Action Potential Morphology of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Does Not Predict Cardiac Chamber Specificity and Is Dependent on Cell Density

Previous studies investigating human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have proposed the distinction of heart chamber-specific (atrial, ventricular, pacemaker) electrophysiological phenotypes based on action potential (AP) morphology. This suggestion has been based on data acquired using techniques that allow measurements from only a small number of cells and at low seeding densities. It has also been observed that density of culture affects the properties of iPSC-CMs. Here we systematically analyze AP morphology from iPSC-CMs at two seeding densities: 60,000 cells/well (confluent monolayer) and 15,000 cells/well (sparsely-seeded) using a noninvasive optical method. The confluent cells (n = 360) demonstrate a series of AP morphologies on a normally distributed spectrum with no evidence for specific subpopulations. The AP morphologies of sparsely seeded cells (n = 32) displayed a significantly different distribution, but even in this case there is no clear evidence of chamber-specificity. Reduction in gap junction conductance using carbenoxolone only minimally affected APD distribution in confluent cells. These data suggest that iPSC-CMs possess a sui generis AP morphology, and when observed in different seeding densities may encompass any shape including those resembling chamber-specific subtypes. These results may be explained by different functional maturation due to culture conditions.     

How the Location of Superoxide Generation Influences the β-Cell Response to Nitric Oxide

Cytokines impair the function and decrease the viability of insulin-producing β-cells by a pathway that requires the expression of inducible nitric oxide synthase (iNOS) and generation of high levels of nitric oxide. In addition to nitric oxide, excessive formation of reactive oxygen species, such as superoxide and hydrogen peroxide, has been shown to cause β-cell damage. Although the reaction of nitric oxide with superoxide results in the formation of peroxynitrite, we have shown that β-cells do not have the capacity to produce this powerful oxidant in response to cytokines. When β-cells are forced to generate peroxynitrite using nitric oxide donors and superoxide-generating redox cycling agents, superoxide scavenges nitric oxide and prevents the inhibitory and destructive actions of nitric oxide on mitochondrial oxidative metabolism and β-cell viability. In this study, we show that the β-cell response to nitric oxide is regulated by the location of superoxide generation. Nitric oxide freely diffuses through cell membranes, and it reacts with superoxide produced within cells and in the extracellular space, generating peroxynitrite. However, only when it is produced within cells does superoxide attenuate nitric oxide-induced mitochondrial dysfunction, gene expression, and toxicity. These findings suggest that the location of radical generation and the site of radical reactions are key determinants in the functional response of β-cells to reactive oxygen species and reactive nitrogen species. Although nitric oxide is freely diffusible, its biological function can be controlled by the local generation of superoxide, such that when this reaction occurs within β-cells, superoxide protects β-cells by scavenging nitric oxide.     

The identification of new metallo-β-lactamase inhibitor leads from fragment-based screening

The emergence of metallo-β-lactamases (MBLs) capable of hydrolysing a broad spectrum of β-lactam antibiotics is particularly concerning for the future treatment of bacterial infections. This work describes the discovery of lead compounds for the development of new inhibitors using a competitive colorimetric assay based on the chromogenic cephalosporin CENTA, and a 500 compound Maybridge™ library suitable for fragment-based screening. The interactions between identified inhibitory fragments and the active site of the MBL from Klebsiella pneumoniae and Pseudomonas aeruginosa were probed by in silico docking studies.     

CREST maps somatic structural variation in cancer genomes with base-pair resolution

We developed 'clipping reveals structure' (CREST), an algorithm that uses next-generation sequencing reads with partial alignments to a reference genome to directly map structural variations at the nucleotide level of resolution. Application of CREST to whole-genome sequencing data from five pediatric T-lineage acute lymphoblastic leukemias (T-ALLs) and a human melanoma cell line, COLO-829, identified 160 somatic structural variations. Experimental validation exceeded 80%, demonstrating that CREST had a high predictive accuracy.