can trigger four dominant locomotion modes in the microrobot, denoted as translational and rotational, spinning, rotational, and translational modes. Next, the effect of various geometrical and actuation parameters on the control and navigation of the microrobot is investigated. Furthermore, the surface‐slipping multimodal locomotion, flow mixing, particle manipulation capabilities, the effective interaction of high flow rates with cells, and subsequent cancerous cell lysing abilities of the proposed microrobot are demonstrated. Overall, these results introduce a design toolbox for the next generation of acoustic microrobots with higher degrees of freedom with multimodal locomotion in biomedical applications.Item Open Access Frosttoleranz bei Pflanzen - Fliehen, vermeiden oder einfach durchhalten!(2005) Heyer, Arnd G.Nur etwa ein Drittel der Fläche des Planeten Erde ist dauerhaft frostfrei: in den tropischen Regenwaldgebieten des Amazonasbeckens, in Kongo, an der afrikanischen Westküste, auf dem Indomalayischen Archipel, in einem kleinen Teil Australiens und auf den pazifischen Inseln in Äquatornähe fällt die Temperatur nie unter etwa 15 Grad. Der Temperaturunterschied zwischen Tag und Nacht ist hier größer als der zwischen Sommer und Winter. Fast die Hälfte aller Pflanzenarten – 115.000 von 250.000 bekannten Blütenpflanzen – kommen nur hier vor. Daraus lässt sich bereits ablesen, dass der Umgang mit niedrigen Temperaturen eine Herausforderung für lebende Organismen ist, der sich nicht jeder stellen mag. Andererseits locken die kälteren Gegenden mit einem großen Flächen- und Mineralstoffangebot – einer Ressource, die für Pflanzen äußerst attraktiv ist.Item Open Access The effect of pooling on the detection of the nucleocapsid protein of SARS-CoV-2 with rapid antigen tests(2021) Berking, Tim; Lorenz, Sabrina; Ulrich, Alexander; Greiner, Joachim; Kervio, Eric; Bremer, Jennifer; Wege, Christina; Kleinow, Tatjana; Richert, ClemensThe COVID-19 pandemic puts significant stress on the viral testing capabilities of many countries. Rapid point-of-care (PoC) antigen tests are valuable tools but implementing frequent large scale testing is costly. We have developed an inexpensive device for pooling swabs, extracting specimens, and detecting viral antigens with a commercial lateral flow test for the nucleocapsid protein of SARS-CoV-2 as antigen. The holder of the device can be produced locally through 3D printing. The extraction and the elution can be performed with the entire set-up encapsulated in a transparent bag, minimizing the risk of infection for the operator. With 0.35 mL extraction buffer and six swabs, including a positive control swab, 43 ± 6% (n = 8) of the signal for an individual extraction of a positive control standard was obtained. Image analysis still showed a signal-to-noise ratio of approximately 2:1 at 32-fold dilution of the extract from a single positive control swab. The relative signal from the test line versus the control line was found to scale linearly upon dilution (R2 = 0.98), indicating that other pooling regimes are conceivable. A pilot project involving 14 participants and 18 pooled tests in a laboratory course at our university did not give any false positives, and an individual case study confirmed the ability to detect a SARS-CoV-2 infection with five-fold or six-fold pooling, including one swab from a PCR-confirmed COVID patient. These findings suggest that pooling can make frequent testing more affordable for schools, universities, and similar institutions, without decreasing sensitivity to an unacceptable level.