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Affinity-Based Separation Methods for the Study of Biological Interactions: the case of Peroxisome Proliferator-Activated Receptors in drug discovery
Publication date: Available online 17 February 2018 Source:Methods Author(s): Caterina Temporini, Gloria Brusotti, Giorgio Pochetti, Gabriella Massolini, Enrica Calleri Affinity-based methods using immobilized proteins are important approaches for understanding the interactions between small molecules and biological targets. This review is intended to provide an overview of different affinity based separation methods that have been applied to the study of peroxisome proliferator activated receptors (PPARs). The screening of compound to increase screening rates for synthetic and natural ligands of PPAR are reported. Pros a...
Source: Methods - February 18, 2018 Category: Molecular Biology Source Type: research

Designing fluorescent biosensors using circular permutations of riboswitches
Publication date: Available online 16 February 2018 Source:Methods Author(s): Johnny Truong, Yu-Fang Hsieh, Lynda Truong, Guifang Jia, Ming C. Hammond RNA-based fluorescent (RBF) biosensors have been applied to detect a variety of metabolites in vitro and in live cells. They are designed by combining the ligand sensing domain of natural riboswitches with in vitro selected fluorogenic aptamers. Different biosensor topologies have been developed to accommodate the diversity of riboswitch structures. Here we show that circular permutation of the riboswitch ligand sensing domain also gives functional biosensors, using the SAM...
Source: Methods - February 17, 2018 Category: Molecular Biology Source Type: research

Methods of reconstitution to investigate membrane protein function
Publication date: Available online 16 February 2018 Source:Methods Author(s): Ruth Skrzypek, Shagufta Iqbal, Richard Callaghan Membrane proteins are notoriously difficult to investigate in isolation. The focus of this chapter is the key step following extraction and purification of membrane proteins; namely reconstitution. The process of reconstitution re-inserts proteins into a lipid bilayer that partly resembles their native environment. This native environment is vital to the stability of membrane proteins, ensuring that they undergo vital conformational transitions and maintain optimal interaction with their substrate...
Source: Methods - February 16, 2018 Category: Molecular Biology Source Type: research

Velocity Landscape Correlation Resolves Multiple Flowing Protein Populations From Fluorescence Image Time Series
Publication date: Available online 16 February 2018 Source:Methods Author(s): Elvis Pandžić, Asmahan Abu-Arish, Renee W. Whan, John W. Hanrahan, Paul W. Wiseman Molecular, vesicular and organellar flows are of fundamental importance for the delivery of nutrients and essential components used in cellular functions such as motility and division. With recent advances in fluorescence/super-resolution microscopy modalities we can resolve the movements of these objects at higher spatio-temporal resolutions and with better sensitivity. Previously, spatio-temporal image correlation spectroscopy has been applied to map molecular...
Source: Methods - February 16, 2018 Category: Molecular Biology Source Type: research

Fluorescence Lifetime Correlation Spectroscopy: Basics and Applications
Publication date: Available online 16 February 2018 Source:Methods Author(s): Arindam Ghosh, Narain Karedla, Jan Christoph Thiele, Ingo Gregor, Jörg Enderlein This chapter presents a concise introduction into the method of Fluorescence Lifetime Correlation Spectroscopy (FLCS). This is an extension of Fluorescence Correlation Spectroscopy (FCS) that analyses fluorescence intensity fluctuations from small detection volumes in samples of ultra-low concentration. FCS has been widely used for investigating diffusion, conformational changes, molecular binding/unbinding equilibria, or chemical reaction kinetics, at single m...
Source: Methods - February 16, 2018 Category: Molecular Biology Source Type: research

A straightforward STED-background corrected fitting model for unbiased STED-FCS analyses
Publication date: Available online 14 February 2018 Source:Methods Author(s): Ruixing Wang, Sophie Brustlein, Sébastien Mailfert, Roxane Fabre, Mathieu Fallet, Siddharth Sivankutty, Hervé Rigneault, Didier Marguet Combining stimulated emission depletion and fluorescence correlation spectroscopy (STED-FCS) provides a powerful and sensitive tool for studying the molecular dynamics in live cells with high spatio-temporal resolution. STED-FCS gives access to molecular diffusion characteristic at the nanoscale occurring within short period of times. However due to the incomplete suppression of fluorescence in the...
Source: Methods - February 15, 2018 Category: Molecular Biology Source Type: research

Advanced flow cytometry techniques for clinical detection
Publication date: 1 February 2018 Source:Methods, Volumes 134–135 Author(s): Brian K. McFarlin, Erin M. Bowman (Source: Methods)
Source: Methods - February 13, 2018 Category: Molecular Biology Source Type: research

Quantifying Membrane Protein Oligomerization with Fluorescence Cross-Correlation Spectroscopy
In this study, we describe a method to quantify the oligomerization of membrane proteins tagged with two commonly used fluorescent probes, mCherry (mCH) and enhanced green (eGFP) fluorescent proteins. A mathematical model is described that relates the relative cross-correlation value (fc ) to the degree of oligomerization. This treatment accounts for mismatch in the confocal volumes, combinatoric effects of using two fluorescent probes, and the presence of non-fluorescent probes. Using this model, we calculate a ladder of fc values which can be used to determine the oligomer state of membrane proteins from live-cell experi...
Source: Methods - February 13, 2018 Category: Molecular Biology Source Type: research

Temperature controlled ionic liquid aqueous two phase system combined with affinity capillary electrophoresis for rapid and precise pharmaceutical-protein binding measurements
Publication date: Available online 10 February 2018 Source:Methods Author(s): Deia Abd El-Hady, Hassan M. Albishri Temperature controlled ionic liquid aqueous two phase system (ILATPS) was used to improve the precision of pharmaceutical-AGP (human alpha (α1)-acid glycoprotein) binding measurements by affinity capillary electrophoresis (ACE). The effect of different types of short-chain alkyl imidazolium ILs within the concentration range of 10.0–1000.0 μmol L-1 on a propranolol (PRO)-AGP model was firstly investigated by ILATPS/ACE system. Use of 100.0 μmol L-1 1-butyl-3-methylimidazolium chloride (BMImC...
Source: Methods - February 11, 2018 Category: Molecular Biology Source Type: research

Sample and substrate preparation for exploring living neurons in culture with quantitative-phase imaging
Publication date: Available online 10 February 2018 Source:Methods Author(s): Sébastien A. Lévesque, Jean-Michel Mugnes, Erik Bélanger, Pierre Marquet Quantitative phase imaging (QPI) has recently emerged as a powerful new quantitative microscopy technique suitable for the noninvasive exploration of the structure and dynamics of transparent specimens, including living cells in culture. Indeed, the quantitative phase signal (QPS), induced by transparent living cells, can be detected with a nanometric axial sensitivity, and contains a wealth of information about both cell morphology and content. However...
Source: Methods - February 11, 2018 Category: Molecular Biology Source Type: research

Bi-clustering of metabolic data using matrix factorization tools
Publication date: Available online 10 February 2018 Source:Methods Author(s): Quan Gu, Kirill Veselkov Metabolic phenotyping technologies based on Nuclear Magnetic Spectroscopy (NMR) and Mass Spectrometry (MS) generate vast amounts of unrefined data from biological samples. Clustering strategies are frequently employed to provide insight into patterns of relationships between samples and metabolites. Here, we propose the use of a non-negative matrix factorization driven bi-clustering strategy for metabolic phenotyping data in order to discover subsets of interrelated metabolites that exhibit similar behaviour across sampl...
Source: Methods - February 11, 2018 Category: Molecular Biology Source Type: research

Thread- Paper, and Fabric Enzyme-Linked Immunosorbent Assays (ELISA)
Publication date: Available online 10 February 2018 Source:Methods Author(s): Ariana Gonzalez, Michelle Gaines, Laura Y. Gallegos, Ricardo Guevara, Frank A. Gomez Enzyme-linked immunosorbent assay (ELISA) is an immunological assay commonly used to measure antibodies, antigens, proteins, and glycoproteins in biological samples. While the procedure is routine and straightforward, there are a number of variables (reagent selection, volume measurement, temperature, and time) that if not carefully considered, can affect the test outcome. Herein, we describe the development of microfluidic thread/paper-based analytical devices ...
Source: Methods - February 11, 2018 Category: Molecular Biology Source Type: research

Thermophoresis for characterizing biomolecular interaction
Publication date: Available online 10 February 2018 Source:Methods Author(s): Mufarreh Asmari, Ratih Ratih, Hassan A. Alhazmi, Sami El Deeb The study of biomolecular interactions is crucial to get more insight into the biological system. The interactions of protein-protein, protein-nucleic acids, protein-sugars, nucleic acid-nucleic acids and protein-small molecules are supporting therapeutics and technological developments. Recently, the development in a large number of analytical techniques for characterizing biomolecular interactions reflect the promising research investments in this field. In this review, microscale t...
Source: Methods - February 11, 2018 Category: Molecular Biology Source Type: research

HiCTMap: Detection and analysis of chromosome territory structure and position by high-throughput imaging
Publication date: Available online 10 February 2018 Source:Methods Author(s): Ziad Jowhar, Prabhakar R. Gudla, Sigal Shachar, Darawalee Wangsa, Jill L. Russ, Gianluca Pegoraro, Thomas Ried, Armin Raznahan, Tom Misteli The spatial organization of chromosomes in the nuclear space is an extensively studied field that relies on measurements of structural features and 3D positions of chromosomes with high precision and robustness. However, no tools are currently available to image and analyze chromosome territories in a high-throughput format. Here, we have developed High-throughput Chromosome Territory Mapping (HiCTMap), a me...
Source: Methods - February 11, 2018 Category: Molecular Biology Source Type: research

A high-throughput and rapid computational method for screening of RNA post-transcriptional modifications that can be recognized by target proteins
Publication date: Available online 1 February 2018 Source:Methods Author(s): Asuka A. Orr, Juan C. Gonzalez-Rivera, Mark Wilson, P. Reena Bhikha, Daiqi Wang, Lydia M. Contreras, Phanourios Tamamis There are over 150 currently known, highly diverse chemically modified RNAs, which are dynamic, reversible, and can modulate RNA-protein interactions. Yet, little is known about the wealth of such interactions. This can be attributed to the lack of tools that allow the rapid study of all the potential RNA modifications that might mediate RNA-protein interactions. As a promising step toward this direction, here we present a compu...
Source: Methods - February 8, 2018 Category: Molecular Biology Source Type: research

Scanning fluorescence correlation spectroscopy comes full circle
Publication date: Available online 7 February 2018 Source:Methods Author(s): German Gunther, David M. Jameson, Joao Aguilar, Susana A. Sánchez In this article, we review the application of fluorescence correlation spectroscopy (FCS) methods to studies on live cells. We begin with a brief overview of the theory underlying FCS, highlighting the type of information obtainable. We then focus on circular scanning FCS. Specifically, we discuss instrumentation and data analysis and offer some considerations regarding sample preparation. Two examples from the literature are discussed in detail. First, we show how this meth...
Source: Methods - February 8, 2018 Category: Molecular Biology Source Type: research

Neural stem cells in health and disease
Publication date: 15 January 2018 Source:Methods, Volume 133 Author(s): Philip H. Schwartz (Source: Methods)
Source: Methods - February 3, 2018 Category: Molecular Biology Source Type: research

StructureFold2: Bringing chemical probing data into the computational fold of RNA structural analysis
Publication date: Available online 2 February 2018 Source:Methods Author(s): David C. Tack, Yin Tang, Laura E. Ritchey, Sarah M. Assmann, Philip C. Bevilacqua The secondary structure of an RNA is often implicit to its function. Recently, various high-throughput RNA structure probing techniques have been developed to elucidate important RNA structure-function relationships genome-wide. These techniques produce unwieldy experimental data sets that require evaluation with unique computational pipelines. Herein, we present StructureFold2, a user-friendly set of analysis tools that makes precise data processing and detailed do...
Source: Methods - February 3, 2018 Category: Molecular Biology Source Type: research

Analysis of Solute-Protein Interactions and Solute-Solute Competition by Zonal Elution Affinity Chromatography
Publication date: Available online 2 February 2018 Source:Methods Author(s): Pingyang Tao, Saumen Poddar, Zuchen Sun, David S. Hage, Jianzhong Chen Many biological processes involve solute-protein interactions and solute-solute competition for protein binding. One method that has been developed to examine these interactions is zonal elution affinity chromatography. This review discusses the theory and principles of zonal elution affinity chromatography, along with its general applications. Examples of applications that are examined include the use of this method to estimate the relative extent of solute-protein binding, t...
Source: Methods - February 3, 2018 Category: Molecular Biology Source Type: research

Spatio-Temporal Image Correlation Spectroscopy and Super-Resolution Microscopy to Quantify Molecular Dynamics in T cells
Publication date: Available online 2 February 2018 Source:Methods Author(s): George W. Ashdown, Dylan M. Owen Many cellular processes are regulated by the spatio-temporal organisation of signalling complexes, cytoskeletal components and membranes. One such example is at the T cell immunological synapse where the retrograde flow of cortical filamentous (F)-actin from the synapse periphery drives signalling protein microclusters towards the synapse centre. The density of this mesh however, makes visualisation and analysis of individual actin fibres difficult due to the resolution limit of conventional microscopy. Recently, ...
Source: Methods - February 3, 2018 Category: Molecular Biology Source Type: research

A reconstitution method for integral membrane proteins in hybrid lipid-polymer vesicles for enhanced functional durability
Publication date: Available online 2 February 2018 Source:Methods Author(s): Rashmi Seneviratne, Sanobar Khan, Ellen Moscrop, Michael Rappolt, Stephen P. Muench, Lars J.C. Jeuken, Paul A. Beales Hybrid vesicles composed of lipids and block copolymers hold promise for increasing liposome stability and providing a stable environment for membrane proteins. Recently we reported the successful functional reconstitution of the integral membrane protein cytochrome bo3 (ubiquinol oxidase) into hybrid vesicles composed of a blend of phospholipids and a block copolymer (PBd-PEO). We demonstrated that these novel membrane environmen...
Source: Methods - February 3, 2018 Category: Molecular Biology Source Type: research

Corrigendum to “Molecular signatures in breast cancer” [Methods 131 (2017) 135–146]
Publication date: Available online 3 February 2018 Source:Methods Author(s): Samir Lal, Amy E. McCart Reed, Xavier M. de Luca, Peter T. Simpson (Source: Methods)
Source: Methods - February 3, 2018 Category: Molecular Biology Source Type: research

“A high-throughput and rapid computational method for screening of RNA post-transcriptional modifications that can be recognized by target proteins”
Publication date: Available online 1 February 2018 Source:Methods Author(s): Asuka A. Orr, Juan C. Gonzalez-Rivera, Mark Wilson, P. Reena Bhikha, Daiqi Wang, Lydia M. Contreras, Phanourios Tamamis There are over 150 currently known, highly diverse chemically modified RNAs, which are dynamic, reversible, and can modulate RNA–protein interactions. Yet, little is known about the wealth of such interactions. This can be attributed to the lack of tools that allow the rapid study of all the potential RNA modifications that might mediate RNA-protein interactions. As a promising step toward this direction, here we present a...
Source: Methods - February 2, 2018 Category: Molecular Biology Source Type: research

Systematic optimization of cell-free synthesized human endothelin B receptor folding
Publication date: Available online 2 February 2018 Source:Methods Author(s): Ralf-Bernhardt Rues, Fang Dong, Volker Dötsch, Frank Bernhard Cell-free production of G-protein coupled receptors is becoming attractive for biochemical characterization, ligand screening or even structural purposes. However, despite high production levels within the range of mg/mL, the fraction of functionally folded receptor is frequently below 1%. In synthetic cell-free reactions, numerous factors that affect the efficient folding and stability of translated membrane proteins can be addressed by the appropriate design of the synthetic exp...
Source: Methods - February 2, 2018 Category: Molecular Biology Source Type: research

Potentials and pitfalls of inverse fluorescence correlation spectroscopy
Publication date: Available online 2 February 2018 Source:Methods Author(s): Stefan Wennmalm Inverse Fluorescence Correlation Spectroscopy (iFCS) is a variant of FCS where unlabeled particles in solution, or domains in membranes, displace their surrounding, signal-generating molecules and thereby generate fluctuations. iFCS has to date been applied to unlabeled as well as labeled particles and protein molecules, using fluorescence as well as Raman scattering as a signal source, in diffraction-limited detection volumes as well as in nano-wells, and on fixed surfaces as well as in lipid bilayers. This review describes these...
Source: Methods - February 2, 2018 Category: Molecular Biology Source Type: research

The Importance of Area Scaling With FACS DIVA Software
Publication date: Available online 31 January 2018 Source:Methods Author(s): Amy L. Hazen, Timothy Bushnell, David L. Haviland With the release and use of the Becton Dickenson FACS Diva Software, the use of Area as the default parameter came into play. As such, the use of area as a calculated parameter, methods were needed to be employed to ensure doublet discrimination and proper display on standard FSC/SSC. Improper setting of forward area scaling can alter the display cell populations. This combined with improper area gating strategy can lead to doublet inclusion which in sorting rare events can compromise sort purity....
Source: Methods - February 1, 2018 Category: Molecular Biology Source Type: research

Using DNase Hi-C techniques to map global and local three-dimensional genome architecture at high resolution
Publication date: Available online 31 January 2018 Source:Methods Author(s): Wenxiu Ma, Ferhat Ay, Choli Lee, Gunhan Gulsoy, Xinxian Deng, Savannah Cook, Jennifer Hesson, Christopher Cavanaugh, Carol B. Ware, Anton Krumm, Jay Shendure, C. Anthony Blau, Christine M. Disteche, William S. Noble, ZhiJun Duan The folding and three-dimensional (3D) organization of chromatin in the nucleus critically impacts genome function. The past decade has witnessed rapid advances in genomic tools for delineating 3D genome architecture. Among them, chromosome conformation capture (3C)-based methods such as Hi-C are the most widely used tech...
Source: Methods - February 1, 2018 Category: Molecular Biology Source Type: research

Phase Contrast Tomography at Lab on Chip scale by Digital Holography
Publication date: Available online 16 January 2018 Source:Methods Author(s): 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 rollin...
Source: Methods - January 25, 2018 Category: Molecular Biology Source Type: research

Direct Detection of Carbon and Nitrogen Nuclei for High-Resolution Analysis of Intrinsically Disordered Proteins using NMR Spectroscopy
Publication date: Available online 16 January 2018 Source:Methods Author(s): 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 ow...
Source: Methods - January 25, 2018 Category: Molecular Biology Source Type: research

Using the Ribodeblur pipeline to recover A-sites from yeast ribosome profiling data
Publication date: Available online 9 January 2018 Source:Methods Author(s): 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 likelih...
Source: Methods - January 10, 2018 Category: Molecular Biology Source Type: research

Design rules of synthetic non-coding RNAs in bacteria
Publication date: Available online 5 January 2018 Source:Methods Author(s): 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 ...
Source: Methods - January 6, 2018 Category: Molecular Biology Source Type: research

Tracking the m7G-cap during translation initiation by crosslinking methods
Publication date: Available online 4 January 2018 Source:Methods Author(s): 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 cycl...
Source: Methods - January 5, 2018 Category: Molecular Biology Source Type: research

Phase sensitivity evaluation and its application to phase shifting interferometry
Publication date: Available online 4 January 2018 Source:Methods Author(s): 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...
Source: Methods - January 4, 2018 Category: Molecular Biology Source Type: research

Alternatives to current flow cytometry data analysis for clinical and research studies
Publication date: Available online 4 January 2018 Source:Methods Author(s): 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...
Source: Methods - January 4, 2018 Category: Molecular Biology Source Type: research

Enhanced spectral density mapping through combined multiple-field deuterium 13CH2D methyl spin relaxation NMR spectroscopy
Publication date: Available online 27 December 2017 Source:Methods Author(s): 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 N...
Source: Methods - January 3, 2018 Category: Molecular Biology Source Type: research

The cell free protein synthesis system from the model filamentous fungus Neurospora crassa
Publication date: Available online 30 December 2017 Source:Methods Author(s): 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-f...
Source: Methods - December 31, 2017 Category: Molecular Biology Source Type: research

Enhanced Spectral Density Mapping through Combined Multiple-Field Deuterium mCH2D Methyl Spin Relaxation NMR Spectroscopy
Publication date: Available online 27 December 2017 Source:Methods Author(s): 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...
Source: Methods - December 29, 2017 Category: Molecular Biology Source Type: research

High Throughput Automated Analysis of Big Flow Cytometry Data
Publication date: Available online 27 December 2017 Source:Methods Author(s): 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 analyze...
Source: Methods - December 27, 2017 Category: Molecular Biology Source Type: research

In vitro reconstitution of translational arrest pathways
Publication date: Available online 23 December 2017 Source:Methods Author(s): 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 ...
Source: Methods - December 24, 2017 Category: Molecular Biology Source Type: research

The use of unfixed bone marrow trephines for multicolour flow cytometry
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. Our aims were: 1) To compare cell markers results between bone marrow trephine (BMT)and bone marrow aspirate (BMA) 24 cases and BMT with peripheral blood (PB) 14 cases in paired samples to establish if they were reproducible with results of the unfixed bone marrow trephine biopsies. 2) To ascertain a precise diagnosis in 27 (42%) of the cases in which only a bone marrow trephine was available. We demonstrated that unfixed bone marr...
Source: Methods - December 22, 2017 Category: Molecular Biology Source Type: research

Application of Paramagnetic Relaxation Enhancements to Accelerate the Acquisition of 2D and 3D Solid-State NMR Spectra of Oriented Membrane Proteins
Publication date: Available online 22 December 2017 Source:Methods Author(s): 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 exploitin...
Source: Methods - December 22, 2017 Category: Molecular Biology Source Type: research

A sensitive flow cytometric method for multi-parametric analysis of microRNA, messenger RNA and protein in single cells.
In this study, we used mouse bone marrow-derived macrophages to demonstrate that our method allows for analysis of the activation and polarization status of cells using expression patterns of protein and RNA. We then performed analysis of four cell subsets of mouse resident peritoneal cells and showed that the two macrophage populations present in this compartment are relatively heterogeneous in terms of expression of two M2 markers: Arg1, Retnla, and a B-cell attractant chemokine Cxcl13. In addition, we profiled the expression of a panel of microRNA in the four peritoneal cell subsets, showing that the assay can be readil...
Source: Methods - December 21, 2017 Category: Molecular Biology Source Type: research

Cell sorting of various cell types from mouse and human skeletal muscle
We present here a method for the concomitant isolation of 4 cell types present in the regenerating skeletal muscle: muscle stem cells, endothelial cells, fibro-adipogenic precursor cells and macrophages. (Source: Methods)
Source: Methods - December 19, 2017 Category: Molecular Biology Source Type: research

No lyse no wash flow cytometry for maximizing minimal sample preparation
Publication date: Available online 18 December 2017 Source:Methods Author(s): 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 spark...
Source: Methods - December 18, 2017 Category: Molecular Biology Source Type: research

Assessing multiparametric drug response in tissue engineered tumor microenvironment models
Publication date: Available online 16 December 2017 Source:Methods Author(s): 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. Her...
Source: Methods - December 17, 2017 Category: Molecular Biology Source Type: research

Circle scanning STED fluorescence correlation spectroscopy to quantify membrane dynamics and compartmentalization
Publication date: Available online 16 December 2017 Source:Methods Author(s): 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 qu...
Source: Methods - December 17, 2017 Category: Molecular Biology Source Type: research

Flow Virometry as a Tool to Study Viruses
Publication date: Available online 16 December 2017 Source:Methods Author(s): 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 o...
Source: Methods - December 17, 2017 Category: Molecular Biology Source Type: research

Mapping the dynamical organization of the cell nucleus through fluorescence correlation spectroscopy
Publication date: Available online 15 December 2017 Source:Methods Author(s): 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 part...
Source: Methods - December 16, 2017 Category: Molecular Biology Source Type: research

Determining mRNA half-lives on a transcriptome-wide scale
Publication date: Available online 13 December 2017 Source:Methods Author(s): Andrew Lugowski, Beth Nicholson, Olivia S. Rissland Every step in the life cycle of an RNA transcript provides opportunity for regulation. One important aspect of post-transcriptional control is the regulation of RNA stability. Of the many strategies for determining mRNA stability, transcription inhibition and metabolic labeling have proved the most amenable to high-throughput analysis and have opened the door to dissecting mRNA decay transcriptome-wide. Here, we describe experimental and computational methods to determine transcriptome-wide RNA...
Source: Methods - December 14, 2017 Category: Molecular Biology Source Type: research

Ensemble and single-molecule FRET studies of protein synthesis
We describe a number of experimental strategies that can be used to introduce fluorophores into the ribosome, tRNA, mRNA and protein factors of translation. Alternative approaches to tethering of translation components to the microscope slide in smFRET experiments are also reviewed. Finally, we discuss possible challenges in the interpretation of FRET data and ways to address these challenges. (Source: Methods)
Source: Methods - December 14, 2017 Category: Molecular Biology Source Type: research