The multi PAM2 protein Upa2 functions as novel core component of endosomal mRNA transport.

mRNA transport determines spatiotemporal protein expression. Transport units are higher-order ribonucleoprotein complexes containing cargo mRNAs, RNA-binding proteins and accessory proteins. Endosomal mRNA transport in fungal hyphae belongs to the best-studied translocation mechanisms. Although several factors are known, additional core components are missing. Here, we describe the 232 kDa protein Upa2 containing multiple PAM2 motifs (poly[A]-binding protein [Pab1]-associated motif 2) as a novel core component. Loss of Upa2 disturbs transport of cargo mRNAs and associated Pab1. Upa2 is present on almost all transport endosomes in an mRNA-dependent manner. Surprisingly, all four PAM2 motifs are dispensable for function during unipolar hyphal growth. Instead, Upa2 harbours a novel N-terminal effector domain as important functional determinant as well as a C-terminal GWW motif for specific endosomal localisation. In essence, Upa2 meets all the criteria of a novel core component of endosomal mRNA transport and appears to carry out crucial scaffolding functions.

Endosomal assembly and transport of heteromeric septin complexes promote septin cytoskeleton formation.

Septins are conserved cytoskeletal structures functioning in a variety of biological processes including cytokinesis and cell polarity. A wealth of information exists on the heterooligomeric architecture of septins and their subcellular localization at distinct sites. However, the precise mechanisms of their subcellular assembly and their intracellular transport are unknown. Here, we demonstrate that endosomal transport of septins along microtubules is crucial for formation of higher-order structures in the fungus Ustilago maydis Importantly, endosomal septin transport is dependent on each individual septin providing strong evidence that septin heteromeric complexes are assembled on endosomes. Furthermore, endosomal trafficking of all four septin mRNAs is required for endosomal localization of their translation products. Based on these results, we propose that local translation promotes the assembly of newly synthesized septins in heteromeric structures on the surface of endosomes. This is important for the long-distance transport of septins and the efficient formation of the septin cytoskeleton.

Compulsory teaching of occupational health: impact on attitude of medical students in Brazil and Germany.

PURPOSE: To assess medical students' attitude toward occupational health (OH) in Brazil and Germany before and after a compulsory OH course. METHODS: A questionnaire regarding the attitude toward OH was completed by medical students at the beginning of the OH teaching term at the Federal University of Paraná in Curitiba, Brazil (n = 79), and at both medical schools in Munich, Germany (n = 266), in 2009/2010. The change in the assessed qualities over the course period was measured repeating the survey at the end of the teaching term (n = 193; 56%). RESULTS: Overall, attitude toward OH was relatively low in both countries (mean 3.0 ± SD 1.2 on a scale from 1 = very low to 6 = very high) and students did not consider OH as a career option. However, Brazilian medical students rated the importance of OH statistically significantly higher than German medical students. Students in both countries considered their knowledge of OH low (2.2 ± 0.9). Not only self-assessed knowledge of OH (mean difference 1.5; 95% confidence interval 1.3-1.6) but also interest in OH (mean difference 0.2; 95% CI 0.06-0.4) improved statistically significantly over the teaching term. CONCLUSIONS: A negative attitude toward OH accompanied by little knowledge of OH seems to be a global problem. However, we have shown that this might be improved through adequate training in OH. The long-term effect still needs to be evaluated. Nevertheless, our results are promising and should encourage medical schools around the globe to include OH as a compulsory subject in medical training.

The germinal centre kinase Don3 is crucial for unconventional secretion of chitinase Cts1 in Ustilago maydis.

Unconventional secretion has emerged as an increasingly important cellular process in eukaryotic cells. The underlying translocation mechanisms are diverse and often little understood. We study unconventional secretion of chitinase Cts1 in the corn smut fungus Ustilago maydis. This protein participates in the cytokinesis of yeast cells. During budding it localizes to the septated fragmentation zone where it presumably functions in the degradation of remnant chitin to allow separation of mother and daughter cell. However, the mechanistic details of Cts1 export remain unclear. Here we investigated the mechanism of unconventional Cts1 secretion with a focus on cytokinesis. Cell-cycle inhibition experiments supported the hypothesis that Cts1 export is connected to cytokinesis. To substantiate this finding we analysed gene deletion mutants impaired in cell separation and discovered that strains defective in secondary septum formation were affected in Cts1 export. The germinal centre kinase Don3 had a particularly strong influence on unconventional secretion. Using a synthetic switch, we unambiguously verified an essential role of Don3 for cytokinesis-dependent Cts1 export via the fragmentation zone. Thus, we gained novel insights into the mechanism of unconventional secretion and discovered the first regulatory component of this process.

RNA Live Imaging in the Model Microorganism Ustilago maydis.

An essential feature of protein expression is the tight regulation of when and where a protein is translated from its cognate mRNA. This spatiotemporal expression is particularly important in guaranteeing the correct and efficient targeting of proteins to defined subcellular sites. In order to achieve local translation, mRNAs must be deposited at specific locations. A common mechanism is the active transport of mRNAs along the actin or microtubule cytoskeleton. To study such dynamic transport processes in vivo RNA live imaging is the method of choice. This method is based on the principle that defined binding sites for a heterologous RNA-binding protein (RBP) are inserted in the 3' UTR of target mRNAs. Coexpression of the RBP fused to a fluorescent protein enables mRNA detection in vivo using fluorescence microscopy techniques. In this chapter we describe the well-established method of studying microtubule-dependent mRNA transport in the eukaryotic model microorganism Ustilago maydis. The presented experimental design and the microscopic techniques are applicable to a broad range of other organisms.

Acute prognosis of critically ill patients with secondary peritonitis: the impact of the number of surgical revisions, and of the duration of surgical therapy.

BACKGROUND: Duration of surgical therapy and the number of surgical revisions performed to control the focus may be important prognostic variables. Association of such time-dependent therapies with survival, however, has not yet been studied. METHODS: We analyzed survival times of adult patients (n = 283) who were suffering from secondary peritonitis and associated organ failure. Cox-type additive hazard regression models were used to analyze associations of surgical variables with survival time. RESULTS: Seventy-two patients (25.4%) survived the period of excess mortality after intensive care unit admission. A total of 79.5% of the 283 patients required one or more surgical revisions. Besides the underlying disease and disease severity at intensive care unit admission, there was a nonlinear smoothed association between a poorer outcome and the duration of surgical therapy, and the number of surgical revisions. For the latter, hazard ratios increased sharply between 1 and 5 revisions, and remained largely constant later on. CONCLUSIONS: In critically ill patients with peritonitis, a long therapy and the necessity for a high number of reoperations is related inversely to acute survival.