Identifying and characterizing functional 3’ nucleotide addition in the miRNA pathway Methods (IF 3.998) Pub Date : 2018-08-20 A. Maxwell Burroughs, Yoshinari Ando
Over the past decade, modifications to microRNAs (miRNAs) via 3’ end nucleotide addition have gone from a deep-sequencing curiosity to experimentally confirmed drivers of a range of regulatory activities. Here we overview the methods that have been deployed by researchers seeking to untangle these diverse functional roles and include characterizing not only the nucleotidyl transferases catalyzing the additions but also the nucleotides being added, and the timing of their addition during the miRNA pathway. These methods and their further development are key to clarifying the diverse and sometimes contradictory functional findings presently attributed to these nucleotide additions.
Nuclear export of mRNA molecules studied by SPEED microscopy Methods (IF 3.998) Pub Date : 2018-08-18 Yichen Li, Samuel L. Junod, Andrew Ruba, Joseph M. Kelich, Weidong Yang
Molecular Engineering Strategies and Methods for the Expression and Purification of IgG1-based Bispecific Bivalent Antibodies Methods (IF 3.998) Pub Date : 2018-08-11 N. Dimasi, R. Fleming, H. Wu, C. Gao
In recent years, bispecific antibodies (BisAbs) have emerged as novel pharmaceutical candidates owing to their ability to engage two disease mediators simultaneously, thus providing a possible alternative therapeutic approach in complex diseases such as cancer and inflammation. Here we provide an overview of the molecular design, recombinant expression in mammalian cells and purification of BisAbs based on full-length IgG-scFv formats. Practical considerations and strategies to optimize transient expression and purification are also discussed.
Using Bioreactors to Study the Effects of Drugs on the Human Microbiota Methods (IF 3.998) Pub Date : 2018-08-11 Mabel Guzman-Rodriguez, Julie A.K. McDonald, Richard Hyde, Emma Allen-Vercoe, Erika C. Claud, Prameet M. Sheth, Elaine O. Petrof
The study of complex microbial communities has become a major research focus as mounting evidence suggests the pivotal role microbial communities play in host health and disease. Microbial communities of the gastrointestinal tract, known as the gut microbiota, have been implicated in aiding the host with vitamin biosynthesis, regulation of host energy metabolism, immune system development, and resistance to pathogen invasion. Conversely, disruptions of the gut microbiota have been linked to host morbidity, including the development of inflammatory diseases, metabolic disorders, increased cardiovascular risk, and increased risk of infectious diseases. However, studying the gut microbiota in humans and animals is challenging, as many microorganisms are fastidious with unique nutritional or environmental requirements that are often not met using conventional culture techniques. Bioreactors provide a unique solution to overcome some of the limitations of conventional culture techniques. Bioreactors have been used to propagate and establish complex microbial communities in vitro by recapitulating the physiological conditions found in the GI tract. These systems further our understanding of microbial physiology and facilitate our understanding of the impact of medications and xenobiotics on microbial communities. Here, we review the versatility and breadth of bioreactor systems that are currently available and how they are being used to study faecal and defined microbial communities. Bioreactors provide a unique opportunity to study complex microbial interactions and perturbations in vitro in a controlled environment without confounding biotic and abiotic variables.
Speech analysis for health: current state-of-the-art and the increasing impact of deep learning Methods (IF 3.998) Pub Date : 2018-08-10 Nicholas Cummins, Alice Baird, Björn Schuller
Due to the complex and intricate nature associated with their production, the acoustic-prosodic properties of a speech signal are modulated with a range of health related effects. There is an active and growing area of machine learning research in this speech and health domain, focusing on developing paradigms to objectively extract and measure such effects. Concurrently, deep learning is transforming intelligent signal analysis, such that machines are now reaching near human capabilities in a range of recognition and analysis tasks. Herein, we review current state-of-the-art approaches with speechbased health detection, placing a particular focus on the impact of deep learning within this domain. Based on this overview, it is evident while that deep learning based solutions be become more present in the literature, it has not had the same overall dominating effect seen in other related fields. In this regard, we suggest some possible research directions aimed at fully leveraging the advantages that deep learning can offer speech-based health detection.
A guide to nucleic acid detection by single-molecule kinetic fingerprinting Methods (IF 3.998) Pub Date : 2018-08-10 Alexander Johnson-Buck, Jieming Li, Muneesh Tewari, Nils G. Walter
Conventional methods for detecting small quantities of nucleic acids require amplification by the polymerase chain reaction (PCR), which necessitates prior purification and introduces copying errors. While amplification-free methods do not have these shortcomings, they are generally orders of magnitude less sensitive and specific than PCR-based methods. In this review, we provide a practical guide to a novel amplification-free method, single-molecule recognition through equilibrium Poisson sampling (SiMREPS), that provides both single-molecule sensitivity and single-base selectivity by monitoring the repetitive interactions of fluorescent probes to immobilized targets. We demonstrate how this kinetic fingerprinting filters out background arising from the inevitable nonspecific binding of probes, yielding virtually zero background signal. As practical applications of this digital detection methodology, we present the quantification of microRNA miR-16 and the detection of the mutation EGFR L858R with an apparent single-base discrimination factor of over 3 million.
Preparation of bispecific antibody-protein adducts by site-specific chemo-enzymatic conjugation Methods (IF 3.998) Pub Date : 2018-08-03 Lina Bartels, Hidde L. Ploegh, Hergen Spits, Koen Wagner
Historically, bispecific antibodies have been constructed through the genetic fusion of additional binding domains to the constant domains of the antibody heavy- or light chains. We present an alternative method for the introduction of additional functional domains to an antibody: site-specific chemo-enzymatic conjugation. This method relies on the combination of site-specific transpeptidases and bioorthogonal chemistry. Transpeptidases are used to site-specifically introduce chemical handles, which can then be used to couple new functional groups by means of a bioorthogonal chemical reaction. We demonstrate site-specific chemo-enzymatic linkage using the transpeptidase sortase (hereafter: sortase) and either a strain-promoted alkyne-azide cycloaddition (SPAAC) or an inverse-electron demand Diels-Alder reaction. Other transpeptidases and bioorthogonal reactions suitable for this purpose exist. Site-specific chemo-enzymatic linkage is a modular method. After introduction of a chemical handle in the antibody, any functional group of interest may then be attached. The modularity of this conjugation method allows for a ‘plug-and-play’ approach to prepare new antibody conjugates, thus bypassing the need for (potentially) laborious genetic fusions. Moreover, as sortase is used to specifically modify the exact C-termini of the antibody chains, the final product will be fused in a C-to-C orientation, which is impossible to achieve by genetic manipulations alone. Here we demonstrate the utility of site-specific chemo-enzymatic conjugation to prepare antibody heterodimers, bispecific T-cell engager antibodies, and immunocytokines, discussing purification methods and describing possible pitfalls.
Generation of Fabs-in-Tandem Immunoglobulin Molecules for Dual-Specific Targeting Methods (IF 3.998) Pub Date : 2018-08-03 Shiyong Gong, Chengbin Wu
Bispecific antibody (BsAb) has become an important trend in developing next generation biologics therapies. By simultaneously engaging two molecular targets, BsAbs show distinctive mechanism of actions that could lead to clinical benefits unattainable by conventional monoclonal antibodies (mAbs). Successful launch provided clinical validation and encourage more BsAb development in the pipeline of pharmaceutical companies. Fabs-in-tandem immunoglobulin (FIT-IgTM) format was initially described in 2017. This unique design provides a symmetrical and tetravalent IgG-like bispecific molecule with correct association of 2 sets of VH/VL pairs, where two Fabs are fused directly in a crisscross orientation without any mutations or use of peptide linkers. FIT-Ig can be readily made from 2 existing monoclonal antibodies by basic molecular biology techniques with high expression level in mammalian cells, and easily purified to homogeneity using standard approaches without extensive optimization. FIT-Ig molecules exhibit favorable drug-like properties, in vitro and in vivo functions, as well as manufacturing efficiency for commercial development. Here, we provide an example of construction and preliminary characterization of a FIT-Ig molecule with discussions on optimization and general utility.
Biotagging, an in vivo biotinylation approach for cell-type specific subcellular profiling in zebrafish Methods (IF 3.998) Pub Date : 2018-08-02 Le A. Trinh, Vanessa Chong-Morrison, Tatjana Sauka-Spengler
Interrogation of gene regulatory circuits in complex organisms requires precise and robust methods to label cell-types for profiling of target proteins in a tissue-specific fashion as well as data analysis to understand interconnections within the circuits. There are several strategies for obtaining cell-type and subcellular specific genome-wide data. We have developed a methodology, termed “biotagging” that uses tissue-specific, genetically encoded components to biotinylate target proteins, enabling in depth genome-wide profiling in zebrafish. We have refined protocols to use the biotagging approach that led to enhanced isolation of coding and non-coding RNAs from ribosomes and nuclei of genetically defined cell-types. The ability to study both the actively translated and transcribed transcriptome in the same cell population, coupled to genomic accessibility assays has enabled the study of cell-type specific gene regulatory circuits in zebrafish due to the high signal-to-noise achieved via its stringent purification protocol. Here, we provide detailed methods to isolate, profile and analyze cell-type specific polyribosome and nuclear transcriptome in zebrafish.
Programmable base editing in zebrafish using a modified CRISPR-Cas9 system Methods (IF 3.998) Pub Date : 2018-08-02 Wei Qin, Xiaochan Lu, Shuo Lin
The use of CRISPR/Cas9 to knockout genes in zebrafish has been well established. However, to better model many human diseases that are caused by point mutations, a robust methodology for generating desirable DNA base changes is still needed. Recently, Cas9-linked cytidine deaminases (base editors) evolved as a strategy to introduce single base mutations in model organisms. They have been used to convert cytidine to thymine at specific genomic loci. Here we describe a protocol for using the base editing system in zebrafish and its application to reproduce a single base mutation observed in human Ablepharon-Macrostomia Syndrome.
Fishing for understanding: Unlocking the zebrafish gene editor’s toolbox Methods (IF 3.998) Pub Date : 2018-08-02 Brandon W. Simone, Gabriel Martínez-Gálvez, Zachary WareJoncas, Stephen C. Ekker
The rapid growth of the field of gene editing can largely be attributed to the discovery and optimization of designer endonucleases. These include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regular interspersed short palindromic repeat (CRISPR) systems including Cas9, Cas12a, and structure-guided nucleases. Zebrafish (Danio rerio) have proven to be a powerful model system for genome engineering testing and applications due to their external development, high fecundity, and ease of housing. As the zebrafish gene editing toolkit continues to grow, it is becoming increasingly important to understand when and how to utilize which of these technologies for maximum efficacy in a particular project. While CRISPR-Cas9 has brought broad attention to the field of genome engineering in recent years, designer endonucleases have been utilized in genome engineering for more than two decades. This chapter provides a brief overview of designer endonuclease and other gene editing technologies in zebrafish as well as some of their known functional benefits and limitations depending on specific project goals. Finally, selected prospects for additional gene editing tools are presented, promising additional options for directed genomic programming of this versatile animal model system.
Imaging Cell-Type-Specific Dynamics of mRNAs in Living Mouse Brain Methods (IF 3.998) Pub Date : 2018-07-29 Chiso Nwokafor, Robert H. Singer, Hyungsik Lim
We describe a method for visualizing mRNAs in living mouse. Nascent transcripts and cytoplasmic mRNAs were labeled via lentiviral expression of MS2 coat protein (MCP) tagged with fluorescent protein (MCP-XFP) in knock-in mice whose β-actin mRNAs contained MCP binding stem loops (MBS). Then the mRNA molecules were imaged in the live cerebral cortex through an optical cranial window by intravital two-photon microscopy. By means of the controlled expression of MCP-XFP, single mRNA particles could be detected differentially in the nucleus and cytoplasm of a specific cell type. Consequently, this method is useful for investigating the cell-type-dependent dynamics of mRNAs underlying the structure and function of the brain.
Time-lapse imaging beyond the diffraction limit Methods (IF 3.998) Pub Date : 2018-07-27 Ajay Chitnis, Damian Dalle Nogare
The zebrafish, with its rapid external development, optical transparency, and the relative ease with which transgenic lines can be created, is rapidly becoming the model of choice for examining developmental processes via time-lapse microscopy. The recent proliferation of techniques for super-resolution imaging now allows for an unprecedented view of embryonic development at high spatial and temporal resolution in live tissues. This review examines both the theoretical basis and practical application of a number of established and emerging super-resolution microscopy techniques, focusing on their application in time-lapse imaging of live zebrafish embryos.
Bi-clustering of metabolic data using matrix factorization tools Methods (IF 3.998) 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 subsets of 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.
Temperature controlled ionic liquid aqueous two phase system combined with affinity capillary electrophoresis for rapid and precise pharmaceutical-protein binding measurements Methods (IF 3.998) Pub Date : 2018-02-10 Deia Abd El-Hady, Hassan M. Albishri
Thread- paper, and fabric enzyme-linked immunosorbent assays (ELISA) Methods (IF 3.998) 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.
Thermophoresis for characterizing biomolecular interaction Methods (IF 3.998) 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.
Methods of reconstitution to investigate membrane protein function Methods (IF 3.998) 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.
Affinity-based separation methods for the study of biological interactions: The case of peroxisome proliferator-activated receptors in drug discovery Methods (IF 3.998) 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.
Fragment screening for drug leads by weak affinity chromatography (WAC-MS) Methods (IF 3.998) Pub Date : 2018-02-23 Sten Ohlson, Minh-Dao Duong-Thi
The yin and yang of solubilization and stabilization for wild-type and full-length membrane protein Methods (IF 3.998) 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.
Toward Comprehensive Measurement of Protein Hydration Dynamics: Facilitation of NMR-based Methods by Reverse Micelle Encapsulation Methods (IF 3.998) Pub Date : 2018-07-24 Pamela N. Gallo, Joseph C. Iovine, Nathaniel V. Nucci
Protein-water interactions are a fundamental determinant of protein structure and function. Despite their importance, the molecular details of water orientations and dynamics near protein surfaces remain poorly understood, largely due to the difficulty of measuring local water mobility near the protein in a site-resolved fashion. Solution NMR-based measurement of water mobility via the nuclear Overhauser effect was presented as a method for performing comprehensive, site-resolved measurements of water dynamics many years ago. Though this approach yielded extensive insight on the dynamics and locations of waters buried within proteins, its promise for measuring surface hydration dynamics was impeded by various technical barriers. Over the past several years, however, this approach has been pursued anew with the aid of reverse micelle encapsulation of proteins of interest. The confined environment of the reverse micelle resolves many of these barriers and permits site-resolved measurement of relative water dynamics across much of the protein surface. Here, the development of this strategy for measuring hydration dynamics is reviewed with particular focus on the important remaining challenges to its widespread application.
Probing membrane protein ground and conformationally excited states using dipolar- and J-coupling mediated MAS solid state NMR experiments Methods (IF 3.998) Pub Date : 2018-07-23 T. Gopinath, Gianluigi Veglia
The intrinsic conformational plasticity of membrane proteins directly influences the magnitude of the orientational-dependent NMR interactions such as dipolar couplings (DC) and chemical shift anisotropy (CSA). As a result, the conventional cross-polarization (CP)-based techniques mainly capture the more rigid regions of membrane proteins, while the most dynamic regions are essentially invisible. Nonetheless, dynamic regions can be detected using experiments in which polarization transfer takes place via J-coupling interactions. Here, we review our recent efforts to develop single and dual acquisition pulse sequences with either 1H or 13C detection that utilize both DC and J-coupling mediated transfer to detect both rigid and mobile regions of membrane proteins in native-like lipid environments. We show the application of these new methods for studying the conformational equilibrium of a single-pass membrane protein, phospholamban, which regulates the calcium transport across the sarcoplasmic reticulum (SR) membrane by interacting with the SR Ca2+-ATPase. We anticipate that these methods will be ideal to portray the complex dynamics of membrane proteins in their native environments.
Determination of the conformational states of strychnine using NMR residual dipolar couplings in a tensor-free approach Methods (IF 3.998) Pub Date : 2018-07-21 Giulia Tomba, Carlo Camilloni, Michele Vendruscolo
Small molecules with rotatable bonds can occupy different conformational states as a consequence of their thermal fluctuations. The accurate determination of the structures of such states, as well as of their statistical weights, has been challenging because of the technical difficulties in extracting information from experimental measurements, which are normally averaged over the conformational space available. Here, to achieve this objective, we present an approach based on a recently proposed tensor-free method for incorporating NMR residual dipolar couplings as structural restraints in replica-averaged molecular dynamics simulations. This approach enables the information provided by the experimental data to be used in the spirit of the maximum entropy principle. Furthermore, in order to enhance the sampling of the conformational space we incorporated the metadynamics method in the simulations. We illustrate the method in the case of strychnine, determining the three major conformational states and their associated occupation probabilities.
Advances in enzyme substrate analysis with capillary electrophoresis Methods (IF 3.998) Pub Date : 2018-02-27 Srikanth Gattu, Cassandra L. Crihfield, Grace Lu, Lloyd Bwanali, Lindsay M. Veltri, Lisa A. Holland
Capillary electrophoresis provides a rapid, cost-effective platform for enzyme and substrate characterization. The high resolution achievable by capillary electrophoresis enables the analysis of substrates and products that are indistinguishable by spectroscopic techniques alone, while the small volume requirement enables analysis of enzymes or substrates in limited supply. Furthermore, the compatibility of capillary electrophoresis with various detectors makes it suitable for KM determinations ranging from nanomolar to millimolar concentrations. Capillary electrophoresis fundamentals are discussed with an emphasis on the separation mechanisms relevant to evaluate sets of substrate and product that are charged, neutral, and even chiral. The basic principles of Michaelis-Menten determinations are reviewed and the process of translating capillary electrophoresis electropherograms into a Michaelis-Menten curve is outlined. The conditions that must be optimized in order to couple off-line and on-line enzyme reactions with capillary electrophoresis separations, such as incubation time, buffer pH and ionic strength, and temperature, are examined to provide insight into how the techniques can be best utilized. The application of capillary electrophoresis to quantify enzyme inhibition, in the form of KI or IC50 is detailed. The concept and implementation of the immobilized enzyme reactor is described as a means to increase enzyme stability and reusability, as well as a powerful tool for screening enzyme substrates and inhibitors. Emerging techniques focused on applying capillary electrophoresis as a rapid assay to obtain structural identification or sequence information about a substrate and in-line digestions of peptides and proteins coupled to mass spectrometry analyses are highlighted.
Characterization of solution-phase drug-protein interactions by ultrafast affinity extraction Methods (IF 3.998) Pub Date : 2018-03-03 Sandya R. Beeram, Xiwei Zheng, Kyungah Suh, David S. Hage
A number of tools based on high-performance affinity separations have been developed for studying drug-protein interactions. An example of one recent approach is ultrafast affinity extraction. This method has been employed to examine the free (or non-bound) fractions of drugs and other solutes in simple or complex samples that contain soluble binding agents. These free fractions have also been used to determine the binding constants and rate constants for the interactions of drugs with these soluble agents. This report describes the general principles of ultrafast affinity extraction and the experimental conditions under which it can be used to characterize such interactions. This method will be illustrated by utilizing data that have been obtained when using this approach to measure the binding and dissociation of various drugs with the serum transport proteins human serum albumin and alpha1-acid glycoprotein. A number of practical factors will be discussed that should be considered in the design and optimization of this approach for use with single-column or multi-column systems. Techniques will also be described for analyzing the resulting data for the determination of free fractions, rate constants and binding constants. In addition, the extension of this method to complex samples, such as clinical specimens, will be considered.
Functional characterisation of G protein-coupled receptors Methods (IF 3.998) Pub Date : 2018-03-03 Romez Uddin, John Simms, David Poyner
Characterisation of receptors can involve either assessment of their ability to bind ligands or measure receptor activation as a result of agonist or inverse agonist interactions. This review focuses on G protein-coupled receptors (GPCRs), examining techniques that can be applied to both receptors in membranes and after solubilisation. Radioligand binding remains a widely used technique, although there is increasing use of fluorescent ligands. These can be used in a variety of experimental designs, either directly monitoring ligand itself with techniques such as fluorescence polarisation or indirectly via resonance energy transfer (fluorescence/Forster resonance energy transfer, FRET and bioluminescence resonance energy transfer, BRET). Label free techniques such as isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) are also increasingly being used. For GPCRs, the main measure of receptor activation is to investigate the association of the G protein with the receptor. The chief assay measures the receptor-stimulated binding of GTP or a suitable analogue to the receptor. The direct association of the G protein with the receptor has been investigated via resonance energy techniques. These have also been used to measure ligand-induced conformational changes within the receptor; a variety of experimental techniques are available to incorporate suitable donors and acceptors within the receptor.
Studying structure and function of membrane proteins with PELDOR/DEER spectroscopy – A crystallographers’ perspective Methods (IF 3.998) Pub Date : 2018-03-03 Janin Glaenzer, Martin F. Peter, Gregor Hagelueken
In 1985, the first X-ray structure of a membrane protein was determined. Today, more than 30 years later, many more structures have been solved. Nevertheless, studying the structure of membrane proteins remains a very challenging task. Due to their inherent conformational flexibility, having a single X-ray structure is usually only the first step towards truly understanding the function of these dynamic molecules. For this reason, additional methods are needed that can provide complementary information, especially about conformational flexibility. Pulsed electron-electron double resonance spectroscopy (PELDOR, also known as DEER) is such a method. It can be used to precisely measure nanometer distance distributions between intrinsic or artificially introduced spin-centers in macromolecules and thereby to probe the conformational state of the macromolecule. PELDOR can be applied in solution, in detergent, in lipid bilayers and even within cells. However, PELDOR is an advanced spectroscopy technique and requires specialised equipment and training. This chapter aims to be a starting point for crystallographers and other structural biologists who want to get a better understanding of PELDOR spectroscopy and its application. It gives an insight into the planning stages of the experiment (i.e., which spin labels are possible and where to place them), how a PELDOR experiment is conducted and how the results are interpreted. For this purpose, the substrate binding protein (SBP) from a Vibrio cholerae TRAP transporter is used as a step-by-step example. Further, the chapter gives examples of how PELDOR spectroscopy has previously been applied to overcome known limitations of X-ray crystallography in modern integrative structural biology approaches.
Mass spectrometry-enabled structural biology of membrane proteins Methods (IF 3.998) Pub Date : 2018-03-03 Antonio N. Calabrese, Sheena E. Radford
The last ∼25 years has seen mass spectrometry (MS) emerge as an integral method in the structural biology toolkit. In particular, MS has enabled the structural characterization of proteins and protein assemblies that have been intractable by other methods, especially those that are large, heterogeneous or transient, providing experimental evidence for their structural organization in support of, and in advance of, high resolution methods. The most recent frontier conquered in the field of MS-based structural biology has been the application of established methods for studying water soluble proteins to the more challenging targets of integral membrane proteins. The power of MS in obtaining structural information has been enabled by advances in instrumentation and the development of hyphenated mass spectrometry-based methods, such as ion mobility spectrometry-MS, chemical crosslinking-MS and other chemical labelling/footprinting-MS methods. In this review we detail the insights garnered into the structural biology of membrane proteins by applying such techniques. Application and refinement of these methods has yielded unprecedented insights in many areas, including membrane protein conformation, dynamics, lipid/ligand binding, and conformational perturbations due to ligand binding, which can be challenging to study using other methods.
Planar lipid bilayers in recombinant ion channel research Methods (IF 3.998) Pub Date : 2018-03-09 Jacqueline Maher, Marcus Allen
There are a number of methods of investigating the function of recombinant proteins such as ion channels. However, after channel purification there are few methods to guarantee that the protein still functions. For ion channels, reconstituting back into planar lipid bilayers and demonstrating preserved function is a convenient and trusted method. It is cell free and even inaccessible, intracellular ion channels can be studied. We have used this method to study the function of recombinant channels of known subunit composition and have found it convenient for investigating the mode of action of ion channel modulators.
Studies of antibody-antigen interactions by capillary electrophoresis: A review Methods (IF 3.998) Pub Date : 2018-03-15 Annette C. Moser, Sidney Trenhaile, Kati Frankenberg
Antibody-antigen interactions are vital in immunoassay development and can determine detection limits and analysis times. Capillary electrophoresis (CE) is a powerful technique that can be used to quantify antibody-antigen interactions. These CE methods range from simple separations of a premixed antibody and antigen sample applied as a short plug to allow for separation of complex, free antibody, and free antigen to more complex systems which inject complexed samples in the presence of antibody or antigen; or even injections of antibody and antigen sequentially. The objective of this review is to identify and describe various CE techniques which have been used to study antibody-antigen interactions. A brief discussion of linear and nonlinear curve fitting is also included.
Tumor classification with MALDI-MSI data of tissue microarrays: A case study Methods (IF 3.998) Pub Date : 2018-04-12 Nadine E. Mascini, Jannis Teunissen, Rob Noorlag, Stefan M. Willems, Ron M.A. Heeren
With mass spectrometry imaging (MSI) on tissue microarrays (TMAs) a large number of biomolecules can be studied for many patients at the same time, making it an attractive tool for biomarker discovery. Here we investigate whether lymph node metastasis can be predicted from MALDI-MSI data. Measurements are performed on TMAs and then filtered based on spectral intensity and the percentage of tumor cells, after which the resulting data for 122 patients is further preprocessed. We assume differences between patients with and without metastasis are expressed in a limited number of features. Two univariate feature selection methods are applied to reduce the dimensionality of the MALDI-MSI data. The selected features are then used in combination with three classifiers. The best classification scores are obtained with a decision tree classifier, which classifies about 72% of patients correctly. Almost all the predictive power comes from a single peak (m/z 718.4). The sensitivity of our classification approach, which can be generically used to search for biomarkers, is investigated using artificially modified data.
Theory and practice of using solvent paramagnetic relaxation enhancement to characterize protein conformational dynamics Methods (IF 3.998) Pub Date : 2018-04-12 Zhou Gong, Charles D. Schwieters, Chun Tang
Paramagnetic relaxation enhancement (PRE) has been established as a powerful tool in NMR for investigating protein structure and dynamics. The PRE is usually measured with a paramagnetic probe covalently attached at a specific site of an otherwise diamagnetic protein. The present work provides the numerical formulation for probing protein structure and conformational dynamics based on the solvent PRE (sPRE) measurement, using two alternative approaches. An inert paramagnetic cosolute randomly collides with the protein, and the resulting sPRE manifests the relative solvent exposure of protein nuclei. To make the back-calculated sPRE values most consistent with the observed values, the protein structure is either refined against the sPRE, or an ensemble of conformers is selected from a pre-generated library using a Monte Carlo algorithm. The ensemble structure comprises either N conformers of equal occupancy, or two conformers with different relative populations. We demonstrate the sPRE method using GB1, a structurally rigid protein, and calmodulin, a protein comprising two domains and existing in open and closed states. The sPRE can be computed with a stand-alone program for rapid evaluation, or with the invocation of a module in the latest release of the structure calculation software Xplor-NIH. As a label-free method, the sPRE measurement can be readily integrated with other biophysical techniques. The current limitations of the sPRE method are also discussed, regarding accurate measurement and theoretical calculation, model selection and suitable timescale.
An NMR strategy to detect conformational differences in a protein complexed with highly analogous inhibitors in solution Methods (IF 3.998) Pub Date : 2018-04-12 John D. Persons, Shahid N. Khan, Rieko Ishima
This manuscript presents an NMR strategy to investigate conformational differences in protein-inhibitor complexes, when the inhibitors tightly bind to a protein at sub-nanomolar dissociation constants and are highly analogous to each other. Using HIV-1 protease (PR), we previously evaluated amide chemical shift differences, ΔCSPs, of PR bound to darunavir (DRV) compared to PR bound to several DRV analogue inhibitors, to investigate subtle but significant long-distance conformation changes caused by the inhibitor’s chemical moiety variation [Khan, S. N., Persons, J. D. Paulsen, J. L., Guerrero, M., Schiffer, C. A., Kurt-Yilmaz, N., and Ishima, R., Biochemistry, (2018), 57, 1652–1662]. However, ΔCSPs are not ideal for investigating subtle PR-inhibitor interface differences because intrinsic differences in the electron shielding of the inhibitors affect protein ΔCSPs. NMR relaxation is also not suitable as it is not sensitive enough to detect small conformational differences in rigid regions among similar PR-inhibitor complexes. Thus, to gain insight into conformational differences at the inhibitor-protein interface, we recorded 15N-half filtered NOESY spectra of PR bound to two highly analogous inhibitors and assessed NOEs between PR amide protons and inhibitor protons, between PR amide protons and hydroxyl side chains, and between PR amide protons and water protons. We also verified the PR amide-water NOEs using 2D water-NOE/ROE experiments. Differences in water-amide proton NOE peaks, possibly due to amide-protein hydrogen bonds, were observed between subunit A and subunit B, and between the DRV-bound form and an analogous inhibitor-bound form, which may contribute to remote conformational changes.
Microbial expression systems for membrane proteins Methods (IF 3.998) Pub Date : 2018-04-12 Marvin V. Dilworth, Mathilde S. Piel, Kim E. Bettaney, Pikyee Ma, Ji Luo, David Sharples, David R. Poyner, Stephane R. Gross, Karine Moncoq, Peter J.F. Henderson, Bruno Miroux, Roslyn M. Bill
Despite many high-profile successes, recombinant membrane protein production remains a technical challenge; it is still the case that many fewer membrane protein structures have been published than those of soluble proteins. However, progress is being made because empirical methods have been developed to produce the required quantity and quality of these challenging targets. This review focuses on the microbial expression systems that are a key source of recombinant prokaryotic and eukaryotic membrane proteins for structural studies. We provide an overview of the host strains, tags and promoters that, in our experience, are most likely to yield protein suitable for structural and functional characterization. We also catalogue the detergents used for solubilization and crystallization studies of these proteins. Here, we emphasize a combination of practical methods, not necessarily high-throughput, which can be implemented in any laboratory equipped for recombinant DNA technology and microbial cell culture.
MS-based conformation analysis of recombinant proteins in design, optimization and development of biopharmaceuticals Methods (IF 3.998) Pub Date : 2018-04-18 Devrishi Goswami, Jun Zhang, Pavel V. Bondarenko, Zhongqi Zhang
Mass spectrometry (MS)-based methods for analyzing protein higher order structures have gained increasing application in the field of biopharmaceutical development. The predominant methods used in this area include native MS, hydrogen deuterium exchange-MS, covalent labeling, cross-linking and limited proteolysis. These MS-based methods will be briefly described in this article, followed by a discussion on how these methods contribute at different stages of discovery and development of protein therapeutics.
Folding and stabilizing membrane proteins in amphipol A8-35 Methods (IF 3.998) Pub Date : 2018-04-18 Christel Le Bon, Anaïs Marconnet, Sandrine Masscheleyn, Jean-Luc Popot, Manuela Zoonens
Membrane proteins (MPs) are important pharmacological targets because of their involvement in many essential cellular processes whose dysfunction can lead to a large variety of diseases. A detailed knowledge of the structure of MPs and the molecular mechanisms of their activity is essential to the design of new therapeutic agents. However, studying MPs in vitro is challenging, because it generally implies their overexpression under a functional form, followed by their extraction from membranes and purification. Targeting an overexpressed MP to a membrane is often toxic and expression yields tend to be limited. One alternative is the formation of inclusion bodies (IBs) in the cytosol of the cell, from which MPs need then to be folded to their native conformation before structural and functional analysis can be contemplated. Folding MPs targeted to IBs is a difficult task. Specially designed amphipathic polymers called ‘amphipols’ (APols), which have been initially developed with the view of improving the stability of MPs in aqueous solutions compared to detergents, can be used to fold both α-helical and β-barrel MPs. APols represent an interesting novel amphipathic medium, in which high folding yields can be achieved. In this review, the properties of APol A8-35 and of the complexes they form with MPs are summarized. An overview of the most important studies reported so far using A8-35 to fold MPs is presented. Finally, from a practical point of view, a detailed description of the folding and trapping methods is given.
How to Run Molecular Dynamics Simulations on Electrospray Droplets and Gas Phase Proteins: Basic Guidelines and Selected Applications Methods (IF 3.998) Pub Date : 2018-04-18 Lars Konermann, Haidy Metwally, Robert G. McAllister, Vlad Popa
The ability to transfer intact proteins and protein complexes into the gas phase by electrospray ionization (ESI) has opened up numerous mass spectrometry (MS)-based avenues for exploring biomolecular structure and function. However, many details regarding the ESI process and the properties of gaseous analyte ions are difficult to decipher when relying solely on experimental data. Molecular dynamics (MD) simulations can provide additional insights into the behavior of ESI droplets and protein ions. This review is geared primarily towards experimentalists who wish to adopt MD simulations as a complementary research tool. We touch on basic points such as force fields, the choice of a proper water model, GPU-acceleration, possible artifacts, as well as shortcomings of current MD models. Following this technical overview, we highlight selected applications. Simulations on aqueous droplets confirm that “native” ESI culminates in protein ion release via the charged residue model. MD-generated charge states and collision cross sections match experimental data. Gaseous protein ions produced by native ESI retain much of their solution structure. Moving beyond classical fixed-charge algorithms, we discuss a simple strategy that captures the mobile nature of H+ within gaseous biomolecules. These mobile proton simulations confirm the high propensity of gaseous proteins to form salt bridges, as well as the occurrence of charge migration during collision-induced unfolding and dissociation. It is hoped that this review will promote the use of MD simulations in ESI-related research. We also hope to encourage the development of improved algorithms for charged droplets and gaseous biomolecular ions.
Methodological considerations for the identification of choline and carnitine-degrading bacteria in the gut Methods (IF 3.998) Pub Date : 2018-04-19 Eleanor Jameson, Mussa Quareshy, Yin Chen
The bacterial formation of trimethylamine (TMA) has been linked to cardiovascular disease. This review focuses on the methods employed to investigate the identity of the bacteria responsible for the formation of TMA from dietary choline and carnitine in the human gut. Recent studies have revealed the metabolic pathways responsible for bacterial TMA production, primarily the anaerobic glycyl radical-containing, choline-TMA lyase, CutC and the aerobic carnitine monooxygenase, CntA. Identification of these enzymes has enabled bioinformatics approaches to screen both human-associated bacterial isolate genomes and whole gut metagenomes to determine which bacteria are responsible for TMA formation in the human gut. We centre on several key methodological aspects for identifying the TMA-producing bacteria and report how these pathways can be identified in human gut microbiota through bioinformatics analysis of available bacterial genomes and gut metagenomes.
An integrative approach to investigate the association among high-sensitive C-reactive protein, body fat mass distribution, and other cardiometabolic risk factors in young healthy women Methods (IF 3.998) Pub Date : 2018-04-24 Bin Wu, Jingshan Huang, Lihua Zhang, Mohan Vamsi Kasukurthi, Fangwan Huang, Jiang Bian, Keisuke Fukuo, Tsutomu Kazumi
Prior research has indicated that as an important biomarker of chronic low-grade inflammation, high-sensitivity C-reactive protein (hs-CRP) can play important roles on the onset of metabolic syndrome and cardiovascular diseases (CVD). We conducted an integrative approach, which combines biological wet-lab experiments, statistical analysis, and semantics-oriented bioinformatics & computational analysis, to investigate the association among hs-CRP, body fat mass (FM) distribution, and other cardiometabolic risk factors in young healthy women. Research outcomes in this study resulted in two novel discoveries. Discovery 1: There are four primary determinants for hs-CRP, i.e., central/abdominal FM (a.k.a. trunk FM) accumulation, leptin, high density lipoprotein cholesterol (HDL-C), and plasminogen activator inhibitior-1 (PAI-1). Discovery 2: Chronic inflammation may involve in adipocyte-cytokine interaction underlying the metabolic derangement in healthy young women.
Hidden motions and motion-induced invisibility: Dynamics-based spectral editing in solid-state NMR Methods (IF 3.998) Pub Date : 2018-04-24 Irina Matlahov, Patrick C.A. van der Wel
Solid-state nuclear magnetic resonance (ssNMR) spectroscopy enables the structural characterization of a diverse array of biological assemblies that include amyloid fibrils, non-amyloid aggregates, membrane-associated proteins and viral capsids. Such biological samples feature functionally relevant molecular dynamics, which often affect different parts of the sample in different ways. Solid-state NMR experiments’ sensitivity to dynamics represents a double-edged sword. On the one hand, it offers a chance to measure dynamics in great detail. On the other hand, certain types of motion lead to signal loss and experimental inefficiencies that at first glance interfere with the application of ssNMR to overly dynamic proteins. Dynamics-based spectral editing (DYSE) ssNMR methods leverage motion-dependent signal losses to simplify spectra and enable the study of sub-structures with particular motional properties.
SigEMD: A powerful method for differential gene expression analysis in single-cell RNA sequencing data Methods (IF 3.998) Pub Date : 2018-04-24 Tianyu Wang, Sheida Nabavi
Differential gene expression analysis is one of the significant efforts in single cell RNA sequencing (scRNAseq) analysis to discover the specific changes in expression levels of individual cell types. Since scRNAseq exhibits multimodality, large amounts of zero counts, and sparsity, it is different from the traditional bulk RNA sequencing (RNAseq) data. The new challenges of scRNAseq data promote the development of new methods for identifying differentially expressed (DE) genes. In this study, we proposed a new method, SigEMD, that combines a data imputation approach, a logistic regression model and a nonparametric method based on the Earth Mover’s Distance, to precisely and efficiently identify DE genes in scRNAseq data. The regression model and data imputation are used to reduce the impact of large amounts of zero counts, and the nonparametric method is used to improve the sensitivity of detecting DE genes from multimodal scRNAseq data. By additionally employing gene interaction network information to adjust the final states of DE genes, we further reduce the false positives of calling DE genes. We used simulated datasets and real datasets to evaluate the detection accuracy of the proposed method and to compare its performance with those of other differential expression analysis methods. Results indicate that the proposed method has an overall powerful performance in terms of precision in detection, sensitivity, and specificity.
Engineering expression and function of membrane proteins Methods (IF 3.998) Pub Date : 2018-04-24 Min-Kyoung Kang, Danielle Tullman-Ercek
Membrane proteins are involved in a diverse array of cellular functions and part of many important metabolic pathways. As such, they are attractive targets in the pharmaceutical and bio-based chemical industries. Despite their great potential, many challenges remain before membrane proteins gain widespread success in biotechnology. The two biggest issues are that expression of membrane proteins leads to inhibition of cellular growth and metabolism, and native membrane proteins often lack a desired function or specificity for use in engineered processes. To address these issues, protein engineering and synthetic biology approaches are leading the charge to develop membrane proteins for biotechnological applications. Here, we describe current methods for engineering membrane proteins and optimizing their expression levels in bacteria. We highlight success stories and describe challenges that still face this growing field.
Cryo-electron microscopy of membrane proteins Methods (IF 3.998) Pub Date : 2018-04-25 Nopnithi Thonghin, Vasileios Kargas, Jack Clews, Robert C. Ford
Membrane proteins represent a large proportion of the proteome, but have characteristics that are problematic for many methods in modern molecular biology (that have often been developed with soluble proteins in mind). For structural studies, low levels of expression and the presence of detergent have been thorns in the flesh of the membrane protein experimentalist. Here we discuss the use of cryo-electron microscopy in breakthrough studies of the structures of membrane proteins. This method can cope with relatively small quantities of sample and with the presence of detergent. Until recently, cryo-electron microscopy could not deliver high-resolution structures of membrane proteins, but recent developments in transmission electron microscope technology and in the image processing of single particles imaged in the microscope have revolutionized the field, allowing high resolution structures to be obtained. Here we focus on the specific issues surrounding the application of cryo-electron microscopy to the study of membrane proteins, especially in the choice of a system to keep the protein soluble.
LC/MS at the whole protein level: Studies of biomolecular structure and interactions using native LC/MS and cross-path reactive chromatography (XP-RC) MS Methods (IF 3.998) Pub Date : 2018-04-25 Igor A. Kaltashov, Jake W. Pawlowski, Wenhua Yang, Khaja Muneeruddin, Honglin Yao, Cedric E. Bobst, Andrei N. Lipatnikov
Interfacing liquid chromatography (LC) with electrospray ionization (ESI) to enable on-line MS detection had been initially implemented using reversed phase LC, which in the past three decades remained the default type of chromatography used for LC/MS and LC/MS/MS studies of protein structure. In contrast, the advantages of other types of LC as front-ends for ESI MS, particularly those that allow biopolymer higher order structure to be preserved throughout the separation process, enjoyed relatively little appreciation until recently. However, the past few years witnessed a dramatic surge of interest in the so-called “native” (with “non-denaturing” being perhaps a more appropriate adjective) LC/MS and LC/MS/MS analyses within the bioanalytical and biophysical communities. This review focuses on recent advances in this field, with an emphasis on size exclusion and ion exchange chromatography as front-end platforms for protein characterization by LC/MS. Also discussed are the benefits provided by the integration of chemical reactions in the native LC/MS analyses, including both ion chemistry in the gas phase (e.g., limited charge reduction for characterization of highly heterogeneous biopolymers) and solution-phase reactions (using the recently introduced technique cross-path reactive chromatography).
Mass spectrometry approaches to metabolic profiling of microbial communities within the human gastrointestinal tract Methods (IF 3.998) Pub Date : 2018-04-26 Simon J.S. Cameron, Zoltán Takáts
The interaction between microbial communities and their environment, such as the human gastrointestinal tract, has been an area of microbiology rapidly advanced, by developments in sequencing technology. However, these techniques are largely limited to the detection of the taxonomic composition of a microbial community and/or its genetic functional capacity. Here, we discuss a range of mass spectrometry-based approaches which researchers can employ to explore the host-microbiome interactions at the metabolic level. Traditional approaches to mass spectrometry are detailed, alongside new developments in the field, namely ambient ionisation mass spectrometry and imaging mass spectrometry, which we believe will prove to be important to future work in this field. We further discuss considerations for experimental workflows, data analysis options and propose a methodology for the establishment of causal relationships between functional host-microbiome interactions with regards to health and disease in the human gastrointestinal tract.
Contemporary hydrogen deuterium exchange mass spectrometry Methods (IF 3.998) Pub Date : 2018-04-26 Irina Oganesyan, Cristina Lento, Derek J. Wilson
Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) emerged as a tool for biochemistry and structural biology around 25 years ago. It has since become a key approach for studying protein dynamics, protein-ligand interactions, membrane proteins and intrinsically disordered proteins (IDPs). In HDX labeling, proteins are exposed to deuterated solvent (usually D2O) for a variable ‘labeling time’, resulting in isotope exchange of unprotected labile protons on the amide backbone and amino acid side chains. By comparing the levels of deuterium uptake in different regions of a protein, information on conformational and dynamic changes in the system can be acquired. When coupled with MS, HDX is suitable for probing allosteric effects in catalysis and ligand binding, epitope mapping, validation of biosimilars, drug candidate screening and mapping membrane-protein interactions among many other bioanalytical applications. This review introduces HDX-MS via a brief description of HDX-MS development, followed by an overview of HDX theory and ultimately an outline of methods and procedures involved in performing HDX-MS experiments.
Resolving biomolecular motion and interactions by R2 and R1ρ relaxation dispersion NMR Methods (IF 3.998) Pub Date : 2018-04-26 Erik Walinda, Daichi Morimoto, Kenji Sugase
Among the tools of structural biology, NMR spectroscopy is unique in that it not only derives a static three-dimensional structure, but also provides an atomic-level description of the local fluctuations and global dynamics around this static structure. A battery of NMR experiments is now available to probe the motions of proteins and nucleic acids over the whole biologically relevant timescale from picoseconds to hours. Here we focus on one of these methods, relaxation dispersion, which resolves dynamics on the micro- to millisecond timescale. Key biological processes that occur on this timescale include enzymatic catalysis, ligand binding, and local folding. In other words, relaxation-dispersion-resolved dynamics are often closely related to the function of the molecule and therefore highly interesting to the structural biochemist. With an astounding sensitivity of ∼0.5%, the method detects low-population excited states that are invisible to any other biophysical method. The kinetics of the exchange between the ground state and excited states are quantified in the form of the underlying exchange rate, while structural information about the invisible excited state is obtained in the form of its chemical shift. Lastly, the population of the excited state can be derived. This diversity in the information that can be obtained makes relaxation dispersion an excellent method to study the detailed mechanisms of conformational transitions and molecular interactions. Here we describe the two branches of relaxation dispersion, R2 and R1ρ, discussing their applicability, similarities, and differences, as well as recent developments in pulse sequence design and data processing.
How can native mass spectrometry contribute to characterization of biomacromolecular higher-order structure and interactions? Methods (IF 3.998) Pub Date : 2018-04-26 Wenjun Tong, Guanbo Wang
Native mass spectrometry (MS) is an emerging approach for characterizing biomacromolecular structure and interactions under physiologically relevant conditions. In native MS measurement, intact macromolecules or macromolecular complexes are directly ionized from a non-denaturing solvent, and key noncovalent interactions that hold the complexes together can be preserved for MS analysis in the gas phase. This technique provides unique multi-level structural information such as conformational changes, stoichiometry, topology and dynamics, complementing conventional biophysical techniques. Despite the maturation of native MS and greatly expanded range of applications in recent decades, further dissemination is needed to make the community aware of such a technique. In this review, we attempt to provide an overview of the current body of knowledge regarding major aspects of native MS and explain how such technique contributes to the characterization of biomacromolecular higher-order structure and interactions.
Synthesis of multi-omic data and community metabolic models reveals insights into the role of hydrogen sulfide in colon cancer Methods (IF 3.998) Pub Date : 2018-04-26 Vanessa L. Hale, Patricio Jeraldo, Michael Mundy, Janet Yao, Gary Keeney, Nancy Scott, E. Heidi Cheek, Jennifer Davidson, Megan Green, Christine Martinez, John Lehman, Chandra Pettry, Erica Reed, Kelly Lyke, Bryan A. White, Christian Diener, Osbaldo Resendis-Antonio, Jaime Gransee, Nicholas Chia
Multi-omic data and genome-scale microbial metabolic models have allowed us to examine microbial communities, community function, and interactions in ways that were not available to us historically. Now, one of our biggest challenges is determining how to integrate data and maximize data potential. Our study demonstrates one way in which to test a hypothesis by combining multi-omic data and community metabolic models. Specifically, we assess hydrogen sulfide production in colorectal cancer based on stool, mucosa, and tissue samples collected on and off the tumor site within the same individuals. 16S rRNA microbial community and abundance data were used to select and inform the metabolic models. We then used MICOM, an open source platform, to track the metabolic flux of hydrogen sulfide through a defined microbial community that either represented on-tumor or off-tumor sample communities. We also performed targeted and untargeted metabolomics, and used the former to quantitatively evaluate our model predictions. A deeper look at the models identified several unexpected but feasible reactions, microbes, and microbial interactions involved in hydrogen sulfide production for which our 16S and metabolomic data could not account. These results will guide future in vitro, in vivo, and in silico tests to establish why hydrogen sulfide production is increased in tumor tissue.
Functional microbiomics: Evaluation of gut microbiota-bile acid metabolism interactions in health and disease Methods (IF 3.998) Pub Date : 2018-04-26 Benjamin H. Mullish, Alexandros Pechlivanis, Grace F. Barker, Mark R. Thursz, Julian R. Marchesi, Julie A.K. McDonald
There is an ever-increasing recognition that bile acids are not purely simple surfactant molecules that aid in lipid digestion, but are a family of molecules contributing to a diverse range of key systemic functions in the host. It is now also understood that the specific composition of the bile acid milieu within the host is related to the expression and activity of bacterially-derived enzymes within the gastrointestinal tract, as such creating a direct link between the physiology of the host and the gut microbiota. Coupled to the knowledge that perturbation of the structure and/or function of the gut microbiota may contribute to the pathogenesis of a range of diseases, there is a high level of interest in the potential for manipulation of the gut microbiota-host bile acid axis as a novel approach to therapeutics. Much of the growing understanding of the biology of this area reflects the recent development and refinement of a range of novel techniques; this study applies a number of those techniques to the analysis of human samples, aiming to illustrate their strengths, drawbacks and biological significance at all stages. Specifically, we used microbial profiling (using 16S rRNA gene sequencing), bile acid profiling (using liquid chromatography–mass spectrometry), bsh and baiCD qPCR, and a BSH enzyme activity assay to demonstrate differences in the gut microbiota and bile metabolism in stool samples from healthy and antibiotic-exposed individuals.
Basic experiments in 2H static NMR for the characterization of protein side-chain dynamics Methods (IF 3.998) Pub Date : 2018-04-27 Liliya Vugmeyster, Dmitry Ostrovsky
The focus of this review is the basic methodology for applications of static deuteron NMR for studies of dynamics in the side chains of proteins. We review experimental approaches for the measurements of static line shapes and relaxation rates as well as signal enhancement strategies using the multiple echo acquisition scheme. Further, we describe computational strategies for modeling jump and diffusive motions underlying experimental data. Applications are chosen from studies of amyloid fibrils comprising the amyloid-β protein.
Variable selection in heterogeneous datasets: A truncated-rank sparse linear mixed model with applications to genome-wide association studies Methods (IF 3.998) Pub Date : 2018-04-27 Haohan Wang, Bryon Aragam, Eric P. Xing
A fundamental and important challenge in modern datasets of ever increasing dimensionality is variable selection, which has taken on renewed interest recently due to the growth of biological and medical datasets with complex, non-i.i.d. structures. Naïvely applying classical variable selection methods such as the Lasso to such datasets may lead to a large number of false discoveries. Motivated by genome-wide association studies in genetics, we study the problem of variable selection for datasets arising from multiple subpopulations, when this underlying population structure is unknown to the researcher. We propose a unified framework for sparse variable selection that adaptively corrects for population structure via a low-rank linear mixed model. Most importantly, the proposed method does not require prior knowledge of sample structure in the data and adaptively selects a covariance structure of the correct complexity. Through extensive experiments, we illustrate the effectiveness of this framework over existing methods. Further, we test our method on three different genomic datasets from plants, mice, and human, and discuss the knowledge we discover with our method.
Selective recovery of RNAs from bacterial pathogens after their internalization by human host cells Methods (IF 3.998) Pub Date : 2018-04-27 Simon Raynaud, Hélène Le Pabic, Brice Felden
Selective RNA extractions are required when studying bacterial gene expression within complex mixtures of pathogens and human cells, during adhesion, internalization and survival within the host. New technologies should be developed and implemented to enrich the amount of bacterial RNAs since the majority of RNAs are from the eukaryotic host cells, requiring high read depth coverage to capture the bacterial transcriptomes in dual-RNAseq studies. This will improve our understanding about bacterial adaptation to the host cell defenses, and about how they will adapt to an intracellular life. Here we present an RNA extraction protocol to selectively enrich the lowest bacterial RNA fraction from a mixture of human and bacterial cells, using zirconium beads, with minimal RNA degradation. Zirconium beads have higher capacity to extract bacterial RNAs than glass beads after pathogen internalization. We optimized the beads size and composition for an optimal bacterial lysis and RNA extraction. The protocol was validated on two human cell lines, differentiated macrophages and osteoblasts, with either Gram-positive (Staphylococcus aureus) or -negative (Salmonella typhimurium) bacteria. Relative to other published protocols, yield of total RNA recovery was significantly improved, while host cell infection was performed with a lower bacterial inoculum. Within the host, bacterial RNA recovery yields were about six-fold lower than an RNA extraction from pure bacteria, but the quality of the RNA recovered was essentially similar. Bacterial RNA recovery was more efficient for S. aureus than for S. typhimurium, probably due to their higher protection by the Gram-positive cell walls during the early step of eukaryotic cell lysis. These purified bacterial RNAs allow subsequent genes expression studies in the course of host cell-bacteria interactions.
High-dimensional NMR methods for intrinsically disordered proteins studies Methods (IF 3.998) Pub Date : 2018-04-27 Katarzyna Grudziąż, Anna Zawadzka-Kazimierczuk, Wiktor Koźmiński
Intrinsically disordered proteins (IDPs) are getting more and more interest of the scientific community. Nuclear magnetic resonance (NMR) is often a technique of choice for these studies, as it provides atomic-resolution information on structure, dynamics and interactions of IDPs. Nonetheless, NMR spectra of IDPs are typically extraordinary crowded, comparing to those of structured proteins. To overcome this problem, high-dimensional NMR experiments can be used, which allow for a better peak separation. In the present review different aspects of such experiments are discussed, from data acquisition and processing to analysis, focusing on experiments for resonance assignment.
Assessment of microbiota:host interactions at the vaginal mucosa interface Methods (IF 3.998) Pub Date : 2018-04-27 Pamela Pruski, Holly V. Lewis, Yun S. Lee, Julian R. Marchesi, Phillip R. Bennett, Zoltan Takats, David A. MacIntyre
There is increasing appreciation of the role that vaginal microbiota play in health and disease throughout a woman’s lifespan. This has been driven partly by molecular techniques that enable detailed identification and characterisation of microbial community structures. However, these methods do not enable assessment of the biochemical and immunological interactions between host and vaginal microbiota involved in pathophysiology. This review examines our current knowledge of the relationships that exist between vaginal microbiota and the host at the level of the vaginal mucosal interface. We also consider methodological approaches to microbiomic, immunologic and metabolic profiling that permit assessment of these interactions. Integration of information derived from these platforms brings the potential for biomarker discovery, disease risk stratification and improved understanding of the mechanisms regulating vaginal microbial community dynamics in health and disease.
Multiplex confounding factor correction for genomic association mapping with squared sparse linear mixed model Methods (IF 3.998) Pub Date : 2018-04-27 Haohan Wang, Xiang Liu, Yunpeng Xiao, Ming Xu, Eric P. Xing
Genome-wide Association Study has presented a promising way to understand the association between human genomes and complex traits. Many simple polymorphic loci have been shown to explain a significant fraction of phenotypic variability. However, challenges remain in the non-triviality of explaining complex traits associated with multifactorial genetic loci, especially considering the confounding factors caused by population structure, family structure, and cryptic relatedness. In this paper, we propose a Squared-LMM (LMM2) model, aiming to jointly correct population and genetic confounding factors. We offer two strategies of utilizing LMM2 for association mapping: 1) It serves as an extension of univariate LMM, which could effectively correct population structure, but consider each SNP in isolation. 2) It is integrated with the multivariate regression model to discover association relationship between complex traits and multifactorial genetic loci. We refer to this second model as sparse Squared-LMM (sLMM2). Further, we extend LMM2/sLMM2 by raising the power of our squared model to the LMMn/sLMMn model. We demonstrate the practical use of our model with synthetic phenotypic variants generated from genetic loci of Arabidopsis Thaliana. The experiment shows that our method achieves a more accurate and significant prediction on the association relationship between traits and loci. We also evaluate our models on collected phenotypes and genotypes with the number of candidate genes that the models could discover. The results suggest the potential and promising usage of our method in genome-wide association studies.
Gut metabolome meets microbiome: A methodological perspective to understand the relationship between host and microbe Methods (IF 3.998) Pub Date : 2018-04-30 Santosh Lamichhane, Partho Sen, Alex M. Dickens, Matej Orešič, Hanne Christine Bertram
It is well established that gut microbes and their metabolic products regulate host metabolism. The interactions between the host and its gut microbiota are highly dynamic and complex. In this review we present and discuss the metabolomic strategies to study the gut microbial ecosystem. We highlight the metabolic profiling approaches to study faecal samples aimed at deciphering the metabolic product derived from gut microbiota. We also discuss how metabolomics data can be integrated with metagenomics data derived from gut microbiota and how such approaches may lead to better understanding of the microbial functions. Finally, the emerging approaches of genome-scale metabolic modelling to study microbial co-metabolism and host–microbe interactions are highlighted.
Domain Intelligible Models Methods (IF 3.998) Pub Date : 2018-07-05 Sultan Imangaliyev, Andrei Prodan, Max Nieuwdorp, Albert K. Groen, Natal A.W. van Riel, Evgeni Levin
Mining biological information from rich ”-omics” datasets is facilitated by organizing features into groups that are related to a biological phenomenon or clinical outcome. For example, microorganisms can be grouped based on a phylogenetic tree that depicts their similarities regarding genetic or physical characteristics. Here, we describe algorithms that incorporate auxiliary information in terms of groups of predictors and the relationships between them into the metagenome learning task to build intelligible models. In particular, our cost function guides the feature selection process using auxiliary information by requiring related groups of predictors to provide similar contributions to the final response. We apply the developed algorithms to a recently published dataset analyzing the effects of fecal microbiota transplantation (FMT) in order to identify factors that are associated with improved peripheral insulin sensitivity, leading to accurate predictions of the response to the FMT.
Guidelines and best practices in successfully using zebrabow for lineage tracing multiple cells within tissues Methods (IF 3.998) Pub Date : 2018-07-03 Phong D. Nguyen, Peter D. Currie
Labelling cells and following their progeny, also known as lineage tracing has provided important insights on the origins of tissues and how they behave in their environment. Traditional lineage tracing experiments have been limited to following single or small groups of cells with classic techniques such as dye injections and Cre/LoxP labelling of cells of interest. In order to allow a broader visualization and analysis of multiple cells within a tissue, the recent combination of fluorophores, genetic manipulation has developed Brainbow, a fluorescent dependent method of permanently labelling multiple cells and following their progeny. This technique has now been adapted to zebrafish (Zebrabow) and takes advantages in imaging capabilities this animal model system provides over other animal models. In this paper we shall describe how Zebrabow is performed as well as some guides on some of the common pitfalls on this labelling strategy.
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