Identification of Long Noncoding RNAs in the Developing Endosperm of Maize.
Abstract Maize endosperm consists of three distinct types of tissues, including the starchy endosperm (SE), the basal endosperm transfer cell layer (BETL), and the aleurone cell layer (AL). Compartmentalization of these tissues during endosperm differentiation makes the endosperm development an excellent model to study changes in gene expression during development. By utilizing cryo-dissection of developing endosperm, morphologically distinct samples can be obtained for transcriptome and epigenome analysis. Here, we describe methods for the isolation of tissues from developing maize endosperm and for the transcrip...
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Kim E, Xiong Y, Kang BH, Sung S Tags: Methods Mol Biol Source Type: research

Medium-Throughput RNA In Situ Hybridization of Serial Sections from Paraffin-Embedded Tissue Microarrays.
Abstract (m)RNA spatiotemporal pattern of distribution is of key importance to decipher gene function. In this post-genomic era, numerous transcriptomic studies are made publicly available, sometimes reaching a tissular resolution and even more rarely the cellular level. This "one tissue-numerous genes" information can be completed by the reverse "one gene-numerous tissues" picture through traditional RNA in situ hybridization (ISH). Here, we present a method including (1) principles of transcriptomic data mining to be performed prior and following ISH and (2) a detailed step-by-step medium-thr...
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Francoz E, Ranocha P, Dunand C, Burlat V Tags: Methods Mol Biol Source Type: research

Purification and Functional Analysis of Plant Long Noncoding RNAs (lncRNA).
Abstract More than 70% of eukaryotic genomes are transcribed into RNA transcripts, the majority of these transcripts are noncoding protein, and their biological functions are largely unknown. Over the last decade, the application of high-throughput sequencing technologies has led to the description of almost all cellular coding and noncoding RNA transcripts except perhaps for those transcripts that are lowly abundant or those present only in specific cells that are underrepresented in sampled tissue(s). An often underrepresented class of noncoding are long noncoding RNAs (lncRNAs), and these often play key regulat...
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Do T, Qu Z, Searle I Tags: Methods Mol Biol Source Type: research

An Easy-to-Follow Pipeline for Long Noncoding RNA Identification: A Case Study in Diploid Strawberry Fragaria vesca.
Abstract Long noncoding RNAs (lncRNAs), defined as transcripts longer than 200 nucleotides without coding potential, are a new class of regulatory molecules with roles in diverse biological processes. New lncRNAs can readily be identified by mining RNA-seq data from a wide range of plant species. However, challenges remain as to how one can distinguish functional lncRNAs from mRNAs coding for small peptides or products of pseudogenes without any function. In this chapter, stepwise instruction is provided using RNA-seq datasets of developing wild strawberry fruit to illustrate each step. The workflow can be divided...
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Kang C, Liu Z Tags: Methods Mol Biol Source Type: research

Identification of Long Noncoding RNA-Protein Interactions Through In Vitro RNA Pull-Down Assay with Plant Nuclear Extracts.
Abstract Recent advances in next-generation sequencing have revealed that majority of the plant genome is transcribed into long noncoding RNA (lncRNA). Many lncRNAs function by interacting with proteins and forming regulatory complexes. RNA-protein interactions are vital in controlling core cellular processes like transcription and translation. Therefore, identifying proteins that interact with lncRNAs is the first step to deciphering lncRNA functions. Here, we describe an RNA-protein pull-down assay, which enables the identification of proteins that interact with an RNA under study. As an example, we describe pul...
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Seo JS, Chua NH Tags: Methods Mol Biol Source Type: research

In Vivo Genome-Wide RNA Structure Probing with Structure-seq.
Abstract In vivo genome-wide RNA structure probing provides a global view of RNA structure as it occurs in the cell and can assist in elucidating important functional aspects of RNA structure. Structure-seq2 provides high-quality data on transcriptome-wide RNA structure in vivo but contains numerous steps that require technical precision. In this chapter we present the steps needed to produce high-quality structural data with a focus on controls and troubleshooting. Structure-seq2 can be applied to numerous organisms including plants, humans, and bacteria and is amenable to a wide variety of RNA-modifying chemical...
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Ritchey LE, Su Z, Assmann SM, Bevilacqua PC Tags: Methods Mol Biol Source Type: research

Using Protein Interaction Profile Sequencing (PIP-seq) to Identify RNA Secondary Structure and RNA-Protein Interaction Sites of Long Noncoding RNAs in Plants.
Abstract From the moment of transcription, RNA molecules are continuously bound by RNA-binding proteins (RBPs). While the majority of research has focused on how these RBPs regulate posttranscriptional gene regulation of messenger RNAs (mRNAs), the majority of cellular RNAs do not code for proteins, such as ribosomal RNAs, transfer RNAs, and microRNAs. Since these RNAs do not code for protein, their function is mainly determined by their interactions with RBPs as well as their intramolecular base pairing, or RNA secondary structure. One class of noncoding RNAs termed long noncoding RNAs (lncRNAs) have recently bec...
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Kramer MC, Gregory BD Tags: Methods Mol Biol Source Type: research

In Situ Hi-C for Plants: An Improved Method to Detect Long-Range Chromatin Interactions.
Abstract Recently, long noncoding RNAs (lncRNAs) are shown to be implicating nuclear domain organization and gene regulation by mediating long-range chromatin interactions. Chromosome conformation capture (3C) is a method used to study such long-range interaction between two different loci in the 3D nuclear space. Through successive improvement in resolution and throughput, 3C, chromosome conformation capture on chip (4C), and chromosome conformation capture carbon copy (5C) to Hi-C methods were developed to study interactions between loci from one versus one scale to an unprecedented genome-wide resolution. In si...
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Padmarasu S, Himmelbach A, Mascher M, Stein N Tags: Methods Mol Biol Source Type: research

Molecular Dynamic Simulations to Probe Water Permeation Pathways of GPCRs.
Abstract Rhodopsin is a light-driven G protein-coupled receptor mediating signal transduction in eyes. The molecular dynamics (MD) simulations are powerful computational tools to investigate molecular behavior of proteins and internal water molecules which are related to the function of proteins; however, the MD simulations of the rhodopsin require several technical setups for accurate calculations. This chapter discusses practical methods for setting up the MD simulations of the rhodopsin [preparation of initial systems, condition files for MD simulation package GROMACS, and data analysis]. The data analysis incl...
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Tomobe K, Yamamoto E, Yasuoka K Tags: Methods Mol Biol Source Type: research

A Combined Cell-Free Protein Synthesis and Fluorescence-Based Approach to Investigate GPCR Binding Properties.
Abstract Fluorescent labeling of de novo synthesized proteins is in particular a valuable tool for functional and structural studies of membrane proteins. In this context, we present two methods for the site-specific fluorescent labeling of difficult-to-express membrane proteins in combination with cell-free protein synthesis. The cell-free protein synthesis system is based on Chinese Hamster Ovary Cells (CHO) since this system contains endogenous membrane structures derived from the endoplasmic reticulum. These so-called microsomes enable a direct integration of membrane proteins into a biological membrane. In th...
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Zemella A, Richter T, Thoring L, Kubick S Tags: Methods Mol Biol Source Type: research

Furan Cross-Linking Technology for Investigating GPCR-Ligand Interactions.
Abstract Interactions between G protein-coupled receptors and their ligands hold extensive potential for drug discovery. Studying these interactions poses technical problems due to their transient nature and the inherent difficulties when working with G protein-coupled receptors (GPCR) that are only functional in a membrane setting. Here, we describe the use of a furan-based chemical cross-linking methodology to achieve selective covalent coupling between a furan-modified peptide ligand and its native GPCR present on the surface of living cells under normal cell culture conditions. This methodology relies on the o...
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Van Troys M, Vannecke W, Ampe C, Madder A Tags: Methods Mol Biol Source Type: research

Chemoselective Acylation of Hydrazinopeptides to Access Fluorescent Probes for Time-Resolved FRET Assays on GPCRs.
net D Abstract Fluorescence techniques represent a powerful tool to investigate dynamic and functional architecture of GPCRs. Thus, fluorescent GPCR ligands have found various applications in cellular imaging, in the development of binding assays as replacements for radioligands in the study of ligand-receptor but also in receptor-receptor interactions at the cell surface or in native tissues. To extend the applicability of these techniques, the design and the synthesis of fluorescent probes are critical steps. As there are numerous peptide receptors in the GPCR family, fluorescent peptide-based probes are of impo...
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Ramanoudjame SM, Esteoulle L, Riché S, Margathe JF, Durroux T, Karpenko IA, Bonnet D Tags: Methods Mol Biol Source Type: research

Time-Resolved FRET-Based Assays to Characterize G Protein-Coupled Receptor Hetero-oligomer Pharmacology.
Abstract Although G protein-coupled receptor (GPCR) oligomerization is a matter of debate, it has been shown that the nature of the GPCR partners within the oligomers can influence the pharmacological properties of the receptors. Therefore, finding specific ligands for homo- or hetero-oligomers opens new perspectives for drug discovery. However, no efficient experimental strategy to screen for such ligands existed yet. Indeed, conventional binding strategies do not discriminate ligand binding on GPCR monomers, homo- or hetero-oligomers. To address this issue, we recently developed a new assay based on a time-resol...
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Heuninck J, Hounsou C, Dupuis E, Trinquet E, Mouillac B, Pin JP, Bonnet D, Durroux T Tags: Methods Mol Biol Source Type: research

Combining Conformational Profiling of GPCRs with CRISPR/Cas9 Gene Editing Approaches.
rt TE Abstract Ligand-biased signaling could have a significant impact on drug discovery programs. As such, many approaches to screening now target a larger section of the signaling responses downstream of an individual G protein-coupled receptor (GPCR). Biosensor-based platforms have been developed to capture signaling signatures. Despite the ability to use such signaling signatures, they may still be particular to an individual cell type and thus such platforms may not be portable from cell to cell, necessitating further cell-specific biosensor development. We have developed a complementary strategy based on cap...
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Bourque K, Devost D, Inoue A, Hébert TE Tags: Methods Mol Biol Source Type: research

Measuring GPCR Stoichiometry Using Types-1, -2, and -3 Bioluminescence Resonance Energy Transfer-Based Assays.
Abstract How G protein-coupled receptors are assembled is a matter of considerable interest owing in large part to their remarkable pharmacological importance. For determining receptor stoichiometry, resonance energy transfer-based methods offer considerable advantages insofar as they provide the necessary spatial resolution, and because measurements can be made in situ, relatively easily. This chapter describes three complementary stoichiometric assays that rely on measurements of bioluminescence resonance energy transfer. These quantitative approaches make it possible to identify true protein-protein interaction...
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Felce JH, James JR, Davis SJ Tags: Methods Mol Biol Source Type: research

Quantification and Comparison of Signals Generated by Different FRET-Based cAMP Reporters.
Abstract A variety of FRET-based biosensors are currently in use for real-time monitoring of dynamic changes of intracellular cAMP. Due to differences in sensor properties, unique features of the cell type under examination and diverse specifications of the imaging setups in different laboratories, data generated using these sensors may not be immediately comparable within the same study or across studies. To facilitate comparison, often FRET data are normalized and expressed as fractional change of the maximal FRET response at sensor saturation. However, this approach may lead to misinterpretation of the underlyi...
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Koschinski A, Zaccolo M Tags: Methods Mol Biol Source Type: research

Measurement of β-Arrestin Recruitment at GPCRs Using the Tango Assay.
Measurement of β-Arrestin Recruitment at GPCRs Using the Tango Assay. Methods Mol Biol. 2019;1947:257-267 Authors: Laroche G, Giguère PM Abstract Intracellular signal transduced by G protein-coupled receptors (GPCRs) is tightly controlled by a guanine nucleotide-binding complex made of G protein Gα, Gβ, and Gγ subunits, as well as a growing array of regulatory and accessory proteins such as arrestins. G protein-independent β-arrestin recruitment at GPCRs is universally accepted as the canonical interactor system and it has been found to be a powerful tracker of most G...
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Laroche G, Giguère PM Tags: Methods Mol Biol Source Type: research

Super-Resolution Imaging of G Protein-Coupled Receptors Using Ground State Depletion Microscopy.
Abstract G protein-coupled receptors (GPCRs) comprise the largest family of integral membrane proteins, which are coupled to heterotrimeric G proteins to influence cell signaling. Subsequent to G protein activation, agonist-stimulated G protein-coupled receptor kinase (GRK) phosphorylation results in the recruitment of β-arrestin proteins, which form both stable and unstable complexes with GPCRs. β-Arrestins when bound to GPCRs not only contribute to the uncoupling of G protein signaling but also to the redistribution of GPCRs to clathrin-coated pits via their association with both clathrin and &bet...
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Caetano Crowley FA, Heit B, Ferguson SSG Tags: Methods Mol Biol Source Type: research

Analysis of Spatial Assembly of GPCRs Using Photoactivatable Dyes and Localization Microscopy.
Abstract Super-resolution imaging has provided unprecedented insight in the molecular complexities of fundamental cell biological questions. For G protein-coupled receptors (GPCRs), its application to the study of receptor homomers and heteromers have unveiled the diversity of complexes these GPCRs can form at the plasma membrane at a structural and functional level. Here, we describe our methodological approach of photoactivated localization microscopy with photoactivatable dyes (PD-PALM) to visualize and quantify the spatial assembly of GPCR heteromers at the plasma membrane. PMID: 30969426 [PubMed - indexe...
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Jonas KC, Hanyaloglu AC Tags: Methods Mol Biol Source Type: research

Imaging of Tissue-Specific and Temporal Activation of GPCR Signaling Using DREADD Knock-In Mice.
Abstract Engineered G protein-coupled receptors (DREADDs, designer receptors exclusively activated by designer drugs) are convenient tools for specific activation of GPCR signaling in many cell types. DREADDs have been utilized as research tools to study numerous cellular and physiologic processes, including regulation of neuronal activity, behavior, and metabolism. Mice with random insertion transgenes and adeno-associated viruses have been widely used to express DREADDs in individual cell types. We recently created and characterized ROSA26-GsDREADD knock-in mice to allow Cre recombinase-dependent expression of a...
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Akhmedov D, Kirkby NS, Mitchell JA, Berdeaux R Tags: Methods Mol Biol Source Type: research

Long non-coding RNA Malat1 activated autophagy, hence promoting cell proliferation and inhibiting apoptosis by sponging miR-101 in colorectal cancer.
Conclusion: Long non-coding RNA Malat1 activated autophagy and promoted cell proliferation, yet inhibited apoptosis by sponging miR-101 in colorectal cancer cells. PMID: 31372165 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - August 4, 2019 Category: Molecular Biology Authors: Si Y, Yang Z, Ge Q, Yu L, Yao M, Sun X, Ren Z, Ding C Tags: Cell Mol Biol Lett Source Type: research

The Contribution of Differential Scanning Calorimetry for the Study of Peptide/Lipid Interactions.
Abstract Membrane-active peptides include a variety of molecules such as antimicrobial (AMP), cell-penetrating (CPP), viral, and amyloid peptides that are implicated in several pathologies. They constitute important targets because they are either at the basis of novel therapies (drug delivery for CPPs or antimicrobial activity for AMPs) or they are the agents causing these pathologies (viral and amyloid peptides). They all share the common property of interacting with the cellular lipid membrane in their mode of action. Therefore, a better understanding of the peptide/lipid (P/L) interaction is essential to help ...
Source: Mol Biol Cell - August 2, 2019 Category: Molecular Biology Authors: Jobin ML, Alves ID Tags: Methods Mol Biol Source Type: research

Intrinsic Thermodynamics of Protein-Ligand Binding by Isothermal Titration Calorimetry as Aid to Drug Design.
Abstract Isothermal titration calorimetry (ITC) is one of the main techniques to determine specific interactions between molecules dissolved in aqueous solution. This technique is commonly used in drug development programs when low-molecular-weight molecules are sought that bind tightly and specifically to a protein (disease target) molecule. The method allows a complete thermodynamic characterization of an interaction, i.e., ITC enables direct determination of the model-independent observed interaction change in enthalpy (ΔH) and a model-dependent observed interaction affinity (change in Gibbs free energy, ...
Source: Mol Biol Cell - August 2, 2019 Category: Molecular Biology Authors: Paketurytė V, Zubrienė A, Ladbury JE, Matulis D Tags: Methods Mol Biol Source Type: research

Thermodynamics of Molecular Machines Using Incremental ITC.
Abstract Molecular biomachines, such as DNA and RNA polymerases or the ribosome, are fascinating biological assemblies able to swiftly perform repeated and highly regulated tasks, with a remarkable accuracy. Significant advances in structural studies during the past 20 years provided a wealth of information regarding their architecture and considerably contributed to a better understanding of their mechanism of action. However, the three-dimensional structure of a biological nanomachine alone does not provide access to its detailed mechanism of action, even when obtained at atomic resolution. When combined wi...
Source: Mol Biol Cell - August 2, 2019 Category: Molecular Biology Authors: Meyer B, da Veiga C, Dumas P, Ennifar E Tags: Methods Mol Biol Source Type: research

Characterization of Microtubule-Associated Proteins (MAPs) and Tubulin Interactions by Isothermal Titration Calorimetry (ITC).
Abstract Microtubules are highly dynamic structures which play a central role in many cellular processes such as cell division, intracellular transport, and migration. Their dynamics is tightly regulated by stabilizing and destabilizing microtubule-associated proteins (MAPs), such as tau and stathmin. Many approaches have been developed to study interactions between tubulin and MAPs. However, isothermal titration calorimetry (ITC) is the only direct thermodynamic method that enables a full thermodynamic characterization of the interaction after a single titration experiment. We provide here the protocols to apply ...
Source: Mol Biol Cell - August 2, 2019 Category: Molecular Biology Authors: Tsvetkov PO, La Rocca R, Malesinski S, Devred F Tags: Methods Mol Biol Source Type: research

Tinkering with Binding Polynomials in Isothermal Titration Calorimetry.
Abstract Isothermal titration calorimetry (ITC) has become the preferred experimental technique for characterizing intermolecular interactions between biological molecules. Among the several advantages, the use of natural non-labeled molecules and the determination of the complete thermodynamic profile for the interaction in solution remain as the primary features that have promoted ITC to the forefront of experimental biophysics. The experimental design in ITC may range from studying a simple direct binary macromolecule-ligand interaction to studying the homotropic or heterotropic cooperative effect between ligan...
Source: Mol Biol Cell - August 2, 2019 Category: Molecular Biology Authors: Claveria-Gimeno R, Vega S, Abian O, Velazquez-Campoy A Tags: Methods Mol Biol Source Type: research

Introduction to the  Immune System.
Introduction to the Immune System. Methods Mol Biol. 2019;2024:1-24 Authors: McComb S, Thiriot A, Akache B, Krishnan L, Stark F Abstract The immune system in a broad sense is a mechanism that allows a living organism to discriminate between "self" and "nonself." Examples of immune systems occur in multicellular organisms as simple and ancient as sea sponges. In fact, complex multicellular life would be impossible without the ability to exclude external life from the internal environment. This introduction to the immune system will explore the cell types and soluble factors inv...
Source: Mol Biol Cell - August 2, 2019 Category: Molecular Biology Authors: McComb S, Thiriot A, Akache B, Krishnan L, Stark F Tags: Methods Mol Biol Source Type: research

Antigen Identification for Cell-Binding Antibodies Using Ligand-Directed Crosslinking and Biotin Transfer.
We describe the synthesis of the ASB crosslinker, labelling of the ligand with ASB, and cell binding of the labelled ligands. Next, biotin affinity purification and trypsin digestion of cell surface proteins that have been crosslinked by ASB are described. Lastly, several hints and tips to improve the proteomic analysis for these types of samples are provided. PMID: 31364049 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - August 2, 2019 Category: Molecular Biology Authors: Tremblay TL, Hill JJ Tags: Methods Mol Biol Source Type: research

Whole-Genome Phage Display Libraries: A Powerful Tool for Antigen Discovery.
Abstract In the last two decades, phage display technology has been used for investigating complex biological processes and isolating molecules of practical value in several applications. Bacteriophage lambda, representing a classical cloning and expression system, has also been exploited for generating display libraries of small peptides and protein domains. More recently, large cDNA and whole-genome lambda display libraries of human pathogens have been generated for the discovery of new antigens for biomedical applications. Here, we describe the construction of a whole-genome library of a common pathogen-Strepto...
Source: Mol Biol Cell - August 2, 2019 Category: Molecular Biology Authors: Beghetto E, Gargano N Tags: Methods Mol Biol Source Type: research

Construction and Screening of an  Antigen-Derived Peptide Library Displayed on Yeast Cell Surface for CD4+ T Cell Epitope Identification.
Construction and Screening of an Antigen-Derived Peptide Library Displayed on Yeast Cell Surface for CD4+ T Cell Epitope Identification. Methods Mol Biol. 2019;2024:213-234 Authors: Wen F, Smith MR, Zhao H Abstract Antigenic peptides (termed T cell epitopes) are assembled with major histocompatibility complex (MHC) molecules and presented on the surface of antigen-presenting cells (APCs) for T cell recognition. T cells engage these peptide-MHCs using T cell receptors (TCRs). Because T cell epitopes determine the specificity of a T cell immune response, their prediction and identification are impo...
Source: Mol Biol Cell - August 2, 2019 Category: Molecular Biology Authors: Wen F, Smith MR, Zhao H Tags: Methods Mol Biol Source Type: research

Enrichment of Phosphorylated MHC Peptides with Immobilized Metal Affinity Chromatography and Titanium Dioxide Particles.
Abstract Phosphorylation is one of the most important forms of posttranslational modification. Dysregulation of phosphorylation is implicated in tumorigenesis, with cancerous signaling pathways activated by kinases. For immunotherapy with neoantigen-based peptides, phosphopeptides derived from aberrantly phosphorylated proteins presented by major histocompatibility complex (MHC) are promising candidates due to their specificity to elicit cytotoxic T-cell responses. Unlike other MHC peptides, phosphorylated MHC peptides cannot be predicted from DNA sequences, and their identification relies on the direct detection ...
Source: Mol Biol Cell - August 2, 2019 Category: Molecular Biology Authors: Chen R, Li J Tags: Methods Mol Biol Source Type: research

Genome-Based Bioinformatic Prediction of Major Histocompatibility (MHC).
Abstract Over the last 17 years, a large amount of knowledge has been accumulated on various aspects of major histocompatibility complex (MHC) molecules. In conjunction, numerous algorithms and tools have been developed to screen protein molecules for these MHC receptor sites. By combining these computational tools and databases with genomic sequence information that is now widely available for a vast range of organisms, it is possible to screen whole genomes for MHC epitopes. By pre-screening these genomes, it allows the researcher to narrow down possible protein targets for further analysis by traditional t...
Source: Mol Biol Cell - August 2, 2019 Category: Molecular Biology Authors: Foote SJ Tags: Methods Mol Biol Source Type: research

Enhancing Mass Spectrometry-Based MHC-I Peptide Identification Through a Targeted Database Search Approach.
Abstract MHC-bound peptide ligands dictate the activation and specificity of CD8+ T- cells-based and thus are important for devising T-cell immunotherapies. In recent times, advances in mass spectrometry (MS) have enabled the precise identification of these peptides, wherein MS/MS spectra are compared against a reference proteome. Unfortunately, matching immunopeptide MS/MS to reference proteome databases is hindered by inflated search spaces attributed to the number of matches that need to be considered due to a lack of enzyme restriction. These large search spaces limit the efficiency with which MHC-I peptides a...
Source: Mol Biol Cell - August 2, 2019 Category: Molecular Biology Authors: Konda P, Murphy JP, Nielsen M, Gujar S Tags: Methods Mol Biol Source Type: research

Prioritization of Therapeutic Targets of Inflammation Using Proteomics, Bioinformatics, and In  Silico Cell-Cell Interactomics.
Prioritization of Therapeutic Targets of Inflammation Using Proteomics, Bioinformatics, and In Silico Cell-Cell Interactomics. Methods Mol Biol. 2019;2024:309-325 Authors: Haqqani AS, Stanimirovic DB Abstract Protein-protein interactions play key roles in leukocyte extravasation process into the brain and have been attractive therapeutic targets for inhibiting brain inflammation using blocking (or neutralizing) antibodies. These targets include protein-protein interactions between cytokines (or chemokines) and their receptors on leukocytes and between adhesion molecules of leukocyte and brain end...
Source: Mol Biol Cell - August 2, 2019 Category: Molecular Biology Authors: Haqqani AS, Stanimirovic DB Tags: Methods Mol Biol Source Type: research

Genomics-Driven Immunoproteomics: An Integrative Platform to Uncover Important Biomarkers for Human Diseases.
Abstract Genomics-driven immunoproteomics (GDI) is a platform that helps identify antigenic protein targets of mutations and other deoxyribonucleic acid (DNA) variations that are commonly associated with pathological states. This platform utilizes data generated from deep sequencing of exomic DNA or ribonucleic acid (RNA) as input to synthesize mutant peptides into microarrays, which then can be used to detect antigenic proteins that invoke immune response in patients. The technology has been used to detect antigenic targets of multiple sclerosis, an autoimmune disease [1], and cancer to identify mutant proteins t...
Source: Mol Biol Cell - August 2, 2019 Category: Molecular Biology Authors: Giri R, Qendro V, Rani P, Jepchumba C, Bugos G, Stadler V, Han DK Tags: Methods Mol Biol Source Type: research

Analysis and Characterization of Immune Cells and Their Activation Status by Whole-Cell MALDI-TOF Mass Spectrometry.
Abstract For 40 years, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been widely used in proteomics and biochemistry. It has been demonstrated in the last decade that MALDI-TOF MS can be used routinely to identify and classify numerous bacterial species or subspecies. We applied MALDI-TOF MS directly to intact mammalian cells, and we found that this method is valuable to identify human circulating cells and cells involved in the immune response including macrophages. We then stimulated human macrophages with cytokines, bacterial products, and a variety of bact...
Source: Mol Biol Cell - August 2, 2019 Category: Molecular Biology Authors: Ouedraogo R, Textoris J, Gorvel L, Daumas A, Capo C, Mege JL Tags: Methods Mol Biol Source Type: research

Proteomics to Predict Loss of RXR- γ During Progression of Epithelial Ovarian Cancer.
Proteomics to Predict Loss of RXR-γ During Progression of Epithelial Ovarian Cancer. Methods Mol Biol. 2019;2019:1-14 Authors: Kalra RS, Bapat SA Abstract Retinoid and rexinoid receptors are known to regulate key processes during development, differentiation, and cell death in vertebrates. However, their contributions to progression of malignant disease remain largely elusive although it is realized that transformed cancer cells, which essentially evade apoptosis, may display altered molecular expressions or functions associated with retinoid signaling. Here, using a progression model of ovarian...
Source: Mol Biol Cell - August 1, 2019 Category: Molecular Biology Authors: Kalra RS, Bapat SA Tags: Methods Mol Biol Source Type: research

Lentiviral-Mediated shRNA Approaches: Applications in Cellular Differentiation and Autophagy.
Abstract Acute myeloid leukemia (AML) is characterized by the accumulation of immature white blood cell precursors in the bone marrow and peripheral circulation. In essence, leukemic cells fail to differentiate and are stalled at a particular step of hematopoietic maturation and are unable to complete their development into functional blood cells with a finite life cycle. They are thus said to possess a "differentiation block." Pharmacological override of this block is one attractive avenue of therapy, termed "differentiation therapy." The most successful example of this therapeutic strategy is...
Source: Mol Biol Cell - August 1, 2019 Category: Molecular Biology Authors: Orfali N, Jeyapalan JN, Woodcock CL, O'Donovan TR, Benjamin D, Cahill M, McKenna S, Gudas LJ, Mongan NP Tags: Methods Mol Biol Source Type: research

Analysis of Retinoic Acid Receptor Signaling in Colorectal Cancer.
Abstract Retinoic acid receptor (RAR) signaling plays an important role in embryonic development and homeostasis of many tissues. At the cellular level, activation of RAR signaling often induces cell cycle arrest, differentiation, and apoptosis in many types of cells. Consequently, loss of normal RAR function in the presence of physiological levels of retinoic acid (RA) is often observed in cancers, and pharmacological reactivation of RAR signaling has been considered a promising strategy for cancer therapy and prevention. One of important mechanisms that regulate RAR activity in cancer cells is cross-talk with gr...
Source: Mol Biol Cell - August 1, 2019 Category: Molecular Biology Authors: Imajo M Tags: Methods Mol Biol Source Type: research

Methods to Assess Activity and Potency of Rexinoids Using Rapid Luciferase-Based Assays: A Case Study with NEt-TMN.
Abstract This chapter outlines the materials, methods, and procedures for the in vitro biological evaluation of retinoid-X-receptor (RXR) agonists including 6-(ethyl(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)amino)nicotinic acid (NEt-TMN), as well as several NEt-TMN analog compounds recently reported by our group. These methods have general applicability beyond this NEt-TMN case study, and can be employed to characterize and biologically evaluate other putative RXR agonists (rexinoids), and benchmarked against perhaps the most common rexinoid known as bexarotene (Bex), a drug awarded FDA approval for t...
Source: Mol Biol Cell - August 1, 2019 Category: Molecular Biology Authors: Jurutka PW, Wagner CE Tags: Methods Mol Biol Source Type: research

Methods to Generate an Array of Novel Rexinoids by SAR on a Potent Retinoid X Receptor Agonist: A Case Study with NEt-TMN.
Abstract The methods described in this chapter concern procedures for the design, synthesis, and in vitro biological evaluation of an array of potent retinoid-X-receptor (RXR) agonists employing 6-(ethyl(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)amino)nicotinic acid (NEt-TMN), and recently reported NEt-TMN analogs, as a case study. These methods have been extensively applied beyond the present case study to generate several analogs of other potent RXR agonists (rexinoids), particularly the RXR agonist known as bexarotene (Bex), a Food and Drug Administration (FDA) approved drug for cutaneous T-cell lym...
Source: Mol Biol Cell - August 1, 2019 Category: Molecular Biology Authors: Wagner CE, Jurutka PW Tags: Methods Mol Biol Source Type: research

Hemodynamics-Based Strategy of Using Retinoic Acid Receptor and Retinoid X Receptor Agonists to Induce MicroRNA-10a and Inhibit Atherosclerotic Lesion.
Abstract The protocols in this chapter describe methods for identifying the functional roles of retinoic acid receptor (RAR) and retinoid X receptor (RXR) signaling in atherosclerosis and developing RARα/RXRα-specific agonists as hemodynamics-based therapeutic components for atherosclerosis treatment. In vitro cell culture flow system is used to elucidate the effects of different flow patterns and shear stresses, i.e., atherogenic oscillatory shear stress (OS) vs. atheroprotective pulsatile shear stress (PS), on RAR/RXR signaling and inflammatory responses in vascular endothelial cells (ECs). Western b...
Source: Mol Biol Cell - August 1, 2019 Category: Molecular Biology Authors: Lee DY, Chiu JJ Tags: Methods Mol Biol Source Type: research

Highly Sensitive Quantitative Determination of Retinoic Acid Levels, Retinoic Acid Synthesis, and Catabolism in Embryonic Tissue Using a Reporter Cell-Based Method.
Abstract The effect of all-trans retinoic acid (RA) on embryogenesis is tissue specific and highly concentration dependent. Using a liquid chromatography/mass spectrometry-based method to quantify trace amounts of RA in embryonic tissue requires expensive specialist facilities. Here, we describe the use of a RA response element (RARE)-lacZ reporter cell-based method, which is simple and cost effective, to measure RA levels in small pieces of tissue from the embryo. We further apply this method to quantitatively assay activities of RA-synthesizing and RA-catabolizing enzymes, the key regulators of RA bioavailabilit...
Source: Mol Biol Cell - August 1, 2019 Category: Molecular Biology Authors: Lee LMY, Tam STK, McCaffery PJ, Shum ASW Tags: Methods Mol Biol Source Type: research

A Behavioral Assay to Study Effects of Retinoid Pharmacology on Nervous System Development in a Marine Annelid.
ert M Abstract Autonomous animal locomotion, such as swimming, is modulated by neuronal networks acting on cilia or muscles. Understanding how these networks are formed and coordinated is a complex scientific problem, which requires various technical approaches. Among others, behavioral studies of developing animals treated with exogenous substances have proven to be a successful approach for studying the functions of neuronal networks. One such substance crucial for the proper development of the nervous system is the vitamin A-derived morphogen retinoic acid (RA). In the larva of the marine annelid Platynereis du...
Source: Mol Biol Cell - August 1, 2019 Category: Molecular Biology Authors: Handberg-Thorsager M, Ulman V, Tomançak P, Arendt D, Schubert M Tags: Methods Mol Biol Source Type: research

Assessing Autophagy During Retinoid Treatment of Breast Cancer Cells.
AM Abstract Retinoids are derived from vitamin A through a multi-step process. Within a target cell, retinoids regulate gene expression by activating the retinoid acid receptors (RAR) and retinoid x receptors (RXR), which are ligand-dependent transcription factors. Besides its therapeutic use in dermatological disorders, all-trans retinoic acid (ATRA) is successfully utilized to treat acute promyelocytic leukemia (APL) patients. The use of ATRA in APL patients is the first example of clinically useful differentiation therapy. Therapeutic strategies aiming at cancer cell differentiation have great potential for so...
Source: Mol Biol Cell - August 1, 2019 Category: Molecular Biology Authors: Parejo S, Tschan MP, Muraro MG, Garattini E, Spagnoli GC, Schläfli AM Tags: Methods Mol Biol Source Type: research

Nonradioactive and Radioactive Telomerase Assays for Detecting Diminished Telomerase Activity in Cancer Cells after Treatment with Retinoid.
Abstract Detection of any decrease in telomerase activity in cancer cells and tumor tissues is an important part in assessing overall therapeutic outcomes of a treatment agent in the laboratory and clinical settings. Almost 85% of cancers have activation of telomerase activity that promotes cell proliferation and discourages differentiation to sustain growth of the cancers. Retinoids are highly regarded as the anti-proliferation and pro-differentiation agents that cause down regulation of telomerase activity in the cancer cells. Two (nonradioactive and radioactive) telomeric repeat amplification protocol (TRAP) as...
Source: Mol Biol Cell - August 1, 2019 Category: Molecular Biology Authors: Ray SK Tags: Methods Mol Biol Source Type: research

Using Functional Genetics in Haploid Cells for Drug Target Identification.
Abstract Pooled genetic screens are a powerful tool to identify targets for drug development as well as chemogenetic interactions. Various complementary methods for mutagenesis are available to generate highly complex cell populations, including mRNA knockdown, directed genome editing, as well as random genome mutagenesis. With the availability of a growing number of haploid mammalian cell lines, random mutagenesis is becoming increasingly powerful and represents an attractive alternative, e.g., to CRISPR-based screening. This chapter provides a step-by-step protocol for performing haploid gene trap screens. ...
Source: Mol Biol Cell - July 19, 2019 Category: Molecular Biology Authors: Volz JC, Schuller N, Elling U Tags: Methods Mol Biol Source Type: research

Unbiased Forward Genetic Screening with Chemical Mutagenesis to Uncover Drug-Target Interactions.
Abstract The steadily increasing throughput in next-generation sequencing technologies is revolutionizing a number of fields in biology. One application requiring massive parallel sequencing is forward genetic screening based on chemical mutagenesis. Such screens interrogate the entire genome in an entirely unbiased fashion and can be applied to a number of research questions. CRISPR/Cas9-based screens, which are largely limited to a gene's loss of function, have already been very successful in identifying drug targets and pathways related to the drug's mode of action. By inducing single nucleotide changes using a...
Source: Mol Biol Cell - July 19, 2019 Category: Molecular Biology Authors: Horn M, Metge F, Denzel MS Tags: Methods Mol Biol Source Type: research

Integration of RNAi and Small Molecule Screens to Identify Targets for Drug Development.
Abstract Cellular models for siRNA and small molecule high-throughput screening have been widely used in the last decade to identify targets for drug discovery. As an example, we present a twofold readout approach based on cell viability and multipolar phenotype. To maximize the discovery of potential targets and at the same time reduce the number of false positives in our dataset, we have combined focused and rationally designed custom siRNA libraries with small molecule inhibitor libraries. Here we describe a cellular model for centrosome amplification as an example of how to design and perform a multiple readou...
Source: Mol Biol Cell - July 19, 2019 Category: Molecular Biology Authors: Drosopoulos K, Linardopoulos S Tags: Methods Mol Biol Source Type: research

CellProfiler and KNIME: Open-Source Tools for High-Content Screening.
We present and discuss the open-source software CellProfiler for image analysis and KNIME for data analysis and data mining that provide software solutions, which increase flexibility and keep costs low. PMID: 30912015 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - July 19, 2019 Category: Molecular Biology Authors: Stöter M, Janosch A, Barsacchi R, Bickle M Tags: Methods Mol Biol Source Type: research