DNA polymerase ϵ links the DNA replication machinery to the S phase checkpoint

AbstractInhibition of DNA synthesis induces transcription of DNA damage -inducible genes and prevents mitotic entry through the action of the S phase checkpoint. We have isolated a mutant, dun2, defective for both of these responses. DUN2 is identical to POL2, encoding DNA polymerase ϵ (pol ϵ). Unlike sadl mutants defective for multiple cell cycle checkpoints, pol2 mutants are defective only for the S phase checkpoint and the activation of DUNI kinase necessary for the transcriptional response to damage. Interallelic complementation and mutation analysis indicate that pol ϵ contains two separable essential domains, an N-terminal polymerase domain and a C-terminal checkpoint domain unique to ϵ polymerases. We propose that DNA pol ϵ acts as a sensor of DNA replication that coordinates the transcriptional and cell cycle responses to replication blocks

Evaluation of optical coherence tomography findings in age-related macular degeneration: a reproducibility study of two independent reading centres

International audienceBackground/aims : To determine the reproducibility among readers of two independent certified centers, the Vienna Reading Center (VRC) and the University of Wisconsin-Madison Reading Center (UW-FPRC) for OCT images in age-related macular degeneration (AMD). Methods : Fast macular thickness scans and 6 mm cross hair scans were obtained from 100 eyes with all subtypes of AMD using Stratus OCT. Consensus readings were performed by two certified OCT readers of each Reading Center using their grading protocol. Common variables of both grading protocols, such as presence of cystoid spaces, subretinal fluid, vitreomacular traction and retinal pigment epithelial detachment were compared using kappa statistics. In addition, the intraclass correlation coefficient (ICC) was calculated for center point thickness (CPT) of values remeasured manually in the presence of alignment errors. Results : The reproducibility was dependent on the variable measured with a kappa value of 0.81 for the presence of cystoid spaces, 0.78 for the presence of subretinal fluid and 0.795 for the presence of vitreomacular traction. The lowest reproducibility was found for the presence of retinal pigment epithelial detachment with a kappa value of 0.51. The CPT was remeasured in 29 out of 100 scans at both sites with an ICC of the remeasured thicknesses of 0.92. Conclusion : OCT scan data are crucial in monitoring treatment efficacy in AMD clinical trials. For comparison of results obtained by different Reading Centers, the inter-Reading Center reproducibility is essential. Although the reproducibility is generally high, the reliability depends on the selected morphological parameters

Development of the (d,n) proton-transfer reaction in inverse kinematics for structure studies

Transfer reactions have provided exciting opportunities to study the structure of exotic nuclei and are often used to inform studies relating to nucleosynthesis and applications. In order to benefit from these reactions and their application to rare ion beams (RIBs) it is necessary to develop the tools and techniques to perform and analyze the data from reactions performed in inverse kinematics, that is with targets of light nuclei and heavier beams. We are continuing to expand the transfer reaction toolbox in preparation for the next generation of facilities, such as the Facility for Rare Ion Beams (FRIB), which is scheduled for completion in 2022. An important step in this process is to perform the (d,n) reaction in inverse kinematics, with analyses that include Q-value spectra and differential cross sections. In this way, proton-transfer reactions can be placed on the same level as the more commonly used neutron-transfer reactions, such as (d,p), (9Be,8Be), and (13C,12C). Here we present an overview of the techniques used in (d,p) and (d,n), and some recent data from (d,n) reactions in inverse kinematics using stable beams of 12C and 16O.Comment: 9 pages, 4 figures, presented at the XXXV Mazurian Lakes Conference on Physics, Piaski, Polan

Academic training in oral and maxillofacial surgery - when and how to enter the pathway

Entering into surgical academia can seem a daunting prospect for an oral and maxillofacial surgery (OMFS) trainee. However, the streamlining of academic training by the NIHR to create the integrated academic training (IAT) pathway has simplified academic training and more clearly defined academic positions and entry points for trainees. In this article we review the current NIHR IAT pathway and the various grades and entry points available to OMF surgeons, both pre- and post-doctoral. We highlight the unique challenges facing OMF trainees and provide advice and insight from both junior and senior OMFS academics. Finally, we focus on the planning and application for a doctoral research fellowship - discussing funding streams available to OMF surgeons

Autoreactivity and Exceptional CDR Plasticity (but Not Unusual Polyspecificity) Hinder Elicitation of the Anti-HIV Antibody 4E10

The broadly-neutralizing anti-HIV antibody 4E10 recognizes an epitope in the membrane-proximal external region of the HIV envelope protein gp41. Previous attempts to elicit 4E10 by vaccination with envelope-derived or reverse-engineered immunogens have failed. It was presumed that the ontogeny of 4E10-equivalent responses was blocked by inherent autoreactivity and exceptional polyreactivity. We generated 4E10 heavy-chain knock-in mice, which displayed significant B cell dysregulation, consistent with recognition of autoantigen/s by 4E10 and the presumption that tolerance mechanisms may hinder the elicitation of 4E10 or 4E10-equivalent responses. Previously proposed candidate 4E10 autoantigens include the mitochondrial lipid cardiolipin and a nuclear splicing factor, 3B3. However, using carefully-controlled assays, 4E10 bound only weakly to cardiolipin-containing liposomes, but also bound negatively-charged, non-cardiolipin-containing liposomes comparably poorly. 4E10/liposome binding was predominantly mediated by electrostatic interactions rather than presumed hydrophobic interactions. The crystal structure of 4E10 free of bound ligands showed a dramatic restructuring of the combining site, occluding the HIV epitope binding site and revealing profound flexibility, but creating an electropositive pocket consistent with non-specific binding of phospholipid headgroups. These results strongly suggested that antigens other than cardiolipin mediate 4E10 autoreactivity. Using a synthetic peptide library spanning the human proteome, we determined that 4E10 displays limited and focused, but unexceptional, polyspecificity. We also identified a novel autoepitope shared by three ER-resident inositol trisphosphate receptors, validated through binding studies and immunohistochemistry. Tissue staining with 4E10 demonstrated reactivity consistent with the type 1 inositol trisphosphate receptor as the most likely candidate autoantigen, but is inconsistent with splicing factor 3B3. These results demonstrate that 4E10 recognition of liposomes competes with MPER recognition and that HIV antigen and autoepitope recognition may be distinct enough to permit eliciting 4E10-like antibodies, evading autoimmunity through directed engineering. However, 4E10 combining site flexibility, exceptional for a highly-matured antibody, may preclude eliciting 4E10 by conventional immunization strategies

Ontogeny of Recognition Specificity and Functionality for the Broadly Neutralizing Anti-HIV Antibody 4E10

The process of antibody ontogeny typically improves affinity, on-rate, and thermostability, narrows polyspecificity, and rigidifies the combining site to the conformer optimal for binding from the broader ensemble accessible to the precursor. However, many broadly-neutralizing anti-HIV antibodies incorporate unusual structural elements and recognition specificities or properties that often lead to autoreactivity. The ontogeny of 4E10, an autoreactive antibody with unexpected combining site flexibility, was delineated through structural and biophysical comparisons of the mature antibody with multiple potential precursors. 4E10 gained affinity primarily by off-rate enhancement through a small number of mutations to a highly conserved recognition surface. Controverting the conventional paradigm, the combining site gained flexibility and autoreactivity during ontogeny, while losing thermostability, though polyspecificity was unaffected. Details of the recognition mechanism, including inferred global effects due to 4E10 binding, suggest that neutralization by 4E10 may involve mechanisms beyond simply binding, also requiring the ability of the antibody to induce conformational changes distant from its binding site. 4E10 is, therefore, unlikely to be re-elicited by conventional vaccination strategies

Low power discriminator for ATLAS pixel chip

The design of the front-end (FE) pixel electronics requires low power, low noise and low threshold dispersion. In this work, we propose a new architecture for the discriminator circuit. It is based on the principle of dynamic biasing and developed for the FE chip of the ATLAS pixel upgrade. This paper presents two discriminator structures where the bias current depends on the presence of a signal at the input of the discriminator. Since the activity in the FE chip is very low, the power consumption is largely reduced allowing the material reduction in the B-layer