Charakterisierung von Trix, einem neuen Guaninnukleotid-Austauschfaktor aus Dictyostelium discoideum

The goal of this project was the characterisation of a new guanine nucleotide exchange factor (GEF protein) for Rac GTPases in Dictyostelium discoideum. GEF proteins function as stimulating proteins of Rac GTPases which are of central importance in the regulation of actin-involving processes. Actin is a major component of the cytoskeleton, constituting a dynamic network of filamentous structures which provide the basis for elementary tasks of growth, differentiation, cell movement and cell division. The ameba D. discoideum is a haploid unicellular eukaryote. It serves as a model organism to study basic actin-involving processes in higher organisms. The D. discoideum genome has been fully sequenced recently and comprises a wide range of regulatory components for the actin cytoskeleton. The gene of a novel GEF protein with a coding sequence of 3597 bp was studied in detail. The gene was cloned and the sequence verified. The protein sequence of 1198 amino acids comprises three Calponin homology domains (CH) and one Dbl homology/Pleckstrin homology tandem domain which is a typical sequence feature of GEF proteins. On the basis of this domain architecture the protein was named 'Trix' in short for 'triple Calponin exchange factor'. The CH domains were classified as type 1 and type 1 CH domain (type 3 - type 3 - type 1). This resembles a novel combination of CH domains in a Rho GEF protein. A recombinant fragment carrying the three CH domains was shown to bind and bundle actin filaments which is not explained by the functions that have been described for CH domains so far. In vivo Trix is localised in the area of the actin-rich cell cortex as well as on the membranes of late endocytic vesicles. This suggests a regulatory role for Trix in the assembly or the disassembly of the actin coat that is associated with endocytic vesicles during the late stages of endocytosis. It was shown that Trix is mainly expressed during the vegetative stadium of D. discoideum which would be consistent with the increased endocytosis during growth. An association of Rac GTPases with late endocytic vesicles in D. discoideum was not described so far. Trix displayed no GDP/MANT-GDP exchange activity with the Rac GTPases Rac1a, RacC and RacE, hence the protein could not be allocated to a Rac GTPase signaling pathway. Some of the interactions between exchange factors and their respective Rac GTPases are of a very specific nature. Thus a potential activity of Trix might be directed against a Rac GTPase that has not yet been tested. The activation of Trix might also depend on further uncharacterised regulatory components. Finally, the results of the in vitro assays might differ from the in vivo situation as it has been demonstrated for other Rho GEFs. The Trix gene was disrupted in AX2 wildtype cells by a gene replacement approach. This allowed detailed characterisation of the protein's function in vivo. Trix is not an essential protein. There were no significant differences in the expression of important marker proteins, in phototaxis, chemotaxis, phagocytosis, cytokinesis, and growth of Trix- mutants. The mutant cells showed subtle changes in the organisation of the actin system as well as a slight delay during the developmental cycle. The most severe phenotypic deviation displayed by Trix- mutants consisted in a marked reduction of exocytosis. This provides further evidence for a regulatory function of the protein in exocytosis. The data suggest that Trix plays a role in the organisation of actin-involving processes in D. discoideum, especially in the regulation of the late endocytic cycle. Trix could not be allocated to a specific Rac GTPase signaling pathway and definite structural or dynamic tasks on the basis of the protein’s actin-binding and actin-bundling properties. The subtle phenotypic alterations in Trix- mutants might be due to a general redundancy in the functions of D. discoideum Rho GEF proteins

Reliability and Validity of a German Standardized Diagnostic Interview Module for ICD-11 Adjustment Disorder

The ICD-11 includes a new definition of adjustment disorder (AjD). The present study aimed to examine interrater reliability, internal consistency, and construct validity of a new diagnostic interview module to assess ICD-11 AjD. Data from two studies that used a standardized diagnostic interview assessment (i.e., DIA-X/M-CIDI and updated DIA-X-5) were used. For interrater reliability, agreement indicators (i.e., κ) were calculated using data from the DIA-X-5 test-retest study (N = 60). To examine internal consistency and construct validity, Cronbach's alpha values and the Kuder-Richardson correlation coefficient were computed along with confirmatory factor and latent class analyses (LCA), using data from the Zurich Adjustment Disorder Study (N = 330). Interrater reliability analyses found an adjusted kappa of 0.807 for the ICD-11 AjD diagnosis. Few items from the impairment criterion of the diagnostic algorithm performed poorly. The internal consistency was acceptable, Cronbach's αs = .43-.80; the lower-bound estimate resulted from the two-item preoccupation symptom pattern. However, both items were significantly associated, OR = 3.14, 95% CI [1.97, 4.99]. Regarding LCA results, a two-class model was favored. We found that 94.3% of all ICD-11 AjD cases belonged to Class 2, OR = 23.69, 95% CI [7.15, 79.54], which was associated with subjectively rated distress, OR = 2.18, 95% CI [1.57, 3.02], and the external measure of the Brief Symptom Inventory global severity index, OR = 2.18, 95% CI [1.57, 3.02]. Overall, the new AjD interview module provided a reliable, valid assessment of the ICD-11 diagnosis; confirmation by other studies is needed

Ticagrelor or Prasugrel in Patients with Acute Coronary Syndromes

Background The relative merits of ticagrelor as compared with prasugrel in patients with acute coronary syndromes for whom invasive evaluation is planned are uncertain. Methods In this multicenter, randomized, open-label trial, we randomly assigned patients who presented with acute coronary syndromes and for whom invasive evaluation was planned to receive either ticagrelor or prasugrel. The primary end point was the composite of death, myocardial infarction, or stroke at 1 year. A major secondary end point (the safety end point) was bleeding. Results A total of 4018 patients underwent randomization. A primary end-point event occurred in 184 of 2012 patients (9.3%) in the ticagrelor group and in 137 of 2006 patients (6.9%) in the prasugrel group (hazard ratio, 1.36; 95% confidence interval [CI], 1.09 to 1.70; P=0.006). The respective incidences of the individual components of the primary end point in the ticagrelor group and the prasugrel group were as follows: death, 4.5% and 3.7%; myocardial infarction, 4.8% and 3.0%; and stroke, 1.1% and 1.0%. Definite or probable stent thrombosis occurred in 1.3% of patients assigned to ticagrelor and 1.0% of patients assigned to prasugrel, and definite stent thrombosis occurred in 1.1% and 0.6%, respectively. Major bleeding (as defined by the Bleeding Academic Research Consortium scale) was observed in 5.4% of patients in the ticagrelor group and in 4.8% of patients in the prasugrel group (hazard ratio, 1.12; 95% CI, 0.83 to 1.51; P=0.46). Conclusions Among patients who presented with acute coronary syndromes with or without ST-segment elevation, the incidence of death, myocardial infarction, or stroke was significantly lower among those who received prasugrel than among those who received ticagrelor, and the incidence of major bleeding was not significantly different between the two groups