Chemische Analytik
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Herein an assay toward a rapid and reliable profiling of extracellular matrix of Escherichia coli (E. coli) utilizing a tandem of GC-MS as a tool for definition of the exact chemical nature of low molecular weight compounds and cyclic voltammetry for their high throughput detection is presented. Briefly, during a set of investigations the formation of glycerol in the extracellular matrix (ECM) of E. coli at physiological relevant conditions of cells was revealed. Based on the obtained knowledge, the electrochemical protocol allowing both qualitative and quantitative analyses of glycerol in E. coli ECMs at palladium ink-modified screen printed electrodes with precision values (RSD) <10 % and recovery rates ranged from 98 % to 102 % was proposed. The provided protocol for a rapid electrochemical profiling of the bacterial ECMs can readily be used as a guideline for the controlled electroanalysis of target electroactive signaling analytes in complex biological samples.
Iron (Fe) toxicity is a major challenge for plant cultivation in acidic water-logged soil environments, where lowland rice is a major staple food crop. Only few studies addressed the molecular characterization of excess Fe tolerance in rice, and these highlight different mechanisms for Fe tolerance in the studied varieties.
Here, we screened 16 lowland rice varieties for excess Fe stress growth responses to identify contrasting lines, Fe-tolerant Lachit and -susceptible Hacha. Hacha and Lachit differed in their physiological and morphological responses to excess Fe, including leaf growth, leaf rolling, reactive oxygen species generation, Fe and metal contents. These responses were mirrored by differential gene expression patterns, obtained through RNA-sequencing, and corresponding GO term enrichment in tolerant versus susceptible lines. From the comparative transcriptomic profiles between Lachit and Hacha in response to excess Fe stress, individual genes of the category metal homeostasis, mainly root-expressed, may contribute to the tolerance of Lachit. 22 out of these 35 metal homeostasis genes are present in selection sweep genomic regions, in breeding signatures and/or differentiated during rice domestication. These findings will serve to design targeted Fe tolerance breeding of rice crops.
SEC14-GOLD protein PATELLIN2 binds IRON-REGULATED TRANSPORTER1 linking root iron uptake to vitamin E
(2023)
Organisms require micronutrients, and Arabidopsis (Arabidopsis thaliana) IRON-REGULATED TRANSPORTER1 (IRT1) is essential for iron (Fe2+) acquisition into root cells. Uptake of reactive Fe2+ exposes cells to the risk of membrane lipid peroxidation. Surprisingly little is known about how this is avoided. IRT1 activity is controlled by an intracellular variable region (IRT1vr) that acts as a regulatory protein interaction platform. Here, we describe that IRT1vr interacted with peripheral plasma membrane SEC14-Golgi dynamics (SEC14-GOLD) protein PATELLIN2 (PATL2). SEC14 proteins bind lipophilic substrates and transport or present them at the membrane. To date, no direct roles have been attributed to SEC14 proteins in Fe import. PATL2 affected root Fe acquisition responses, interacted with ROS response proteins in roots, and alleviated root lipid peroxidation. PATL2 had high affinity in vitro for the major lipophilic antioxidant vitamin E compound α-tocopherol. Molecular dynamics simulations provided insight into energetic constraints and the orientation and stability of the PATL2-ligand interaction in atomic detail. Hence, this work highlights a compelling mechanism connecting vitamin E with root metal ion transport at the plasma membrane with the participation of an IRT1-interacting and α-tocopherol-binding SEC14 protein.
Hydrogel precursors that crosslink within minutes are essential for the development of cell encapsulation matrices and their implementation in automated systems. Such timescales allow sufficient mixing of cells and hydrogel precursors under low shear forces and the achievement of homogeneous networks and cell distributions in the 3D cell culture. The previous work showed that the thiol-tetrazole methylsulfone (TzMS) reaction crosslinks star-poly(ethylene glycol) (PEG) hydrogels within minutes at around physiological pH and can be accelerated or slowed down with small pH changes. The resulting hydrogels are cytocompatible and stable in cell culture conditions. Here, the gelation kinetics and mechanical properties of PEG-based hydrogels formed by thiol-TzMS crosslinking as a function of buffer, crosslinker structure and degree of TzMS functionality are reported. Crosslinkers of different architecture, length and chemical nature (PEG versus peptide) are tested, and degree of TzMS functionality is modified by inclusion of RGD cell-adhesive ligand, all at concentration ranges typically used in cell culture. These studies corroborate that thiol/PEG-4TzMS hydrogels show gelation times and stiffnesses that are suitable for 3D cell encapsulation and tunable through changes in hydrogel composition. The results of this study guide formulation of encapsulating hydrogels for manual and automated 3D cell culture.
Crosslinking chemistries that allow hydrogel formation within minutes are essential to achieve homogeneous networks and cell distributions in 3D cell culture. Thiol-methylsulfone (MS) crosslinking chemistry offers minutes-scale gelation under near-physiological conditions showing many desirable attributes for 3D cell encapsulation. Here we investigate the gelation kinetics and mechanical properties of PEG-based hydrogels formed by thiol-tetrazole methylsulfone (TzMS) crosslinking as a function of buffer, crosslinker structure, and degree of TzMS functionalization. Appropriate buffer selection ensured constant pH throughout crosslinking. The formulation containing cell adhesive ligand RGD and enzymatically-degradable peptide VPM gelled in ca. 4 min at pH 7.5, and stiffness could be increased from hundreds of Pascals to > 1 kPa by using excess VPM. The gelation times and stiffnesses for these hydrogels are highly suitable for 3D cell encapsulations, and pave the way for reliable 3D cell culture workflows in pipetting robots.
We report a simple fast, practical and effective method for the replication of the complex venation patterns of natural leaves into PDMS with accuracy down to a lateral size of 500 nm. Optimising the amount of crosslinker enabled the replication and sealing of the microvascular structures to yield enclosed microfluidic networks. The use of plant leaves as templates for soft lithography was demonstrated across over ten species and included reticulate, arcuate, pinnate, parallel and palmate venation patterns. SEM imaging revealed replication of the plants microscopic and sub-microscopic topography into the PDMS structures, making this method especially attractive for mimicking biological structures for in vitro assays. Flow analysis revealed that the autonomous liquid transport velocity in 1 st -order microchannel was 1.5-2.2 times faster than that in the 2 nd -order microchannels across three leaf types, with the sorptivity rule surprisingly preserved during self-powered flow through leaf-inspired vascularity from Carpinus betulus.
In this study, we propose a simple and rapid technique for characterization of free fatty acids and triacylglycerides (TAG) based on palladium nanoparticular (Pd-NP) surface-assisted laser desorption/ionization (SALDI) mass spectrometry (MS). The implemented Pd-NP material allowed detection of free fatty acids and TAGs exclusively as [M + K] + ions in positive ion mode. Under negative ionization conditions, unusual trimetric structures were generated for free fatty acids, while TAGs underwent irreproducible degradation reactions. Importantly, the mass spectra obtained from Pd-NP targets in positive ion mode were very clean without interferences from matrix-derived ions in the low m/z range and readily enabled the detection of intact TAGs in vegetable oils without major fragmentation reactions as compared to conventional MALDI-MS, requiring only a minimal amount of sample preparation.
In the past decade the significant progress in the cellular stress response was witnessed. Nevertheless, the development of the minimally-invasive and accurate sensing tools for the identification of the increasing number of potentially relevant species in clinical diagnostics, using smaller sample volumes is a major challenge. Herein, the potential of the electroplated nanomaterials towards biomedical sensing and diagnostics is summarized. The key factors affecting the surface functionality, dimensionality, S/N ratio and analytical response of the prepared chips are highlighted. Furthermore, the application of electroplated chips as a fast “read out” platform for profiling of clinical samples was demonstrated.
Background: Quantum dots (QDs) have great potential as fluorescent labels but cytotoxicity relating to extra- and intracellular degradation in biological systems has to be addressed prior to biomedical applications. In this study, human intestinal cells (Caco-2) grown on transwell membranes were used to study penetration depth, intracellular localization, translocation and cytotoxicity of CdSe/ZnS QDs with amino and carboxyl surface modifications. The focus of this study was to compare the penetration depth of QDs in differentiated vs undifferentiated cells using confocal microscopy and image processing. Results: Caco-2 cells were exposed to QDs with amino (NH2) and carboxyl (COOH) surface groups for 3 days using a concentration of 45 µg cadmium ml−1. Image analysis of confocal/multiphoton microscopy z-stacks revealed no penetration of QDs into the cell lumen of differentiated Caco-2 cells. Interestingly, translocation of cadmium ions onto the basolateral side of differentiated monolayers was observed using high resolution inductively coupled plasma mass spectrometry (ICP-MS). Membrane damage was neither detected after short nor long term incubation in Caco-2 cells. On the other hand, intracellular localization of QDs after exposure to undifferentiated cells was observed and QDs were partially located within lysosomes.Conclusions: In differentiated Caco-2 monolayers, representing a model for small intestinal enterocytes, no penetration of amino and carboxyl functionalized CdSe/ZnS QDs into the cell lumen was detected using microscopy analysis and image processing. In contrast, translocation of cadmium ions onto the basolateral side could be detected using ICP-MS. However, even after long term incubation, the integrity of the cell monolayer was not impaired and no cytotoxic effects could be detected. In undifferentiated Caco-2 cells, both QD modifications could be found in the cell lumen. Only to some extend, QDs were localized in endosomes or lysosomes in these cells. The results indicate that the differentiation status of Caco-2 cells is an important factor in internalization and localization studies using Caco-2 cells. Furthermore, a combination of microscopy analysis and sensitive detection techniques like ICP-MS are necessary for studying the interaction of cadmium containing QDs with cells.
Dissection of iron signaling and iron accumulation by overexpression of subgroup Ib bHLH039 protein
(2017)
Iron is an essential growth determinant for plants, and plants acquire this micronutrient in amounts they need in their environment. Plants can increase iron uptake in response to a regulatory transcription factor cascade. Arabidopsis thaliana serves as model plant to identify and characterize iron regulation genes. Here, we show that overexpression of subgroup Ib bHLH transcription factor bHLH039 (39Ox) caused constitutive iron acquisition responses, which resulted in enhanced iron contents in leaves and seeds. Transcriptome analysis demonstrated that 39Ox plants displayed simultaneously gene expression patterns characteristic of iron deficiency and iron stress signaling. Thereby, we could dissect iron deficiency response regulation. The transcription factor FIT, which is required to regulate iron uptake, was essential for the 39Ox phenotype. We provide evidence that subgroup Ib transcription factors are involved in FIT transcriptional regulation. Our findings pose interesting questions to the feedback control of iron homeostasis.
