Purification and proteomics of influenza virions

This chapter describes a basic workflow for analyzing the protein composition of influenza virions. In order to obtain suitable material, the chapter describes how to concentrate influenza virions from the growth media of infected cells and to purify them by ultracentrifugation through a density gradient. This approach is also suitable for purifying influenza virions from the allantoic fluid of embryonated chicken eggs. As a small quantity of microvesicles are co-purified with virions, optional steps are included to increase the stringency of purification by enriching material with viral receptor binding and cleaving activity. Material purified in this way can be used for a variety of downstream applications, including proteomics. As a detailed example of this, the chapter also describes a standard workflow for analyzing the protein composition of concentrated virions by liquid chromatography and tandem mass spectrometry

Familien als Ganzes in den Blick nehmen

Vor dem Hintergrund eines weiten Familienbegriffs werden in diesem Artikel aktuelle Daten und Entwicklungstendenzen zu Familien dargestellt. Eine haushaltsübergreifende Erweiterung des Familienbegriffs öffnet den Blick für Ambivalenzen zwischen familialen Unterstützungsressourcen und Konfliktpotentialen und bietet Anschlussmöglichkeiten für die künftige konzeptionelle Gestaltung der Familienbildung

Facilitation of Digital Change of Teaching in School by Professional Learning Communities: Use of Multiplicators to Establish Learning Communities

Zahlreiche Studien zeigen, dass die wirkungsvolle Implementierung von Innovationen davon profitiert und sich positive Effekte auf die Schüler:innen nachweisen lassen, wenn Lehrpersonen kooperieren. In vielen Fällen einer solchen Zusammenarbeit handelt es sich um professionelle Lerngemeinschaften, typischerweise bestehend aus Lehrpersonen, Schulleitung, Schulverwaltung und Wissenschaft. Unter einer Professionellen Lerngemeinschaft (PLG) wird eine «Gemeinschaft fortlaufender Forschung und Verbesserung» verstanden, in der alle Gruppenmitglieder sowohl miteinander als auch voneinander lernen. Eine offene Frage in der Forschung zu Professionellen Lerngemeinschaften ist, inwieweit anstelle einer intensiven Kooperation mit Vertreter:innen aus der Wissenschaft geschulte Multiplikator:innen die Inhalte aus der Wissenschaft einbringen und die Arbeit in den Lerngemeinschaften fördern können. Dieser Fragestellung wird im Rahmen dieser Pilotstudie nachgegangen. Zu diesem Zweck wurden an drei bayerischen Schulen (zwei Mittelschulen, eine Realschule) professionelle Lerngemeinschaften zur Digitalisierung des MINT-Unterrichts in der Schule im Schuljahr 2020/2021 gegründet. Die Befunde der durchgeführten Interviewstudie deuten darauf hin, dass die wissenschaftliche Unterstützung von Lerngemeinschaften zur Digitalisierung von Unterricht in der Schule mit Ausnahme der evidenzbasierten Arbeit an Unterrichtskonzepten erfolgreich mithilfe von Multiplikator:innen umgesetzt werden kann. Für die fachspezifische Unterstützung sollten jedoch zusätzliche Lehrpersonen (z. B. die Fachleitungen) geschult werden, um die fachbezogene Arbeit an den Unterrichtskonzepten zu unterstützen.Numerous studies provide evidence that successful implementation of innovation as well as learning of students benefit from teacher collaboration. Such collaboration often takes place as part of so-called Professional Learning Communities (PLC). PLCs are communities of learners that continuously do research with the goal of further improvement at three levels: individual, community, and institution. All members of PLC learn from and with each other through collaborative work to reach a joint goal. An open issue in research on PLC is, however, to what extent an intensive collaboration with Science can be compensated by facilitation through specifically trained multiplicators. To what extent can multiplicators place the scientific content and facilitate the collaboration within a PLC? To answer these research questions, PLCs on digitalization of teaching STEM in school were founded in 2020/2021 at three Bavarian secondary schools (two Mittelschulen, one Realschule). The results of the conducted interview study suggest, that multiplicators can successfully establish PLCs. However, not all functions of scientific PLC members can be equally compensated. To fulfill the needs of the PLC, additional multiplicators with focus on STEM didactics (e. g., heads of STEM subjects from the schools themselves) may be trained to support the subject-related work on material for teaching with digital media

Minimum Information about a Biosynthetic Gene cluster

A wide variety of enzymatic pathways that produce specialized metabolites in bacteria, fungi and plants are known to be encoded in biosynthetic gene clusters. Information about these clusters, pathways and metabolites is currently dispersed throughout the literature, making it difficult to exploit. To facilitate consistent and systematic deposition and retrieval of data on biosynthetic gene clusters, we propose the Minimum Information about a Biosynthetic Gene cluster (MIBiG) data standard.Chemistry and Chemical Biolog

Broadband Multi-wavelength Properties of M87 during the 2017 Event Horizon Telescope Campaign

Abstract: In 2017, the Event Horizon Telescope (EHT) Collaboration succeeded in capturing the first direct image of the center of the M87 galaxy. The asymmetric ring morphology and size are consistent with theoretical expectations for a weakly accreting supermassive black hole of mass ∼6.5 × 109 M ⊙. The EHTC also partnered with several international facilities in space and on the ground, to arrange an extensive, quasi-simultaneous multi-wavelength campaign. This Letter presents the results and analysis of this campaign, as well as the multi-wavelength data as a legacy data repository. We captured M87 in a historically low state, and the core flux dominates over HST-1 at high energies, making it possible to combine core flux constraints with the more spatially precise very long baseline interferometry data. We present the most complete simultaneous multi-wavelength spectrum of the active nucleus to date, and discuss the complexity and caveats of combining data from different spatial scales into one broadband spectrum. We apply two heuristic, isotropic leptonic single-zone models to provide insight into the basic source properties, but conclude that a structured jet is necessary to explain M87’s spectrum. We can exclude that the simultaneous γ-ray emission is produced via inverse Compton emission in the same region producing the EHT mm-band emission, and further conclude that the γ-rays can only be produced in the inner jets (inward of HST-1) if there are strongly particle-dominated regions. Direct synchrotron emission from accelerated protons and secondaries cannot yet be excluded

Quantifying plasma membrane protein reduction during immune activation using mass spectrometry

The communication of immune cells through cell surface receptors is crucial for maintaining immune homeostasis, coordinating the immune response and pathogen clearance. The interaction of cell surface receptors with their cognate ligands is dependent on a key-lock principle and therefore only allows binding of partners in their correct conformational state. Reductive cleavage of labile disulfide bonds is a post-translational modification, capable of controlling protein function by mediating a conformational change. Activation of the immune system leads to the secretion of thiol isomerases that catalyse labile disulfide bond reduction and thereby are able to modulate protein function. A bioinformatics study and a mass spectrometry based screen have shown that many different plasma membrane proteins potentially contain labile disulfide bonds. To identify proteins with biologically relevant labile disulfide bonds it is necessary to quantify the reduction occurring as it is expected that only proteins that are reduced to a substantial amount affect cellular function. In this study, a mass spectrometry based method to screen for proteins with labile disulfide bonds and quantify their reduction in primary cells was developed. It was then applied to study the plasma membrane redox proteome during immune activation and identified several proteins, mainly activating (CD132, gp130) and adhesion (integrins, ICAM1 and CD44) molecules, that are reduced to a major degree during immune activation. Furthermore, functional studies on the shared cytokine receptor gp130 showed that thioredoxin reduction inhibits IL-6 signalling. These observations in conjunction with the literature suggest that labile disulfide bond reduction during immune activation is a mechanism to prevent over-activation of the immune system and excessive accumulation of leukocytes in sites of inflammation. Presenting a method to identify altered redox control of proteins in diseases such as autoimmunity provides the basis for the rational design of novel redox drugs.</p

Quantifying plasma membrane protein reduction during immune activation using mass spectrometry

The communication of immune cells through cell surface receptors is crucial for maintaining immune homeostasis, coordinating the immune response and pathogen clearance. The interaction of cell surface receptors with their cognate ligands is dependent on a key-lock principle and therefore only allows binding of partners in their correct conformational state. Reductive cleavage of labile disulfide bonds is a post-translational modification, capable of controlling protein function by mediating a conformational change. Activation of the immune system leads to the secretion of thiol isomerases that catalyse labile disulfide bond reduction and thereby are able to modulate protein function. A bioinformatics study and a mass spectrometry based screen have shown that many different plasma membrane proteins potentially contain labile disulfide bonds. To identify proteins with biologically relevant labile disulfide bonds it is necessary to quantify the reduction occurring as it is expected that only proteins that are reduced to a substantial amount affect cellular function. In this study, a mass spectrometry based method to screen for proteins with labile disulfide bonds and quantify their reduction in primary cells was developed. It was then applied to study the plasma membrane redox proteome during immune activation and identified several proteins, mainly activating (CD132, gp130) and adhesion (integrins, ICAM1 and CD44) molecules, that are reduced to a major degree during immune activation. Furthermore, functional studies on the shared cytokine receptor gp130 showed that thioredoxin reduction inhibits IL-6 signalling. These observations in conjunction with the literature suggest that labile disulfide bond reduction during immune activation is a mechanism to prevent over-activation of the immune system and excessive accumulation of leukocytes in sites of inflammation. Presenting a method to identify altered redox control of proteins in diseases such as autoimmunity provides the basis for the rational design of novel redox drugs.</p

CD44 Binding to Hyaluronic Acid Is Redox Regulated by a Labile Disulfide Bond in the Hyaluronic Acid Binding Site

<div><p>CD44 is the primary leukocyte cell surface receptor for hyaluronic acid (HA), a component of the extracellular matrix. Enzymatic post translational cleavage of labile disulfide bonds is a mechanism by which proteins are structurally regulated by imparting an allosteric change and altering activity. We have identified one such disulfide bond in CD44 formed by Cys77 and Cys97 that stabilises the HA binding groove. This bond is labile on the surface of leukocytes treated with chemical and enzymatic reducing agents. Analysis of CD44 crystal structures reveal the disulfide bond to be solvent accessible and in the–LH hook configuration characteristic of labile disulfide bonds. Kinetic trapping and binding experiments on CD44-Fc chimeric proteins show the bond is preferentially reduced over the other disulfide bonds in CD44 and reduction inhibits the CD44-HA interaction. Furthermore cells transfected with CD44 no longer adhere to HA coated surfaces after pre-treatment with reducing agents. The implications of CD44 redox regulation are discussed in the context of immune function, disease and therapeutic strategies.</p></div

Cell adhesion assay showing the effect of TCEP-HCl and Trx1 reduction of untransfected and hCD44 transfected CHO cells binding to HA coated plates.

<p>Binding data are compositions of three independent experiments all normalised assuming non reduced CHO-hCD44 binding to HA coated wells as 100% response. Mean and SEM of total fluorescence are shown. The differences from control: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ***, P < 0.0001</p

(A) Crystal structure of the HABD of murine CD44 in complex with an HA 8-mer (adapted from PDB ID:2JCQ).

<p>The protein backbone is represented as a blue cartoon. The HA 8-mer is shown as pink sticks. Disulfide bonds are shown as yellow sticks and the cysteine residues from which they are formed are numbered according to their position in the mouse CD44 sequence (human numbering is shown in parenthesis). (B) For each disulfide bond dark grey bars show solvent accessibility and light grey bars show bond strain energy calculated from the PDB coordinates (PDB ID:2JCQ for mouse and 1UUH for human, the coordinates for HA were removed from 2JCQ prior to analysis) <b>using</b> (<a href="http://powcs.med.unsw.edu.au/research/adult-cancer-program/services-resources/disulfide-bond-analysis-tool/disulfide-bond" target="_blank">http://powcs.med.unsw.edu.au/research/adult-cancer-program/services-resources/disulfide-bond-analysis-tool/disulfide-bond</a>).</p