Structural and functional studies of the Dsc1 cell cycle transcription factor complex in fission yeast

The fission yeast DSC 1 (DNA synthesis control) transcription factor complex regulates cell cycle-specific periodic transcription of a group of genes at the G1-S phase transition during the mitotic cell cycle, by binding to MCB (Mlul cell cycle box) sequence elements common to their promoters. Included in this group are several genes whose functions are required for the onset and progression of S phase, such as cdc22+ (the large subunit of ribonucleotide reductase), cig2+ (the major S phase cyclin) and the DNA replication licensing factors cdc18+ and cdt1+. In concert with cyclin-dependent kinase activity, DSCl function is required for passage of START and entry into the mitotic cell division cycle. Similar gene expression programmes exist in both budding yeast and humans controlled by the SBF/MBF and E2F transcription factors, respectively. Fission yeast DSC1 comprises two related DNA-binding subunits, Res1p and Res2p, each bound to a single molecule of the regulatory Cdc10p protein. In addition, the Rep2p protein has a transcriptional activator function (replaced by Rep1p in the meiotic cycle). Knowledge about functional aspects of each of the DSC1 components has been greatly enhanced by genetic and biochemical studies. However, to date, these proteins remain poorly characterised at the atomic level, with little known about structure beyond their amino acid sequence. The aim of this study was to clone and bacterially express the individual DSC1 genes, to provide sufficient protein to carry out more detailed biophysical and functional studies. The cdc10+, res1+, res2+ and rep2+ genes, together with the putative meiotic subunit rep1+, were cloned and overexpressed in E. coli as N-terminal histidine-tagged fusion proteins. Inclusion of the His-tag facilitated purification of the proteins by affinity chromatography. Each recombinant protein (with the exception of His-Cdc10p) was shown to function in vivo-, ectopic expression of His-resl+, His-res2+, His-rep1+ or His-rep2+ rescued the cold-sensitive lethality of the fission yeast Deltares1 mutant strain. Bacterially expressed His-Res1p, His-Res2p and His-Rep2p were recovered in soluble form, whilst His-Rep1p and His-Cdc10p were detergent-solubilised from inclusion bodies. His Res2p was expressed and purified in yields sufficient to undertake biophysical analyses. Both His-Rep1p and His-Cdc10p were solubilised and purified from inclusion bodies in yields sufficient for structural studies, although initial biophysical data suggests that re-folding strategies will be required to obtain active preparations of these proteins. In electrophoretic mobility shift assay experiments, neither His-Res1p nor His-Res2p displayed detectable MCB-specific DNA-binding in vitro. Intriguingly, replacement of the His- tag with an N terminal GST-tag conferred detectable MCB-specific DNA-binding upon both proteins. These results suggest that efficient DNA-binding requires dimerisation, a property that, at least in vitro, is apparently not naturally intrinsic to either Resp protein. Taken together, the results presented in this study provide a significant basis with which to undertake future structural analyses of these proteins. The implications of these results for further in vitro studies are discussed

Using degraded music quality to encourage a health improving walking pace : BeatClearWalker

Meeting the target of 8000 steps/day, as recommended by many national governments and health authorities, can provide considerable physical and mental health benefits and is seen as a key target for reducing obesity levels and improving public health. However, to optimize the health benefits, walking should be performed at a “moderate” intensity. While there are numerous mobile fitness applications that monitor distance walked, none directly support walking at this cadence nor has there been any research into live feedback for walking cadence. We present a smartphone fitness application to help users learn how to walk at a moderate cadence and maintain that cadence. We apply real-time audio effects that diminish the audio quality of music when the target walking cadence is not being reached. This provides an immersive and intuitive application that can easily be integrated into everyday life as allows users to walk while listening to their own music and encourages eyes-free interaction. In this paper, we introduce our approach, design, initial lab evaluation and a controlled outdoor study. Results show that using music degradation decreases the number of below-cadence steps, that users felt they worked harder with our player and would use it while exercise walking

Using degraded music quality to encourage a health improving walking pace : BeatClearWalker

Meeting the target of 8000 steps/day, as recommended by many national governments and health authorities, can provide considerable physical and mental health benefits and is seen as a key target for reducing obesity levels and improving public health. However, to optimize the health benefits, walking should be performed at a "moderate" intensity. While there are numerous mobile fitness applications that monitor distance walked, none directly support walking at this cadence nor has there been any research into live feedback for walking cadence. We present a smartphone fitness application to help users learn how to walk at a moderate cadence and maintain that cadence. We apply real-time audio effects that diminish the audio quality of music when the target walking cadence is not being reached. This provides an immersive and intuitive application that can easily be integrated into everyday life as allows users to walk while listening to their own music and encourages eyes-free interaction. In this paper, we introduce our approach, design, initial lab evaluation and a controlled outdoor study. Results show that using music degradation decreases the number of below-cadence steps, that users felt they worked harder with our player and would use it while exercise walking

The Economic Impact of Regular Season Sporting Competitions : The Glasgow Old Firm Football Spectators as Sports Tourists

This paper considers the economic impact of the sports tourism expenditures associated with regular season sports competition. In particular the sports tourism characteristics of the supporters of two largest football clubs in Scotland – Celtic FC and Rangers FC – are investigated. Both of these clubs are based in Glasgow, but play matches (and draw supporters from) across and outside Scotland. The paper quantifies the direct, indirect and induced effects of this expenditure using a novel extension of a two-region Input Output (IO) table for Glasgow and the rest of Scotland. The IO system is used to calculate net additional economic activity, in terms of GDP and employment, from gross and displaced activity at different regional levels

Global Scale Variation in the Salinity Sensitivity of Riverine Macroinvertebrates: Eastern Australia, France, Israel and South Africa

Salinity is a key abiotic property of inland waters; it has a major influence on biotic communities and is affected by many natural and anthropogenic processes. Salinity of inland waters tends to increase with aridity, and biota of inland waters may have evolved greater salt tolerance in more arid regions. Here we compare the sensitivity of stream macroinvertebrate species to salinity from a relatively wet region in France (Lorraine and Brittany) to that in three relatively arid regions eastern Australia (Victoria, Queensland and Tasmania), South Africa (south-east of the Eastern Cape Province) and Israel using the identical experimental method in all locations. The species whose salinity tolerance was tested, were somewhat more salt tolerant in eastern Australia and South Africa than France, with those in Israel being intermediate. However, by far the greatest source of variation in species sensitivity was between taxonomic groups (Order and Class) and not between the regions. We used a bayesian statistical model to estimate the species sensitivity distributions (SSDs) for salinity in eastern Australia and France adjusting for the assemblages of species in these regions. The assemblage in France was slightly more salinity sensitive than that in eastern Australia. We therefore suggest that regional salinity sensitivity is therefore likely to depend most on the taxonomic composition of respective macroinvertebrate assemblages. On this basis it would be possible to screen rivers globally for risk from salinisation

Dimerization of cAMP phosphodiesterase-4 (PDE4) in living cells requires interfaces located in both the UCR1 and catalytic unit domains

PDE4 family cAMP phosphodiesterases play a pivotal role in determining compartmentalised cAMP signalling through targeted cAMP breakdown. Expressing the widely found PDE4D5 isoform, as both bait and prey in a yeast 2-hybrid system, we demonstrated interaction consistent with the notion that long PDE4 isoforms form dimers. Four potential dimerization sites were uncovered using a scanning peptide array approach, where a recombinant purified PDE4D5 fusion protein was used to probe a 25-mer library of overlapping peptides covering the entire PDE4D5 sequence. Key residues involved in PDE4D5 dimerization were defined using a site-directed mutagenesis programme directed by an alanine scanning peptide array approach. Critical residues stabilising PDE4D5 dimerization were defined within the regulatory UCR1 region found in long, but not short, PDE4 isoforms, namely the Arg173, Asn174 and Asn175 (DD1) cluster. Disruption of the DD1 cluster was not sufficient, in itself, to destabilise PDE4D5 homodimers. Instead, disruption of an additional interface, located on the PDE4 catalytic unit, was also required to convert PDE4D5 into a monomeric form. This second dimerization site on the conserved PDE4 catalytic unit is dependent upon a critical ion pair interaction. This involves Asp463 and Arg499 in PDE4D5, which interact in a trans fashion involving the two PDE4D5 molecules participating in the homodimer. PDE4 long isoforms adopt a dimeric state in living cells that is underpinned by two key contributory interactions, one involving the UCR modules and one involving an interface on the core catalytic domain. We propose that short forms do not adopt a dimeric configuration because, in the absence of the UCR1 module, residual engagement of the remaining core catalytic domain interface provides insufficient free energy to drive dimerization. The functioning of PDE4 long and short forms is thus poised to be inherently distinct due to this difference in quaternary structure

The Ursinus Weekly, March 6, 1970

Light show, Ben Hair Campus Chest events • USGA elects President: Novak, Emig meet press • Haas, Karpinski selected Woodrow Wilson scholars • Student Union to occupy Memorial Library building • Editorial: Student election, Ursinus style • Focus: John Fioravanti • Faculty self-portrait: Dr. Allan Lake Rice • Survey surveyed • Lantern in the limelight • Letters to the editor: Mr. Swarr; Dr. Helfferich; Sorority slander; Michener Forum • Perspectives: Drug forum • First semester Dean\u27s List • Kings and Queens crowned • Contemplations: Tom Rush • Matmen trim PMC for first win • Hoopmen sink Drexel • Badminton netgals prolong streak • Rams syndrome snaps Bearettes Boston-bound • 1970 Ursinus Festival of Artshttps://digitalcommons.ursinus.edu/weekly/1156/thumbnail.jp

A fuzzy levelised energy cost method for renewable energy technology assessment

Renewable energy project development is highly complex and success is by no means guaranteed. Decisions are often made with approximate or uncertain information yet the current methods employed by decision-makers do not necessarily accommodate this. Levelised energy costs (LEC) are one such commonly applied measure utilised within the energy industry to assess the viability of potential projects and inform policy. The research proposes a method for achieving this by enhancing the traditional discounting LEC measure with fuzzy set theory. Furthermore, the research develops the fuzzy LEC (F-LEC) methodology to incorporate the cost of financing a project from debt and equity sources. Applied to an example bioenergy project, the research demonstrates the benefit of incorporating fuzziness for project viability, optimal capital structure and key variable sensitivity analysis decision-making. The proposed method contributes by incorporating uncertain and approximate information to the widely utilised LEC measure and by being applicable to a wide range of energy project viability decisions