The Virtual Ventricular Wall: A Tool for Exploring Cardiac Propagation and Arrhythmogenesis

Methods for the experimental and clinical investigation of cardiac arrhythmias are limited to inferring propagation within the myocardium, from surface measurements, or from electrodes at a few sites within the cardiac wall. Biophysically and anatomically detailed computational models of cardiac tissues offer a powerful way for studying the electrical propagation processes and arrhythmias within the virtual heart. We use virtual tissues to study and visualise the effects of patho- and physiological conditions, and pharmacological interventions on transmural propagation in the virtual ventricular walls. Class III drug actions are quantitatively explained by changes induced in the transmural dispersion of action potential duration. We illustrate the automated construction of a virtual anisotropic ventricle from Diffusion Tensor MRI for individual hearts, and use it to explore mechanisms leading to ventricular fibrillation. The virtual ventricular wall provides an effective tool for exploring, evaluating and visualising processes during the initiation and maintenance of ventricular arrhythmias.     

Properties of a Novel PBP2A Protein Homolog from Staphylococcus aureus Strain LGA251 and Its Contribution to the ��-Lactam-resistant Phenotype

Methicillin-resistant Staphylococcus aureus (MRSA) strains show strain-to-strain variation in resistance level, in genetic background, and also in the structure of the chromosomal cassette (SCCmec) that carries the resistance gene mecA. In contrast, strain-to-strain variation in the sequence of the mecA determinant was found to be much more limited among MRSA isolates examined so far. The first exception to this came with the recent identification of MRSA strain LGA251, which carries a new homolog of this gene together with regulatory elements mecI/mecR that also have novel, highly divergent structures. After cloning and purification in Escherichia coli, PBP2A_LGA, the protein product of the new mecA homolog, showed aberrant mobility in SDS-PAGE, structural instability and loss of activity at 37 °C, and a higher relative affinity for oxacillin as compared with cefoxitin. The mecA homolog free of its regulatory elements was cloned into a plasmid and introduced into the background of the β-lactam-susceptible S. aureus strain COL-S. In this background, the mecA homolog expressed a high-level resistance to cefoxitin (MIC = 400 μg/ml) and a somewhat lower resistance to oxacillin (minimal inhibitory concentration = 200 μg/ml). Similar to PBP2A, the protein homolog PBP2A_LGA was able to replace the essential function of the S. aureus PBP2 for growth. In contrast to PBP2A, PBP2A_LGA did not depend on the transglycosylase activity of the native PBP2 for expression of high level resistance to oxacillin, suggesting that the PBP2A homolog may preferentially cooperate with a monofunctional transglycosylase as the alternative source of transglycosylase activity.     

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.¹ Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.^2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.^6-7Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.     

Obesity-related metabolic dysfunction in dogs: a comparison with human metabolic syndrome

ABSTRACT: BACKGROUND: Recently, metabolic syndrome (MS) has gained attention in human metabolic medicine given its associations with development of type 2 diabetes mellitus and cardiovascular disease. Canine obesity is associated with the development of insulin resistance, dyslipidaemia, and mild hypertension, but the authors are not aware of any existing studies examining the existence or prevalence of MS in obese dogs.Thirty-five obese dogs were assessed before and after weight loss (median percentage loss 29%, range 10-44%). The diagnostic criteria of the International Diabetes Federation were modified in order to define canine obesity-related metabolic dysfunction (ORMD), which included a measure of adiposity (using a 9-point body condition score [BCS]), systolic blood pressure, fasting plasma cholesterol, plasma triglyceride, and fasting plasma glucose. By way of comparison, total body fat mass was measured by dual-energy X-ray absorptiometry, whilst total adiponectin, fasting insulin, and high-sensitivity C-reactive protein (hsCRP) were measured using validated assays. RESULTS: Systolic blood pressure (P = 0.008), cholesterol (P = 0.003), triglyceride (P = 0.018), and fasting insulin (P < 0.001) all decreased after weight loss, whilst plasma total adiponectin increased (P = 0.001). However, hsCRP did not change with weight loss. Prior to weight loss, 7 dogs were defined as having ORMD, and there was no difference in total fat mass between these dogs and those who did not meet the criteria for ORMD. However, plasma adiponectin concentration was less (P = 0.031), and plasma insulin concentration was greater (P = 0.030) in ORMD dogs. CONCLUSIONS: In this study, approximately 20% of obese dogs suffer from ORMD, and this is characterized by hypoadiponectinaemia and hyperinsulinaemia. These studies can form the basis of further investigations to determine path genetic mechanisms and the health significance for dogs, in terms of disease associations and outcomes of weight loss.     

Effect of beta2-adrenoceptor agonists and other cAMP-elevating agents on inflammatory gene expression in human ASM cells: a role for protein kinase A

In diseases such as asthma, airway smooth muscle (ASM) cells play a synthetic role by secreting inflammatory mediators such as granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, or IL-8 and by expressing surface adhesion molecules, including ICAM-1. In the present study, PGE(2), forskolin, and short-acting (salbutamol) and long-acting (salmeterol and formoterol) beta(2)-adrenoceptor agonists reduced the expression of ICAM-1 and the release of GM-CSF evoked by IL-1beta in ASM cells. IL-1beta-induced IL-8 release was also repressed by PGE(2) and forskolin, whereas the beta(2)-adrenoceptor agonists were ineffective. In each case, repression of these inflammatory indexes was prevented by adenoviral overexpression of PKIalpha, a highly selective PKA inhibitor. These data indicate a PKA-dependent mechanism of repression and suggest that agents that elevate intracellular cAMP, and thereby activate PKA, may have a widespread anti-inflammatory effect in ASM cells. Since ICAM-1 and GM-CSF are highly NF-kappaB-dependent genes, we used an adenoviral-delivered NF-kappaB-dependent luciferase reporter to examine the effects of forskolin and the beta(2)-adrenoceptor agonists on NF-kappaB activation. There was no effect on luciferase activity measured in the presence of forskolin or beta(2)-adrenoceptor agonists. This finding is consistent with the observation that IL-1beta-induced expression of IL-6, a known NF-kappaB-dependent gene in ASM, was also unaffected by beta(2)-adrenoceptor agonists, forskolin, PGE(2), 8-bromo-cAMP, or rolipram. Collectively, these results indicate that repression of IL-1beta-induced ICAM-1 expression and GM-CSF release by cAMP-elevating agents, including beta(2)-adrenoceptor agonists, may not occur through a generic effect on NF-kappaB.     

In vivo deuteration strategies for neutron scattering analysis of bacterial polyhydroxyoctanoate

The cultivation of microorganisms on deuterated substrates has allowed us to control deuterium incorporation into biopolymer systems which is important for characterisation using neutron scattering techniques. Bacterial polyhydroxyoctanoate (PHO) is a polyester formed within inclusions inside bacterial cells and was deuterated in vivo under various conditions to characterise the formation of these inclusions by neutron scattering. Manipulation of deuterated media during microbial growth and PHO production phases resulted in polymer with partial or complete substitution of hydrogen by deuterium, as shown by gas chromatography. Sequential feeding of hydrogenated and deuterated forms of the same precursor was used to demonstrate that neutron scattering analysis could be used to differentiate between chemically similar phases in these polymer inclusions.     

A novel streptococcal integrative conjugative element involved in iron acquisition

In this study, we determined the function of a novel non-ribosomal peptide synthetase (NRPS) system carried by a streptococcal integrative conjugative element (ICE), ICESe2. The NRPS shares similarity with the yersiniabactin system found in the high-pathogenicity island of Yersinia sp. and is the first of its kind to be identified in streptococci. We named the NRPS product 'equibactin' and genes of this locus eqbA-N. ICESe2, although absolutely conserved in Streptococcus equi, the causative agent of equine strangles, was absent from all strains of the closely related opportunistic pathogen Streptococcus zooepidemicus. Binding of EqbA, a DtxR-like regulator, to the eqbB promoter was increased in the presence of cations. Deletion of eqbA resulted in a small-colony phenotype. Further deletion of the irp2 homologue eqbE, or the genes eqbH, eqbI and eqbJ encoding a putative ABC transporter, or addition of the iron chelator nitrilotriacetate, reversed this phenotype, implicating iron toxicity. Quantification of (55)Fe accumulation and sensitivity to streptonigrin suggested that equibactin is secreted by S. equi and that the eqbH, eqbI and eqbJ genes are required for its associated iron import. In agreement with a structure-based model of equibactin synthesis, supplementation of chemically defined media with salicylate was required for equibactin production.