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Subcloning

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https://www.readbyqxmd.com/read/28210460/cloning-and-expression-of-clostridium-perfringens-type-d-vaccine-strain-epsilon-toxin-gene-in-e-coli-as-a-recombinant-vaccine-candidate
#1
Parastoo Aziminia, Reza Pilehchian-Langroudi, Kasra Esmaeilnia
BACKGROUND AND OBJECTIVES: Clostridium perfringens, a Gram-positive obligate anaerobic bacterium, is able to form resistant spores which are widely distributed in the environment. C. perfringens is subdivided into five types A to E based on its four major alpha, beta, epsilon and iota toxins. The aim of the present study was cloning and expression of C. perfringens type D vaccine strain epsilon toxin gene. MATERIALS AND METHODS: Genomic DNA was extracted and the epsilon toxin gene was amplified using Pfu DNA polymerase...
August 2016: Iranian Journal of Microbiology
https://www.readbyqxmd.com/read/28194744/heterogeneous-expression-of-dnak-gene-from-alicyclobacillus-acidoterrestris-improves-the-resistance-of-escherichia-coli-against-heat-and-acid-stress
#2
Xixi Xu, Lingxia Jiao, Xin Feng, Junjian Ran, Xinhong Liang, Ruixiang Zhao
Alicyclobacillus acidoterrestris, an acidophilic and thermophilic bacteria, is an important microbial resource for stress resistance genes screening. In this study, DnaK gene from A. acidoterrestris was subcloned to construct the recombinant plasmid pET28a-DnaK. The successful construction of the plasmid was verified by double-enzyme digestion and sequencing analysis. The recombinant plasmid was transformed into Escherichia coli BL21 and isopropy-β-D-thiogalactoside (IPTG) was used to induce recombinant E...
December 2017: AMB Express
https://www.readbyqxmd.com/read/28193548/advances-in-understanding-tumour-evolution-through-single-cell-sequencing
#3
Jack Kuipers, Katharina Jahn, Niko Beerenwinkel
The mutational heterogeneity observed within tumours poses additional challenges to the development of effective cancer treatments. A thorough understanding of a tumour's subclonal composition and its mutational history is essential to open up the design of treatments tailored to individual patients. Comparative studies on a large number of tumours permit the identification of mutational patterns which may refine forecasts of cancer progression, response to treatment and metastatic potential. The composition of tumours is shaped by evolutionary processes...
February 11, 2017: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/28192198/molecular-cloning-purification-and-characterization-of-brugia-malayi-phosphoglycerate-kinase
#4
Ranjeet Kumar, Pawan Kumar Doharey, Jitendra Kumar Saxena, Sushma Rathaur
Phosphoglycerate kinase (PGK) is a glycolytic enzyme present in many parasites. It has been reported as a candidate molecule for drug and vaccine developments. In the present study, a full-length cDNA encoding the Brugia malayi 3-phosphoglycerate kinase (BmPGK) with an open reading frame of 1.3 kb was isolated and PCR amplified and cloned. The exact size of the BmPGK's ORF is 1377 bps. The BmPGK gene was subcloned into pET-28a (+) expression vector, the expressed enzyme was purified by affinity column and characterized...
February 9, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28190919/heterogeneity-of-tumor-cells-in-terms-of-cancer-initiating-cells
#5
REVIEW
Eiichi Morii
Tumors derive from a single cell clone but consist of heterogeneous cell subpopulations with diverse features and functions. A limited number of subclones with a selective advantage can initiate tumors when inoculated into immunocompromised mice, and are called cancer-initiating cells (CICs). CICs can be isolated from the bulk of tumors on the basis of their characteristics, such as high reagent efflux, degradation of reactive oxygen species, and aldehyde dehydrogenase (ALDH) activity. Under normal conditions, new CICs are produced by existing CICs rather than non-CICs...
January 2017: Journal of Toxicologic Pathology
https://www.readbyqxmd.com/read/28187514/aieop-bfm-consensus-guidelines-2016-for-flow-cytometric-immunophenotyping-of-pediatric-acute-lymphoblastic-leukemia
#6
Michael N Dworzak, Barbara Buldini, Giuseppe Gaipa, Richard Ratei, Ondrej Hrusak, Drorit Luria, Eti Rosenthal, Jean-Pierre Bourquin, Mary Sartor, Angela Schumich, Leonid Karawajew, Ester Mejstrikova, Oscar Maglia, Georg Mann, Wolf-Dieter Ludwig, Andrea Biondi, Martin Schrappe, Giuseppe Basso
Immunophenotyping by flow cytometry (FCM) is a worldwide mainstay in leukemia diagnostics. For concordant multicentric application, however, a gap exists between available classification systems, technologic standardization, and clinical needs. The AIEOP-BFM consortium induced an extensive standardization and validation effort between its nine national reference laboratories collaborating in immunophenotyping of pediatric acute lymphoblastic leukemia (ALL). We elaborated common guidelines which take advantage of the possibilities of multi-color FCM: marker panel requirements, immunological blast gating, in-sample controls, tri-partite antigen expression rating (negative vs...
February 10, 2017: Cytometry. Part B, Clinical Cytometry
https://www.readbyqxmd.com/read/28177909/fibulin-4-is-associated-with-prognosis-of-endometrial-cancer-patients-and-inhibits-cancer-cell-invasion-and-metastasis-via-wnt-%C3%AE-catenin-signaling-pathway
#7
Tiantian Wang, Mei Wang, Shuang Fang, Qiang Wang, Rui Fang, Jie Chen
Fibulin-4, an extracellular glycoprotein, which plays significant roles in elastic fiber assembly, is correlated to the progression of some cancers. However, the role of fibulin-4 in endometrial cancer cell invasion and metastasis remains unexplored. In our study, fibulin-4 expression was assessed by immunohistochemistry (IHC) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in normal endometrial tissues and endometrial carcinoma tissues. Using single cell cloning, strongly, and weakly, invasive subclones were derived from KLE and Ishikawa endometrial carcinoma cell lines...
February 4, 2017: Oncotarget
https://www.readbyqxmd.com/read/28168700/progenitor-genotyping-reveals-a-complex-clonal-architecture-in-a-subset-of-calr-mutated-myeloproliferative-neoplasms
#8
Sarah Martin, Casey M Wright, Linda M Scott
The identification of acquired CALR mutations in patients with essential thrombocythaemia (ET) or myelofibrosis (MF) has meant that disease-initiating mutations can now be detected in about 90% of all patients with a myeloproliferative neoplasm (MPN). Here, we show that only those CALR mutations that cause a +1 frameshift, thereby altering the carboxy-terminus of calreticulin, promote cytokine independence in vitro; in-frame deletions were not functional, and are unlikely to be the pathogenetic mutation underlying some MPN cases...
February 7, 2017: British Journal of Haematology
https://www.readbyqxmd.com/read/28167771/ipscs-and-fibroblast-subclones-from-the-same-fibroblast-population-contain-comparable-levels-of-sequence-variations
#9
Erika M Kwon, John P Connelly, Nancy F Hansen, Frank X Donovan, Thomas Winkler, Brian W Davis, Halah Alkadi, Settara C Chandrasekharappa, Cynthia E Dunbar, James C Mullikin, Paul Liu
Genome integrity of induced pluripotent stem cells (iPSCs) has been extensively studied in recent years, but it is still unclear whether iPSCs contain more genomic variations than cultured somatic cells. One important question is the origin of genomic variations detected in iPSCs-whether iPSC reprogramming induces such variations. Here, we undertook a unique approach by deriving fibroblast subclones and clonal iPSC lines from the same fibroblast population and applied next-generation sequencing to compare genomic variations in these lines...
February 6, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28167216/capturing-tumor-heterogeneity-and-clonal-evolution-in-solid-cancers-using-circulating-tumor-dna-analysis
#10
REVIEW
Nieves Perdigones, Muhammed Murtaza
Circulating tumor DNA analysis has emerged as a potential noninvasive alternative to tissue biopsies for tumor genotyping in patients with metastatic cancer. This is particularly attractive in cases where tissue biopsies are contraindicated or repeat genotyping after progression on treatment is required. However, tissue and plasma analysis results are not always concordant and clinical interpretation of discordant results is not completely understood. Discordant results could arise due to analytical limits of assays used for tumor and plasma DNA analysis or due to low overall contribution of tumor-specific DNA in plasma...
February 3, 2017: Pharmacology & Therapeutics
https://www.readbyqxmd.com/read/28164534/expression-of-hcv-alternative-reading-frame-protein-core-1-f-in-baculovirus-expression-system-and-its-evaluation-for-assessment-of-specific-anti-core-1-antibody-in-iranian-hcv-infected-patients
#11
Pooneh Rahimi, Rouhollah Vahabpour, Farideh Sadat Sajadian Fard, Fatemeh Motevalli, Fatemeh Fotouhi Chahouki, Mohammad Reza Aghasadeghi, Azam Bolhassani, Seyed Mehdi Sadat, Ehsan Mostafavi, Nasir Mohajel, Ali Jahanian-Najafabadi, Mohammad Reza Amiran
BACKGROUND: Hepatitis C virus (HCV) genome contains an overlapping reading frame which results in alternative core protein (ARFP). Baculovirus expression system was used as a powerful eukaryotic vector system to express core+1/F protein for the first time. This recombinant core+1/F protein was used to assess the anti-core+1 antibody in anti-HCV drug resistant and sustained virologic response (SVR) patients. METHODS: The core+1 coding sequence from HCV genotype 1 was designed and synthesized in pUC57 vector...
October 1, 2016: Clinical Laboratory
https://www.readbyqxmd.com/read/28161964/in-vitro-evaluation-of-a-polytope-dna-construct-as-a-novel-dna-vaccine-strategy-against-human-cytomegalovirus-associated-diseases
#12
E Sabbaghian, F Roodbari, J Amani, A Rafiei
Human cytomegalovirus (HCMV) establishes severe disease in fetus, newborn and immunocompromised individuals. Polytope DNA vaccine strategy allows us to choose conserved and immunodominant epitopes from different antigens that can stimulate cellular and humoral immune responses simultaneously. In this study, a synthetic chimeric gene fragment was subcloned in to DNA vaccine vector pcDNA3.1+. The recombinant vector was transferred in to suitable eukaryotic cell line HEK 293T and the expression level of polytope construct from HEK 293T-infected cells was determined by western blot...
February 6, 2017: Acta Virologica
https://www.readbyqxmd.com/read/28157837/a-novel-surfactant-protein-c-mutation-resulting-in-aberrant-protein-processing-and-altered-subcellular-localization-causes-infantile-interstitial-lung-disease
#13
Da Hong, Yuanyuan Qi, Jing Liu, Huijun Wang, Libo Wang, Liling Qian
BACKGROUND: Mutations in the surfactant protein C gene (SFTPC) result in interstitial lung disease (ILD). Our objective was to report a novel SFTPC mutation and evaluate the effect of this mutant on protein synthesis and processing. METHODS: Genomic DNA was extracted from whole blood of a Chinese infant with ILD and candidate genes associated with ILD were sequenced by next-generation sequencing. Subclones of wild-type and mutant SFTPC were transiently transfected into A549 cells...
February 3, 2017: Pediatric Research
https://www.readbyqxmd.com/read/28157713/the-subclonal-structure-and-genomic-evolution-of-oral-squamous-cell-carcinoma-revealed-by-ultra-deep-sequencing
#14
Siavosh Tabatabaeifar, Mads Thomassen, Martin J Larsen, Stine R Larsen, Torben A Kruse, Jens A Sørensen
Recent studies suggest that head and neck squamous cell carcinomas are very heterogeneous between patients; however the subclonal structure remains unexplored mainly due to studies using only a single biopsy per patient. To deconvolute the clonal structure and describe the genomic cancer evolution, we applied whole-exome sequencing combined with ultra-deep targeted sequencing on oral squamous cell carcinomas (OSCC). From each patient, a set of biopsies was sampled from distinct geographical sites in primary tumor and lymph node metastasis...
February 2, 2017: Oncotarget
https://www.readbyqxmd.com/read/28154920/high-grade-serous-ovarian-cancer-the-clone-wars
#15
REVIEW
Aleksander Salomon-Perzyński, Magdalena Salomon-Perzyńska, Bogdan Michalski, Violetta Skrzypulec-Plinta
BACKGROUND: The last 5 years' studies using next-generation sequencing provided evidences that many types of solid tumors present spatial and temporal genetic heterogeneity and are composed of multiple populations of genetically distinct subclones that evolve over time following a pattern of branched evolution. The evolutionary nature of cancer has been proposed as the major contributor to drug resistance and treatment failure. In this review, we present the current state of knowledge about the clonal evolution of high-grade serous ovarian cancer and discuss the challenge that clonal evolution poses for efforts to achieve an optimal cancer control...
February 3, 2017: Archives of Gynecology and Obstetrics
https://www.readbyqxmd.com/read/28151710/new-developments-in-diagnosis-prognosis-and-assessment-of-response-in-multiple-myeloma
#16
EDITORIAL
Ola Landgren, S Vincent Rajkumar
Over the past few years, the management of multiple myeloma has changed. We have new guidelines regarding how to set the diagnosis, when to initiate therapy, and how to monitor treatment response. In 2014, the updated International Myeloma Working Group (IMWG) diagnostic criteria changed the definition of multiple myeloma from being a disease defined by symptoms to a disease defined by biomarkers. Today, modern combination therapies have reported up to 60% to 80% of patients reaching a complete response. As a logical and necessary step forward, investigators have explored strategies to detect minimal residual disease (MRD) and its correlation with clinical outcomes...
November 15, 2016: Clinical Cancer Research: An Official Journal of the American Association for Cancer Research
https://www.readbyqxmd.com/read/28149494/cloning-expression-and-immunoreactivity-of-recombinant-toxoplasma-gondii-gra5-protein
#17
Zahra Arab-Mazar, Shirzad Fallahi, Ameneh Koochaki, Hadi Mirahmadi, Seyyed Javad Seyyed Tabaei
BACKGROUND AND OBJECTIVES: Toxoplasma gondii is an obligatory intracellular parasite which causes severe diseases in the fetus of pregnant women and immunocopmromised patients. Serological tests based on recombinant protein are one of the main diagnosis methods for the detection of specific antibodies in serum samples. Dense granule antigenic proteins derived from T. gondii (TgGRAs) are potential antigens for the development of diagnostic tools. MATERIALS AND METHODS: DNA was extracted from T...
October 2016: Iranian Journal of Microbiology
https://www.readbyqxmd.com/read/28145097/multiregion-ultra-deep-sequencing-reveals-early-intermixing-and-variable-levels-of-intratumoral-heterogeneity-in-colorectal-cancer
#18
Yuka Suzuki, Sarah Boonhsi Ng, Clarinda Chua, Wei Qiang Leow, Jermain Chng, Shi Yang Liu, Kalpana Ramnarayanan, Anna Gan, Dan Liang Ho, Rachel Ten, Yan Su, Alexandar Lezhava, Jiunn Herng Lai, Dennis Koh, Kiat Hon Lim, Patrick Tan, Steven G Rozen, Iain Beehuat Tan
Intratumor heterogeneity (ITH) contributes to cancer progression and chemoresistance. We sought to comprehensively describe ITH of somatic mutations, copy number, and transcriptomic alterations involving clinically and biologically relevant gene pathways in colorectal cancer (CRC). We performed multiregion, high-depth (384× on average) sequencing of 799 cancer-associated genes in 24 spatially separated primary tumor and nonmalignant tissues from four treatment-naïve CRC patients. We then used ultra-deep sequencing (17 075× on average) to accurately verify the presence or absence of identified somatic mutations in each sector...
February 2017: Molecular Oncology
https://www.readbyqxmd.com/read/28143511/a-novel-salmonella-strain-inactivated-by-a-regulated-autolysis-system-and-expressing-the-b-subunit-of-shiga-toxin-2e-efficiently-elicits-immune-responses-and-confers-protection-against-virulent-stx2e-producing-escherichia-coli
#19
Gayeon Won, Tae Hoon Kim, John Hwa Lee
BACKGROUND: Salmonella Typhimurium (S. Typhimurium) inactivated by a regulated autolysis system was genetically engineered to express the homo-pentameric B subunit of Shiga toxin 2e (Stx2eB) on its surface. To prepare a strain able to yield autolyzed Salmonella bearing Stx2eB, the plasmid pJHL184 harboring stx 2eB gene was transformed into the attenuated S. Typhimurium strain, JOL1454. Stx2eB subcloned into the antigen delivery cassette of the plasmid was expressed as fusion protein with the outer membrane protein RESULTS: The expression of Stx2eB fused to the signal peptide in JOL1454 was validated by immunoblot analysis...
February 1, 2017: BMC Veterinary Research
https://www.readbyqxmd.com/read/28139641/reconstructing-metastatic-seeding-patterns-of-human-cancers
#20
Johannes G Reiter, Alvin P Makohon-Moore, Jeffrey M Gerold, Ivana Bozic, Krishnendu Chatterjee, Christine A Iacobuzio-Donahue, Bert Vogelstein, Martin A Nowak
Reconstructing the evolutionary history of metastases is critical for understanding their basic biological principles and has profound clinical implications. Genome-wide sequencing data has enabled modern phylogenomic methods to accurately dissect subclones and their phylogenies from noisy and impure bulk tumour samples at unprecedented depth. However, existing methods are not designed to infer metastatic seeding patterns. Here we develop a tool, called Treeomics, to reconstruct the phylogeny of metastases and map subclones to their anatomic locations...
January 31, 2017: Nature Communications
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