Covalent Chemical Cochaperones of the p300/CBP GACKIX Domain.

The GACKIX activator binding domain has been a compelling target for small-molecule probe discovery because of the central role of activator-GACKIX complexes in diseases ranging from leukemia to memory disorders. Additionally, GACKIX is an ideal model to dissect the context-dependent function of activator-coactivator complexes. However, the dynamic and transient protein-protein interactions (PPIs) formed by GACKIX are difficult targets for small molecules. An additional complication is that activator-binding motifs, such as GACKIX, are found in multiple coactivators, making specificity difficult to attain. In this study, we demonstrate that the strategy of tethering can be used to rapidly discover highly specific covalent modulators of the dynamic PPIs between activators and coactivators. These serve as both ortho- and allosteric modulators, enabling the tunable assembly or disassembly of the activator-coactivator complexes formed between the KIX domain and its cognate activator binding partners MLL and CREB. The molecules maintain their function and selectivity, even in human cell lysates and in bacterial cells, and thus, will ultimately be highly useful probes for cellular studies.

BET bromodomain ligands: Probing the WPF shelf to improve BRD4 bromodomain affinity and metabolic stability.

Ligands for the bromodomain and extra-terminal domain (BET) family of bromodomains have shown promise as useful therapeutic agents for treating a range of cancers and inflammation. Here we report that our previously developed 3,5-dimethylisoxazole-based BET bromodomain ligand (OXFBD02) inhibits interactions of BRD4(1) with the RelA subunit of NF-κB, in addition to histone H4. This ligand shows a promising profile in a screen of the NCI-60 panel but was rapidly metabolised (t½â€¯= 39.8 min). Structure-guided optimisation of compound properties led to the development of the 3-pyridyl-derived OXFBD04. Molecular dynamics simulations assisted our understanding of the role played by an internal hydrogen bond in altering the affinity of this series of molecules for BRD4(1). OXFBD04 shows improved BRD4(1) affinity (IC50 = 166 nM), optimised physicochemical properties (LE = 0.43; LLE = 5.74; SFI = 5.96), and greater metabolic stability (t½â€¯= 388 min).

A Bifunctional Amino Acid Enables Both Covalent Chemical Capture and Isolation of in Vivo Protein-Protein Interactions.

In vivo covalent chemical capture by using photoactivatable unnatural amino acids (UAAs) is a powerful tool for the identification of transient protein-protein interactions (PPIs) in their native environment. However, the isolation and characterization of the crosslinked complexes can be challenging. Here, we report the first in vivo incorporation of the bifunctional UAA BPKyne for the capture and direct labeling of crosslinked protein complexes through post-crosslinking functionalization of a bioorthogonal alkyne handle. Using the prototypical yeast transcriptional activator Gal4, we demonstrate that BPKyne is incorporated at the same level as the commonly used photoactivatable UAA pBpa and effectively captures the Gal4-Gal80 transcriptional complex. Post-crosslinking, the Gal4-Gal80 adduct was directly labeled by treatment of the alkyne handle with a biotin-azide probe; this enabled facile isolation and visualization of the crosslinked adduct from whole-cell lysate. This bifunctional amino acid extends the utility of the benzophenone crosslinker and expands our toolbox of chemical probes for mapping PPIs in their native cellular environment.

Impact of Flexible Circuit Bonding and System Integration on Energy Resolution of Cross-Strip CZT Detectors.

Cadmium zinc telluride (CZT) detectors enable high spatial resolution and high detection efficiency and are utilized for many gamma-ray and X-ray spectroscopy applications. In this article, we describe a stable bonding process and report on the characterization of cross-strip CZT detectors before and after bonding to flexible circuit. The bonding process utilizes gold stud bonding and polymer epoxy technique to bond the flexible circuits to two CZT crystals and form a detector module in an anode-cathode-cathode-anode (ACCA) configuration. The readout electronics is optimized in terms of shaper setting and steering electrode voltage. The average full-width half maximum (FWHM) energy resolution at 662 keV of 110 CZT crystals tested individually was 3.5% ± 0.59% and 4.75% ± 0.48% prebonded and post-bonded, respectively. No depth correction was performed in this study. The average FWHM energy resolution at 662 keV of the scaled-up system with 80 CZT crystals was 4.40% ± 0.53%, indicating the scaled-up readout electronics and stacking of the modules does not deteriorate performance. The proper shielding and grounding of the scaled-up system slightly improved the system-wide performance. The FWHM energy resolution at 511 keV of the scaled-up system was 5.85% ± 0.73%.

Hemostasis Checklist Reduces Bleeding and Blood Product Consumption After Cardiac Surgery.

BACKGROUND: Considerable mediastinal bleeding is a recognized complication after cardiac surgery and may require reexploration and blood product transfusion, both of which are associated with inferior clinical outcomes with greater morbidity and mortality. The aim of this study was to develop a hemostasis checklist, with the intention of reducing mediastinal bleeding after cardiac surgery. METHODS: A hemostasis checklist was developed with multidisciplinary collaboration. It contains 2 components: a series of surgical sites and factors affecting coagulation status. The checklist is performed at a time-out before sternal wire insertion. Analysis compared outcomes for patients undergoing cardiac surgery in the 1 year before and 2 years after implementation. RESULTS: A total of 5542 patients underwent surgery during the study. After we implemented the checklist, there was a significant reduction in the reexploration rate (3.5% versus 1.9%; P < .001) and the proportion of patients bleeding greater than 1 L in 12 hours (6.1% versus 2.8%; P < .001). There was a major reduction in consumption of blood products, saving $430,513. There was progressive improvement in the second year after implementation. Checklist implementation was also associated with reduced intensive care unit and hospital length of stay, adding to the financial benefit. CONCLUSIONS: Implementation of a simple and quickly performed hemostasis checklist has had a sustained impact over the 2 years after implementation, reducing the incidence of noteworthy mediastinal bleeding and reexploration, which has resulted in a major reduction in blood product consumption. Together, these have resulted in an associated reduction in intensive care unit and hospital length of stay, and a considerable financial savings. This highlights that perioperative bleeding is a preventable complication.

Cross-Contamination on Atypical Surfaces and Venues in Food Service Environments.

ABSTRACT: Cross-contamination of raw food to other surfaces, hands, and foods is a serious issue in food service. With individuals eating more meals away from home, contracting a foodborne illness from a food service establishment is an increasing concern. However, most studies have concentrated on hands or food contact surfaces and neglected atypical and unusual surfaces (surfaces that are not typically identified as a source of cross-contamination) and venues. This review was conducted to identify atypically cross-contaminated surfaces and atypical venues where cross-contamination could occur that have not been examined thoroughly in the literature. Most surfaces that could be at risk for cross-contamination are frequently touched, are rarely cleaned and sanitized, and can support the persistence and/or growth of foodborne pathogens. These surfaces include menus, spice and condiment containers, aprons and coveralls, mobile devices and tablets, and money. Venues that are explored, such as temporary events, mobile vendors, and markets, are usually limited in space or infrastructure, have low compliance with proper hand washing, and provide the opportunity for raw and ready-to-eat foods to come into contact with one another. These factors create an environment in which cross-contamination can occur and potentially impact food safety. A more comprehensive cleaning and sanitizing regime encompassing these surfaces and venues could help mitigate cross-contamination. This review highlights key surfaces and venues that have the potential to be cross-contaminated and have been underestimated or not fully investigated. These knowledge gaps indicate where further work is needed to fully understand the role of these surfaces and venues in cross-contamination and how it can be prevented.

Controlling Intramolecular Interactions in the Design of Selective, High-Affinity Ligands for the CREBBP Bromodomain.

CREBBP (CBP/KAT3A) and its paralogue EP300 (KAT3B) are lysine acetyltransferases (KATs) that are essential for human development. They each comprise 10 domains through which they interact with >400 proteins, making them important transcriptional co-activators and key nodes in the human protein-protein interactome. The bromodomains of CREBBP and EP300 enable the binding of acetylated lysine residues from histones and a number of other important proteins, including p53, p73, E2F, and GATA1. Here, we report a work to develop a high-affinity, small-molecule ligand for the CREBBP and EP300 bromodomains [(-)-OXFBD05] that shows >100-fold selectivity over a representative member of the BET bromodomains, BRD4(1). Cellular studies using this ligand demonstrate that the inhibition of the CREBBP/EP300 bromodomain in HCT116 colon cancer cells results in lowered levels of c-Myc and a reduction in H3K18 and H3K27 acetylation. In hypoxia (<0.1% O2), the inhibition of the CREBBP/EP300 bromodomain results in the enhanced stabilization of HIF-1α.

Selective Fragments for the CREBBP Bromodomain Identified from an Encoded Self-assembly Chemical Library.

DNA-encoded chemical libraries (DECLs) are collections of chemical moieties individually coupled to distinctive DNA barcodes. Compounds can be displayed either at the end of a single DNA strand (i. e., single-pharmacophore libraries) or at the extremities of two complementary DNA strands (i. e., dual-pharmacophore libraries). In this work, we describe the use of a dual-pharmacophore encoded self-assembly chemical (ESAC) library for the affinity maturation of a known 4,5-dihydrobenzodiazepinone ring (THBD) acetyl-lysine (KAc) mimic for the cyclic-AMP response element binding protein (CREB) binding protein (CREBBP or CBP) bromodomain. The new pair of fragments discovered from library selection showed a sub-micromolar affinity for the CREBBP bromodomain in fluorescence polarization and ELISA assays, and selectivity against BRD4(1).

Dynamic individual vital sign trajectory early warning score (DyniEWS) versus snapshot national early warning score (NEWS) for predicting postoperative deterioration.

AIMS: International early warning scores (EWS) including the additive National Early Warning Score (NEWS) and logistic EWS currently utilise physiological snapshots to predict clinical deterioration. We hypothesised that a dynamic score including vital sign trajectory would improve discriminatory power. METHODS: Multicentre retrospective analysis of electronic health record data from postoperative patients admitted to cardiac surgical wards in four UK hospitals. Least absolute shrinkage and selection operator-type regression (LASSO) was used to develop a dynamic model (DyniEWS) to predict a composite adverse event of cardiac arrest, unplanned intensive care re-admission or in-hospital death within 24 h. RESULTS: A total of 13,319 postoperative adult cardiac patients contributed 442,461 observations of which 4234 (0.96%) adverse events in 24 h were recorded. The new dynamic model (AUC = 0.80 [95% CI 0.78-0.83], AUPRC = 0.12 [0.10-0.14]) outperforms both an updated snapshot logistic model (AUC = 0.76 [0.73-0.79], AUPRC = 0.08 [0.60-0.10]) and the additive National Early Warning Score (AUC = 0.73 [0.70-0.76], AUPRC = 0.05 [0.02-0.08]). Controlling for the false alarm rates to be at current levels using NEWS cut-offs of 5 and 7, DyniEWS delivers a 7% improvement in balanced accuracy and increased sensitivities from 41% to 54% at NEWS 5 and 18% to -30% at NEWS 7. CONCLUSIONS: Using an advanced statistical approach, we created a model that can detect dynamic changes in risk of unplanned readmission to intensive care, cardiac arrest or in-hospital mortality and can be used in real time to risk-prioritise clinical workload.

Reduced re-exploration and blood product transfusion after the introduction of the Papworth haemostasis checklist†.

OBJECTIVES: Between 2% and 8% of patients return to the theatre for mediastinal bleeding following cardiac surgery. In the majority of patients, a surgical source of bleeding is identified. Both mediastinal bleeding and re-exploration are associated with increased morbidity and mortality and the use of blood products. The aim of this study was to develop a 'haemostasis checklist' with the intention of reducing mediastinal bleeding and re-exploration following cardiac surgery. METHODS: The Papworth haemostasis checklist was developed with a multidisciplinary collaboration. It consists of 2 components: surgical sites and coagulation status. The checklist is completed at a 'time-out' prior to sternal wire insertion. The analysis compared the outcomes of patients undergoing cardiac surgery in the 1 year before and after implementation. A propensity analysis assessed the impact of re-exploration on outcomes. RESULTS: Three thousand eight hundred and eleven patients underwent cardiac surgery during the study period. Re-exploration for bleeding was associated with inferior outcomes. Following checklist implementation, there was a significant reduction in the re-exploration rate (3.47% vs 2.08%, P = 0.01) and proportion of patients bleeding >1 l in 12 h (6.1% vs 3.49%, P < 0.001). There was a significant reduction in consumption of blood products saving £102 165 ($134 198). The checklist implementation was associated with reduced intensive care unit length of stay and hospital length of stay, adding to the financial benefit. CONCLUSIONS: The haemostasis checklist represents a simple intervention which is quick and easy to use but has had a substantial impact on clinical outcomes. We have observed a significant reduction in the mediastinal blood loss, return-to-theatre rate and consumption of blood products, which is associated with a significant clinical and financial benefit.

Acute kidney injury after thoracic surgery: a proposal for a multicentre evaluation (MERITS).

OBJECTIVES: Because the mortality rate is very low in thoracic surgery, its use as a quality discriminator is limited. Acute kidney injury (AKI) is a candidate measure because it is associated with increased rates of morbidity and mortality and is partly preventable. The incidence of AKI after thoracic surgery is not well documented. We conducted an audit to determine the incidence and outcomes of AKI. This audit became a pilot project, and the results indicate the feasibility of a larger study. METHODS: Retrospective data on renal function post-thoracic surgery were collected at a tertiary cardiothoracic unit over 12 months. Renal impairment was classified according to the Kidney Disease Improving Global Outcomes criteria. RESULTS: Of 568 patients (mean = 59 ± SD 18; 38% women), AKI was diagnosed in 86 (15.1%) within 72 h post-thoracic surgery based on the Kidney Disease Improving Global Outcomes staging system (stage 1, n = 55; stage 2, n = 25; stage 3, n = 6). Significant differences were found in postoperative length of stay (3 vs 5 days; P < 0.001) of patients with and without AKI. There was a significant difference between the age groups of patients with and without AKI (P < 0.05) in the open surgical group but not in the group having video-assisted thoracoscopic surgery (VATS). There was no significant difference in the mortality rates between patients with and without AKI. CONCLUSIONS: The incidence of AKI after thoracic surgery was 15.1%. AKI was associated with longer hospital stays and was more likely in ≥60-year-old patients after open surgery than after VATS. Reducing AKI could improve patient outcomes. We propose that AKI may be a useful quality measure in thoracic surgery. We are developing a multicentre audit based on this approach.

Conformational switching between diastereoisomeric atropisomers of arenedicarboxamides induced by complexation with Lewis acids.

Tertiary diamides of xanthene-1,8-dicarboxylic acid and biphenyl-2,2'-dicarboxylic acid exhibit a thermodynamic preference for anti stereochemistry which is inverted in the presence of Ti- or Sn-based Lewis acids, allowing interconversion between kinetically stable syn and anti diastereoisomeric atropisomers.

What are the critical steps in processing blood cultures? A prospective audit evaluating current practice of reporting blood cultures in a centralised laboratory serving secondary care hospitals.

AIMS: To assess current procedures of processing positive blood cultures against national standards with an aim to evaluate its clinical impact and to determine the utility of currently available rapid identification and susceptibility tests in processing of blood cultures. METHODS: Blood cultures from three secondary care hospitals, processed at a centralised laboratory, were prospectively audited. Data regarding processing times, communication with prescribers, changes to patient management and mortality within 30 days of a significant blood culture were collected in a preplanned pro forma for a 4-week period. RESULTS: Of 2206 blood cultures, 211 positive blood cultures flagged positive. Sixty-nine (3.1%) of all cultures were considered to be contaminated. Fifty per cent of blood cultures that flagged positive had a Gram stain reported within 2 hours. Two (0.99%) patients with a significant bacteraemia had escalation of antimicrobial treatment at the point of reporting the Gram stain that was subsequently deemed necessary once sensitivity results were known. Most common intervention was de-escalation of therapy for Gram-positive organisms at the point of availability of pathogen identification (25.6% in Gram positive vs 10% in Gram negative; p=0.012). For Gram-negative organisms, the most common intervention was de-escalation of therapy at the point of availability of sensitivity results (43% in Gram negatives vs 17.9% in Gram positive; p=0.0097). Overall mortality within 30 days of a positive blood culture was 10.9% (23/211). Antibiotic resistance may have contributed to mortality in four of these patients (three Gram negative and one Gram positive). CONCLUSION: Gram stain result had the least impact on antibiotic treatment interventions (escalation or de-escalation). Tests that improve identification time for Gram-positive pathogens and sensitivity time for Gram-negative pathogens had the greatest impact in making significant changes to antimicrobial treatment.

Endocarditis caused by Propionibacterium species: a report of three cases and a review of clinical features and diagnostic difficulties.

Propionibacterium species are members of the normal flora of skin and the mouth but their pathogenic potential is often overlooked. Three fatal cases of endocarditis caused by Propionibacterium species over an 8-year period are reported, and a review is presented of a further 33 cases from the world literature. In most cases, infection was protracted, with minimal signs in the early stages. Fourteen cases (42.4%) involved native valves, 16 (48.5%) involved prosthetic valves and three (9.1%) were associated with other intracardiac prosthetic material. Intracardiac abscesses were commonly encountered, with Propionibacterium endocarditis occurring in 28.6% of native valve infections and 52.9% of prosthetic valve infections. A very high proportion of all of the cases (70.6%) required surgical intervention. Several factors appeared to delay institution of appropriate therapy and may have contributed to abscess formation, including an indolent clinical course, negative or delayed culture results, and the tendency to consider this organism as a blood-culture contaminant. The authors recommend careful clinical evaluation before disregarding a blood-culture isolate of Propionibacterium spp. as a skin contaminant, and consideration of this bacterium as a potential cause of apparently culture-negative endocarditis.

Hookworm-related cutaneous larva migrans acquired in the UK.

Hookworm-related cutaneous larva migrans (HrCLM) is a skin disease caused by infection with the larvae of animal hookworms. With conditions for infection more favourable in tropical climates, HrCLM in the UK is classically diagnosed in the returning traveller. We present two cases of clinically diagnosed UK-acquired HrCLM from a district general hospital in the south of England. A 68-year-old woman presented with a pruritic serpiginous tract on the right hand. She was a keen gardener and had been handling compost. A 50-year-old man, a long distance runner, presented with a similar lesion on the dorsum of his foot. Both patients were treated with a single dose of albendazole. These cases may represent an emerging infection in the UK. In the absence of a suggestive travel history, early recognition followed by efficient access to therapy is vital for treating HrCLM transmitted in the UK.