Exopolysaccharide-Repressing Small Molecules with Antibiofilm and Antivirulence Activity against Pseudomonas aeruginosa.

Biofilm formation is a universal virulence strategy in which bacteria grow in dense microbial communities enmeshed within a polymeric extracellular matrix that protects them from antibiotic exposure and the immune system. Pseudomonas aeruginosa is an archetypal biofilm-forming organism that utilizes a biofilm growth strategy to cause chronic lung infections in cystic fibrosis (CF) patients. The extracellular matrix of P. aeruginosa biofilms is comprised mainly of exopolysaccharides (EPS) and DNA. Both mucoid and nonmucoid isolates of P. aeruginosa produce the Pel and Psl EPS, each of which have important roles in antibiotic resistance, biofilm formation, and immune evasion. Given the central importance of the EPS for biofilms, they are attractive targets for novel anti-infective compounds. In this study, we used a high-throughput gene expression screen to identify compounds that repress expression of the pel genes. The pel repressors demonstrated antibiofilm activity against microplate and flow chamber biofilms formed by wild-type and hyperbiofilm-forming strains. To determine the potential role of EPS in virulence, pel/psl mutants were shown to have reduced virulence in feeding behavior and slow killing virulence assays in Caenorhabditis elegans The antibiofilm molecules also reduced P. aeruginosa PAO1 virulence in the nematode slow killing model. Importantly, the combination of antibiotics and antibiofilm compounds increased killing of P. aeruginosa biofilms. These small molecules represent a novel anti-infective strategy for the possible treatment of chronic P. aeruginosa infections.

Fusion Bead Procedure for Nuclear Forensics Employing Synthetic Enstatite to Dissolve Uraniferous and Other Challenging Materials Prior to Laser Ablation Inductively Coupled Plasma Mass Spectrometry.

There is an increasing demand for rapid and effective analytical tools to support nuclear forensic investigations of seized or suspect materials. Some methods are simply adapted from other scientific disciplines and can effectively be used to rapidly prepare complex materials for subsequent analysis. A novel sample fusion method is developed, tested, and validated to produce homogeneous, flux-free glass beads of geochemical reference materials (GRMs), uranium ores, and uranium ore concentrates (UOC) prior to the analysis of 14 rare earth elements (REE) via laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The novelty of the procedure is the production of glass beads using 9 parts high purity synthetic enstatite (MgSiO3) as the glass former with 1 part of sample (sample mass ∼1.5 mg). The beads are rapidly prepared (∼10 min overall time) by fusing the blended mixture on an iridium strip resistance heater in an argon-purged chamber. Many elements can be measured in the glass bead, but the rare earth group in particular is a valuable series in nuclear forensic studies and is well-determined using LA-ICP-MS. The REE data obtained from the GRMs, presented as chondrite normalized patterns, are in very good agreement with consensus patterns. The UOCs have comparable patterns to solution ICP-MS methods and published data. The attractions of the current development are its conservation of sample, speed of preparation, and suitability for microbeam analysis, all of which are favorable for nuclear forensics practitioners and geochemists requiring REE patterns from scarce or valuable samples.

Attenuation of zinc finger nuclease toxicity by small-molecule regulation of protein levels.

Zinc finger nucleases (ZFNs) have been used successfully to create genome-specific double-strand breaks and thereby stimulate gene targeting by several thousand fold. ZFNs are chimeric proteins composed of a specific DNA-binding domain linked to a non-specific DNA-cleavage domain. By changing key residues in the recognition helix of the specific DNA-binding domain, one can alter the ZFN binding specificity and thereby change the sequence to which a ZFN pair is being targeted. For these and other reasons, ZFNs are being pursued as reagents for genome modification, including use in gene therapy. In order for ZFNs to reach their full potential, it is important to attenuate the cytotoxic effects currently associated with many ZFNs. Here, we evaluate two potential strategies for reducing toxicity by regulating protein levels. Both strategies involve creating ZFNs with shortened half-lives and then regulating protein level with small molecules. First, we destabilize ZFNs by linking a ubiquitin moiety to the N-terminus and regulate ZFN levels using a proteasome inhibitor. Second, we destabilize ZFNs by linking a modified destabilizing FKBP12 domain to the N-terminus and regulate ZFN levels by using a small molecule that blocks the destabilization effect of the N-terminal domain. We show that by regulating protein levels, we can maintain high rates of ZFN-mediated gene targeting while reducing ZFN toxicity.

Bioavailable actinide fluxes to the Irish Sea from Sellafield-labelled sediments.

Nuclear discharges to the oceans have given rise to significant accumulations of radionuclides in sediments which can later remobilise back into the water column. A continuing supply of radionuclides to aquatic organisms and the human food chain can therefore exist, despite the absence of ongoing nuclear discharges. Radionuclide remobilisation from sediment is consequently a critical component of the modelled radiation dose to the public. However, radionuclide remobilisation fluxes from contaminated marine sediments have never been quantitatively determined in-situ to provide a valid assessment of the issue. Here, we combine recent advances in the Diffusive Gradients in Thin Films (DGT) sampling technique with ultrasensitive measurement by accelerator mass spectrometry (AMS) to calculate the remobilisation fluxes of plutonium, americium and uranium isotopes from the Esk Estuary sediments (UK), which have accumulated historic discharges from the Sellafield nuclear reprocessing facility. Isotopic evidence indicates the local biota are accumulating remobilised plutonium and demonstrates the DGT technique as a valid bioavailability proxy, which more accurately reflects the elemental fractionation of the actinides in the biota than traditional bulk water sampling. These results provide a fundamental evaluation of the re-incorporation of bioavailable actinides into the biosphere from sediment reservoirs. We therefore anticipate this work will provide a tool and point of reference to improve radiation dose modelling and contribute insight for other environmental projects, such as the near-surface and deep disposal of nuclear waste.

Non-enzymatic hydrolysis of creatine ethyl ester.

The rate of the non-enzymatic hydrolysis of creatine ethyl ester (CEE) was studied at 37 degrees C over the pH range of 1.6-7.0 using (1)H NMR. The ester can be present in solution in three forms: the unprotonated form (CEE), the monoprotonated form (HCEE(+)), and the diprotonated form (H(2)CEE(2+)). The values of pK(a1) and pK(a2) of H(2)CEE(2+) were found to be 2.30 and 5.25, respectively. The rate law is found to be Rate=-dCCEE/dt=k++[H2CEE2+][OH-]+k+[HCEE+][OH-]+k0[CEE][OH-] where the rate constants k(++), k(+), and k(0) are (3.9+/-0.2)x10(6)L mol(-1)s(-1), (3.3+/-0.5)x10(4)L mol(-1)s(-1), and (4.9+/-0.3)x10(4)L mol(-1)s(-1), respectively. Calculations performed at the density functional theory level support the hypothesis that the similarity in the values of k(+) and k(0) results from intramolecular hydrogen bonding that plays a crucial role. This study indicates that the half-life of CEE in blood is on the order of one minute, suggesting that CEE may hydrolyze too quickly to reach muscle cells in its ester form.

Quantifying the medical student learning curve for ECG rhythm strip interpretation using deliberate practice.

Objectives: Obtaining competency in medical skills such as interpretation of electrocardiograms (ECGs) requires repeated practice and feedback. Structured repeated practice and feedback for ECGs is likely not provided to most medical students, so skill development is dependent on opportunistic training during clinical rotations. Our aim was to describe: the amount of deliberate practice completed for learning ECG rhythm strip diagnoses in first year medical students, the learning curve for rhythm strip diagnosis, and student experiences with deliberate practice. Methods: First year medical students from two medical schools were provided with online rhythm strip practice cases. Diagnostic accuracy was measured throughout practice, and students were provided feedback for every case they completed. Total cases practiced and time spent practicing were correlated with their performance during practice and on an exam. Results: 314 of 384 (82%) students consented. The mean number of ECGs each student practiced was 59 (range 0-280), representing 18,466 total instances of deliberate practice. We generated mathematical models that accurately correlated both the number of cases practiced and time spent practicing, with diagnostic accuracy on an exam (p<0.001). For example, students would need to spend on average of 112 minutes and complete 34 practice cases to obtain 75% on an ECG rhythm strip exam. Student satisfaction was high using the online cases. Conclusions: We succeeded in delivering deliberate practice for ECG rhythm strip interpretation to a large cohort of students at 2 medical schools. We quantified a learning curve that estimates the number of cases and practice time required to achieve pre-determined levels of diagnostic accuracy. This data can help inform a competency-based approach to curriculum development.

Cellular Aging Contributes to Failure of Cold-Induced Beige Adipocyte Formation in Old Mice and Humans.

Cold temperatures induce progenitor cells within white adipose tissue to form beige adipocytes that burn energy and generate heat; this is a potential anti-diabesity therapy. However, the potential to form cold-induced beige adipocytes declines with age. This creates a clinical roadblock to potential therapeutic use in older individuals, who constitute a large percentage of the obesity epidemic. Here we show that aging murine and human beige progenitor cells display a cellular aging, senescence-like phenotype that accounts for their age-dependent failure. Activating the senescence pathway, either genetically or pharmacologically, in young beige progenitors induces premature cellular senescence and blocks their potential to form cold-induced beige adipocytes. Conversely, genetically or pharmacologically reversing cellular aging by targeting the p38/MAPK-p16Ink4a pathway in aged mouse or human beige progenitor cells rejuvenates cold-induced beiging. This in turn increases glucose sensitivity. Collectively, these data indicate that anti-aging or senescence modalities could be a strategy to induce beiging, thereby improving metabolic health in aging humans.

Applying multivariate statistics to discriminate uranium ore concentrate geolocations using (radio)chemical data in support of nuclear forensic investigations.

The application of Principal Components Analysis (PCA) to U and Th series gamma spectrometry data provides a discriminatory tool to help determine the provenance of illicitly recovered uranium ore concentrates (UOCs). The PCA is applied to a database of radiometric signatures from 19 historic UOCs from Australia, Canada, and the USA representing many uranium geological deposits. In this study a key process to obtain accurate radiometric data (gamma and alpha) is to digest the U-ores and UOCs using a lithium tetraborate fusion. Six UOCs from the same sample set were analysed 'blind' and compared against the database to identify their geolocation. These UOCs were all accurately linked to their correct geolocations which can aid the forensic laboratory in determining which further analytical techniques should be used to improve the confidence of the particular location.

A rapid dissolution procedure to aid initial nuclear forensics investigations of chemically refractory compounds and particles prior to gamma spectrometry.

A rapid and effective preparative procedure has been evaluated for the accurate determination of low-energy (40-200 keV) gamma-emitting radionuclides ((210)Pb, (234)Th, (226)Ra, (235)U) in uranium ores and uranium ore concentrates (UOCs) using high-resolution gamma ray spectrometry. The measurement of low-energy gamma photons is complicated in heterogeneous samples containing high-density mineral phases and in such situations activity concentrations will be underestimated. This is because attenuation corrections, calculated based on sample mean density, do not properly correct where dense grains are dispersed within a less dense matrix (analogous to a nugget effect). The current method overcomes these problems using a lithium tetraborate fusion that readily dissolves all components including high-density, self-attenuating minerals/compounds. This is the ideal method for dissolving complex, non-volatile components in soils, rocks, mineral concentrates, and other materials where density reduction is required. Lithium borate fusion avoids the need for theoretical efficiency corrections or measurement of matrix matched calibration standards. The resulting homogeneous quenched glass produced can be quickly dissolved in nitric acid producing low-density solutions that can be counted by gamma spectrometry. The effectiveness of the technique is demonstrated using uranium-bearing Certified Reference Materials and provides accurate activity concentration determinations compared to the underestimated activity concentrations derived from direct measurements of a bulk sample. The procedure offers an effective solution for initial nuclear forensic studies where complex refractory minerals or matrices exist. It is also significantly faster, safer and simpler than alternative approaches.

Food allergy training event for restaurant staff; a pilot evaluation.

A previous cross-sectional survey highlighted that restaurant staff in Brighton had gaps in their knowledge of food allergy, which could lead to the provision of unsafe meals to food-allergic customers. A food allergy training event was developed by a multi-disciplinary team (health service researcher, clinician, teacher and patient group representative) to equip restaurant staff with the knowledge and skills necessary to safely serve food-allergic customers. This evaluation summarises the training event's impact on participants' knowledge of food allergy and their satisfaction with the event. No attendee had previously attended any formal training on food allergy. The percentage of participants who answered all true-false questions correctly increased from 82% before the training event to 91% afterwards. The percentage of participants who were able to name at least three common allergens increased from 9% to 64%. Both quantitative and qualitative feedback was positive. Restaurant staff require a good understanding of food allergy to ensure that food-allergic customers are kept safe, and their restaurants operate within the law. This food allergy training event improved participants' absolute knowledge of food allergy, and attendees changed practice. Recommendations are made which could improve the impact and uptake of future food allergy training events.

Unilateral absence of a pulmonary artery: a rare disorder with variable presentation.

Unilateral absence of a pulmonary artery (UAPA) is a rare condition with an estimated prevalence of 1 in 200,000 young adults. Most commonly, UAPA occurs in conjunction with cardiovascular abnormalities such as tetralogy of Fallot or cardiac septal defects, but it can also occur in an isolated manner. Patients with isolated UAPA can remain asymptomatic into late adulthood but usually report symptoms such as dyspnea or chest pain or suffer from hemoptysis or recurrent infections. Diagnosis can be difficult due to the rarity of the condition and its nonspecific presentation. We present a case of a 61-year-old man who presented for lung transplant evaluation and was found to have UAPA. Typical findings on chest radiograph, strategies for diagnosis, and available treatments are discussed.