Mass Spectrometry-based Methods for Phosphorylation Site Mapping of Hyperphosphorylated Proteins Applied to Net1, a Regulator of Exit from Mitosis in Yeast

Prior to anaphase in Saccharomyces cerevisiae, Cdc14 protein phosphatase is sequestered within the nucleolus and inhibited by Net1, a component of the RENT complex in budding yeast. During anaphase the RENT complex disassembles, allowing Cdc14 to migrate to the nucleus and cytoplasm where it catalyzes exit from mitosis. The mechanism of Cdc14 release appears to involve the polo-like kinase Cdc5, which is capable of promoting the dissociation of a recombinant Net1·Cdc14 complex in vitro by phosphorylation of Net1. We report here the phosphorylation site mapping of recombinant Net1 (Net1N) and a mutant Net1N allele (Net1N-19m) with 19 serines or threonines mutated to alanine. A variety of chromatographic and mass spectrometric-based strategies were used, including immobilized metal-affinity chromatography, alkaline phosphatase treatment, matrix-assisted laser-desorption post-source decay, and a multidimensional electrospray mass spectrometry-based approach. No one approach was able to identify all phosphopeptides in the tryptic digests of these proteins. Most notably, the presence of a basic residue near the phosphorylated residue significantly hampered the ability of alkaline phosphatase to hydrolyze the phosphate moiety. A major goal of research in proteomics is to identify all proteins and their interactions and post-translational modification states. The failure of any single method to identify all sites in highly phosphorylated Net1N, however, raises significant concerns about how feasible it is to map phosphorylation sites throughout the proteome using existing technologies

The role of research in higher education: implications and challenges for an active future contribution to human and social development

Mankind is on the brink of a tragic era, in which the anarchic forces of the market and the incessant pressures upon natural resources on the verge of exhaustion will push sovereign states to increasingly dangerous rivalries. What will the role of research in higher education be, in response to the challenge of an active future contribution to human and social development? A good balance must be reached in the basic functions of research in order to avoid governance risks. While focusing only on the transformative function of research may pose dangers to the human dimension and development, unilateral concentration on responsible development aspects may generate reactive approaches and delay economic benefits. Disregarding the inclusiveness function may lead to slower development and even isolationism, and focusing on short-term issues is not good for long-term goals and future generations. We must reinforce research networks between ‘Southern’ and ‘Northern’, rich and poor, and developed and developing countries and institutions in order to bridge the gap between knowledge consumers and producers. This paper reviews aspects such as development, globalization and the inequality of nations; constraints and choices of the orthodox views of research; rethinking research and higher education to contribute to a better future; and knowledge integration for effective action.Peer Reviewe

Observable implications of geometrical and dynamical aspects of freeze-out in heavy ion collisions

Using an analytical parameterization of hadronic freeze-out in relativistic heavy ion collisions, we present a detailed study of the connections between features of the freeze-out configuration and physical observables. We focus especially on anisotropic freeze-out configurations (expected in general for collisions at finite impact parameter), azimuthally-sensitive HBT interferometry, and final-state interactions between non-identical particles. Model calculations are compared with data taken in the first year of running at RHIC; while not perfect, good agreement is found, raising the hope that a consistent understanding of the full freeze-out scenario at RHIC is possible, an important first step towards understanding the physics of the system prior to freeze-out.Comment: 36 pages, 56 figures, 2 tables; version accepted for publication in PRC: some figures, references and discussion added; now also discusses classical versus quantum statistic

Femtoscopy in Relativistic Heavy Ion Collisions: Two Decades of Progress

Analyses of two-particle correlations have provided the chief means for determining spatio-temporal characteristics of relativistic heavy ion collisions. We discuss the theoretical formalism behind these studies and the experimental methods used in carrying them out. Recent results from RHIC are put into context in a systematic review of correlation measurements performed over the past two decades. The current understanding of these results is discussed in terms of model comparisons and overall trends.Comment: 49 pages, 16 figures; to appear in Annual Review of Nuclear and Particle Science; final version includes minor updates in text, a few references added, and two figures updated; Figures and numerical data tables available at http://www.physics.ohio-state.edu/~lisa/FemtoscopyReview2005

On the state dependency of fast feedback processes in (palaeo) climate sensitivity

Palaeo data have been frequently used to determine the equilibrium (Charney) climate sensitivity $S^a$, and - if slow feedback processes (e.g. land ice-albedo) are adequately taken into account - they indicate a similar range as estimates based on instrumental data and climate model results. Most studies implicitly assume the (fast) feedback processes to be independent of the background climate state, e.g., equally strong during warm and cold periods. Here we assess the dependency of the fast feedback processes on the background climate state using data of the last 800 kyr and a conceptual climate model for interpretation. Applying a new method to account for background state dependency, we find $S^a=0.61\pm0.06$ K(Wm$^{-2}$)$^{-1}$ using the latest LGM temperature reconstruction and significantly lower climate sensitivity during glacial climates. Due to uncertainties in reconstructing the LGM temperature anomaly, $S^a$ is estimated in the range $S^a=0.55-0.95$ K(Wm$^{-2}$)$^{-1}$.Comment: submitted to Geophysical Research Letter

Embedding the concept of ecosystems services:The utilisation of ecological knowledge in different policy venues

The concept of ecosystem services is increasingly being promoted by academics and policy makers as a means to protect ecological systems through more informed decision making. A basic premise of this approach is that strengthening the ecological knowledge base will significantly enhance ecosystem health through more sensitive decision making. However, the existing literature on knowledge utilisation, and many previous attempts to improve decision making through better knowledge integration, suggest that producing ‘more knowledge’ is only ever a necessary but insufficient condition for greater policy success. We begin this paper by reviewing what is already known about the relationship between ecological knowledge development and utilisation, before introducing a set of theme issue papers that examine—for the very first time—how this politically and scientifically salient relationship plays out across a number of vital policy venues such as land-use planning, policy-level impact assessment, and cost–benefit analysis. Following a detailed synthesis of the key findings of all the papers, this paper identifies and explores new research and policy challenges in this important and dynamic area of environmental governance

Effectiveness of the Malnutrition eLearning Course for Global Capacity Building in the Management of Malnutrition: Cross-Country Interrupted Time-Series Study.

BACKGROUND: Scaling up improved management of severe acute malnutrition has been identified as the nutrition intervention with the largest potential to reduce child mortality, but lack of operational capacity at all levels of the health system constrains scale-up. We therefore developed an interactive malnutrition eLearning course that is accessible at scale to build capacity of the health sector workforce to manage severely malnourished children according to the guidelines of the World Health Organization. OBJECTIVE: The aim of this study was to test whether the malnutrition eLearning course improves knowledge and skills of in-service and preservice health professionals in managing children with severe acute malnutrition and enables them to apply the gained knowledge and skills in patient care. METHODS: This 2-year prospective, longitudinal, cross-country, interrupted time-series study took place in Ghana, Guatemala, El Salvador, and Colombia between January 2015 and February 2017. A subset of 354 in-service health personnel from 12 hospitals and 2 Ministries of Health, 703 preservice trainees from 9 academic institutions, and 204 online users participated. Knowledge gained after training and retention over time was measured through pre- and postassessments comprising questions pertaining to screening, diagnosis, pathophysiology and treatment, and prevention of malnutrition. Comprehension, application, and integration of knowledge were tested. Changes in perception, confidence, and clinical practice were assessed through questionnaires and interviews. RESULTS: Before the course, awareness of the World Health Organization guidelines was 36.73% (389/1059) overall, and 26.3% (94/358) among in-service professionals. The mean score gain in knowledge after access to the course in 606 participants who had pre- and postassessment data was 11.8 (95% CI 10.8-12.9; P<.001)-a relative increase of 41.5%. The proportion of participants who achieved a score above the pass mark posttraining was 58.7% (356/606), compared with 18.2% (110/606) in pretraining. Of the in-service professionals, 85.9% (128/149) reported applying their knowledge by changing their clinical practice in screening, assessment, diagnosis, and management. This group demonstrated significantly increased retained knowledge 6 months after training (mean difference [SD] from preassessment of 12.1 [11.8]), retaining 65.8% (12.1/18.4) of gained knowledge from the training. Changes in the management of malnutrition were reported by trained participants, and institutional, operational, and policy changes were also found. CONCLUSIONS: The malnutrition eLearning course improved knowledge, understanding, and skills of health professionals in the diagnosis and management of children with severe acute malnutrition, and changes in clinical practice and confidence were reported following the completion of the course

Linking Community Participatory Research to Global Policymaking: Lessons Learned

The past two decades saw a proliferation of opportunities for the perspectives of people experiencing poverty and marginalisation to input into global policymaking spaces. So far these efforts have been contested, with attempts to embed participatory methodologies facilitating only limited consultation and falling into many pitfalls. While Participate was built on learning from previous attempts to influence global policy, we aimed to further understand participatory processes, and advance practical mechanisms for participation at every level of decision-making, from local to national and global. Participate emphasises the interactions between these levels and the importance of impacting multiple levels in order to create lasting, transformative change. This chapter describes what has been learnt about bringing the voices on the ground into global policymaking. We examine how and by whom the knowledge and evidence created were used, and the barriers and challenges to embedding this knowledge into global policy processes, through the following questions: • What have been the challenges to embedding participatory processes into global policy spaces? • What have these pathways of influence looked like, and what are some of the key lessons learned from these

Major flaws in conflict prevention policies towards Africa : the conceptual deficits of international actors’ approaches and how to overcome them

Current thinking on African conflicts suffers from misinterpretations oversimplification, lack of focus, lack of conceptual clarity, state-centrism and lack of vision). The paper analyses a variety of the dominant explanations of major international actors and donors, showing how these frequently do not distinguish with sufficient clarity between the ‘root causes’ of a conflict, its aggravating factors and its triggers. Specifically, a correct assessment of conflict prolonging (or sustaining) factors is of vital importance in Africa’s lingering confrontations. Broader approaches (e.g. “structural stability”) offer a better analytical framework than familiar one-dimensional explanations. Moreover, for explaining and dealing with violent conflicts a shift of attention from the nation-state towards the local and sub-regional level is needed.Aktuelle Analysen afrikanischer Gewaltkonflikte sind häufig voller Fehlinterpretationen (Mangel an Differenzierung, Genauigkeit und konzeptioneller Klarheit, Staatszentriertheit, fehlende mittelfristige Zielvorstellungen). Breitere Ansätze (z. B. das Modell der Strukturellen Stabilität) könnten die Grundlage für bessere Analyseraster und Politiken sein als eindimensionale Erklärungen. häufig differenzieren Erklärungsansätze nicht mit ausreichender Klarheit zwischen Ursachen, verschärfenden und auslösenden Faktoren. Insbesondere die richtige Einordnung konfliktverlängernder Faktoren ist in den jahrzehntelangen gewaltsamen Auseinandersetzungen in Afrika von zentraler Bedeutung. Das Diskussionspapier stellt die große Variationsbreite dominanter Erklärungsmuster der wichtigsten internationalen Geber und Akteure gegenüber und fordert einen Perspektivenwechsel zum Einbezug der lokalen und der subregionalen Ebene für die Erklärung und Bearbeitung gewaltsamer Konflikte

Targeted online liquid chromatography electron capture dissociation mass spectrometry for the localization of sites of in vivo phosphorylation in human Sprouty2

We demonstrate a strategy employing collision-induced dissociation for phosphopeptide discovery, followed by targeted electron capture dissociation (ECD) for site localization. The high mass accuracy and low background noise of the ECD mass spectra allow facile sequencing of coeluting isobaric phosphopeptides, with up to two isobaric phosphopeptides sequenced from a single mass spectrum. In contrast to the previously described neutral loss of dependent ECD method, targeted ECD allows analysis of both phosphotyrosine peptides and lower abundance phosphopeptides. The approach was applied to phosphorylation analysis of human Sprouty2, a regulator of receptor tyrosine kinase signaling. Fifteen sites of phosphorylation were identified, 11 of which are novel