Regulation of inflammatory responses by mRNA decay

TTP ist ein mRNS-destabilisierendes Protein, welches in Zusammenarbeit mit anderen Faktoren den Abbau von Entzündungsmediatoren, ausgehend von der Binding zur Ziel-3´UTR, hervorruft. Die Regulierung von TTP´s mRNS-destabilisierender Aktivität ist komplex und involviert verschiedenste Aspekte wie Expression, zelluläre Lokalisation, posttranslationale Modifizierungen und Interaktion mit anderen trans-agierenden Faktoren, welche cis-regulatorische Elemente binden. Interessanterweise können TTP und das mRNS-stabilisierende Protein HuR ein überlappendes Repertoire an mRNS-Zielen während einer Entzündungsreaktion binden, darunter wichtige pro-inflammatorische Zytokine (Tnf, Cxcl1, Cxcl2, etc.). Daher wollten wir einen tieferen Einblick in die kooperative Wechselwirkung von TTP mit anderen trans-agierenden Faktoren und deren Einfluss auf die Entzündungsreaktion gewinnen. Durch Immunopräzipitation konnte gezeigt werden, dass TTP mit HuR mittels Ziel-RNS interagiert; eine Interaktion zwischen TTP und Ago2 konnte allerdings nicht festgestellt werden. Darüber hinaus konnte nach der Etablierung eines sequentiellen RNS-IP-Protokolls gezeigt werden, dass mehrere mRNS-Sequenzen (z.B. Tnf, Cxcl2) simultan von TTP und HuR gebunden werden. Die funktionalen Konsequenzen des gleichzeitigen Bindens an mRNS wurde mittels CRISPR/Cas-9 Genom-editierten RAW 264.7 Makrophagen, welche Inaktivierung von TTP, HuR oder beider Gene für TTP und HuR aufweisen, untersucht. Im Allgemeinen hat HuR einen geringen Einfluss auf die mRNS-Stabilität von inflammatorischen Mediatoren und nur Tnf mRNS konnte als eindeutiges Ziel, reguliert durch direkte Konkurrenz zwischen TTP und HuR, identifiziert werden. Weiters wurden RAW 264.7 Makrophagen, welche markiertes TTP vom endogenen Lokus exprimieren, gewonnen. Da TTP in diesen Zellen inhärent exprimiert wird erlaubt dieses System verschiedene Interaktions-Analysen, ohne die Risiken Artefakt-anfälliger Überexpression berücksichtigen zu müssen.TTP is an mRNA-destabilizing protein that regulates in concert with other factors decay of transcripts of inflammatory mediators upon binding to the target 3´UTR. However, the regulation of TTP´s induced mRNA-destabilizing activity is complex and involves many different facets including expression, cellular localization, posttranslational modifications and interaction with other trans-acting factors. Interestingly, within the inflammatory response TTP and the mRNA-stabilizing protein HuR can bind an overlapping repertoire of mRNA targets, including important pro-inflammatory cytokines (Tnf, Cxcl1, Cxcl2, etc.). We aimed to obtain deeper insight into the cooperative action of TTP with other factors, which help shaping the inflammatory response. Immunoprecipitation revealed that TTP interacts with HuR in presence of target RNA; however, no interaction of TTP with Ago2 could be detected. Moreover, upon establishment of a novel sequential RNA-IP protocol we could show that some mRNAs (e.g. Tnf, Cxcl2) were simultaneously bound by TTP and HuR. The functional consequences of concomitant binding were examined in CRISPR/Cas-9 genome edited RAW 264.7 macrophages, bearing inactivation of TTP, HuR or both TTP and HuR genes. In general HuR has a minor effect on mRNA stability of inflammatory mediators and only Tnf mRNA was unambiguously identified as target regulated by the direct competition of TTP and HuR. Further, RAW 264.7 cells, expressing tagged TTP from its genomic locus were attained. Since TTP is natively expressed in those cells, this system will allow various interaction analysis without risks associated with artifact-prone overexpression studies

The Legation of Johann Rudolf Schmid zum Schwarzenhorn (1649)

Quellenedition im Rahmen des FWF Projektes P30091-G28 zur Medialität diplomatischer Kommunikation, das den Schriftverkehr habsburgischer Gesandter in Konstantinopel mit dem Kaiserhof in Wien in der Mitte des 17. Jahrhunderts untersucht. Der erste Teil des Reisetagebuchs von Johann Georg Metzer (1650) als digitale Edition zur Verfügung. Der lange Zeit verschollen geglaubte und im Zuge der Editionsarbeit wiederentdeckte Bericht über seine Reise ins Osmanische Reich wurde im Rahmen des von Prof. Strohmeyer (Uni Salzburg) geleiteten FWF-Projekts in Kooperation mit dem Zentrum für Informationsmodellierung (Uni Graz) wissenschaftlich aufbereitet und ist jetzt als Betaversion online frei zugänglich.Digital edition in the context of the FWF project "Mediality of Diplomatic Communication" (P30091-G28). The first book of Johann Georg Metzger’s travelogue (1650) is now available as a digital edition. The itinerary about his journey to Constantinople was long believed to be lost and was rediscovered in the course of the project “The Mediality of Diplomatic Communication” led by Prof. Strohmeyer (funded by the Austrian Science Fund). The scholarly edition was created in cooperation with the Centre of Information Modelling (Uni Graz) and is available as a beta version

CEBPE-Mutant Specific Granule Deficiency Correlates With Aberrant Granule Organization and Substantial Proteome Alterations in Neutrophils

Specific granule deficiency (SGD) is a rare disorder characterized by abnormal neutrophils evidenced by reduced granules, absence of granule proteins, and atypical bilobed nuclei. Mutations in CCAAT/enhancer-binding protein-ε (CEBPE) are one molecular etiology of the disease. Although C/EBPε has been studied extensively, the impact of CEBPE mutations on neutrophil biology remains elusive. Here, we identified two SGD patients bearing a previously described heterozygous mutation (p.Val218Ala) in CEBPE. We took this rare opportunity to characterize SGD neutrophils in terms of granule distribution and protein content. Granules of patient neutrophils were clustered and polarized, suggesting that not only absence of specific granules but also defects affecting other granules contribute to the phenotype. Our analysis showed that remaining granules displayed mixed protein content and lacked several glycoepitopes. To further elucidate the impact of mutant CEBPE, we performed detailed proteomic analysis of SGD neutrophils. Beside an absence of several granule proteins in patient cells, we observed increased expression of members of the linker of nucleoskeleton and cytoskeleton complex (nesprin-2, vimentin, and lamin-B2), which control nuclear shape. This suggests that absence of these proteins in healthy individuals might be responsible for segmented shapes of neutrophilic nuclei. We further show that the heterozygous mutation p.Val218Ala in CEBPE causes SGD through prevention of nuclear localization of the protein product. In conclusion, we uncover that absence of nuclear C/EBPε impacts on spatiotemporal expression and subsequent distribution of several granule proteins and further on expression of proteins controlling nuclear shape

Diagnostic Accuracy of the Amsler Grid Test for Detecting Neovascular Age-Related Macular Degeneration: A Systematic Review and Meta-analysis

Importance: Patients with nonneovascular age-related macular degeneration (AMD) are encouraged to use the Amsler grid test for self-assessment to facilitate early diagnosis. The test is widely recommended, suggesting a belief that it signals worsening AMD, warranting its use in home monitoring. Objective: To systematically review studies of the diagnostic test accuracy of the Amsler grid in the diagnosis of neovascular AMD and to perform diagnostic test accuracy meta-analyses. Data Sources: A systematic literature search was conducted in 12 databases for relevant titles from database inception until May 7, 2022. Study Selection: Studies included those with groups defined as having (1) neovascular AMD and (2) either healthy eyes or eyes with nonneovascular AMD. The index test was the Amsler grid. The reference standard was ophthalmic examination. After removal of obviously irrelevant reports, 2 authors (J.B. and M.S.) independently screened the remaining references in full text for potential eligibility. Disagreements were resolved by a third author (Y.S.). Data Extraction and Synthesis: Two authors (J.B. and I.P.) independently extracted all data and evaluated quality and applicability of eligible studies using the Quality Assessment of Diagnostic Accuracy Studies 2. Disagreements were resolved by a third author (Y.S.). Main Outcomes and Measures: Sensitivity and specificity of the Amsler grid for detecting neovascular AMD with comparators being either healthy control participants or patients with nonneovascular AMD. Results: Of 523 records screened, 10 studies were included with a total of 1890 eyes (mean participant age ranging from 62 to 83 years). Sensitivity and specificity to diagnose neovascular AMD were 67% (95% CI, 51%-79%) and 99% (95% CI, 85%-100%), respectively, when comparators were healthy control participants and 71% (95% CI, 60%-80%) and 63% (95% CI, 49%-51%), respectively, when control participants were patients with nonneovascular AMD. Overall, potential sources of bias were low across studies. Conclusions and Relevance: Although the Amsler grid is easy and inexpensive to use for detection of metamorphopsia, its sensitivity may be at levels typically not recommended for monitoring. Coupling this lower sensitivity with only moderate specificity to identify neovascular AMD in a population at risk, these findings suggest that such patients typically should be encouraged to undergo ophthalmic examination regularly, regardless of any results of Amsler grid self-assessment

Tristetraprolin binding site atlas in the macrophage transcriptome reveals a switch for inflammation resolution

Abstract Precise regulation of mRNA decay is fundamental for robust yet not exaggerated inflammatory responses to pathogens. However, a global model integrating regulation and functional consequences of inflammation‐associated mRNA decay remains to be established. Using time‐resolved high‐resolution RNA binding analysis of the mRNA‐destabilizing protein tristetraprolin (TTP), an inflammation‐limiting factor, we qualitatively and quantitatively characterize TTP binding positions in the transcriptome of immunostimulated macrophages. We identify pervasive destabilizing and non‐destabilizing TTP binding, including a robust intronic binding, showing that TTP binding is not sufficient for mRNA destabilization. A low degree of flanking RNA structuredness distinguishes occupied from silent binding motifs. By functionally relating TTP binding sites to mRNA stability and levels, we identify a TTP‐controlled switch for the transition from inflammatory into the resolution phase of the macrophage immune response. Mapping of binding positions of the mRNA‐stabilizing protein HuR reveals little target and functional overlap with TTP, implying a limited co‐regulation of inflammatory mRNA decay by these proteins. Our study establishes a functionally annotated and navigable transcriptome‐wide atlas (http://ttp-atlas.univie.ac.at) of cis‐acting elements controlling mRNA decay in inflammation

Morphological profiling of human T and NK lymphocytes by high-content cell imaging

International audienceThe immunological synapse is a complex structure that decodes stimulatory signals into adapted lymphocyte responses. It is a unique window to monitor lymphocyte activity because of development of systematic quantitative approaches. Here we demonstrate the applicability of high-content imaging to human T and natural killer (NK) cells and develop a pipeline for unbiased analysis of high-definition morphological profiles. Our approach reveals how distinct facets of actin cytoskeleton remodeling shape immunological synapse architecture and affect lytic granule positioning. Morphological profiling of CD8+ T cells from immunodeficient individuals allows discrimination of the roles of the ARP2/3 subunit ARPC1B and the ARP2/3 activator Wiskott-Aldrich syndrome protein (WASP) in immunological synapse assembly. Single-cell analysis further identifies uncoupling of lytic granules and F-actin radial distribution in ARPC1B-deficient lymphocytes. Our study provides a foundation for development of morphological profiling as a scalable approach to monitor primary lymphocyte responsiveness and to identify complex aspects of lymphocyte micro-architecture

The role and activities of employment agencies

This study provides an overview of the importance and activities of employment agencies as well as their legal framework (WTO, ILO, EU) in the EU Member States and closely examines their role in selected countries, while focusing on temporary work agencies, a significantly growing market within the EU. Due to limited data, there is no clear-cut result on the agencies’ longer-term impact. However, the four identified market types (market driven, social dialogue based, legislator driven and emerging markets) are analysed through country cases regarding national regulations, the treatment of workers and everyday functioning of the agencies. It becomes evident that there is a wide diversity of the branch, which needs to be taken into account when reviewing EU Directive 2008/104/EC

Exudative Progression of Treatment-Naïve Non-Exudative Macular Neovascularization in Age-Related Macular Degeneration: A Systematic Review with Meta-Analyses

Purpose: To systematically review and report the rate of exudative progression over time in patients with non-exudative macular neovascularization (MNV) in age-related macular degeneration (AMD). Design: Systematic review with prevalence meta-analyses and individual participant meta-analysis. Methods: We searched 10 literature databases on March 26, 2023, for studies of consecutive patients with treatment-naïve non-exudative MNV in AMD. The primary outcome of interest was time from diagnosis to exudative progression. We conducted meta-analyses on the prevalence of exudative progression at 1 and 2 years. Where possible, we extracted individual participant data from studies and conducted an individual participant meta-analysis and explored the exudative progression using a time-to-event curve. Results: We identified 16 eligible studies with a total of 384 eyes with non-exudative MNV. Exudative progression had occurred in 20.9 % (95 % CI: 13.1-29.8) of eyes at 1 year and in 30.7% (95%CI: 21.8-40.4%) at 2 years. Similar results were observed in the individual participant meta-analysis, showing exudative progression in 18.9% (95%CI: 13.5-26.3%) of eyes at 1 year and 31.3% (95%CI: 24.2-40.0%) at 2 years. Risk factors for a fast exudative progression were the presence of subretinal lipid globules, large MNV areas, rapid MNV growth, growth in pigment epithelium detachment height and width, appearance of a branching pattern, and development of a hyporeflective halo around the MNV. Conclusions: Non-exudative MNVs in AMD are at high risk of exudative progression. Recognition of these lesions may allow for better individualized follow-up regimens in which closer monitoring may facilitate earlier diagnosis of exudative progression