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  • A detailed cell-free transcription-translation-based assay to decipher CRISPR protospacer-adjacent motifs
    Methods (IF 3.802) Pub Date : 2018-02-24
    Colin S. Maxwell, Thomas Jacobsen, Ryan Marshall, Vincent Noireaux, Chase L. Beisel

    The RNA-guided nucleases derived from the CRISPR-Cas systems in bacteria and archaea have found numerous applications in biotechnology, including genome editing, imaging, and gene regulation. However, the discovery of novel Cas nucleases has outpaced their characterization and subsequent exploitation. A key step in characterizing Cas nucleases is determining which protospacer-adjacent motif (PAM) sequences they recognize. Here, we report advances to an in vitro method based on an E. coli cell-free transcription-translation system (TXTL) to rapidly elucidate PAMs recognized by Cas nucleases. The method obviates the need for cloning Cas nucleases or gRNAs, does not require the purification of protein or RNA, and can be performed in less than a day. To advance our previously published method, we incorporated an internal GFP cleavage control to assess the extent of library cleavage as well as Sanger sequencing of the cleaved library to assess PAM depletion prior to next-generation sequencing. We also detail the methods needed to construct all relevant DNA constructs, and how to troubleshoot the assay. We finally demonstrate the technique by determining PAM sequences recognized by the Neisseria meningitidis Cas9, revealing subtle sequence requirements of this highly specific PAM. The overall method offers a rapid means to identify PAMs recognized by diverse CRISPR nucleases, with the potential to greatly accelerate our ability to characterize and harness novel CRISPR nucleases across their many uses.

  • The yin and yang of solubilization and stabilization for wild-type and full-length membrane protein
    Methods (IF 3.802) Pub Date : 2018-02-23
    David Hardy, Elodie Desuzinges Mandon, Alice Rothnie, Anass Jawhari

    Membrane proteins (MP) are stable in their native lipid environment. To enable structural and functional investigations, MP need to be extracted from the membrane. This is a critical step that represents the main obstacle for MP biochemistry and structural biology. General guidelines and rules for membrane protein solubilization remain difficult to establish. This review aims to provide the reader with a comprehensive overview of the general concepts of MP solubilization and stabilization as well as recent advances in detergents innovation. Understanding how solubilization and stabilization are intimately linked is key to facilitate MP isolation toward fundamental structural and functional research as well as drug discovery applications. How to manage the tour de force of destabilizing the lipid bilayer and stabilizing MP at the same time is the holy grail of successful isolation and investigation of such a delicate and fascinating class of proteins.

  • Fragment screening for drug leads by weak affinity chromatography (WAC-MS)
    Methods (IF 3.802) Pub Date : 2018-02-23
    Sten Ohlson, Minh-Dao Duong-Thi
  • Crosstalk-free multicolor RICS using spectral weighting
    Methods (IF 3.802) Pub Date : 2018-02-22
    Waldemar Schrimpf, Veerle Lemmens, Nick Smisdom, Marcel Ameloot, Don C. Lamb, Jelle Hendrix
  • Solid-State NMR of highly 13C-enriched cholesterol in lipid bilayers
    Methods (IF 3.802) Pub Date : 2018-02-21
    Lisa A. Della Ripa, Zoe A. Petros, Alexander G. Cioffi, Dennis W. Piehl, Joseph M. Courtney, Martin D. Burke, Chad M. Rienstra

    Cholesterol (Chol) is vital for cell function as it is essential to a myriad of biochemical and biophysical processes. The atomistic details of Chol’s interactions with phospholipids and proteins is therefore of fundamental interest, and NMR offers unique opportunities to interrogate these properties at high resolution. Towards this end, here we describe approaches for examining the structure and dynamics of Chol in lipid bilayers using high levels of 13C enrichment in combination with magic-angle spinning (MAS) methods. We quantify the incorporation levels and demonstrate high sensitivity and resolution in 2D 13C-13C and 1H-13C spectra, enabling de novo assignments and site-resolved order parameter measurements obtained in a fraction of the time required for experiments with natural abundance sterols. We envision many potential future applications of these methods to study sterol interactions with drugs, lipids and proteins.

  • Intermolecular Interactions Determined by NOE Build-up in Macromolecules from Hyperpolarized Small Molecules
    Methods (IF 3.802) Pub Date : 2018-02-19
    Chia-Hsiu Chen, Yunyi Wang, Christian Hilty

    The nuclear Overhauser effect (NOE) is a primary means to characterize intermolecular interactions using modern NMR spectroscopy. Multiple experiments measured using different mixing time can be used for quantifying NOE buildup and measuring cross-relaxation rates. However, this approach using conventional multi-dimensional NMR is time consuming. Hyperpolarization by dissolution dynamic nuclear polarization (D-DNP) can generate deviations from equilibrium spin polarization by orders of magnitude, thereby enhancing signals and allowing to characterize NOE build up in real-time. Since most small molecules can be hyperpolarized using D-DNP, this method is applicable to the study of intermolecular interactions between small molecules and macromolecules. This application is demonstrated using a model system for host-guest interactions including the third generation polyamidoamine dendrimer (G3 PAMAM) and the pharmaceutical phenylbutazone (PBZ). After mixing 1H hyperpolarized PBZ with PAMAM, the NOE build up is directly observed at different sites of the dendrimer in series of one-dimensional NMR spectra. Cross relaxation rates specific to individual source and target spins are determined from the build up curves. Further, the polarization enhancement is shown to be sufficiently large to allow identification of cross-peaks not observed in a conventional 2D-NOESY spectrum. The improved signal-to-noise ratio provided by hyperpolarization allows for characterizing the intermolecular interaction in an almost instantaneous measurement, opening an application to macromolecular and biomacromolecular NMR.

  • Affinity-Based Separation Methods for the Study of Biological Interactions: the case of Peroxisome Proliferator-Activated Receptors in drug discovery
    Methods (IF 3.802) Pub Date : 2018-02-17
    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 and cons of the approaches in ligand discovery initiatives. are discussed.

  • Designing fluorescent biosensors using circular permutations of riboswitches
    Methods (IF 3.802) Pub Date : 2018-02-16
    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-I riboswitch as our model. We reveal that this design can enhance fluorescence turn-on and ligand binding affinity compared to the non-permuted topology.

  • Velocity Landscape Correlation Resolves Multiple Flowing Protein Populations From Fluorescence Image Time Series
    Methods (IF 3.802) Pub Date : 2018-02-16
    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 flows by correlation analysis of fluorescence fluctuations in image series. However, an underlying assumption of this approach is that the sampled time windows contain one dominant flowing component. Although this was true for most of the cases analyzed earlier, in some situations two or more different flowing populations can be present in the same spatio-temporal window. We introduce an approach, termed velocity landscape correlation (VLC), which detects and extracts multiple flow components present in a sampled image region via an extension of the correlation analysis of fluorescence intensity fluctuations. First we demonstrate theoretically how this approach works, test the performance of the method with a range of computer simulated image series with varying flow dynamics. Finally we apply VLC to study variable fluxing of STIM1 proteins on microtubules connected to the plasma membrane of Cystic Fibrosis Bronchial Epithelial (CFBE) cells.

  • Fluorescence Lifetime Correlation Spectroscopy: Basics and Applications
    Methods (IF 3.802) Pub Date : 2018-02-16
    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 molecule sensitivity. In FCS, this is done by calculating intensity correlation curves for the measured intensity fluctuations. FLCS extends this idea by calculating fluorescence-lifetime specific intensity correlation curves. Thus, FLCS is the method of choice for all studies where a parameter of interest (conformational state, spatial position, molecular environmental condition) is connected with a change in the fluorescence lifetime. After presenting the theoretical and experimental basis of FLCS, the chapter gives an overview of its various applications.

  • Methods of reconstitution to investigate membrane protein function
    Methods (IF 3.802) Pub Date : 2018-02-16
    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 substrates. Reconstitution may take many forms and these have been classified into two broad categories. Symmetric systems enable unfettered access to both sides of a bilayer. Compartment containing systems contain a lumen and are ideally suited to measurement of transport processes. The investigator is encouraged to ascertain what aspects of protein function will be undertaken and to apply the most advantageous reconstitution system or systems. It is important to note that the process of reconstitution is not subject to defined protocols and requires empirical optimisation to specific targets.

  • A straightforward STED-background corrected fitting model for unbiased STED-FCS analyses
    Methods (IF 3.802) Pub Date : 2018-02-14
    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 STED process, the STED-FCS point spread function (PSF) deviates from a Gaussian shape and challenges the analysis of the auto-correlation curves obtained by FCS. Here, we model the effect of the incomplete fluorescence suppression in STED-FCS experiments and propose a new fitting model improving the accuracy of the diffusion times and average molecule numbers measurements. The implementation of a STED module with pulsed laser source on a commercial confocal / FCS microscope allowed us to apply the STED-background corrected model to fit the STED-FCS measurements. The experimental results are in good accordance with the theoretical analysis both for the number of molecules and the diffusion time which decrease accordingly with the STED power.

  • Quantifying Membrane Protein Oligomerization with Fluorescence Cross-Correlation Spectroscopy
    Methods (IF 3.802) Pub Date : 2018-02-13
    Megan J. Kaliszewski, Xiaojun Shi, Yixuan Hou, Ryan Lingerak, Soyeon Kim, Paul Mallory, Adam W. Smith

    Fluorescence cross-correlation spectroscopy (FCCS) is an advanced fluorescence technique that can quantify protein-protein interactions in vivo. Due to the dynamic, heterogeneous nature of the membrane, special considerations must be made to interpret FCCS data accurately. 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 experimental data. Additionally, a probabilistic mathematical simulation is described to resolve the affinity of different dimeric and oligomeric protein controls.

  • Bi-clustering of metabolic data using matrix factorization tools
    Methods (IF 3.802) Pub Date : 2018-02-10
    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 samples. The proposed strategy incorporates bi-cross validation and statistical segmentation techniques to automatically determine the number and structure of bi-clusters. This alternative approach is in contrast to the widely used conventional clustering approaches that incorporate all molecular peaks for clustering in metabolic studies and require a priori specification of the number of clusters. We perform the comparative analysis of the proposed strategy with other bi-clustering approaches, which were developed in the context of genomics and transcriptomics research. We demonstrate the superior performance of the proposed bi-clustering strategy on both simulated (NMR) and real (MS) bacterial metabolic data.

  • Thermophoresis for characterizing biomolecular interaction
    Methods (IF 3.802) Pub Date : 2018-02-10
    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 thermophoresis technology (MST) is presented as an analytical technique for characterizing biomolecular interactions. Recent years have seen much progress and several applications established. MST is a powerful technique in quantitation of binding events based on the movement of molecules in microscopic temperature gradient. Simplicity, free solutions analysis, low sample volume, short analysis time, and immobilization free are the MST advantages over other competitive techniques. A wide range of studies in biomolecular interactions have been successfully carried out using MST, which tend to the versatility of the technique to use in screening binding events in order to save time, cost and obtained high data quality.

  • HiCTMap: Detection and analysis of chromosome territory structure and position by high-throughput imaging
    Methods (IF 3.802) Pub Date : 2018-02-10
    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 method for the robust and rapid analysis of 2D and 3D chromosome territory positioning in mammalian cells. HiCTMap is a high-throughput imaging-based chromosome detection method which enables routine analysis of chromosome structure and nuclear position. Using an optimized FISH staining protocol in a 384-well plate format in conjunction with a bespoke automated image analysis workflow, HiCTMap faithfully detects chromosome territories and their position in 2D and 3D in a large population of cells per experimental condition. We apply this novel technique to visualize chromosomes 18, X, and Y in male and female primary human skin fibroblasts, and show accurate detection of the correct number of chromosomes in the respective genotypes. Given the ability to visualize and quantitatively analyze large numbers of nuclei, we use HiCTMap to measure chromosome territory area and volume with high precision and determine the radial position of chromosome territories using either centroid or equidistant-shell analysis. The HiCTMap protocol is also compatible with RNA FISH as demonstrated by simultaneous labeling of X chromosomes and Xist RNA in female cells. We suggest HiCTMap will be a useful tool for routine precision mapping of chromosome territories in a wide range of cell types and tissues.

  • Sample and substrate preparation for exploring living neurons in culture with quantitative-phase imaging
    Methods (IF 3.802) Pub Date : 2018-02-10
    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, QPS is also sensitive to various sources of experimental noise. In this chapter, we emphasize how to properly and specifically measure the cell-mediated QPS in a wet-lab environment, when measuring with a quantitative phase digital holographic microscope (QP-DHM). First, we present the substrate-requisite characteristics for properly achieving such cell-mediated QPS measurements at single-cell level. Then, we describe how QP-DHM can be used to numerically process holograms and subsequently reshape wavefronts in association with post-processing algorithms, thereby allowing for highly stable QPS obtainable over extended periods of time. Such stable QPS is a prerequisite for exploring the dynamics of specific cellular processes. We also describe experimental procedures that make it possible to extract important biophysical cellular parameters from QPS including absolute cell volume, transmembrane water permeability, and the movements of water in and out of the cell. To illustrate how QP-digital holographic microscopy can reveal the dynamics of specific cellular processes, we show how the monitoring of transmembrane water movements can be used to resolve the neuronal network dynamics at single-cell level. This is possible because QPS can measure the activity of electroneutral cotransports, including NKCC1 and KCC2, during a neuronal firing mediated by glutamate, the main excitatory neurotransmitter in the brain. Finally, we added a supplemental section, with more technical details, for readers who are interested in troubleshooting live-cell QP-DHM imaging.

  • Thread- Paper, and Fabric Enzyme-Linked Immunosorbent Assays (ELISA)
    Methods (IF 3.802) Pub Date : 2018-02-10
    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 (µTPAD), microfluidic fabric-based analytical devices (µFAD), and microfluidic thread-based analytical devices (µTAD) as new platforms for ELISA. The quantitative detection of biotinylated goat anti-mouse IgG (system one) and rabbit IgG (system two) antibodies via colorimetric analysis is detailed. We explain the design and fabrication of the devices and the step-by-step protocol for the ELISA. A comparison between the techniques is described and the results obtained from them elucidated.

  • 更新日期:2018-02-10
  • Structure determination of protein-ligand complexes by NMR in solution
    Methods (IF 3.802) Pub Date : 2018-02-08
    Julien Orts, Alvar D. Gossert

    In this paper, we discuss methods for determining structures of protein-ligand complexes by NMR in solution. Our discussion is based on small ligands (< 2 kDa) as for example drugs, metabolites or oligo-peptides, but most of the considerations also apply to more general cases. In NMR in solution, the kinetics of association and dissociation of the complex – the exchange rate – determines the optimal sample preparation and the NMR experimental approach. Additionally, depending on the part of the complex that will be studied (only the bound ligand, the protein, the protein-ligand interface or the entire protein-ligand complex structure), different types of NMR experiments are needed. Therefore, the choice of a combination of the appropriate experiment and a suitable sample preparation in terms of ligand to protein ratios are discussed in detail. Also, considerations for practically preparing samples of protein-ligand complexes and carrying out experiments including trouble shooting are described. For structure determination, the scope of this paper is limited to NOE-based methods and some of the most recent approaches will be covered.

  • Scanning fluorescence correlation spectroscopy comes full circle
    Methods (IF 3.802) Pub Date : 2018-02-07
    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 method, coupled with the photon counting histogram analysis, can provide information on yeast ribosomal structures in live cells. The combination of scanning FCS with dual channel detection in the study of lipid domains in live cells is also illustrated.

  • Analysis of Solute-Protein Interactions and Solute-Solute Competition by Zonal Elution Affinity Chromatography
    Methods (IF 3.802) Pub Date : 2018-02-02
    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, to examine solute-solute competition and displacement from proteins, and to measure the strength of these interactions. It is also shown how zonal elution affinity chromatography can be used in solvent and temperature studies and to characterize the binding sites for solutes on proteins. In addition, several alternative applications of zonal elution affinity chromatography are discussed, which include the analysis of binding by a solute with a soluble binding agent and studies of allosteric effects. Other recent applications that are considered are the combined use of immunoextraction and zonal elution for drug-protein binding studies, and binding studies that are based on immobilized receptors or small targets.

  • 更新日期:2018-02-02
  • A reconstitution method for integral membrane proteins in hybrid lipid-polymer vesicles for enhanced functional durability
    Methods (IF 3.802) Pub Date : 2018-02-02
    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 environments stabilise the enzymes’ activity, prolonging their functional lifetime [Chem Commun. 52 (2016) 11020-11023]. This approach holds great promise for applications of membrane proteins where enhanced durability, stability and shelf-life will be essential to creating a viable technology. Here we present a detailed account of our methods for membrane protein reconstitution into hybrid vesicles and discuss tips and challenges when using block copolymers compared to pure phospholipid systems that are more common materials for this purpose. We also extend the characterisation of these hybrid vesicles beyond what we have previously reported and show: (i) hybrid membranes are less permeable to protons than phospholipid bilayers; (ii) extended enzyme activity data is presented over a period of 500 days, which fully reveals the truly remarkable enhancement in functional lifetime that hybrid vesicles facilitate.

  • StructureFold2: Bringing chemical probing data into the computational fold of RNA structural analysis
    Methods (IF 3.802) Pub Date : 2018-02-02
    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 downstream analyses of such data sets both available and practical. StructureFold2 processes high-throughput reads sequenced from libraries prepared after experimental probing for reverse transcription (RT) stops generated by chemical modification of RNA at solvent accessible residues. This pipeline is able to analyze reads generated from a variety of structure-probing chemicals (e.g. DMS, glyoxal, SHAPE). Notably, StructureFold2 offers a new fully featured suite of utilities and tools to guide a user through multiple types of analyses. A particular emphasis is placed on analyzing the reactivity patterns of transcripts, complementing their use as folding restraints for predicting RNA secondary structure. StructureFold2 is hosted as a Github repository and is available at (https://github.com/StructureFold2/StructureFold2).

  • Using DNase Hi-C techniques to map global and local three-dimensional genome architecture at high resolution
    Methods (IF 3.802) Pub Date : 2018-01-31
    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 techniques for mapping chromatin interactions. However, traditional Hi-C protocols rely on restriction enzymes (REs) to fragment chromatin and are therefore limited in resolution. We recently developed DNase Hi-C for mapping 3D genome organization, which uses DNase I for chromatin fragmentation. DNase Hi-C overcomes RE-related limitations associated with traditional Hi-C methods, leading to improved methodological resolution. Furthermore, combining this method with DNA capture technology provides a high-throughput approach (targeted DNase Hi-C) that allows for mapping fine-scale chromatin architecture at exceptionally high resolution. Hence, targeted DNase Hi-C will be valuable for delineating the physical landscapes of cis-regulatory networks that control gene expression and for characterizing phenotype-associated chromatin 3D signatures. Here, we provide a detailed description of method design and step-by-step working protocols for these two methods.

  • The Importance of Area Scaling With FACS DIVA Software
    Methods (IF 3.802) Pub Date : 2018-01-31
    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. In extreme cases where area scaling with the individual lasers is ignored, differences can exist between Area and Height where compensation will likely not be optimal, particularly if one parameter – usually height is saturated. In addition, area scaling can impact population grouping. As FSC and individual laser area scaling is a function of event size, the most common error is to accept the setting determined by CS&T, which are 3.2 micron particles and proceed with the sample(s) without regard to the sample’s actual size. With cellular events smaller or more likely larger than the CS&T beads, this will make the area scaling settings less than optimal. Analysis and sorting rare events with populations larger than the CS&T beads can be compromised if adjustments in FSC area scaling are not addressed. Proper FSC and laser area scaling must be determined empirically for each sample. Examples of the effects of sample size on area scaling will be presented in addition to gating and templates for determining area scaling.

  • “A high-throughput and rapid computational method for screening of RNA post-transcriptional modifications that can be recognized by target proteins”
    Methods (IF 3.802) Pub Date : 2018-02-01
    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 computational protocol for the characterization of interactions between proteins and RNA containing post-transcriptional modifications. Given an RNA–protein complex structure, potential RNA modified ribonucleoside positions, and (already existing) molecular mechanics parameters for capturing energetics of RNA modifications, our protocol operates in two stages. In the first stage, a decision-making tool, comprising short simulations and interaction energy calculations, performs a fast and efficient search in a high-throughput fashion, through a list of different types of RNA modifications categorized into trees according to their structural and physicochemical properties, and selects a subset of RNA modifications prone to interact with the target protein. In the second stage, RNA modifications that are selected as recognized by the protein are examined in-detail using all-atom simulations and free energy calculations. We implement and experimentally validate this protocol in a test case involving the study of RNA modifications in complex with Escherichia coli (E. coli) protein Polynucleotide Phosphorylase (PNPase), depicting the favorable interaction between 8-oxo-7,8-dihydroguanosine (8-oxoG) RNA modification and PNPase. Further advancement of the protocol can broaden our understanding of protein interactions with all known RNA modifications in several systems.

  • Systematic optimization of cell-free synthesized human endothelin B receptor folding
    Methods (IF 3.802) Pub Date : 2018-02-02
    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 expression environment. We demonstrate the systematic quality optimization of the cell-free synthesized human endothelin B receptor by a combined approach of lipid screening, redox optimization, and molecular engineering. Key parameters for receptor folding are the implementation of nanodiscs, the selection of suitable lipid environments for co-translational solubilization, as well as providing an optimized redox system for essential disulfide bridge formation. In addition, enrichment with chaperones as well as receptor engineering by thermostabilization further supported the folding into ligand binding conformation. In summary, we provide evidence that the initial co-translational folding process rather than long-term stability of the receptor is limiting. The folding efficiency could be improved by more than 103-fold and under optimized conditions, up to 1.6 nmol or ∼100 µg of ligand binding competent receptor could be produced per mL of reaction mixture in a timescale of less than 24 h. The identified parameters affect rather common characteristics of G-protein receptors and are thus likely to improve the folding of similar targets as well. The optimized process provides full-length receptors embedded in defined membrane environments and in quantities and quality sufficient for throughput screening applications.

  • Potentials and pitfalls of inverse fluorescence correlation spectroscopy
    Methods (IF 3.802) Pub Date : 2018-02-02
    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 applications and discusses the potentials and pitfalls when using iFCS.

  • 更新日期:2018-01-17
  • Phase Contrast Tomography at Lab on Chip scale by Digital Holography
    Methods (IF 3.802) Pub Date : 2018-01-16
    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. Thus, a-priori knowledge of such information is no longer needed. This approach extremely simplifies the optical system avoiding any mechanical/optical scanning of light source. The proof is given for different classes of biosamples, red-blood-cells (RBCs) and diatom algae. Accurate characterization of each type of cells is reported and compared to that obtained by other tomographic techniques.

  • Using the Ribodeblur pipeline to recover A-sites from yeast ribosome profiling data
    Methods (IF 3.802) Pub Date : 2018-01-09
    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. Ribodeblur is simple to install, and can be run on an average modern Mac or Linux-based laptop. We detail the process of applying the pipeline to high-coverage ribosome profiling data in yeast, and discuss important considerations for potential extension to other organisms.

  • Design rules of synthetic non-coding RNAs in bacteria
    Methods (IF 3.802) Pub Date : 2018-01-05
    Young Je Lee, Tae Seok Moon
  • Tracking the m7G-cap during translation initiation by crosslinking methods
    Methods (IF 3.802) Pub Date : 2018-01-04
    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. The principle is to use the m7G cap located at the 5’ extremity of mRNAs as a tracker to monitor RNA and protein components that are in its vicinity. Cap-binding molecules are trapped by chemical and UV crosslinking. The combination of cap crosslinking methods in cell-free translation systems with the use of specific translation inhibitors for different steps such as edeine, GMP-PNP or cycloheximide allowed assessing the cap fate during eukaryotic translation. Here, we followed the position of the cap in the histone H4 mRNA and the cap binding proteins during H4 mRNA translation.

  • Alternatives to current flow cytometry data analysis for clinical and research studies
    Methods (IF 3.802) Pub Date : 2018-01-04
    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. Further, because the user must carefully define the layout of the plate, this information is already defined when considering the analytical process, expanding the opportunities for automated analysis. Advances in multi-parametric data collection, as demonstrated by the development of hyperspectral flow-cytometry, 20-40 color polychromatic flow cytometry, and mass cytometry (CyTOF), are game-changing. As data and assay complexity increase, so too does the complexity of data analysis. Complex data analysis is already a challenge to traditional flow cytometry software. New methods for reviewing large and complex data sets can provide rapid insight into processes difficult to define without more advanced analytical tools. In settings such as clinical labs where rapid and accurate data analysis is a priority, rapid, efficient and intuitive software is needed. This paper outlines opportunities for analysis of complex data sets using examples of multiplexed bead-based assays, drug screens and cell cycle analysis – any of which could become integrated into the clinical environment.

  • Phase sensitivity evaluation and its application to phase shifting interferometry
    Methods (IF 3.802) Pub Date : 2018-01-04
    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. In discussions of specific phase shifting algorithms, we focus on the shot noise-limited model. This simplified model not only reflects the rapid developments in modern detectors that are often dominated by shot noise, but also permits the calculation of theoretical sensitivities based on measured data, which is important in evaluating experimental performance. As examples, we study several common phase shifting interferometric techniques. The results of different techniques are compared to provide insights into algorithm optimization and energy efficiency of sensitivity. A normalized algorithm sensitivity table is also provided for readers to conveniently estimate their system’s algorithm sensitivity and compare against experiments. © 2017 Elsevier Science. All rights reserved

  • The cell free protein synthesis system from the model filamentous fungus Neurospora crassa
    Methods (IF 3.802) Pub Date : 2017-12-30
    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. Methods to accomplish cell-free translation and analyze protein synthesis, and to map positions of ribosomes on mRNAs by toeprinting, are described.

  • Enhanced Spectral Density Mapping through Combined Multiple-Field Deuterium mCH2D Methyl Spin Relaxation NMR Spectroscopy
    Methods (IF 3.802) Pub Date : 2017-12-27
    Andrew Hsu, Paul A. O'Brien, Shibani Bhattacharya, Mark Rance, Arthur G. Palmer
  • High Throughput Automated Analysis of Big Flow Cytometry Data
    Methods (IF 3.802) Pub Date : 2017-12-27
    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.g., cleaning the raw files, event outlier detection, extracting immunophenotypes). We review the components of a customized automated analysis pipeline that can be generally applied to large scale flow cytometry data. We demonstrate these methodologies on data collected by the International Mouse Phenotyping Consortium (IMPC).

  • In vitro reconstitution of translational arrest pathways
    Methods (IF 3.802) Pub Date : 2017-12-23
    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. Here, we describe principles and methods for reconstituting and analyzing translational arrest pathways in cell-free translation systems using rabbit reticulocyte lysate as an example. These approaches can be exploited to dissect various fundamental, regulatory, and quality control mechanisms of eukaryotic protein translation.

  • Application of Paramagnetic Relaxation Enhancements to Accelerate the Acquisition of 2D and 3D Solid-State NMR Spectra of Oriented Membrane Proteins
    Methods (IF 3.802) Pub Date : 2017-12-22
    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. We demonstrate the efficacy of this method for a small membrane protein, sarcolipin, reconstituted in DMPC/POPC/DHPC oriented bicelles. In particular, using Cu2+-DMPE-DTPA as a dopant, we observed a decrease of 1H T1 of sarcolipin by 2/3, allowing us to reduce the recycle delay up to 3 times. We anticipate that these new developments will enable the routine acquisition of multidimensional OS-ssNMR experiments.

  • The use of unfixed bone marrow trephines for multicolour flow cytometry
    Methods (IF 3.802) Pub Date : 2017-12-21
    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. 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 marrow trephine provides an adequate and representative cell suspension for flow cytometry and it is a powerful tool when no other material (bone marrow aspirate or peripheral blood) is available to make a rapid diagnosis. Furthermore when marrow aspirate or peripheral blood paired samples were available, flow cytometry results obtained were identical across all the sample types. Applicability to the clinical laboratory: We described a method to obtain a cell suspension from core biopsies that can easily be implemented routinely in a laboratory that performs diagnostic flow cytometry immunophenotyping. This method is simple, inexpensive and it doesn’t require extra equipment.

  • A sensitive flow cytometric method for multi-parametric analysis of microRNA, messenger RNA and protein in single cells.
    Methods (IF 3.802) Pub Date : 2017-12-19
    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. 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 readily adapted to parallel, high-throughput screening of multiple cell populations. This new method allows for single cell analysis of multiple RNA targets without the need for cell sorting, enables direct correlation between RNA and protein expression, and promises to accelerate biomarker and drug discovery.

  • Cell sorting of various cell types from mouse and human skeletal muscle
    Methods (IF 3.802) Pub Date : 2017-12-19
    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. 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.

  • No lyse no wash flow cytometry for maximizing minimal sample preparation
    Methods (IF 3.802) Pub Date : 2017-12-18
    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. No lyse no wash protocols reduce sample handling and are procedurally less complex than lysis protocols, but the impact of keeping intact red blood cells that grossly outnumber the target white blood cells, must be understood to fully take advantage of this simplicity. Presented here are theories and methods for executing and interpreting no lyse no wash assays in whole blood. Methods for distinguishing white blood cells and platelets from red blood cells and improving scatter data by combining 405 nm and 488 nm side scatter are shown. Methods for assessing white blood cell light scattering profiles for individual instruments and sample treatments are discussed within the context of example profiles for no lysis and hypotonic and ammonium chloride lysis treatments. The utility of overcoming no lyse no wash scatter and fluorescence background limitations using alternate scatter and fluorescence thresholding strategies is also discussed in the context of application examples.

  • Flow Virometry as a Tool to Study Viruses
    Methods (IF 3.802) Pub Date : 2017-12-16
    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. We also discuss the various flow virometry methods used to identify and analyze viruses. We briefly summarize other applications of flow virometry, including: virus detection, quantification, population discrimination, and viral particles’ antigenic properties. Finally, we summarize how viral sorting will allow further progress of flow virometry to relate viral surface characteristics to infectivity properties.

  • Circle scanning STED fluorescence correlation spectroscopy to quantify membrane dynamics and compartmentalization
    Methods (IF 3.802) Pub Date : 2017-12-16
    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. We show how to use model membranes to calibrate and test the technique and how to apply it in the context of living cells to quantify membrane dynamics with high spatiotemporal resolution and good statistics.

  • Assessing multiparametric drug response in tissue engineered tumor microenvironment models
    Methods (IF 3.802) Pub Date : 2017-12-16
    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. We show our physiologically-relevant systems used to interrogate a variety of parameters associated with chemotherapeutic efficacy, including cell death, proliferation, drug uptake, and invasion of cancer and stromal cell populations. The use of flow cytometry allows for single cell, quantitative, and fast assessments of multiple outcomes affecting anti-tumor therapy failure. Our system can be modified to add and remove cellular components with ease, thereby enabling the study of individual cellular contributions in the tumor microenvironment. Together, our models and analysis methods illustrate the importance of developing fast, cost-effective, and reproducible methods to model complex human systems in a physiologically-relevant manner that may prove useful for drug screening efforts in the future.

  • Mapping the dynamical organization of the cell nucleus through fluorescence correlation spectroscopy
    Methods (IF 3.802) Pub Date : 2017-12-15
    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. Here, we describe how FCS methods can be applied to reveal the dynamical organization of the nucleus in live cells. Specifically, we provide instructions for the preparation of cellular samples with fluorescent tagged proteins and detail how FCS can be easily instrumented in commercial confocal microscopes. In addition, we describe general rules to set the parameters for one and two-color experiments and the required controls for these experiments. Finally, we review the statistical analysis of the FCS data and summarize the use of numerical simulations as a complementary approach that helps us to understand the complex matrix of molecular interactions network within the nucleus.

  • Structural dynamics of protein S1 on the 70S ribosome visualized by ensemble cryo-EM
    Methods (IF 3.802) Pub Date : 2017-12-14
    Anna B. Loveland, Andrei A. Korostelev

    Bacterial ribosomal protein S1 is the largest and highly flexible protein of the 30S subunit, and one of a few core ribosomal proteins for which a complete structure is lacking. S1 is thought to participate in transcription and translation. Best understood is the role of S1 in facilitating translation of mRNA with structured 5́ UTRs. Here, we present cryo-EM analyses of the 70S ribosome that reveal multiple conformations of S1. Based on comparison of several 3D maximum likelihood classification approaches in Frealign, we propose a streamlined strategy for visualizing a highly dynamic component of a large macromolecular assembly that itself exhibits high compositional and conformational heterogeneity. The resulting maps show how S1 docks at the ribosomal protein S2 near the mRNA exit channel. The globular OB-fold domains sample a wide area around the mRNA exit channel and interact with mobile tails of proteins S6 and S18. S1 also interacts with the mRNA entrance channel, where an OB-fold domain can be localized near S3 and S5. Our analyses suggest that S1 cooperates with other ribosomal proteins to form a dynamic mesh near the mRNA exit and entrance channels to modulate the binding, folding and movement of mRNA.

  • Ensemble and single-molecule FRET studies of protein synthesis
    Methods (IF 3.802) Pub Date : 2017-12-13
    Christine (Wan-Jung) Lai, Dmitri N. Ermolenko

    Protein synthesis is a complex, multi-step process that involves large conformational changes of the ribosome and protein factors of translation. Over the last decade, Förster resonance energy transfer (FRET) has become instrumental for studying structural rearrangements of the translational apparatus. Here, we discuss the design of ensemble and single-molecule (sm) FRET assays of translation. 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.

  • Determining mRNA half-lives on a transcriptome-wide scale
    Methods (IF 3.802) Pub Date : 2017-12-13
    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 stabilities using both pharmacological inhibition of transcription and metabolic labeling. To aid in the analysis of these experiments, we discuss key characteristics of high-quality experiments and address other experimental and computational considerations for the analysis of mRNA stability. Broader application of these approaches will further our understanding of mRNA decay and illuminate its contribution to different biological processes.

  • Quantitative image mean squared displacement (iMSD) analysis of the dynamics of Profilin 1 at the membrane of live cells
    Methods (IF 3.802) Pub Date : 2017-12-11
    Rhonda J. Davey, Michelle A. Digman, Enrico Gratton, Pierre D.J. Moens

    Image mean square displacement analysis (iMSD) is a method allowing the mapping of diffusion dynamics of molecules in living cells. However, it can also be used to obtain quantitative information on the diffusion processes of fluorescently labelled molecules and how their diffusion dynamics change when the cell environment is modified. In this paper, we describe the use of iMSD to obtain quantitative data of the diffusion dynamics of a small cytoskeletal protein, profilin 1 (pfn1), at the membrane of live cells and how its diffusion is perturbed when the cells are treated with Cytochalasin D and/or the interactions of pfn1 are modified when its actin and polyphosphoinositide binding sites are mutated (pfn1-R88A). Using total internal reflection fluorescence microscopy images, we obtained data on isotropic and confined diffusion coefficients, the proportion of cell areas where isotropic diffusion is the major diffusion mode compared to the confined diffusion mode, the size of the confinement zones and the size of the domains of dynamic partitioning of pfn1. Using these quantitative data, we could demonstrate a decreased isotropic diffusion coefficient for the cells treated with Cytochalasin D and for the pfn1-R88A mutant. We could also see changes in the modes of diffusion between the different conditions and changes in the size of the zones of pfn1 confinements for the pfn1 treated with Cytochalasin D. All of this information was acquired in only a few minutes of imaging per cell and without the need to record thousands of single molecule trajectories.

  • Detecting protein aggregation and interaction in live cells: A guide to number and brightness
    Methods (IF 3.802) Pub Date : 2017-12-06
    Rory Nolan, Maro Iliopoulou, Luis Alvarez, Sergi Padilla-Parra

    The possibility to detect and quantify protein-protein interactions with good spatial and temporal resolutions in live cells is crucial in biology. Number and brightness is a powerful approach to detect both protein aggregation/desegregation dynamics and stoichiometry in live cells. Importantly, this technique can be applied in commercial set ups: both camera based and laser scanning microscopes. It provides pixel-by-pixel information on protein oligomeric states. If performed with two colours, the technique can retrieve the stoichiometry of the reaction under study. In this review, we discuss the strengths and weaknesses of the technique, stressing which are the correct acquisition parameters for a given microscope, the main challenges in analysis, and the limitations of the technique.

  • Unraveling co-translational protein folding: Concepts and methods
    Methods (IF 3.802) Pub Date : 2017-12-06
    Anton A. Komar

    Advances in techniques such as nuclear magnetic resonance spectroscopy, cryo-electron microscopy, and single-molecule and time-resolved fluorescent approaches are transforming our ability to study co-translational protein folding both in vivo in living cells and in vitro in reconstituted cell-free translation systems. These approaches provide comprehensive information on the spatial organization and dynamics of nascent polypeptide chains and the kinetics of co-translational protein folding. This information has led to an improved understanding of the process of protein folding in living cells and should allow remaining key questions in the field, such as what structures are formed within nascent chains during protein synthesis and when, to be answered. Ultimately, studies using these techniques will facilitate development of a unified concept of protein folding, a process that is essential for proper cell function and organism viability. This review describes current methods for analysis of co-translational protein folding with an emphasis on some of the recently developed techniques that allow monitoring of co-translational protein folding in real-time.

  • The imaging FCS diffusion law in the presence of multiple diffusive modes
    Methods (IF 3.802) Pub Date : 2017-12-05
    Sapthaswaran Veerapathiran, Thorsten Wohland

    The cellular plasma membrane is the barrier over which cells exchange materials and communicate with their surroundings, and thus plays the central role in cellular sensing and metabolism. Therefore, the investigation of plasma membrane organization and dynamics is required for understanding of cellular functions. The plasma membrane is a heterogeneous matrix. The presence of structures such as lipid and protein domains and the cytoskeleton meshwork poses a hindrance to the free diffusion of membrane associated biomolecules. However, these domains and the cytoskeleton meshwork barriers are below the optical diffraction limit with potentially short lifetimes and are not easily detected even in super-resolution microscopy. Therefore, dynamic measurements are often used to indirectly prove the existence of domains and barriers by analyzing the mode of diffusion of probe molecules. One of these tools is the Fluorescence Correlation Spectroscopy (FCS) diffusion law. The FCS diffusion law is a plot of diffusion time (τd) versus observation area. For at least three different diffusive modes – free, domain confined, and meshwork hindered hop diffusion – the expected plots have been characterized, typically by its y-intercept (τ0) when fit with a linear model, and have been verified in many cases. However, a description of τ0 has only been given for pure diffusive modes. But in many experimental cases it is not evident that a protein will undergo only one kind of diffusion, and thus the interpretation of the τ0 value is problematic. Here, we therefore address the question about the absolute value of τ0 in the case of complex diffusive modes, i.e. when either one molecule is domain confined and cytoskeleton hindered or when two molecules exhibit different diffusive behavior at the same position in a sample. In addition, we investigate how τ0 changes when the diffusive mode of a probe alters upon disruption of domains or the cytoskeleton by drug treatments. By a combination of experimental studies and simulations, we show that τ0 is not influenced equally by the different diffusive modes as typically found in cellular environments, and that it is the relative change of τ0 rather than its absolute value that provides information on the mode of diffusion.

  • A high content imaging flow cytometry approach to study mitochondria in T cells: MitoTracker Green FM dye concentration optimization
    Methods (IF 3.802) Pub Date : 2017-12-02
    Namrata Gautam, Shvetha Sankaran, John A. Yason, Kevin S.W. Tan, Nicholas R.J. Gascoigne

    Mitochondria, the powerhouse of the cell, are known to remodel their membrane structures through the process of fusion or fission. Studies have indicated that T cells adopt different energy metabolic phenotypes, namely oxidative phosphorylation and glycolysis depending on whether they are naïve, effector and memory T cells. It has recently been shown that changes in mitochondrial morphology dictate T cell fate via regulation of their metabolism. Our keen interest in T cell function and metabolism led us to explore and establish a method to study mitochondria in live T cells through a novel high content approach called Imaging Flow Cytometry (IFC). The focus of our current study was on developing a protocol to standardize the concentration of MitoTracker Green FM dye to observe mitochondria in live T cells using IFC. We began the study by using widefield microscopy to confirm the localisation of MitoTracker Green FM labelled mitochondria in live T cells. This was followed by testing various concentrations of the dye to achieve a similar labelling pattern using IFC while eliminating false positive or negative staining. The optimization of the method used to label the mitochondria by IFC for analysis included standardisation of a number of important parameters such as dye concentration, voltage, fluorescence intensity values for acquisition and processing. IFC could potentially be a powerful method to study T cells in a relatively high throughput manner.

  • Exploring viral reservoir: The combining approach of cell sorting and droplet digital PCR
    Methods (IF 3.802) Pub Date : 2017-12-02
    Lara Gibellini, Simone Pecorini, Sara De Biasi, Marcello Pinti, Elena Bianchini, Anna De Gaetano, Margherita Digaetano, Rosalberta Pullano, Domenico Lo Tartaro, Anna Iannone, Cristina Mussini, Andrea Cossarizza, Milena Nasi

    Combined antiretroviral therapy (cART) blocks different steps of HIV replication and maintains plasma viral RNA at undetectable levels. The virus can remain in long-living cells and create a reservoir where HIV can restart replicating after cART discontinuation. A persistent viral production triggers and maintains a persistent immune activation, which is a well-known feature of chronic HIV infection, and contributes either to precocious aging, or to the increased incidence of morbidity and mortality of HIV positive patients. The new frontier of the treatment of HIV infection is nowadays eradication of the virus from all host cells and tissues. For this reason, it is crucial to have a clear and precise idea of where the virus hides, and which are the cells that keep it silent. Important efforts have been made to improve the detection of viral reservoirs, and new techniques are now giving the opportunity to characterize viral reservoirs. Among these techniques, a strategic approach based upon cell sorting and droplet digital PCR (ddPCR) is opening new horizons and opportunities of research. This review provides an overview of the methods that combine cell sorting and ddPCR for the quantification of HIV DNA in different cell types, and for the detection of its maintenance.

  • Isolation of mammalian stress granule cores for RNA-Seq analysis
    Methods (IF 3.802) Pub Date : 2017-12-01
    Anthony Khong, Saumya Jain, Tyler Matheny, Joshua R. Wheeler, Roy Parker

    Stress granules are dynamic, conserved non-translating RNA-protein assemblies that form during cellular stress and are related to pathological aggregates in many neurodegenerative diseases. Mammalian stress granules contain stable structures, referred to as “cores” that can be biochemically purified. Herein, we describe a step-by-step guide on how to isolate RNA from stress granule cores for RNA-Seq analysis. We also describe a methodology for validating the RNA-Seq results by single molecule FISH and how to quantify the single molecule FISH results. These protocols provide a starting point for describing the RNA content of stress granules and may assist in the discovery of the assembly mechanisms and functions of stress granules in a variety of biological contexts.

Some contents have been Reproduced with permission of the American Chemical Society.
Some contents have been Reproduced by permission of The Royal Society of Chemistry.
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