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Methods: a Companion to Methods in Enzymology

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https://www.readbyqxmd.com/read/29792918/translational-omics-future-potential-and-current-challenges-in-precision-medicine
#1
Arsalan Wafi, Reza Mirnezami
Rapid advances in computational science and biotechnology are paving the way for precision medicine - a vision in next-generation healthcare that promises to provide a care package uniquely tailored to each individual's molecular make-up. Until relatively recently, the focus has been firmly centred around the genome; however, over the past two decades there has been a surge in the study of molecular activity within other biological domains (proteome/transcriptome/metabolome) involved in health and pathogenesis...
May 21, 2018: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/29792917/storage-visualization-and-navigation-of-3d-genomics-data
#2
Jérôme Waldispühl, Eric Zhang, Alexander Butyaev, Elena Nazarova, Yan Cyr
The field of 3D genomics grew at increasing rates in the last decade. The volume and complexity of 2D and 3D data produced is progressively outpacing the capacities of the technology previously used for distributing genome sequences. The emergence of new technologies provides also novel opportunities for the development of innovative approaches. In this paper, we review the state-of-the-art computing technology, as well as the solutions adopted by the platforms currently available.
May 21, 2018: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/29778647/expression-of-recombinant-proteins-in-insect-and-mammalian-cells
#3
REVIEW
Edward A McKenzie, W Mark Abbott
Purified recombinant proteins are key reagents in academic and industrial research. The ability to make these proteins quickly often relies on the availability of higher eukaryotic cell hosts such as insect and mammalian cells where there is a very wide range of post-translational modifications, protein folding and trafficking pathways. This enables the generation of many proteins that cannot be made in microbial hosts. In this article we outline some of the most commonly used methods to express recombinant proteins in insect and mammalian cells...
May 17, 2018: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/29778646/the-fine-art-of-integral-membrane-protein-crystallisation
#4
REVIEW
James Birch, Danny Axford, James Foadi, Arne Meyer, Annette Eckardt, Yvonne Thielmann, Isabel Moraes
Integral membrane proteins are among the most fascinating and important biomolecules as they play a vital role in many biological functions. Knowledge of their atomic structures is fundamental to the understanding of their biochemical function and key in many drug discovery programs. However, over the years, structure determination of integral membrane proteins has proven to be far from trivial, hence they are underrepresented in the protein data bank. Low expression levels, insolubility and instability are just a few of the many hurdles one faces when studying these proteins...
May 17, 2018: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/29778645/screening-and-selection-of-artificial-riboswitches
#5
Svetlana V Harbaugh, Jennifer Martin, Jenna Weinstein, Grant Ingram, Nancy Kelley-Loughnane
Synthetic riboswitches are engineered to regulate gene expression in response to a variety of non-endogenous small molecules, and a challenge to select this engineered response requires robust screening tools. A new synthetic riboswitch can be created by linking an in vitro-selected aptamer library with a randomized expression platform followed by in vivo selection and screening. In order to determine response to analyte, we developed a dual-color reporter comprising elements of the E. coli fimbriae phase variation system: recombinase FimE controlled by a synthetic riboswitch and an invertible DNA segment (fimS) containing a constitutively active promoter placed between two fluorescent protein genes...
May 17, 2018: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/29777751/evaluating-the-susceptibility-of-ago2-loaded-micrornas-to-degradation-by-nucleases-in-vitro
#6
Reyad Elbarbary, Lynne E Maquat
MicroRNAs (miRNAs) comprise a class of small non-coding RNAs that regulate the stability and/or translatability of most protein-coding transcripts. Steady-state levels of mature miRNAs can be controlled through mechanisms that influence their biogenesis and/or decay rates. Pathways that mediate mature miRNA decay are less well understood than those that mediate miRNA biogenesis. We recently described Tudor-staphylococcal/micrococcal-like nuclease (TSN)-mediated miRNA decay (TumiD) as a cellular pathway that promotes the sequence-specific endonucleolytic decay of miRNAs that harbor a CA and/or UA dinucleotide...
May 16, 2018: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/29777750/post-a-framework-for-set-based-association-analysis-in-high-dimensional-data
#7
Xueyuan Cao, E Olusegun George, Mingjuan Wang, Dale B Armstrong, Cheng Cheng, Susana Raimondi, Jeffrey E Rubnitz, James R Downing, Mondira Kundu, Stanley B Pounds
Evaluating the differential expression of a set of genes belonging to a common biological process or ontology has proven to be a very useful tool for biological discovery. However, existing gene-set association methods are limited to applications that evaluate differential expression across k⩾2 treatment groups or biological categories. This limitation precludes researchers from most effectively evaluating the association with other phenotypes that may be more clinically meaningful, such as quantitative variables or censored survival time variables...
May 16, 2018: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/29772275/5c-id-increased-resolution-chromosome-conformation-capture-carbon-copy-with-in-situ-3c-and-double-alternating-primer-design
#8
Ji Hun Kim, Katelyn R Titus, Wanfeng Gong, Jonathan A Beagan, Zhendong Cao, Jennifer E Phillips-Cremins
Mammalian genomes are folded in a hierarchy of compartments, topologically associating domains (TADs), subTADs, and looping interactions. Currently, there is a great need to evaluate the link between chromatin topology and genome function across many biological conditions and genetic perturbations. Hi-C can generate genome-wide maps of looping interactions but is intractable for high-throughput comparison of loops across multiple conditions due to the enormous number of reads (>6 Billion) required per library...
May 14, 2018: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/29758273/in-silico-experiment-system-for-testing-hypothesis-on-gene-functions-using-three-condition-specific-biological-networks
#9
Chai-Jin Lee, Dongwon Kang, Sangseon Lee, Sunwon Lee, Jaewoo Kang, Sun Kim
Determining functions of a gene requires time consuming, expensive biological experiments. Scientists can speed up this experimental process if the literature information and biological networks can be adequately provided. In this paper, we present a web-based information system that can perform in silico experiments of computationally testing hypothesis on the function of a gene. A hypothesis that is specified in English by the user is converted to genes using a literature and knowledge mining system called BEST...
May 11, 2018: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/29753788/installation-validation-and-application-examples-of-two-instrumental-setups-for-gas-phase-hdx-ms-analysis-of-peptides-and-proteins
#10
Ulrik H Mistarz, Kasper D Rand
Gas-phase hydrogen/deuterium exchange measured by mass spectrometry in a millisecond timeframe after ESI (gas-phase HDX-MS) is a fast and sensitive, yet unharnessed method to analyze the primary- and higher-order structure, intramolecular and intermolecular interactions, surface properties, and charge location of peptides and proteins. During a gas-phase HDX-MS experiment, heteroatom-bound non-amide hydrogens are made to exchange with deuterium during a millisecond timespan after electrospray ionization (ESI) by reaction with the highly basic reagent ND3 , enabling conformational analysis of protein states that are pertinent to the native solution-phase...
May 10, 2018: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/29753787/studying-sparsely-populated-conformational-states-in-rna-combining-chemical-synthesis-and-solution-nmr-spectroscopy
#11
Elisabeth Strebitzer, Felix Nußbaumer, Johannes Kremser, Martin Tollinger, Christoph Kreutz
Using chemical synthesis and solution NMR spectroscopy, RNA structural ensembles including a major ground state and minor populated excited states can be studied at atomic resolution. In this work, atom-specific 13 C labeled RNA building blocks - a 5-13 C-uridine and a 2,8-13 C2 -adenosine building block - are used to introduce isolated 13 C-1 H-spin topologies into a target RNA to probe such structural ensembles via NMR spectroscopy. First, the 5-13 C-uridine 2'-O-TBDMS-phosphoramidite building block was introduced into a 21 nucleotide (nt) tP5c stem construct of the tP5abc subdomain of the Tetrahymena group I ribozyme...
May 10, 2018: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/29753786/affinity-capillary-electrophoresis-for-studying-interactions-in-life-sciences
#12
REVIEW
Mais Olabi, Matthias Stein, Hermann Wätzig
Affinity capillary electrophoresis (ACE) analyzes noncovalent interactions between ligands and analytes based on changes in their electrophoretic mobility. This technique has been widely used to investigate various biomolecules, mainly proteins, polysaccharides and hormones. ACE is becoming a technique of choice to validate high throughput screening results, since it is very predictively working in realistic and relevant media, e.g. in body fluids. It is highly recommended to incorporate ACE as a powerful analytical tool to properly prepare animal testing and preclinical studies...
May 10, 2018: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/29753003/bifunctional-cross-linking-approaches-for-mass-spectrometry-based-investigation-of-nucleic-acids-and-protein-nucleic-acid-assemblies
#13
M Scalabrin, S M Dixit, M M Makshood, C E Krzemien, Daniele Fabris
With the goal of expanding the very limited toolkit of cross-linking agents available for nucleic acids and their protein complexes, we evaluated the merits of a wide range of bifunctional agents that may be capable of reacting with the functional groups characteristic of these types of biopolymers. The survey specifically focused on the ability of test reagents to produce desirable inter-molecular conjugates, which could reveal the identity of interacting components and the position of mutual contacts, while also considering a series of practical criteria for their utilization as viable nucleic acid probes...
May 9, 2018: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/29753002/nmr-based-investigations-into-target-dna-search-processes-of-proteins
#14
REVIEW
Junji Iwahara, Levani Zandarashvili, Catherine A Kemme, Alexandre Esadze
To perform their function, transcription factors and DNA-repair/modifying enzymes must first locate their targets in the vast presence of nonspecific, but structurally similar sites on genomic DNA. Before reaching their targets, these proteins stochastically scan DNA and dynamically move from one site to another on DNA. Solution NMR spectroscopy provides unique atomic-level insights into the dynamic DNA-scanning processes, which are difficult to gain by any other experimental means. In this review, we provide an introductory overview on the NMR methods for the structural, dynamic, and kinetic investigations of target DNA search by proteins...
May 9, 2018: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/29730250/efficient-computation-of-co-transcriptional-rna-ligand-interaction-dynamics
#15
Michael T Wolfinger, Christoph Flamm, Ivo L Hofacker
Riboswitches form an abundant class of cis-regulatory RNA elements that mediate gene expression by binding a small metabolite. For synthetic biology applications, they are becoming cheap and accessible systems for selectively triggering transcription or translation of downstream genes. Many riboswitches are kinetically controlled, hence knowledge of their co-transcriptional mechanisms is essential. We present here an efficient implementation for analyzing co-transcriptional RNA-ligand interaction dynamics. This approach allows for the first time to model concentration-dependent metabolite binding/unbinding kinetics...
May 3, 2018: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/29723572/iteratively-improving-hi-c-experiments-one-step-at-a-time
#16
Rosela Golloshi, Jacob Sanders, Rachel Patton McCord
The 3D organization of eukaryotic chromosomes affects key processes such as gene expression, DNA replication, cell division, and response to DNA damage. The genome-wide chromosome conformation capture (Hi-C) approach can characterize the landscape of 3D genome organization by measuring interaction frequencies between all genomic regions. Hi-C protocol improvements and rapid advances in DNA sequencing power have made Hi-C useful to study diverse biological systems, not only to elucidate the role of 3D genome structure in proper cellular function, but also to characterize genomic rearrangements, assemble new genomes, and consider chromatin interactions as potential biomarkers for diseases...
April 30, 2018: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/29715508/gut-metabolome-meets-microbiome-a-methodological-perspective-to-understand-the-relationship-between-host-and-microbe
#17
REVIEW
Santosh Lamichhane, Partho Sen, Alex M Dickens, Matej Orešič, Hanne Christine Bertram
It is well established that gut microbes and their metabolic products regulate host metabolism. The interactions between the host and its gut microbiota are highly dynamic and complex. In this review we present and discuss the metabolomic strategies to study the gut microbial ecosystem. We highlight the metabolic profiling approaches to study faecal samples aimed at deciphering the metabolic product derived from gut microbiota. We also discuss how metabolomics data can be integrated with metagenomics data derived from gut microbiota and how such approaches may lead to better understanding of the microbial functions...
April 28, 2018: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/29709561/selective-recovery-of-rnas-from-bacterial-pathogens-after-their-internalization-by-human-host-cells
#18
Simon Raynaud, Hélène LePabic, Brice Felden
Selective RNA extractions are required when studying bacterial gene expression within complex mixtures of pathogens and human cells, during adhesion, internalization and survival within the host. New technologies should be developed and implemented to enrich the amount of bacterial RNAs since the majority of RNAs are from the eukaryotic host cells, requiring high read depth coverage to capture the bacterial transcriptomes in dual-RNAseq studies. This will improve our understanding about bacterial adaptation to the host cell defenses, and about how they will adapt to an intracellular life...
April 27, 2018: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/29705212/variable-selection-in-heterogeneous-datasets-a-truncated-rank-sparse-linear-mixed-model-with-applications-to-genome-wide-association-studies
#19
Haohan Wang, Bryon Aragam, Eric P Xing
A fundamental and important challenge in modern datasets of ever increasing dimensionality is variable selection, which has taken on renewed interest recently due to the growth of biological and medical datasets with complex, non-i.i.d. structures. Naïvely applying classical variable selection methods such as the Lasso to such datasets may lead to a large number of false discoveries. Motivated by genome-wide association studies in genetics, we study the problem of variable selection for datasets arising from multiple subpopulations, when this underlying population structure is unknown to the researcher...
April 26, 2018: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/29705211/assessment-of-microbiota-host-interactions-at-the-vaginal-mucosa-interface
#20
REVIEW
Pamela Pruski, Holly V Lewis, Yun S Lee, Julian R Marchesi, Phillip R Bennett, Zoltan Takats, David A MacIntyre
There is increasing appreciation of the role that vaginal microbiota play in health and disease throughout a woman's lifespan. This has been driven partly by molecular techniques that enable detailed identification and characterisation of microbial community structures. However, these methods do not enable assessment of the biochemical and immunological interactions between host and vaginal microbiota involved in pathophysiology. This review examines our current knowledge of the relationships that exist between vaginal microbiota and the host at the level of the vaginal mucosal interface...
April 26, 2018: Methods: a Companion to Methods in Enzymology
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