Identification of Regulators of Polyploidization Presents Therapeutic Targets for Treatment of AMKL

The mechanism by which cells decide to skip mitosis to become polyploid is largely undefined. Here we used a high-content image-based screen to identify small-molecule probes that induce polyploidization of megakaryocytic leukemia cells and serve as perturbagens to help understand this process. Our?study implicates five networks of kinases that?regulate the switch to polyploidy. Moreover, we find that dimethylfasudil (diMF, H-1152P) selectively increased polyploidization, mature cell-surface marker expression, and apoptosis of malignant megakaryocytes. An integrated target identification approach employing proteomic and shRNA screening revealed that a major target of diMF is Aurora kinase A (AURKA). We further find that MLN8237 (Alisertib), a selective inhibitor of AURKA, induced polyploidization and expression of mature megakaryocyte markers in acute megakaryocytic leukemia (AMKL) blasts and displayed potent anti-AMKL activity in?vivo. Our findings provide a rationale to support clinical trials of MLN8237 and other inducers of polyploidization and differentiation in AMKL.     

A web server to locate periodicities in a sequence

Summary : FT is a tool written in C++, which implements the Fourier analysis method to locate periodicities in aminoacid or DNA sequences. It is provided for free public use on a WWW server with a Java interface. Availability : The server address is http://o2.db. uoa.gr/FT Contact : shamodr@atlas.uoa.gr      

Direct evidence for a critical role of myosin II in budding yeast cytokinesis and the evolvability of new cytokinetic mechanisms in the absence of myosin II

In the budding yeast Saccharomyces cerevisiae, an actomyosin-based contractile ring is present during cytokinesis, as occurs in animal cells. However, the precise requirement for this structure during budding yeast cytokinesis has been controversial. Here we show that deletion of MYO1, the single myosin II gene, is lethal in a commonly used strain background. The terminal phenotype of myo1Delta is interconnected chains of cells, suggestive of a cytokinesis defect. To further investigate the role of Myo1p in cytokinesis, we conditionally disrupted Myo1 function by using either a dominant negative Myo1p construct or a strain where expression of Myo1p can be shut-off. Both ways of disruption of Myo1 function result in a failure in cytokinesis. Additionally, we show that a myo1Delta strain previously reported to grow nearly as well as the wild type contains a single genetic suppressor that alleviates the severe cytokinesis defects of myo1Delta. Using fluorescence time-lapse imaging and electron microscopy techniques, we show that cytokinesis in this strain is achieved through formation of multiple aberrant septa. Taken together, these results strongly suggest that the actomyosin ring is crucial for successful cytokinesis in budding yeast, but new cytokinetic mechanisms can evolve through genetic changes when myosin II function is impaired.     

In situ hybridization at the electron microscope level: hybrid detection by autoradiography and colloidal gold

In situ hybridization has become a standard method for localizing DNA or RNA sequences in cytological preparations. We developed two methods to extend this technique to the transmission electron microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope in situ hybridization. Radioactively labeled complementary RNA (cRNA) is hybridized to metaphase chromosomes deposited on electron microscope grids and fixed in 70 percent ethanol vapor; hybridixation site are detected by autoradiography. Specific and intense labeling of chromosomal centromeric regions is observed even after relatively short exposure times. Inerphase nuclei present in some of the metaphase chromosome preparations also show defined paatterms of satellite DNA labeling which suggests that satellite-containing regions are associate with each other during interphase. The sensitivity of this method is estimated to at least as good as that at the light microscope level while the resolution is improved at least threefold. The second method, which circumvents the use of autoradiogrphic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction is improved at least threefold. The second method, which circumvents the use of autoradiographic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction with an antibody against biotin and secondary antibody adsorbed to the surface of over centromeric heterochromatin and along the associated peripheral fibers. Labeling is on average ten times that of background binding. This method is rapid and possesses the potential to allow precise ultrastructual localization of DNA sequences in chromosomes and chromatin.     

Rho1 directs formin-mediated actin ring assembly during budding yeast cytokinesis

In eukaryotic cells, dynamic rearrangement of the actin cytoskeleton is critical for cell division. In the yeast Saccharomyces cerevisiae, three main structures constitute the actin cytoskeleton: cortical actin patches, cytoplasmic actin cables, and the actin-based cytokinetic ring. The conserved Arp2/3 complex and a WASP-family protein mediate actin patch formation, whereas the yeast formins (Bni1 and Bnr1) promote assembly of actin cables. However, the mechanism of actin ring formation is currently unclear. Here, we show that actin filaments are required for cytokinesis in S. cerevisiae, and that the actin ring is a highly dynamic structure that undergoes constant turnover. Assembly of the actin ring requires the formin-like proteins and profilin, but is not Arp2/3-mediated. Furthermore, the formin-dependent actin ring assembly pathway is regulated by the Rho-type GTPase Rho1 but not Cdc42. Finally, we show that the formins are not required for localization of Cyk1/Iqg1, an IQGAP-like protein previously shown to be required for actin ring formation, suggesting that formin-like proteins and Cyk1 act synergistically but independently in assembly of the actin ring.     

A novel mechanism of autophagic cell death in dystrophic muscle regulated by P2RX7 receptor large-pore formation and HSP90

P2RX7 is an ATP-gated ion channel, which can also exhibit an open state with a considerably wider permeation. However, the functional significance of the movement of molecules through the large pore (LP) and the intracellular signaling events involved are not known. Here, analyzing the consequences of P2RX7 activation in primary myoblasts and myotubes from the Dmd^mdx mouse model of Duchenne muscular dystrophy, we found ATP-induced P2RX7-dependent autophagic flux, leading to CASP3-CASP7-independent cell death. P2RX7-evoked autophagy was triggered by LP formation but not Ca²⁺ influx or MAPK1-MAPK3 phosphorylation, 2 canonical P2RX7-evoked signals. Phosphoproteomics, protein expression inference and signaling pathway prediction analysis of P2RX7 signaling mediators pointed to HSPA2 and HSP90 proteins. Indeed, specific HSP90 inhibitors prevented LP formation, LC3-II accumulation, and cell death in myoblasts and myotubes but not in macrophages. Pharmacological blockade or genetic ablation of p2rx7 also proved protective against ATP-induced death of muscle cells, as did inhibition of autophagy with 3-MA. The functional significance of the P2RX7 LP is one of the great unknowns of purinergic signaling. Our data demonstrate a novel outcome—autophagy—and show that molecules entering through the LP can be targeted to phagophores. Moreover, we show that in muscles but not in macrophages, autophagy is needed for the formation of this LP. Given that P2RX7-dependent LP and HSP90 are critically interacting in the ATP-evoked autophagic death of dystrophic muscles, treatments targeting this axis could be of therapeutic benefit in this debilitating and incurable form of muscular dystrophy.     

Mannose-binding lectin deficiency is associated with early onset of polyarticular juvenile rheumatoid arthritis: a cohort study

Background  

Mannose-binding lectin (MBL) is an innate immune protein. The aim of our study was to determine whether genetically determined MBL deficiency is associated with susceptibility to juvenile rheumatoid arthritis (JRA) and whether MBL2 genotypes are associated with JRA severity.     

Cell-Based Assays for High-Throughput Screening

Cell-based assays represent approximately half of all high-throughput screens currently performed. Here, we review in brief the history and status of high-throughput screening (HTS), and summarize some of the challenges and benefits associated with the use of cell-based assays in HTS. Approaches for successful experimental design and execution of cell-based screens are introduced, including strategies for assay development, implementation of primary and secondary screens, and target identification. In doing so, we hope to provide a comprehensive review of the cell-based HTS process and an introduction to the methodologies and techniques used.