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Methods: a Companion to Methods in Enzymology

Tianle Ma, Aidong Zhang
Reconstructing context-specific transcriptional regulatory network is crucial for deciphering principles of regulatory mechanisms underlying various conditions. Recently studies that reconstructed transcriptional networks have focused on individual organisms or cell types and relied on data repositories of context-free regulatory relationships. Here we present a comprehensive framework to systematically derive putative regulator-target pairs in any given context by integrating context-specific transcriptional profiling and public data repositories of gene regulatory networks...
May 18, 2017: Methods: a Companion to Methods in Enzymology
Andreas Paulus, Wouter van Marken Lichtenbelt, Felix M Mottaghy, Matthias Bauwens
PURPOSE: Brown adipose tissue (BAT) research has evolved from an underestimated to a fast developing field. Its assumed curing properties for the world wide epidemic obesity, and its related diseases, makes this tissue an interesting target for a broad amount of non-invasive molecular BAT tracers. Apart from (18)F-FDG PET/CT there are several methods to detect BAT and measure its metabolism in a more appropriate way. Especially interesting is the measure of lipid turnover, because fatty acids comprise the main fuel for active BAT...
May 18, 2017: Methods: a Companion to Methods in Enzymology
Laura S Bisogno, Jack D Keene
Post-transcriptional regulation of gene expression by RNA binding proteins (RBPs) and non-coding RNAs plays an important role in global gene expression. Many post-transcriptional regulators are misexpressed and misregulated in cancers, resulting in altered programs of protein biosynthesis that can drive tumor progression. While comparative studies of several RBPs and microRNAs expressed in various cancer types have been reported, a model system that can be used to quantify RBP regulation and functional outcomes during the initiation and early stages of tumorigenesis is lacking...
May 18, 2017: Methods: a Companion to Methods in Enzymology
Clarisse van der Feltz, Aaron A Hoskins
The spliceosome is an extraordinarily dynamic molecular machine in which significant changes in composition as well as protein and RNA conformation are required for carrying out pre-mRNA splicing. Single-molecule fluorescence resonance energy transfer (smFRET) can be used to elucidate these dynamics both in well-characterized model systems and in entire spliceosomes. These types of single-molecule data provide novel information about spliceosome components and can be used to identify sub-populations of molecules with unique behaviors...
May 18, 2017: Methods: a Companion to Methods in Enzymology
Inbar Nevo-Yassaf, Marcos Lovelle, Yaacov Nahmias, Koret Hirschberg, Ella H Sklan
Lipid droplets (LDs) are regulated neutral lipid storage organelles having a central role in numerous cellular processes as well as in various pathologies such as metabolic disorders, immune responses and during pathogen infection. Several viruses were found to use the interface between the endoplasmic reticulum (ER) and LDs as the site for replication and assembly. Due to the growing significance of LDs, extensive efforts are made to study the mechanism and the dynamics of their formation and life history and how are these diverted or modified by pathogens...
May 16, 2017: Methods: a Companion to Methods in Enzymology
Bas G J Surewaard, Paul Kubes
It is central to the field of bacterial pathogenesis to define how bacteria are killed by phagocytic cells. During phagocytosis, the microbe is localized to the phagolysosome where crucial defense mechanisms such as acidification and production of reactive oxygen species (ROS) are initiated. This process has extensively been studied in vitro, however many resident tissue phagocytes will phenotypically change upon isolation from their natural environment. Therefore, interrogation of phagocytosis and phagosomal function of cells in the context of their natural tissue environment enhances our understanding of the biological process in vivo...
May 15, 2017: Methods: a Companion to Methods in Enzymology
Saniye Yumlu, Jürgen Stumm, Sanum Bashir, Anne-Kathrin Dreyer, Pawel Lisowski, Eric Danner, Ralf Kühn
Human induced pluripotent stem cells (hiPSCs) represent an ideal in vitro platform to study human genetics and biology. The recent advent of programmable nucleases makes also the human genome amenable to experimental genetics through either the correction of mutations in patient-derived iPSC lines or the de novo introduction of mutations into otherwise healthy iPSCs. The production of specific and sometimes complex genotypes in multiple cell lines requires efficient and streamlined gene editing technologies...
May 15, 2017: Methods: a Companion to Methods in Enzymology
Fabio Vanoli, Maria Jasin
Recurrent chromosomal translocations often lead to expression of fusion proteins associated with oncogenic transformation. To study translocations and downstream events, genome editing techniques have been developed to generate chromosomal translocations through non-homologous end joining of DNA double-strand breaks introduced at the two participating endogenous loci. However, the frequencies at which these events occur is usually too low to efficiently clone cells carrying the translocation. This article provides a detailed method using CRISPR-Cas9 technology and homology-directed repair to efficiently isolate cells harboring a chromosomal translocation...
May 15, 2017: Methods: a Companion to Methods in Enzymology
Luci A Witcomb, Julie Czupryna, Kevin P Francis, Gad Frankel, Peter W Taylor
In contrast to two-dimensional bioluminescence imaging, three dimensional diffuse light imaging tomography with integrated micro-computed tomography (DLIT-μCT) has the potential to realise spatial variations in infection patterns when imaging experimental animals dosed with derivatives of virulent bacteria carrying bioluminescent reporter genes such as the lux operon from the bacterium Photorhabdus luminescens. The method provides an opportunity to precisely localise the bacterial infection sites within the animal and enables the generation of four-dimensional movies of the infection cycle...
May 15, 2017: Methods: a Companion to Methods in Enzymology
Joana Tavares, David M Costa, Ana Rafaela Teixeira, Anabela Cordeiro-da-Silva, Rogerio Amino
Hematogenous dissemination followed by tissue tropism is a characteristic of the infectious process of many pathogens including those transmitted by blood-feeding vectors. After entering into the blood circulation, these pathogens must arrest in the target organ before they infect a specific tissue. Here, we describe a non-invasive method to visualize and quantify the homing of pathogens to the host tissues. By using in vivo bioluminescence imaging we quantify the accumulation of luciferase-expressing parasites in the host organs during the first minutes following their intravascular inoculation in mice...
May 15, 2017: Methods: a Companion to Methods in Enzymology
Noelia Lopez-Montero, Yuen-Yan Chang, Anna Sartori-Rupp, Sonja Kühn, Jost Enninga
Macropinocytosis is the uptake of extracellular fluid within vesicles of varying size that takes place during numerous cellular processes in a large variety of cells. A growing number of pathogens, including viruses, parasites, and bacteria are known to induce macropinocytosis during their entry into targeted host cells. We have recently discovered that the human enteroinvasive, bacterial pathogen Shigella causes in situ macropinosome formation during its entry into epithelial cells. These infection-associated macropinosomes are not generated to ingest the bacteria, but are instead involved in Shigella's intracellular niche formation...
May 15, 2017: Methods: a Companion to Methods in Enzymology
Sara Espinosa, Lingdi Zhang, Xueni Li, Rui Zhao
Crystallography is a powerful tool to determine the atomic structures of proteins and RNAs. X-ray crystallography has been used to determine the structure of many splicing related proteins and RNAs, making major contributions to our understanding of the molecular mechanism and regulation of pre-mRNA splicing. Compared to other structural methods, crystallography has its own advantage in the high-resolution structural information it can provide and the unique biological questions it can answer. In addition, two new crystallographic methods - the serial femtosecond crystallography and 3D electron crystallography - were developed to overcome some of the limitations of traditional X-ray crystallography and broaden the range of biological problems that crystallography can solve...
May 12, 2017: Methods: a Companion to Methods in Enzymology
M Wassef, A Luscan, A Battistella, S Le Corre, H Li, M R Wallace, M Vidaud, R Margueron
The advent of programmable nucleases such as ZFNs, TALENs and CRISPR/Cas9 has brought the power of genetic manipulation to widely used model systems. In mammalian cells, nuclease-mediated DNA double strand break is mainly repaired through the error-prone non-homologous end-joining (NHEJ) repair pathway, eventually leading to accumulation of small deletions or insertions (indels) that can inactivate gene function. However, due to the variable size of the indels and the polyploid status of many cell lines (e...
May 10, 2017: Methods: a Companion to Methods in Enzymology
Cécile Collonnier, Anouchka Guyon-Debast, François Maclot, Kostlend Mara, Florence Charlot, Fabien Nogué
Beyond its predominant role in human and animal therapy, the CRISPR-Cas9 system has also become an essential tool for plant research and plant breeding. Agronomic applications rely on the mastery of gene inactivation and gene modification. However, if the knock-out of genes by non-homologous end-joining (NHEJ)-mediated repair of the targeted double-strand breaks (DSBs) induced by the CRISPR-Cas9 system is rather well mastered, the knock-in of genes by homology-driven repair or end-joining remains difficult to perform efficiently in higher plants...
May 3, 2017: Methods: a Companion to Methods in Enzymology
Scott Ronquist, Walter Meixner, Indika Rajapakse, John Snyder
The human genome is dynamic in structure, complicating researcher's attempts at fully understanding it. Time series "Fluorescent in situ Hybridization" (FISH) imaging has increased our ability to observe genome structure, but due to cell type and experimental variability this data is often noisy and difficult to analyze. Furthermore, computational analysis techniques are needed for homolog discrimination and canonical framework detection, in the case of time-series images. In this paper we introduce novel ideas for nucleus imaging analysis, present findings extracted using dynamic genome imaging, and propose an objective algorithm for high-throughput, time-series FISH imaging...
April 29, 2017: Methods: a Companion to Methods in Enzymology
Joshua R Wheeler, Saumya Jain, Anthony Khong, Roy Parker
Stress granules are dynamic, conserved RNA-protein (RNP) assemblies that form when translation is limiting; and are related to pathological aggregates in degenerative disease. Mammalian stress granules are comprised of two structures - an unstable shell and more stable cores. Herein we describe methodology for isolation of stress granule cores from both yeast and mammalian cells. The protocol consists of first enriching for stress granule cores using centrifugation and then further purifying stress granule cores using immunoprecipitation...
April 27, 2017: Methods: a Companion to Methods in Enzymology
A Mandic, D Strebinger, C Regali, N E Phillips, D M Suter
Gene expression is at the heart of virtually any biological process, and its deregulation is at the source of numerous pathological conditions. While impressive progress has been made in genome-wide measurements of mRNA and protein expression levels, it is still challenging to obtain highly quantitative measurements in single living cells. Here we describe a novel approach based on internal tagging of endogenous proteins with a reporter allowing luminescence and fluorescence time-lapse microscopy. Using luminescence microscopy, fluctuations of protein expression levels can be monitored in single living cells with high sensitivity and temporal resolution over extended time periods...
April 26, 2017: Methods: a Companion to Methods in Enzymology
Katherine I Zhou, Nian Liu, Tao Pan
The reversible N(6)-methyladenosine (m(6)A) modification of eukaryotic messenger RNAs (mRNAs) is a widespread regulatory mechanism that impacts every step in the mRNA life cycle. The effect of m(6)A on mRNA fate depends on the binding of "m(6)A reader" proteins - RNA binding proteins that specifically bind to RNAs containing m(6)A. Here, we describe an RNA pull-down method that can be used to identify novel m(6)A reader proteins starting from a known m(6)A-modified site in cellular or viral RNA. We further describe how a combination of immunoprecipitation-based sequencing methods can be used to identify m(6)A-modified sites bound by an m(6)A reader protein on a transcriptome-wide level...
April 26, 2017: Methods: a Companion to Methods in Enzymology
Dazhi Jiao, Wontack Han, Yuzhen Ye
Recent years have witnessed unprecedented accumulation of DNA sequences and therefore protein sequences (predicted from DNA sequences), due to the advances of sequencing technology. One of the major sources of the hypothetical proteins is the metagenomics research. Current annotation of metagenomes (collections of short metagenomic sequences or assemblies) relies on similarity searches against known gene/protein families, based on which functional profiles of microbial communities can be built. This practice, however, leaves out the hypothetical proteins, which may outnumber the known proteins for many microbial communities...
April 25, 2017: Methods: a Companion to Methods in Enzymology
Frank Eggert, Katharina Kulikov, Christof Domnick, Philipp Leifels, Stephanie Kath-Schorr
The synthesis of sequence-specifically modified long RNA molecules, which cannot entirely be prepared via solid phase synthesis methods is experimentally challenging. We are using a new approach based on an expanded genetic alphabet preparing site-specifically modified RNA molecules via standard in vitro transcription. In this report, the site-specific labeling of functional RNAs, in particular ribozymes and a long non-coding RNA with cyclopropene moieties, is presented. We provide detailed instructions for RNA labeling via in vitro transcription and include required analytical methods to verify production and identity of the transcript...
April 25, 2017: Methods: a Companion to Methods in Enzymology
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