Molecular Biology

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    Protocols in Current Issue
    Fluorescence Screens for Identifying Central Nervous System–Acting Drug–Biosensor Pairs for Subcellular and Supracellular Pharmacokinetics

    Subcellular pharmacokinetic measurements have informed the study of central nervous system (CNS)–acting drug mechanisms. Recent investigations have been enhanced by the use of genetically encoded fluorescent biosensors for drugs of interest at the

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    Proteome Integral Solubility Alteration (PISA) Assay in Mammalian Cells for Deep, High-Confidence, and High-Throughput Target Deconvolution

    Chemical proteomics focuses on the drug–target–phenotype relationship for target deconvolution and elucidation of the mechanism of action—key and bottleneck in drug development and repurposing. Majorly due to the limits of using

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    Fluorescent Binding Protein Sensors for Detection and Quantification of Biochemicals, Metabolites, and Natural Products
    Authors:  Salete M. Newton and Phillip E. Klebba, date: 11/20/2022, view: 805, Q&A: 0

    Membrane transporters and soluble binding proteins recognize particular nutrients, metabolites, vitamins, or ligands. By modifying genetically engineered single cysteine residues near the active sites of such proteins with extrinsic maleimide

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    Fluorescence Screens for Identifying Central Nervous System–Acting Drug–Biosensor Pairs for Subcellular and Supracellular Pharmacokinetics
    [Abstract]

    Subcellular pharmacokinetic measurements have informed the study of central nervous system (CNS)–acting drug mechanisms. Recent investigations have been enhanced by the use of genetically encoded fluorescent biosensors for drugs of interest at the plasma membrane and in organelles. We describe screening and validation protocols for identifying hit

    ...
    Proteome Integral Solubility Alteration (PISA) Assay in Mammalian Cells for Deep, High-Confidence, and High-Throughput Target Deconvolution
    [Abstract]

    Chemical proteomics focuses on the drug–target–phenotype relationship for target deconvolution and elucidation of the mechanism of action—key and bottleneck in drug development and repurposing. Majorly due to the limits of using chemically modified ligands in affinity-based methods, new, unbiased, proteome-wide, and MS-based

    ...
    Fluorescent Binding Protein Sensors for Detection and Quantification of Biochemicals, Metabolites, and Natural Products
    Authors:  Salete M. Newton and Phillip E. Klebba, date: 11/20/2022, view: 805, Q&A: 0
    [Abstract]

    Membrane transporters and soluble binding proteins recognize particular nutrients, metabolites, vitamins, or ligands. By modifying genetically engineered single cysteine residues near the active sites of such proteins with extrinsic maleimide fluorophores, the approaches that we report create sensitive fluorescent sensors that detect, quantify,

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    OxiDIP-Seq for Genome-wide Mapping of Damaged DNA Containing 8-Oxo-2'-Deoxyguanosine
    Authors:  Francesca Gorini, Giovanni Scala, Susanna Ambrosio, Barbara Majello and Stefano Amente, date: 11/05/2022, view: 549, Q&A: 0
    [Abstract]

    8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) is considered to be a premutagenic DNA lesion generated by 2'-deoxyguanosine (dG) oxidation due to reactive oxygen species (ROS). In recent years, the 8-oxodG distribution in human, mouse, and yeast genomes has been underlined using various next-generation sequencing (NGS)–based strategies.

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    Single-molecule Force Spectroscopy on Biomembrane Force Probe to Characterize Force-dependent Bond Lifetimes of Receptor–ligand Interactions on Living Cells
    Authors:  Tongtong Zhang, Chenyi An, Wei Hu and Wei Chen, date: 10/20/2022, view: 881, Q&A: 0
    [Abstract]

    The transmembrane receptor–ligand interactions play a vital role in the physiological and pathological processes of living cells, such as immune cell activation, neural synapse formation, or viral invasion into host cells. Mounting evidence suggests that these processes involve mechanosensing and mechanotransduction, which are directly

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    A Novel Method of Inducible Directed Evolution to Evolve Complex Phenotypes
    Authors:  Ibrahim S. Al’Abri, Zidan Li, Daniel J. Haller and Nathan Crook, date: 10/20/2022, view: 1089, Q&A: 0
    [Abstract]

    Directed evolution is a powerful technique for identifying beneficial mutations in defined DNA sequences with the goal of improving desired phenotypes. Recent methodological advances have made the evolution of short DNA sequences quick and easy. However, the evolution of DNA sequences >5kb in length, notably gene clusters, is still a challenge

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    Extraction and Quantification of Plant Hormones and RNA from Pea Axillary Buds
    Authors:  Da Cao, Francois Barbier, Kaori Yoneyama and Christine A. Beveridge, date: 10/05/2022, view: 750, Q&A: 0
    [Abstract] The quantification of plant hormones and related gene expression is essential to improve the understanding of the molecular regulation of plant growth and development. However, plant hormone quantification is still challenging due to extremely low endogenous levels and high chemical diversity. In this study, we present a convenient extraction ...
    Tissue-Specific, Genome-wide Mapping of R-loops in Drosophila Using MapR
    Authors:  Juan Jauregui-Lozano, Kendall Cottingham and Hana Hall, date: 09/20/2022, view: 697, Q&A: 0
    [Abstract]

    R-loops, or RNA:DNA hybrids, are structures that arise co-transcriptionally when a nascent RNA hybridizes back with the template ssDNA, leading to a displaced ssDNA. Because accumulation of R-loops can lead to genomic instability and loss of cellular homeostasis, it is important to determine the genome-wide distribution of R-loops in

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    A Novel Imaging Technique for The On-site Assessment of Renal Biopsy Specimens
    Authors:  Tomoaki Takata, Hajime Isomoto, Takuji Iyama and Kentaro Yamada, date: 09/20/2022, view: 528, Q&A: 0
    [Abstract]

    When performing renal biopsy, it is necessary to identify the cortex, where glomeruli are exclusively distributed, to ensure the quality of the specimen for histological diagnosis. However, conventional methods using a stereomicroscope or magnifying lens often fail to clarify the quality of the specimen. We have established a fluorescent-based

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    Carbamoyltransferase Enzyme Assay: In vitro Modification of 5-hydroxymethylcytosine (5hmC) to 5-carbamoyloxymethylcytosine (5cmC)
    [Abstract]

    Nucleic acids in living organisms are more complex than the simple combinations of the four canonical nucleotides. Recent advances in biomedical research have led to the discovery of numerous naturally occurring nucleotide modifications and enzymes responsible for the synthesis of such modifications. In turn, these enzymes can be leveraged towards

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