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single b cell cloning

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https://www.readbyqxmd.com/read/28212528/the-crispr-cas9%C3%A2-system-efficiently-reverts-the-tumorigenic-ability-of-bcr-abl-in-vitro-and-in-a-xenograft-model-of-chronic-myeloid-leukemia
#1
Ignacio García-Tuñón, María Hernández-Sánchez, José Luis Ordoñez, Veronica Alonso-Pérez, Miguel Álamo-Quijada, Rocio Benito, Carmen Guerrero, Jesús María Hernández-Rivas, Manuel Sánchez-Martín
CRISPR/Cas9 technology was used to abrogate p210 oncoprotein expression in the Boff-p210 cell line, a pro-B line derived from interlukin-3-dependent Baf/3, that shows IL-3-independence arising from the constitutive expression of BCR-ABL p210. Using this approach, pools of Boff-p210-edited cells and single edited cell-derived clones were obtained and functionally studied in vitro. The loss of p210 expression in Boff-p210 cells resulted in the loss of ability to grow in the absence of IL-3, as the Baf/3 parental line, showing significantly increased apoptosis levels...
February 9, 2017: Oncotarget
https://www.readbyqxmd.com/read/28202626/cancer-cells-exhibit-clonal-diversity-in-phenotypic-plasticity
#2
Robert Austin Mathis, Ethan S Sokol, Piyush B Gupta
Phenotypic heterogeneity in cancers is associated with invasive progression and drug resistance. This heterogeneity arises in part from the ability of cancer cells to switch between phenotypic states, but the dynamics of this cellular plasticity remain poorly understood. Here we apply DNA barcodes to quantify and track phenotypic plasticity across hundreds of clones in a population of cancer cells exhibiting epithelial or mesenchymal differentiation phenotypes. We find that the epithelial-to-mesenchymal cell ratio is highly variable across the different clones in cancer cell populations, but remains stable for many generations within the progeny of any single clone-with a heritability of 0...
February 2017: Open Biology
https://www.readbyqxmd.com/read/28179494/hierarchical-clustering-can-identify-b-cell-clones-with-high-confidence-in-ig-repertoire-sequencing-data
#3
Namita T Gupta, Kristofor D Adams, Adrian W Briggs, Sonia C Timberlake, Francois Vigneault, Steven H Kleinstein
Adaptive immunity is driven by the expansion, somatic hypermutation, and selection of B cell clones. Each clone is the progeny of a single B cell responding to Ag, with diversified Ig receptors. These receptors can now be profiled on a large scale by next-generation sequencing. Such data provide a window into the microevolutionary dynamics that drive successful immune responses and the dysregulation that occurs with aging or disease. Clonal relationships are not directly measured, but they must be computationally inferred from these sequencing data...
February 8, 2017: Journal of Immunology: Official Journal of the American Association of Immunologists
https://www.readbyqxmd.com/read/28125682/the-number-of-overlapping-aid-hotspots-in-germline-ighv-genes-is-inversely-correlated-with-mutation-frequency-in-chronic-lymphocytic-leukemia
#4
Chaohui Yuan, Charles C Chu, Xiao-Jie Yan, Davide Bagnara, Nicholas Chiorazzi, Thomas MacCarthy
The targeting of mutations by Activation-Induced Deaminase (AID) is a key step in generating antibody diversity at the Immunoglobulin (Ig) loci but is also implicated in B-cell malignancies such as chronic lymphocytic leukemia (CLL). AID has previously been shown to preferentially deaminate WRC (W = A/T, R = A/G) hotspots. WGCW sites, which contain an overlapping WRC hotspot on both DNA strands, mutate at much higher frequency than single hotspots. Human Ig heavy chain (IGHV) genes differ in terms of WGCW numbers, ranging from 4 for IGHV3-48*03 to as many as 12 in IGHV1-69*01...
2017: PloS One
https://www.readbyqxmd.com/read/28114943/joining-the-in-vitro-immunization-of-alpaca-lymphocytes-and-phage-display-rapid-and-cost-effective-pipeline-for-sdab-synthesis
#5
Lubos Comor, Saskia Dolinska, Katarina Bhide, Lucia Pulzova, Irene Jiménez-Munguía, Elena Bencurova, Zuzana Flachbartova, Lenka Potocnakova, Evelina Kanova, Mangesh Bhide
BACKGROUND: Camelids possess unique functional heavy chain antibodies, which can be produced and modified in vitro as a single domain antibody (sdAb or nanobody) with full antigen binding ability. Production of sdAb in conventional manner requires active immunization of Camelidae animal, which is laborious, time consuming, costly and in many cases not feasible (e.g. in case of highly toxic or infectious antigens). RESULTS: In this study, we describe an alternative pipeline that includes in vitro stimulation of naïve alpaca B-lymphocytes by antigen of interest (in this case endothelial cell binding domain of OspA of Borrelia) in the presence of recombinant alpaca interleukins 2 and 4, construction of sdAb phage library, selection of antigen specific sdAb expressed on phages (biopanning) and confirmation of binding ability of sdAb to the antigen...
January 23, 2017: Microbial Cell Factories
https://www.readbyqxmd.com/read/28109146/-cloning-expression-and-activity-identification-of-human-innate-immune-protein-apolipoprotein-b-mrna-editing-enzyme-catalytic-subunit-3a-apobec3a
#6
Shan Cheng, Liyan Cao, Juan Du, Wenyong Wang, Yanhai Guo
Objective To construct the expression vector of apolipoprotein B mRNA editing enzyme catalytic subunit 3A (APOBEC3A), express APOBEC3A in eukaryotic cells and identify its cytosine deaminase activity. Methods The APOBEC3A gene was obtained by PCR and inserted into the eukaryotic expression vector pcDNA3.0(+). The recombinant vector pcDNA3.0-APOBEC3A was then transfected into HEK293T and HepG2 cells after confirmed by DNA sequencing. The recombinant protein was purified by Ni-NTA HisBind affinity column. Western blot analysis was used to detect the expression of APOBEC3A protein...
February 2017: Xi Bao Yu Fen Zi Mian Yi Xue za Zhi, Chinese Journal of Cellular and Molecular Immunology
https://www.readbyqxmd.com/read/28107386/regulation-of-pi-2b-pilus-expression-in-hypervirulent-streptococcus-agalactiae-st-17-bm110
#7
Bruno Périchon, Noémi Szili, Laurence du Merle, Isabelle Rosinski-Chupin, Myriam Gominet, Samuel Bellais, Claire Poyart, Patrick Trieu-Cuot, Shaynoor Dramsi
The widely spread Streptococcus agalactiae (also known as Group B Streptococcus, GBS) "hypervirulent" ST17 clone is strongly associated with neonatal meningitis. The PI-2b locus is mainly found in ST17 strains but is also present in a few non ST17 human isolates such as the ST-7 prototype strain A909. Here, we analysed the expression of the PI-2b pilus in the ST17 strain BM110 as compared to the non ST17 A909. Comparative genome analyses revealed the presence of a 43-base pair (bp) hairpin-like structure in the upstream region of PI-2b operon in all 26 ST17 genomes, which was absent in the 8 non-ST17 strains carrying the PI-2b locus...
2017: PloS One
https://www.readbyqxmd.com/read/28093446/tumor-infiltrating-merkel-cell-polyomavirus-specific-t-cells-are-diverse-and-associated-with-improved-patient-survival
#8
Natalie J Miller, Candice D Church, Lichun Dong, David Crispin, Matthew P Fitzgibbon, Kristina Lachance, Lichen Jing, Michi Shinohara, Ioannis Gavvovidis, Gerald Willimsky, Martin McIntosh, Thomas Blankenstein, David M Koelle, Paul Nghiem
Tumor-infiltrating CD8(+) T cells are associated with improved survival of patients with Merkel cell carcinoma (MCC), an aggressive skin cancer causally linked to Merkel cell polyomavirus (MCPyV). However, CD8(+) T-cell infiltration is robust in only 4% to 18% of MCC tumors. We characterized the T-cell receptor (TCR) repertoire restricted to one prominent epitope of MCPyV (KLLEIAPNC, "KLL") and assessed whether TCR diversity, tumor infiltration, or T-cell avidity correlated with clinical outcome. HLA-A*02:01/KLL tetramer(+) CD8(+) T cells from MCC patient peripheral blood mononuclear cells (PBMC) and tumor-infiltrating lymphocytes (TIL) were isolated via flow cytometry...
January 16, 2017: Cancer Immunology Research
https://www.readbyqxmd.com/read/28089880/a-combined-approach-for-enhancing-the-stability-of-recombinant-cis-dihydrodiol-naphthalene-dehydrogenase-from-pseudomonas-putida-g7-allowed-for-the-structural-and-kinetic-characterization-of-the-enzyme
#9
Débora Maria Abrantes Costa, Mariana Amalia Figueiredo Costa, Samuel Leite Guimarães, Juliana Barbosa Coitinho, Stefanya Velásquez Gómez, Tiago Antônio da Silva Brandão, Ronaldo Alves Pinto Nagem
The second enzyme of the naphthalene degradation pathway in Pseudomonas putida G7 is NahB, a dehydrogenase that converts cis-1,2-dihydroxy-1,2-dihydronaphthalene to 1,2-dihydroxynaphthalene. We report the cloning, optimization of expression, purification, kinetic studies and preliminary structural characterization of the recombinant NahB. The nahB gene was cloned into a T7 expression vector and the enzyme was overexpressed in Escherichia coli Rosetta (DE3) as an N-terminal hexa-histidine-tagged protein (6xHis-NahB)...
January 9, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28075174/analysis-of-sivmac-envelope-specific-antibodies-selected-via-phage-display
#10
Sergio Ita, Mayara Rovariz Agostinho, Katherine Sullivan, Seung Yub Han, Rana Akleh, Welkin Johnson, Ismael Ben Farouck Fofana
We have constructed a single chain Fv (scFv) phage display library from an SIV-infected rhesus macaque that developed unusually high-titer neutralizing antibody responses against tier-3, neutralization-resistant SIVmac239. The library was screened using trimeric (gp140) and monomeric (gp120) forms of the SIVmac239 envelope (Env) glycoprotein. We also cloned variable-heavy and variable-light (VH-VL) antibody fragments from 7 previously described rhesus macaque B-cell lines (BLCL) that produce SIV gp120-specific monoclonal antibodies (mAbs)...
January 11, 2017: AIDS Research and Human Retroviruses
https://www.readbyqxmd.com/read/28073404/isolation-and-characterization-of-hiv-1-envelope-glycoprotein-specific-b-cell-from-immortalized-human-na%C3%A3-ve-b-cell-library
#11
Zehua Sun, Shiqiang Lu, Zheng Yang, Jingjing Li, Meiyun Zhang
With the recent development of single B cell cloning techniques, an increasing number of HIV-1-specific broadly neutralizing antibodies (bNAbs) have been isolated since 2009. However, knowledge regarding HIV-1-specific B cells in vivo is limited. In this study, an HIV-1-specific B cell line has been established using healthy PBMC donors by the highly efficient EBV transformation method to generate immortalized human naïve B cell libraries. The enrichment of HIV-1 envelope-specific B cells was observed after four rounds of cell panning with the HIV-1 envelope glycoprotein...
January 10, 2017: Journal of General Virology
https://www.readbyqxmd.com/read/28038927/cellular-immune-response-to-%C3%AE-2-glycoprotein-i-valine-leucine-247-phenotypes-in-mexican-patients-with-primary-antiphospholipid-syndrome
#12
Carlos A Núñez-Álvarez, Diego F Hernández-Ramírez, Araceli Martinez-Castillo, Virginia Pascual Ramos, Javier Cabiedes, Alicia Ortega, Antonio R Cabral
: Homozygote genotype V(247) of the β2-glycoprotein-I (β2GP-I) gene has been associated with anti-β2GP-I and thrombosis in patients with primary anti-phospholipid syndrome APS (PAPS). However, the cellular immune response to β2GP-I(247) has been little studied. OBJECTIVE: To evaluate the immune cellular proliferation in response to native and non-native β2GP-I(247) valine/leucine phenotype from Mexican patients with PAPS. METHODS: We studied 10 patients with PAPS and 10 healthy control subjects (HC)...
December 27, 2016: Human Immunology
https://www.readbyqxmd.com/read/28017968/mutations-in-notch1-pest-domain-orchestrate-ccl19-driven-homing-of-chronic-lymphocytic-leukemia-cells-by-modulating-the-tumor-suppressor-gene-dusp22
#13
F Arruga, B Gizdic, C Bologna, S Cignetto, R Buonincontri, S Serra, T Vaisitti, K Gizzi, N Vitale, G Garaffo, E Mereu, F Diop, F Neri, D Incarnato, M Coscia, J Allan, R Piva, S Oliviero, R R Furman, D Rossi, G Gaidano, S Deaglio
Even if NOTCH1 is commonly mutated in Chronic Lymphocytic Leukemia (CLL), its functional impact in the disease remains unclear. Using CRISPR/Cas9-generated Mec-1 cell line models, we show that NOTCH1 regulates growth and homing of CLL cells by dictating expression levels of the tumor suppressor gene DUSP22. Specifically, NOTCH1 affects the methylation of DUSP22 promoter by modulating a nuclear complex, which tunes the activity of DNA methyltransferase 3A (DNMT3A). These effects are enhanced by PEST-domain mutations, which stabilize the molecule and prolong signaling...
December 26, 2016: Leukemia: Official Journal of the Leukemia Society of America, Leukemia Research Fund, U.K
https://www.readbyqxmd.com/read/28008177/mass-cytometry-analysis-reveals-hyperactive-nf-kappa-b-signaling-in-myelofibrosis-and-secondary-acute-myeloid-leukemia
#14
D A C Fisher, O Malkova, E K Engle, C A Miner, M C Fulbright, G K Behbehani, T B Collins, S Bandyopadhyay, A Zhou, G P Nolan, S T Oh
Myeloproliferative neoplasms (MPNs) feature a malignant clone containing the JAK2 V617F mutation, or another mutation causing dysregulated JAK2 kinase activity. The multiple disease phenotypes of MPNs, and their tendency to transform phenotypically, suggest pathophysiologic heterogeneities beyond a common phenomenon of JAK2 hyperactivation. JAK2 has the potential to activate multiple other signaling molecules, either directly through downstream effectors, or indirectly through induction of target gene expression...
February 3, 2017: Leukemia: Official Journal of the Leukemia Society of America, Leukemia Research Fund, U.K
https://www.readbyqxmd.com/read/27997564/nonstructural-protein-11-of-porcine-reproductive-and-respiratory-syndrome-virus-suppresses-both-mavs-and-rig-i-expression-as-one-of-the-mechanisms-to-antagonize-type-i-interferon-production
#15
Yan Sun, Hanzhong Ke, Mingyuan Han, Ning Chen, Weihuan Fang, Dongwan Yoo
Type I interferons (IFN-α/β) play a key role in antiviral defense, and porcine reproductive and respiratory syndrome virus (PRRSV) is known to down-regulate the IFN response in virus-infected cells and pigs. In this study, we showed that the overexpression of nsp11 of PRRSV induced a strong suppression of IFN production. Nsp11 suppressed both IRF3 and NF-κB activities when stimulated with a dsRNA analogue and TNF-α, respectively. This suppression was RLR dependent, since the transcripts and proteins of MAVS and RIG-I, two critical factors in RLR-mediated pathway, were both found to be reduced in the presence of overexpressed nsp11...
2016: PloS One
https://www.readbyqxmd.com/read/27982015/evolution-of-multiple-cell-clones-over-a-29-year-period-of-a-cll-patient
#16
Zhikun Zhao, Lynn Goldin, Shiping Liu, Liang Wu, Weiyin Zhou, Hong Lou, Qichao Yu, Shirley X Tsang, Miaomiao Jiang, Fuqiang Li, MaryLou McMaster, Yang Li, Xinxin Lin, Zhifeng Wang, Liqin Xu, Gerald Marti, Guibo Li, Kui Wu, Meredith Yeager, Huanming Yang, Xun Xu, Stephen J Chanock, Bo Li, Yong Hou, Neil Caporaso, Michael Dean
Chronic lymphocytic leukaemia (CLL) is a frequent B-cell malignancy, characterized by recurrent somatic chromosome alterations and a low level of point mutations. Here we present single-nucleotide polymorphism microarray analyses of a single CLL patient over 29 years of observation and treatment, and transcriptome and whole-genome sequencing at selected time points. We identify chromosome alterations 13q14-, 6q- and 12q+ in early cell clones, elimination of clonal populations following therapy, and subsequent appearance of a clone containing trisomy 12 and chromosome 10 copy-neutral loss of heterogeneity that marks a major population dominant at death...
December 16, 2016: Nature Communications
https://www.readbyqxmd.com/read/27975311/detection-of-hepatocyte-clones-containing-integrated-hepatitis-b-virus-dna-using-inverse-nested-pcr
#17
Thomas Tu, Allison R Jilbert
Chronic hepatitis B virus (HBV) infection is a major cause of liver cirrhosis and hepatocellular carcinoma (HCC), leading to ~600,000 deaths per year worldwide. Many of the steps that occur during progression from the normal liver to cirrhosis and/or HCC are unknown. Integration of HBV DNA into random sites in the host cell genome occurs as a by-product of the HBV replication cycle and forms a unique junction between virus and cellular DNA. Analyses of integrated HBV DNA have revealed that HCCs are clonal and imply that they develop from the transformation of hepatocytes, the only liver cell known to be infected by HBV...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27975303/ntcp-reconstituted-in-vitro-hbv-infection-system
#18
Yinyan Sun, Yonghe Qi, Bo Peng, Wenhui Li
Sodium taurocholate cotransporting polypeptide (NTCP) has been identified as a functional receptor for hepatitis B virus (HBV). Expressing human NTCP in human hepatoma HepG2 cells (HepG2-NTCP) renders these cells susceptible for HBV infection. The HepG2-NTCP stably transfected cell line provides a much-needed and easily accessible platform for studying the virus. HepG2-NTCP cells could also be used to identify chemicals targeting key steps of the virus life cycle including HBV covalent closed circular (ccc) DNA, and enable the development of novel antivirals against the infection...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27975259/finding-mapt-mutations-in-frontotemporal-dementia-and-other-tauopathies
#19
Carol Dobson-Stone, John B J Kwok
Sanger sequencing is a classic technique in molecular genetics to detect single nucleotide DNA variants in genomic DNA. Here we describe the detection of MAPT mutations by polymerase chain reaction amplification of patient genomic DNA followed by bidirectional Sanger sequencing. Exon trapping is a technique whereby genomic DNA covering the exon of interest and flanking intronic sequence is cloned into the intron of an expression vector and transfected into human cell lines. RNA is extracted and splicing products are examined by reverse-transcriptase PCR and agarose gel electrophoresis...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27957393/stress-responses-of-the-oil-producing-green-microalga-botryococcus-braunii-race-b
#20
Ivette Cornejo-Corona, Hem R Thapa, Daniel R Browne, Timothy P Devarenne, Edmundo Lozoya-Gloria
Plants react to biotic and abiotic stresses with a variety of responses including the production of reactive oxygen species (ROS), which may result in programmed cell death (PCD). The mechanisms underlying ROS production and PCD have not been well studied in microalgae. Here, we analyzed ROS accumulation, biomass accumulation, and hydrocarbon production in the colony-forming green microalga Botryococcus braunii in response to several stress inducers such as NaCl, NaHCO3, salicylic acid (SA), methyl jasmonate, and acetic acid...
2016: PeerJ
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