Structural and Functional Dissection of Mif2p, a Conserved DNA-binding Kinetochore Protein

Mif2p is the budding-yeast orthologue of the mammalian centromere-binding protein CENP-C. We have mapped domains of Saccharomyces cerevisiae Mif2p and studied the phenotyptic consequences of their deletion. Using chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays, we have further shown that Mif2p binds in the CDEIII region of the budding-yeast centromere, probably in close spatial association with Ndc10p. Moreover, ChIP experiments show that Mif2p recruits to yeast kinetochores a substantial subset of inner and outer kinetochore proteins, but not the Ndc80 or Spc105 complexes. We have determined the crystal structure of the C-terminal, dimerization domain of Mif2p. It has a "cupin" fold, extremely similar both in polypeptide chain conformation and in dimer geometry to the dimerization domain of a bacterial transcription factor. The Mif2p dimer seems to be part of an enhanceosome-like structure that nucleates kinetochore assembly in budding yeast.Molecular and Cellular Biolog

Effects of Bulk and Surface Conductivity on the Performance of CdZnTe Pixel Detectors

We studied the effects of bulk and surface conductivity on the performance of high-resistivity CdZnTe (CZT) pixel detectors with Pt contacts. We emphasize the difference in mechanisms of the bulk and surface conductivity as indicated by their different temperature behaviors. In addition, the existence of a thin (10-100 A) oxide layer on the surface of CZT, formed during the fabrication process, affects both bulk and surface leakage currents. We demonstrate that the measured I-V dependencies of bulk current can be explained by considering the CZT detector as a metal-semiconductor-metal system with two back-to-back Schottky-barrier contacts. The high surface leakage current is apparently due to the presence of a low-resistivity surface layer that has characteristics which differ considerably from those of the bulk material. This surface layer has a profound effect on the charge collection efficiency in detectors with multi-contact geometry; some fraction of the electric field lines originated on the cathode intersects the surface areas between the pixel contacts where the charge produced by an ionizing particle gets trapped. To overcome this effect we place a grid of thin electrodes between the pixel contacts; when the grid is negatively biased, the strong electric field in the gaps between the pixels forces the electrons landing on the surface to move toward the contacts, preventing the charge loss. We have investigated these effects by using CZT pixel detectors indium bump bonded to a custom-built VLSI readout chip

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion events, making it difficult to quantitatively analyze the sequence of the molecular steps involved. We have developed an in vitro, two-color fluorescence assay to monitor kinetics of single virus particles fusing with a target bilayer on an essentially fluid support

Greenhouse gas balance over thaw-freeze cycles in discontinuous zone permafrost

Peat in the discontinuous permafrost zone contains a globally significant reservoir of carbon that has undergone multiple permafrost-thaw cycles since the end of the mid-Holocene (~3700 years before present). Periods of thaw increase C decomposition rates which leads to the release of CO2 and CH4 to the atmosphere creating potential climate feedback. To determine the magnitude and direction of such feedback, we measured CO2 and CH4 emissions and modeled C accumulation rates and radiative fluxes from measurements of two radioactive tracers with differing lifetimes to describe the C balance of the peatland over multiple permafrost-thaw cycles since the initiation of permafrost at the site. At thaw features, the balance between increased primary production and higher CH4 emission stimulated by warmer temperatures and wetter conditions favors C sequestration and enhanced peat accumulation. Flux measurements suggest that frozen plateaus may intermittently (order of years to decades) act as CO2 sources depending on temperature and net ecosystem respiration rates, but modeling results suggest that—despite brief periods of net C loss to the atmosphere at the initiation of thaw—integrated over millennia, these sites have acted as net C sinks via peat accumulation. In greenhouse gas terms, the transition from frozen permafrost to thawed wetland is accompanied by increasing CO2 uptake that is partially offset by increasing CH4 emissions. In the short-term (decadal time scale) the net effect of this transition is likely enhanced warming via increased radiative C emissions, while in the long-term (centuries) net C deposition provides a negative feedback to climate warming

Functional and Biochemical Alterations of the Medial Frontal Cortex in Obsessive-Compulsive Disorder

Context: The medial frontal cortex (MFC), including the dorsal anterior cingulate (dAC) and supplementary motor area (SMA), is critical for adaptive and inhibitory control of behaviour. Abnormally high MFC activity has been a consistent finding in functional neuroimaging studies of obsessive-compulsive disorder (OCD). However, the precise regions and the neural alterations associated with this abnormality remain unclear. Objective: To examine the functional and biochemical properties of the MFC in patients with OCD. Design: Cross-sectional design combining volume localized proton magnetic resonance spectroscopy (1H-MRS) and functional MRI (fMRI) with an inhibitory control paradigm (the Multi-Source Interference Task; MSIT) designed to activate the MFC. Setting: Healthy control participants and OCD patients recruited from the general community. Participants: Nineteen OCD patients (10 male, and 9 female) and nineteen age, gender, education and intelligence-matched healthy control participants. Main Outcome Measures: Psychometric measures of symptom severity, MSIT behavioural performance, blood-oxygen-level-dependent (BOLD) activation and 1H-MRS brain metabolite concentrations. Results: MSIT behavioural performance did not differ between OCD patients and control subjects. Reaction-time interference and response errors were correlated with BOLD activation in the dAC region in both groups. Relative to control subjects, OCD patients showed hyper- activation of the SMA during high response-conflict (incongruent > congruent) trials and hyper-activation of the rostral anterior cingulate (rAC) region during low response- conflict (incongruent < congruent) trials. OCD patients also showed reduced levels of neuronal N-acetylaspartate in the dAC region, which was negatively correlated with their BOLD activation of the region. Conclusions: Our findings suggest that hyper-activation of the medial frontal cortex in OCD patients may be a compensatory response to neural pathology in the region. This relationship may partly explain the nature of inhibitory control deficits that are frequently seen in this group and may serve as a focus of future treatment studies

RNA Synthesis in a Cage—Structural Studies of Reovirus Polymerase λ3

AbstractThe reovirus polymerase and those of other dsRNA viruses function within the confines of a protein capsid to transcribe the tightly packed dsRNA genome segments. The crystal structure of the reovirus polymerase, λ3, determined at 2.5 Å resolution, shows a fingers-palm-thumb core, similar to those of other viral polymerases, surrounded by major N- and C-terminal elaborations, which create a cage-like structure, with four channels leading to the catalytic site. This “caged” polymerase has allowed us to visualize the results of several rounds of RNA polymerization directly in the crystals. A 5′ cap binding site on the surface of λ3 suggests a template retention mechanism by which attachment of the 5′ end of the plus-sense strand facilitates insertion of the 3′ end of the minus-sense strand into the template channel

Identifying what makes a good question in a mechanics diagnostic test

Many students entering engineering degrees encounter problems with the mathematics involved. More recently, research has shown that freshers may have insuffi cient knowledge of mechanics. In order to assess this, the authors created and administered a multiple-choice mechanics diagnostic test. This paper gives details of the test, and evaluates, using item analysis, how students performed on the questions and on the topics assessed by it. It also makes recommendations for devising questions which allow a diagnostic test to discriminate between students

Stu2p binds tubulin and undergoes an open-to-closed conformational change

Stu2p from budding yeast belongs to the conserved Dis1/XMAP215 family of microtubule-associated proteins (MAPs). The common feature of proteins in this family is the presence of HEAT repeat–containing TOG domains near the NH2 terminus. We have investigated the functions of the two TOG domains of Stu2p in vivo and in vitro. Our data suggest that Stu2p regulates microtubule dynamics through two separate activities. First, Stu2p binds to a single free tubulin heterodimer through its first TOG domain. A large conformational transition in homodimeric Stu2p from an open structure to a closed one accompanies the capture of a single free tubulin heterodimer. Second, Stu2p has the capacity to associate directly with microtubule ends, at least in part, through its second TOG domain. These two properties lead to the stabilization of microtubules in vivo, perhaps by the loading of tubulin dimers at microtubule ends. We suggest that this mechanism of microtubule regulation is a conserved feature of the Dis1/XMAP215 family of MAPs