Cardiovascular disease is the primary cause of morbidity and mortality in the United States. The majority of patients who survive an initial myocardial infarction (M.I.) do not have a positive prognosis due to the increased work load that is placed upon the heart and this is compounded by the inability to regenerate the lost myocardium. Heart failure therefore is at its essence a loss of cardiomyocytes. This problem has stimulated an intense research effort to develop the means to replace the damaged or dead cardiomyocytes with transplanted cells, such as human Embryonic Stem Cell Derived Cardiomyocytes (hESCDCs) or through expansion of putative adult cardiomyocytes, progenitors or stem cells. In the current study we utilized Invitrogen's microRNA (miR) microarrays to examine the miR profile of hESCDCs in order to identify novel miRs that regulate the process of differentiation to the cardiomyocyte lineage in addition to miRs that are involved in maintaining the proliferative capacity of immature hESCDCs. With the same tools we are also analyzing Neonatal Rat Ventricular Cardiomyocytes (NRVCs) that have been induced to re-enter the cell cycle through over-expression of components of the Notch pathway. Here, we are looking also for novel miRs that are involved in Notch replication of NRVC cell cycle, either as agonists or antagonists. Identification of such miRs could increase our understanding of cardiomyocytes cell cycle and might lead to strategies to increase the pool of cardiomyogenic cells for therapeutic purposes. From these microarrays, we have identified several miRs that are involved in the processes before mentioned and are therefore being characterized functionally by means of high throughput screens and other studies to determine relevant targets

The mechanism of permanganate-mediated dual C-H oxidation of complex diketopiperazines has been examined with density functional theory computations. The products of these oxidations are enabling intermediates in the synthesis of structurally diverse ETP natural products. We evaluated, for the first time, the impact of ion-pairing and aggregation states of the permanganate ion and counter-cations, such as bis(pyridine)-silver(I) (Ag⁺) and tetra- n-butylammonium (TBA⁺), on the C-H oxidation mechanism. The C-H abstraction occurs through an open shell singlet species, as noted previously, followed by O-rebound and a competing OH-rebound pathway. The second C-H oxidation proceeds with a second equivalent of oxidant with lower free energy barriers than the first C-H oxidation due to directing effects and the generation of a more reactive oxidant species after the first C-H oxidation. The success and efficiency of the second C-H oxidation are found to be critically dependent on the presence of an ion-paired oxidant. We used the developed mechanistic knowledge to rationalize an experimentally observed oxidation pattern for C³-indole-substituted diketopiperazine (+)-5 under optimal oxidation conditions: namely, the formation of diol (-)-6 as a single diastereomer and lack of the ketone products. We proposed two factors that may impede the ketone formation: (i) the conformational flexibility of the diketopiperazine ring, and (ii) hindrance of this site, making it less accessible to the ion-paired oxidant species.

The role of alternative splicing in self-renewal, pluripotency and tissue lineage specification of human embryonic stem cells (hESCs) is largely unknown. To better define these regulatory cues, we modified the H9 hESC line to allow selection of pluripotent hESCs by neomycin resistance and cardiac progenitors by puromycin resistance. Exon-level microarray expression data from undifferentiated hESCs and cardiac and neural precursors were used to identify splice isoforms with cardiac-restricted or common cardiac/neural differentiation expression patterns. Splice events for these groups corresponded to the pathways of cytoskeletal remodeling, RNA splicing, muscle specification, and cell cycle checkpoint control as well as genes with serine/threonine kinase and helicase activity. Using a new program named AltAnalyze (http://www.AltAnalyze.org), we identified novel changes in protein domain and microRNA binding site architecture that were predicted to affect protein function and expression. These included an enrichment of splice isoforms that oppose cell-cycle arrest in hESCs and that promote calcium signaling and cardiac development in cardiac precursors. By combining genome-wide predictions of alternative splicing with new functional annotations, our data suggest potential mechanisms that may influence lineage commitment and hESC maintenance at the level of specific splice isoforms and microRNA regulation.