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Kazunari Nakaishi, Satoshi Watabe, Toshiyuki Kitagawa, Etsuro Ito
We propose a new detection method for a tuberculosis-specific protein, MPB64, obtained from active bacillus Calmette-Guérin (BCG) by heating. When BCG was included in solution at a concentration >2.75 × 104 CFU/ml, our method for collecting MPB64 through heating active BCG combined with an immunochromatographic assay detected active bacilli within 2.5 h. By contrast, a culture test, which is the gold standard for tuberculosis diagnosis, does not provide results for between 1 week and 2 months. The rapid tests based on PCR have some drawbacks, for example they detect DNA from both active and latent (or even dead) tubercle bacilli...
April 8, 2019: BioTechniques
Wei Jiang, Wei Lian, Jieting Chen, Wenlei Li, Jieyong Huang, Baoyu Lai, Lingyun Li, Zhong Huang, Jianyong Xu
Mesenchymal stem cells (MSCs) have been intensively investigated and widely applied in regenerative medicine and immune modulation. However, their efficacy declines during the aging or disease process. Thus, genome-edited MSCs with over-expression or inhibition of specific genes hold a great deal of promise in terms of their therapeutic application. Here we optimized the direct PCR approach for rapid identification of genome-edited MSCs with only ten cells required, which reduces the time and labor to expand the MSC colonies...
March 29, 2019: BioTechniques
Jo-Ann L Stanton, Abishek Muralidhar, Christy J Rand, David J Saul
BACKGROUND: PDQeX is a novel, single-step DNA extraction method that purifies nucleic acid from sample in under 30 min. MATERIALS & METHODS: Six bacterial suspensions from species with different cell morphologies and growth optima were made. DNA from half the suspension was purified using PDQeX and the other half using a conventional column purification method. Sequencing and analyses using Ion PGM were performed, blinded to extraction method and species. RESULTS: Genomes extracted with either method sequenced successfully...
February 28, 2019: BioTechniques
Mieko Kato, Yoshiro Hanyu
We present a simple colony assay method for screening antibody libraries based on autoinduction of antibody fragment expression. This protocol eliminates the need for colony size monitoring and a separate induction step for single-chain Fv (scFv) antibody fragment expression. Here, scFvs are expressed in an automatic and timely fashion during the assay, resulting in high yields of positive clones and substantial time savings. The method was used successfully to establish monoclonal scFvs with high affinity and specificity against human IgG...
February 20, 2019: BioTechniques
Tulsi Ram Damase, Peter B Allen
To explore thermofluorimetric analysis (TFA) in detail, we compared two related aptamers. The first, LINN2, is a DNA aptamer previously selected against EGFR recombinant protein. In this work we selected a second aptamer, KM4, against EGFR-overexpressing A549 cells. The two aptamers were derived from the same pool and bind the same target but behave differently in TFA. Our results suggest four overall conclusions about TFA of aptamers: 1. Some aptamers show reduced fluorescence upon target binding suggesting that target-bound aptamer is not always fluorescent...
February 15, 2019: BioTechniques
Helena Čelešnik, Uroš Potočnik
We report two restriction enzyme-based approaches for generating clean locus-specific unmethylated controls for methylation-sensitive high-resolution melting (MS-HRM) analyses. These unmethylated standards are derived from DNA treated with the demethylating agent 5-aza-2-deoxycytidine (5-Aza-dc). By using them, we overcome a limitation of 5-Aza-dc treatment - incomplete demethylation at various genomic regions. When 5-Aza-dc-treated DNA is used directly as unmethylated MS-HRM standard, partially demethylated DNA can give false methylation results...
February 14, 2019: BioTechniques
Michel Vivaudou
We present here a software program dedicated to the fitting of experimental dose-response data, which integrates seamlessly with Excel and allows curve fitting plots and results to reside alongside data within Excel spreadsheets. The program, named eeFit, for Excel-Embedded Fitting software, requires no advanced knowledge of Excel or non-linear least-squares fitting. Any experimental data present in an Excel file, such as dose-effect data obtained with membrane receptor or ion channel ligands, can be graphed and fitted interactively with standard Hill models for activation or inhibition, or with more complex models for biphasic effects resulting from combinations of activation and inhibition...
April 2019: BioTechniques
Nikolay Zolotarev, Pavel Georgiev, Oksana Maksimenko
The CRISPR/Cas9 system has recently emerged as a powerful tool for functional genomic studies and has been adopted for many organisms, including Drosophila. Previously, an efficient two-step strategy was developed to engineer the fly genome by combining CRISPR/Cas9 with recombinase-mediated cassette exchange (RMCE). This strategy allows the introduction of designed mutations into a gene of interest in vivo. However, the loxP or frt site remains in the edited locus. Here, we propose a modification of this approach for rapid and efficient seamless genome editing with CRISPR/Cas9 and site-specific recombinase-mediated integration (SSRMI) combined with recombination between homologous sequences induced by the rare-cutting endonuclease I-SceI...
April 2019: BioTechniques
Gilles Malherbe, David Paul Humphreys, Emma Davé
Fractionation in Gram-negative bacteria is used to identify the subcellular localization of proteins, in particular the localization of exported recombinant proteins. The process of cell fractionation can be fraught with cross-contamination issues and often lacks supporting data for fraction purity. Here, we compare three periplasm extraction and two cell disruption techniques in different combinations to investigate which process gives uncontaminated compartments from Escherichia coli. From these data, a robust method named PureFrac was compiled that gives pure periplasmic fractions and a superior recovery of soluble cytoplasmic proteins...
April 2019: BioTechniques
Joseph Martin, Abigail Sawyer
No abstract text is available yet for this article.
April 2019: BioTechniques
(no author information available yet)
No abstract text is available yet for this article.
April 2019: BioTechniques
Vera DesMarais, Robert J Eddy, Ved P Sharma, Orrin Stone, John S Condeelis
We systematically evaluated the performance and reliability of several widely used, commercially available actin-filament probes in a highly motile breast adenocarcinoma cell line to optimize the visualization of F-actin-rich dynamic lamellipodia. We evaluated four Phalloidin-fluorophores, two anti-actin antibodies, and three live-cell actin probes in five fixation conditions across three imaging platforms as a basis for the design of optimized protocols. Of the fluorescent phalloidin-dye conjugates tested, Alexa Fluor-488 Phalloidin ranked best in overall labeling of the actin cytoskeleton and maintenance of the fluorescence signal over time...
March 2019: BioTechniques
Pranvera Hiseni, Robert C Wilson, Ola Storrø, Roar Johnsen, Torbjørn Øien, Knut Rudi
We present a novel liquid array diagnostics (LAD) method, which enables rapid and inexpensive detection of microbial markers in a single-tube multiplex reaction. We evaluated LAD both on pure cultures, and on infant gut microbiota for a 15-plex reaction. LAD showed more than 80% accuracy of classification and a detection limit lower than 2% of the Illumina reads per sample. The results on the clinical dataset showed that there was a rapid decrease of staphylococci from 10-day- to 4-month-old children, a peak of bifidobacteria at 4 months, and a peak of Bacteroides in 2-year-old children, which is in accordance with findings described in the literature...
March 2019: BioTechniques
Kazuhiro Nakagawa, Takuya Kishimoto
The need for technologies to monitor cell health is increasing with advancements in the field of cell therapy and regenerative medicine. In this study, we demonstrated unlabeled optical metabolic imaging of cultured living cells. This imaging technique is based on motion vector analysis with a block-matching algorithm to compare sequential time-lapse images. Motion vector analysis evaluates the movement of intracellular granules observed with a phase-contrast microscope. We demonstrated that the motion speed of intracellular movement reflects adenosine triphosphate (ATP)-dependent intracellular trafficking in cells...
March 2019: BioTechniques
Zhi Li, Yael Zilberman, Qing-Bin Lu, Xiaowu Shirley Tang
An electrochemical approach was devised for detecting DNA damage and differentiating two DNA damage mechanisms, which is important to the design of new chemotherapeutics. This approach combined two platforms, based on the detection of base damage and DNA strand cleavage. In this work, our approach was demonstrated for the detection of cisplatin-induced DNA damage and the enhancement effects of two electron donors, N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) and reduced graphene oxide (rGO). Our results demonstrated that TMPD enhanced DNA strand cleavage, supporting the proposed dissociative electron transfer mechanism...
March 2019: BioTechniques
Jenny Straiton
Jenny Straiton explores the miniature world of organoids and discusses how these small models are making big changes in the world of neurological research.
March 2019: BioTechniques
Ryan Sangston, Jay Hirsh
Controlling the environment of an organism has many biologically relevant applications. Temperature-dependent inducible biological reagents have proven invaluable for elucidating signaling cascades and dissection of neural circuits. Here we develop a simple and affordable system for rapidly changing temperature in a chamber housing adult Drosophila melanogaster. Utilizing flies expressing the temperature-inducible channel dTrpA1 in dopaminergic neurons we show rapid and reproducible changes in locomotor behavior...
March 2019: BioTechniques
(no author information available yet)
No abstract text is available yet for this article.
March 2019: BioTechniques
Jenny Straiton, Tristan Free, Abigail Sawyer, Joseph Martin
We look at how next-generation sequencing has advanced research across different disease fields, and the growing importance of open access genomic databases.
February 2019: BioTechniques
Pawan K Pandoh, Richard D Corbett, Helen McDonald, Miguel Alcaide, Heather Kirk, Eva Trinh, Simon Haile, Tina MacLeod, Duane Smailus, Steve Bilobram, Andrew J Mungall, Yussanne Ma, Richard A Moore, Robin Coope, Yongjun Zhao, Steven Jm Jones, Robert A Holt, Aly Karsan, Ryan D Morin, Marco A Marra
The analysis of cell-free circulating tumor DNA (ctDNA) is potentially a less invasive, more dynamic assessment of cancer progression and treatment response than characterizing solid tumor biopsies. Standard isolation methods require separation of plasma by centrifugation, a time-consuming step that complicates automation. To address these limitations, we present an automatable magnetic bead-based ctDNA isolation method that eliminates centrifugation to purify ctDNA directly from peripheral blood (PB). To develop and test our method, ctDNA from cancer patients was purified from PB and plasma...
February 2019: BioTechniques
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