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Crispr primary t cell

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https://www.readbyqxmd.com/read/28344889/loss-of-tapasin-in-human-lung-and-colon-cancer-cells-and-escape-from-tumor-associated-antigen-specific-ctl-recognition
#1
Yosuke Shionoya, Takayuki Kanaseki, Sho Miyamoto, Serina Tokita, Ayumi Hongo, Yasuhiro Kikuchi, Vitaly Kochin, Kazue Watanabe, Ryota Horibe, Hiroshi Saijo, Tomohide Tsukahara, Yoshihiko Hirohashi, Hiroki Takahashi, Noriyuki Sato, Toshihiko Torigoe
Cytotoxic T-lymphocytes (CTLs) lyse target cells after recognizing the complexes of peptides and MHC class I molecules (pMHC I) on cell surfaces. Tapasin is an essential component of the peptide-loading complex (PLC) and its absence influences the surface repertoire of MHC class I peptides. In the present study, we assessed tapasin expression in 85 primary tumor lesions of non-small cell lung cancer (NSCLC) patients, demonstrating that tapasin expression positively correlated with patient survival. CD8(+) T-cell infiltration of tumor lesions was synergistically observed with tapasin expression and correlated positively with survival...
2017: Oncoimmunology
https://www.readbyqxmd.com/read/28246360/multiple-histone-lysine-methyltransferases-are-required-for-the-establishment-and-maintenance-of-hiv-1-latency
#2
Kien Nguyen, Biswajit Das, Curtis Dobrowolski, Jonathan Karn
We showed previously that the histone lysine methyltransferase (HKMT) H3K27me3 (EZH2) is the catalytic subunit of Polycomb repressive complex 2 (PRC2) and is required for the maintenance of HIV-1 latency in Jurkat T cells. Here we show, by using chromatin immunoprecipitation experiments, that both PRC2 and euchromatic histone-lysine N-methyltransferase 2 (EHMT2), the G9a H3K9me2-3 methyltransferase, are highly enriched at the proviral 5' long terminal repeat (LTR) and rapidly displaced upon proviral reactivation...
February 28, 2017: MBio
https://www.readbyqxmd.com/read/28152074/multiple-ubxn-family-members-inhibit-retrovirus-and-lentivirus-production-and-canonical-nf%C3%AE%C2%BA%C3%AE-signaling-by-stabilizing-i%C3%AE%C2%BAb%C3%AE
#3
Yani Hu, Kaitlin O'Boyle, Jim Auer, Sagar Raju, Fuping You, Penghua Wang, Erol Fikrig, Richard E Sutton
UBXN proteins likely participate in the global regulation of protein turnover, and we have shown that UBXN1 interferes with RIG-I-like receptor (RLR) signaling by interacting with MAVS and impeding its downstream effector functions. Here we demonstrate that over-expression of multiple UBXN family members decreased lentivirus and retrovirus production by several orders-of-magnitude in single cycle assays, at the level of long terminal repeat-driven transcription, and three family members, UBXN1, N9, and N11 blocked the canonical NFκB pathway by binding to Cullin1 (Cul1), inhibiting IκBα degradation...
February 2017: PLoS Pathogens
https://www.readbyqxmd.com/read/28110021/the-application-of-crispr-technology-to-high-content-screening-in-primary-neurons
#4
Ben L Callif, Brian Maunze, Nick L Krueger, Matthew T Simpson, Murray G Blackmore
Axon growth is coordinated by multiple interacting proteins that remain incompletely characterized. High content screening (HCS), in which manipulation of candidate genes is combined with rapid image analysis of phenotypic effects, has emerged as a powerful technique to identify key regulators of axon outgrowth. Here we explore the utility of a genome editing approach referred to as CRISPR (Clustered Regularly Interspersed Palindromic Repeats) for knockout screening in primary neurons. In the CRISPR approach a DNA-cleaving Cas enzyme is guided to genomic target sequences by user-created guide RNA (sgRNA), where it initiates a double-stranded break that ultimately results in frameshift mutation and loss of protein production...
April 2017: Molecular and Cellular Neurosciences
https://www.readbyqxmd.com/read/28102301/corrigendum-crispr-cas9-mediated-efficient-pd-1-disruption-on-human-primary-t-cells-from-cancer-patients
#5
Shu Su, Bian Hu, Jie Shao, Bin Shen, Juan Du, Yinan Du, Jiankui Zhou, Lixia Yu, Lianru Zhang, Fangjun Chen, Huizi Sha, Lei Cheng, Fanyan Meng, Zhengyun Zou, Xingxu Huang, Baorui Liu
No abstract text is available yet for this article.
January 19, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28025172/crispr-cas9-system-mediated-impairment-of-synaptobrevin-vamp-function-in-postmitotic-hippocampal-neurons
#6
Patricia M Horvath, Ege T Kavalali, Lisa M Monteggia
BACKGROUND: The use of the CRISPR/Cas9 system is becoming widespread, however current studies have predominantly focused on dividing cells. It is currently unknown if CRISPR/Cas9 can be used in a postmitotic setting to examine non-cell autonomous/presynaptic phenotypes in the resulting genetically heterogeneous cell population. NEW METHOD: A single CRISPR/Cas9 lentivirus was used to transfect a high percentage of primary cultured neurons and target synaptobrevin 2 (Syb2, also called VAMP2)...
December 23, 2016: Journal of Neuroscience Methods
https://www.readbyqxmd.com/read/27992415/a-genome-wide-crispr-screen-identifies-a-restricted-set-of-hiv-host-dependency-factors
#7
Ryan J Park, Tim Wang, Dylan Koundakjian, Judd F Hultquist, Pedro Lamothe-Molina, Blandine Monel, Kathrin Schumann, Haiyan Yu, Kevin M Krupzcak, Wilfredo Garcia-Beltran, Alicja Piechocka-Trocha, Nevan J Krogan, Alexander Marson, David M Sabatini, Eric S Lander, Nir Hacohen, Bruce D Walker
Host proteins are essential for HIV entry and replication and can be important nonviral therapeutic targets. Large-scale RNA interference (RNAi)-based screens have identified nearly a thousand candidate host factors, but there is little agreement among studies and few factors have been validated. Here we demonstrate that a genome-wide CRISPR-based screen identifies host factors in a physiologically relevant cell system. We identify five factors, including the HIV co-receptors CD4 and CCR5, that are required for HIV infection yet are dispensable for cellular proliferation and viability...
February 2017: Nature Genetics
https://www.readbyqxmd.com/read/27889530/identification-of-proximal-biomarkers-of-pkc-agonism-and-evaluation-of-their-role-in-hiv-reactivation
#8
Sai Vikram Vemula, Jill W Maxwell, Alexey Nefedov, Bang-Lin Wan, Justin Steve, William Newhard, Rosa I Sanchez, David Tellers, Richard J Barnard, Wade Blair, Daria Hazuda, Andrea L Webber, Bonnie J Howell
DESIGN: The HIV latent CD4(+) T cell reservoir is broadly recognized as a barrier to HIV cure. Induction of HIV expression using protein kinase C (PKC) agonists is one approach under investigation for reactivation of latently infected CD4(+) T cells (Beans et al., 2013; Abreu et al., 2014; Jiang et al., 2014; Jiang and Dandekar, 2015). We proposed that an increased understanding of the molecular mechanisms of action of PKC agonists was necessary to inform on biological signaling and pharmacodynamic biomarkers...
November 23, 2016: Antiviral Research
https://www.readbyqxmd.com/read/27883055/gapmer-cellular-internalization-by-macropinocytosis-induces-sequence-specific-gene-silencing-in-human-primary-t-cells
#9
Mobashar Hussain Urf Turabe Fazil, Seow Theng Ong, Madhavi Latha Somaraju Chalasani, Jian Hui Low, Atish Kizhakeyil, Akshay Mamidi, Carey Fang Hui Lim, Graham D Wright, Rajamani Lakshminarayanan, Dermot Kelleher, Navin Kumar Verma
Post-transcriptional gene silencing holds great promise in discovery research for addressing intricate biological questions and as therapeutics. While various gene silencing approaches, such as siRNA and CRISPR-Cas9 techniques, are available, these cannot be effectively applied to "hard-to-transfect" primary T-lymphocytes. The locked nucleic acid-conjugated chimeric antisense oligonucleotide, called "GapmeR", is an emerging new class of gene silencing molecule. Here, we show that GapmeR internalizes into human primary T-cells through macropinocytosis...
November 24, 2016: Scientific Reports
https://www.readbyqxmd.com/read/27803388/viral-vectors-the-road-to-reducing-genotoxicity
#10
Rhiannon M David, Ann T Doherty
Viral vector use in gene therapy has highlighted several safety concerns, including genotoxic events. Generally, vector-mediated genotoxicity results from upregulation of cellular proto-oncogenes via promoter insertion, promoter activation, or gene transcript truncation, with enhancer-mediated activation of nearby genes the primary mechanism reported in gene therapy trials. Vector-mediated genotoxicity can be influenced by virus type, integration target site, and target cell type; different vectors have distinct integration profiles which are cell-specific...
November 1, 2016: Toxicological Sciences: An Official Journal of the Society of Toxicology
https://www.readbyqxmd.com/read/27783955/a-cas9-ribonucleoprotein-platform-for-functional-genetic-studies-of-hiv-host-interactions-in-primary-human-t-cells
#11
Judd F Hultquist, Kathrin Schumann, Jonathan M Woo, Lara Manganaro, Michael J McGregor, Jennifer Doudna, Viviana Simon, Nevan J Krogan, Alexander Marson
New genetic tools are needed to understand the functional interactions between HIV and human host factors in primary cells. We recently developed a method to edit the genome of primary CD4(+) T cells by electroporation of CRISPR/Cas9 ribonucleoproteins (RNPs). Here, we adapted this methodology to a high-throughput platform for the efficient, arrayed editing of candidate host factors. CXCR4 or CCR5 knockout cells generated with this method are resistant to HIV infection in a tropism-dependent manner, whereas knockout of LEDGF or TNPO3 results in a tropism-independent reduction in infection...
October 25, 2016: Cell Reports
https://www.readbyqxmd.com/read/27780223/lim-only-protein-4-lmo4-and-lim-domain-binding-protein-1-ldb1-promote-growth-and-metastasis-of-human-head-and-neck-cancer-lmo4-and-ldb1-in-head-and-neck-cancer
#12
Elizabeth A Simonik, Ying Cai, Katherine N Kimmelshue, Dana M Brantley-Sieders, Holli A Loomans, Claudia D Andl, Grant M Westlake, Victoria M Youngblood, Jin Chen, Wendell G Yarbrough, Brandee T Brown, Lalitha Nagarajan, Stephen J Brandt
Squamous cell carcinoma of the head and neck (HNSCC) accounts for more than 300,000 deaths worldwide per year as a consequence of tumor cell invasion of adjacent structures or metastasis. LIM-only protein 4 (LMO4) and LIM-domain binding protein 1 (LDB1), two directly interacting transcriptional adaptors that have important roles in normal epithelial cell differentiation, have been associated with increased metastasis, decreased differentiation, and shortened survival in carcinoma of the breast. Here, we implicate two LDB1-binding proteins, single-stranded binding protein 2 (SSBP2) and 3 (SSBP3), in controlling LMO4 and LDB1 protein abundance in HNSCC and in regulating specific tumor cell functions in this disease...
2016: PloS One
https://www.readbyqxmd.com/read/27729526/efficient-crispr-mediated-mutagenesis-in-primary-immune-cells-using-crisprgold-and-a-c57bl-6-cas9-transgenic-mouse-line
#13
Van Trung Chu, Robin Graf, Tristan Wirtz, Timm Weber, Jeremy Favret, Xun Li, Kerstin Petsch, Ngoc Tung Tran, Michael H Sieweke, Claudia Berek, Ralf Kühn, Klaus Rajewsky
Applying clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9)-mediated mutagenesis to primary mouse immune cells, we used high-fidelity single guide RNAs (sgRNAs) designed with an sgRNA design tool (CrispRGold) to target genes in primary B cells, T cells, and macrophages isolated from a Cas9 transgenic mouse line. Using this system, we achieved an average knockout efficiency of 80% in B cells. On this basis, we established a robust small-scale CRISPR-mediated screen in these cells and identified genes essential for B-cell activation and plasma cell differentiation...
November 1, 2016: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/27703011/a-role-for-ceramides-but-not-sphingomyelins-as-antagonists-of-insulin-signaling-and-mitochondrial-metabolism-in-c2c12-myotubes
#14
Min Park, Vincent Kaddai, Jianhong Ching, Kevin T Fridianto, Ryan J Sieli, Shigeki Sugii, Scott A Summers
The accumulation of sphingolipids in obesity leads to impairments in insulin sensitivity and mitochondrial metabolism, but the precise species driving these defects is unclear. We have modeled these obesity-induced effects in cultured C2C12 myotubes, using BSA-conjugated palmitate to increase synthesis of endogenous sphingolipids and to inhibit insulin signaling and oxidative phosphorylation. Palmitate (a) induced the accumulation of sphingomyelin (SM) precursors such as sphinganine, dihydroceramide, and ceramide; (b) inhibited insulin stimulation of a central modulator of anabolic metabolism, Akt/PKB; (c) inhibited insulin-stimulated glycogen synthesis; and (d) decreased oxygen consumption and ATP synthesis...
November 11, 2016: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/27661404/pcm1-depletion-inhibits-glioblastoma-cell-ciliogenesis-and-increases-cell-death-and-sensitivity-to-temozolomide
#15
Lan B Hoang-Minh, Loic P Deleyrolle, Nariaki S Nakamura, Alexander K Parker, Regina T Martuscello, Brent A Reynolds, Matthew R Sarkisian
A better understanding of the molecules implicated in the growth and survival of glioblastoma (GBM) cells and their response to temozolomide (TMZ), the standard-of-care chemotherapeutic agent, is necessary for the development of new therapies that would improve the outcome of current GBM treatments. In this study, we characterize the role of pericentriolar material 1 (PCM1), a component of centriolar satellites surrounding centrosomes, in GBM cell proliferation and sensitivity to genotoxic agents such as TMZ...
October 2016: Translational Oncology
https://www.readbyqxmd.com/read/27606350/sendai-virus-an-rna-virus-with-no-risk-of-genomic-integration-delivers-crispr-cas9-for-efficient-gene-editing
#16
Arnold Park, Patrick Hong, Sohui T Won, Patricia A Thibault, Frederic Vigant, Kasopefoluwa Y Oguntuyo, Justin D Taft, Benhur Lee
The advent of RNA-guided endonuclease (RGEN)-mediated gene editing, specifically via CRISPR/Cas9, has spurred intensive efforts to improve the efficiency of both RGEN delivery and targeted mutagenesis. The major viral vectors in use for delivery of Cas9 and its associated guide RNA, lentiviral and adeno-associated viral systems, have the potential for undesired random integration into the host genome. Here, we repurpose Sendai virus, an RNA virus with no viral DNA phase and that replicates solely in the cytoplasm, as a delivery system for efficient Cas9-mediated gene editing...
2016: Molecular Therapy. Methods & Clinical Development
https://www.readbyqxmd.com/read/27531490/immunogenomic-engineering-of-a-plug-and-dis-play-hybridoma-platform
#17
Mark Pogson, Cristina Parola, William J Kelton, Paul Heuberger, Sai T Reddy
Hybridomas, fusions of primary mouse B cells and myelomas, are stable, rapidly-proliferating cell lines widely utilized for antibody screening and production. Antibody specificity of a hybridoma clone is determined by the immunoglobulin sequence of the primary B cell. Here we report a platform for rapid reprogramming of hybridoma antibody specificity by immunogenomic engineering. Here we use CRISPR-Cas9 to generate double-stranded breaks in immunoglobulin loci, enabling deletion of the native variable light chain and replacement of the endogenous variable heavy chain with a fluorescent reporter protein (mRuby)...
2016: Nature Communications
https://www.readbyqxmd.com/read/27530326/crispr-cas9-mediated-trp53-and-brca2-knockout-to-generate-improved-murine-models-of-ovarian-high-grade-serous-carcinoma
#18
Josephine Walton, Julianna Blagih, Darren Ennis, Elaine Leung, Suzanne Dowson, Malcolm Farquharson, Laura A Tookman, Clare Orange, Dimitris Athineos, Susan Mason, David Stevenson, Karen Blyth, Douglas Strathdee, Frances R Balkwill, Karen Vousden, Michelle Lockley, Iain A McNeish
There is a need for transplantable murine models of ovarian high-grade serous carcinoma (HGSC) with regard to mutations in the human disease to assist investigations of the relationships between tumor genotype, chemotherapy response, and immune microenvironment. In addressing this need, we performed whole-exome sequencing of ID8, the most widely used transplantable model of ovarian cancer, covering 194,000 exomes at a mean depth of 400× with 90% exons sequenced >50×. We found no functional mutations in genes characteristic of HGSC (Trp53, Brca1, Brca2, Nf1, and Rb1), and p53 remained transcriptionally active...
October 15, 2016: Cancer Research
https://www.readbyqxmd.com/read/27486782/whole-exome-sequencing-identifies-loci-associated-with-blood-cell-traits-and-reveals-a-role-for-alternative-gfi1b-splice-variants-in-human-hematopoiesis
#19
Linda M Polfus, Rajiv K Khajuria, Ursula M Schick, Nathan Pankratz, Raha Pazoki, Jennifer A Brody, Ming-Huei Chen, Paul L Auer, James S Floyd, Jie Huang, Leslie Lange, Frank J A van Rooij, Richard A Gibbs, Ginger Metcalf, Donna Muzny, Narayanan Veeraraghavan, Klaudia Walter, Lu Chen, Lisa Yanek, Lewis C Becker, Gina M Peloso, Aoi Wakabayashi, Mart Kals, Andres Metspalu, Tõnu Esko, Keolu Fox, Robert Wallace, Nora Franceshini, Nena Matijevic, Kenneth M Rice, Traci M Bartz, Leo-Pekka Lyytikäinen, Mika Kähönen, Terho Lehtimäki, Olli T Raitakari, Ruifang Li-Gao, Dennis O Mook-Kanamori, Guillaume Lettre, Cornelia M van Duijn, Oscar H Franco, Stephen S Rich, Fernando Rivadeneira, Albert Hofman, André G Uitterlinden, James G Wilson, Bruce M Psaty, Nicole Soranzo, Abbas Dehghan, Eric Boerwinkle, Xiaoling Zhang, Andrew D Johnson, Christopher J O'Donnell, Jill M Johnsen, Alexander P Reiner, Santhi K Ganesh, Vijay G Sankaran
Circulating blood cell counts and indices are important indicators of hematopoietic function and a number of clinical parameters, such as blood oxygen-carrying capacity, inflammation, and hemostasis. By performing whole-exome sequence association analyses of hematologic quantitative traits in 15,459 community-dwelling individuals, followed by in silico replication in up to 52,024 independent samples, we identified two previously undescribed coding variants associated with lower platelet count: a common missense variant in CPS1 (rs1047891, MAF = 0...
August 4, 2016: American Journal of Human Genetics
https://www.readbyqxmd.com/read/27226607/the-activity-of-menkes-disease-protein-atp7a-is-essential-for-redox-balance-in-mitochondria
#20
Ashima Bhattacharjee, Haojun Yang, Megan Duffy, Emily Robinson, Arianrhod Conrad-Antoville, Ya-Wen Lu, Tony Capps, Lelita Braiterman, Michael Wolfgang, Michael P Murphy, Ling Yi, Stephen G Kaler, Svetlana Lutsenko, Martina Ralle
Copper-transporting ATPase ATP7A is essential for mammalian copper homeostasis. Loss of ATP7A activity is associated with fatal Menkes disease and various other pathologies. In cells, ATP7A inactivation disrupts copper transport from the cytosol into the secretory pathway. Using fibroblasts from Menkes disease patients and mouse 3T3-L1 cells with a CRISPR/Cas9-inactivated ATP7A, we demonstrate that ATP7A dysfunction is also damaging to mitochondrial redox balance. In these cells, copper accumulates in nuclei, cytosol, and mitochondria, causing distinct changes in their redox environment...
August 5, 2016: Journal of Biological Chemistry
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