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single b cell cloning

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https://www.readbyqxmd.com/read/29650674/germinal-center-antibody-mutation-trajectories-are-determined-by-rapid-self-foreign-discrimination
#1
Deborah L Burnett, David B Langley, Peter Schofield, Jana R Hermes, Tyani D Chan, Jennifer Jackson, Katherine Bourne, Joanne H Reed, Kate Patterson, Benjamin T Porebski, Robert Brink, Daniel Christ, Christopher C Goodnow
Antibodies have the specificity to differentiate foreign antigens that mimic self antigens, but it remains unclear how such specificity is acquired. In a mouse model, we generated B cells displaying an antibody that cross-reacts with two related protein antigens expressed on self versus foreign cells. B cell anergy was imposed by self antigen but reversed upon challenge with high-density foreign antigen, leading to germinal center recruitment and antibody gene hypermutation. Single-cell analysis detected rapid selection for mutations that decrease self affinity and slower selection for epistatic mutations that specifically increase foreign affinity...
April 13, 2018: Science
https://www.readbyqxmd.com/read/29644860/up-regulation-of-t-cell-activation-micrornas-in-drug-specific-cd4-t-cells-from-hypersensitive-patients
#2
Alejandra Monroy-Arreola, Noe Duran-Figueroa, Silvia Mendez-Flores, Judith Dominguez-Cherit, Joel Watkinson, Agustin Badillo-Corona, Paul Whitaker, Dean Naisbitt, Luis Castrejon Flores
Dysregulation in the expression of microRNAs (miRNAs), single stranded RNAs which regulate gene expression, has been associated with diseases such as Stevens-Johnson syndrome (SJS) / toxic epidermal necrolysis (TEN), although their cellular origin has not been explored. Thus, the focus of this work was to study expression patterns of reported miRNAs involved in T - cell activation following drug-specific stimulation in peripheral blood mononuclear cells (PBMCs) and drug specific CD4+ T-cell clones (TCC) from patients with different cutaneous manifestations of delayed-type drug hypersensitivity reactions...
April 12, 2018: Chemical Research in Toxicology
https://www.readbyqxmd.com/read/29628923/evaluation-of-antigen-conjugated-fluorescent-beads-to-identify-antigen-specific-b-cells
#3
Isabel Correa, Kristina M Ilieva, Silvia Crescioli, Sara Lombardi, Mariangela Figini, Anthony Cheung, James F Spicer, Andrew N J Tutt, Frank O Nestle, Panagiotis Karagiannis, Katie E Lacy, Sophia N Karagiannis
Selection of single antigen-specific B cells to identify their expressed antibodies is of considerable interest for evaluating human immune responses. Here, we present a method to identify single antibody-expressing cells using antigen-conjugated fluorescent beads. To establish this, we selected Folate Receptor alpha (FRα) as a model antigen and a mouse B cell line, expressing both the soluble and the membrane-bound forms of a human/mouse chimeric antibody (MOv18 IgG1) specific for FRα, as test antibody-expressing cells...
2018: Frontiers in Immunology
https://www.readbyqxmd.com/read/29624616/sequence-genome-organization-annotation-and-proteomics-of-the-thermophilic-47-7-kb-geobacillus-stearothermophilus-bacteriophage-tp-84-and-its-classification-in-the-new-tp84virus-genus
#4
Piotr M Skowron, Andrew M Kropinski, Joanna Zebrowska, Lukasz Janus, Kasjan Szemiako, Edyta Czajkowska, Natalia Maciejewska, Malgorzata Skowron, Joanna Łoś, Marcin Łoś, Agnieszka Zylicz-Stachula
Bacteriophage TP-84 is a well-characterized bacteriophage of historical interest. It is a member of the Siphoviridae, and infects a number of thermophilic Geobacillus (Bacillus) stearothermophilus strains. Its' 47.7-kbp double-stranded DNA genome revealed the presence of 81 coding sequences (CDSs) coding for polypeptides of 4 kDa or larger. Interestingly, all CDSs are oriented in the same direction, pointing to a dominant transcription direction of one DNA strand. Based on a homology search, a hypothetical function could be assigned to 31 CDSs...
2018: PloS One
https://www.readbyqxmd.com/read/29608918/a-new-multiplex-pcr-protocol-to-detect-mixed-trypanosomatid-infections-in-species-of-apis-and-bombus
#5
Carolina Bartolomé, María Buendía, María Benito, Pilar De la Rúa, Concepción Ornosa, Raquel Martín-Hernández, Mariano Higes, Xulio Maside
Trypanosomatids are highly prevalent pathogens of Hymenoptera; however, most molecular methods used to detect them in Apis and Bombus spp. do not allow the identification of the infecting species, which then becomes expensive and time consuming. To overcome this drawback, we developed a multiplex PCR protocol to readily identify in a single reaction the main trypanosomatids present in these hymenopterans (Lotmaria passim, Crithidia mellificae and Crithidia bombi), which will facilitate the study of their epidemiology and transmission dynamics...
March 30, 2018: Journal of Invertebrate Pathology
https://www.readbyqxmd.com/read/29608237/designer-nuclease-mediated-gene-correction-via-homology-directed-repair-in-an-in-vitro-model-of-canine-hemophilia-b
#6
Thorsten Bergmann, Eric Ehrke-Schulz, Jian Gao, Maren Schiwon, Verena Schildgen, Stephan David, Oliver Schildgen, Anja Ehrhardt
BACKGROUND: Gene correction at specific target loci provides a powerful strategy to overcome genetic diseases. Here we aimed at using an in vitro model for canine hemophilia B containing a single point mutation in the catalytic domain of the canine coagulation factor IX (cFIX) gene. To correct the defective gene via homology-directed repair (HDR), we designed transcription-activator like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats including Cas9 (CRISPR/Cas9) for introduction of double strand breaks at the mutation site...
April 2, 2018: Journal of Gene Medicine
https://www.readbyqxmd.com/read/29582535/nucleocytoplasmic-shuttling-of-the-west-nile-virus-rna-dependent-rna-polymerase-ns5-is-critical-to-infection
#7
Adam J Lopez-Denman, Alice Russo, Kylie M Wagstaff, Peter A White, David A Jans, Jason M Mackenzie
West Nile Virus (WNV) is a single stranded, positive sense RNA virus of the family Flaviviridae, and is a significant pathogen of global medical importance. Flavivirus replication is known to be exclusively cytoplasmic, but we show here for the first time that access to the nucleus of the WNV strain Kunjin (WNVKUN ) RNA-dependent RNA polymerase (protein NS5) is central to WNVKUN virus production. We show that treatment of cells with the specific nuclear export inhibitor leptomycin B (LMB) results in increased NS5 nuclear accumulation in WNVKUN -infected cells and NS5 transfected cells, indicative of nucleocytoplasmic shuttling under normal conditions...
March 26, 2018: Cellular Microbiology
https://www.readbyqxmd.com/read/29561802/selection-and-characterization-of-single-stranded-dna-aptamers-binding-human-b-cell-surface-protein-cd20-by-cell-selex
#8
Mansoureh Haghighi, Hossein Khanahmad, Abbasali Palizban
The B-lymphocyte antigen (CD20) is a suitable target for single-stranded (ss) nucleic acid oligomer (aptamers). The aim of study was selection and characterization of a ssDNA aptamer against CD20 using Cell-Systematic Evolution of Ligands by Exponential Enrichment (Cell-SELEX). The cDNA clone of CD20 (pcDNA-CD20) was transfected to human embryonic kidney (HEK293T) cells. Ten rounds of Cell-SELEX was performed on recombinant HEK-CD20 cells. The final eluted ssDNA pool was amplified and ligated in T/A vector for cloning...
March 21, 2018: Molecules: a Journal of Synthetic Chemistry and Natural Product Chemistry
https://www.readbyqxmd.com/read/29514901/importance-of-neutralizing-monoclonal-antibodies-targeting-multiple-antigenic-sites-on-mers-cov-spike-to-avoid-neutralization-escape
#9
Lingshu Wang, Wei Shi, James D Chappell, M Gordon Joyce, Yi Zhang, Masaru Kanekiyo, Michelle M Becker, Neeltje van Doremalen, Robert Fischer, Nianshuang Wang, Kizzmekia S Corbett, Misook Choe, Rosemarie D Mason, Joseph G Van Galen, Tongqing Zhou, Kevin O Saunders, Kathleen M Tatti, Lia M Haynes, Peter D Kwong, Kayvon Modjarrad, Wing-Pui Kong, Jason S McLellan, Mark R Denison, Vincent J Munster, John R Mascola, Barney S Graham
Middle East Respiratory Syndrome coronavirus (MERS-CoV) causes a highly lethal pulmonary infection with ∼35% mortality. The potential for a future pandemic originating from animal reservoirs or healthcare-associated events is a major public health concern. There are no vaccines or therapeutic agents currently available for MERS-CoV. Using a probe-based single B cell-cloning strategy, we have identified and characterized multiple neutralizing mAbs specifically binding to the receptor binding domain (RBD) or S1 (non-RBD) regions from a convalescent MERS-CoV-infected patient and from immunized rhesus macaques...
March 7, 2018: Journal of Virology
https://www.readbyqxmd.com/read/29512823/variable-domain-n-linked-glycosylation-and-negative-surface-charge-are-key-features-of-monoclonal-acpa-implications-for-b-cell-selection
#10
Katy A Lloyd, Johanna Steen, Khaled Amara, Philip J Titcombe, Lena Israelsson, Susanna L Lundström, Diana Zhou, Roman A Zubarev, Evan Reed, Luca Piccoli, Cem Gabay, Antonio Lanzavecchia, Dominique Baeten, Karin Lundberg, Daniel L Mueller, Lars Klareskog, Vivianne Malmström, Caroline Grönwall
Autoreactive B cells have a central role in the pathogenesis of rheumatoid arthritis (RA), and recent findings have proposed that anti-citrullinated protein autoantibodies (ACPA) may be directly pathogenic. Herein, we demonstrate the frequency of variable-region glycosylation in single-cell cloned mAbs. A total of 14 ACPA mAbs were evaluated for predicted N-linked glycosylation motifs in silico, and compared to 452 highly-mutated mAbs from RA patients and controls. Variable region N-linked motifs (N-X-S/T) were strikingly prevalent within ACPA (100%) compared to somatically hypermutated (SHM) RA bone marrow plasma cells (21%), and synovial plasma cells from seropositive (39%) and seronegative RA (7%)...
March 7, 2018: European Journal of Immunology
https://www.readbyqxmd.com/read/29510906/reconstruction-of-the-repetitive-antifreeze-glycoprotein-genomic-loci-in-the-cold-water-gadids-boreogadus-saida-and-microgadus-tomcod
#11
Xuan Zhuang, Katherine R Murphy, Laura Ghigliotti, Eva Pisano, C-H Christina Cheng
Antifreeze glycoproteins (AFGPs) are a novel evolutionary innovation in members of the northern cod fish family (Gadidae), crucial in preventing death from inoculative freezing by environmental ice in their frigid Arctic and sub-Arctic habitats. However, the genomic origin and molecular mechanism of evolution of this novel life-saving adaptive genetic trait remained to be definitively determined. To this end, we constructed large insert genomic DNA BAC (bacterial artificial chromosome) libraries for two AFGP-bearing gadids, the high-Arctic polar cod Boreogadus saida and the cold-temperate Atlantic tomcod Microgadus tomcod, to isolate and sequence their AFGP genomic regions for fine resolution evolutionary analyses...
March 3, 2018: Marine Genomics
https://www.readbyqxmd.com/read/29510760/cysteine-peptidases-of-eudiplozoon-nipponicum-a-broad-repertoire-of-structurally-assorted-cathepsins-l-in-contrast-to-the-scarcity-of-cathepsins-b-in-an-invasive-species-of-haematophagous-monogenean-of-common-carp
#12
Lucie Jedličková, Hana Dvořáková, Jan Dvořák, Martin Kašný, Lenka Ulrychová, Jiří Vorel, Vojtěch Žárský, Libor Mikeš
BACKGROUND: Cysteine peptidases of clan CA, family C1 account for a major part of proteolytic activity in the haematophagous monogenean Eudiplozoon nipponicum. The full spectrum of cysteine cathepsins is, however, unknown and their particular biochemical properties, tissue localisation, and involvement in parasite-host relationships are yet to be explored. METHODS: Sequences of cathepsins L and B (EnCL and EnCB) were mined from E. nipponicum transcriptome and analysed bioinformatically...
March 6, 2018: Parasites & Vectors
https://www.readbyqxmd.com/read/29497015/yopt-domain-of-the-pfhb2-toxin-from-pasteurella-multocida-protein-expression-characterization-crystallization-and-crystallographic-analysis
#13
Sanjeev Kumar, Victoria Hedrick, Seema Mattoo
Pasteurella multocida causes respiratory-tract infections in a broad range of animals, as well as opportunistic infections in humans. P. multocida secretes a multidomain toxin called PfhB2, which contains a YopT-like cysteine protease domain at its C-terminus. The YopT domain of PfhB2 contains a well conserved Cys-His-Asp catalytic triad that defines YopT family members, and shares high sequence similarity with the prototype YopT from Yersinia sp. To date, only one crystal structure of a YopT family member has been reported; however, additional structural information is needed to help characterize the varied substrate specificity and enzymatic action of this large protease family...
March 1, 2018: Acta Crystallographica. Section F, Structural Biology Communications
https://www.readbyqxmd.com/read/29483496/comparison-of-chromosomal-rearrangements-in-bone-marrow-cells-and-blast-transformed-b-cells-in-relapse-of-b-cell-chronic-lymphocytic-leukemia-small-lymphocytic-lymphoma
#14
S V Andreieva, K V Korets, O E Ruzhinska, I M Skorokhod, O G Alkhimova
AIM: The genetic mechanisms of resistance to chemotherapy in B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma (B-CLL/SLL) are not clear. We aimed to determine the peculiarities of abnormal karyotype formation in bone marrow (BM) cells and peripheral blood (PB) blast transformed B-cells in relapse of B-CLL/SLL. MATERIALS AND METHODS: Cytogenetic GTG banding technique and molecular cytogenetic in interphase cells (i-FISH) studies of BM cells and PB blast transformed B-lymphocytes were performed in 14 patients (10 males and 4 females) with B-CLL/SLL...
July 2017: Experimental Oncology
https://www.readbyqxmd.com/read/29431697/convergent-therapeutic-strategies-to-overcome-the-heterogeneity-of-acquired-resistance-in-brafv600e-colorectal-cancer
#15
Mehlika Hazar-Rethinam, Marianna Kleyman, G Celine Han, David Liu, Leanne G Ahronian, Heather A Shahzade, Lifeng Chen, Aparna R Parikh, Jill N Allen, Jeffrey W Clark, Eunice L Kwak, Jason E Faris, Janet E Murphy, Theodore S Hong, Emily E Van Seventer, Brandon Nadres, Catriona B Hong, Joseph M Gurski, Nicholas A Jessop, Dora Dias-Santagata, A John Iafrate, Eliezer M Van Allen, Ryan B Corcoran
Clonal heterogeneity associated with acquired resistance presents a critical therapeutic challenge. Whole-exome sequencing of paired tumor biopsies and targeted sequencing of cell-free DNA (cfDNA) from BRAFV600E colorectal cancer patients receiving BRAF inhibitor combinations identified 14 distinct alterations in MAPK pathway components driving acquired resistance, with as many as eight alterations in a single patient. We developed a novel pooled clone system to study clonal outgrowth during acquired resistance, in vitro and in vivo...
February 5, 2018: Cancer Discovery
https://www.readbyqxmd.com/read/29403498/two-distinct-pathways-in-mice-generate-antinuclear-antigen-reactive-b-cell-repertoires
#16
Martin Faderl, Fabian Klein, Oliver F Wirz, Stefan Heiler, Llucia Albertí-Servera, Corinne Engdahl, Jan Andersson, Antonius Rolink
The escape of anti-self B cells from tolerance mechanisms like clonal deletion, receptor editing, and anergy results in the production of autoantibodies, which is a hallmark of many autoimmune disorders. In this study, we demonstrate that both germline sequences and somatic mutations contribute to autospecificity of B cell clones. For this issue, we investigated the development of antinuclear autoantibodies (ANAs) and their repertoire in two different mouse models. First, in aging mice that were shown to gain several autoimmune features over time including ANAs...
2018: Frontiers in Immunology
https://www.readbyqxmd.com/read/29400437/different-bioactive-neuropeptides-are-expressed-in-two-sub-classes-of-gabaergic-rme-nerve-ring-motorneurons-inascaris-suum
#17
Jennifer J Knickelbine, Christopher J Konop, India R Viola, Colette B Rogers, Lynn A Messinger, Martha M Vestling, Antony O W Stretton
Neuropeptides can have significant effects on neurons and synapses, but among the ~250 predicted peptides in nematodes, few have been functionally characterized. Here, we report new neuropeptides in the 4 RME nerve ring motorneurons of the nematode Ascaris suum. These GABAergic neurons are involved in three-dimensional head movement. Mass spectrometry (MS) of single dissected RMEs detected a total of 12 neuropeptides (encoded by 5 genes), 9 of which are novel. None of these are expressed in the DI/VI inhibitory GABAergic motorneurons that synapse onto body wall muscle...
February 5, 2018: ACS Chemical Neuroscience
https://www.readbyqxmd.com/read/29391594/contraction-of-t-cell-richness-in-lung-cancer-brain-metastases
#18
Aaron S Mansfield, Hongzheng Ren, Shari Sutor, Vivekananda Sarangi, Asha Nair, Jaime Davila, Laura R Elsbernd, Julia B Udell, Roxana S Dronca, Sean Park, Svetomir N Markovic, Zhifu Sun, Kevin C Halling, Wendy K Nevala, Marie Christine Aubry, Haidong Dong, Jin Jen
Very little is known about how the adaptive immune system responds to clonal evolution and tumor heterogeneity in non-small cell lung cancer. We profiled the T-cell receptor β complementarity determining region 3 in 20 patients with fully resected non-small cell lung cancer primary lesions and paired brain metastases. We characterized the richness, abundance and overlap of T cell clones between pairs, in addition to the tumor mutation burden and predicted neoantigens. We found a significant contraction in the number of unique T cell clones in brain metastases compared to paired primary cancers...
February 1, 2018: Scientific Reports
https://www.readbyqxmd.com/read/29388096/activation-induced-cytidine-deaminase-aided-in-vitro-antibody-evolution
#19
Lili An, Chuan Chen, Ruiqi Luo, Yun Zhao, Haiying Hang
Activation-induced cytidine deaminase (AID) initiates somatic hypermutation (SHM) by converting deoxycytidines (dC) to deoxyuracils (dU) which then can induce other mutations, and plays a central role in introducing diversification of the antibody repertoire in B cells. Ectopic expression of AID in bacteria and non-B cells can also lead to frequent mutations in highly expressed genes. Taking advantage of this feature of AID, in recent years, systems coupling in vitro somatic hypermutation and mammalian cell surface display have been developed, with unique benefits in antibody discovery and optimization in vitro...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29379065/preferential-amplification-of-a-human-mitochondrial-dna-deletion-in-vitro-and-in-vivo
#20
Oliver M Russell, Isabelle Fruh, Pavandeep K Rai, David Marcellin, Thierry Doll, Amy Reeve, Mitchel Germain, Julie Bastien, Karolina A Rygiel, Raffaele Cerino, Andreas W Sailer, Majlinda Lako, Robert W Taylor, Matthias Mueller, Robert N Lightowlers, Doug M Turnbull, Stephen B Helliwell
We generated induced pluripotent stem cells (iPSCs) from patient fibroblasts to yield cell lines containing varying degrees of heteroplasmy for a m.13514 A > G mtDNA point mutation (2 lines) and for a ~6 kb single, large scale mtDNA deletion (3 lines). Long term culture of the iPSCs containing a single, large-scale mtDNA deletion showed consistent increase in mtDNA deletion levels with time. Higher levels of mtDNA heteroplasmy correlated with increased respiratory deficiency. To determine what changes occurred in deletion level during differentiation, teratomas comprising all three embryonic germ layers were generated from low (20%) and intermediate heteroplasmy (55%) mtDNA deletion clones...
January 29, 2018: Scientific Reports
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