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

Leadership for health improvement: implementation and evaluation

Purpose The purpose of this paper is to present a co-authored reflection on the health improvement leadership development programme and the key evaluation messages derived from piloting in an English National Health Service region. It highlights the specific attributes of this approach to health improvement leadership development and clarifies health improvement development issues. Design/methodology/approach Appreciative inquiry and soft systems methodology are combined in an evaluation approach designed to capture individual as well as organisation learning and how it impacts on leadership in specific contexts. Findings The evaluation exposes the health improvement leadership needs of a multi-organisation cohort, offers some explanations for successful achievement of learning needs while also exposing of the challenges and paradoxes faced in this endeavour. Originality/value There are limited reported templates of how to develop leadership for health improvement. This paper details a whole systems approach, acknowledging the impact of context on leadership and an approach to evaluating such complex initiatives

Prevalence and determinants of hepatitis C virus infection among female drug injecting sex workers in Glasgow

Background: Few studies of the prevalence of hepatitis C virus (HCV) infection have focussed on women who work as street sex workers to finance their drug use. Methods: The investigators report the survey findings of such a population in Glasgow. All women attending the health and social care drop-in centre, situated in Glasgow's "Red Light Area", during a four-week period in 1999 were invited to participate in a survey involving theprovision of a saliva sample for anonymous HCV testing and the self-completion of a questionnaire seeking demographic, sexual and injecting practice data. Of the 223 women who attended, 51% agreed to participate. Of the 98 women who provided a sufficient saliva sample, 64% (95% CI: 54%-74%) tested HCV antibody positive; 98% of those who tested positive had ever injected drugs. Adjusting for the 85% sensitivity of the saliva test, the HCV antibody prevalence among IDU sex workers sampled was 81%; a rate which is considerably higher than those recorded, contemporaneously, among Glasgow IDUs generally. Two factors were independently associated with HCV antibody positivity in saliva: ever shared needles and syringes (adjusted OR 5.7, 95% CI 2-16) and number of times imprisoned (adjusted OR 7.3, 95% CI 1.4-39, for more than five times compared to zero times). Women who engage in street sex work to finance their drug habit are a particularly desperate, chaotic and vulnerable population. This study demonstrates that their HCV infection risk may be greater than that for other IDUs. Those responsible for designing interventions to prevent HCV infection among IDUs should consider the special needs of this group

2003, UMaine News Press Releases

This is a catalog of press releases put out by the University of Maine Division of Marketing and Communications between January 21, 2003 and September 15, 2003

Cdc5 influences phosphorylation of Net1 and disassembly of the RENT complex

BACKGROUND: In S. cerevisiae, the mitotic exit network (MEN) proteins, including the Polo-like protein kinase Cdc5 and the protein phosphatase Cdc14, are required for exit from mitosis. In pre-anaphase cells, Cdc14 is sequestered to the nucleolus by Net1 as a part of the RENT complex. When cells are primed to exit mitosis, the RENT complex is disassembled and Cdc14 is released from the nucleolus. RESULTS: Here, we show that Cdc5 is necessary to free nucleolar Cdc14 in late mitosis, that elevated Cdc5 activity provokes ectopic release of Cdc14 in pre-anaphase cells, and that the phosphorylation state of Net1 is regulated by Cdc5 during anaphase. Furthermore, recombinant Cdc5 and Xenopus Polo-like kinase can disassemble the RENT complex in vitro by phosphorylating Net1 and thereby reducing its affinity for Cdc14. Surprisingly, although RENT complexes containing Net1 mutants (Net1(7m) and Net1(19m') lacking sites phosphorylated by Cdc5 in vitro are refractory to disassembly by Polo-like kinases in vitro, net1(7m) and net1(19m') cells grow normally and exhibit only minor defects in releasing Cdc14 during anaphase. However, net1(19m') cells exhibit a synergistic growth defect when combined with mutations in CDC5 or DBF2 (another MEN gene). CONCLUSIONS: We propose that although Cdc5 potentially disassembles RENT by directly phosphorylating Net1, Cdc5 mediates exit from mitosis primarily by phosphorylating other targets. Our study suggests that Cdc5/Polo is unusually promiscuous and highlights the need to validate Cdc5/Polo in vitro phosphorylation sites by direct in vivo mapping experiments

A Scenario-Based Methodology for Exploring Risks:Children and Programmable IoT

In this paper we report on research exploring the privacy, security and safety implications of children being able to program Internet of Things devices. We present our methodology for understanding the contexts in which children may wish to use programmable IoT, identifying risks that emerge in such contexts, and creating a set of questions that might guide design of such technologies so that they are safe for child users. We evaluate the success of the methodology, discuss the limitations of the approach, and describe future work

Formation and degradation of chaotic terrain in the Galaxias regions of Mars: implications for near-surface storage of ice

Galaxias Chaos is a region of low plateaus separated by narrow fractures – a chaotic terrain. Galaxias Mensae and Galaxias Colles are characterised by mesa and knobby terrains of individual landforms, or small assemblages, separated by plains. Galaxias Chaos has been attributed to ground disturbance due to sublimation in shallow subsurface ice-rich deposits, Galaxias Mensae and Galaxias Colles to sublimation and degradation of icy surface materials, without production of chaotic terrain. Liquid water has not been regarded as a product of the degradation of these icy terrains. This paper asks two research questions: (1) what was the total extent of the different modes of landscape degradation, especially chaotic terrain, involved in producing the present landscapes of Galaxias Chaos and Galaxias Mensae–Colles; (2) can the generation of liquid water as a product of landscape degradation be ruled-out? Using a morphological-statistical approach, including power spectrum analysis of relief, our observations and analyses show that present mesa-knobby terrains of Galaxias Mensae–Colles evolved from a landscape that had the same directional pattern and relief as presently found in Galaxias Chaos. This terrain extended across ∼440,000 km2 but ∼22,000 km3 (average thickness, 77 m) have been lost across ∼285,000 km2. This represents a significant loss of ice-bearing deposits. Moreover, this surface degradation was spatially partitioned by landforms associated with elevated ground heating and the transmission of a fluid in the shallow subsurface towards a distal channel. In answer to research question 2, it cannot be determined definitively if the fluid involved was groundwater, generated by the thermal destabilisation of the icy deposits, or low viscosity lava. However, it is likely that the degradation of Galaxias Mensae–Colles was not a consequence of sublimation alone. These findings underscore the significance of cryo-volcanic interactions in the cycling of water between the Martian surface and the atmosphere

Unexpected Consequences: Women’s experiences of a self-hypnosis intervention to help with pain relief during labour.

Background Self-hypnosis is becoming increasingly popular as a means of labour pain management. Previous studies have produced mixed results. There are very few data on women’s views and experiences of using hypnosis in this context. As part of a randomized controlled trial of self-hypnosis for intra-partum pain relief (the SHIP Trial) we conducted qualitative interviews with women randomized to the intervention arm to explore their views and experiences of using self-hypnosis during labour and birth. Methods Participants were randomly selected from the intervention arm of the study, which consisted of two antenatal self-hypnosis training sessions and a supporting CD that women were encouraged to listen to daily from 32 weeks gestation until the birth of their baby. Those who consented were interviewed in their own homes 8-12 weeks after birth. Following transcription, the interviews were analysed iteratively and emerging concepts were discussed amongst the authors to generate organizing themes. These were then used to develop a principal organizing metaphor or global theme, in a process known as thematic networks analysis. Results Of the 343 women in the intervention group, 48 were invited to interview, and 16 were interviewed over a 12 month period from February 2012 to January 2013. Coding of the data and subsequent analysis revealed a global theme of ‘unexpected consequences’, supported by 5 organising themes, ‘calmness in a climate of fear’, ‘from sceptic to believer’, ‘finding my space’, ‘delays and disappointments’ and ‘personal preferences’. Most respondents reported positive experiences of self-hypnosis and highlighted feelings of calmness, confidence and empowerment. They found the intervention to be beneficial and used a range of novel strategies to personalize their self-hypnosis practice. Occasionally women reported feeling frustrated or disappointed when their relaxed state was misinterpreted by midwives on admission or when their labour and birth experiences did not match their expectations. Conclusion The women in this study generally appreciated antenatal self-hypnosis training and found it to be beneficial during labour and birth. The state of focused relaxation experienced by women using the technique needs to be recognized by providers if the intervention is to be implemented into the maternity service