Identifizierung und Charakterisierung des Mais-spezifischen Signalpeptids Zip1 und dessen Aktivierung durch Cysteinproteasen

Während ihrer Entwicklung haben Pflanzen mit einer hohen Vielfalt von Pathogenen zu kämpfen. Pflanzen wirken einem Pathogenbefall entgegen, indem sie abhängig vom Lebensstils des Pathogens eine entgegengesetzte Immunreaktion, Zelltod oder Zellüberleben, induzieren. In diesem Zusammenhang spielen Papain-ähnliche Cysteinproteasen (PLCPs) eine entscheidene Rolle bei der Regulation der pflanzlichen Immunantwort und der Einleitung des Zelltods. In Mais (Zea mays) konnten fünf apoplastische PLCPs (CP1A, CP1B, CP2, XCP2 und CatB) identifiziert werden, die für diesen Prozess und die Inszenierung der Salicylsäure (SA)-abhängigen Immunantwort entscheidend sind. Interessanterweise ist das Zusammenspiel von PLPCs und SA-Immunreaktionen nicht linear, sondern zeigt eine reziproke Amplifikation. Im Gegensatz zu phänotypischen Beobachtungen sind die zugrundeliegenden, biochemischen Mechanismen weitgehend unerforscht. In dieser Arbeit konnte mittels massenspektrometrischer Analysen und Bioassays ein Peptid in SA-behandelten Maisblättern identifiziert werden, dass als Zip1 (Zea mays immune signaling peptide 1) bezeichnet wurde. Zip1 induziert die Expression SA-assoziierter PR (pathogenesis-related)-Gene und aktiviert, ähnlich wie SA, apoplastische PLCPs. In vitro-Studien konnten zeigen, dass zwei der zuvor identifizierten PLCPs, CP1 und CP2, immunogene Peptidsignale aus dem Zip1-Vorläuferprotein (PROZIP1) proteolytisch freisetzen. Weiterhin bestätigen RNAseq-basierte Transkriptomanalysen, dass Zip1- und SA-behandelte Maisblätter einen Großteil ähnlicher Immunantworten auf Transkriptebene teilen, weshalb bei Zip1 von einem SA-analogen Signalpeptid gesprochen werden kann. Bemerkenswerterweise und mit den RNAseq-Daten einhergehend konnte außerdem gezeigt werden, dass Zip1 die de novo Synthese von SA induziert. Darauf aufbauend erhöht Zip1 die Suszeptibilität von Mais gegenüber dem nekrotrophen Pathogen Botrytis cinerea. Mit der Identifizierung von Zip1, einem endogenen Maispeptid, welches von SA-ativierten PLCPs freigesetzt wird, ist das erste pflanzliche Peptidsignal charakteriseriert worden, das an der SA-abhänigen Immunantwort und dessen sequentiellen Amplifikation beteiligt ist

Vascular histone deacetylation by pharmacological HDAC inhibition

HDAC inhibitors can regulate gene expression by post-translational modification of histone as well as nonhistone proteins. Often studied at single loci, increased histone acetylation is the paradigmatic mechanism of action. However, little is known of the extent of genome-wide changes in cells stimulated by the hydroxamic acids, TSA and SAHA. In this article, we map vascular chromatin modifications including histone H3 acetylation of lysine 9 and 14 (H3K9/14ac) using chromatin immunoprecipitation (ChIP) coupled with massive parallel sequencing (ChIP-seq). Since acetylation-mediated gene expression is often associated with modification of other lysine residues, we also examined H3K4me3 and H3K9me3 as well as changes in CpG methylation (CpG-seq). RNA sequencing indicates the differential expression of ∼30% of genes, with almost equal numbers being up- and down-regulated. We observed broad deacetylation and gene expression changes conferred by TSA and SAHA mediated by the loss of EP300/CREBBP binding at multiple gene promoters. This study provides an important framework for HDAC inhibitor function in vascular biology and a comprehensive description of genome-wide deacetylation by pharmacological HDAC inhibition

Low Energy Electron Irradiation Is a Potent Alternative to Gamma Irradiation for the Inactivation of (CAR-)NK-92 Cells in ATMP Manufacturing

Background: With increasing clinical use of NK-92 cells and their CAR-modified derivatives in cancer immunotherapy, there is a growing demand for efficient production processes of these “off-the-shelf” therapeutics. In order to ensure safety and prevent the occurrence of secondary tumors, (CAR-)NK-92 cell proliferation has to be inactivated before transfusion. This is commonly achieved by gamma irradiation. Recently, we showed proof of concept that low energy electron irradiation (LEEI) is a new method for NK-92 inactivation. LEEI has several advantages over gamma irradiation, including a faster reaction time, a more reproducible dose rate and much less requirements on radiation shielding. Here, LEEI was further evaluated as a promising alternative to gamma irradiation yielding cells with highly maintained cytotoxic effector function. Methods: Effectiveness and efficiency of LEEI and gamma irradiation were analyzed using NK-92 and CD123-directed CAR-NK-92 cells. LEE-irradiated cells were extensively characterized and compared to gamma-irradiated cells via flow cytometry, cytotoxicity assays, and comet assays, amongst others. Results: Our results show that both irradiation methods caused a progressive decrease in cell viability and are, therefore, suitable for inhibition of cell proliferation. Notably, the NKmediated specific lysis of tumor cells was maintained at stable levels for three days postirradiation, with a trend towards higher activities after LEEI treatment as compared to gamma irradiation. Both gamma irradiation as well as LEEI led to substantial DNA damage and an accumulation of irradiated cells in the G2/M cell cycle phases. In addition, transcriptomic analysis of irradiated cells revealed approximately 12-fold more differentially expressed genes two hours after gamma irradiation, compared to LEEI. Analysis of surface molecules revealed an irradiation-induced decrease in surface expression of CD56, but no changes in the levels of the activating receptors NKp46, NKG2D, or NKp30. Conclusions: The presented data show that LEEI inactivates (CAR-)NK-92 cells as efficiently as gamma irradiation, but with less impact on the overall gene expression. Due to logistic advantages, LEEI might provide a superior alternative for the manufacture of (CAR-)NK-92 cells for clinical application

Proteases Underground: Analysis of the Maize Root Apoplast Identifies Organ Specific Papain-Like Cysteine Protease Activity

Plant proteases are key regulators of plant cell processes such as seed development, immune responses, senescence and programmed cell death (PCD). Apoplastic papain-like cysteine proteases (PL) are hubs in plant-microbe interactions and play an important role during abiotic stresses. The apoplast is a crucial interface for the interaction between plant and microbes. So far, apoplastic maize PL and their function have been mostly described for aerial parts. In this study, we focused on apoplastic PLCPs in the roots of maize plants. We have analyzed the phylogeny of maize PLCPs and investigated their protein abundance after salicylic acid (SA) treatment. Using activity-based protein profiling (ABPP) we have identified a novel root-specific PLCP belonging to the RD21-like subfamily, as well as three SA activated PLCPs. The root specific PLCP CP1C shares sequence and structural similarities to known CP1-like proteases. Biochemical analysis of recombinant CP1C revealed different substrate specificities and inhibitor affinities compared to the related proteases. This study characterized a root-specific PLCP and identifies differences between the SA-dependent activation of PLCPs in roots and leaves

Dendrimer-Based Fluorescent Indicators: In Vitro and In Vivo Applications

BACKGROUND: The development of fluorescent proteins and synthetic molecules whose fluorescence properties are controlled by the environment makes it possible to monitor physiological and pathological events in living systems with minimal perturbation. A large number of small organic dyes are available and routinely used to measure biologically relevant parameters. Unfortunately their application is hindered by a number of limitations stemming from the use of these small molecules in the biological environment. PRINCIPAL FINDINGS: We present a novel dendrimer-based architecture leading to multifunctional sensing elements that can overcome many of these problems. Applications in vitro, in living cells and in vivo are reported. In particular, we image for the first time extracellular pH in the brain in a mouse epilepsy model. CONCLUSION: We believe that the proposed architecture can represent a useful and novel tool in fluorescence imaging that can be widely applied in conjunction with a broad range of sensing dyes and experimental setups

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Identifizierung und Charakterisierung des Mais-spezifischen Signalpeptids Zip1 und dessen Aktivierung durch Cysteinproteasen

Während ihrer Entwicklung haben Pflanzen mit einer hohen Vielfalt von Pathogenen zu kämpfen. Pflanzen wirken einem Pathogenbefall entgegen, indem sie abhängig vom Lebensstils des Pathogens eine entgegengesetzte Immunreaktion, Zelltod oder Zellüberleben, induzieren. In diesem Zusammenhang spielen Papain-ähnliche Cysteinproteasen (PLCPs) eine entscheidene Rolle bei der Regulation der pflanzlichen Immunantwort und der Einleitung des Zelltods. In Mais (Zea mays) konnten fünf apoplastische PLCPs (CP1A, CP1B, CP2, XCP2 und CatB) identifiziert werden, die für diesen Prozess und die Inszenierung der Salicylsäure (SA)-abhängigen Immunantwort entscheidend sind. Interessanterweise ist das Zusammenspiel von PLPCs und SA-Immunreaktionen nicht linear, sondern zeigt eine reziproke Amplifikation. Im Gegensatz zu phänotypischen Beobachtungen sind die zugrundeliegenden, biochemischen Mechanismen weitgehend unerforscht. In dieser Arbeit konnte mittels massenspektrometrischer Analysen und Bioassays ein Peptid in SA-behandelten Maisblättern identifiziert werden, dass als Zip1 (Zea mays immune signaling peptide 1) bezeichnet wurde. Zip1 induziert die Expression SA-assoziierter PR (pathogenesis-related)-Gene und aktiviert, ähnlich wie SA, apoplastische PLCPs. In vitro-Studien konnten zeigen, dass zwei der zuvor identifizierten PLCPs, CP1 und CP2, immunogene Peptidsignale aus dem Zip1-Vorläuferprotein (PROZIP1) proteolytisch freisetzen. Weiterhin bestätigen RNAseq-basierte Transkriptomanalysen, dass Zip1- und SA-behandelte Maisblätter einen Großteil ähnlicher Immunantworten auf Transkriptebene teilen, weshalb bei Zip1 von einem SA-analogen Signalpeptid gesprochen werden kann. Bemerkenswerterweise und mit den RNAseq-Daten einhergehend konnte außerdem gezeigt werden, dass Zip1 die de novo Synthese von SA induziert. Darauf aufbauend erhöht Zip1 die Suszeptibilität von Mais gegenüber dem nekrotrophen Pathogen Botrytis cinerea. Mit der Identifizierung von Zip1, einem endogenen Maispeptid, welches von SA-ativierten PLCPs freigesetzt wird, ist das erste pflanzliche Peptidsignal charakteriseriert worden, das an der SA-abhänigen Immunantwort und dessen sequentiellen Amplifikation beteiligt ist