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Recent Publications

A 17-gene stemness score for rapid determination of risk in acute leukaemia.

A 17-gene stemness score for rapid determination of risk in acute leukaemia.

Nature. 2016 Dec 07;:

Authors: Ng SW, Mitchell A, Kennedy JA, Chen WC, McLeod J, Ibrahimova N, Arruda A, Popescu A, Gupta V, Schimmer AD, Schuh AC, Yee KW, Bullinger L, Herold T, Görlich D, Büchner T, Hiddemann W, Berdel WE, Wörmann B, Cheok M, Preudhomme C, Dombret H, Metzeler K, Buske C, Löwenberg B, Valk PJ, Zandstra PW, Minden MD, Dick JE, Wang JC

Abstract
Refractoriness to induction chemotherapy and relapse after achievement of remission are the main obstacles to cure in acute myeloid leukaemia (AML). After standard induction chemotherapy, patients are assigned to different post-remission strategies on the basis of cytogenetic and molecular abnormalities that broadly define adverse, intermediate and favourable risk categories. However, some patients do not respond to induction therapy and another subset will eventually relapse despite the lack of adverse risk factors. There is an urgent need for better biomarkers to identify these high-risk patients before starting induction chemotherapy, to enable testing of alternative induction strategies in clinical trials. The high rate of relapse in AML has been attributed to the persistence of leukaemia stem cells (LSCs), which possess a number of stem cell properties, including quiescence, that are linked to therapy resistance. Here, to develop predictive and/or prognostic biomarkers related to stemness, we generated a list of genes that are differentially expressed between 138 LSC(+) and 89 LSC(-) cell fractions from 78 AML patients validated by xenotransplantation. To extract the core transcriptional components of stemness relevant to clinical outcomes, we performed sparse regression analysis of LSC gene expression against survival in a large training cohort, generating a 17-gene LSC score (LSC17). The LSC17 score was highly prognostic in five independent cohorts comprising patients of diverse AML subtypes (n = 908) and contributed greatly to accurate prediction of initial therapy resistance. Patients with high LSC17 scores had poor outcomes with current treatments including allogeneic stem cell transplantation. The LSC17 score provides clinicians with a rapid and powerful tool to identify AML patients who do not benefit from standard therapy and who should be enrolled in trials evaluating novel upfront or post-remission strategies.

PMID: 27926740 [PubMed - as supplied by publisher]



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Genome-wide changes in lncRNA, splicing, and regional gene expression patterns in autism.

Genome-wide changes in lncRNA, splicing, and regional gene expression patterns in autism.

Nature. 2016 Dec 05;:

Authors: Parikshak NN, Swarup V, Belgard TG, Irimia M, Ramaswami G, Gandal MJ, Hartl C, Leppa V, Ubieta LT, Huang J, Lowe JK, Blencowe BJ, Horvath S, Geschwind DH

Abstract
Autism spectrum disorder (ASD) involves substantial genetic contributions. These contributions are profoundly heterogeneous but may converge on common pathways that are not yet well understood. Here, through post-mortem genome-wide transcriptome analysis of the largest cohort of samples analysed so far, to our knowledge, we interrogate the noncoding transcriptome, alternative splicing, and upstream molecular regulators to broaden our understanding of molecular convergence in ASD. Our analysis reveals ASD-associated dysregulation of primate-specific long noncoding RNAs (lncRNAs), downregulation of the alternative splicing of activity-dependent neuron-specific exons, and attenuation of normal differences in gene expression between the frontal and temporal lobes. Our data suggest that SOX5, a transcription factor involved in neuron fate specification, contributes to this reduction in regional differences. We further demonstrate that a genetically defined subtype of ASD, chromosome 15q11.2-13.1 duplication syndrome (dup15q), shares the core transcriptomic signature observed in idiopathic ASD. Co-expression network analysis reveals that individuals with ASD show age-related changes in the trajectory of microglial and synaptic function over the first two decades, and suggests that genetic risk for ASD may influence changes in regional cortical gene expression. Our findings illustrate how diverse genetic perturbations can lead to phenotypic convergence at multiple biological levels in a complex neuropsychiatric disorder.

PMID: 27919067 [PubMed - as supplied by publisher]



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Proteomic analysis of the human KEOPS complex identifies C14ORF142 as a core subunit homologous to yeast Gon7.

Related Articles

Proteomic analysis of the human KEOPS complex identifies C14ORF142 as a core subunit homologous to yeast Gon7.

Nucleic Acids Res. 2016 Nov 29;:

Authors: Wan LC, Maisonneuve P, Szilard RK, Lambert JP, Ng TF, Manczyk N, Huang H, Laister R, Caudy AA, Gingras AC, Durocher D, Sicheri F

Abstract
The KEOPS/EKC complex is a tRNA modification complex involved in the biosynthesis of N(6)-threonylcarbamoyladenosine (t(6)A), a universally conserved tRNA modification found on ANN-codon recognizing tRNAs. In archaea and eukaryotes, KEOPS is composed of OSGEP/Kae1, PRPK/Bud32, TPRKB/Cgi121 and LAGE3/Pcc1. In fungi, KEOPS contains an additional subunit, Gon7, whose orthologs outside of fungi, if existent, remain unidentified. In addition to displaying defective t(6)A biosynthesis, Saccharomyces cerevisiae strains harboring KEOPS mutations are compromised for telomere homeostasis, growth and transcriptional co-activation. To identify a Gon7 ortholog in multicellular eukaryotes as well as to uncover KEOPS-interacting proteins that may link t(6)A biosynthesis to the diverse set of KEOPS mutant phenotypes, we conducted a proteomic analysis of human KEOPS. This work identified 152 protein interactors, one of which, C14ORF142, interacted strongly with all four KEOPS subunits, suggesting that it may be a core component of human KEOPS. Further characterization of C14ORF142 revealed that it shared a number of biophysical and biochemical features with fungal Gon7, suggesting that C14ORF142 is the human ortholog of Gon7. In addition, our proteomic analysis identified specific interactors for different KEOPS subcomplexes, hinting that individual KEOPS subunits may have additional functions outside of t(6)A biosynthesis.

PMID: 27903914 [PubMed - as supplied by publisher]



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Mitochondrial control through nutritionally regulated global histone H3 lysine-4 demethylation.

Mitochondrial control through nutritionally regulated global histone H3 lysine-4 demethylation.

Sci Rep. 2016 Nov 29;6:37942

Authors: Soloveychik M, Xu M, Zaslaver O, Lee K, Narula A, Jiang R, Rosebrock AP, Caudy AA, Meneghini MD

Abstract
Histone demethylation by Jumonji-family proteins is coupled with the decarboxylation of α-ketoglutarate (αKG) to yield succinate, prompting hypotheses that their activities are responsive to levels of these metabolites in the cell. Consistent with this paradigm we show here that the Saccharomyces cerevisiae Jumonji demethylase Jhd2 opposes the accumulation of H3K4me3 in fermenting cells only when they are nutritionally manipulated to contain an elevated αKG/succinate ratio. We also find that Jhd2 opposes H3K4me3 in respiratory cells that do not exhibit such an elevated αKG/succinate ratio. While jhd2∆ caused only limited gene expression defects in fermenting cells, transcript profiling and physiological measurements show that JHD2 restricts mitochondrial respiratory capacity in cells grown in non-fermentable carbon in an H3K4me-dependent manner. In association with these phenotypes, we find that JHD2 limits yeast proliferative capacity under physiologically challenging conditions as measured by both replicative lifespan and colony growth on non-fermentable carbon. JHD2's impact on nutrient response may reflect an ancestral role of its gene family in mediating mitochondrial regulation.

PMID: 27897198 [PubMed - in process]



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Brief Report: Stage-specific differences in secretory profile of mesenchymal stromal cells (MSCs) subjected to early-vs. late-stage OA synovial fluid.

Brief Report: Stage-specific differences in secretory profile of mesenchymal stromal cells (MSCs) subjected to early-vs. late-stage OA synovial fluid.

Osteoarthritis Cartilage. 2016 Nov 25;:

Authors: Gómez-Aristizábal A, Sharma A, Bakooshli MA, Kapoor M, Gilbert PM, Viswanathan S, Gandhi R

Abstract
OBJECTIVE: Although, mesenchymal stromal cells (MSCs) are being clinically investigated for their use in osteoarthritis (OA), it is unclear whether their postulated therapeutic properties are equally effective in the early- and late-stages of OA. In this study we investigated MSC cytokine secretion post-exposure to synovial fluid (SF), obtained from early-vs. late-stage knee OA patients to justify a potential patient stratification strategy to maximize MSC-mediated treatment effects.
METHOD: Subjects were recruited and categorized into early- [Kellgren-Lawrence (KL) grade I/II, n = 12] and late-stage (KL-III/IV, n = 12) knee OA groups. SF samples were obtained, and their proteome was tested using multiplex assays, after three-days culture, with and without MSCs. SFs cultured without MSCs were used as a baseline to identify MSC-secreted factors into SFs cultured with MSCs. Linear mixed-effect models and non-parametric tests were used to identify alterations in the MSC secretome during exposure to OA SF (three-days). MSCs cultured for three-days in 0.5% fetal bovine serum-supplemented medium were used to compare SF results with culture medium.
RESULTS: Following exposure to OA SF, the MSC secretome contained proteins that are involved in tissue repair, angiogenesis, chemotaxis, matrix remodeling and the clotting process. However, CXCL8 (chemokine (C-X-C motif) ligand-8; chemoattractant), interleukin-6 and CCL2 (chemokine (C-C motif) ligand 2) were elevated in the MSC-secretome in response to early-vs. late-stage OA SF.
CONCLUSION: Early-vs. late-stage OA SF samples elicit a differential MSC secretome response, arguing for stratification of OA patients to maximize MSC-mediated therapeutic effects.

PMID: 27894935 [PubMed - as supplied by publisher]



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A Highly Diverse and Functional Naïve Ubiquitin Variant Library for Generation of Intracellular Affinity Reagents.

A Highly Diverse and Functional Naïve Ubiquitin Variant Library for Generation of Intracellular Affinity Reagents.

J Mol Biol. 2016 Nov 22;:

Authors: Leung I, Jarvik N, Sidhu S

Abstract
We report the design, construction and validation of a highly diverse phage-displayed naïve ubiquitin variant (Ubv) library. We first conducted a mutation tolerance scan of 27 residues and confirmed that 24 of these could be substituted by chemically diverse amino acids without compromising the display of Ubvs on phage. Subsequently, we constructed a library containing 6.8×10(10) unique members in which these 24 positions were diversified with a degenerate codon that encodes for six amino acids that are prevalent in protein interaction sites. To ensure optimal structural stability of the Ubvs, the library was constructed in a two-step process whereby 12 positions were randomized first, and following selection for displayed Ubvs, the resulting pool was further diversified at the other 12 positions. The resulting library was validated by conducting binding selections against a panel of 40 diverse protein antigens and was found to be as functional as a highly validated synthetic antibody library, yielding binders against 30 of the antigens. Detailed characterization of an Ubv that bound to the cell-surface receptor Her3 revealed tight binding in the single-digit nanomolar range. Moreover, Ubvs that bound to two distinct sites on the intracellular adapter Grb2 could be combined to generate a potent inhibitor that functioned in cells. These results validate ubiquitin as a robust scaffold for the construction of naïve libraries that can be used to generate Ubvs that target signaling networks both outside and inside cells.

PMID: 27887869 [PubMed - as supplied by publisher]



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Protein-phosphotyrosine proteome profiling by superbinder-SH2 domain affinity purification mass spectrometry, sSH2-AP-MS.

Protein-phosphotyrosine proteome profiling by superbinder-SH2 domain affinity purification mass spectrometry, sSH2-AP-MS.

Proteomics. 2016 Nov 23;:

Authors: Tong J, Cao B, Martyn GD, Krieger JR, Taylor P, Yates B, Sidhu SS, Li SS, Mao X, Moran MF

Abstract
Recently, "superbinder" SH2 domain variants with three amino acid substitutions (sSH2) were reported to have 100-fold or greater affinity for protein-phosphotyrosine (pY) than natural SH2 domains. Here we report a protocol in which His-tagged Src sSH2 efficiently captures pY-peptides from protease-digested HeLa cell total protein extracts. Affinity purification of pY-peptides by this method shows little bias for pY-proximal amino acid sequences, comparable to that achieved by using antibodies to pY, but with equal or higher yield. Superbinder-SH2 affinity purification mass spectrometry (sSH2-AP-MS) therefore provides an efficient and economical approach for unbiased pY-directed phospho-proteome profiling without the use of antibodies. This article is protected by copyright. All rights reserved.

PMID: 27880036 [PubMed - as supplied by publisher]



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Large-Scale Interaction Profiling of Protein Domains Through Proteomic Peptide-Phage Display Using Custom Peptidomes.

Related Articles

Large-Scale Interaction Profiling of Protein Domains Through Proteomic Peptide-Phage Display Using Custom Peptidomes.

Methods Mol Biol. 2017;1518:213-226

Authors: Seo MH, Nim S, Jeon J, Kim PM

Abstract
Protein-protein interactions are essential to cellular functions and signaling pathways. We recently combined bioinformatics and custom oligonucleotide arrays to construct custom-made peptide-phage libraries for screening peptide-protein interactions, an approach we call proteomic peptide-phage display (ProP-PD). In this chapter, we describe protocols for phage display for the identification of natural peptide binders for a given protein. We finally describe deep sequencing for the analysis of the proteomic peptide-phage display.

PMID: 27873209 [PubMed - in process]



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Genome-wide CRISPR screens reveal a Wnt-FZD5 signaling circuit as a druggable vulnerability of RNF43-mutant pancreatic tumors.

Genome-wide CRISPR screens reveal a Wnt-FZD5 signaling circuit as a druggable vulnerability of RNF43-mutant pancreatic tumors.

Nat Med. 2016 Nov 21;:

Authors: Steinhart Z, Pavlovic Z, Chandrashekhar M, Hart T, Wang X, Zhang X, Robitaille M, Brown KR, Jaksani S, Overmeer R, Boj SF, Adams J, Pan J, Clevers H, Sidhu S, Moffat J, Angers S

Abstract
Forward genetic screens with CRISPR-Cas9 genome editing enable high-resolution detection of genetic vulnerabilities in cancer cells. We conducted genome-wide CRISPR-Cas9 screens in RNF43-mutant pancreatic ductal adenocarcinoma (PDAC) cells, which rely on Wnt signaling for proliferation. Through these screens, we discovered a unique requirement for a Wnt signaling circuit: engaging FZD5, one of the ten Frizzled receptors encoded in the human genome. Our results uncover an underappreciated level of context-dependent specificity at the Wnt receptor level. We further derived a panel of recombinant antibodies that reports the expression of nine FZD proteins and confirms that FZD5 functional specificity cannot be explained by protein expression patterns. Additionally, antibodies that specifically bind FZD5 and FZD8 robustly inhibited the growth of RNF43-mutant PDAC cells grown in vitro and as xenografts in vivo, providing orthogonal support for the functional specificity observed genetically. Proliferation of a patient-derived PDAC cell line harboring an RNF43 variant was also selectively inhibited by the FZD5 antibodies, further demonstrating their use as a potential targeted therapy. Tumor organoid cultures from colorectal carcinoma patients that carried RNF43 mutations were also sensitive to the FZD5 antibodies, highlighting the potential generalizability of these findings beyond PDAC. Our results show that CRIPSR-based genetic screens can be leveraged to identify and validate cell surface targets for antibody development and therapy.

PMID: 27869803 [PubMed - as supplied by publisher]



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Engineering cell signaling modulators from native protein-protein interactions.

Engineering cell signaling modulators from native protein-protein interactions.

Curr Opin Struct Biol. 2016 Nov 17;45:25-35

Authors: Zhang W, Ben-David M, Sidhu SS

Abstract
Recent studies on genome sequencing and genetic screens with RNAi and CRISPR technology have revolutionized our understanding of aberrant signaling networks in human diseases. A strategy combining both genetic and protein-based technologies should be at the heart of modern drug-development efforts, particularly in the era of precision medicine. Thus, there is an urgent need for efficient platforms to develop probes that can modulate protein function in cells to validate drug targets and to develop therapeutic leads. Advanced protein engineering has enabled the rapid production of monoclonal antibodies and small protein scaffold affinity reagents for diverse protein targets. Here, we review the most recent progress on engineering natural protein-protein interactions for modulation of cell signaling.

PMID: 27866084 [PubMed - as supplied by publisher]



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