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Methods: a Companion to Methods in Enzymology

E B Gibbs, R W Kriwacki
Nuclear magnetic resonance spectroscopy (NMR) is a powerful technique for characterizing the structural and dynamic properties of intrinsically disordered proteins and protein regions (IDPs & IDRs). However, the application of NMR to IDPs has been limited by poor chemical shift dispersion in two-dimensional (2D) 1H-15N heteronuclear correlation spectra. Among the various detection schemes available for heteronuclear correlation spectroscopy, 13C direct-detection has become a mainstay for investigations of IDPs owing to the favorable chemical shift dispersion in 2D 13C'-15N correlation spectra...
January 13, 2018: Methods: a Companion to Methods in Enzymology
F Merola, P Memmolo, L Miccio, M Mugnano, P Ferraro
High-throughput single-cell analysis is a challenging target for implementing advanced biomedical applications. An excellent candidate for this aim is label-free tomographic phase microscopy (TPM). In this paper, some of the methods used to obtain TPM are reviewed, analyzing advantages and disadvantages of each of them. Moreover, an alternative tomographic technique is described for live cells analysis, and future trends of the method are foreseen. In particular, by exploiting random rolling of cells while they are flowing along a microfluidic channel, it is possible to obtain phase-contrast tomography thus obtaining complete retrieval of both 3D-position and orientation of rotating cells...
January 13, 2018: Methods: a Companion to Methods in Enzymology
Hao Wang, Carl Kingsford, C Joel McManus
Ribosome profiling has emerged as a powerful technique to study mRNA translation. Ribosome profiling has the potential to determine the relative quantities and locations of ribosomes on mRNA genome wide. Taking full advantage of this approach requires accurate measurement of ribosome locations. However, experimental inconsistencies often obscure the positional information encoded in ribosome profiling data. Here, we describe the Ribodeblur pipeline, a computational analysis tool that uses a maximum likelihood framework to infer ribosome positions from heterogeneous datasets...
January 9, 2018: Methods: a Companion to Methods in Enzymology
Young Je Lee, Tae Seok Moon
One of the long-term goals of synthetic biology is to develop designable genetic parts with predictable behaviors that can be utilized to implement diverse cellular functions. The discovery of non-coding RNAs and their importance in cellular processing have rapidly attracted researchers' attention towards designing functional non-coding RNA molecules. These synthetic non-coding RNAs have simple design principles governed by Watson-Crick base pairing, but exhibit increasingly complex functions. Importantly, due to their specific and modular behaviors, synthetic non-coding RNAs have been widely adopted to modulate transcription and translation of target genes...
January 5, 2018: Methods: a Companion to Methods in Enzymology
Lauriane Gross, Laure Schaeffer, Fatima Alghoul, Hassan Hayek, Christine Allmang, Gilbert Eriani, Franck Martin
In eukaryotes, cap-dependent translation initiation is a sophisticated process that requires numerous trans-acting factors, the eukaryotic Initiation Factors (eIFs). Their main function is to assist the ribosome for accurate AUG start codon recognition. The whole process requires a 5'-3' scanning step and is therefore highly dynamic. Therefore translation requires a complex interplay between eIFs through assembly/release cycles. Here, we describe an original approach to assess the dynamic features of translation initiation...
January 4, 2018: Methods: a Companion to Methods in Enzymology
Carmen Gondhalekar, Bartek Rajwa, Valery Patsekin, Kathy Ragheb, Jennifer Sturgis, J Paul Robinson
Flow cytometry has well-established methods for data analysis based on traditional data collection techniques. These techniques typically involved manual insertion of tube samples into an instrument that, historically, could only measure 1-3 colors. The field has since evolved to incorporate new technologies for faster and highly automated sample preparation and data collection. For example, the use of microwell plates on benchtop instruments is now a standard on virtually every new instrument, and so users can easily accumulate multiple data sets quickly...
January 3, 2018: Methods: a Companion to Methods in Enzymology
Shichao Chen, Yizheng Zhu
In quantitative phase imaging, sensitivity is a key measure of system reproducibility. Despite continuous experimental breakthroughs in achieving highly sensitive detection, in-depth studies of theoretical constraints on sensitivity are inadequate and comparisons between different techniques are difficult. In this paper, we introduce the method to evaluate the sensitivity of phase shifting interferometry which is a major category of quantitative phase imaging techniques. The method discusses in detail several key concepts of sensitivity evaluation, including a general three-level evaluation framework, a complete interference signal model, Cramér-Rao bound and algorithm sensitivity, algorithm and system efficiencies, as well as energy efficiency of an algorithm...
January 3, 2018: Methods: a Companion to Methods in Enzymology
Cheng Wu, Ananya Dasgupta, Lunda Shen, Deborah Bell-Pedersen, Matthew S Sachs
Cell-free protein synthesis (CFPS) can be used in many applications to produce polypeptides and to analyze mechanisms of mRNA translation. Here we describe how to make and use a CPFS system from the model filamentous fungus Neurospora crassa. The extensive genetic resources available in this system provide capacities to exploit robust CFPS for understanding translational control. Included are procedures for the growth and harvesting of cells, the preparation of cell-free extracts that serve as the source of the translational machinery in the CFPS and the preparation of synthetic mRNA to program the CFPS...
December 30, 2017: Methods: a Companion to Methods in Enzymology
Andrew Hsu, Paul A O'Brien, Shibani Bhattacharya, Mark Rance, Arthur G Palmer
Quadrupolar relaxation of 2H (D) nuclear spins is a powerful probe of conformational dynamics in biological macromolecules. Deuterium relaxation rate constants are determined by the spectral density function for reorientation of the C-D bond vector at zero, single-quantum, and double-quantum 2H frequencies. In the present work, 2H relaxation rate constants were measured for an E. coli ribonuclease H [U-2H, 15N] ILV-[13CH2D] sample using 400, 500, 800, and 900 MHz NMR spectrometers and analyzed by three approaches to determine spectral density values...
December 27, 2017: Methods: a Companion to Methods in Enzymology
Albina Rahim, Justin Meskas, Sibyl Drissler, Alice Yue, Anna Lorenc, Adam Laing, Namita Saran, Jacqui White, Lucie Abeler-Dörner, Adrian Hayday, Ryan R Brinkman
The rapid expansion of flow cytometry applications has outpaced the functionality of traditional manual analysis tools used to interpret flow cytometry data. Scientists are faced with the daunting prospect of manually identifying interesting cell populations in 50-dimensional datasets, equalling the complexity previously only reached in mass cytometry. Data can no longer be analyzed or interpreted fully by manual approaches. While automated gating has been the focus of intense efforts, there are many significant additional steps to the analytical pipeline (e...
December 26, 2017: Methods: a Companion to Methods in Enzymology
Qing Feng, Sichen Shao
Protein translation is tightly regulated to ensure high-fidelity expression of genetic information. Various conditions cause ribosomes to stall while synthesizing new proteins. Different types of translational arrest initiate specific mRNA surveillance, protein quality control, and stress response pathways that directly impact gene expression and protein homeostasis. Our understanding of these pathways is greatly enhanced by reconstituting these processes in cell-free systems. The high degree of biochemical manipulability of in vitro systems facilitates the identification of key machineries, mechanistic dissection of their functional roles, and structural analysis of intermediate complexes...
December 22, 2017: Methods: a Companion to Methods in Enzymology
Songlin Wang, T Gopinath, Gianluigi Veglia
Oriented sample solid-state NMR (OS-ssNMR) spectroscopy is uniquely suited to determine membrane protein topology at the atomic resolution in liquid crystalline bilayers under physiological temperature. However, the inherent low sensitivity of this technique has hindered the throughput of multidimensional experiments necessary for resonance assignments and structure determination. In this work, we show that doping membrane protein bicelle preparations with paramagnetic ion chelated lipids and exploiting paramagnetic relaxation effects it is possible to accelerate the acquisition of both 2D and 3D multidimensional experiments with significant saving in time...
December 21, 2017: Methods: a Companion to Methods in Enzymology
R Morilla, K Moss, V Nikolova, K Marquardt, S Duke, K Adamowska, L Fuller, A Taifoor, N Johnson, A Zeisig, A Morilla, A Atra, D C Taussig
An adequate bone marrow aspirate is essential for a rapid diagnosis of acute leukaemia by multicolour flow cytometry enabling the simultaneous assessment of multiple antigens on the cell surface as well as intracellular or nuclear ones. In the context of acute leukaemia, it is important to have a diagnosis of the blasts lineage as soon as possible to decide the appropriate treatment. This is sometimes delayed due to difficulties in of obtaining a bone marrow aspirate due to a "dry tap". In this study we evaluated retrospectively cell markers results by flow cytometry of unfixed bone marrow trephines of 65 patients with leukaemia at diagnosis and including a few after treatment...
December 21, 2017: Methods: a Companion to Methods in Enzymology
Chunfai Lai, Dariusz Stepniak, Leslie Sias, Castle Funatake
Analysis of RNA expression in mixed cell populations often requires laborious and costly cell sorting. Here we describe a flow cytometric assay that combines antibody staining and in situ hybridization for multi-parametric analysis of single cells. This method, referred to as the PrimeFlowTM RNA Assay, enables simultaneous detection of protein markers and RNA targets in mixed cell populations. Both coding and non-coding RNA sequences can be measured with a limit of detection of approximately 10 copies of mRNA and 20 copies of microRNA per cell...
December 19, 2017: Methods: a Companion to Methods in Enzymology
Claire Latroche, Michèle Weiss-Gayet, Cyril Gitiaux, Bénédicte Chazaud
Muscle stem cells or satellite cells are required for skeletal muscle regeneration. It has been shown that the satellite cell microenvironment, including neighboring cells such as endothelial cells, macrophages or fibroblasts are essential for complete and efficient regeneration. A deficient behavior of these cells compromises regeneration. Therefore, there is a strong interest in understanding the cellular and molecular interactions at work between these cell types during muscle regeneration. Fluorescence-activated cell sorting allows to isolate these four cell types at different time points of regeneration, for further high throughput or behavioral experiments...
December 18, 2017: Methods: a Companion to Methods in Enzymology
Jordi Petriz, Jolene A Bradford, Michael D Ward
Red blood cell lysis is an integral part of many flow cytometry protocols. It's potential to cause artifacts has been known for decades, but lysis free sample preparation has failed to replace lysis in most applications. Studies of various lysing protocols on cell losses and effects on phenotypic markers and cell function began early in the history of immunophenotyping and continue to this day. Opportunities to combine live cell response and functional assessment with phenotyping have sparked increasing interest in no lyse no wash protocols, with minimizing sample preparation effects on the cell biology as the primary goal...
December 18, 2017: Methods: a Companion to Methods in Enzymology
Alexandra R Harris, Jessica X Yuan, Jennifer M Munson
The tumor microenvironment is important in promoting treatment resistance of tumor cells via multiple mechanisms. However, studying this interaction often proves difficult. In vivo animal models are costly, time-consuming, and often fail to adequately predict human response to treatment. Conversely, testing drug response on human tumor cells in vitro in 2D cell culture excludes the important contribution of stromal cells and biophysical forces seen in the in vivo tumor microenvironment. Here, we present tissue-engineered models of both human brain and breast tumor microenvironments incorporating key stromal cell populations for assessing multiple mechanisms of therapeutic response using flow cytometry...
December 16, 2017: Methods: a Companion to Methods in Enzymology
Riccardo Maraspini, Oliver Beutel, Alf Honigmann
Quantifying molecular dynamics of cell membrane constituents is required to understand organization and function of biological membranes. Because of its complex structure unambiguous interpretation of molecular membrane dynamics requires high spatial and temporal resolution measurements. In this paper, we provide a comprehensive description of circle scanning fluorescence correlation spectroscopy and its combination with stimulated emission depletion microscopy (CS-STED-FCS). This method allows quantification of sub-diffusion processes and direct mapping of heterogeneities in membranes with high spatiotemporal resolution...
December 16, 2017: Methods: a Companion to Methods in Enzymology
J Lizbeth Zamora Reyes, Hector C Aguilar
In the last few decades, flow cytometry has redefined the field of biology, exponentially enhancing our understanding of cells, immunology, and microbiology. Flow cytometry recently gave birth to flow virometry, a new way to detect, analyze, and characterize single viral particles. Detection of viruses by flow cytometry is possible due to improvements in current flow cytometers, calibration and tuning methods. We summarize the recent birth and novel uses of flow virometry and the progressive evolution of this tool to advance the field of virology...
December 16, 2017: Methods: a Companion to Methods in Enzymology
Martin Stortz, Juan Angiolini, Esteban Mocksos, Alejandro Wolosiuk, Adali Pecci, Valeria Levi
The hierarchical organization of the cell nucleus into specialized open reservoirs and the nucleoplasm overcrowding impose restrictions to the mobility of biomolecules and their interactions with nuclear targets. These properties determine that many nuclear functions such as transcription, replication, splicing or DNA repair are regulated by complex, dynamical processes that do not follow simple rules. Advanced fluorescence microscopy tools and, in particular, fluorescence correlation spectroscopy (FCS) provide complementary and exquisite information on the dynamics of fluorescent labeled molecules moving through the nuclear space and are helping us to comprehend the complexity of the nuclear structure...
December 15, 2017: Methods: a Companion to Methods in Enzymology
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