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Plants grown under iron (Fe)-deficient conditions induce a set of genes that enhance the efficiency of Fe uptake by the roots. In Arabidopsis (Arabidopsis thaliana), the central regulator of this response is the basic helix-loop-helix transcription factor FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT). FIT activity is regulated by protein-protein interactions, which also serve to integrate external signals that stimulate and possibly inhibit Fe uptake. In the search of signaling components regulating FIT function, we identified ZINC FINGER OF ARABIDOPSIS THALIANA12 (ZAT12), an abiotic stress-induced transcription factor. ZAT12 interacted with FIT, dependent on the presence of the ethylene-responsive element-binding factor-associated amphiphilic repression motif. ZAT12 protein was found expressed in the root early differentiation zone, where its abundance was modulated in a root layer-specific manner. In the absence of ZAT12, FIT expression was upregulated, suggesting a negative effect of ZAT12 on Fe uptake. Consistently, zat12 loss-of-function mutants had higher Fe content than the wild type at sufficient Fe. We found that under Fe deficiency, hydrogen peroxide (H 2 O 2 ) levels were enhanced in a FIT-dependent manner. FIT protein, in turn, was stabilized by H 2 O 2 but only in the presence of ZAT12, showing that H 2 O 2 serves as a signal for Fe deficiency responses. We propose that oxidative stress-induced ZAT12 functions as a negative regulator of Fe acquisition. A model where H 2 O 2 mediates the negative regulation of plant responses to prolonged stress might be applicable to a variety of stress conditions.
Es ist gelungen, silberhalogenidhaltige Materialien (Pulver und dünne Schichten) mit photochromen Eigenschaften über den Sol-Gel-Prozeß zu synthetisieren. Zuerst wurden AgCl0.11Br0.89-dotierte Borosilikatglaspulver hergestellt. Dazu wurden kleine Silberkolloide spontan in einem Natriumalumoborosilikatsol gebildet und durch Überführung in den Gelzustand stabilisiert. Die Umwandlung zu Silberhalogenid-Kristalliten der Form AgCl0.11Br0.89 erfolgte im Gel durch Rühren in chlorid- und bromidhaltiger Lösung. Nach Temperung zum Glas zeigen diese AgCl0.11Br0.89-haltigen Pulver photochrome
Eigenschaften. Es wurde dargelegt, daß mit steigender Silberkonzentration (von 0.32 bis 4.8 % Massenanteil AgCl0.11Br0.89 je 100 g Oxide) die Kristallitgröße (dAgCl0.11Br0.89 von 4.6
bis 56 nm) zunimmt. Je größer die Kristallite, umso schneller und tiefer dunkeln die Pulver ein. Das Bleichen setzt erst thermisch aktiviert ein (bei Temperaturen von 150 bis 400 °C), dabei hellen die Pulver, die nicht so stark eindunkeln, bei geringerer Temperatur auf. Die hergestellten Pulver mit einem AgCl0.11Br0.89-Massenanteil < 0.32 % je 100 g Oxide dunkeln durch die Temperaturbehand lung zum thermischen Bleichen erst nach (bis ca. 150 °C), bevor sie aufhellen. Die beste Kinetik hinsichtlich des thermischen Bleichens zeigt das Glaspulver mit einer Silberkonzentration von 0.32 % Massenanteil AgCl0.11Br0.89 bezogen auf 100 g Oxide. Für dieses Pulver sinkt die Reflexion nach5 Minuten UV-Bestrahlung von 85 auf 36 %, die Sättigungseindunkelung ist nach 25 Minuten Belichten erreicht (R = 30 %) und kann durch Temperaturbehandlung 1 Stunde bei 150 °C vollständig gebleicht werden. Die kalkulierte Aktivierungsenergie für die thermische Bleiche beträgt für dieses Pulver 19 kJ/ mol. Nach der erfolgreichen Pulversynthese wurden dünne Schichten mit photochromen Eigenschaften über die Sol-Gel-Route erzeugt. Beim ersten Weg wurde das Silber über Ionenaustauschreaktionen mit den Natrium-Ionen aus der Gelschicht eingebracht und nachfolgend chloriert bzw. bromiert. Beim zweiten und dritten Weg wurden das Halogenid über ein modifiziertes Alkoxysilan und das Silber über DIAMO komplex gebunden in das Beschichtungssol eingebracht, wobei es sich um ein Borosilikatsol oder ein Ormocervorhydrolysat handelte. Die Tabelle 36 faßt die wichtigsten Ergebnisse der in dieser Arbeit erzeugten Schichten zusammen. Die silberchloridhaltigen Borosilikatschichten (Kapitel 4.2.1.), in die das Silber über Ionenaustauschreaktionen eingebracht wurde, sind synthesebedingt leicht eingetrübt. Sie zeigen die tiefste Sättigungseindunkelung, für das thermisch aktivierte Bleichen sind Temperaturen von 400 °C erforderlich. Die Reversibilität des photochromen Effektes wurde in 30 Zyklen verfolgt, dabei wurde nur eine geringe Transmissionserhöhung (+ 4.2 %) im eingedunkelten Zustand festgestellt. Die mittlere Kristallitgröße beträgt 31 nm und liegt fast im Größenordnungsbereich herkömmlicher photochromer Gläser ( 10-30 nm).
Peptide drugs have seen rapid advancement in biopharmaceutical development, with over 80 candidates approved globally. Despite their therapeutic potential, the clinical translation of peptide drugs is hampered by challenges in production yields and stability. Engineered bacterial therapeutics is a unique approach being explored to overcome these issues by using bacteria to produce and deliver therapeutic compounds at the body site of use. A key advan‑ tage of this technology is the possibility to control drug delivery within the body in real time using genetic switches. However, the performance of such genetic switches suffers when used to control drugs that require post‑translational modifications or are toxic to the host. In this study, these challenges were experienced when attempting to establish a thermal switch for the production of a ribosomally synthesized and post‑translationally modified peptide antibiotic, darobactin, in probiotic E. coli. These challenges were overcome by developing a thermo‑amplifier circuit that combined the thermal switch with a T7 RNA Polymerase. Due to the orthogonality of the Polymerase, this strategy overcame limitations imposed by the host transcriptional machinery. This circuit enabled production of pathogen‑inhibitory levels of darobactin at 40 °C while maintaining leakiness below the detection limit at 37 °C. Furthermore, the thermo‑amplifier circuit sustained gene expression beyond the thermal induction duration such that with only 2 h of induction, the bacteria were able to produce pathogen‑inhibitory levels of darobactin. This performance was maintained even in physiologically relevant simulated conditions of the intestines that include bile salts and low nutrient levels.
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.
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.
Peptide drugs have seen rapid advancement in biopharmaceutical development, with over 80 candidates approved globally. Despite their therapeutic potential, the clinical translation of peptide drugs is hampered by challenges in production yields and stability. Engineered bacterial therapeutics is a unique approach being explored to overcome these issues by using bacteria to produce and deliver therapeutic compounds at the body site of use. A key advantage of this technology is the possibility to control drug delivery within the body in real time using genetic switches. However, the performance of such genetic switches suffers when used to control drugs that require post-translational modifications or are toxic to the host. In this study, these challenges were experienced when attempting to establish a thermal switch for the production of a ribosomally synthesized and post-translationally modified peptide antibiotic, darobactin, in probiotic E. coli. These challenges were overcome by developing a thermo-amplifier circuit that combined the thermal-switch with a T7 RNA Polymerase and its promoter that overcame limitations imposed by the host transcriptional machinery due to its orthogonality to it. This circuit enabled production of pathogen-inhibitory levels of darobactin at 40°C while maintaining leakiness below the detection limit at 37°C. More impressively, the thermo-amplifier circuit sustained production beyond the thermal induction duration. Thus, raised temperature for 2 h was sufficient for the bacteria to produce pathogen-inhibitory levels of darobactin even in the physiologically relevant simulated conditions of the intestines that include bile salts and low nutrient levels.
Herein, a study dealing with a progress on palladium (Pd) electrocatalysts for an efficient glycerol electrooxidation in model aqueous and real fermentation solutions with special focus on some physicochemical parameters (e.g., the impact of adsorption stage of multiple species, presence of oxygen, influence of anodic limits and Pd-size) was conducted. During the course of investigations by tandem of an optical oxygen minisensor and cyclic voltammetry a significant impact of oxygen on the efficiency of glycerol electrooxidation on Pd electrocatalysts at alkaline pH in model aqueous and yeast fermentation media was revealed. The obtained knowledge was used for the optimization of an assay utilizing Pd-sensing layers for glycerol determination and quantification in yeast fermentation medium. Received results showed a satisfactory agreement with a control measurement carried out by gas chromatography mass-spectrometry.
Hydrogels are natural/synthetic polymer-based materials with a large percentage of water content, usually above 80 %, and are suitable for many application fields such as wearable sensors, biomedicine, cosmetics, agriculture, etc. However, their performance is susceptible to environmental changes in temperature, relative humidity, and mechanical deformation due to their aqueous and soft nature. We investigate the mechanical response of both filled and unfilled alginate/gellan hydrogels using a combined axial-torsional rheometric approach with cylindrical samples of large length/diameter ratio under controlled temperature and relative humidity. Dynamic Mechanical Analysis (DMA) is performed on the same specimen in both torsion and extension under identical experimental conditions. This rheometric approach ensures consistent initial and boundary conditions, which are essential for a reliable estimation of viscoelastic moduli G* and E*, and their dependence on temperature, frequency, and relative humidity. Our findings indicate that humidity critically affects the mechanical response of the material due to sample volume shrinkage, necessitating corrections to the viscoelastic moduli. We also find temperature plays a role only at low/medium relative humidity values. The inclusion of fillers leads to a modest increase in the elasticity of the hydrogel, probably due to restricted water diffusion out of the sample. In connection with the latest, unfilled samples in breaking tests present only slippage due to twist-induced surface water excess, opposite to breakage events shown by filled samples, probably linked to restricted water diffusion.
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.
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.
