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  • Deep-Resp-Forest: A deep forest model to predict anti-cancer drug response
    Methods (IF 3.998) Pub Date : 2019-02-14
    Ran Su, Xinyi Liu, Leyi Wei, Quan Zou

    The identification of therapeutic biomarkers predictive of drug response is crucial in personalized medicine. A number of computational models to predict response of anti-cancer drugs have been developed as the establishment of several pharmacogenomics screening databases. In our study, we proposed a deep cascaded forest model, Deep-Resp-Forest, to classify the anti-cancer drug response as “sensitive” or “resistant”. We made three contributions in this study. Firstly, diverse molecular data could be effectively integrated to provide more information than single type of data for the classification. Combination of two types of data were tested here. Secondly, two structures based on the multi-grained scanning to transform the raw features into high-dimensional feature vectors and integrate the diverse data were proposed in our study. Thirdly, the original deep and time-consuming architecture of cascade forest was improved by a feature optimization operation, which emphasized the most discriminative features across layers. We evaluated the proposed method on the Cancer Cell Line Encyclopedia (CCLE) and Genomics of Drug Sensitivity in Cancer (GDSC) data sets and then compared with the Support Vector Machine. The proposed Deep-Resp-Forest has demonstrated the promising use of deep learning and deep forest approach on the drug response prediction tasks. The R implementation for running our experiments is available at https://github.com/RanSuLab/Deep-Resp-Forest.

  • Use and reliability of multiplex bead-based assays (Luminex) at Containment Level 4
    Methods (IF 3.998) Pub Date : 2019-02-13
    Stuart D. Dowall, Victoria A. Graham, Tom Fletcher, Roger Hewson

    In the UK, research on hazard group 4 (HG4) pathogens requires specialised Containment Level 4 (CL4) facilities. These differ from Biosafety Level 4 (BSL4) conditions in that work is conducted in class III microbiological safety cabinets for primary containment instead of using positive pressure suits. This presents unique challenges associated with the physical restrictions of working in a limited space, and prohibits the use of many techniques and specialist equipment. In consequence, detailed studies on the biology of HG4 pathogens and in particular their immunological relationships with the host are understudied in the UK; for example, the majority of immunological assays with which the immune system is interrogated require specialist equipment that is unsuitable for CL4.Multiplexing to simultaneously measure multiple analytes is increasingly being used in immunological studies. This assay is attractive for CL4 work because it reduces the time spent in the laboratory whilst maximising the use of valuable sample volume. The Luminex microsphere approach allows for the determination of many cytokines and chemokines, however, the detection system uses fixed aligned lasers and integrated computer systems which are unsuitable for use at CL4. Therefore, we have developed an approach in which the Luminex assay is conducted within the CL4 laboratory and a formalin-fixation stage is introduced to allow for analysis to be undertaken outside of containment. Quality control preparations allow the assay characteristics to be monitored and analysis of assay performance to be evaluated. Our data demonstrate that Luminex is an applicable tool for use at CL4 and that assays can be run reliably to generate reproducible standardised data across different plates and individual experiments.

  • Computer-aided detection of mass in digital breast tomosynthesis using a faster region-based convolutional neural network
    Methods (IF 3.998) Pub Date : 2019-02-13
    Ming Fan, Yuanzhe Li, Shuo Zheng, Weijun Peng, Wei Tang, Lihua Li

    Digital breast tomosynthesis (DBT) is a newly developed three-dimensional tomographic imaging modality in the field of breast cancer screening designed to alleviate the limitations of conventional digital mammography-based breast screening methods. A computer-aided detection (CAD) system was designed for masses in DBT using a faster region-based convolutional neural network (faster-RCNN). To this end, a data set was collected, including 89 patients with 105 masses. An efficient detection architecture of convolution neural network with a region proposal network (RPN) was used for each slice to generate region proposals (i.e., bounding boxes) with a mass likelihood score. In each DBT volume, a slice fusion procedure was used to merge the detection results on consecutive 2D slices into one 3D DBT volume. The performance of the CAD system was evaluated using free-response receiver operating characteristic (FROC) curves. Our RCNN-based CAD system was compared with a deep convolutional neural network (DCNN)-based CAD system. The RCNN-based CAD generated a performance with an area under the ROC (AUC) of 0.96, whereas the DCNN-based CAD achieved a performance with AUC of 0.92. For lesion-based mass detection, the sensitivity of RCNN-based CAD was 90% at 1.54 false positive (FP) per volume, whereas the sensitivity of DCNN-based CAD was 90% at 2.81 FPs/volume. For breast-based mass detection, RCNN-based CAD generated a sensitivity of 90% at 0.76 FP/breast, which is significantly increased compared with the DCNN-based CAD with a sensitivity of 90% at 2.25 FPs/breast. The results suggest that the faster R-CNN has the potential to augment the prescreening and FP reduction in the CAD system for masses.

  • Analysis of RNA polymerase II ubiquitylation and proteasomal degradation
    Methods (IF 3.998) Pub Date : 2019-02-13
    Ana Tufegdzic Vidakovic, Michelle Harreman, A. Barbara Dirac-Svejstrup, Stefan Boeing, Anindya Roy, Vesela Encheva, Michelle Neumann, Marcus Wilson, Ambrosius P. Snijders, Jesper Q. Svejstrup

    Transcribing RNA polymerase II (RNAPII) is decorated by a plethora of post-translational modifications that mark different stages of transcription. One important modification is RNAPII ubiquitylation, which occurs in response to numerous different stimuli that cause RNAPII stalling, such as DNA damaging agents, RNAPII inhibitors, or depletion of the nucleotide pool. Stalled RNAPII triggers a so-called “last resort pathway”, which involves RNAPII poly-ubiquitylation and proteasome-mediated degradation. Different approaches have been described to study RNAPII poly-ubiquitylation and degradation, each method with its own advantages and caveats. Here, we describe optimised strategies for detecting ubiquitylated RPB1 and studying its degradation, but these protocols are suitable for studying other ubiquitylated proteins as well.

  • Method to Quantify Cytokines and Chemokines in Mouse Brain Tissue Using Bio-Plex Multiplex Immunoassays
    Methods (IF 3.998) Pub Date : 2019-02-10
    Monica Manglani, Rejane Rua, Amy Hendricksen, Daniel Braunschweig, Qian Gao, Woei Tan, Brett Houser, Dorian B. McGavern, Kenneth Oh

    This protocol describes how to prepare mouse brain tissue for quantification of multiple inflammatory mediators using a multiplex bead-based immunoassay. It is important to have methods that allow quantification of multiple analytes from small amounts of tissue. Bio-Plex is a Luminex xMAP-based multiplex bead-based immunoassay technology that permits simultaneous analysis of up to 100 analytes from a single tissue sample. This assay has been used extensively to investigate analytes in plasma and serum samples as well as cultured and primary cells. Here, we describe a method for simultaneous analysis of 33 different inflammatory cytokines and chemokines from mouse brain tissue using the Bio-Plex Pro Mouse Chemokine Panel 33-Plex.

  • Deriving a sub-nanomolar affinity peptide from TAP to enable smFRET analysis of RNA polymerase II complexes
    Methods (IF 3.998) Pub Date : 2019-02-10
    Jheng-Syong Wu, Tzu-Yun Chen, Sam Song-Yao Lin, Shu-Yu Lin, Cheng-Yu Hung, I-Ping Tu, Hung-Ta Chen, Wei-Hau Chang

    Our capability to visualize protein complexes such as RNA polymerase II (pol II) by single-molecule imaging techniques has largely been hampered by the absence of a simple bio-orthogonal approach for selective labeling with a fluorescent probe. Here, we modified the existing calmodulin-binding peptide (CBP) in the widely used Tandem Affinity Purification (TAP) tag to endow it with a high affinity for calmodulin (CaM) and used dye-CaM to conduct site-specific labeling of pol II. To demonstrate the single molecule applicability of this approach, we labeled the C-terminus of the Rpb9 subunit of pol II with donor-CaM and a site in TFIIF with an acceptor to generate a FRET (fluorescence resonance energy transfer) pair in the pol II-TFIIF complex. We then used total internal reflection fluorescence microscopy (TIRF) with alternating excitation to measure the single molecule FRET (smFRET) efficiency between these two sites in pol II-TFIIF. We found they exhibited a proximity consistent with that observed in the transcription pre-initiation complex by cryo-electron microscopy (cryo-EM). We further compared our non-covalent labeling approach with an enzyme-enabled covalent labeling method. The virtually indistinguishable results validate our smFRET approach and show that the observed proximity between the two sites represents a hallmark of the pol II-TFIIF complex. Taken together, we present a simple and versatile bio-orthogonal method derived from TAP to enable selective labeling of a protein complex. This method is suitable for analyzing dynamic relationships among proteins involved in transcription and it can be readily extended to many other biological processes.

  • Genome-wide analysis of RNA and protein localization and local translation in mESC-derived neurons
    Methods (IF 3.998) Pub Date : 2019-02-08
    Katarzyna A. Ludwik, Nicolai von Kuegelgen, Marina Chekulaeva

    The subcellular localization and translation of mRNAs are fundamental biological processes. In neurons, they underlie cell growth and synaptic plasticity, which serves as a foundation of learning and memory. Multiple approaches have been developed to separate neurons on subcellular compartments – cell bodies (soma) and cell extensions (axons and dendrites) – for further biochemical analyses. Here we describe neurite/soma separation approach in combination with RNA sequencing and proteomic analyses to identify localized and locally translated RNAs and proteins. This approach allows quantification of around 7,000 of local proteins and the entire local transcriptome. It provides a powerful tool for investigation of the mechanisms underlying RNA localization and local translation in neurons.

  • Tracking of single tRNAs for translation kinetics measurements in chloramphenicol treated bacteria
    Methods (IF 3.998) Pub Date : 2019-02-08
    Ivan L. Volkov, A. Carolin Seefeldt, Magnus Johansson

    Chloramphenicol is a broad-spectrum antibiotic targeting the protein synthesis machinery by binding to the bacterial ribosome. Chloramphenicol has been considered a classic general inhibitor of translation, blocking the accommodation of aa-tRNA into the A site of the large ribosomal subunit. However, recent studies suggest that this proposed mechanism is a simplification and that the effect of chloramphenicol on mRNA translation is much more dynamic. By tracking single dye-labelled elongator and initiator tRNAs in Escherichia coli cells treated with chloramphenicol, we observe the direct effect of chloramphenicol on translation kinetics. We find clear indications of slow but significant mRNA translation on drug bound ribosomes.

  • 更新日期:2019-02-11
  • Multiplexing Protein and Gene Level Measurements on a Single Luminex Platform
    Methods (IF 3.998) Pub Date : 2019-02-08
    Damon B. Cook, Brian C McLucas, Leticia A Montoya, Chris M Brotski, Shelley Das, Markus Miholits, Thao H Sebata
  • Applications of high-throughput sequencing to analyze and engineer ribozymes
    Methods (IF 3.998) Pub Date : 2019-02-06
    Yohei Yokobayashi

    A large number of catalytic RNAs, or ribozymes, have been identified in the genomes of various organisms and viruses. Ribozymes are involved in biological processes such as regulation of gene expression and viral replication, but biological roles of many ribozymes still remain unknown. Ribozymes have also inspired researchers to engineer synthetic ribozymes that function as sensors or gene switches. To gain deeper understanding of the sequence-function relationship of ribozymes and to efficiently engineer synthetic ribozymes, a large number of ribozyme variants need to be examined which was limited to hundreds of sequences by Sanger sequencing. The advent of high-throughput sequencing technologies, however, has allowed us to sequence millions of ribozyme sequences at low cost. This review focuses on the recent applications of high-throughput sequencing to both characterize and engineer ribozymes, to highlight how the large-scale sequence data can advance ribozyme research and engineering.

  • Measuring RNA polymerase activity genome-wide with high-resolution run-on-based methods
    Methods (IF 3.998) Pub Date : 2019-02-02
    Antonio Jordán-Pla, Maria E. Pérez-Martínez, José E. Pérez-Ortín

    The biogenesis of RNAs is a multi-layered and highly regulated process that involves a diverse set of players acting in an orchestrated manner throughout the transcription cycle. Transcription initiation, elongation and termination factors act on RNA polymerases to modulate their movement along the DNA template in a very precise manner, more complex than previously anticipated. Genome-scale run-on-based methodologies have been developed to study in detail the position of transcriptionally-engaged RNA polymerases. Genomic run-on (GRO), and its many variants and refinements made over the years, are helping the community to address an increasing amount of scientific questions, spanning an increasing range of organisms and systems. In this review, we aim to summarize the most relevant high throughput methodologies developed to study nascent RNA by run-on methods, compare their main features, advantages and limitations, while putting them in context with alternative ways of studying the transcriptional process.

  • The development of a multiplex serological assay for avian influenza based on Luminex technology
    Methods (IF 3.998) Pub Date : 2019-01-30
    E.A. Evelien Germeraad, R.P. René Achterberg, S. Sandra Venema, J. Jacob Post, O. Olav de Leeuw, G. Guus Koch, F.J. Fimme Jan van der Wal, N. Nancy Beerens
  • A Universal Method for the Functionalization of Dyed Magnetic Microspheres with Peptides
    Methods (IF 3.998) Pub Date : 2019-01-30
    Matthew B. Coppock, Dimitra N. Stratis-Cullum

    The need for the functionalization of magnetic, water-soluble dyed microspheres with peptides is apparent with the ever-growing biointeraction capabilities and the increased use of dyed microspheres in multiplex, microsphere-based detection assays. This method describes the attachment of any peptide to dyed magnetic microspheres regardless of peptide length, size, or sequence. The method exploits ‘click’ chemistry with short reaction times in a mixed organic/water system for simultaneous selective surface functionalization and reduction of microsphere dye leaching. All optimization studies were performed using a Luminex 200 assay platform, but the functionalized microspheres are capable of use in any similar multiplex format.

  • Time-Resolved Analysis of Transcription through Chromatin
    Methods (IF 3.998) Pub Date : 2019-01-29
    Han-Wen Chang, Fu-Kai Hsieh, Smita S. Patel, Vasily M. Studitsky

    During transcription along nucleosomal DNA, RNA polymerase II (Pol II) pauses at multiple positions and induces formation of multiple intermediates that aid in maintaining proper chromatin structure. To describe the kinetics of this multiple-step reaction, we utilized a computational model-based approach and KinTek Explorer software to analyze the time courses. Here we describe the stepwise protocol for analysis of the kinetics of transcription through a nucleosome that provides the rate constants for each step of this complex process. We also present an example where this time-resolved approach was applied to study the mechanism of histone chaperone FACT action during Pol II transcription through a single nucleosome by comparing the rate constants derived in the presence or in the absence of FACT.

  • Validation of monoplex assays detecting antibodies against Corynebacterium diphtheriae and Clostridium tetani toxins, Rubella virus and Parvovirus B19 for incorporation into Multiplex Serology
    Methods (IF 3.998) Pub Date : 2019-01-28
    Nicole Brenner, Julia Butt, Izaura Lima Bomfim, Julia Tabatabai, Michael Pawlita, Paul Schnitzler, Tim Waterboer

    Serological assays detecting antibodies in serum or plasma samples are useful and versatile instruments to investigate an individual’s infection and vaccination history, e.g. for clinical diagnosis, personal risk evaluation, and seroepidemiological studies. Multiplex Serology is a suspension bead array-based high-throughput methodology for simultaneous measurement of antibodies against multiple pathogens in a single reaction vessel, thus economizing sample volume, measurement time, and costs. We developed and validated bead-based pathogen-specific Monoplex Serology assays, i.e. assays including only antigens for the respective pathogen, to detect antibodies against Corynebacterium diphtheriae and Clostridium tetani toxins, rubella virus and parvovirus B19. The developed assays expand the portfolio of existing pathogen-specific bead-based serology assays and can be efficiently incorporated into larger Multiplex Serology panels. The newly developed Monoplex Serology assays consist of only one antigen per infectious agent, expressed as Glutathione S-transferase-fusion proteins in E. coli. Specificity, sensitivity and Cohen’s kappa statistics in comparison with routine clinical diagnostic assays were calculated for serum dilutions 1:100 and 1:1000. All pathogen-specific assays were successfully validated at both serum dilutions with the exception of rubella Monoplex Serology which showed impaired sensitivity (57.6%) at dilution 1:1000. Specificities of successfully validated Monoplex Serology assays ranged from 85.6% to 100.0% (median: 91.7%), and sensitivities from 81.3% to 95.8% (median: 90.9%); agreement with the reference assays ranged from substantial to almost perfect (kappa: 0.66-0.86, median: 0.78). Statistical performance and slim assay design enable efficient incorporation of the developed assays into Multiplex Serology.

  • Development and Validation of Magnetic Bead Pentaplex Immunoassay for Simultaneous Quantification of murine serum IgG antibodies to Acellular Pertussis, Diphtheria and Tetanus Antigens used in combination vaccines
    Methods (IF 3.998) Pub Date : 2019-01-25
    Laxmikant Kadam, Krunal Patel, Manish Gautam, Shrikant Thorat, Prathamesh Kale, Arvind Kumar Ghule, Harish Rao, Yojana Shinde, Umesh Shaligram, Sunil Gairola

    We describe here a magnetic bead-based multiplex (pentaplex) immunoassay (MIA) platform developed as an alternative to enzyme-linked immunosorbent assays (ELISA) used in immunogenicity testing of DTaP/TdaP vaccine in animals. MIA simultaneously measures the concentration of serum (IgG) antibodies against B. Pertussis antigens; pertussis toxin, , filamentous hemagglutinin (FHA), pertactin (PRN) and tetanus (T) and diphtheria (D) toxoid in the Tdap vaccine immunized animals. Assay validation experiments were done using a panel of serum samples. The results are expressed in IU/ml using WHO reference mice serum. The standard curve was linear with 4PL logistic fit over an eight 2-fold dilution range with LOQ of 0.003,0.022, 0.005 IU/ml for PT, FHA and PRN and 0.016 U/ml for T and D antigens indicating sensitivity. No interference was observed in monoplex versus multiplex measurements. Specificity was demonstrated by ≥ 90 % homologous and ≤ 15% heterologous inhibition for all the antigens. The assay was reproducible, with a mean coefficient of variation (CV) of ≤ 10 % for intra-assay duplicates and ≤ 25 % for interassays using different lots of beads and analyst. Accuracy was demonstrated wherein the ratio of observed vs. assigned unitages were within 80-120 %. The study suggests that the Pentaplex (MIA) platform meets all the criteria for the serological assay combination vaccines with additional advantages of high throughput, reduced sample volumes, faster analysis with reduced manpower in contrast to conventional monoplex ELISA.

  • Genetic analysis of the RNA polymerase II CTD in Drosophila
    Methods (IF 3.998) Pub Date : 2019-01-24
    Feiyue Lu, David S. Gilmour

    The Carboxy-terminal Domain (CTD) of RNA polymerase II (Pol II) plays essential roles in regulating gene expression in eukaryotes. Here, we describe multiple genetic approaches for studying the CTD in Drosophila that complement pre-existing molecular analyses of the Pol II CTD in other experimental models. These approaches will allow one to assess the effects of any CTD mutations in a developmentally complex organism. The approaches discussed in this work can in principle, be applied to analyze other transcription components in eukaryotes.

  • Biochemical Methods to Characterize RNA Polymerase II Elongation Complexes
    Methods (IF 3.998) Pub Date : 2019-01-24
    J. Brooks Crickard, Joseph C. Reese

    Transcription of DNA into RNA is critical for all life, and RNA polymerases are enzymes tasked with this activity. In eukaryotes, RNA Polymerase II (RNAPII) is responsible for transcription of all protein coding genes and many non-coding RNAs. RNAPII carries out the remarkable feat of unwinding the stable double-stranded DNA template, synthesizing the transcript and re-forming the double helix behind it with great precision and speed. In vitro, RNAPII is capable of carrying out templated RNA chain elongation in the absence of any accessory proteins. However, in cells, the transcription of genes is influenced by several factors, including DNA structure, chromatin, co-transcriptional processes, and DNA binding proteins, which impede the smooth progression of RNAPII down the template. Many transcription elongation proteins have evolved to mitigate the complications and barriers encountered by polymerase during transcription. Many of these elongation factors physically interact with components of the RNAPII elongation complex, including the growing RNA transcript and the DNA template entering and exiting RNAPII. To better understand how transcription elongation factors (EFs) regulate RNAPII, elegant methods are required to probe the structure of the elongation complex. Here, we describe a collection of biochemical assays to interrogate the structure of the RNAPII elongation complex of Saccharomyces cerevisiae that are capable of providing insights into the function of EFs and the elongation process.

  • 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.

  • 更新日期:2019-01-18
  • “Second-generation” fluorogenic RNA-based sensors
    Methods (IF 3.998) Pub Date : 2019-01-17
    Aruni P.K.K. Karunanayake Mudiyanselage, Rigumula Wu, Mark A. Leon-Duque, Kewei Ren, Mingxu You

    A fluorogenic aptamer can specifically interact with a fluorophore to activate its fluorescence. These nucleic acid-based fluorogenic modules have been dramatically developed over the past decade, and have been used as versatile reporters in the sensor development and for intracellular imaging. In this review, we summarize the design principles, applications, and challenges of the first-generation fluorogenic RNA-based sensors. Moreover, we discuss some strategies to develop next-generation biosensors with improved sensitivity, selectivity, quantification property, and eukaryotic robustness. Using genetically encoded catalytic hairpin assembly strategy as an example, we further introduce a standard protocol to design, characterize, and apply these fluorogenic RNA-based sensors for in vitro detection and cellular imaging of target biomolecules. By incorporating natural RNA machineries, nucleic acid nanotechnology, and systematic evolution approaches, next-generation fluorogenic RNA-based devices can be potentially engineered to be widely applied in cell biology and biomedicine.

  • Single-molecule FRET method to investigate the dynamics of transcription elongation through the nucleosome by RNA polymerase II
    Methods (IF 3.998) Pub Date : 2019-01-17
    Jaehyoun Lee, J. Brooks Crickard, Joseph C. Reese, Tae-Hee Lee

    Transcription elongation through the nucleosome is a precisely coordinated activity to ensure timely production of RNA and accurate regulation of co-transcriptional histone modifications. Nucleosomes actively participate in transcription regulation at various levels and impose physical barriers to RNA polymerase II (RNAPII) during transcription elongation. Despite its high significance, the detailed dynamics of how RNAPII translocates along nucleosomal DNA during transcription elongation and how the nucleosome structure dynamically conforms to the changes necessary for RNAPII progression remain poorly understood. Transcription elongation through the nucleosome is a complex process and investigating the changes of the nucleosome structure during this process by ensemble measurements is daunting. This is because it is nearly impossible to synchronize elongation complexes within a nucleosome or a sub-nucleosome to a designated location at a high enough efficiency for desired sample homogeneity. Here we review our recently developed single-molecule FRET experimental system and method that has fulfilled this deficiency. With our method, one can follow the changes in the structure of individual nucleosomes during transcription elongation. We demonstrated that this method enables the detailed measurements of the kinetics of transcription elongation through the nucleosome and its regulation by a transcription factor, which can be easily extended to investigations of the roles of environmental variables and histone post-translational modifications in regulating transcription elongation.

  • Multiplexed detection and identification of respiratory pathogens using the NxTAG® Respiratory Pathogen Panel
    Methods (IF 3.998) Pub Date : 2019-01-17
    Sarah Gonsalves, James Mahony, Arundhati Rao, Sherry Dunbar, Stefan Juretschko

    The Luminex® NxTAG® Respiratory Pathogen Panel (NxTAG RPP) is an IVD-cleared assay for the simultaneous detection and identification of nucleic acids from 18 respiratory viruses and 2 (or 3 outside of the U.S.) atypical bacterial pathogens in nasopharyngeal swabs. Its scalability allows concurrent testing of up to 96 samples in a single batch. Nucleic acid extracted from 200 µl of raw specimen using the easyMAG® extractor is added directly to pre-plated, lyophilized bead reagents (LBRs), where multiplexed RT-PCR and hybridization to MagPlex-TAG™ microspheres occurs within a sealed reaction well using a single cycling program. Data acquisition is done on the MAGPIX® instrument which reads and sorts the reaction products directly from the sealed well following transfer of the assay plate from the thermal cycler. NxTAG is the newest innovation in bead-based nucleic acid chemistry developed by Luminex. Here we provide the detailed assay protocol and present data which describe the clinical and analytical performance characteristics of NxTAG RPP.

  • The Genesis and Evolution of Bead-Based Multiplexing
    Methods (IF 3.998) Pub Date : 2019-01-17
    Hilary Graham, Don J. Chandler, Sherry A Dunbar

    Multiplexed analysis has the advantage of allowing for simultaneous detection of multiple analytes in a single reaction vessel which reduces time, labor, and cost as compared to single-reaction-based detection methods. Microsphere-based suspension array technologies, such as the Luminex® xMAP® system, offer high-throughput detection of both protein and nucleic acid targets in multiple assay chemistries. After Luminex’s founding in 1995, it quickly became the leader in bead-based multiplexing solutions. Today, xMAP Technology is the most widely adopted bead-based multiplexing platform with over 35,000 peer-reviewed publications, an installed base of approximately 15,500 instruments, and over 70 Luminex Partners offering more than 1,300 research use kits as well as custom assay solutions. Because of the open architecture of the xMAP platform it has been implemented in a variety of applications that range from transplant medicine, biomarker discovery and validation, pathogen detection, drug discovery, vaccine development, personalized medicine, neurodegeneration, and cancer research.

  • Neomycin-dependent Hammerhead Ribozymes for the Direct Control of Gene Expression in Saccharomyces cerevisiae
    Methods (IF 3.998) Pub Date : 2019-01-09
    Monika Sack, Julia Stifel, Stefan G. Kreft, Elke Deuerling, Jörg S. Hartig

    Hammerhead ribozyme-based RNA switches have been proven to be powerful tools for conditional gene regulation in various organisms. We present neomycin-dependent hammerhead ribozymes (HHR) that influence gene expression in a ligand- and dose-dependent manner in S. cerevisiae. We utilized a novel design of fusing the aptamer domain to the HHR enabling for the first time the identification of genetic ON- and OFF-switches within the same library. For this purpose a neomycin aptamer was fused to stem 1 of a type 3 hammerhead ribozyme via an addressable three-way junction that shows high flexibility at the connection site. An in vivo screening approach identified sequences that allow to induce or repress gene expression 2- to 3-fold in response to neomycin addition. The ribozyme switches operate at neomycin concentrations that show no toxic effect on cell growth and pose powerful genetic tools to study and modulate cellular function in yeast.

  • A fluorescent assay for the genetic dissection of the RNA polymerase II termination machinery
    Methods (IF 3.998) Pub Date : 2019-01-05
    Daniel Reines

    RNA polymerase II is a highly processive enzyme that synthesizes mRNAs and some non-protein coding RNAs. Termination of transcription, which entails release of the transcript and disengagement of the polymerase, requires an active process. In yeast, there are at least two multi-protein complexes needed for termination of transcription, depending upon which class of RNAs are being acted upon. In general, the two classes are relatively short non-coding RNAs (e.g. snoRNAs) and relatively long mRNAs, although there are exceptions. Here, a procedure is described in which defective termination can be detected in living cells, resulting in a method that allows strains with mutations in termination factors or cis-acting sequences, to be identified and recovered. The strategy employs a reporter plasmid with a galactose inducible promoter driving transcription of green fluorescent protein which yields highly fluorescent cells. When a test terminator is inserted between the promoter and the fluorescent protein reading frame, cells fail to fluoresce. Mutant strains that have lost termination capability, so called terminator-override mutants, gain expression of the fluorescent protein and can be collected by fluorescence activated cell sorting. The strategy is robust since acquisition of fluorescence is a positive trait that has a low probability of happening adventitiously. Live mutant cells can easily be cloned from the population of positive candidates. Flow sorting is a sensitive, high-throughput detection step capable of discovering spontaneous mutations in yeast with high fidelity.

  • Development of Poly(A)-ClickSeq as a Tool Enabling Simultaneous Genome-wide Poly(A)-site identification and Differential Expression Analysis
    Methods (IF 3.998) Pub Date : 2019-01-06
    Nathan R. Elrod, Elizabeth A. Jaworski, Ping Ji, Eric J. Wagner, Andrew Routh

    The use of RNA-seq as a generalized tool to measure the differential expression of genes has essentially replaced the use of the microarray. Despite the acknowledged technical advantages to this approach, RNA-seq library preparation remains mostly conducted by core facilities rather than in the laboratory due to the infrastructure, expertise and time required per sample. We have recently described two ‘click-chemistry’ based library construction methods termed ClickSeq and poly(A)-click-seq (PAC-seq) as alternatives to conventional RNA-seq that are both cost effective and rely on straightforward reagents readily available to most labs. ClickSeq is random-primed and can sequence any (unfragmented) RNA template, while PAC-seq is targeted to poly(A) tails of mRNAs. Here, we further develop PAC-seq as a platform that allows for simultaneous mapping of poly(A) sites and the measurement of differential expression of genes. We provide a detailed protocol, descriptions of appropriate computational pipelines, and a proof-of-principle dataset to illustrate the technique. PAC-seq offers a unique advantage over other 3’ end mapping protocols in that it does not require additional purification, selection, or fragmentation steps allowing sample preparation directly from crude total cellular RNA. We have shown that PAC-seq is able to accurately and sensitively count transcripts for differential gene expression analysis, as well as identify alternative poly(A) sites and determine the precise nucleotides of the poly(A) tail boundaries.

  • Inhibiting transcription in cultured metazoan cells with actinomycin D to monitor mRNA turnover
    Methods (IF 3.998) Pub Date : 2019-01-06
    Wi S. Lai, Rene M. Arvola, Aaron C. Goldstrohm, Perry J. Blackshear

    Decay of transcribed mRNA is a key determinant of steady state mRNA levels in cells. Global analysis of mRNA decay in cultured cells has revealed amazing heterogeneity in rates of decay under normal growth conditions, with calculated half-lives ranging from several minutes to many days. The factors that are responsible for this wide range of decay rates are largely unknown, although our knowledge of trans-acting RNA binding proteins and non-coding RNAs that can control decay rates is increasing. Many methods have been used to try to determine mRNA decay rates under various experimental conditions in cultured cells, and transcription inhibitors like actinomycin D have probably the longest history of any technique for this purpose. Despite this long history of use, the actinomycin D method has been criticized as prone to artifacts, and as ineffective for some promoters. With appropriate guidelines and controls, however, it can be a versatile, effective technique for measuring endogenous mRNA decay in cultured mammalian and insect cells, as well as the decay of exogenously-expressed transcripts. It can be used readily on a genome-wide level, and is remarkably cost-effective. In this short review, we will discuss our utilization of this approach in these cells; we hope that these methods will allow more investigators to apply this useful technique to study mRNA decay under the appropriate conditions.

  • A Humanized Yeast System to Analyze Cleavage of Prelamin A by ZMPSTE24
    Methods (IF 3.998) Pub Date : 2019-01-06
    Eric D. Spear, Rebecca F. Alford, Tim D. Babatz, Kaitlin M. Wood, Otto W. Mossberg, Kamsi Odinammadu, Khurts Shilagardi, Jeffrey J. Gray, Susan Michaelis

    The nuclear lamins A, B, and C are intermediate filament proteins that form a nuclear scaffold adjacent to the inner nuclear membrane in higher eukaryotes, providing structural support for the nucleus. In the past two decades it has become evident that the final step in the biogenesis of the mature lamin A from its precursor prelamin A by the zinc metalloprotease ZMPSTE24 plays a critical role in human health. Defects in prelamin A processing by ZMPSTE24 result in premature aging disorders including Hutchinson Gilford Progeria Syndrome (HGPS) and related progeroid diseases. Additional evidence suggests that defects in prelamin A processing, due to diminished ZMPSTE24 expression or activity, may also drive normal physiological aging. Because of the important connection between prelamin A processing and human aging, there is increasing interest in how ZMPSTE24 specifically recognizes and cleaves its substrate prelamin A, encoded by LMNA. Here, we describe two humanized yeast systems we have recently developed to examine ZMPSTE24 processing of prelamin A. These systems differ from one another slightly. Version 1.0 is optimized to analyze ZMPSTE24 mutations, including disease alleles that may affect the function or stability of the protease. Using this system, we previously showed that some ZMPSTE24 disease alleles that affect stability can be rescued by the proteasome inhibitor bortezomib, which may have therapeutic implications. Version 2.0 is designed to analyze LMNA mutations at or near the ZMPSTE24 processing site to assess whether they permit or impede prelamin A processing. Together these systems offer powerful methodology to study ZMPSTE24 disease alleles and to dissect the specific residues and features of the lamin A tail that are required for recognition and cleavage by the ZMPSTE24 protease.

  • 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.

  • Using Time-lapse Fluorescence Microscopy to Study Gene Regulation
    Methods (IF 3.998) Pub Date : 2018-12-29
    Fan Zou, Lu Bai

    Time-lapse fluorescence microscopy is a powerful tool to study gene regulation. By probing fluorescent signals in single cells over extended period of time, this method can be used to study the dynamics, noise, movement, memory, inheritance, and coordination, of gene expression during cell growth, development, and differentiation. In combination with a flow-cell device, it can also measure gene regulation by external stimuli. Due to the single cell nature and the spatial / temporal capacity, this method can often provide information that is hard to get using other methods. Here, we review the standard experimental procedures and new technical developments in this field.

  • Selective profiling of ribosomes associated with yeast Upf proteins
    Methods (IF 3.998) Pub Date : 2018-12-26
    Robin Ganesan, John Leszyk, Allan Jacobson

    Ribosomes associated with nonsense-mediated decay factors Upf1, Upf2, or Upf3 were purified by immunoprecipitation, and enrichment and stoichiometry of Upf factors and ribosomal proteins were analyzed by western blot and mass spectrometry. Using a small RNA library preparation protocol that eliminates in-gel RNA and cDNA size selection and incorporates four random nucleotides on each side of the ribosome-protected RNA fragment allowed recovery, detection, and analysis of all size classes of protected fragments from a sample simultaneously.

  • Manipulating the mechanics of extracellular matrix to study effects on the nucleus and its structure
    Methods (IF 3.998) Pub Date : 2018-12-26
    Yuntao Xia, Sangkyun Cho, Manasvita Vashisth, Irena L. Ivanovska, P.C. Dave P. Dingal, Dennis E. Discher

    Tissues such as brain, muscle, and bone differ greatly not only in their biological functions but also in their mechanical properties. Stiffness of the extracellular microenvironment affects processes such as cell polarization and DNA replication, thereby regulating nuclear size, shape, and levels of nuclear proteins such as the lamins that can modulate gene expression. Reductionist approaches have helped dissect the effects of matrix mechanics away from confounding biochemical signals. Here, we summarize materials and methods for synthesizing and characterizing soft and stiff synthetic hydrogels widely used for mechanobiological studies. Such gels are easily made to mimic the mechanical heterogeneity of fibrotic tissues. We also describe a nano-thin collagen film system, which enables control of fiber isotropy in addition to bulk material stiffness. With the different systems, we illustrate the effects of matrix stiffness on nuclear size, shape, and proteins including the lamins.

  • A lentivirus-based system for Cas9/gRNA expression and subsequent removal by Cre-mediated recombination
    Methods (IF 3.998) Pub Date : 2018-12-19
    Michael A. Carpenter, Emily K. Law, Artur Serebrenik, William L. Brown, Reuben S. Harris

    A major concern of CRISPR and related genome engineering technologies is off-target mutagenesis from prolonged exposure to Cas9 and related editing enzymes. To help mitigate this concern we added a loxP site to the 3’-LTR of an HIV-based lentiviral vector capable of expressing Cas9/gRNA complexes in a wide variety of mammalian cell types. Transduction of susceptible target cells yields an integrated provirus that expresses the desired Cas9/gRNA complex. The reverse transcription process also results in duplication of the 3’-LTR such that the integrated provirus becomes flanked by loxP sites (floxed). Subsequent expression of Cre recombinase results in loxP-to-loxP site-specific recombination that deletes the Cas9/gRNA payload and effectively prevents additional Cas9-mediated mutations. This construct also expresses a gRNA with a single transcription termination sequence, which results in higher expression levels and more efficient genome engineering as evidenced by disruption of the SAMHD1 gene. This hit-and-run CRISPR approach was validated by recreating a natural APOBEC3B deletion and by disrupting the mismatch repair gene MSH2. This hit-and-run strategy may have broad utility in many areas and especially those where cell types are difficult to engineer by transient delivery of ribonucleoprotein complexes.

  • Transcriptome-wide identification of A-to-I RNA editing sites using ICE-seq
    Methods (IF 3.998) Pub Date : 2018-12-20
    Shunpei Okada, Hiroki Ueda, Yuta Noda, Tsutomu Suzuki

    In A-to-I RNA editing, adenosine is converted to inosine in double-stranded regions of RNAs. Inosine, an abundant epitranscriptomic mark, contributes to a wide range of biological processes by regulating gene expression post-transcriptionally. To understand the effect of A-to-I RNA editing on regulation of the epitranscriptome, accurate mapping of inosines is necessary. To this end, we established a biochemical method called inosine chemical erasing sequencing (ICE-seq) that enables unbiased and reliable identification of A-to-I RNA editing sites throughout the transcriptome. Here, we describe our updated protocol for ICE-seq in the human transcriptome.

  • Defining nonsense-mediated mRNA decay intermediates in human cells
    Methods (IF 3.998) Pub Date : 2018-12-19
    Tatsuaki Kurosaki, Jason R. Myers, Lynne E. Maquat

    Nonsense-mediated mRNA decay (NMD) is a cellular mRNA degradation mechanism that inhibits the expression of aberrant mRNAs harboring premature termination codons (PTCs). Recent progress in transcriptome-wide sequencing techniques has revealed that NMD also degrades approximately 5-30% of non-mutated cellular mRNAs in a way that can be regulated in response to various cellular signals. In mammals, NMD is governed by the central NMD factor UPF1, which is activated by phosphorylation after translation terminates at a nonsense codon that triggers NMD. We have found that immunoprecipitation using an antibody that is specific for phosphorylated UPF1 is a useful tool to define not only cellular NMD targets but also the nature of NMD decay intermediates and, thus, the process of NMD. To this end, we describe here a detailed protocol for what we call “NMD degradome sequencing” using high-throughput technology.

  • Global analysis of RNA metabolism using bio-orthogonal labeling coupled with next-generation RNA sequencing
    Methods (IF 3.998) Pub Date : 2018-12-06
    Michael B. Wolfe, Aaron C. Goldstrohm, Peter L. Freddolino

    Many open questions in RNA biology relate to the kinetics of gene expression and the impact of RNA binding regulatory factors on processing or decay rates of particular transcripts. Steady state measurements of RNA abundance obtained from RNA-seq approaches are not able to separate the effects of transcription from those of RNA decay in the overall abundance of any given transcript, instead only giving information on the (presumed steady-state) abundances of transcripts. Through the combination of metabolic labeling and high-throughput sequencing, several groups have been able to measure both transcription rates and decay rates of the entire transcriptome of an organism in a single experiment. This review focuses on the methodology used to specifically measure RNA decay at a global level. By comparing and contrasting approaches and describing the experimental protocols in a modular manner, we intend to provide both experienced and new researchers to the field the ability to combine aspects of various protocols to fit the unique needs of biological questions not addressed by current methods.

  • Practical considerations on performing and analyzing CLIP-seq experiments to identify transcriptomic-wide RNA-Protein interactions
    Methods (IF 3.998) Pub Date : 2018-12-06
    Xiaoli Chen, Sarah A. Castro, Qiuying Liu, Wenqian Hu, Shaojie Zhang

    RNA-binding proteins are important players in post-transcriptional regulation, such as modulating mRNA splicing, translation, and degradation under diverse biological settings. Identifying and characterizing the RNA substrates is a critical step in deciphering the function and molecular mechanisms of the target RNA-binding proteins. High-throughput sequencing of the RNA fragments isolated by crosslinking immunoprecipitation (CLIP-seq) is one of the standard techniques to identify the in vivo transcriptome-wide binding sites of the target RNA-binding protein. This method is widely used in functional and mechanistic characterizations of RNA-binding proteins. In this review, we provide several practical considerations on performing and analyzing CLIP-seq experiments. Particularly, we focus on how to perform CLIP-seq experiments on endogenous RNA-binding proteins. In addition, we provide a practical summary on how to choose and use computational pipelines from an increasing number of computational methods and packages that are available for analyzing the sequencing datasets from the CLIP-seq experiments. We hope these practical considerations will facilitate experimental biologists in performing and analyzing CLIP-seq experiment to obtain biologically relevant mechanistic insights.

  • Engineered Viral RNA Decay Intermediates to Assess XRN1-mediated decay
    Methods (IF 3.998) Pub Date : 2018-12-03
    Joseph Russo, Cary T. Mundell, Phillida A. Charley, Carol Wilusz, Jeffrey Wilusz

    Both RNA synthesis and decay must be balanced within a cell to achieve proper gene expression. Additionally, modulation of RNA decay specifically offers the cell an opportunity to rapidly reshape the transcriptome in response to specific stimuli or cues. Therefore, it is critical to understand the underlying mechanisms through which RNA decay contribute to gene expression homeostasis. Cell-free reconstitution approaches have been used successfully to reveal mechanisms associated with numerous post-transcriptional RNA processes. Historically, it has been difficult to examine all aspects of RNA decay in such an in vitro setting due, in part, to limitations on the ability to resolve larger RNAs through denaturing polyacrylamide gels. Thus, in vitro systems to study RNA decay rely on smaller, less biologically relevant RNA fragments. Herein, we present an approach to more confidently examine RNA decay parameters of large mRNA size transcripts through the inclusion of an engineered XRN1-resistant reporter RNA (xrRNA). By placing a 67 nucleotide xrRNA near the 3’ end of any in vitro transcribed RNA with variable size or sequence context, investigators can observe the accumulation of the xrRNA as a readout of exoribonuclease-mediated 5’-3’ decay. This approach may allow in vitro RNA decay assays to include full biologically relevant mRNA/mRNPs, extending their utility and allow improved experimental design considerations to promote biologically relevant outcomes.

  • Genome-wide probing RNA structure with the modified DMS-MaPseq in Arabidopsis
    Methods (IF 3.998) Pub Date : 2018-11-29
    Zhiye Wang, Meiyue Wang, Tian Wang, Yijing Zhang, Xiuren Zhang

    Transcripts have intrinsic propensity to form stable secondary structure that is fundamental to regulate RNA transcription, splicing, translation, RNA localization and turnover. Numerous methods that integrate chemical reactions with next-generation sequencing (NGS) have been applied to study in vivo RNA structure, providing new insights into RNA biology. Dimethyl sulfate (DMS) probing coupled with mutational profiling through NGS (DMS-MaPseq) is a newly developed method for revealing genome-wide or target-specific RNA structure. Herein, we present our experimental protocol of a modified DMS-MaPseq method for plant materials. The DMS treatment condition was optimized, and library preparation procedures were simplified. We also provided custom scripts for bioinformatic analysis of genome-wide DMS-MaPseq data. Bioinformatic results showed that our method could generate high-quality and reproducible data. Further, we assessed sequencing depth and coverage for genome-wide RNA structure profiling in Arabidopsis, and provided two examples of in vivo structure of mobile RNAs. We hope that our modified DMS-MaPseq method will serve as a powerful tool for analyzing in vivo RNA structurome in plants.

  • Sequencing-based methods for detection and quantitation of ribose methylations in RNA
    Methods (IF 3.998) Pub Date : 2018-11-29
    Nicolai Krogh, Henrik Nielsen

    Ribose methylation is one of the most abundant RNA modifications and is found in all domains of life and all major classes of RNA (rRNA, tRNA, and mRNA). Ribose methylations are introduced by stand-alone enzymes or by generic enzymes guided to the target by small RNA guides. Recent years have seen the development of several sequencing-based methods for RNA modifications relying on different principles. In this review, we compare mapping and quantitation studies of ribose methylations from yeast and human culture cells. The emphasis is on ribosomal RNA for which the results can be compared to results from RNA fingerprinting and mass spectrometry. One sequencing approach is consistent with these methods and paints a conservative picture of rRNA modifications. Other approaches detect many more sites. Similar discrepancies are found in measurements of modification stoichiometry. The results are discussed in relation to the more challenging task of mapping ribose methylations in mRNA.

  • 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-Ig™) 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.

  • 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 W. 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 speech-based 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.

  • 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.

  • 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
  • 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.

  • Single molecule analysis of lamin dynamics
    Methods (IF 3.998) Pub Date : 2018-08-24
    Leonid A. Serebryannyy, David A. Ball, Tatiana S. Karpova, Tom Misteli

    The nuclear envelope (NE) is an essential cellular structure that contributes to nuclear stability, organization, and function. Mutations in NE-associated proteins result in a myriad of pathologies with widely diverse clinical manifestations, ages of onsets, and affected tissues. Notably, several hundred disease-causing mutations have been mapped to the LMNA gene, which encodes the intermediate filament proteins lamin A and C, two of the major architectural components of the nuclear envelope. However, how NE dysfunction leads to the highly variable pathologies observed in patient cells and tissues remains poorly understood. One model suggests alterations in the dynamic properties of the nuclear lamina and its associated proteins contribute to disease phenotype. Here, we describe the application of single molecule tracking (SMT) methodology to characterize the behavior of nuclear envelope transmembrane proteins and nuclear lamins in their native cellular environment at the single molecule level. As proof-of-concept, we demonstrate by SMT that Halo-tagged lamin B1, Samp1, lamin A, and lamin AΔ50 have distinct binding and kinetic properties, and we identify several disease-relevant mutants which exhibit altered binding dynamics. SMT is also able to separately probe the dynamics of the peripheral and the nucleoplasmic populations of lamin A mutants. We suggest that SMT is a robust and sensitive method to investigate the relationship between pathogenic mutations or cellular processes and protein dynamics at the NE.

  • An IgG1-like bispecific antibody targeting CD52 and CD20 for the treatment of B-cell malignancies
    Methods (IF 3.998) Pub Date : 2018-08-24
    Junpeng Qi, Shih-Shih Chen, Nicholas Chiorazzi, Christoph Rader

    Bispecific antibodies (biAb) targeting two different antigens or two distinct epitopes on the same antigen have demonstrated broad therapeutic utility. CD52 and CD20 are co-expressed on the cell surface of malignant B cells in B-cell non-Hodgkin lymphoma (B-NHL) and chronic lymphocytic leukemia (CLL) and increased expression of both antigens is detected on dividing or recently divided cells (“proliferative fraction”) in CLL. The CD52-targeting monoclonal antibody (mAb) alemtuzumab (atz) not only depletes malignant B cells but also healthy CD52+ B and T lymphocytes and monocytes, causing severe immunosuppression. Loss of CD20 can occur in CLL after treatment with rituximab (rtx) and other CD20-targeting mAbs. To broaden the benefit of atz and rtx, we engineered an IgG1-like biAb, atz × rtx scFv-Fc. The Fc fragment of the biAb facilitates purification by Protein A affinity chromatography and supports a longer circulatory half-life. While atz × rtx scFv-Fc retained both antigen binding specificities, it showed superior binding to CD52+CD20+ B cells compared to CD52+CD20− T cells. Moreover, atz × rtx scFv-Fc mediated potent complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) in vitro and exhibited B-cell depleting but T-cell sparing activities in vivo in a CLL patient-derived xenograft model. B-cell depletion was more pronounced for cells of the proliferative fraction.

  • 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.

  • Current Strategies for Site-Directed RNA editing using ADARs
    Methods (IF 3.998) Pub Date : 2018-11-29
    Maria Fernanda Montiel-Gonzalez, Juan Felipe Diaz Quiroz, Joshua J.C. Rosenthal

    Adenosine Deaminases that Act on RNA (ADARs) are a group of enzymes that catalyze the conversion of adenosines (A’s) to inosines (I’s) in a process known as RNA editing. Though ADARs can act on different types of RNA, editing events in coding regions of mRNA are of particular interest as I’s base pair like guanosines (G’s). Thus, every A-to-I change catalyzed by ADAR is read as an A-to-G change during translation, potentially altering protein sequence and function. This ability to re-code makes ADAR an attractive therapeutic tool to correct genetic mutations within mRNA. The main challenge in doing so is to re-direct ADAR’s catalytic activity towards A’s that are not naturally edited, a process termed Site-Directed RNA Editing (SDRE). Recently, a handful of labs have taken up this challenge and two basic strategies have emerged. The first involves redirecting endogenous ADAR to new sites by making editable structures using antisense RNA oligonucleotides. The second also utilizes antisense RNA oligonucleotides, but it uses them as guides to deliver the catalytic domain of engineered ADARs to new sites, much as CRISPR guides deliver Cas nucleases. In fact, despite the intense current focus on CRISPR-Cas9 genome editing, SDRE offers a number of distinct advantages. In the present review we will discuss these strategies in greater detail, focusing on the concepts on which they are based, how they were developed and tested, and their respective advantages and disadvantages. Though the precise and efficient re-direction of ADAR activity still remains a challenge, the systems that are being developed lay the foundation for SDRE as a powerful tool for transient genome editing.

  • Cell-type specific polysome profiling from mammalian tissues
    Methods (IF 3.998) Pub Date : 2018-11-27
    Joseph Seimetz, Waqar Arif, Sushant Bangru, Mikel Hernaez, Auinash Kalsotra

    The regulation of gene expression occurs through complex relationships between transcription, processing, turnover, and translation, which are only beginning to be elucidated. We know that at least for certain messenger (m) RNAs, processing, modifications, and sequence elements can greatly influence their translational output through recognition by translation and turn-over machinery. Recently, we and others have combined high-throughput sequencing technologies with traditional biochemical methods of studying translation to extend our understanding of these relationships. Additionally, there is growing importance given to how these processes may be regulated across varied cell types as a means to achieve tissue-specific expression of proteins. Here, we provide an in-depth methodology for polysome profiling to dissect the composition of mRNAs and proteins that make up the translatome from both whole tissues and a specific cell type isolated from mammalian tissue. Also, we provide a detailed computational workflow for the analysis of the next-generation sequencing data generated from these experiments.

  • Combined Bead-Based Multiplex Detection of RNA and Protein Biomarkers: Implications for Understanding the Time Course of Skeletal Muscle Injury and Repair
    Methods (IF 3.998) Pub Date : 2018-11-22
    Melody A. Gary, Elizabeth A. Tanner, Asheal A. Davis, Brian K. McFarlin

    Biological response to skeletal muscle injury time course is generally classified as initial (elevated within first 4-h), delayed (elevated at 24-h), and/or prolonged (elevated at 4-h and sustained to 24-h). Accurate description of this process requires the ability to measure a robust set of RNA and protein biomarkers, yet such an approach is not common and not always feasible. This method proposes a novel experimental approach that focuses on the use of bead-based multiplex detection to measure mRNA, lncRNA, cytokines, soluble cytokine receptors, and myokines at 4-h and 24-h post muscle injury. We used an extreme aerobic exercise session (half-marathon race) to create a consistent muscle injury stimulus via oxidative stress and eccentric contractions. Venous blood samples were analyzed to determine the change in 90 targets. Specifically, we identified 14 mRNA, 2 lncRNA, 4 cytokines, and 5 myokines that had only an initial response (change at 4-h). We identified 2 mRNA, 2 cytokines, 13 soluble cytokine receptors, and 1 myokine that had only a delayed response (change at 24-h). Finally, we identified 18 mRNA, 4 lncRNA, 6 myokines and 15 cytokines that had a prolonged response (change at 4-h and sustained at 24-h). We found 4 targets to be undetectable or having no response relative to muscle injury recovery. These findings demonstrate the interplay between RNA and protein biomarkers in response to skeletal muscle injury. This novel experimental application of bead-based multiplexing is applicable to a variety of clinical models that involve muscle injury and/or wasting.

  • Single base resolution mapping of 2’-O-methylation sites in human mRNA and in 3’ terminal ends of small RNAs
    Methods (IF 3.998) Pub Date : 2018-11-22
    Phillip J. Hsu, Qili Fei, Qing Dai, Hailing Shi, Dan Dominissini, Lijia Ma, Chuan He

    The post-transcriptional modification 2’-O-Methyl (2’OMe) could be present on the ribose of all four ribonucleosides, and is highly prevalent in a wide variety of RNA species, including the 5’ RNA cap of viruses and higher eukaryotes, as well as internally in transfer RNA and ribosomal RNA. Recent studies have suggested that 2’OMe is also located internally in low-abundance RNA species such as viral RNA and mRNA. To profile 2’OMe on different RNA species, we have developed Nm-seq, which could identify 2’OMe sites at single base resolution. Nm-seq is particularly useful for identifying 2’OMe sites located at the 3’ terminal ends of small RNAs. Here, we present an optimized protocol for Nm-seq and a protocol for applying Nm-seq to identify 2’OMe sites on small RNA 3’ terminal ends.

  • Ancestral Transcriptome Inference Based on RNA-Seq and ChIP-seq Data
    Methods (IF 3.998) Pub Date : 2018-11-22
    Jingwen Yang, Hang Ruan, Yangyun Zou, Zhixi Su, Xun Gu

    With the help of high-throughput NGS (next-generation sequencing) technologies, ancestral transcriptome reconstruction is helpful to understand the complexity of transcriptional regulatory systems that underlies the evolution of multiple cellular metazoans with sophisticated functions and distinctive morphologies. To this end, we report a new method of ancestral state inference. The new method used Ornstein-Uhlenbeck (OU) model, which is more biologically realistic, to replace the Brownian motion (BM) model and is suitable for multi-transcriptome data. Implemented in the free R package, AnceTran is specially designed for RNA-seq and ChIP-seq data, which is feasible. It should be noticed that our work will be integrated to a unified, statistically-sound phylogenetic framework to study the evolution of many other molecular phenomes such as proteomics, chromatin accessibility, methylation status, and metabolomics. We exemplify our method by a case study, using the ChIP-seq binding data of three liver-specific transcription factors and the RNA-seq liver expression data in four closely related mice species, and some technical issues are discussed.

  • Combining Single Molecule Counting with Bead-Based Multiplexing to Quantify Biological Inflammation Time Course Following Skeletal Muscle Injury
    Methods (IF 3.998) Pub Date : 2018-11-22
    Elizabeth A. Tanner, Melody A. Gary, Asheal A. Davis, Brian K. McFarlin

    Bead-based analysis methods allow for the exploration of a variety of complex biological processes. In particular, these techniques can be applied to better understand how peripheral muscle injury contributes to systemic inflammation. Understanding how these two processes affect one another can give additional insight concerning how changes in inflammation effect readiness to perform in exercise and work environments. The present method sought to combine the strengths of bead-based multiplexing with the precision and low-end detection of single molecule counting (SMC) methods. We used performance of an extreme aerobic exercise session (i.e. half-marathon race) to cause a defined quantity of lower body muscle injury and a systemic inflammatory response lasting up to 24 hours. Using a high-sensitivity, multiplex assay (Milliplex; Millipore-Sigma) we were able to identify 9 of 21 cytokines that were significantly elevated at either 4 or 24 hours post half-marathon performance. Despite the known role of IL-1β, IL-6, and TNF-α in the pro-inflammatory response, they did not appear to change based on the multiplex analysis. We thus, conducted further analysis using an SMC assay and found increases in IL-1β, IL-6, and TNF-α at 4h compared to 24h post exercise. This method approach demonstrates how combining two common, bead-based protein assays can increase the amount of meaningful biological information that can be collected. We anticipate that this approach will be useful in a variety of inflammation-associated disease states.

  • Analysis of Spliceosome Dynamics by Maximum Likelihood Fitting of Dwell Time Distributions
    Methods (IF 3.998) Pub Date : 2018-11-23
    Harpreet Kaur, Fatemehsadat Jamalidinan, Samson G.F. Condon, Alessandro Senes, Aaron A. Hoskins

    Colocalization single-molecule methods can provide a wealth of information concerning the ordering and dynamics of biomolecule assembly. These have been used extensively to study the pathways of spliceosome assembly in vitro. Key to these experiments is the measurement of binding times—either the dwell times of a multi-molecular interaction or times in between binding events. By analyzing hundreds of these times, many new insights into the kinetic pathways governing spliceosome assembly have been obtained. Collections of binding times are often plotted as histograms and can be fit to kinetic models using a variety of methods. Here, we describe the use of maximum likelihood methods to fit dwell time distributions without binning. In addition, we discuss several aspects of analyzing these distributions with histograms and pitfalls that can be encountered if improperly binned histograms are used. We have automated several aspects of maximum likelihood fitting of dwell time distributions in the AGATHA software package.

  • Computational approaches for detection and quantification of A-to-I RNA-editing
    Methods (IF 3.998) Pub Date : 2018-11-20
    Yishay Pinto, Erez Y. Levanon

    Adenosine deaminases that act on RNA (ADARs) catalyze adenosine-to-inosine (A-to-I) RNA editing in double-stranded RNA. Such editing is important for protection against false activation of the immune system, but also confers plasticity on the transcriptome by generating several versions of a transcript from a single genomic locus. Recently, great efforts were made in developing computational methods for detecting editing events directly from RNA-sequencing (RNA-seq) data. These efforts have led to an improved understanding of the makeup of the editome in various genomes. Here we review recent advances in editing detection based on the data available to the researcher, with emphasis on the principles underlying the various methods and the limitations they were designed to overcome. We also discuss the available various methods for analyzing and quantifying editing levels. This review collects and organizes the available approaches for analyzing RNA editing and discuss the current status of the different A-to-I detection methods with possible directions for extending these approaches.

  • 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.

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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