Stereolithographic 3D printing of extrinsically self-healing composites

We demonstrate for the first time the direct stereolithographic 3D printing of an extrinsically self-healing composite, comprised of commercial photocurable resin modified with anisole and PMMA-filled microcapsules. The composites demonstrate solvent-welding based autonomous self-healing to afford 87% recovery of the initial critical toughness. This work illustrates the potential of stereolithographic printing to fabricate self-healing composites with user-defined structures, avoiding the need for extensive rheological optimization of printing inks, like in direct-write 3D printing. Importantly, this work also demonstrates the inclusion of microcapsules into 3D printing resins to incorporate additional functionality into printed composites, which could be adapted for applications beyond self-healing materials

Structural activation of the transcriptional repressor EthR from Mycobacterium tuberculosis by single amino acid change mimicking natural and synthetic ligands

Ethionamide is an antituberculous drug for the treatment of multidrug-resistant Mycobacterium tuberculosis. This antibiotic requires activation by the monooxygenase EthA to exert its activity. Production of EthA is controlled by the transcriptional repressor EthR, a member of the TetR family. The sensitivity of M. tuberculosis to ethionamide can be artificially enhanced using synthetic ligands of EthR that allosterically inactivate its DNA-binding activity. Comparison of several structures of EthR co-crystallized with various ligands suggested that the structural reorganization of EthR resulting in its inactivation is controlled by a limited portion of the ligand-binding-pocket. In silico simulation predicted that mutation G106W may mimic ligands. X-ray crystallography of variant G106W indeed revealed a protein structurally similar to ligand-bound EthR. Surface plasmon resonance experiments established that this variant is unable to bind DNA, while thermal shift studies demonstrated that mutation G106W stabilizes EthR as strongly as ligands. Proton NMR of the methyl regions showed a lesser contribution of exchange broadening upon ligand binding, and the same quenched dynamics was observed in apo-variant G106W. Altogether, we here show that the area surrounding Gly106 constitutes the molecular switch involved in the conformational reorganization of EthR. These results also shed light on the mechanistic of ligand-induced allosterism controlling the DNA binding properties of TetR family repressors

Regulation of the Fruit-Specific PEP Carboxylase SlPPC2 Promoter at Early Stages of Tomato Fruit Development

The SlPPC2 phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) gene from tomato (Solanum lycopersicum) is differentially and specifically expressed in expanding tissues of developing tomato fruit. We recently showed that a 1966 bp DNA fragment located upstream of the ATG codon of the SlPPC2 gene (GenBank AJ313434) confers appropriate fruit-specificity in transgenic tomato. In this study, we further investigated the regulation of the SlPPC2 promoter gene by analysing the SlPPC2 cis-regulating region fused to either the firefly luciferase (LUC) or the β-glucuronidase (GUS) reporter gene, using stable genetic transformation and biolistic transient expression assays in the fruit. Biolistic analyses of 5′ SlPPC2 promoter deletions fused to LUC in fruits at the 8th day after anthesis revealed that positive regulatory regions are mostly located in the distal region of the promoter. In addition, a 5′ UTR leader intron present in the 1966 bp fragment contributes to the proper temporal regulation of LUC activity during fruit development. Interestingly, the SlPPC2 promoter responds to hormones (ethylene) and metabolites (sugars) regulating fruit growth and metabolism. When tested by transient expression assays, the chimeric promoter:LUC fusion constructs allowed gene expression in both fruit and leaf, suggesting that integration into the chromatin is required for fruit-specificity. These results clearly demonstrate that SlPPC2 gene is under tight transcriptional regulation in the developing fruit and that its promoter can be employed to drive transgene expression specifically during the cell expansion stage of tomato fruit. Taken together, the SlPPC2 promoter offers great potential as a candidate for driving transgene expression specifically in developing tomato fruit from various tomato cultivars

Redoxnetzwerke des Malariaerregers Plasmodium : Validierung von Schlüsselenzymen für neue chemotherapeutische Ansätze

Die Malaria-assozierte Pathologie wird durch die Entwicklung von Plasmodien in den Erythrozyten ihres Wirtes verursacht. Um ein reduzierendes intrazelluläres Milieu in diesem Sauerstoff-reichen Umfeld zu erhalten, haben Malaria-Parasiten ein komplexes antioxidatives Netzwerk entwickelt, welches auf zwei zentralen Elektronen-Donatoren beruht, dem Glutathion und dem Thioredoxin. Die intrazellulären Spiegel dieser beiden redox-aktiven Peptide in reduzierter – und damit reduzierender Form – werden durch die entsprechenden NADPH-abhängigen Flavoenzyme Thioredoxinreduktase (TrxR) und Glutathionreduktase (GR) aufrechterhalten. Da Katalase und eine klassische Selen-abhängige Glutathionperoxidase in Plasmodium fehlen, spielen die auf den Enzymen Thioredoxin- und Glutathionreduktase basierenden Systeme eine besondere Rolle. Weitere Bestandteile der antioxidativen Abwehr sind verschiedene Mitglieder der Thioredoxin-Familie, vor allem das Plasmodien-spezifische Plasmoredoxin (Plrx), welches als zusätzliche Verteidigungslinie der Parasiten gegen oxidativen Stress diskutiert wird. Im Rahmen der vorliegenden Arbeit wurden antioxidative Proteine der Parasiten untersucht, die als Zielmoleküle für die rationale Medikamentenentwicklung von Bedeutung sind. Um zu klären, welche der beteiligten Proteine sich aufgrund einer essentiellen Funktion in den Blutstadien-Parasiten insbesondere dafür eignen, wurden die zugrunde liegenden Gene dreier Redox-Proteine mit Methoden der reversen Genetik untersucht. Dies geschah durch stabile Gen-Inaktivierung unter Nutzung von Integrations- und replacement-Strategien, im Falle der Glutathionreduktase ergänzt durch Gen-Komplementation mit dem entsprechenden Ortholog. Die Targetvalidierung individueller redox-aktiver Proteine ist eine unerlässliche Voraussetzung für die rationale Entwicklung neuer effektiver Antimalaria-Medikamente. Die erfolgreiche Herstellung von Plasmoredoxin knock out-Mutanten in Nagetiermalaria-Modellparasiten und die phänotypischen Analysen im Verlauf des Lebenszyklus offenbarten in vitro und in vivo keine essentiellen Funktionen dieses Proteins. Dieses Ergebnis kann mit funktioneller Redundanz innerhalb der Mitglieder der Thioredoxin-Familie erklärt werden, somit bietet sich eine weitere Arzneimittelentwicklung allein auf der Basis Plasmoredoxin-spezifischer Inhibitoren nicht an. Weiterhin konnten Thioredoxinreduktase-defiziente P. berghei-Mutanten erzeugt werden. Eine systematische Analyse dieser Parasiten im Verlauf des Plasmodium-Lebenszyklus zeigte an keiner Stelle eine lebensnotwendige Funktion der Thioredoxinreduktase. Dies steht im Gegensatz zu früheren in vitro-Studien bei P. falciparum, welche diesem Enzym eine essentielle Funktion zuschrieben. Da ein praktikables in vivo-Modell für humanpathogene Malaria-Parasiten nicht verfügbar ist, verdeutlicht das hier vorgestellte Ergebnis der Targetvalidierung für Thioredoxinreduktase die Bedeutung funktioneller Studien in Nagetiermalaria-Modellen für die präklinische Entwicklung neuer Medikamente gegen Malaria. Für eines der viel versprechendsten drug targets von Malaria-Parasiten, der Glutathionreduktase, konnte auf genetischer Ebene der Beweis erbracht werden, dass durch den Verlust der Funktion dieses Enzyms die Parasiten nicht lebensfähig sind. Somit nimmt die Glutathionreduktase in dem komplexen antioxidativen Netzwerk von Plasmodium eine zentrale und essentielle Rolle ein. Zusätzlich wurde in dieser Arbeit die molekulare Wirkungsweise des seit mehr als 100 Jahren klinisch genutzten Medikaments Methylenblau auf Malaria-Parasiten untersucht. Hierbei zeigte sich, dass diese Verbindung neben ihren inhibierenden Eigenschaften interessante Charakteristika als subversives Substrat verschiedener Disulfidreduktasen besitzt. Bei Anwesenheit von Methylenblau werden diese Enzyme zu pro-oxidativen, H2O2-produzierenden Verbindungen, welche die reduzierenden zellulären Bedingungen herausfordern, statt sie zu bewahren.Malaria-associated pathology is caused by the continuous expansion of Plasmodium parasites inside host erythrocytes. To maintain a reducing intracellular milieu in this oxygen-rich environment, malaria parasites have evolved a complex antioxidative network based on two central electron donors, glutathione and thioredoxin. The intracellular levels of these redox-active peptides in reduced and thus reducing forms are maintained by the respective NADPH-dependent flavoenzymes thioredoxin reductase (TrxR) and glutathione reductase (GR). As catalase and classical selenium-dependant glutathione peroxidase are absent in Plasmodium, the systems based on thioredoxin reductase and glutathione reductase play a prominent role. Further components of the antioxidative defense comprise different members of the thioredoxin family, especially Plasmodium-specific plasmoredoxin which has been discussed as an additional defense line against oxidative stress. In the framework of this thesis, antioxidative parasite proteins that are of interest as targets for rational drug development were investigated. To clarify which proteins are particularly suitable as drug target due to an essential function for blood stage parasites, the respective genes of three redox proteins were studied employing reverse genetics. This was achieved by stable gene inactivation with integration and replacement strategies, in the case of glutathione reductase supplemented by gene complementation of the respective ortholog. Target validation of individual redox-active proteins is an indispensable prerequisite for the rational development of new and effective antimalarial drugs. The successful generation of plasmoredoxin knockout mutants in the rodent model malaria parasite and phenotypic analysis during life cycle progression revealed both in vitro and in vivo a non-vital function of this protein. This finding can be explained by functional redundancy among the members of the thioredoxin family and discourages future drug discovery efforts that aim at specifically targeting plasmoredoxin. Furthermore P. berghei thioredoxin reductase-deficient parasites could be generated in this thesis. A systematic phenotypic analysis of this mutant throughout the Plasmodium life cycle demonstrated no essential function for thioredoxin reductase at any phase of the parasite life cycle. This is in contrast to previous in vitro studies which attributed an essential role to thioredoxin reductase in P. falciparum. As a feasible in vivo model for human pathogen malaria parasites is not available, the presented target validation result of thioredoxin reductase highlights the importance of functional studies in rodent malaria models to guide preclinical development of novel antimalaria intervention strategies. For one of the most promising drug targets of malaria parasites, glutathione reductase, this work provides the genetic proof that in the case of this enzyme loss of function results in non-viable malaria parasites in vivo. Hence, glutathione reductase occupies an essential position in the complex antioxidant network of Plasmodium parasites. Furthermore, the molecular mode of action of methylene blue, which has been clinically used as an antimalarial drug for over 100 years, against malaria parasites has been investigated. It could be shown that this compound – besides its inhibitory potential – has interesting characteristics as a subversive substrate of different disulfide reductases. In the presence of methylene blue, they turn into pro-oxidant, H2O2-producing enzymes which challenge the reducing cellular milieu that they are meant to protect in the absence of this perturbing drug

A first update on mapping the human genetic architecture of COVID-19

peer reviewe

Lithium-ion capacitor safety assessment under electrical abuse tests based on ultrasound characterization and cell opening

International audienc

Nanocast Mixed Ni–Co–Mn Oxides with Controlled Surface and Pore Structure for Electrochemical Oxygen Evolution Reaction

Nanocasting or hard-templating is a versatile method to produce ordered mesoporous mixed transition metal oxides (MTMOs) with promising potential for both oxygen evolution reaction (OER) and oxygen reduction reaction (ORR). Herein, a comprehensive investigation was conducted on various NixCoyMnzO4 replicated from large pore KIT-6 silica. The materials were calcined at different temperatures to study the influence of the oxide formation and the resulting pore structure ordering, as well as surface properties, on the electrochemical activity and stability of the catalysts. After a comprehensive characterization, electrocatalytic performances of the materials were investigated in detail for OER to find a structure–activity relationship. In OER, a correlation was established between calcination temperature, pore and surface properties, and the overall efficiency and stability. The best sample, NixCoyMnzO4 calcined at 300 °C, provided a reasonable current density (25 mA/cm2 at 1.7 V vs RHE) and an overpotential of 400 mV at 10 mA/cm2, and demonstrated increased current density (above 200 mA/cm2 at 1.7 V vs RHE) once loaded into a Ni foam compared to the bare foam. This sample also remained stable over 15 h. Our results indicate that the calcination temperature greatly affects the porosity, crystalline structure, phase composition, and the activity of the catalysts toward OER