Bio-electrospraying primary cardiac cells: in vitro tissue creation and functional study

Manifestations of myocardial infarctions have been recognized as one of the major killers in the Western world. Therefore, advancing and developing novel cardiac tissue repair and replacement therapeutics have great implications to our health sciences and well-being. There are several approaches for forming cardiac tissues, non-jet-based and jet-based methodologies. A unique advantage of jet-based approaches is the possibility to handle living cells with a matrix for cell distribution and deposition in suspension, either as single or heterogeneous cell populations. Our previous studies on bio-electrospraying of cardiac cells have shown great promise. Here, we show for the first time the ability to bio-electrospray the three major cell types of the myocardium, both independently and simultaneously, for forming a fully functional cardiac tissue. Several samples are characterized in vitro and found to be indistinguishable in comparison to controls. Thus, we are describing a swiftly emerging novel biotechnique for direct cardiac tissue generation. Moreover, the present investigations pave the way for the development and optimization of a bio-patterning approach for the fabrication of biologically viable cardiac tissue grafts for the potential treatment of severe heart failure after myocardial infarction.     

Prediction of novel microRNA genes in cancer-associated genomic regions—a combined computational and experimental approach

The majority of existing computational tools rely on sequence homology and/or structural similarity to identify novel microRNA (miRNA) genes. Recently supervised algorithms are utilized to address this problem, taking into account sequence, structure and comparative genomics information. In most of these studies miRNA gene predictions are rarely supported by experimental evidence and prediction accuracy remains uncertain. In this work we present a new computational tool (SSCprofiler) utilizing a probabilistic method based on Profile Hidden Markov Models to predict novel miRNA precursors. Via the simultaneous integration of biological features such as sequence, structure and conservation, SSCprofiler achieves a performance accuracy of 88.95% sensitivity and 84.16% specificity on a large set of human miRNA genes. The trained classifier is used to identify novel miRNA gene candidates located within cancer-associated genomic regions and rank the resulting predictions using expression information from a full genome tiling array. Finally, four of the top scoring predictions are verified experimentally using northern blot analysis. Our work combines both analytical and experimental techniques to show that SSCprofiler is a highly accurate tool which can be used to identify novel miRNA gene candidates in the human genome. SSCprofiler is freely available as a web service at http://www.imbb.forth.gr/SSCprofiler.html.     

Cardiac release of urocortin precedes the occurrence of irreversible myocardial damage in the rat heart exposed to ischemia/reperfusion injury

This study evaluates whether cardiac ischemia induces release of urocortin, before and independently from myocyte cell death. Urocortin levels rose after 5-min ischemia and peaked after 10-min ischemia, when cell death was not detected. However, myocyte apoptosis and/or necrosis occurred following 20- and 30-min ischemia, which paralleled a fall in urocortin levels, suggesting that urocortin expression and release are mainly sustained by metabolically challenged, though still viable myocytes. Hence, since cardiac release of urocortin, unlike that of conventional biomarkers, occurs before and apart from cell death, urocortin levels may be clinically useful in the diagnosis of sublethal myocardial ischemia.     

Antiapoptotic activity of the free caspase recruitment domain of procaspase-9: a novel endogenous rescue pathway in cell death

Mitochondrial injury initiates proteolytic processing of procaspase-9 into the large and small subunits, leading to apoptotic cell death. Here we show that the free caspase recruitment domain (CARD) released by procaspase-9 processing activates nuclear factor kappaB expression. A procaspase-9 construct with a point mutation that abrogates the release of the CARD abolished nuclear factor kappaB activation. Most importantly, the free CARD is shown to enhance the expression of the gene encoding the antiapoptotic Bcl-x protein and to strongly inhibit apoptosis. This is the first demonstration that different domains of the same caspase protein have proapoptotic and antiapoptotic effects and suggests that the relative effects of these domains are important in regulating the balance between death and survival.     

MultiElec: A MATLAB Based Application for MEA Data Analysis

We present MultiElec, an open source MATLAB based application for data analysis of microelectrode array (MEA) recordings. MultiElec displays an extremely user-friendly graphic user interface (GUI) that allows the simultaneous display and analysis of voltage traces for 60 electrodes and includes functions for activation-time determination, the production of activation-time heat maps with activation time and isoline display. Furthermore, local conduction velocities are semi-automatically calculated along with their corresponding vector plots. MultiElec allows ad hoc signal suppression, enabling the user to easily and efficiently handle signal artefacts and for incomplete data sets to be analysed. Voltage traces and heat maps can be simply exported for figure production and presentation. In addition, our platform is able to produce 3D videos of signal progression over all 60 electrodes. Functions are controlled entirely by a single GUI with no need for command line input or any understanding of MATLAB code. MultiElec is open source under the terms of the GNU General Public License as published by the Free Software Foundation, version 3. Both the program and source code are available to download from http://www.cancer.manchester.ac.uk/MultiElec/.