The properties of Msh2-Msh6 ATP binding mutants suggest a signal amplification mechanism in DNA mismatch repair.

DNA mismatch repair (MMR) corrects mispaired DNA bases and small insertion/deletion loops generated by DNA replication errors. After binding a mispair, the eukaryotic mispair recognition complex Msh2-Msh6 binds ATP in both of its nucleotide-binding sites, which induces a conformational change resulting in the formation of an Msh2-Msh6 sliding clamp that releases from the mispair and slides freely along the DNA. However, the roles that Msh2-Msh6 sliding clamps play in MMR remain poorly understood. Here, using Saccharomyces cerevisiae, we created Msh2 and Msh6 Walker A nucleotide-binding site mutants that have defects in ATP binding in one or both nucleotide-binding sites of the Msh2-Msh6 heterodimer. We found that these mutations cause a complete MMR defect in vivo The mutant Msh2-Msh6 complexes exhibited normal mispair recognition and were proficient at recruiting the MMR endonuclease Mlh1-Pms1 to mispaired DNA. At physiological (2.5 mm) ATP concentration, the mutant complexes displayed modest partial defects in supporting MMR in reconstituted Mlh1-Pms1-independent and Mlh1-Pms1-dependent MMR reactions in vitro and in activation of the Mlh1-Pms1 endonuclease and showed a more severe defect at low (0.1 mm) ATP concentration. In contrast, five of the mutants were completely defective and one was mostly defective for sliding clamp formation at high and low ATP concentrations. These findings suggest that mispair-dependent sliding clamp formation triggers binding of additional Msh2-Msh6 complexes and that further recruitment of additional downstream MMR proteins is required for signal amplification of mispair binding during MMR

Cellular interaction with novel biomaterials

The objective of this thesis is to report the behaviour of mammalian cells with biocompatible synthetic polymers with potential for applications to the human body. Composite hydrogel materials were tested as possible keratoprosthetic devices. It was found that surface topography is an important consideration, pores, channels and fibres exposed on the surface of the hydrogels tested can have significant effects on the extent of cell adheson and proliferation. It is recommended that the core component is fabricated out of one of the following to provide a non cell adhesive base; A8, A11, A13, A22, A23. The haptic periphery fabricated out of one of the following would provide a cell adhesive composite; A16, A30, A33, A37, A38, A42, A43, A44. The presence of vitronectin in the ocular tissue appears to lead to higher cell adhesion to the posterior surface of a contact lens when compared to the anterior surface. Group IV contact lenses adhere more cells than Group II contact lenses - this may indicate that more protein (including vitronectin) is able to adhere to the contact lens due to the Group IV contact lenses high water content and ionic hydrogel matrix. Artificial lung surfactant analogues were found to be non cytotoxic but also decreased cell proliferation when tested at higher concentrations. Poly(lysine ethyl ester adipamide) [PLETESA] had the most favourable response on cell proliferation and commercial styrene/maleic anhydride (pMA/STY sp2) the most pronounced inhibitory response. The mode of action that decreases cell proliferation appears to be through membrane destabilization. Tissue culture well plates coated with PLETESA allowed cells to adhere in a concentration dependent manner, multilaminar liposomes possibly of PLETESA were observed in solution in PLETESA coated wells. Polyhydroxybutryate (PHB) and polyhydroxyvalerate (PHV) blends that contained hydroxyapatite were found to be the most cell adhesive material of those materials tested. The blends that were most susceptible to degradation adhered the most cells in initial stages of degradation. The initial slight increase in cell adhesion may be due to the increased rugosity of the material. As the degradation continued the number of cells adhering to the samples decreased, this may indicate that the polarity was inhibitory to cell adhesion during the later stages of degradation

Imaging of the Stellar Population of IC10 with Laser Guide Star Adaptive Optics and the Hubble Space Telescope

We present adaptive optics (AO) images of the central starburst region of the dwarf irregular galaxy IC10. The Keck 2 telescope laser guide star was used to achieve near diffraction-limited performance at H and K' (Strehls of 18% and 32%, respectively). The images are centered on the putative Wolf-Rayet (W-R) object [MAC92]24. We combine our AO images with F814W data from HST. By comparing the K' vs. [F814W]-K' color-magnitude diagram (CMD) with theoretical isochrones, we find that the stellar population is best represented by at least two bursts of star formation, one ~ 10 Myr ago and one much older (150-500 Myr). Young, blue stars are concentrated in the vicinity of [MAC92]24. This population represents an OB association with a half-light radius of about 3 pc. We resolve the W-R object [MAC92]24 into at least six blue stars. Four of these components have near-IR colors and luminosities that make them robust WN star candidates. By matching the location of C-stars in the CMD with those in the SMC we derive a distance modulus for IC10 of about 24.5 mag. and a foreground reddening of E(B-V) = 0.95. We find a more precise distance by locating the tip of the giant branch in the F814W, H, and K' luminosity functions. We find a weighted mean distance modulus of 24.48 +/- 0.08. The systematic error in this measurement, due to a possible difference in the properties of the RGB populations in IC10 and the SMC, is +/- 0.16 mag.Comment: 13 pages, 13 figures, ApJ in pres

Variable Drive Vehicle

The versatility of current rovers and exploratory vehicles is limited by a single drive system. The Variable Drive Vehicle (VDV) employs a actuated systemcapable of switching between wheeled and tracked drive modes. This allows the vehicle to travel quickly and efficiently over smooth terrain and to traverse more arduous terrain by switching between these two systems. The small scale prototype built over the course of this project is equipped with two modular wheel driven track units to demonstrate the viability of the system. Electric linear actuators and servo motors allow for simple control and a smooth transition between each drive system. These devices allow the modular tracks to be rotated out from under the wheels, and stowed on the vehicle when not in use. Finite element analysis ensured that the VDV’s switchingmechanism maintains safe loading at its most critical points during a drive system transition. The VDV was tested on smooth concrete to determine its maximum wheel speed, track speed, and how fast the drive system could be switched. Experiments yielded a top speed of 11.5 mph in the wheel mode, 0.8mph in the track mode, and a switching time of 6.4 seconds. The vehicle’smaximumobstacle clearance, 1 inch in track mode and 2 inches in wheel mode, and slope, 5 degrees in track mode and 22 degrees in wheel mode, fell short of expected values. These shortcomings resulted from a poor frictional power transfer when attempting to power the tracks using the wheels. However, this prototype provides a proof of concept for a variable drive system successfully incorporating two drive systems, and future improvements may yield a promising platformfor future robotics research

Testing models of inflation with CMB non-gaussianity

Two different predictions for the primordial curvature fluctuation bispectrum are compared through their effects on the Cosmic Microwave Background temperature fluctuations. The first has a local form described by a single parameter f_{NL}. The second is based on a prediction from the warm inflationary scenario, with a different dependence on wavenumber and a parameter f_{WI}. New expressions are obtained for the angular bispectra of the temperature fluctuations and for the estimators used to determine $f_{NL}$ and f_{WI}. The standard deviation of the estimators in an ideal experiment is roughly 5 times larger for f_{WI} than for f_{NL}. Using 3 year WMAP data gives limits -375<f_{WI}<36.8, but there is a possibility of detecting a signal for f_{WI} from the Planck satellite.Comment: 13 pages, 5 figures in ReVTe

Structure and functional motifs of GCR1, the only plant protein with a GPCR fold?

Whether GPCRs exist in plants is a fundamental biological question. Interest in deorphanizing new G protein coupled receptors (GPCRs), arises because of their importance in signaling. Within plants, this is controversial as genome analysis has identified 56 putative GPCRs, including GCR1 which is reportedly a remote homologue to class A, B and E GPCRs. Of these, GCR2, is not a GPCR; more recently it has been proposed that none are, not even GCR1. We have addressed this disparity between genome analysis and biological evidence through a structural bioinformatics study, involving fold recognition methods, from which only GCR1 emerges as a strong candidate. To further probe GCR1, we have developed a novel helix alignment method, which has been benchmarked against the the class A – class B - class F GPCR alignments. In addition, we have presented a mutually consistent set of alignments of GCR1 homologues to class A, class B and class F GPCRs, and shown that GCR1 is closer to class A and /or class B GPCRs than class A, class B or class F GPCRs are to each other. To further probe GCR1, we have aligned transmembrane helix 3 of GCR1 to each of the 6 GPCR classes. Variability comparisons provide additional evidence that GCR1 homologues have the GPCR fold. From the alignments and a GCR1 comparative model we have identified motifs that are common to GCR1, class A, B and E GPCRs. We discuss the possibilities that emerge from this controversial evidence that GCR1 has a GPCR fol

The Millennium Galaxy Catalogue: The connection between close pairs and asymmetry; implications for the galaxy merger rate

We compare the use of galaxy asymmetry and pair proximity for measuring galaxy merger fractions and rates for a volume limited sample of 3184 galaxies with -21 < M(B) -5 log h < -18 mag. and 0.010 < z < 0.123 drawn from the Millennium Galaxy Catalogue. Our findings are that: (i) Galaxies in close pairs are generally more asymmetric than isolated galaxies and the degree of asymmetry increases for closer pairs. At least 35% of close pairs (with projected separation of less than 20 h^{-1} kpc and velocity difference of less than 500 km s^{-1}) show significant asymmetry and are therefore likely to be physically bound. (ii) Among asymmetric galaxies, we find that at least 80% are either interacting systems or merger remnants. However, a significant fraction of galaxies initially identified as asymmetric are contaminated by nearby stars or are fragmented by the source extraction algorithm. Merger rates calculated via asymmetry indices need careful attention in order to remove the above sources of contamination, but are very reliable once this is carried out. (iii) Close pairs and asymmetries represent two complementary methods of measuring the merger rate. Galaxies in close pairs identify future mergers, occurring within the dynamical friction timescale, while asymmetries are sensitive to the immediate pre-merger phase and identify remnants. (iv) The merger fraction derived via the close pair fraction and asymmetries is about 2% for a merger rate of (5.2 +- 1.0) 10^{-4} h^3 Mpc^{-3} Gyr^{-1}. These results are marginally consistent with theoretical simulations (depending on the merger time-scale), but imply a flat evolution of the merger rate with redshift up to z ~1.Comment: 10 pages, 10 figures, emulateapj format. ApJ, accepte

Implementing health research through academic and clinical partnerships : a realistic evaluation of the Collaborations for Leadership in Applied Health Research and Care (CLAHRC)

Background: The English National Health Service has made a major investment in nine partnerships between higher education institutions and local health services called Collaborations for Leadership in Applied Health Research and Care (CLAHRC). They have been funded to increase capacity and capability to produce and implement research through sustained interactions between academics and health services. CLAHRCs provide a natural ‘test bed’ for exploring questions about research implementation within a partnership model of delivery. This protocol describes an externally funded evaluation that focuses on implementation mechanisms and processes within three CLAHRCs. It seeks to uncover what works, for whom, how, and in what circumstances. Design and methods: This study is a longitudinal three-phase, multi-method realistic evaluation, which deliberately aims to explore the boundaries around knowledge use in context. The evaluation funder wishes to see it conducted for the process of learning, not for judging performance. The study is underpinned by a conceptual framework that combines the Promoting Action on Research Implementation in Health Services and Knowledge to Action frameworks to reflect the complexities of implementation. Three participating CLARHCS will provide indepth comparative case studies of research implementation using multiple data collection methods including interviews, observation, documents, and publicly available data to test and refine hypotheses over four rounds of data collection. We will test the wider applicability of emerging findings with a wider community using an interpretative forum. Discussion: The idea that collaboration between academics and services might lead to more applicable health research that is actually used in practice is theoretically and intuitively appealing; however the evidence for it is limited. Our evaluation is designed to capture the processes and impacts of collaborative approaches for implementing research, and therefore should contribute to the evidence base about an increasingly popular (e.g., Mode two, integrated knowledge transfer, interactive research), but poorly understood approach to knowledge translation. Additionally we hope to develop approaches for evaluating implementation processes and impacts particularly with respect to integrated stakeholder involvement

Impact of Incremental Perfusion Loss on Oxygen Transport in a Capillary Network Mathematical Model.

OBJECTIVES: To quantify how incremental capillary perfusion loss, such as that seen in experimental models of sepsis, affects tissue oxygenation using a computation model of oxygen transport. METHODS: A computational model was applied to capillary networks with dimensions 84x168x342 (NI) and 70x157x268 (NII) μm, reconstructed in vivo from rat skeletal muscle. Functional capillary density (FCD) loss was applied incrementally up to ~40% and combined with high tissue oxygen consumption to simulate severe sepsis. RESULTS: A loss of ~40% FCD loss decreased median tissue PO2 to 22.9 and 20.1 mmHg in NI and NII compared to 28.1 and 27.5 mmHg under resting conditions. Increasing red blood cell supply rate (SR) to baseline levels returned tissue PO2 to within 5% of baseline. High consumption combined with a 40% FCD loss, resulted in tissue anoxia in both network volumes and median tissue PO2 of 11.5 and 8.9 mmHg in NI and NII respectively; median tissue PO2 was recovered to baseline levels by increasing total SR 3 - 4 fold. CONCLUSIONS: These results suggest a substantial increase in total SR is required in order to compensate for impaired oxygen delivery as a result of loss of capillary perfusion and increased oxygen consumption during sepsis. This article is protected by copyright. All rights reserved

Further discussion of a preliminary study of sleep quality in functional neurological disorders : a reply to Professor Kawada

We would like to thank Prof. Kawada for his interest in our manuscript (1), which describes a preliminary investigation of sleep quality in people with functional neurological disorders (FND). Below we respond to Prof. Kawada's comments