Saccharomyces cerevisiae Rad9 Acts as a Mec1 Adaptor to Allow Rad53 Activation

SummaryBackground: The DNA damage checkpoint is a protein kinase-based signaling system that detects and signals physical alterations in DNA. Despite having identified many components of this signaling cascade, the exact mechanisms by which checkpoint kinases are activated after DNA damage, as well as the role of the checkpoint mediators, remain poorly understood.Results: To elucidate the mechanisms that underlie the MEC1 and RAD9-dependent activation of Rad53, the Saccharomyces cerevisiae ortholog of Chk2, we mapped and characterized in vivo phosphorylation sites present on Rad53 after DNA damage by mass spectrometry. We find that Rad53 requires for its activation multisite phosphorylation on a number of typical and atypical Mec1 phosphorylation sites, thus confirming that Rad53 is a direct target of Mec1, the mammalian ATR homolog. Moreover, by using biochemical reconstitution experiments, we demonstrate that efficient and direct phosphorylation of Rad53 by Mec1 is only observed in the presence of purified Rad9, the archetypal checkpoint mediator. We find that the stimulatory activity of Rad9 requires a phospho- and FHA-dependent interaction with Rad53, which allows Rad53 to be recognized as a substrate for Mec1.Conclusions: Our results indicate that Rad9 acts as a bona fide signaling adaptor that enables Rad53 phosphorylation by Mec1. Given the high degree of conservation of checkpoint signaling in eukaryotes, we propose that one of the critical functions of checkpoint mediators such as MDC1, 53BP1, or Brca1 is to act as PIKK adaptors during the DNA damage response

Indexing multi-dimensional uncertain data with arbitrary probability density functions

Research Session 26: Spatial and Temporal DatabasesIn an "uncertain database", an object o is associated with a multi-dimensional probability density function (pdf), which describes the likelihood that o appears at each position in the data space. A fundamental operation is the "probabilistic range search" which, given a value p q and a rectangular area r q, retrieves the objects that appear in r q with probabilities at least p q. In this paper, we propose the U-tree, an access method designed to optimize both the I/O and CPU time of range retrieval on multi-dimensional imprecise data. The new structure is fully dynamic (i.e., objects can be incrementally inserted/deleted in any order), and does not place any constraints on the data pdfs. We verify the query and update efficiency of U-trees with extensive experiments.postprintThe 31st International Conference on Very Large Data Bases (VLDB 2005), Trondheim, Norway, 30 August-2 September 2005. In Proceedings of 31st VLDB, 2005, v. 3, p. 922-93

Increasing peptide identifications and decreasing search times for ETD spectra by pre-processing and calculation of parent precursor charge

<p>Abstract</p> <p>Background</p> <p>Electron Transfer Dissociation [ETD] can dissociate multiply charged precursor polypeptides, providing extensive peptide backbone cleavage. ETD spectra contain charge reduced precursor peaks, usually of high intensity, and whose pattern is dependent on its parent precursor charge. These charge reduced precursor peaks and associated neutral loss peaks should be removed before these spectra are searched for peptide identifications. ETD spectra can also contain ion-types other than c and z<b>˙</b>. Modifying search strategies to accommodate these ion-types may aid in increased peptide identifications. Additionally, if the precursor mass is measured using a lower resolution instrument such as a linear ion trap, the charge of the precursor is often not known, reducing sensitivity and increasing search times. We implemented algorithms to remove these precursor peaks, accommodate new ion-types in noise filtering routine in OMSSA and to estimate any unknown precursor charge, using Linear Discriminant Analysis [LDA].</p> <p>Results</p> <p>Spectral pre-processing to remove precursor peaks and their associated neutral losses prior to protein sequence library searches resulted in a 9.8% increase in peptide identifications at a 1% False Discovery Rate [FDR] compared to previous OMSSA filter. Modifications to the OMSSA noise filter to accommodate various ion-types resulted in a further 4.2% increase in peptide identifications at 1% FDR. Moreover, ETD spectra when searched with charge states obtained from the precursor charge determination algorithm is shown to be up to 3.5 times faster than the general range search method, with a minor 3.8% increase in sensitivity.</p> <p>Conclusion</p> <p>Overall, there is an 18.8% increase in peptide identifications at 1% FDR by incorporating the new precursor filter, noise filter and by using the charge determination algorithm, when compared to previous versions of OMSSA.</p

Modelling Canopy Flows over Complex Terrain

Recent studies of flow over forested hills have been motivated by a number of important applications including understanding CO22 and other gaseous fluxes over forests in complex terrain, predicting wind damage to trees, and modelling wind energy potential at forested sites. Current modelling studies have focussed almost exclusively on highly idealized, and usually fully forested, hills. Here, we present model results for a site on the Isle of Arran, Scotland with complex terrain and heterogeneous forest canopy. The model uses an explicit representation of the canopy and a 1.5-order turbulence closure for flow within and above the canopy. The validity of the closure scheme is assessed using turbulence data from a field experiment before comparing predictions of the full model with field observations. For near-neutral stability, the results compare well with the observations, showing that such a relatively simple canopy model can accurately reproduce the flow patterns observed over complex terrain and realistic, variable forest cover, while at the same time remaining computationally feasible for real case studies. The model allows closer examination of the flow separation observed over complex forested terrain. Comparisons with model simulations using a roughness length parametrization show significant differences, particularly with respect to flow separation, highlighting the need to explicitly model the forest canopy if detailed predictions of near-surface flow around forests are required

Influence of topography on tide propagation and amplification in semi-enclosed basins

An idealized model for tide propagation and amplification in semi-enclosed rectangular basins is presented, accounting for depth differences by a combination of longitudinal and lateral topographic steps. The basin geometry is formed by several adjacent compartments of identical width, each having either a uniform depth or two depths separated by a transverse topographic step. The problem is forced by an incoming Kelvin wave at the open end, while allowing waves to radiate outward. The solution in each compartment is written as the superposition of (semi)-analytical wave solutions in an infinite channel, individually satisfying the depth-averaged linear shallow water equations on the f plane, including bottom friction. A collocation technique is employed to satisfy continuity of elevation and flux across the longitudinal topographic steps between the compartments. The model results show that the tidal wave in shallow parts displays slower propagation, enhanced dissipation and amplified amplitudes. This reveals a resonance mechanism, occurring when\ud the length of the shallow end is roughly an odd multiple of the quarter Kelvin wavelength. Alternatively, for sufficiently wide basins, also Poincaré waves may become resonant. A transverse step implies different wavelengths of the incoming and reflected Kelvin wave, leading to increased amplitudes in shallow regions and a shift of amphidromic points in the direction of the deeper part. Including the shallow parts near the basin’s closed end (thus capturing the Kelvin resonance mechanism) is essential to reproduce semi-diurnal and diurnal\ud tide observations in the Gulf of California, the Adriatic Sea and the Persian Gulf

Energy- and flux-budget (EFB) turbulence closure model for the stably stratified flows. Part I: Steady-state, homogeneous regimes

We propose a new turbulence closure model based on the budget equations for the key second moments: turbulent kinetic and potential energies: TKE and TPE (comprising the turbulent total energy: TTE = TKE + TPE) and vertical turbulent fluxes of momentum and buoyancy (proportional to potential temperature). Besides the concept of TTE, we take into account the non-gradient correction to the traditional buoyancy flux formulation. The proposed model grants the existence of turbulence at any gradient Richardson number, Ri. Instead of its critical value separating - as usually assumed - the turbulent and the laminar regimes, it reveals a transition interval, 0.1< Ri <1, which separates two regimes of essentially different nature but both turbulent: strong turbulence at Ri<<1; and weak turbulence, capable of transporting momentum but much less efficient in transporting heat, at Ri>1. Predictions from this model are consistent with available data from atmospheric and lab experiments, direct numerical simulation (DNS) and large-eddy simulation (LES).Comment: 40 pages, 6 figures, Boundary-layer Meteorology, resubmitted, revised versio

Plugging a hole and lightening the burden: A process evaluation of a practice education team

Aim: To investigate the perceptions of clinical and senior managers about the role of Practice Educators employed in one acute hospital in the UK. Background: Producing nurses who are fit for practice, purpose and academic award is a key issue for nurse education partnership providers in the UK. Various new models for practice learning support structures and new roles within health care institutions have been established. To sustain funding and policy support for these models, there is a need for evaluation research. Design: A process evaluation methodology was employed to determine the current value of a practice education team and to provide information to guide future direction. Methods: Data were collected through semi-structured telephone interviews using a previously designed schedule. All senior nurse managers (N=5) and a purposive sample of clinical managers (n=13) who had personal experience of and perceptions about the role of practice educators provided the data. Interview notes were transcribed, coded and a thematic framework devised to present the results. Results: A number of key themes emerged including: qualities needed for being a successful practice educator; visibility and presence of practice educators; providing a link with the university; ‘plugging a hole’ in supporting learning needs; providing relief to practitioners in dealing with ‘the burden of students’; alleviating the ‘plight of students’; and effects on student attrition. Conclusions: Findings provided evidence for the continued funding of the practice educator role with improvements to be made in dealing with stakeholder expectations and outcomes. Relevance to clinical practice: In the UK, there still remain concerns about the fitness for practice of newly registered nurses, prompting a recent national consultation by the professional regulating body. Despite fiscal pressures, recommendations for further strengthening of all systems that will support the quality of practice learning may continue to sustain practice learning support roles

Ucma/GRP inhibits phosphate-induced vascular smooth muscle cell calcification via SMAD-dependent BMP signalling

Vascular calcification (VC) is the process of deposition of calcium phosphate crystals in the blood vessel wall, with a central role for vascular smooth muscle cells (VSMCs). VC is highly prevalent in chronic kidney disease (CKD) patients and thought, in part, to be induced by phosphate imbalance. The molecular mechanisms that regulate VC are not fully known. Here we propose a novel role for the mineralisation regulator Ucma/GRP (Upper zone of growth plate and Cartilage Matrix Associated protein/Gla Rich Protein) in phosphate-induced VSMC calcification. We show that Ucma/GRP is present in calcified atherosclerotic plaques and highly expressed in calcifying VSMCs in vitro. VSMCs from Ucma/GRP(-/-) mice showed increased mineralisation and expression of osteo/chondrogenic markers (BMP-2, Runx2, beta-catenin, p-SMAD1/5/8, ALP, OCN), and decreased expression of mineralisation inhibitor MGP, suggesting that Ucma/GRP is an inhibitor of mineralisation. Using BMP signalling inhibitor noggin and SMAD1/5/8 signalling inhibitor dorsomorphin we showed that Ucma/GRP is involved in inhibiting the BMP-2-SMAD1/5/8 osteo/chondrogenic signalling pathway in VSMCs treated with elevated phosphate concentrations. Additionally, we showed for the first time evidence of a direct interaction between Ucma/GRP and BMP-2. These results demonstrate an important role of Ucma/GRP in regulating osteo/chondrogenic differentiation and phosphate-induced mineralisation of VSMCs.NWO ZonMw [MKMD 40-42600-98-13007]; FCT [SFRH/BPD/70277/2010]info:eu-repo/semantics/publishedVersio