Clustering of Nck by a 12-residue Tir phosphopeptide is sufficient to trigger localized actin assembly

Enteropathogenic Escherichia coli (EPEC) translocates effector proteins into mammalian cells to promote reorganization of the cytoskeleton into filamentous actin pedestals. One effector, Tir, is a transmembrane receptor for the bacterial surface adhesin intimin, and intimin binding by the extracellular domain of Tir is required for actin assembly. The cytoplasmic NH2 terminus of Tir interacts with focal adhesion proteins, and its tyrosine-phosphorylated COOH terminus binds Nck, a host adaptor protein critical for pedestal formation. To define the minimal requirements for EPEC-mediated actin assembly, Tir derivatives were expressed in mammalian cells in the absence of all other EPEC components. Replacement of the NH2 terminus of Tir with a viral membrane-targeting sequence promoted efficient surface expression of a COOH-terminal Tir fragment. Artificial clustering of this fusion protein revealed that the COOH terminus of Tir, by itself, is sufficient to initiate a complete signaling cascade leading to pedestal formation. Consistent with this finding, clustering of Nck by a 12-residue Tir phosphopeptide triggered actin tail formation in Xenopus egg extracts

SC3: consensus clustering of single-cell RNA-seq data

Single-cell RNA-seq enables the quantitative characterization of cell types based on global transcriptome profiles. We present single-cell consensus clustering (SC3), a user-friendly tool for unsupervised clustering, which achieves high accuracy and robustness by combining multiple clustering solutions through a consensus approach (http://bioconductor.org/packages/SC3). We demonstrate that SC3 is capable of identifying subclones from the transcriptomes of neoplastic cells collected from patients.V.Y.K., T.A., A.Y. and M.H. are supported by Wellcome Trust Grants. K.N.N. is supported by the Wellcome Trust Strategic Award 'Single cell genomics of mouse gastrulation'. M.T.S. acknowledges support from FRS-FNRS; the Belgian Network DYSCO (Dynamical Systems, Control and Optimisation), funded by the Interuniversity Attraction Poles Programme initiated by the Belgian State Science Policy Office; and the ARC (Action de Recherche Concerte) on Mining and Optimization of Big Data Models, funded by the Wallonia-Brussels Federation. M.B. acknowledges support from EPSRC (grant EP/N014529/1). T.C. was funded through a core funded fellowship by the Sanger Institute and a Chancellor′s fellowship from the University of Edinburgh. K.K. and A.R.G. are supported by Bloodwise (grant ref. 13003), the Wellcome Trust (grant ref. 104710/Z/14/Z), the Medical Research Council, the Kay Kendall Leukaemia Fund, the Cambridge NIHR Biomedical Research Center, the Cambridge Experimental Cancer Medicine Centre, the Leukemia and Lymphoma Society of America (grant ref. 07037) and a core support grant from the Wellcome Trust and MRC to the Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute. W.R. was supported by BBSRC (grant ref. BB/K010867/1), the Wellcome Trust (grant ref. 095645/Z/11/Z), EU BLUEPRINT and EpiGeneSys

Ruxolitinib-induced defects in DNA repair cause sensitivity to PARP inhibitors in myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPNs) often carry JAK2(V617F), MPL(W515L), or CALR(del52) mutations. Current treatment options for MPNs include cytoreduction by hydroxyurea and JAK1/2 inhibition by ruxolitinib, both of which are not curative. We show here that cell lines expressing JAK2(V617F), MPL(W515L), or CALR(del52) accumulated reactive oxygen species-induced DNA double-strand breaks (DSBs) and were modestly sensitive to poly-ADP-ribose polymerase (PARP) inhibitors olaparib and BMN673. At the same time, primary MPN cell samples from individual patients displayed a high degree of variability in sensitivity to these drugs. Ruxolitinib inhibited 2 major DSB repair mechanisms, BRCA-mediated homologous recombination and DNA-dependent protein kinase-mediated nonhomologous end-joining, and, when combined with olaparib, caused abundant accumulation of toxic DSBs resulting in enhanced elimination of MPN primary cells, including the disease-initiating cells from the majority of patients. Moreover, the combination of BMN673, ruxolitinib, and hydroxyurea was highly effective in vivo against JAK2(V617F)+ murine MPN-like disease and also against JAK2(V617F)+, CALR(del52)+, and MPL(W515L)+ primary MPN xenografts. In conclusion, we postulate that ruxolitinib-induced deficiencies in DSB repair pathways sensitized MPN cells to synthetic lethality triggered by PARP inhibitors

Multiplexing for Oxidative Bisulfite Sequencing (oxBS-seq).

DNA modifications, especially methylation, are known to play a crucial part in many regulatory processes in the cell. Recently, 5-hydroxymethylcytosine (5hmC) was discovered, a DNA modification derived as an intermediate of 5-methylcytosine (5mC) oxidation. Efforts to gain insights into function of this DNA modification are underway and several methods were recently described to assess 5hmC levels using sequencing approaches. Here we integrate adaptation based multiplexing and high-efficiency library prep into the oxidative Bisulfite Sequencing (oxBS-seq) workflow reducing the starting amount and cost per sample to identify 5hmC levels genome-wide

Producing *Class: Indie Rock and Cultural Studies

229 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2002.This study examines the operational logic of the indie rock formation. Indie rock, a relatively autonomous sphere of rock culture, is defined as a cultural formation. Using a cultural studies approach I show how the operational logic (the complex articulation of forces that organize cultural formations) of indie rock produces class elitism in its participants. Chapter one introduces indie rock as a cultural formation and cultural studies as an interdisciplinary intellectual practice. Chapter two critiques interpretive models of academic popular music scholarship, arguing that a contextually-based, analytical method is more useful for cultural studies. Chapter three deals with theoretical and methodological concerns, promoting cosmopolitan ethnography as the best way to understand the material forces that structure the indie rock formation. Chapter four is a Foucauldian genealogy of indie rock that reconstructs the contingent set of contextual conditions that produce the indie rock formation. Chapter five details the operational logic of the indie rock formation, demonstrating how it produces class difference. Chapter six uses ethnographic data to understand the status of gender in the indie rock formation.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD