Functions and cell biology of the sphingolipids sphingosine and sphinganine in cells are not well understood. While some signaling roles for sphingosine have been elucidated, the closely related ...sphinganine has been described only insofar as it does not elicit many of the same signaling responses. Here, we prepared multifunctionalized derivatives of the two lipid species that differ only in a single double bond of the carbon backbone. Using these novel probes, we were able to define their spatiotemporal distributions within cells. Furthermore, we used these tools to systematically map the protein interactomes of both lipids. The lipid–protein conjugates, prepared through photo-crosslinking in live cells and extraction via click chemistry to azide beads, revealed significant differences in the captured proteins, highlighting their distinct roles in various cellular processes. This work elucidates mechanistic differences between these critical lipids and sets the foundation for further studies of the cellular functions of sphingosine and sphinganine.
Modelling of solidification is of industrial and theoretical relevance. An accurate estimation of the actual liquidus and solidus temperatures leads to significant improvements in quality and ...efficiency of steel production as well as substantially reduces the energy consumption and the ecological footprint.
An optimisation of the Scheil–Gulliver model for solidification with the aim to predict the solidus temperature of steels (Scheil–Gulliver for Steel, SGS) is presented. The SGS model allows an easy and accurate simulation of the solidification interval using software based on the CALPHAD approach. Based only on the steel composition, the model consistently choses between full equilibrium for ferrite and partial redistribution of alloying elements (Scheil–Gulliver approach) for austenite. The predictions of the model were compared to differential thermal analysis (DTA) measurements of industrial heats, which represent a wide range of compositions. The agreement of the data calculated with the SGS model with the values measured by DTA represents an improvement compared to existing models.
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•The Scheil–Gulliver for Steel (SGS) model for solidification is presented.•SGS consistently reconciles equilibrium (ferrite) and Scheil–Gulliver (austenite).•SGS model predicts solidus temperatures of technical steels reliably.
Abstract
Regarding the effect of composition on the mechanical properties of intermetallic phases such as Laves phases, there is conflicting information in the literature. Some authors observed ...defect hardening when deviating from stoichiometric Laves phase composition, whereas others find defect softening. Here, we present a systematic investigation of the defect state, hardness, and elastic modulus of cubic and hexagonal NbCo
2
Laves phases as a function of crystal structure and composition. For this purpose, diffusion couples were prepared which exhibit diffusion layers of the cubic C15 and hexagonal C14 and C36 NbCo
2
Laves phases, with concentration gradients covering their entire homogeneity ranges from 24 to 37 at.% Nb. Direct observations of dislocations and stacking faults in the diffusion layers as a function of composition were performed by electron channeling contrast imaging, and the hardness and elastic modulus were probed in the diffusion layers along the concentration gradients by nanoindentation.
Recent developments in high-throughput reverse genetics
have revolutionized our ability to map gene function and interactions
. The power of these approaches depends on their ability to identify ...functionally associated genes, which elicit similar phenotypic changes across several perturbations (chemical, environmental or genetic) when knocked out
. However, owing to the large number of perturbations, these approaches have been limited to growth or morphological readouts
. Here we use a high-content biochemical readout, thermal proteome profiling
, to measure the proteome-wide protein abundance and thermal stability in response to 121 genetic perturbations in Escherichia coli. We show that thermal stability, and therefore the state and interactions of essential proteins, is commonly modulated, raising the possibility of studying a protein group that is particularly inaccessible to genetics. We find that functionally associated proteins have coordinated changes in abundance and thermal stability across perturbations, owing to their co-regulation and physical interactions (with proteins, metabolites or cofactors). Finally, we provide mechanistic insights into previously determined growth phenotypes
that go beyond the deleted gene. These data represent a rich resource for inferring protein functions and interactions.
The concept of template-assembled synthetic proteins (TASP) describes a central scaffold that predefines the three dimensional structure for diverse molecules linked to this platform. Cyclic ...β-tripeptides are interesting candidates for use as templates due to their conformationally defined structure, stability to enzymatic degradation, and ability to form intermolecular stacked tubular structures. To validate the applicability of cyclic β-tripeptides within the TASP concept, an efficient synthesis of the cyclopeptide with orthogonal functionalization of the side chains is desired. A solid-phase-supported route with on-resin cyclization is described, employing the aryl hydrazide linker cleavable by oxidation. An orthogonal protection-group strategy allows functionalization of the central cyclic β-tripeptide with up to three different peptide fragments or fluorescent labels.
Mapping of weak and hence transient interactions between low-abundance interacting molecules is still a major challenge in systems biology and protein biochemistry. Therefore, additional system-wide ...acting tools are needed to determine protein interactomics. Most important are reagents that can be applied at any kind of protein interface and the possibility to enrich cross-linked fragments with high efficiency. In this study, we report the synthesis of a novel noncanonical amino acid that features a diazirine group for ultraviolet cross-linking as well as an alkyne group for labeling by click chemistry. This bifunctional amino acid, called PrDiAzK, may be inserted into almost any protein interface with minimal structural perturbation using genetic code expansion. We demonstrate that PrDiAzK can be site-selectively incorporated into proteins in both bacterial and mammalian cell cultures, and we show that PrDiAzK allows protein labeling as well as cross-linking. In addition, we tested PrDiAzK for proteome-wide incorporation via stochastic orthogonal recoding of translation, implying potential applications in system-wide mapping of protein–protein interactions in the future.
Functionalized lipid probes are a critical new tool to interrogate the function of individual lipid species, but the structural parameters that constrain their utility have not been thoroughly ...described. Here, we synthesize three palmitic acid derivatives with a diazirine at different positions on the acyl chain and examine their metabolism, subcellular localization, and protein interactions. We demonstrate that while they produce very similar metabolites and subcellular distributions, probes with the diazirine at either end pulldown distinct subsets of proteins after photo-crosslinking. This highlights the importance of thoughtful diazirine placement when developing probes based on biological molecules.
Functionalized lipid probes are a critical new tool to interrogate the function of individual lipid species, but the structural parameters that constrain their utility have not been thoroughly described.
Retrons are prokaryotic genetic retroelements encoding a reverse transcriptase that produces multi-copy single-stranded DNA
(msDNA). Despite decades of research on the biosynthesis of msDNA
, the ...function and physiological roles of retrons have remained unknown. Here we show that Retron-Sen2 of Salmonella enterica serovar Typhimurium encodes an accessory toxin protein, STM14_4640, which we renamed as RcaT. RcaT is neutralized by the reverse transcriptase-msDNA antitoxin complex, and becomes active upon perturbation of msDNA biosynthesis. The reverse transcriptase is required for binding to RcaT, and the msDNA is required for the antitoxin activity. The highly prevalent RcaT-containing retron family constitutes a new type of tripartite DNA-containing toxin-antitoxin system. To understand the physiological roles of such toxin-antitoxin systems, we developed toxin activation-inhibition conjugation (TAC-TIC), a high-throughput reverse genetics approach that identifies the molecular triggers and blockers of toxin-antitoxin systems. By applying TAC-TIC to Retron-Sen2, we identified multiple trigger and blocker proteins of phage origin. We demonstrate that phage-related triggers directly modify the msDNA, thereby activating RcaT and inhibiting bacterial growth. By contrast, prophage proteins circumvent retrons by directly blocking RcaT. Consistently, retron toxin-antitoxin systems act as abortive infection anti-phage defence systems, in line with recent reports
. Thus, RcaT retrons are tripartite DNA-regulated toxin-antitoxin systems, which use the reverse transcriptase-msDNA complex both as an antitoxin and as a sensor of phage protein activities.
The present work addresses the microstructure evolution of refractory Me-Si-B (Me = Mo, V) alloys consisting of a refractory metal solid solution phase (MeSS) and two intermetallic phases Me5SiB2 and ...Me3Si. The aim of the present study is to find the ternary eutectic composition in such systems which are expected to combine a well-defined eutectic microstructure with properties such as high strength, an excellent creep resistance at high temperatures and acceptable oxidation behavior. Two refractory metal systems based on molybdenum and vanadium are investigated. Various alloy compositions located in different primary solidification areas were produced by arc-melting and analyzed via SEM. The obtained results are discussed in the light of published liquidus projections. By carrying out these experiments, a MeSS-Me5SiB2-Me3Si ternary eutectic could be determined in both Me-Si-B systems.
BACKGROUND: Endothelial cells (ECs) are primed to respond to various signaling cues. For example, TGF (transforming growth factor)-β has major effects on EC function and phenotype by driving ECs ...towards a more mesenchymal state (ie, triggering endothelial to mesenchymal activation), a dynamic process associated with cardiovascular diseases. Although transcriptional regulation triggered by TGF-β in ECs is well characterized, post-transcriptional regulatory mechanisms induced by TGF-β remain largely unknown. METHODS: Using RNA interactome capture, we identified global TGF-β driven changes in RNA-binding proteins in ECs. We investigated specific changes in the RNA-binding patterns of hnRNP H1 (heterogeneous nuclear ribonucleoprotein H1) and Csde1 (cold shock domain containing E1) using RNA immunoprecipitation and overlapped this with RNA-sequencing data after knockdown of either protein for functional insight. Using a modified proximity ligation assay, we visualized the specific interactions between hnRNP H1 and Csde1 and target RNAs in situ both in vitro and in mouse heart sections. RESULTS: Characterization of TGF-β–regulated RBPs (RNA-binding proteins) revealed hnRNP H1 and Csde1 as key regulators of the cellular response to TGF-β at the post-transcriptional level, with loss of either protein-promoting mesenchymal activation in ECs. We found that TGF-β drives an increase in binding of hnRNP H1 to its target RNAs, offsetting mesenchymal activation, but a decrease in Csde1 RNA-binding, facilitating this process. Both, hnRNP H1 and Csde1, dynamically bind and regulate specific subsets of mRNAs related to mesenchymal activation and endothelial function. CONCLUSIONS: Together, we show that RBPs play a key role in the endothelial response to TGF-β stimulation at the post-transcriptional level and that the RBPs hnRNP H1 and Csde1 serve to maintain EC function and counteract mesenchymal activation. We propose that TGF-β profoundly modifies RNA-protein interaction entailing feedback and feed-forward control at the post-transcriptional level, to fine-tune mesenchymal activation in ECs.
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