NMR assignment of the conserved bacterial DNA replication protein DnaA domain IV.

Chromosomal replication is a ubiquitous and essential cellular process. In bacteria, the master replication initiator DnaA plays a key role in promoting an open complex at the origin (oriC) and recruiting helicase in a tightly regulated process. The C-terminal domain IV specifically recognises consensus sequences of double-stranded DNA in oriC, termed DnaA-boxes, thereby facilitating the initial engagement of DnaA to oriC.

In Situ Quantification of Carbonate Species Concentrations, pH, and pCO in Calcite Fluid Inclusions Using Confocal Raman Spectroscopy.

Carbonate minerals are globally distributed on the modern and ancient Earth and are abundant in terrestrial and marine depositional environments. Fluid inclusions hosted by calcite retain primary signatures of the source fluid geochemistry at the time of mineral formation (i.e.

The top 10 priorities in adults living with type 1 diabetes in Ireland and the United Kingdom - A James Lind Alliance priority setting partnership.

Aims: To undertake a Priority Setting Partnership (PSP), identifying the most important unanswered questions in type 1 diabetes in Ireland and the United Kingdom and to compare these to priorities identified in a 2011 PSP.

Methods: A steering committee (including eight individuals with lived experience/charity representatives and six clinicians) designed a survey which asked stakeholders to list three questions about type 1 diabetes. This was disseminated through social media, direct email contact, and printed posters.

The bacterial replication origin BUS promotes nucleobase capture.

Genome duplication is essential for the proliferation of cellular life and this process is generally initiated by dedicated replication proteins at chromosome origins. In bacteria, DNA replication is initiated by the ubiquitous DnaA protein, which assembles into an oligomeric complex at the chromosome origin (oriC) that engages both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) to promote DNA duplex opening. However, the mechanism of DnaA specifically opening a replication origin was unknown.

Backbone resonance assignments of the catalytic and regulatory domains of Ca/calmodulin-dependent protein kinase 1D.

The CaMK subfamily of Ser/Thr kinases are regulated by calmodulin interactions with their C-terminal regions. They are exemplified by Ca/calmodulin dependent protein kinase 1δ which is known as CaMK1D, CaMKIδ or CKLiK. CaMK1D mediates intracellular signalling downstream of Ca influx and thereby exhibits amplifications of Casignals and polymorphisms that have been implicated in breast cancer and diabetes.

Production and Crystallization of Full-Length Human AMP-Activated Protein Kinase (α1β1γ1).

Determination of the crystal structure of AMP-activated protein kinase (AMPK) is fundamental to understanding its biological function and role in a number of diseases related to energy metabolism including type 2 diabetes, obesity, and cancer. We describe methods for the expression and purification of a human full-length active AMPK complex that is suitable for biochemical and structural analyses, followed by methods for its crystallization in complex with small molecule activators. Quality control of the purified protein by functional and biophysical analysis was an essential part of the process enabling the achievement of crystals of the full-length protein capable of being used for high-resolution structure determination by X-ray diffraction.

Phosphorylation of AMPK by upstream kinases is required for activity in mammalian cells.

AMP-activated protein kinase (AMPK) plays a major role in regulating metabolism and has attracted significant attention as a therapeutic target for treating metabolic disorders. AMPK activity is stimulated more than 100-fold by phosphorylation of threonine 172 (Thr). Binding of AMP to the γ subunit allosterically activates the kinase.

Unlocking the secrets of the gatekeeper: methods for stabilizing and crystallizing GPCRs.

G-protein coupled receptors (GPCRs) are integral membrane cell surface receptors with key roles in mediating the cellular responses to a wide range of biologically relevant molecules including hormones, neurotransmitters and importantly the majority of currently available drugs. The first high-resolution, X-ray crystallographic structure of a GPCR, that of rhodopsin, was obtained in 2000. It took a further seven years for the next structure, that of the β2 adrenergic receptor.

Loss of constitutive activity is correlated with increased thermostability of the human adenosine A2A receptor.

Background And Purpose: Thermostabilization by mutagenesis is one method which has facilitated the determination of high-resolution structures of the adenosine A2A receptor (A(2A)R). Sets of mutations were identified, which both thermostabilized the receptor and resulted in preferential agonist (Rag23 mutant) or antagonist (Rant5 and Rant21) binding forms as assessed by radioligand binding analysis. While the ligand-binding profiles of these mutants are known, the effects these mutations have on receptor activation and downstream signalling are less well characterized.

Homeostasis 4: nurses as agents of control in myocardial infarction.

The role of health practitioners in attempting to reverse homeostatic imbalances essentially makes them external agents of homeostatic control-they are replacing the assessment, controlling and effector mechanisms that operate during health (homeostasis), but have failed in ill-health (homeostatic imbalances). Myocardial infarction (MI) is the homeostatic imbalance examined in this article, which aims to apply the analogy between the components of homeostatic theory and the components of the nursing process (Clancy and McVicar, 2011b) to the condition. After reading the article, nurses should be able to understand that: the components of homeostasis are associated with health, and the failure of one or more of these components results in illness; illness arises from a cellular, hence chemical, homeostatic imbalance(s); MI results from a cellular imbalance leading to a restriction in blood flow to the myocardium, and is identified by signs and symptoms (i.

Novel macrocyclic HCV NS3 protease inhibitors derived from α-amino cyclic boronates.

A novel series of P2-P4 macrocyclic HCV NS3/4A protease inhibitors with α-amino cyclic boronates as warheads at the P1 site was designed and synthesized. When compared to their linear analogs, these macrocyclic inhibitors exhibited a remarkable improvement in cell-based replicon activities, with compounds 9a and 9e reaching sub-micromolar potency in replicon assay. The SAR around α-amino cyclic boronates clearly established the influence of ring size, chirality and of the substitution pattern.

Synthesis and evaluation of novel alpha-amino cyclic boronates as inhibitors of HCV NS3 protease.

We have designed and synthesized a novel series of alpha-amino cyclic boronates and incorporated them successfully in several acyclic templates at the P1 position. These compounds are inhibitors of the HCV NS3 serine protease, and structural studies show that they inhibit the NS3 protease by trapping the Ser-139 hydroxyl group in the active site. Synthetic methodologies and SARs of this series of compounds are described.

Second generation of BACE-1 inhibitors part 2: Optimisation of the non-prime side substituent.

Our first generation of hydroxyethylamine transition-state mimetic BACE-1 inhibitors allowed us to validate BACE-1 as a key target for Alzheimer's disease by demonstrating amyloid lowering in an animal model, albeit at rather high doses. Finding a molecule from this series which was active at lower oral doses proved elusive and demonstrated the need to find a novel series of inhibitors with improved pharmacokinetics. This Letter describes the discovery of such inhibitors.

Second generation of BACE-1 inhibitors. Part 1: The need for improved pharmacokinetics.

Inhibition of the aspartyl protease BACE-1 has the potential to deliver a disease-modifying therapy for Alzheimer's disease. We have recently disclosed a series of transition-state mimetic BACE-1 inhibitors showing nanomolar potency in cell-based assays. Amongst them, GSK188909 (compound 2) had favorable pharmacokinetics and was the first orally bioavailable inhibitor reported to demonstrate brain amyloid lowering in an animal model.

Second generation of BACE-1 inhibitors part 3: Towards non hydroxyethylamine transition state mimetics.

Our first generation of hydroxyethylamine BACE-1 inhibitors proved unlikely to provide molecules that would lower amyloid in an animal model at low oral doses. This observation led us to the discovery of a second generation of inhibitors having nanomolar activity in a cell-based assay and with the potential for improved pharmacokinetic profiles. In this Letter, we describe our successful strategy for the optimization of oral bioavailability and also give insights into the design of compounds with the potential for improved brain penetration.

Selective and dual action orally active inhibitors of thrombin and factor Xa.

The synthetic entry to new classes of dual fXa/thrombin and selective thrombin inhibitors with significant oral bioavailability is described. This was achieved through minor modifications to the sulfonamide group in our potent and selective fXa inhibitor (E)-2-(5-chlorothien-2-yl)-N-{(3S)-1-[(1S)-1-methyl-2-(morpholin-4-yl)-2-oxoethyl]-2-oxopyrrolidin-3-yl}ethenesulfonamide and these observed activity changes have been rationalised using structural studies.

Sulfonamide-related conformational effects and their importance in structure-based design.

Structure-based design (SBD) is a challenging endeavour since even localised SAR can hardly ever be explained by the variation of just one dominating factor. Here, we present a rare example where structural information combined with ab initio calculations clearly indicate that the observed difference in biological activity is dominated by conformational effects. The learnings discussed are successfully put to the test and have the potential to be of general use as a qualitative guide in SBD efforts.

Global analysis of proteins synthesized by Mycobacterium smegmatis provides direct evidence for physiological heterogeneity in stationary-phase cultures.

We have characterized the induction kinetics of approximately 1,700 proteins during entry into and survival in carbon-starved stationary phase by Mycobacterium smegmatis. Strikingly, among the patterns of expression observed were a group of proteins that were expressed in exponential-phase cultures and severely repressed in 48-h stationary-phase cultures (Spr or stationary-phase-repressed proteins) but were synthesized again at high levels in > or =128-day stationary-phase cultures (Spr(128) proteins). A number of Spr(128) proteins were identified, and they included the heat shock protein DnaK, the tricarboxylic acid cycle enzyme succinyl coenzyme A synthase, a FixA-like flavoprotein, a single-stranded DNA binding protein, and elongation factor Tu (EF-Tu).

Management of atrial fibrillation.

Atrial fibrillation (AF) is one of the most common arrhythmias encountered in the adult population. It can be an isolated event or a chronic lifelong rhythm disturbance. The common causes of AF and the presenting electrocardiogram characteristics are outlined.

An overview of permanent and temporary cardiac pacemakers.

As pacemaker technology comes closer to mimicking the heart's own pacing system, patients in need of such devices can hope to live increasingly normal and productive lives. This article outlines common types of cardiac pacing and indications for pacemaker insertion. Nursing considerations are outlined both during and following the insertion of a pacemaker.

Nuclear magnetic resonance spectroscopy reveals the functional state of the signalling protein CheY in vivo in Escherichia coli.

Two-component signal transduction (TCST) pathways are regulatory systems that are highly homologous throughout the bacterial kingdom. Their established role in virulence and absence in vertebrates has made TCST an attractive target for therapeutic intervention. However, such systems have yet to yield success in the development of novel antibiotics.

Common biochemical markers for diagnosing heart disease.

An electrocardiogram (ECG) and clinical features alone are used to diagnose almost 75 per cent of patients presenting with an acute myocardial infarction (MI). In these cases elevated cardiac enzyme levels merely confirm the diagnosis. However, when the clinical picture is less characteristic and the ECG is normal or has nonspecific changes, verification may rely on the detection of enzyme elevation.

Complications associated with myocardial infarction.

The incidence of complications after acute myocardial infarction (MI) has been estimated to range from 14-95 per cent, with an overall one-month mortality of 30 per cent. Early treatment, as advocated by the National Service Framework for Heart Disease, has brought about some reduction in associated morbidity and mortality after MI. This article reviews the common complications associated with an acute MI, such as cardiogenic shock, pericarditis and heart failure.

Myocardial infarction: signs symptoms and treatment.

From early middle age onwards myocardial infarction is recognised as being a major cause of death and disability in this country. Approximately 300,000 people in the UK experience an MI each year and about 140,000 will die as a result. The rapid management of patients with chest pain should ensure that there is no more than 30 minutes delay in decision-making and initiation of thrombolytic therapy.