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  • Analysis of post-transcriptional RNA metabolism in prokaryotes
    Methods (IF 3.998) Pub Date : 2018-11-15
    Bijoy K. Mohanty, Sidney R. Kushner

    Post-transcriptional RNA metabolic pathways play important roles in permitting prokaryotes to operate under a variety of environmental conditions. Although significant progress has been made during the last decade in deciphering RNA processing pathways in a number of bacteria, a complete understanding of post-transcriptional RNA metabolism in any single microorganism is far from reality. Here we describe multiple experimental approaches that can be used to study mRNA stability, tRNA and rRNA processing, sRNA metabolism, and polyadenylation in prokaryotes. The methods described here can be readily utilized in both Gram-negative and Gram-positive bacteria with simple modifications.

    更新日期:2018-11-16
  • Round Optimization for Improved Discovery of Native Bispecific Antibodies
    Methods (IF 3.998) Pub Date : 2018-11-16
    F. Gueneau, U. Ravn, N. Fischer

    The assembly of bispecific antibodies (bsAb) that retain the structure of a standard IgG can be challenging as the correct pairing of the different heavy and light chains has to be ensured while unwanted side products kept to a minimum. The use of antibodies sharing a common chain facilitates assembly of such bsAb formats but requires additional efforts during the initial discovery phase. We have developed a native bsAb format called κλ body based on antibodies that, while being specific for different antigens, share the same heavy chain. Such antibodies can readily be isolated from antibody libraries incorporating a single VH combined with light chain diversity. However, in order to improve the discovery process of such fixed VH antibodies, we developed a method to optimize populations of light chains by recovering and shuffling CDRL3 sequences that have been enriched for antigen binding by phage display selection. This approach allowed for the isolation of a more diverse and potent panel of antibodies blocking the interaction between PD-1 and PD-L1 when compared to our standard in vitro selection approach, thus providing better building blocks for subsequent bsAb generation.

    更新日期:2018-11-16
  • Engineering therapeutic bispecific antibodies using CrossMab technology
    Methods (IF 3.998) Pub Date : 2018-11-16
    Christian Klein, Wolfgang Schaefer, Joerg T Regula, Charles Dumontet, Ulrich Brinkmann, Marina Bacac, Pablo Umaña

    Bispecific antibodies have recently gained major interest as they allow novel mechanisms-of-action and/or therapeutic applications that cannot be achieved using conventional IgG-based antibodies. A major issue in engineering IgG-based bispecific antibodies has been to enable the correct association of heavy and light chains resulting in correct assembly of the desired bispecific antibody in sufficient yield. Various approaches have been described during recent years to tackle this challenge. We have developed the so-called CrossMab technology that enforces correct light chain association based on the domain crossover of immunoglobulin domains in the Fab region of the bispecific antibody. This versatile technology allows the generation of different bispecific antibody formats including asymmetric heterodimeric monovalent 1+1 bispecific antibodies and asymmetric heterodimeric bispecific antibodies with 2+1 valency in combination with approaches enabling Fc-hetermodimerization like knob-into-hole technology as well as the generation of tetravalent symmetric bispecific antibodies with 2+2 valency, also known as Tandem-Fab based IgG antibodies, using processes suitable for the large scale production of therapeutic bispecific antibodies. Notably, as of now, at least eight different bispecific antibodies using CrossMab technology entered clinical development, and additional CrossMabs are in late preclinical development. This review provides a summary of the status and progress with the engineering and generation of CrossMab technology based bispecific antibodies as well as their therapeutic application.

    更新日期:2018-11-16
  • Optimization of DamID for use in primary cultures of mouse hepatocytes
    Methods (IF 3.998) Pub Date : 2018-11-13
    Leonardo Gatticchi, Jose I. de las Heras, Rita Roberti, Eric C. Schirmer

    DamID, a method to identify DNA associating with a particular protein, was originally developed for use in immortalized tissue culture lines. The power of this technique has led to its adaptation for a number of additional systems. Here we report adaptations for its use in primary cells isolated from rodents with emphasis on the challenges this presents. Specifically, we present several modifications that allow the method to be performed in mouse acutely isolated primary hepatocytes while seemingly maintaining tissue genome architecture. We also describe the downstream bioinformatic analysis necessary to identify LADs and discuss some of the parameters and their effects with regards to the sensitivity of the method.

    更新日期:2018-11-13
  • Analysis of RNA 5′ ends: phosphate enumeration and cap characterization
    Methods (IF 3.998) Pub Date : 2018-11-10
    Daniel J. Luciano, Joel G. Belasco

    The function and fate of cellular RNAs are often governed by the phosphorylation state at the 5′ end or the identity of whatever cap may be present there. Here we described methods for examining these important 5′-terminal features on any cellular or synthetic RNA of interest that can be detected by Northern blotting. One such method, PABLO, is a splinted ligation assay that makes it possible to accurately quantify the percentage of 5′ ends that are monophosphorylated. Another, PACO, is a capping assay that reveals the percentage of 5′ ends that are diphosphorylated. A third, boronate gel electrophoresis in conjunction with deoxyribozyme-mediated cleavage, enables different types of caps (e.g., m7Gppp caps versus NAD caps) to be distinguished from one another and the percentage of each to be determined. After completing all three tests, the percentage of 5′ ends that are triphosphorylated can be deduced by process of elimination. Together, this battery of assays allows the 5′ terminus of an RNA to be profiled in unprecedented detail.

    更新日期:2018-11-10
  • HiPLA: High-throughput imaging Proximity Ligation Assay
    Methods (IF 3.998) Pub Date : 2018-11-10
    Leonid A. Serebryannyy, Tom Misteli

    Protein-protein interactions are essential for cellular structure and function. To delineate how the intricate assembly of protein interactions contribute to cellular processes in health and disease, new methodologies that are both highly sensitive and can be applied at large scale are needed. Here, we develop HiPLA (high-throughput imaging proximity ligation assay), a method that employs the antibody-based proximity ligation assay in a high-throughput imaging screening format as a novel means to systematically visualize changes to protein interactomes. Using HiPLA with a library of antibodies, we probe the interaction of 60 proteins and associated post-translational modifications (PTMs) with the nuclear lamina in a model of the premature aging disorder Hutchinson-Gilford progeria syndrome (HGPS). We identify a subset of proteins that differentially interact with the nuclear lamina in HGPS. In combination with quantitative indirect immunofluorescence, we find that the majority of differential interactions were accompanied by corresponding changes in expression of the interacting protein. Taken together, HiPLA offers a novel approach to probe cellular protein-protein interaction at a large scale and reveals mechanistic insights into the assembly of protein complexes.

    更新日期:2018-11-10
  • Towards Improving Proximity Labeling by the Biotin Ligase BirA
    Methods (IF 3.998) Pub Date : 2018-11-10
    Luke T. Oostdyk, Leonard Shank, Kasey Jividen, Natalia Dworak, Nicholas E. Sherman, Bryce M. Paschal
    更新日期:2018-11-10
  • Fluorescence-based Quantification of Nucleocytoplasmic Transport
    Methods (IF 3.998) Pub Date : 2018-11-10
    Joshua B. Kelley, Bryce M. Paschal

    The sequestration of DNA within the membrane-bound nucleus is a defining characteristic of eukaryotic cells. Replication and transcription are therefore restricted to the nucleus, however, the regulation of these events relies on cytoplasmic processes including protein synthesis and signal transduction pathways. Because a variety of cellular activities depend on nuclear transport, researchers from diverse fields have found it useful to examine the nuclear localization of proteins of interest. Here we present some important technical considerations for studying nuclear and cytoplasmic localization, and provide guidance for quantifying protein levels using fluorescence microscopy and ImageJ software. We include discussion of the use of regions of interest and image segmentation for quantification of protein localization. Nucleocytoplasmic transport is fundamentally important for controlling protein levels and activity in the nucleus or cytoplasm, and quantitative analysis can provide insight into how biological output is achieved.

    更新日期:2018-11-10
  • Determining degradation intermediates and the pathway of 3’ to 5’ degradation of histone mRNA using high-throughput sequencing
    Methods (IF 3.998) Pub Date : 2018-11-05
    Christopher E. Holmquist, William F. Marzluff

    The half-life of an mRNA is an important parameter contributing to the steady-state level of the mRNA. Rapid changes in mRNA levels can result from decreasing the half-life of an mRNA. Establishing the detailed pathway of mRNA degradation for a particular class of mRNAs requires the ability to isolate mRNA degradation intermediates. High-throughput sequencing provides a method for detecting these intermediates. Here we describe a method for determining the intermediates in 3’ to 5’ degradation. Characterizing these intermediates requires not only determining the precise 3’ end of the molecule to a single nucleotide resolution, but also the ability to detect and characterize any untemplated nucleotides present on the intermediates. We achieve this by ligating a known sequence to all the 3’ termini in the cell, and then sequence the 3’ termini and the ligated linker to identify any alterations to the genomic reference sequence. We have applied this method to characterize the intermediates in histone mRNA metabolism, allowing us to deduce the pathway of 3’ to 5’ degradation. This method can potentially be applied to any RNA, and we discuss possible strategies for extending the method to include simultaneous determination of the 3’ and 5’ end of the same RNA molecule.

    更新日期:2018-11-05
  • RPAD (RNase R Treatment, Polyadenylation, and Poly(A)+ RNA Depletion) Method to Isolate Highly Pure Circular RNA
    Methods (IF 3.998) Pub Date : 2018-11-02
    Poonam R. Pandey, Pranita K. Rout, Aniruddha Das, Myriam Gorospe, Amaresh C. Panda

    Recent developments in high-throughput RNA sequencing methods coupled with innovative bioinformatic tools have uncovered thousands of circular (circ)RNAs. CircRNAs have emerged as a vast and novel class of regulatory RNAs with potential to modulate gene expression by acting as sponges for microRNAs (miRNAs) and RNA-binding proteins (RBPs). The biochemical enrichment of circRNAs by exoribonuclease treatment or by depletion of polyadenylated RNAs coupled with deep-sequencing is widely used for the systematic identification of circRNAs. Although these methods enrich circRNAs substantially, they do not eliminate efficiently non-polyadenylated and highly-structured RNAs. Here, we describe a method we termed RPAD, based on initial RNase R treatment followed by Polyadenylation and poly(A)+ RNA Depletion. These joint interventions drastically depleted linear RNAs leading to isolation of highly pure circRNAs from total RNA pools. By facilitating the isolation of highly pure circRNAs, RPAD enables the elucidation of circRNA biogenesis, sequence, and function.

    更新日期:2018-11-05
  • Chemical Probing for Examining the Structure of Modified RNAs and Ligand Binding to RNA
    Methods (IF 3.998) Pub Date : 2018-11-02
    Prabuddha Waduge, Yogo Sakakibara, Christine S. Chow
    更新日期:2018-11-05
  • Surpassing limits of static RNA modification analysis with dynamic NAIL-MS
    Methods (IF 3.998) Pub Date : 2018-11-03
    Valentin F. Reichle, Steffen Kaiser, Matthias Heiss, Felix Hagelskamp, Kayla Borland, Stefanie Kellner
    更新日期:2018-11-05
  • Unraveling 3’-end RNA Uridylation at Nucleotide Resolution
    Methods (IF 3.998) Pub Date : 2018-11-03
    Mehdi Pirouz, Aref G. Ebrahimi, Richard I. Gregory

    Post-transcriptional modification of RNA, the so-called ‘Epitranscriptome’, can regulate RNA structure, stability, localization, and function. Numerous modifications have been identified in virtually all classes of RNAs, including messenger RNAs (mRNAs), transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), microRNAs (miRNAs), and other noncoding RNAs (ncRNAs). These modifications may occur internally (by base or sugar modifications) and include RNA methylation at different nucleotide positions, or by the addition of various nucleotides at the 3’-end of certain transcripts by a family of terminal nucleotidylyl transferases. Developing methods to specifically and accurately detect and map these modifications is essential for understanding the molecular function(s) of individual RNA modifications and also for identifying and characterizing the proteins that may read, write, or erase them. Here, we focus on the characterization of RNA species targeted by 3’ terminal uridylyl transferases (TUTases) (TUT4/7, also known as Zcchc11/6) and a 3’-5’ exoribonuclease, Dis3l2, in the recently identified Dis3l2-mediated decay (DMD) pathway - a dedicated quality control pathway for a subset of ncRNAs. We describe the detailed methods used to precisely identify 3’-end modifications at nucleotide level resolution with a particular focus on the U1 and U2 small nuclear RNA (snRNA) components of the Spliceosome. These tools can be applied to investigate any RNA of interest and should facilitate studies aimed at elucidating the functional relevance of 3’-end modifications.

    更新日期:2018-11-05
  • Bispecific T-cell engagers: towards understanding variables influencing the in vitro potency and tumor selectivity and their modulation to enhance their efficacy and safety
    Methods (IF 3.998) Pub Date : 2018-11-03
    Diego Ellerman

    Bispecific molecules redirecting the cytotoxicity of T-cells are a growing class of therapeutics with numerous molecules being tested in clinical trials. However, it has been a long way since the proof of concept studies in the mid 1980’s. In the process we have learnt about the impact of different variables related to the bispecific molecule and the target antigen on the potency of this type of drugs. This work reviews the insights gained and how that knowledge has been used to design more potent bispecific T-cell engagers. The more recent advancement of antibodies with this modality into safety studies in non-human primates and as well as in clinical studies has revealed potential toxicity liabilities for the mode of action. Modifications in existing antibody formats and new experimental molecules designed to mitigate these problems are discussed.

    更新日期:2018-11-05
  • Quantitation of low abundant soluble biomarkers using high sensitivity Single Molecule Counting technology
    Methods (IF 3.998) Pub Date : 2018-10-28
    Joseph Hwang, Munmun Banerjee, Adam S. Venable, Zara Walden, John Jolly, Cathleen Zimmerman, Elizabeth Adkisson, Qiang Xiao

    Quantitation of biomarkers in biofluids plays a central role in basic research to management of patient care and is routinely used in clinical laboratories and academic institutions. Standard immunoassays, such as an enzyme-linked immunosorbent assay (ELISA), have provided understanding of both normal and pathological processes for many decades. However, in more recent decades, new immunoassay technologies have uncovered numerous analytes in blood that were once undetectable using traditional ELISAs. To meet this new challenge for quantifying low abundant proteins in biofluids, Single Molecule Counting (SMCTM) technology was developed. This new technology is a combination of improvements to both the immunoassay procedure as well as the instrument. The aim of this article is to introduce the new SMCxPROTM instrument, xPRO Acquisition and Analysis software, and the high sensitivity immunoassay kits validated on this instrument for the detection of low abundant proteins in biofluids, such as serum and plasma. Using this new technology platform, biomarkers that were once unquantifiable can now be quantitated in both normal and diseased biofluids.

    更新日期:2018-10-30
  • Determination of enrichment factors for modified RNA in MeRIP experiments
    Methods (IF 3.998) Pub Date : 2018-10-28
    Kaouthar Slama, Aurellia Galliot, Franziska Weichmann, Jasmin Hertler, Regina Feederle, Gunter Meister, Mark Helm

    In the growing field of RNA modification, precipitation techniques using antibodies play an important role. However, little is known about their specificities and protocols are missing to assess their effectiveness. Here we present a method to assess enrichment factors after MeRIP-type pulldown experiments, here exemplified with a commercial antibody against N6-methyladenosine (m6A). Testing different pulldown and elution conditions, we measure enrichment factors of 4-5 using m6A-containing mRNAs against an unmodified control of identical sequence. Both types of mRNA carry 32P labels at different nucleotides, allowing their relative quantification in a mixture after digestion to nucleotides, separation by TLC and quantitative phosphorimaging of the labels.

    更新日期:2018-10-30
  • RNA modifications in structure prediction - status quo and future challenges
    Methods (IF 3.998) Pub Date : 2018-10-30
    Andrea Tanzer, Ivo L. Hofacker, Ronny Lorenz

    Chemical modifications of RNA nucleotides change their identity and characteristics and thus alter genetic and structural information encoded in the genomic DNA. tRNA and rRNA are probably the most heavily modified genes, and often depend on derivatization or isomerization of their nucleobases in order to correctly fold into their functional structures. Recent RNomics studies, however, report transcriptome wide RNA modification and suggest a more general regulation of structuredness of RNAs by this so called epitranscriptome. Modification seems to require specific substrate structures, which in turn are stabilized or destabilized and thus promote or inhibit refolding events of regulatory RNA structures. In this review, we revisit RNA modifications and the related structures from a computational point of view. We discuss known substrate structures, their properties such as sub-motifs as well as consequences of modifications on base pairing patterns and possible refolding events. Given that efficient RNA structure prediction methods for canonical base pairs have been established several decades ago, we review to what extend these methods allow the inclusion of modified nucleotides to model and study epitranscriptomic effects on RNA structures.

    更新日期:2018-10-30
  • Nucleoside Analogs in the Study of the Epitranscriptome
    Methods (IF 3.998) Pub Date : 2018-10-26
    Cody M. Palumbo, Peter A. Beal

    Over 150 unique RNA modifications are now known including several nonstandard nucleotides present in the body of messenger RNAs. These modifications can alter a transcript’s function and are collectively referred to as the epitrancriptome. Chemically modified nucleoside analogs are poised to play an important role in the study of these epitranscriptomic marks. Introduced chemical features on nucleic acid strands provide unique structures or reactivity that can be used for downstream detection or quantification. Three methods are commonly used in the field to synthesize oligonucleotides containing chemically modified nucleoside analogs. Nucleoside analogs can be introduced via phosphoramidite-based chemical synthesis, via polymerases with modified nucleotide triphosphates or by metabolic labeling. In this review, these methods for incorporation of nucleoside analogs will be discussed with specific recently published examples pertaining to the study of the epitranscriptome.

    更新日期:2018-10-27
  • Using Mouse Models to Unlock the Secrets of Non-Synonymous RNA Editing
    Methods (IF 3.998) Pub Date : 2018-10-26
    Hussain Jinnah, Randi J. Ulbricht

    The deamination of adenosine to inosine by RNA editing is a widespread post-transcriptional process that expands genetic diversity. Selective substitution of inosine for adenosine in pre-mRNA transcripts can alter splicing, mRNA stability, and the amino acid sequence of the encoded protein. The functional consequences of RNA editing-dependent amino acid substitution are known for only a handful of RNA editing substrates. Many of these studies began in heterologous mammalian expression systems; however, the gold-standard for determining the functional significance of transcript-specific re-coding A-to-I editing events is the generation of a mouse model that expresses only one RNA editing-dependent isoform. The frequency of site-specific RNA editing varies spatially, temporally, and in some diseases, therefore, determining the profile of RNA editing frequency is also an important element of research. Here we review the strengths and weaknesses of existing mouse models for the study of RNA editing, as well as methods for quantifying RNA editing frequencies in vivo. Importantly, we highlight opportunities for future RNA editing studies in mice, projecting that improvements in genome editing and high-throughput sequencing technologies will allow the field to excel in coming years.

    更新日期:2018-10-27
  • Improved Application of RNAModMapper – an RNA Modification Mapping Software Tool – for Analysis of Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) Data
    Methods (IF 3.998) Pub Date : 2018-10-24
    Peter A. Lobue, Ningxi Yu, Manasses Jora, Scott Abernathy, Patrick A. Limbach

    Research into post-transcriptional processing and modification of RNA continues to speed forward, as their ever-emerging role in the regulation of gene expression in biological systems continues to unravel. Liquid chromatography tandem mass spectrometry (LC-MS/MS) has proven for over two decades to be a powerful ally in the elucidation of RNA modification identity and location, but the technique has not proceeded without its own unique technical challenges. The throughput of LC-MS/MS modification mapping experiments continues to be impeded by tedious and time-consuming spectral interpretation, particularly during for the analysis of complex RNA samples. RNAModMapper was recently developed as a tool to improve the interpretation and annotation of LC-MS/MS data sets from samples containing post-transcriptionally modified RNAs. Here, we delve deeper into the methodology and practice of RNAModMapper to provide greater insight into its utility, and remaining hurdles, in current RNA modification mapping experiments.

    更新日期:2018-10-24
  • BisAMP: A web-based pipeline for targeted RNA cytosine-5 methylation analysis
    Methods (IF 3.998) Pub Date : 2018-10-24
    Felix Bormann, Francesca Tuorto, Cansu Cirzi, Frank Lyko, Carine Legrand

    RNA cytosine-5 methylation (m5C) has emerged as a key epitranscriptomic mark, which fulfills multiple roles in structural modulation, stress signaling and the regulation of protein translation. Bisulfite sequencing is currently the most accurate and reliable method to detect m5C marks at nucleotide resolution. Targeted bisulfite sequencing allows m5C detection at single base resolution, by combining the use of tailored primers with bisulfite treatment. A number of computational tools currently exist to analyse m5C marks in DNA bisulfite sequencing. However, these methods are not directly applicable to the analysis of RNA m5C marks, because DNA analysis focuses on CpG methylation, and because artifactual unconversion and misamplification in RNA can obscure actual methylation signals. We describe a pipeline designed specifically for RNA cytosine-5 methylation analysis in targeted bisulfite sequencing experiments. The pipeline is directly applicable to Illumina MiSeq (or equivalent) sequencing datasets using a web interface (https://bisamp.dkfz.de), and is defined by optimized mapping parameters and the application of tailored filters for the removal of artifacts. We provide examples for the application of this pipeline in the unambiguous detection of m5C marks in tRNAs from mouse embryonic stem cells and neuron-differentiated stem cells as well as in 28S rRNA from human fibroblasts. Finally, we also discuss the adaptability of BisAMP to the analysis of DNA methylation. Our pipeline provides an accurate, fast and user-friendly framework for the analysis of cytosine-5 methylation in amplicons from bisulfite-treated RNA.

    更新日期:2018-10-24
  • A robust heterodimeric Fc platform engineered for efficient development of bispecific antibodies of multiple formats
    Methods (IF 3.998) Pub Date : 2018-10-23
    Gregory L. Moore, Matthew J. Bernett, Rumana Rashid, Erik W. Pong, Duc-Hanh T. Nguyen, Jonathan Jacinto, Araz Eivazi, Alex Nisthal, Juan E. Diaz, Seung Y. Chu, Umesh S. Muchhal, John R. Desjarlais

    Bispecific monoclonal antibodies can bind two protein targets simultaneously and enable therapeutic modalities inaccessible by traditional mAbs. Bispecific formats containing a heterodimeric Fc region are of particular interest, as a heterodimeric Fc empowers both bispecificity and altered valencies while retaining the developability and druggability of a monoclonal antibody. We present a robust heterodimeric Fc platform, called the XmAb® bispecific platform, engineered for efficient development of bispecific antibodies and Fc fusions of multiple formats. First, we engineer a purification solution for proteins containing a heterodimeric Fc using engineered isoelectric point differences in the Fc region that enable straightforward purification of the heterodimeric species. Then, we combine this purification solution with a novel set of Fc substitutions capable of achieving heterodimer yields over 95% with little change in thermostability. Next, we illustrate the flexibility of our heterodimeric Fc with a case study in which a wide range of tumor-associated antigen x CD3 bispecifics are generated, differing in choice of tumor antigen, affinities for both tumor antigen and CD3, and tumor antigen valency. Finally, we present manufacturing data reinforcing the robustness of the heterodimeric Fc platform at scale.

    更新日期:2018-10-23
  • Misincorporation signatures for detecting modifications in mRNA: Not as simple as it sounds
    Methods (IF 3.998) Pub Date : 2018-10-23
    Aldema Sas-Chen, Schraga Schwartz

    Post-transcriptional modification on mRNA has become a field of intense interest in recent years, and next-generation sequencing based technologies are constantly emerging to detect an increasing number of modifications at a transcriptome-wide level. Some of these approaches are based on identification of misincorporation events induced by reverse transcriptase at modified sites. Although conceptually trivial, sensitive and specific identification of such events is a challenge prone to a surprising number of artifacts, which can lead to substantially inflated estimates of the abundance of diverse modifications. Here we discuss the sources of some of these artifacts and delineate approaches to overcome them.

    更新日期:2018-10-23
  • A versatile image analysis platform for three-dimensional nuclear reconstruction
    Methods (IF 3.998) Pub Date : 2018-10-22
    Jessica F. Johnston, Simon G.J. Mochrie, Megan C. King

    Nuclear morphology is indicative of cellular health in many contexts. In order to robustly and quantitatively measure nuclear size and shape, numerous experimental methods leveraging fluorescence microscopy have been developed. While these methods are useful for quantifying two-dimensional morphology, they often fail to accurately represent the three-dimensional structure of the nucleus, thus omitting important spatial and volumetric information. To address the need for a more accurate image analysis modality, we have developed a software platform that faithfully reconstructs membrane surfaces in three dimensions with sub-pixel resolution. Here, we showcase its broad applicability across species and nuclear scale, as well as provide information on how to employ this platform for diverse experimental systems.

    更新日期:2018-10-22
  • Bispecific antibodies: potential immunotherapies for HIV treatment
    Methods (IF 3.998) Pub Date : 2018-10-22
    Giulia Fabozzi, Amarendra Pegu, Richard A. Koup, Constantinos Petrovas

    Bispecific (bs) antibodies (Abs, bsAbs) are engineered immunoglobulins that contain two different antigen-binding sites in one molecule. bsAbs can be divided in two molecular formats; the IgG-like and non-IgG like. The structural elements of each format have implications for engaging the immune system. Elimination of HIV will need sophisticated approaches with immunotherapies being one of the strategies under investigation. Furthermore, HIV genetic variability and functional compromise of the adaptive CTL response complicate the potential usefulness of some immunotherapeutic strategies. Inclusion of novel HIV neutralizing Abs with high potency and breadth as components of bsAbs could represent alternative strategies for virus elimination by harnessing the adaptive immune response in vivo.

    更新日期:2018-10-22
  • Bead-based RNA multiplex panels for biomarker detection in oncology samples
    Methods (IF 3.998) Pub Date : 2018-10-22
    Jeanesse Scerri, Shawn Baldacchino, Christian Saliba, Christian Scerri, Godfrey Grech

    Patient stratification, prognosis and disease monitoring are three important aspects of personalized cancer medicine. With traditional serum tumour protein biomarkers showing lack of specificity and sensitivity, and tumour heterogeneity affecting the response to targeted therapy based on tissue biomarkers, the focus shifted to the use of molecular tumour signatures as specific biomarkers. Multiplex microsphere-based panels are robust and cost-effective, high throughput molecular assays, which can accurately characterize tumours even from small amounts of poor quality nucleic acids. Only few studies have reported the use of microspheres (beads) to quantify RNA expression of targets of interest simultaneously (multiplexing). This review is an overview of the various applications of bead-based RNA panels in molecular oncology, with focus on the Invitrogen™ QuantiGene™ Plex Assay (Thermo Fisher Scientific), and provides a comparison with PCR-based and other methodologies. The advantages of multiplex bead assays are exemplified by the quantification of RNA expression in formalin-fixed, paraffin embedded (FFPE) archival tissue and the simultaneous detection of biomarkers in low input samples, including quantification of markers in microdissected tissue material, to characterise heterogeneous tumour sites within a sample, and detection of markers in low numbers of circulating tumour cells.

    更新日期:2018-10-22
  • Engineering a bispecific antibody with a common light chain: Identification and optimization of an anti-CD3 epsilon and anti-GPC3 bispecific antibody, ERY974
    Methods (IF 3.998) Pub Date : 2018-10-13
    Hirotake Shiraiwa, Atsushi Narita, Mika Kamata-Sakurai, Takahiro Ishiguro, Yuji Sano, Naoka Hironiwa, Takashi Tsushima, Hiroaki Segawa, Toshiaki Tsunenari, Yosuke Ikeda, Yoko Kayukawa, Mizuho Noguchi, Tetsuya Wakabayashi, Akihisa Sakamoto, Hiroko Konishi, Taichi Kuramochi, Mika Endo, Kunihiro Hattori, Tomoyuki Igawa

    The antibody drug market is rapidly expanding, and various antibody engineering technologies are being developed to create antibodies that can provide better benefit to patients. Although bispecific antibody drugs have been researched for more than 30 years, currently only a limited number of bispecific antibodies have achieved regulatory approval. Of the few successful examples of industrially manufacturing a bispecific antibody, the “common light chain format” is an elegant technology that simplifies the purification of a whole IgG–type bispecific antibody. Using this IgG format, the bispecific function can be introduced while maintaining the natural molecular shape of the antibody. In this article, we will first introduce the outline, prospects, and limitations of the common light chain format. Then, we will describe the identification and optimization process for ERY974, an anti-glypican-3 × anti-CD3ε T cell–redirecting bispecific antibody with a common light chain. This format includes one of Chugai’s proprietary technologies, termed ART-Ig technology, which consists of a method to identify a common light chain, isoelectric point (pI) engineering to purify the desired bispecific IgG antibody from byproducts, and Fc heterodimerization by an electrostatic steering effect. Furthermore, we describe some tips for de-risking the antibody when engineering a T cell redirecting antibody.

    更新日期:2018-10-14
  • META RNA profiling: Multiplexed quantitation of targeted RNAs across large numbers of samples
    Methods (IF 3.998) Pub Date : 2018-10-09
    Azeet Narayan, Rofina Johnkennedy, Maheen Zakaria, Victor Lee, Abhijit A. Patel

    META RNA profiling is a simple and inexpensive method to measure the expression of multiple targeted RNAs across many samples. By assigning sample-specific tags up-front during reverse-transcription, cDNAs from multiple samples can be pooled prior to amplification and deep sequencing. Such early parallelization of samples simplifies the workflow, minimizes cross-sample experimental variability, and reduces reagent and sequencing costs. Herein we describe the theoretical framework of the method and provide a detailed protocol to facilitate its implementation.

    更新日期:2018-10-09
  • Catalytic crosslinking-based methods for enzyme-specified profiling of RNA ribonucleotide modifications
    Methods (IF 3.998) Pub Date : 2018-10-09
    Shobbir Hussain

    Well over a hundred types of naturally occurring covalent modifications can be made to ribonucleotides in RNA molecules. Moreover, several types of such modifications are each known to be catalysed by multiple enzymes which largely appear to modify distinct sites within the cellular RNA. In order to aid functional investigations of such multi-enzyme RNA modification types in particular, it is important to determine which enzyme is responsible for catalysing modification at each site. Two methods, Aza-IP and methylation-iCLIP, were developed and used to map genome-wide locations of methyl-5-cytosine (m5C) RNA modifications inherently in an enzyme specific context. Though the methods are quite distinct, both rely on capturing catalytic intermediates of RNA m5C methyltransferases in a state where the cytosine undergoing methylation is covalently crosslinked to the enzyme. More recently the fundamental methylation-iCLIP principle has also been applied to map methyl-2-adenosine sites catalysed by the E.Coli RlmN methylsynthase. Here I describe the ideas on which the two basic methods hinge, and summarise what has been achieved by them thus far. I also discuss whether and how such principles may be further exploited for profiling of other RNA modification types, such as methyl-5-uridine and pseudouridine.

    更新日期:2018-10-09
  • Naturally split intein Npu DnaE mediated rapid generation of bispecific IgG antibodies
    Methods (IF 3.998) Pub Date : 2018-10-09
    Lei Han, Huifang Zong, Yuexian Zhou, Zhidi Pan, Jie Chen, Kai Ding, Yueqing Xie, Hua Jiang, Baohong Zhang, Huili Lu, John Gilly, Jianwei Zhu
    更新日期:2018-10-09
  • Shedding light on microRNA function via microscopy-based screening
    Methods (IF 3.998) Pub Date : 2018-10-05
    Ines Rodrigues Lopes, Ricardo Jorge Silva, Ines Caramelo, Ana Eulalio, Miguel Mano

    MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally modulate gene expression and orchestrate a wide range of biological and pathological processes. The use of high-throughput screening technologies, in particular microscopy-based screenings (also known as high-content screenings), coupled with genome-wide libraries for modulation of miRNA levels, allow for comprehensive functional analysis of each member of the miRNome in different phenotypic cell-based assays. The wealth of information obtained from such screenings spans across various fields of research, including cancer, cardiovascular, cell reprogramming, and infection biology. Here, we provide an overview of the rationale for performing screenings using synthetic libraries of miRNA mimics and inhibitors, and of the microscopy-based miRNA screenings performed to date. Moreover, a list of resources available for such endeavor is provided. Finally, we describe a detailed procedure for a case study where microscopy-based screening using a library of miRNA mimics was performed to identify miRNAs that control infection of epithelial cells by the bacterial pathogen Salmonella. The methodologies described here can be easily adapted for screenings addressing other biological questions.

    更新日期:2018-10-05
  • Bispecific anti-mPDGFRβ x cotinine scFv-Cκ-scFv fusion protein and cotinine-duocarmycin can form antibody-drug conjugate-like complexes that exert cytotoxicity against mPDGFRβ expressing cells
    Methods (IF 3.998) Pub Date : 2018-10-04
    Soohyun Kim, Hyori Kim, Dong Hyun Jo, Jeong Hun Kim, Su Ree Kim, Dongmin Kang, Dobeen Hwang, Junho Chung

    Antibody selection for antibody-drug conjugates (ADCs) has traditionally depended on its internalization into the target cell, although ADC efficacy also relies on recycling of the receptor-ADC complex, endo-lysosomal trafficking, and subsequent linker/antibody proteolysis. In this study, we observed that a bispecific anti-murine platelet-derived growth factor receptor beta (mPDGFRβ) x cotinine single-chain variable fragment (scFv)-kappa constant region (Cκ)-scFv fusion protein and cotinine-duocarmycin can form an ADC-like complex to induce cytotoxicity against mPDGFRβ expressing cells. Multiple anti-mPDGFRβ antibody candidates can be produced in this bispecific scFv-Cκ-scFv fusion protein format and tested for their ability to deliver cotinine-conjugated cytotoxic drugs, thus providing an improved approach for antibody selection in ADC development.

    更新日期:2018-10-04
  • Bispecific applications of non-immunoglobulin scaffold binders
    Methods (IF 3.998) Pub Date : 2018-10-01
    Sophia Hober, Sarah Lindbo, Johan Nilvebrant

    Non-immunoglobulin scaffolds represent a proven group of small affinity proteins that can be engineered in vitro to similar affinity and potency as monoclonal antibodies. Several novel candidate biotherapeutics that exploit the potential advantages scaffold proteins hold over larger and more complex antibodies have been developed over the past decade. The ease of using small and robust binding proteins as flexible and modular building blocks has led to the development of a wide range of innovative approaches to combine them in various bi- and multispecific formats. This progress is expected to aid the ongoing challenge of identifying niche applications where clear differentiation from antibody-based molecules will be key to success. Given the many engineering options that are available for non-immunoglobulin scaffold proteins, they have potential to not only complement but probably also surpass antibodies in certain applications.

    更新日期:2018-10-02
  • Precision gaming for health: Computer games as digital medicine
    Methods (IF 3.998) Pub Date : 2018-09-29
    James M Kinross

    Health based games have significant potential as therapeutic interventions due to the inherent mechanisms associated with social and individual game play and their capacity for sensor integration, data capture and mechanism. Moreover, they are low cost, they can be deployed at the point of care across an evolving digital ecosystem. However, a robust evidence base to support their wider adoption as a clinical intervention for chronic diseases is lacking and significant methodological barriers exist for health games developers creating efficacious ‘digital medicines’. Game design is complex and it must utilise validated game mechanics balanced with a creative and engaging game design. The aim of this review is therefore to outline the fundamental steps of game development for health professionals and to critically appraise the methodology for assessing health games as medical interventions. This requires 1) The adoption of clearly defined global language for health games development based on a targeted function as therapeutic agents. 2). The development of multidisciplinary teams with a broad portfolio of development and clinical skill sets. 3. The creation health game engines specifically built to facilitate clinical game development. 4. Robust trial design and assessment of translational impact: If games are to be prescribed, their efficacy and toxicity must be based on a rigorous assessment of their use within a real world clinical environment. Trials for precision health games have specific challenges around blinding, learning curves, bias and confounding that are particularly problematic. We propose the adoption of the IDEAL-GAMES framework for game development that systematically assess and validates games through open registries. In conclusion we propose a new framework for assessing the robustness and clinical efficacy of games for health as clinical interventions in the clinical environment.

    更新日期:2018-09-29
  • A user-interactive algorithm quantifying nuclear pore complex distribution within the nuclear lamina network in single molecular localization microscopic image
    Methods (IF 3.998) Pub Date : 2018-09-27
    John S.Y. Lim, Graham D. Wright, Brian Burke, Wei Xie

    For decades, components of the mammalian nuclear envelope (NE), such as the nuclear lamina and nuclear pore complexes (NPCs), have been largely resistant to quantitative cell biological analysis using conventional fluorescence microscopy. This is in part due to their sub diffraction limit dimensions. Super-resolution microscopy, a major advancement in cell biology research, has now made possible the acquisition of images in which nuclear lamin networks and single NPCs can be resolved in intact mammalian somatic cells. In particular, single molecule localization microscopy is able to generate data sets that are accurate enough to allow detailed quantitative analysis. Here we describe an algorithm that will identify the centroid of single NPCs and will determine their localization relative to the distribution of lamin protein filaments. Using this algorithm, a percentage of NPCs localized within the nuclear lamin network was accurately calculated, that could be compared between cells expressing different lamin complements. With modifications tweaked according to user specified sample images, this algorithm serves as a semi-automatic and fast computational tool to quantify and compare the localization and distribution of two or more cellular components at the nanometre scale.

    更新日期:2018-09-28
  • Protein oligomerization and mobility within the nuclear envelope evaluated by the time-shifted mean-segmented Q factor
    Methods (IF 3.998) Pub Date : 2018-09-28
    Jared Hennen, Kwang-Ho Hur, Siddarth Reddy Karuka, G.W. Gant Luxton, Joachim D. Mueller

    Analysis of fluorescence fluctuation experiments by the mean-segmented Q (MSQ) method was recently used to successfully characterize the oligomeric state and mobility of proteins within the nuclear envelope (NE) of living cells. However, two significant shortcomings of MSQ were recognized. Non-ideal detector behavior due to dead-time and afterpulsing as well as the lack of error analysis currently limit the potential of MSQ. This paper presents time-shifted MSQ (tsMSQ), a new formulation of MSQ that is robust with respect to dead-time and afterpulsing. In addition, a protocol for performing error analysis on tsMSQ data is introduced to assess the quality of fit models and estimate the uncertainties of fit parameters. Together, these developments significantly simplify and improve the analysis of fluorescence fluctuation data taken within the NE. To demonstrate these new developments, tsMSQ was used to characterize the oligomeric state and mobility of the luminal domains of two inner nuclear membrane SUN proteins. The results for the luminal domain of SUN2 obtained through tsMSQ without correction for non-ideal detector effects agree with a recent study that was conducted using the original MSQ formulation. Finally, tsMSQ was applied to characterize the oligomeric state and mobility of the luminal domain of the germline-restricted SUN3.

    更新日期:2018-09-28
  • Structural diversity and flexibility of diabodies
    Methods (IF 3.998) Pub Date : 2018-09-25
    Na-Young Kwon, Youngjin Kim, Jie-Oh Lee

    Diabodies are bispecific antibody fragments that have two antigen binding Fv domains. They are unique among hundreds of different formats of bispecific antibodies because they are small and rigid enough to be crystallized. Diabodies are generated by connecting variable regions of heavy and light chains by a peptide linker. Because of the short length of the linker, intramolecular association of the variable regions is not allowed. Instead, the variable regions from the different peptide chains associate together, forming a dimeric complex with two antigen binding sites. Previous crystallographic studies of diabodies demonstrate the extraordinary structural diversity of diabodies. They have also shown that the relative orientation and interaction of the two Fv domains in diabodies have substantial flexibility due to instability of the Fv interface. Introduction of site specific mutations and disulfide bridges can reduce flexibility and therefore increase rigidity and predictability of the diabody structures. These stabilized diabodies will be useful for future application to structural biology and protein nanotechnology.

    更新日期:2018-09-25
  • miARma-Seq, a comprehensive pipeline for the simultaneous study and integration of miRNA and mRNA expression data
    Methods (IF 3.998) Pub Date : 2018-09-22
    Eduardo Andrés-León, Ana M. Rojas

    Messenger RNAs (mRNAs) fulfil specific biological roles in cells and, thus, their expression may be adapted to suit specific circumstances. This is in part achieved through selective gene transcription and post-transcriptional events, the regulation of which must be tightly integrated and controlled. To comprehensively study the coordinated effects of transcriptional and post-transcriptional regulatory elements, and to obtain coherent results, it is advisable to use different methodologies. Adequately integrating the data derived from these distinct methodologies then becomes critical to elucidating the relationships between the coordinated cellular effects assayed, particularly when applied to normal and disease states. Such integrated studies are likely to be particularly useful to identify markers suitable for early detection of diseases and to devise strategies for therapeutic interventions. Throughout this chapter, we will focus on the methods currently available to analyse mRNA and microRNA (miRNA) expression, paying special attention to the influence of miRNAs on mRNA metabolism. We will introduce miARma-Seq, a comprehensive pipeline that facilitates the simultaneous integration of mRNA and miRNA expression data. For illustrative purposes, we include a case study that incorporates data from RNASeq and small-RNASeq, detailing all the steps necessary to define the differential expression of both mRNA- and miRNA-encoding genes. Finally, we explore the possible regulatory relationships that drive significant and potentially relevant changes in mRNA and miRNA gene expression.

    更新日期:2018-09-25
  • A CRISPR/Cas9 platform for MS2-labelling of single mRNA in live stem cells
    Methods (IF 3.998) Pub Date : 2018-09-12
    Jan-Hendrik Spille, Micca Hecht, Valentin Grube, Won-ki Cho, Choongman Lee, Ibrahim I. Cissé
    更新日期:2018-09-12
  • Generation of Fcabs targeting human and murine LAG-3 as building blocks for novel bispecific antibody therapeutics
    Methods (IF 3.998) Pub Date : 2018-09-09
    Katy L. Everett, Matthew Kraman, Francisca P.G. Wollerton, Carlo Zimarino, Katarzyna Kmiecik, Miguel Gaspar, Sarka Pechouckova, Natalie L. Allen, Jacqueline F. Doody, Mihriban Tuna

    The immunoglobulin superfamily protein lymphocyte-activation gene 3 (LAG-3) participates in immune suppression and has been identified as a suitable target for cancer therapies. In order to generate bispecific antibodies targeting LAG-3, Fcabs (Fc-region with antigen binding) targeting human and murine LAG-3 were generated from phage libraries. These Fcabs bind to LAG-3, inhibiting its interaction with MHC class II, and induce IL-2 production in a T cell assay. Bispecific antibodies, known as mAb2, were produced by replacing the Fc region of a monoclonal antibody with Fcab sequences in the CH3 domain. mAb2 containing anti-LAG-3 Fcabs have mAb-like biophysical characteristics and retain LAG-3 binding and functional activity. mAb2 can thus be generated using multiple Fabs to investigate bispecific parings and develop novel therapeutics.

    更新日期:2018-09-10
  • Systematic optimization of cell-free synthesized human endothelin B receptor folding
    Methods (IF 3.998) 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.

    更新日期:2018-09-09
  • A reconstitution method for integral membrane proteins in hybrid lipid-polymer vesicles for enhanced functional durability
    Methods (IF 3.998) 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.

    更新日期:2018-09-09
  • Observing and tracking single small ribosomal subunits in vivo
    Methods (IF 3.998) Pub Date : 2018-09-06
    Lisa Landvogt, Jan Andreas Ruland, Christian Montellese, Jan Peter Siebrasse, Ulrike Kutay, Ulrich Kubitscheck
    更新日期:2018-09-06
  • Optogenetic precision toolkit to reveal form, function and connectivity of single neurons
    Methods (IF 3.998) Pub Date : 2018-09-05
    Dominique Förster, Anna Kramer, Herwig Baier, Fumi Kubo

    All-optical methods enable the control and monitoring of neuronal activity with minimal perturbation of the system. Although imaging and optogenetic manipulations can be performed at cellular resolution, the morphology of single cells in a dense neuronal population has often remained unresolvable. Here we describe in detail two recently established optogenetic protocols for systematic description of function and morphology of single neurons in zebrafish. First, the Optobow toolbox allows unbiased mapping of excitatory functional connectivity. Second, the FuGIMA technique enables selective labeling and anatomical tracing of neurons that are responsive to a given sensory stimulus or correlated with a specific behavior. Both strategies can be genetically targeted to a neuronal population of choice using the Gal4/UAS system. As these in vivo approaches are non-invasive, we envision useful applications for the study of neuronal structure, function and connectivity during development and behavior.

    更新日期:2018-09-05
  • Building Blocks for Bispecific and Trispecific Antibodies
    Methods (IF 3.998) Pub Date : 2018-08-30
    Xiufeng Wu, Stephen J. Demarest

    Bispecific antibodies (BsAbs), which target two antigens or epitopes, incorporate the specificities and properties of two distinct monoclonal antibodies (mAbs) into a single molecule. As such, BsAbs can elicit synergistic activities and provide the capacity for enhanced therapeutic efficacy and/or safety compared to what can be achieved with conventional monospecific IgGs. There are many building block formats to generate BsAbs and Trispecific antibodies (TsAbs) based on combining the antigen recognition domains of monoclonal antibodies (mAbs). This review describes the many and varied antibody-based building blocks used to achieve multivalency and multispecificity. These diverse building blocks provide opportunities to tailor the design of BsAbs and TsAbs to match the desired applications.

    更新日期:2018-08-30
  • 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.

    更新日期:2018-07-15
  • 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.

    更新日期:2018-07-15
  • 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.

    更新日期:2018-07-15
  • 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.

    更新日期:2018-07-14
  • 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.

    更新日期:2018-07-14
  • 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.

    更新日期:2018-07-14
  • Efficient computation of co-transcriptional RNA-ligand interaction dynamics
    Methods (IF 3.998) Pub Date : 2018-05-04
    Michael T. Wolfinger, Christoph Flamm, Ivo L. Hofacker

    Riboswitches form an abundant class of cis-regulatory RNA elements that mediate gene expression by binding a small metabolite. For synthetic biology applications, they are becoming cheap and accessible systems for selectively triggering transcription or translation of downstream genes. Many riboswitches are kinetically controlled, hence knowledge of their co-transcriptional mechanisms is essential. We present here an efficient implementation for analyzing co-transcriptional RNA-ligand interaction dynamics. This approach allows for the first time to model concentration-dependent metabolite binding/unbinding kinetics. We exemplify this novel approach by means of the recently studied I-A 2’-deoxyguanosine (2’dG)-sensing riboswitch from Mesoplasma florum.

    更新日期:2018-05-04
  • Iteratively improving Hi-C experiments one step at a time
    Methods (IF 3.998) Pub Date : 2018-04-30
    Rosela Golloshi, Jacob Sanders, Rachel Patton McCord

    The 3D organization of eukaryotic chromosomes affects key processes such as gene expression, DNA replication, cell division, and response to DNA damage. The genome-wide chromosome conformation capture (Hi-C) approach can characterize the landscape of 3D genome organization by measuring interaction frequencies between all genomic regions. Hi-C protocol improvements and rapid advances in DNA sequencing power have made Hi-C useful to study diverse biological systems, not only to elucidate the role of 3D genome structure in proper cellular function, but also to characterize genomic rearrangements, assemble new genomes, and consider chromatin interactions as potential biomarkers for diseases. Yet, the Hi-C protocol is still complex and subject to variations at numerous steps that can affect the resulting data. Thus, there is still a need for better understanding and control of factors that contribute to Hi-C experiment success and data quality. Here, we evaluate recently proposed Hi-C protocol modifications as well as often overlooked variables in sample preparation and examine their effects on Hi-C data quality. We examine artifacts that can occur during Hi-C library preparation, including microhomology-based artificial template copying and chimera formation that can add noise to the downstream data. Exploring the mechanisms underlying Hi-C artifacts pinpoints steps that should be further optimized in the future. To improve the utility of Hi-C in characterizing the 3D genome of specialized populations of cells or small samples of primary tissue, we identify steps prone to DNA loss which should be considered to adapt Hi-C to lower cell numbers.

    更新日期:2018-05-01
  • 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 LePabic, 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.

    更新日期:2018-04-28
  • Mass Spectrometry Approaches to Metabolic Profiling of Microbial Communities within the Human Gastrointestinal Tract
    Methods (IF 3.998) Pub Date : 2018-04-26
    Simon JS 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.

    更新日期:2018-04-26
  • 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.

    更新日期:2018-04-26
  • 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.

    更新日期:2018-04-26
  • 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.

    更新日期:2018-04-26
  • 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, Tumpa Dutta, Xuan-Mai Petterson, Lisa Boardman, David Larson, Heidi Nelson, 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.

    更新日期:2018-04-26
  • 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.

    更新日期:2018-04-25
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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