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Subcloning

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https://www.readbyqxmd.com/read/28535087/cytotoxic-t-cells-in-chronic-idiopathic-neutropenia-express-restricted-antigen-receptors
#1
Semeli Mastrodemou, Evangelia Stalika, Anna Vardi, Katerina Gemenetzi, Michalis Spanoudakis, Maria Karypidou, Irene Mavroudi, Anastasia Hadzidimitriou, Catherine Stavropoulos-Giokas, Helen A Papadaki, Kostas Stamatopoulos
Chronic idiopathic neutropenia (CIN) is an acquired disorder of granulopoiesis characterized by female predominance and mostly uncomplicated course. Crucial to CIN pathophysiology is the presence of activated T lymphocytes with myelosuppressive properties in both peripheral blood (PB) and bone marrow (BM). We systematically profiled the T cell receptor beta chain (TRB) gene repertoire in CD8(+) cells of 34 CIN patients through subcloning/Sanger sequencing analysis of TRBV-TRBD-TRBJ gene rearrangements. Remarkable repertoire skewing and oligoclonality were observed, along with shared clonotypes between different patients, alluding to antigen selection...
May 23, 2017: Leukemia & Lymphoma
https://www.readbyqxmd.com/read/28534229/production-of-a-gonadotropin-releasing-hormone-2-receptor-knockdown-gnrhr2-kd-swine-line
#2
A T Desaulniers, R A Cederberg, G A Mills, C A Lents, B R White
Swine are the only livestock species that produce both the second mammalian isoform of gonadotropin-releasing hormone (GNRH2) and its receptor (GNRHR2). Previously, we reported that GNRH2 and GNRHR2 mediate LH-independent testosterone secretion from porcine testes. To further explore this ligand-receptor complex, a pig model with reduced GNRHR2 expression was developed. Small hairpin RNA sequences targeting porcine GNRHR2 were subcloned into a lentiviral-based vector, lentiviral particles were generated and microinjected into the perivitelline space of zygotes, and embryos were transferred into a recipient...
May 22, 2017: Transgenic Research
https://www.readbyqxmd.com/read/28533233/epidemic-emergence-in-the-united-states-of-escherichia-coli-sequence-type-131-h30-2000-2009
#3
James R Johnson, Stephen Porter, Paul Thuras, Mariana Castanheira
The H30 subclone of Escherichia coli sequence type 131 (ST131) has become the leading antimicrobial-resistance E. coli lineage in the U.S., and often exhibits resistance to one or both of two key antimicrobial classes for treating Gram-negative infections, extended-spectrum cephalosporins (ESCs) and fluoroquinolones (FQs). However, the timing of and reasons for its recent emergence are inadequately defined. Accordingly, from E. coli clinical isolates collected systematically across the U.S. by the SENTRY Antimicrobial Surveillance Programs in 2000, 2003, 2006, and 2009, 234 isolates were selected randomly, stratified by year, within three resistance categories: (i) ESC-reduced susceptibility, regardless of FQ phenotype (hereafter, ESC-RS), (ii) FQ-resistant, ESC-susceptible (hereafter, FQ-R), and (iii) FQ-susceptible, ESC-susceptible (hereafter, FQ-S)...
May 22, 2017: Antimicrobial Agents and Chemotherapy
https://www.readbyqxmd.com/read/28533157/characterization-of-the-cellulosomal-scaffolding-protein-cbpc-from-clostridium-cellulovorans-743b
#4
Daichi Nakajima, Toshiyuki Shibata, Reiji Tanaka, Kouichi Kuroda, Mitsuyoshi Ueda, Hideo Miyake
Clostridium cellulovorans 743B, an anaerobic and mesophilic bacterium, produces an extracellular enzyme complex called the cellulosome on the cell surface. Recently, we have reported the whole genome sequence of C. cellulovorans, which revealed that a total of 4 cellulosomal scaffolding proteins: CbpA, HbpA, CbpB, and CbpC were encoded in the C. cellulovorans genome. In particular, cbpC encoded a 429-residue polypeptide that includes a carbohydrate-binding module (CBM), an S-layer homology module, and a cohesin...
May 19, 2017: Journal of Bioscience and Bioengineering
https://www.readbyqxmd.com/read/28531253/gender-related-prognostic-value-and-genomic-pattern-of-intra-tumor-heterogeneity-ith-in-colorectal-cancer
#5
Jieyun Zhang, Shican Yan, Xiyu Liu, Lu Gan, Zhenhua Wu, Yiwei Gong, Mingzhu Huang, Xiaowei Zhang, Weijian Guo
Intra-tumor heterogeneity (ITH) is crucial in tumorigenesis and resistance to target therapy. Here, we used mutant-allele tumor heterogeneity (MATH) to measure ITH based on next-generation sequencing data and high MATH was proven as an independent risk prognostic factor in male CRC patients in both a training set of 284 colorectal cancer (CRC) patients with from The Cancer Genome Atlas (TCGA) and a validating set of 187 CRC patients from International Cancer Genome Consortium (ICGC). Further, the genomic pattern according to MATH demonstrated that mutation rates of TP53, IRF5 and KRAS were independently associated with MATH, and the latter two were only significant in male patients...
May 20, 2017: Carcinogenesis
https://www.readbyqxmd.com/read/28523377/mining-lipolytic-enzymes-in-community-dna-from-high-andean-soils-using-a-targeted-approach
#6
Daniel Borda-Molina, José Salvador Montaña, María Mercedes Zambrano, Sandra Baena
Microbial enrichments cultures are a useful strategy to speed up the search for enzymes that can be employed in industrial processes. Lipases have gained special attention because they show unique properties such as: broad substrate specificity, enantio- and regio-selectivity and stability in organic solvents. A major goal is to identify novel lipolytic enzymes from microorganisms living in cold extreme environments such as high Andean soils, of relevance to our study being their capability be used in industrial processes...
May 18, 2017: Antonie Van Leeuwenhoek
https://www.readbyqxmd.com/read/28521463/endoplasmic-reticulum-protein-erp46-in-prostate-adenocarcinoma
#7
Wilhelmina C M Duivenvoorden, Sarah N Hopmans, Richard C Austin, Jehonathan H Pinthus
Endoplasmic reticulum (ER) protein ERp46 is a member of the protein disulfide isomerase family of oxidoreductases, which facilitates the reduction of disulfides in proteins and their folding. Accumulation of misfolded proteins has been implicated in cancer. The objectives of the present study were to investigate the role of ERp46 in prostate cancer, its expression and its effects on prostate cancer growth. A tissue microarray with human prostate cancer and normal prostate tissue samples was stained for ERp46 followed by image analysis...
May 2017: Oncology Letters
https://www.readbyqxmd.com/read/28514780/two-distinct-bcl2-rearrangements-each-observed-in-2-independent-subclones-evolving-from-a-founder-clone-with-trisomy-12-in-a-unique-case-of-chronic-lymphocytic-leukemia-small-lymphocytic-lymphoma
#8
https://www.readbyqxmd.com/read/28513614/recurrent-somatic-jak-stat-pathway-variants-within-a-runx1-mutated-pedigree
#9
Kiran Tawana, Jun Wang, Péter A Király, Krisztián Kállay, Gábor Benyó, Marianna Zombori, Judit Csomor, Ahad Al Seraihi, Ana Rio-Machin, András Matolcsy, Claude Chelala, Jamie Cavenagh, Jude Fitzgibbon, Csaba Bödör
Germline variants within the transcription factor RUNX1 are associated with familial platelet disorder and acute leukemia in over 40% of carriers. At present, the somatic events triggering leukemic transformation appear heterogeneous and profiles of leukemia initiation across family members are poorly defined. We report a new RUNX1 family where three sisters harboring a germline nonsense RUNX1 variant, c.601C>T (p.(Arg201*)), developed acute myelomonocytic leukemia (AML) at 5 years of age. Whole-exome sequencing of tumor samples revealed all three siblings independently acquired variants within the JAK-STAT pathway, specifically targeting JAK2 and SH2B3 (a negative regulator of JAK2), while also sharing the 46/1 haplotype linked with sporadic JAK2-positive myeloproliferative neoplasms...
May 17, 2017: European Journal of Human Genetics: EJHG
https://www.readbyqxmd.com/read/28512562/morphologic-confounders-and-cd19-negativity-in-a-case-of-hairy-cell-leukemia
#10
Pulkit Rastogi, Sreejesh Sreedharanunni, Uday Yanamandra, Man Updesh Singh Sachdeva, Neelam Varma
OBJECTIVES: We report a case of hairy cell leukemia (HCL) initially misdiagnosed as plasma cell dyscrasia due to various clinical, morphological and immunophenotypic confounders. METHODS AND RESULTS: In a patient diagnosed of marrow plasmacytosis and serum monoclonal protein elsewhere and referred to our hospital, morphological evaluation of bone marrow aspirate smears and trephine biopsy, immunophenotyping, and molecular testing (BRAFV600E mutation) were done. Clinically, the patient was asymptomatic; bone marrow revealed plasmacytosis, mastocytosis, and lymphocytosis with a few "hairy" cells...
2017: Mediterranean Journal of Hematology and Infectious Diseases
https://www.readbyqxmd.com/read/28510221/development-of-a-dual-expression-vector-facilitated-with-selection-free-pcr-recombination-cloning-strategy
#11
Liting Cao, Yancheng Zhou, Lin Huang, Shiqi Dong, Yue Ma
The conventional procedure for the construction of recombinant expression vector of a target gene includes PCR cloning and restriction enzyme mediated subcloning, which is time-consuming and sometimes troublesome because of the inefficiency of ligation. A variety of ligase-independent PCR cloning strategies have been developed, but they either involve complicated PCR procedures or need other DNA modifying enzymes. In this study, we report the design, and construction of an omnipotent expression vector pOmni, with which a target gene can be easily cloned through innovative selection-free PCR recombination cloning strategy with only one pair of primer and two times of PCR in one work day, without using any restriction enzymes, ligase and other DNA modifying enzymes...
December 2017: AMB Express
https://www.readbyqxmd.com/read/28508214/efficient-reverse-genetic-systems-for-rapid-genetic-manipulation-of-emergent-and-preemergent-infectious-coronaviruses
#12
Adam S Cockrell, Anne Beall, Boyd Yount, Ralph Baric
Emergent and preemergent coronaviruses (CoVs) pose a global threat that requires immediate intervention. Rapid intervention necessitates the capacity to generate, grow, and genetically manipulate infectious CoVs in order to rapidly evaluate pathogenic mechanisms, host and tissue permissibility, and candidate antiviral therapeutic efficacy. CoVs encode the largest viral RNA genomes at about 28-32,000 nucleotides in length, and thereby complicate efficient engineering of the genome. Deconstructing the genome into manageable fragments affords the plasticity necessary to rapidly introduce targeted genetic changes in parallel and assort mutated fragments while maximizing genome stability over time...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28504724/single-cell-transcriptomics-uncovers-distinct-molecular-signatures-of-stem-cells-in-chronic-myeloid-leukemia
#13
Alice Giustacchini, Supat Thongjuea, Nikolaos Barkas, Petter S Woll, Benjamin J Povinelli, Christopher A G Booth, Paul Sopp, Ruggiero Norfo, Alba Rodriguez-Meira, Neil Ashley, Lauren Jamieson, Paresh Vyas, Kristina Anderson, Åsa Segerstolpe, Hong Qian, Ulla Olsson-Strömberg, Satu Mustjoki, Rickard Sandberg, Sten Eirik W Jacobsen, Adam J Mead
Recent advances in single-cell transcriptomics are ideally placed to unravel intratumoral heterogeneity and selective resistance of cancer stem cell (SC) subpopulations to molecularly targeted cancer therapies. However, current single-cell RNA-sequencing approaches lack the sensitivity required to reliably detect somatic mutations. We developed a method that combines high-sensitivity mutation detection with whole-transcriptome analysis of the same single cell. We applied this technique to analyze more than 2,000 SCs from patients with chronic myeloid leukemia (CML) throughout the disease course, revealing heterogeneity of CML-SCs, including the identification of a subgroup of CML-SCs with a distinct molecular signature that selectively persisted during prolonged therapy...
May 15, 2017: Nature Medicine
https://www.readbyqxmd.com/read/28504647/identification-of-a-nucleoside-analog-active-against-adenosine-kinase-expressing-plasma-cell-malignancies
#14
Utthara Nayar, Jouliana Sadek, Jonathan Reichel, Denise Hernandez-Hopkins, Gunkut Akar, Peter J Barelli, Michelle A Sahai, Hufeng Zhou, Jennifer Totonchy, David Jayabalan, Ruben Niesvizky, Ilaria Guasparri, Duane Hassane, Yifang Liu, Shizuko Sei, Robert H Shoemaker, J David Warren, Olivier Elemento, Kenneth M Kaye, Ethel Cesarman
Primary effusion lymphoma (PEL) is a largely incurable malignancy of B cell origin with plasmacytic differentiation. Here, we report the identification of a highly effective inhibitor of PEL. This compound, 6-ethylthioinosine (6-ETI), is a nucleoside analog with toxicity to PEL in vitro and in vivo, but not to other lymphoma cell lines tested. We developed and performed resistome analysis, an unbiased approach based on RNA sequencing of resistant subclones, to discover the molecular mechanisms of sensitivity...
May 15, 2017: Journal of Clinical Investigation
https://www.readbyqxmd.com/read/28504024/detection-of-driver-and-subclonal-mutations-in-myelofibrosis-clinical-impact-on-pharmacologic-and-transplant-based-treatment-strategies
#15
Maria Chiara Finazzi, Federico Lussana, Silvia Salmoiraghi, Orietta Spinelli, Alessandro Rambaldi
Myelofibrosis (MF) is the most aggressive form among Philadelphia negative (Ph-) myeloproliferative neoplasms (MPNs). In the last years, the mutational landscape of MF has expanded remarkably by the identification of additional recurrent mutations, called subclonal mutations. Areas covered. Here we describe the available data about the currently identified subclonal mutations and their prognostic value in MF patients. We also review the practical value of including such molecular information in available prognostic models for both outcome prediction and possibly treatment decision with regards to transplant indication...
May 15, 2017: Expert Review of Hematology
https://www.readbyqxmd.com/read/28502749/a-relevant-ige-reactive-28kda-protein-identified-from-salsola-kali-pollen-extract-by-proteomics-is-a-natural-degradation-product-of-an-integral-47kda-polygalaturonase
#16
Salvador Mas, Carmen Oeo-Santos, Javier Cuesta-Herranz, Araceli Díaz-Perales, Carlos Colás, Javier Fernández, Domingo Barber, Rosalía Rodríguez, V de Los Ríos, Rodrigo Barderas, Mayte Villalba
A highly prevalent IgE-binding protein band of 28kDa is observed when Salsola kali pollen extract is incubated with individual sera from Amaranthaceae pollen sensitized patients. By an immunoproteomic analysis of S. kali pollen extract, we identified this protein band as an allergenic polygalacturonase enzyme. The allergen, named Sal k 6, exhibits a pI of 7.14 and a molecular mass of 39,554.2Da. It presents similarities to Platanaceae, Poaceae, and Cupressaceae allergenic polygalacturonases. cDNA-encoding sequence was subcloned into the pET41b vector and produced in bacteria as a His-tag fusion recombinant protein...
May 10, 2017: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/28496009/dynamic-changes-in-clonal-cytogenetic-architecture-during-progression-of-chronic-lymphocytic-leukemia-in-patients-and-patient-derived-murine-xenografts
#17
Nicholas J Davies, Marwan Kwok, Clive Gould, Ceri E Oldreive, Jingwen Mao, Helen Parry, Edward Smith, Angelo Agathanggelou, Guy Pratt, Alexander Malcolm R Taylor, Paul Moss, Mike Griffiths, Tatjana Stankovic
Subclonal heterogeneity and clonal selection influences disease progression in chronic lymphocytic leukemia (CLL). It is therefore important that therapeutic decisions are made based on an understanding of the CLL clonal architecture and its dynamics in individual patients. Identification of cytogenetic abnormalities by FISH remains the cornerstone of contemporary clinical practice and provides a simple means for prognostic stratification. Here, we demonstrate that multiplexed-FISH can enhance recognition of CLL subclonal repertoire and its dynamics during disease progression, both in patients and CLL patient-derived xenografts (PDX)...
April 26, 2017: Oncotarget
https://www.readbyqxmd.com/read/28495913/longitudinal-fluorescence-in-situ-hybridization-reveals-cytogenetic-evolution-in-myeloma-relapsing-after-autologous-transplantation
#18
Maximilian Merz, Anna Jauch, Thomas Hielscher, Elias K Mai, Anja Seckinger, Dirk Hose, Uta Bertsch, Kai Neben, Marc S Raab, Hans Salwender, Igor W Blau, Hans-Walter Lindemann, Ingo Schmidt-Wolf, Christof Scheid, Mathias Haenel, Katja Weisel, Hartmut Goldschmidt, Jens Hillengass
To investigate cytogenetic evolution after upfront autologous stem cell transplantation for newly diagnosed myeloma we retrospectively analyzed fluorescence in situ hybridization results of 128 patients with paired bone marrow samples at primary diagnosis and relapse. High-risk cytogenetic abnormalities (deletion 17p and/or gain1q21) occurred more frequently after relapse (odds ratio: 6.33; 95% confidence interval: 1.86,33.42;p<0.001). No significant changes were observed for defined IgH translocations (t(4;14); t(11;14); t(14;16)) or hyperdiploid karyotypes between primary diagnosis and relapse...
May 11, 2017: Haematologica
https://www.readbyqxmd.com/read/28492226/circulating-tumour-dna-sequence-analysis-as-an-alternative-to-multiple-myeloma-bone-marrow-aspirates
#19
Olena Kis, Rayan Kaedbey, Signy Chow, Arnavaz Danesh, Mark Dowar, Tiantian Li, Zhihua Li, Jessica Liu, Mark Mansour, Esther Masih-Khan, Tong Zhang, Scott V Bratman, Amit M Oza, Suzanne Kamel-Reid, Suzanne Trudel, Trevor J Pugh
The requirement for bone-marrow aspirates for genomic profiling of multiple myeloma poses an obstacle to enrolment and retention of patients in clinical trials. We evaluated whether circulating cell-free DNA (cfDNA) analysis is comparable to molecular profiling of myeloma using bone-marrow tumour cells. We report here a hybrid-capture-based Liquid Biopsy Sequencing (LB-Seq) method used to sequence all protein-coding exons of KRAS, NRAS, BRAF, EGFR and PIK3CA in 64 cfDNA specimens from 53 myeloma patients to >20,000 × median coverage...
May 11, 2017: Nature Communications
https://www.readbyqxmd.com/read/28489852/recurrent-promoter-mutations-in-melanoma-are-defined-by-an-extended-context-specific-mutational-signature
#20
Nils Johan Fredriksson, Kerryn Elliott, Stefan Filges, Jimmy Van den Eynden, Anders Ståhlberg, Erik Larsson
Sequencing of whole tumor genomes holds the promise of revealing functional somatic regulatory mutations, such as those described in the TERT promoter. Recurrent promoter mutations have been identified in many additional genes and appear to be particularly common in melanoma, but convincing functional data such as influence on gene expression has been more elusive. Here, we show that frequently recurring promoter mutations in melanoma occur almost exclusively at cytosines flanked by a distinct sequence signature, TTCCG, with TERT as a notable exception...
May 10, 2017: PLoS Genetics
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