Large scale localization of protein phosphorylation by use of electron capture dissociation mass spectrometry.

We used on-line electron capture dissociation (ECD) for the large scale identification and localization of sites of phosphorylation. Each FT-ICR ECD event was paired with a linear ion trap collision-induced dissociation (CID) event, allowing a direct comparison of the relative merits of ECD and CID for phosphopeptide identification and site localization. Linear ion trap CID was shown to be most efficient for phosphopeptide identification, whereas FT-ICR ECD was superior for localization of sites of phosphorylation. The combination of confident CID and ECD identification and confident CID and ECD localization is particularly valuable in cases where a phosphopeptide is identified just once within a phosphoproteomics experiment

SILAC-based phosphoproteomics reveals an inhibitory role of KSR1 in p53 transcriptional activity via modulation of DBC1

BACKGROUND We have previously identified kinase suppressor of ras-1 (KSR1) as a potential regulatory gene in breast cancer. KSR1, originally described as a novel protein kinase, has a role in activation of mitogen-activated protein kinases. Emerging evidence has shown that KSR1 may have dual functions as an active kinase as well as a scaffold facilitating multiprotein complex assembly. Although efforts have been made to study the role of KSR1 in certain tumour types, its involvement in breast cancer remains unknown. METHODS A quantitative mass spectrometry analysis using stable isotope labelling of amino acids in cell culture (SILAC) was implemented to identify KSR1-regulated phosphoproteins in breast cancer. In vitro luciferase assays, co-immunoprecipitation as well as western blotting experiments were performed to further study the function of KSR1 in breast cancer. RESULTS Of significance, proteomic analysis reveals that KSR1 overexpression decreases deleted in breast cancer-1 (DBC1) phosphorylation. Furthermore, we show that KSR1 decreases the transcriptional activity of p53 by reducing the phosphorylation of DBC1, which leads to a reduced interaction of DBC1 with sirtuin-1 (SIRT1); this in turn enables SIRT1 to deacetylate p53. CONCLUSION Our findings integrate KSR1 into a network involving DBC1 and SIRT1, which results in the regulation of p53 acetylation and its transcriptional activity

Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Serine⁶⁵

We have previously reported that the Parkinson's disease-associated kinase PINK1 (PTEN-induced putative kinase 1) is activated by mitochondrial depolarization and stimulates the Parkin E3 ligase by phosphorylating Ser(65) within its Ubl (ubiquitin-like) domain. Using phosphoproteomic analysis, we identified a novel ubiquitin phosphopeptide phosphorylated at Ser(65) that was enriched 14-fold in HEK (human embryonic kidney)-293 cells overexpressing wild-type PINK1 stimulated with the mitochondrial uncoupling agent CCCP (carbonyl cyanide m-chlorophenylhydrazone), to activate PINK1, compared with cells expressing kinase-inactive PINK1. Ser(65) in ubiquitin lies in a similar motif to Ser(65) in the Ubl domain of Parkin. Remarkably, PlNK1 directly phosphorylates Ser(65) of ubiquitin in vitro. We undertook a series of experiments that provide striking evidence that Ser(65)-phosphorylated ubiquitin (ubiquitin(Phospho-Ser65)) functions as a critical activator of Parkin. First, we demonstrate that a fragment of Parkin lacking the Ubl domain encompassing Ser(65) (Delta Ubl-Parkin) is robustly activated by ubiquitin(Phospho-Ser65), but not by non-phosphorylated ubiquitin. Secondly, we find that the isolated Parkin Ubl domain phosphorylated at Ser(65) (Ubl(phospho-Ser65)) can also activate Delta Ubl-Parkin similarly to ubiquitin(PhosPh-Ser65). Thirdly, we establish that ubiquitin(PhosPh-Ser65), but not non-phosphorylated ubiquitin or Ubl(PhosPh-Ser65) activates full-length wild-type Parkin as well as the non-phosphorylatable S65A Parkin mutant. Fourthly, we provide evidence that optimal activation of full-length Parkin E3 ligase is dependent on PINK1-mediated phosphorylation of both Parkin at Ser(65) and ubiquitin at Ser(65), since only mutation of both proteins at Ser(65) completely abolishes Parkin activation. In conclusion, the findings of the present study reveal that PINK1 controls Parkin E3 ligase activity not only by phosphorylating Parkin at Ser(65), but also by phosphorylating ubiquitin at Ser(65). We propose that phosphorylation of Parkin at Ser(65) serves to prime the E3 ligase enzyme for activation by ubiquitin(PhosPh-Ser65), suggesting that small molecules that mimic ubiquitin(PhosPh-Ser65) could hold promise as novel therapies for Parkinson's disease

A novel, non-canonical mechanism of regulation of MST3 (mammalian Sterile20-related kinase 3)

The canonical pathway of regulation of the germinal centre kinase (GCK) III subgroup member, mammalian Sterile20-related kinase 3 (MST3), involves a caspase-mediated cleavage between N-terminal catalytic and C-terminal regulatory domains with possible concurrent autophosphorylation of the activation loop MST3(Thr178-), induction of Ser-/Thr-protein kinase activity and nuclear localisation. We identified an alternative ‘non-canonical’ pathway of MST3 activation (regulated primarily through dephosphorylation) which may also be applicable to other GCKIII (and GCKVI) subgroup members. In the basal state, inactive MST3 co-immunoprecipitated with the Golgi protein, GOLGA2/gm130. Activation of MST3 by calyculin A (a protein Ser-/Thr- phosphatase 1/2A inhibitor) stimulated (auto)phosphorylation of MST3(Thr178-) in the catalytic domain with essentially simultaneous cis-autophosphorylation of MST3(Thr328-) in the regulatory domain, an event also requiring the MST3(341-376) sequence which acts as a putative docking domain. MST3(Thr178-) phosphorylation increased MST3 kinase activity but this activity was independent of MST3(Thr328-) phosphorylation. Interestingly, MST3(Thr328-) lies immediately C-terminal to a STRAD pseudokinase-like site recently identified as being involved in binding of GCKIII/GCKVI members to MO25 scaffolding proteins. MST3(Thr178- /Thr328-) phosphorylation was concurrent with dissociation of MST3 from GOLGA2/gm130 and association of MST3 with MO25, and MST3(Thr328-) phosphorylation was necessary for formation of the activated MST3-MO25 holocomplex

Takeaway Teaching - A design for redesign

This paper introduces a new teaching design called Takeaway Teaching. The design consists of pre-designed generic themes and activities that support the development of study strategies in higher education. These themes and activities are easily redesigned by the teacher and integrated into the academic curriculum. The purpose of the design is twofold. It supports the didactic reflection and development of the teacher and the development of the students’ study competence. This paper presents the theoretical framework of the design, based on a theoretical approach of metacognition and second-order teaching and learning, and an example of a redesign of one of the generic themes; Literature Search

The association between paternal diabetes mellitus and successful pregnancy—Examined in a nationwide population undergoing reproductive treatment

Background: About 15% of all pregnancies end in pregnancy loss. As most studies have focused on maternal factors little is known regarding the influence of paternal factors on the chance of successful pregnancy. Objectives: This cohort study aims to assess the chance of biochemical pregnancy, clinical pregnancy, and live-born children in couples where the male partner has diabetes mellitus (DM). Materials and methods: We performed a nationwide cohort study. Couples undergoing assisted reproductive technology treatment from 2006 to 2019 were included. The exposed cohorts comprised embryo transfers in couples with paternal type 1 DM (T1DM), type 2 DM (T2DM), or mixed type DM (TMDM). The unexposed cohort included embryo transfers in couples without paternal DM. Results: A total of 101,875 embryo transfers were included. Of these, 503 males had T1DM, 225 males had T2DM, 263 males had TMDM, and 100,884 did not have DM. For paternal T1DM, the adjusted OR for achieving a biochemical pregnancy, clinical pregnancy, and live-born child were 0.97 (95% CI 0.77–1.23), 1.08 (95% CI 0.65–1.79), and 0.75 (95% CI 0.49–1.14), respectively. For paternal T2DM, the adjusted OR for achieving a biochemical pregnancy, clinical pregnancy, and live-born child were 0.80 (95% CI 0.56;1.16), 0.67 (95% CI 0.32–1.41), and 1.03 (95% CI 0.48–2.20), respectively. For the paternal TMDM, the adjusted OR for achieving a biochemical pregnancy, clinical pregnancy and livebirth were 0.95 (95% CI 0.67–1.33), 1.31 (95% CI 0.56–2.92), and 1.19 (95% CI 0.59–2.38), respectively. Conclusion: Paternal DM was not associated with a statistically significant decreased chance of biochemical pregnancy, clinical pregnancy, or live birth.</p

Navigating in Higher Education – NiHE: Et blik fra studerende og undervisere på faglige, sociale og personlige perspektiver på undervisningen

Denne rapport er skrevet på baggrund af spørgeskemaundersøgelsen – Navigating in Higher Education (NiHE) – der rummer besvarelser fra 1410 bachelorstuderende og 283 undervisere fordelt på ni uddannelser fra Aarhus Universitet: Uddannelsesvidenskab, Historie, Nordisk sprog og litteratur, Informationsteknologi, Biologi, Fysik, Medicin, Odontologi og Folkesundhedsvidenskab. NiHE undersøgelsen er gennemført i efteråret 2015 og vinter 2016, og den har til formål at generere data til almen undervisningsudvikling og rummer derfor både faglige, sociale og personlige perspektiver på undervisning.

Om undervisning af første og anden orden

Artiklen beskriver og diskuterer undervisningsbegrebet, sådan som det tager sig ud med afsæt i Niklas Luhmanns sociologiske systemteori. Formålet med artiklen er at vise, hvorledes det systemteoretiske blik giver mulighed for at reflektere over nogle af de didaktiske usandsynligheder, som underviseren håndterer i dagligdagen, men som ikke skrives tydeligt frem i andre didaktiske positioner. Undervisningsbegrebet kobles til tre centrale didaktiske temaer: Første tema omhandler spørgsmålene om undervisningens hensigt. Andet tema omhandler iagttagelse af elevens læring. Tredje tema omhandler det, vi med afsæt i systemteorien vil kalde andenordensundervisning eller undervisning om undervisning og læring

Minor compositional alterations in faecal microbiota after five weeks and five months storage at room temperature on filter papers

The gut microbiota is recognized as having major impact in health and disease. Sample storage is an important aspect to obtain reliable results. Mostly recommended is immediate freezing, however, this is not always feasible. Faecal occult blood test (FOBT) papers are an appealing solution in such situations, and most studies find these to be applicable, showing no major changes within 7 days storage at room temperature (RT). As fieldwork often requires RT storage for longer periods, evaluation of this is warranted. We performed 16S rRNA gene sequencing of 19 paired faecal samples immediately frozen or kept five weeks and five months at RT on FOBT papers. Alpha-diversity evaluation revealed no effect of FOBT storage, and evaluation of beta-diversity showed that host explained 65% of community variation, while storage method explained 5%. Evaluation of community dispersion and the Firmicutes/Bacteroidetes ratio revealed a larger effect of storage time for fresh-frozen samples. Single taxa evaluation (order-to-genus level) showed significant alterations of four (of 37) genera after five weeks and five genera after five months. When comparing the two timepoints, alterations were only detectable for fresh-frozen samples. Our findings reveal that long term storage on FOBT papers is an applicable approach for microbiota research.</p