Analyzing mRNA-protein complexes using a yeast three-hybrid system.

Abstract RNA-protein interactions are essential for the proper execution and regulation of every step in the life of a eukaryotic mRNA. Here we describe a three-hybrid system in which RNA-protein interactions can be analyzed using simple phenotypic or enzymatic assays in Saccharomyces cerevisiae. The system can be used to detect or confirm an RNA-protein interaction, to analyze RNAprotein interactions genetically, and to discover new protein or RNA partners when only one is known. Multicomponent complexes containing more than one protein can be detected, identified, and analyzed. We describe the method and how to use it, and discuss applications that bear particularly on eukaryotic mRNAs.

Vergleich von Bodenbewegungen mit In-situ Messungen zur Überwachung von Deformationen an Staubauwerken in Thüringen

In dieser Studie wurden Satellitenbeobachtungen mittels Persistent Scatterer Interferometrie (PSI) an der Tal-sperre Leibis/Lichte im Thüringer Schiefergebirge mit In-situ-Lotdaten verglichen, um Deformationen an Stau-bauwerken zu überwachen. Es zeigte sich eine starke Korrelation zwischen den Satellitendaten des Boden Bewegungsdienstes Deutschland (BBD) und den In-situ-Messungen, insbesondere spiegelten die BBD-Daten den saisonalen Deformationsverlauf der Staumauer genau wider. Die Analyse ergab hohe R²-Werte, was auf eine starke lineare Beziehung hinweist. 90% der p-Werte lagen unter 0,05, was hohe statistische Signifikanz anzeigt. Diese Ergebnisse bestätigen das Potenzial der PSI-Satellitentechnologie als ergänzendes Instrument zur Überwachung von Staubauwerken, wobei eine sorgfältige Datenanalyse entscheidend für die Genauigkeit ist.In this study, satellite observations using Persistent Scatterer Interferometry (PSI) at the Leibis/Lichte dam in the Thuringian Slate Mountains were compared with in-situ plumb data to monitor deformations on dams. A strong correlation was observed between the German Ground Motion Service (BBD) satellite data and the in-situ measurements, especially reflecting the seasonal deformation pattern of the dam accurately. The analysis revealed high R² values, indicating a strong linear relationship. 90% of the p-values were below 0.05, demonstrating high statistical significance. These results confirm the potential of PSI satellite technology as a complementary tool for monitoring dams, with precise data analysis being crucial for accuracy

Analyzing mRyzingCF:3 complexes using a yeast

RR)C)C)C) interactions are essential for the proper execution and regulation of every step in the life of a eukaryoticmRka Here we describe a three-hybrid system in whichRchC[1FI[C interactions can be analyzed using simple phenotypic or enzymatic assays in Saccharomyces cerevisiae. The system can be used to detect or confirm anR1:F]FCE1: interaction, to analyzeRal protein interactions genetically, and to discover new protein or RC partners when only one is known. Multicomponent complexes containing more than one protein can be detected, identified, and analyzed. We describe the method and how to use it, and discuss applications that bear particularly on eukaryoticmRkar # 2002 Elsevier Science (USA). All rights reserved

A high-salt diet compromises antibacterial neutrophil responses through hormonal perturbation

The Western diet is rich in salt, which poses various health risks. A high-salt diet (HSD) can stimulate immunity through the nuclear factor of activated T cells 5 (Nfat5)-signaling pathway, especially in the skin, where sodium is stored. The kidney medulla also accumulates sodium to build an osmotic gradient for water conservation. Here, we studied the effect of an HSD on the immune defense against uropathogenic E. coli-induced pyelonephritis, the most common kidney infection. Unexpectedly, pyelonephritis was aggravated in mice on an HSD by two mechanisms. First, on an HSD, sodium must be excreted; therefore, the kidney used urea instead to build the osmotic gradient. However, in contrast to sodium, urea suppressed the antibacterial functionality of neutrophils, the principal immune effectors against pyelonephritis. Second, the body excretes sodium by lowering mineralocorticoid production via suppressing aldosterone synthase. This caused an accumulation of aldosterone precursors with glucocorticoid functionality, which abolished the diurnal adrenocorticotropic hormone-driven glucocorticoid rhythm and compromised neutrophil development and antibacterial functionality systemically. Consistently, under an HSD, systemic Listeria monocytogenes infection was also aggravated in a glucocorticoid-dependent manner. Glucocorticoids directly induced Nfat5 expression, but pharmacological normalization of renal Nfat5 expression failed to restore the antibacterial defense. Last, healthy humans consuming an HSD for 1 week showed hyperglucocorticoidism and impaired antibacterial neutrophil function. In summary, an HSD suppresses intrarenal neutrophils Nfat5-independently by altering the local microenvironment and systemically by glucocorticoid-mediated immunosuppression. These findings argue against high-salt consumption during bacterial infections

Pathogen-induced tissue-resident memory T(H)17 (T(RM)17) cells amplify autoimmune kidney disease

Although it is well established that microbial infections predispose to autoimmune diseases, the underlying mechanisms remain poorly understood. After infection, tissue-resident memory T (T-RM) cells persist in peripheral organs and provide immune protection against reinfection. However, whether T-RM cells participate in responses unrelated to the primary infection, such as autoimmune inflammation, is unknown. By using high-dimensional single-cell analysis, we identified CD4(+) T-RM cells with a T(H)17 signature (termed T(RM)17 cells) in kidneys of patients with ANCA-associated glomerulonephritis. Experimental models demonstrated that renal T(RM)17 cells were induced by pathogens infecting the kidney, such as Staphylococcus aureus, Candida albicans, and uropathogenic Escherichia coli, and persisted after the clearance of infections. Upon induction of experimental glomerulonephritis, these kidney T(RM)17 cells rapidly responded to local proinflammatory cytokines by producing IL-17A and thereby exacerbate renal pathology. Thus, our data show that pathogen-induced T(RM)17 cells have a previously unrecognized function in aggravating autoimmune disease.erman Research Foundation (DFG) SFB1192 SFB1286 SFBTR57 Deutsche Nierenstiftung Deutsche Gesellschaft fur Nephrologie Werner Otto Stiftung eMed Consortia "Fibromap" from the Federal Ministry of Education and Research Comision Nacional de Investigacion Cientifica y Tecnologica (CONICYT) AFB 170004 Conicyt/FONDEQUIP/EQM140016 Else Kroner-Fresenius Foundatio