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  1. Article ; Online: Mapping the in vivo fitness landscape of a tethered ribosome.

    Radford, Felix / Rinehart, Jesse / Isaacs, Farren J

    Science advances

    2023  Volume 9, Issue 17, Page(s) eade8934

    Abstract: Fitness landscapes are models of the sequence space of a genetic element that map how each sequence corresponds to its activity and can be used to guide laboratory evolution. The ribosome is a macromolecular machine that is essential for protein ... ...

    Abstract Fitness landscapes are models of the sequence space of a genetic element that map how each sequence corresponds to its activity and can be used to guide laboratory evolution. The ribosome is a macromolecular machine that is essential for protein synthesis in all organisms. Because of the prevalence of dominant lethal mutations, a comprehensive fitness landscape of the ribosomal peptidyl transfer center (PTC) has not yet been attained. Here, we develop a method to functionally map an orthogonal tethered ribosome (oRiboT), which permits complete mutagenesis of nucleotides located in the PTC and the resulting epistatic interactions. We found that most nucleotides studied showed flexibility to mutation, and identified epistatic interactions between them, which compensate for deleterious mutations. This work provides a basis for a deeper understanding of ribosome function and malleability and could be used to inform design of engineered ribosomes with applications to synthesize next-generation biomaterials and therapeutics.
    MeSH term(s) Ribosomes/genetics ; Ribosomes/metabolism ; Protein Biosynthesis ; Mutation ; Nucleotides/metabolism
    Chemical Substances Nucleotides
    Language English
    Publishing date 2023-04-28
    Publishing country United States
    Document type Journal Article
    ZDB-ID 2810933-8
    ISSN 2375-2548 ; 2375-2548
    ISSN (online) 2375-2548
    ISSN 2375-2548
    DOI 10.1126/sciadv.ade8934
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article ; Online: Targeted editing and evolution of engineered ribosomes in vivo by filtered editing.

    Radford, Felix / Elliott, Shane D / Schepartz, Alanna / Isaacs, Farren J

    Nature communications

    2022  Volume 13, Issue 1, Page(s) 180

    Abstract: Genome editing technologies introduce targeted chromosomal modifications in organisms yet are constrained by the inability to selectively modify repetitive genetic elements. Here we describe filtered editing, a genome editing method that embeds group 1 ... ...

    Abstract Genome editing technologies introduce targeted chromosomal modifications in organisms yet are constrained by the inability to selectively modify repetitive genetic elements. Here we describe filtered editing, a genome editing method that embeds group 1 self-splicing introns into repetitive genetic elements to construct unique genetic addresses that can be selectively modified. We introduce intron-containing ribosomes into the E. coli genome and perform targeted modifications of these ribosomes using CRISPR/Cas9 and multiplex automated genome engineering. Self-splicing of introns post-transcription yields scarless RNA molecules, generating a complex library of targeted combinatorial variants. We use filtered editing to co-evolve the 16S rRNA to tune the ribosome's translational efficiency and the 23S rRNA to isolate antibiotic-resistant ribosome variants without interfering with native translation. This work sets the stage to engineer mutant ribosomes that polymerize abiological monomers with diverse chemistries and expands the scope of genome engineering for precise editing and evolution of repetitive DNA sequences.
    MeSH term(s) Anti-Bacterial Agents/pharmacology ; CRISPR-Cas Systems ; Escherichia coli/drug effects ; Escherichia coli/genetics ; Escherichia coli/metabolism ; Exons ; Gene Editing/methods ; Genetic Engineering ; Genome, Bacterial ; Introns ; Mutagenesis, Site-Directed/methods ; Polymers/chemistry ; Protein Biosynthesis ; RNA Splicing ; RNA, Ribosomal, 16S/genetics ; RNA, Ribosomal, 16S/metabolism ; RNA, Ribosomal, 23S/genetics ; RNA, Ribosomal, 23S/metabolism ; Repetitive Sequences, Nucleic Acid ; Ribosomes/genetics ; Ribosomes/metabolism
    Chemical Substances Anti-Bacterial Agents ; Polymers ; RNA, Ribosomal, 16S ; RNA, Ribosomal, 23S
    Language English
    Publishing date 2022-01-10
    Publishing country England
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
    ZDB-ID 2553671-0
    ISSN 2041-1723 ; 2041-1723
    ISSN (online) 2041-1723
    ISSN 2041-1723
    DOI 10.1038/s41467-021-27836-x
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  3. Article: Flow Cytometric Characterization of Antigen-Specific T Cells Based on RNA and Its Advantages in Detecting Infections and Immunological Disorders.

    Radford, Felix / Tyagi, Sanjay / Gennaro, Maria Laura / Pine, Richard / Bushkin, Yuri

    Critical reviews in immunology

    2017  Volume 36, Issue 5, Page(s) 359–378

    Abstract: Fluorescence in situ hybridization coupled with flow cytometry (FISH-Flow) is a highly quantitative, high-throughput platform allowing precise quantification of total mRNA transcripts in single cells. In undiagnosed infections posing a significant health ...

    Abstract Fluorescence in situ hybridization coupled with flow cytometry (FISH-Flow) is a highly quantitative, high-throughput platform allowing precise quantification of total mRNA transcripts in single cells. In undiagnosed infections posing a significant health burden worldwide, such as latent tuberculosis or asymptomatic recurrent malaria, an important challenge is to develop accurate diagnostic tools. Antigen-specific T cells create a persistent memory to pathogens, making them useful for diagnosis of infection. Stimulation of memory response initiates T-cell transitions between functional states. Numerous studies have shown that changes in protein levels lag real-time T-cell transitions. However, analysis at the single-cell transcriptional level can determine the differences. FISH-Flow is a powerful tool with which to study the functional states of T-cell subsets and to identify the gene expression profiles of antigen-specific T cells during disease progression. Advances in instrumentation, fluorophores, and FISH methodologies will broaden and deepen the use of FISH-Flow, changing the immunological field by allowing determination of functional immune signatures at the mRNA level and the development of new diagnostic tools.
    MeSH term(s) Animals ; Antigens/immunology ; Cell Separation ; Flow Cytometry/methods ; High-Throughput Screening Assays ; Humans ; Immune System Diseases/diagnosis ; Immune System Diseases/immunology ; Immunologic Memory ; In Situ Hybridization, Fluorescence/methods ; Infections/diagnosis ; Infections/immunology ; RNA/analysis ; T-Cell Antigen Receptor Specificity ; T-Lymphocyte Subsets/physiology ; T-Lymphocytes/physiology ; Transcriptome
    Chemical Substances Antigens ; RNA (63231-63-0)
    Keywords covid19
    Language English
    Publishing date 2017-06-12
    Publishing country United States
    Document type Journal Article
    ZDB-ID 1353116-5
    ISSN 1040-8401
    ISSN 1040-8401
    DOI 10.1615/CritRevImmunol.2017018316
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    In stock of ZB MED Cologne/Königswinter

    Zs.A 1699: Show issues Location:
    Je nach Verfügbarkeit (siehe Angabe bei Bestand)
    bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular
    Jg. 1995 - 2021: Lesesall (1.OG)
    ab Jg. 2022: Lesesaal (EG)

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  4. Article ; Online: Profiling T cell activation using single-molecule fluorescence in situ hybridization and flow cytometry.

    Bushkin, Yuri / Radford, Felix / Pine, Richard / Lardizabal, Alfred / Mangura, Bonita T / Gennaro, Maria Laura / Tyagi, Sanjay

    Journal of immunology (Baltimore, Md. : 1950)

    2015  Volume 194, Issue 2, Page(s) 836–841

    Abstract: Flow cytometric characterization of Ag-specific T cells typically relies on detection of protein analytes. Shifting the analysis to detection of RNA would provide several significant advantages, which we illustrate by developing a new host immunity-based ...

    Abstract Flow cytometric characterization of Ag-specific T cells typically relies on detection of protein analytes. Shifting the analysis to detection of RNA would provide several significant advantages, which we illustrate by developing a new host immunity-based platform for detection of infections. Cytokine mRNAs synthesized in response to ex vivo stimulation with pathogen-specific Ags are detected in T cells with single-molecule fluorescence in situ hybridization followed by flow cytometry. Background from pre-existing in vivo analytes is lower for RNAs than for proteins, allowing greater sensitivity for detection of low-frequency cells. Moreover, mRNA analysis reveals kinetic differences in cytokine expression that are not apparent at the protein level but provide novel insights into gene expression programs expected to define different T cell subsets. The utility of probing immunological memory of infections is demonstrated by detecting T cells that recognize mycobacterial and viral Ags in donors exposed to the respective pathogens.
    MeSH term(s) Adult ; Aged ; Antigens, Bacterial/immunology ; Cytokines/blood ; Cytokines/immunology ; Female ; Flow Cytometry/methods ; Humans ; In Situ Hybridization, Fluorescence/methods ; Lymphocyte Activation ; Male ; Middle Aged ; RNA, Messenger/blood ; RNA, Messenger/immunology ; T-Lymphocytes/immunology ; T-Lymphocytes/pathology ; Tuberculosis, Pulmonary/blood ; Tuberculosis, Pulmonary/immunology ; Tuberculosis, Pulmonary/pathology
    Chemical Substances Antigens, Bacterial ; Cytokines ; RNA, Messenger
    Language English
    Publishing date 2015-01-15
    Publishing country United States
    Document type Clinical Trial ; Journal Article ; Research Support, N.I.H., Extramural
    ZDB-ID 3056-9
    ISSN 1550-6606 ; 0022-1767 ; 1048-3233 ; 1047-7381
    ISSN (online) 1550-6606
    ISSN 0022-1767 ; 1048-3233 ; 1047-7381
    DOI 10.4049/jimmunol.1401515
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    In stock of ZB MED Cologne/Königswinter

    Ud II Zs.37: Show issues Location:
    Je nach Verfügbarkeit (siehe Angabe bei Bestand)
    bis Jg. 2021: Bestellungen von Artikeln über das Online-Bestellformular
    ab Jg. 2022: Lesesaal (EG)
    Zs.MG 28: Show issues

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  5. Article ; Online: FISH-Flow, a protocol for the concurrent detection of mRNA and protein in single cells using fluorescence in situ hybridization and flow cytometry.

    Arrigucci, Riccardo / Bushkin, Yuri / Radford, Felix / Lakehal, Karim / Vir, Pooja / Pine, Richard / Martin, December / Sugarman, Jeffrey / Zhao, Yanlin / Yap, George S / Lardizabal, Alfred A / Tyagi, Sanjay / Gennaro, Maria Laura

    Nature protocols

    2017  Volume 12, Issue 6, Page(s) 1245–1260

    Abstract: We describe a flow-cytometry-based protocol for intracellular mRNA measurements in nonadherent mammalian cells using fluorescence in situ hybridization (FISH) probes. The method, which we call FISH-Flow, allows for high-throughput multiparametric ... ...

    Abstract We describe a flow-cytometry-based protocol for intracellular mRNA measurements in nonadherent mammalian cells using fluorescence in situ hybridization (FISH) probes. The method, which we call FISH-Flow, allows for high-throughput multiparametric measurements of gene expression, a task that was not feasible with earlier, microscopy-based approaches. The FISH-Flow protocol involves cell fixation, permeabilization and hybridization with a set of fluorescently labeled oligonucleotide probes. In this protocol, surface and intracellular protein markers can also be stained with fluorescently labeled antibodies for simultaneous protein and mRNA measurement. Moreover, a semiautomated, single-tube version of the protocol can be performed with a commercially available cell-wash device that reduces cell loss, operator time and interoperator variability. It takes ∼30 h to perform this protocol. An example of FISH-Flow measurements of cytokine mRNA induction by ex vivo stimulation of primed T cells with specific antigens is described.
    MeSH term(s) Automation, Laboratory/methods ; Flow Cytometry/methods ; Humans ; In Situ Hybridization, Fluorescence/methods ; Leukocytes, Mononuclear/chemistry ; Proteins/analysis ; RNA, Messenger/analysis ; Single-Cell Analysis/methods
    Chemical Substances Proteins ; RNA, Messenger
    Language English
    Publishing date 2017-06
    Publishing country England
    Document type Journal Article
    ZDB-ID 2244966-8
    ISSN 1750-2799 ; 1754-2189
    ISSN (online) 1750-2799
    ISSN 1754-2189
    DOI 10.1038/nprot.2017.039
    Database MEDical Literature Analysis and Retrieval System OnLINE

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