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Past Issue in 2021
Volume: 11, Issue: 5
Biochemistry
Simultaneous Imaging of Single Protein Size, Charge, and Binding Using A Protein Oscillation Approach
Electrophoresis and Western blot are important tools in protein research for detection and identification of proteins. These traditional techniques separate the proteins based on size and charge differences and identify the proteins by antibody binding. Over the past decade, the emergence of single-molecule techniques has shown great potential in improving the resolution of the traditional protein analysis methods to the single-molecule level. However, such single-molecule techniques measure either size or charge, and it is challenging to measure both at the same time. Recently, we have developed a single-molecule approach to address this problem. We tether the single proteins to a surface with a polymer linker and drive them into oscillation with an electric field. By tracking the electromechanical response of the proteins to the field using an optical imaging method, the size and charge can be obtained simultaneously. Binding of antibodies or ions to the tethered protein also changes the size and charge, which allows us to probe the interactions. This protocol includes fabrication of protein oscillators, configuration of the optical detection system, and analysis of the oscillation signal for quantification of protein size and charge. We wish this protocol will enable researchers to perform comprehensive single-protein analysis on a single platform.
Identification of Intrinsic RNA Binding Specificity of Purified Proteins by in vitro RNA Immunoprecipitation (vitRIP)
RNA-protein interactions are often mediated by dedicated canonical RNA binding domains. However, interactions through non-canonical domains with unknown specificity are increasingly observed, raising the question how RNA targets are recognized. Knowledge of the intrinsic RNA binding specificity contributes to the understanding of target selectivity and function of an individual protein.The presented in vitro RNA immunoprecipitation assay (vitRIP) uncovers intrinsic RNA binding specificities of isolated proteins using the total cellular RNA pool as a library. Total RNA extracted from cells or tissues is incubated with purified recombinant proteins, RNA-protein complexes are immunoprecipitated and bound transcripts are identified by deep sequencing or quantitative RT-PCR. Enriched RNA classes and the nucleotide frequency in these RNAs inform on the intrinsic specificity of the recombinant protein. The simple and versatile protocol can be adapted to other RNA binding proteins and total RNA libraries from any cell type or tissue.Graphic abstract:Figure 1. Schematic of the in vitro RNA immunoprecipitation (vitRIP) protocol
Biophysics
Automated Analysis of Cerebrospinal Fluid Flow and Motile Cilia Properties in The Central Canal of Zebrafish Embryos
Circulation of cerebrospinal fluid (CSF) plays an important role during development. In zebrafish embryo, the flow of CSF has been found to be bidirectional in the central canal of the spinal cord. In order to compare conditions and genetic mutants across each other, we recently automated the quantification of the velocity profile of exogenous fluorescent particles in the CSF. We demonstrated that the beating of motile and tilted cilia localized on the ventral side of the central canal was sufficient to generate locally such bidirectionality. Our approach can easily be extended to characterize CSF flow in various genetic mutants. We provide here a detailed protocol and a user interface program to quantify CSF dynamics. In order to interpret potential changes in CSF flow profiles, we provide additional tools to measure the central canal diameter, characterize cilia dynamics and compare experimental data with our theoretical model in order to estimate the impact of cilia in generating a volume force in the central canal. Our approach can also be of use for measuring particle velocity in vivo and modeling flow in diverse biological solutions.
Characterize the Interaction of the DNA Helicase PriA with the Stalled DNA Replication Fork Using Atomic Force Microscopy
In bacteria, the restart of stalled DNA replication forks requires the DNA helicase PriA. PriA can recognize and remodel abandoned DNA replication forks, unwind DNA in the 3'-to-5' direction, and facilitate the loading of the helicase DnaB onto the DNA to restart replication. ssDNA-binding protein (SSB) is typically present at the abandoned forks, protecting the ssDNA from nucleases. Research that is based on the assays for junction dissociation, surface plasmon resonance, single-molecule FRET, and x-ray crystal structure has revealed the helicase activity of PriA, the SSB-PriA interaction, and structural information of PriA helicase. Here, we used Atomic Force Microscopy (AFM) to visualize the interaction between PriA and DNA substrates with or without SSB in the absence of ATP to delineate the substrate recognition pattern of PriA before its ATP-catalyzed DNA-unwinding reaction. The protocol describes the steps to obtain high-resolution AFM images and the details of data analysis and presentation.
Cancer Biology
Strategy of Isolating ‘Primed’ Tumor Initiating Cells Based on Mitochondrial Transmembrane Potential
Various stem cells have been found to be dependent on mitochondrial energetics. The role of mitochondria in regulating the self-renewal of normal stem cells and stem-like tumor initiating cells (TICs) is increasingly being appreciated. We proposed that TIC populations have a sub population of cells that are “primed” by mitochondria for self-renewal. Using ovarian cancer model, we have developed a protocol to identify and isolate these “primed” cells using Fluorescence-Assisted Cell Sorting (FACS). We combined live cell stains for a functional marker of TICs and for mitochondrial transmembrane potential to enrich TICs with higher mitochondrial potential that form in vitro spheroids 10-fold more than the other TICs with lower mitochondrial potential. This protocol can be directly used or modified to be used in various cell types. Thus, this protocol is anticipated to be invaluable for the basic understanding of mitochondrial and energetic heterogeneity within stem cell population, and may also prove valuable in translational studies in regenerative medicine and cancer biology.
Cell Biology
Retention Using Selective Hooks (RUSH) Cargo Sorting Assay for Protein Vesicle Tracking in HeLa Cells
Monitoring vesicle trafficking is an excellent tool for the evaluation of protein dynamics in living cells. Such study is key for the understanding of protein sorting and secretion. Recent developments in microscopy, as well as new methodologies developed to study synchronized trafficking of proteins, allowed a better understanding of signaling, regulation and trafficking dynamics at the secretory pathway. One of the most helpful tools so far developed is the Retention Using Selective Hooks (RUSH) system, a methodology that facilitates the evaluation of synchronized cargo trafficking by monitoring fluorescent vesicles in cells upon biotin addition. Here we present a protocol that allows the quantitative evaluation of protein cargo trafficking at different fixed time points and an analytic approach that enables a better examination of specific cargo trafficking dynamics at the secretory pathway.Graphic abstract:Schematic representation of RUSH sorting assay in mammalian cells
Live Intravital Intestine with Blood Flow Visualization in Neonatal Mice Using Two-photon Laser Scanning Microscopy
This protocol describes a novel technique to investigate the microcirculation dynamics underlying the pathology in the small intestine of neonatal mice using two-photon laser-scanning microscopy (TPLSM). Recent technological advances in multi-photon microscopy allow intravital analysis of different organs such as the liver, brain and intestine. Despite these advances, live visualization and analysis of the small intestine in neonatal rodents remain technically challenging. We herein provide a detailed description of a novel method to capture high resolution and stable images of the small intestine in neonatal mice as early as postnatal day 0. This imaging technique allows a comprehensive understanding of the development and blood flow dynamics in small intestine microcirculation.
Microbiology
Primer ID Next-Generation Sequencing for the Analysis of a Broad Spectrum Antiviral Induced Transition Mutations and Errors Rates in a Coronavirus Genome
Next generations sequencing (NGS) has become an important tool in biomedical research. The Primer ID approach combined with the MiSeq platform overcomes the limitation of PCR errors and reveals the true sampling depth of population sequencing, making it an ideal tool to study mutagenic effects of potential broad-spectrum antivirals on RNA viruses. In this report we describe a protocol using Primer ID sequencing to study the mutations induced by antivirals in a coronavirus genome from an in vitro cell culture model and an in vivo mouse model. Viral RNA or total lung tissue RNA is tagged with Primer ID-containing cDNA primers during the initial reverse transcription step, followed by two rounds of PCR to amplify viral sequences and incorporate sequencing adaptors. Purified and pooled libraries are sequenced using the MiSeq platform. Sequencing data are processed using the template consensus sequence (TCS) web-app. The Primer ID approach provides an accurate sequencing protocol to measure mutation error rates in viral RNA genomes and host mRNA. Sequencing results suggested that β-D-N4-hydroxycytidine (NHC) greatly increased the transition substitution rate but not the transversion substitution rate in the viral RNA genomes, and cytosine (C) to uridine (U) was found as the most frequently seen mutation.
Assay for Assessing Mucin Binding to Bacteria and Bacterial Proteins
Legionella pneumophila, a Gram-negative bacterium and the causative agent of Legionnaires’ disease, exports over 300 effector proteins/virulence factors, through its type II (T2SS) and type IV secretion systems (T4SS). One such T2SS virulence factor, ChiA, not only functions as a chitinase, but also as a novel mucinase, which we believe aids ChiA-dependent virulence during lung infection. Previously published protocols manipulated wild-type L. pneumophila strain 130b and its chiA mutant to express plasmid-encoded GFP. Similarly, earlier studies demonstrated that wheat germ agglutinin (WGA) can be fluorescently labeled and can bind to mucins. In the current protocol, GFP-labeled bacteria were incubated with type II and type III porcine stomach mucins, which were then labeled with TexasRed-tagged WGA and analyzed by flow-cytometry to measure the binding of bacteria to mucins in the presence or absence of endogenous ChiA. In addition, we analysed binding of purified ChiA to type II and type III porcine stomach mucins. This protocol couples both bacterial and direct protein binding to mucins and is the first to measure Gram-negative bacterial binding to mucins using WGA and flow-cytometric analysis.Graphic abstract:Strategy for assessing bacterial and protein binding to mucins
Generation and Implementation of Reporter BHK-21 Cells for Live Imaging of Flavivirus Infection
The genus Flavivirus within the family Flaviviridae includes many viral species of medical importance, such as yellow fever virus (YFV), Zika virus (ZIKV), and dengue virus (DENV), among others. Presently, the identification of flavivirus-infected cells is based on either the immunolabeling of viral proteins, the application of recombinant reporter replicons and viral genomes, or the use of cell-based molecular reporters of the flaviviral protease NS2B-NS3 activity. Among the latter, our flavivirus-activatable GFP and mNeptune reporters contain a quenching peptide (QP) joined to the fluorescent protein by a linker consisting of a cleavage site for the flavivirus NS2B-NS3 proteases (AAQRRGRIG). When the viral protease cleaves the linker, the quenching peptide is removed, and the fluorescent protein adopts a conformation promoting fluorescence. Here we provide a detailed protocol for the generation, selection and implementation of stable BHK-21 cells expressing our flavivirus genetically-encoded molecular reporters, suitable to monitor the viral infection by live-cell imaging. We also describe the image analysis procedures and provide the required software pipelines. Our reporter cells allow the implementation of single-cell infection kinetics as well as plaque assays for both reference and native strains of flaviviruses by live-cell imaging.Graphic abstract:Workflow for the generation and implementation of reporter BHK-21 cells for live imaging of flavivirus infection.
Candida Biofilm Formation Assay on Essential Oil Coated Silicone Rubber
Development of biofilm associated candidemia for patients with implanted biomaterials causes an urgency to develop antimicrobial and biofilm inhibitive coatings in the management of recalcitrant Candida infections. Recently, there is an increase in the number of patients with biofilm formation and resistance to antifungal therapy. Therefore, there is a growing interest to use essential oils as coating agents in order to prevent biomaterial-associated Candida infections. Often high costs, complicated and laborious technologies are used for both applying the coating and determination of the antibiofilm effects hampering a rapid screening of essential oils. In order to determine biofilm formation of Candida on essential oil coated surfaces easier, cheaper and faster, we developed an essential oil (lemongrass oil) coated surface (silicone-rubber) by using a hypromellose ointment/essential oil mixture. Furthermore, we modified the “crystal violet binding assay” to quantify the biofilm mass of Candida biofilm formed on the lemongrass oil coated silicone rubber surface. The essential oil coating and the biomass determination of biofilms on silicone rubber can be easily applied with simple and accessible equipment, and will therefore provide rapid information about whether or not a particular essential oil is antiseptic, also when it is used as a coating agent.
Molecular Biology
Defined Mutant Library Sequencing (DML-Seq) for Identification of Conditional Essential Genes
Transposon insertion sequencing (TIS) is an emerging technique which utilizes a massive transposon mutant library to screen specific phenotype and determine the conditional essential genetic requirements for bacterial fitness under distinct conditions combined with high-throughput parallel sequencing technology. Compared with a massive mutant library in traditional TIS, the defined mutant library sequencing (DML-Seq) has advantages as: 1) efficient mutagenesis; 2) low bottleneck effects; 3) avoid hotpots caused by screening; 4) can be directly used in the following experiments. Here, we described an optimized procedure of DML-Seq for fitness screen to supply classical TIS using the marine pathogenic bacterium Edwardsiella piscicida as an example.
Trypanosomatid, fluorescence-based in vitro U-insertion/U-deletion RNA-editing (FIDE)
Gene expression within the mitochondria of African trypanosomes and other protozoan organisms relies on a nucleotide-specific RNA-editing reaction. In the process exclusively uridine (U)-nucleotides are site-specifically inserted into and deleted from sequence-deficient primary transcripts to convert them into translatable mRNAs. The reaction is catalyzed by a 0.8 MDa multiprotein complex termed the editosome. Here we describe an improved in vitro test to quantitatively explore the catalytic activity of the editosome. The assay uses synthetic, fluorophore-derivatized oligoribonucleotides as editing substrates, which enable the automated electrophoretic separation of the reaction products by capillary electrophoresis (CE) coupled to laser-induced fluorescence (LIF) detection systems. The assay is robust, it requires only nanogram amounts of materials and by using multicapillary CE/LIF-instruments it can be executed in a highly parallel layout. Further improvements include the usage of phosphorothioate-modified and thus RNase-resistant substrate RNAs as well as multiplex-type fluorophore labeling strategies to monitor the U-insertion and U-deletion reaction simultaneously. The assay is useful for investigating the mechanism and enzymology of the editosome. However, it can also be executed in high-throughput to screen for RNA editing-specific inhibitors.Graphic abstract:Characteristics of the fluorescence-based in vitro U-insertion/U-deletion RNA-editing (FIDE) assay
Neuroscience
Transcardiac Perfusion of the Mouse for Brain Tissue Dissection and Fixation
Transcardiac perfusion with saline followed by 4% paraformaldehyde (PFA) is widely used to clear blood and preserve brain for immunostaining or in situ hybridization. PFA breaks into formaldehyde in solution, which cross-link protein and DNA molecules to preserve tissue and cell structure. Here we provide a step by step guide for performing this procedure in mouse.
Generation of Mouse Primary Hypothalamic Neuronal Cultures for Circadian Bioluminescence Assays
An endogenous circadian clock system enables organisms to adapt to time-of-day dependent environmental changes. In consequence, most physiological processes exhibit daily rhythms of, e.g., energy metabolism, immune function, sleep, or hormone production. Hypothalamic circadian clocks have been identified to play a particular role in coordinating many of these processes. Primary neuronal cultures are widely used as a physiologically relevant model to study molecular events within neurons. However, as circadian rhythms include dynamic molecular changes over longer timescales that vary between individual cells, longitudinal measurement methods are essential to investigate the regulation of circadian clocks of hypothalamic neurons. Here we provide a protocol for generating primary hypothalamic neuronal cultures expressing a circadian luciferase reporter. Such reporter cells can be used to longitudinally monitor cellular circadian rhythms at high temporal resolution by performing bioluminescence measurements.
Dissociating Behavior and Spatial Working Memory Demands Using an H Maze
The development of mazes for animal experiments has allowed for the investigation of cognitive maps and place cells, spatial working memory, naturalistic navigation, perseverance, exploration, and choice and motivated behavior. However, many mazes, such as the T maze, currently developed to test learning and memory, do not distinguish temporally and spatially between the encoding and recall periods, which makes it difficult to study these stages separately when analyzing animal behavior and electrophysiology. Other mazes, such as the radial maze, rely on single visits to portions of the maze, making maze coverage sparse for place cell and electrophysiology experiments. In this protocol, we present instructions for building and training an animal on a spatial appetitive choice task on a low-cost double-sided T (or H) maze. This maze has several advantages over the traditional T maze and radial mazes. This maze is unique in that it temporally and directionally dissociates the memory encoding and retrieval periods, while requiring the same behaviors of the animal during both periods. This design allows for independent investigation of brain mechanisms, such as cross-region theta coordination, during memory encoding and retrieval, while at least partially dissociating these stages from behavior. This maze has been previously used in our laboratory to investigate cell firing, single-region local field potential (LFP) patterns, and cross region LFP coherence in the hippocampus, lateral septum, prefrontal cortex, and ventral tegmental area, as well as to investigate the effects of hippocampal theta perturbations on task performance.
Plant Science
Chromatographic Analysis for Targeted Metabolomics of Antioxidant and Flavor-Related Metabolites in Tomato
Targeted metabolomics is a useful approach to evaluate crop breeding studies. Antioxidant and flavor-related traits are of increasing interest and are considered quality traits in tomato breeding. The present study presents chromatographic methods to study antioxidants (carotenoids, vitamin C, vitamin E, phenolic compounds, and glutathione) and flavor-related characters (sugars and organic acids) in tomato. Two different extraction methods (for polar and apolar entities) were applied to isolate the targeted compounds. The extraction methods developed in this work were time and cost-effective since no further purification was needed. Carotenoids, vitamin C, glutathione, and phenolic acids were analyzed by HPLC-PDA using a RP C18 column at an appropriate wavelength for each compound. Vitamin E and sugars were analyzed by HPLC with RP C18 and NH2 columns and detected by FLD and RI detectors, respectively. In addition, organic acids were analyzed with GC-FID using a Rtx 5DA column after derivatization with MSTFA. As a result, sensitive analytical methods to quantify important plant metabolites were developed and are described herein. These methods are not only applicable in tomato but are also useful to characterize other species for flavor-related and antioxidant compounds. Thus, these protocols can be used to guide selection in crop breeding.
Stem Cell
Generation of Human iPSC-derived Neural Progenitor Cells (NPCs) as Drug Discovery Model for Neurological and Mitochondrial Disorders
The high attrition rate in drug development processes calls for additional human-based model systems. However, in the context of brain disorders, sampling live neuronal cells for compound testing is not applicable. The use of human induced pluripotent stem cells (iPSCs) has revolutionized the field of neuronal disease modeling and drug discovery. Thanks to the development of iPSC-based neuronal differentiation protocols, including tridimensional cerebral organoids, it is now possible to molecularly dissect human neuronal development and human brain disease pathogenesis in a dish. These approaches may allow dissecting patient-specific treatment efficacy in a disease-relevant cellular context. For drug discovery approaches, however, a highly reproducible and cost-effective cell model is desirable. Here, we describe a step-by-step process for generating robust and expandable neural progenitor cells (NPCs) from human iPSCs. NPCs generated with this protocol are homogeneous and highly proliferative. These features make NPCs suitable for the development of high-throughput compound screenings for drug discovery. Human iPSC-derived NPCs show a metabolism dependent on mitochondrial activity and can therefore be used also to investigate neurological disorders in which mitochondrial function is affected. The protocol covers all steps necessary for the preparation, culture, and characterization of human iPSC-derived NPCs.Graphic abstract:Schematic of the protocol for the generation of human iPSC-derived NPCs