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  1. Article ; Online: Next-generation single virus tracking.

    Padilla-Parra, Sergi

    Nature methods

    2022  Volume 19, Issue 12, Page(s) 1524–1525

    MeSH term(s) High-Throughput Nucleotide Sequencing
    Language English
    Publishing date 2022-11-10
    Publishing country United States
    Document type Journal Article
    ZDB-ID 2169522-2
    ISSN 1548-7105 ; 1548-7091
    ISSN (online) 1548-7105
    ISSN 1548-7091
    DOI 10.1038/s41592-022-01670-5
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  2. Article ; Online: Time-resolved single virus tracking and spectral imaging to understand HIV-1 entry and fusion.

    Padilla-Parra, Sergi

    Biology of the cell

    2022  Volume 115, Issue 3, Page(s) e2200082

    Abstract: Single Virus Tracking (SVT) is a key technique to understand how individual viral particles evolve during the infection cycle. In the case of the human immunodeficiency virus (HIV-1), this technology, which can be employed using a simple and affordable ... ...

    Abstract Single Virus Tracking (SVT) is a key technique to understand how individual viral particles evolve during the infection cycle. In the case of the human immunodeficiency virus (HIV-1), this technology, which can be employed using a simple and affordable wide-field microscope, has proven to be very useful in the first steps of infection, such as the kinetics of the fusion reaction or the point of fusion within live cells. Here, we describe how SVT in combination with other spectral imaging approaches is a powerful technique to illuminate crucial mechanistic aspects of the HIV-1 fusion reaction. We also stress the role of our laboratory in elucidating a few mechanistic aspects of retroviral fusion employing SVT such as: (i) the role of dynamin, (ii) how metabolism modulates membrane composition and cholesterol and its impact in fusion, (iii) the importance of envelope glycoprotein (Env) intra- and inter-molecular dynamics for neutralization, or (iv) the time-resolved fusion stoichiometry in three characteristic steps for the HIV-1 prefusion step. These observations constitute a good testimony of the complexity of retroviral fusion and show the strength of SVT when applied to live cells and combined with quantitative spectral approaches. Finally, we propose several crucial remaining questions around HIV-1 fusion and how the combined use of these technologies, always in live cells, will be able to shed light into the intricacies of arguably the most important step of the HIV-1 infection cycle.
    MeSH term(s) Humans ; HIV-1 ; HIV Infections ; Virus Internalization ; Membrane Fusion
    Language English
    Publishing date 2022-12-15
    Publishing country England
    Document type Journal Article ; Review
    ZDB-ID 245745-3
    ISSN 1768-322X ; 0399-0311 ; 0248-4900
    ISSN (online) 1768-322X
    ISSN 0399-0311 ; 0248-4900
    DOI 10.1111/boc.202200082
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  3. Article: HIV-1 Induced Cell-to-Cell Fusion or Syncytium Formation.

    Starling, Tobias / Padilla-Parra, Sergi

    Results and problems in cell differentiation

    2023  Volume 71, Page(s) 319–328

    Abstract: HIV-1 cell-free infection has been thoroughly investigated; however, its relevance and importance in vitro are questionable. Cell-cell transmission is now thought to be the dominant mode of transmission within the host; however precise molecular details ... ...

    Abstract HIV-1 cell-free infection has been thoroughly investigated; however, its relevance and importance in vitro are questionable. Cell-cell transmission is now thought to be the dominant mode of transmission within the host; however precise molecular details remain elusive. The considerable potency of cell-cell transmission hinges upon its ability to hijack and manipulate host immunological function to target uninfected cells, along with overcoming restriction factors and increasing the speed of latent pool formation. Another question of relevance is virus induced cell-cell fusion and how this process is regulated. How often HIV-1 induces the formation of syncytia? Is cell-cell function a potential process for HIV-1 transmission? These questions are discussed and reviewed together with a description of the most common ways of HIV-1 entry and transinfection.
    MeSH term(s) HIV-1/physiology ; Cell Fusion ; Giant Cells/physiology
    Language English
    Publishing date 2023-11-24
    Publishing country Germany
    Document type Journal Article
    ISSN 0080-1844
    ISSN 0080-1844
    DOI 10.1007/978-3-031-37936-9_15
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  4. Article ; Online: HIV-1 transmission: modelling and direct visualization in the third dimension.

    Coomer, Charles A / Padilla-Parra, Sergi

    Microscopy (Oxford, England)

    2023  Volume 72, Issue 3, Page(s) 164–177

    Abstract: Identifying initial events of mucosal entry of human immunodeficiency virus type-1 (HIV-1) in laboratory-based, physiologically relevant and high-throughput contexts may aid in designing effective strategies to block local transmission and spread of HIV- ... ...

    Abstract Identifying initial events of mucosal entry of human immunodeficiency virus type-1 (HIV-1) in laboratory-based, physiologically relevant and high-throughput contexts may aid in designing effective strategies to block local transmission and spread of HIV-1. Several paradigms have been posited for how HIV-1 crosses mucosal barriers to establish infection based on two dimensional (2D) culture-based or animal-based models. Nevertheless, despite these models stemming from 2D culture and animal studies, monolayers of cells poorly replicate the complex niche that influences viral entry at mucosal surfaces, whereas animal models often inadequately reproduce human disease pathophysiology and are prohibitively expensive. Organoids, having never been directly utilized in HIV-1 transmission investigations, may offer a compromise between 2D culture and animal models as they provide a platform that mimics the biophysical and biochemical niche of mucosal tissues. Importantly, observation of events downstream of viral inoculation is potentially accessible to researchers via an array of microscopy techniques. Because of the potential insights organoids may provide in this context, we offer this review to highlight key physiological factors of HIV-1 transmission at common mucosal sites and a discussion to highlight how many of these factors can be recapitulated in organoids, their current limitations and what questions can initially be addressed, particularly using a selective inclusion of quantitative light microscopy techniques. Harnessing organoids for direct observation of HIV-1 entry at mucosal sites may uncover potential therapeutic targets which prevent the establishment of HIV-1 infection.
    MeSH term(s) Animals ; Humans ; HIV-1/physiology ; HIV Infections/prevention & control ; Mucous Membrane ; Microscopy
    Language English
    Publishing date 2023-01-23
    Publishing country England
    Document type Review ; Journal Article
    ZDB-ID 2707496-1
    ISSN 2050-5701 ; 2050-5698
    ISSN (online) 2050-5701
    ISSN 2050-5698
    DOI 10.1093/jmicro/dfad014
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  5. Article ; Online: Structure dynamics of HIV-1 Env trimers on native virions engaged with living T cells.

    Carlon-Andres, Irene / Malinauskas, Tomas / Padilla-Parra, Sergi

    Communications biology

    2021  Volume 4, Issue 1, Page(s) 1228

    Abstract: The HIV-1 envelope glycoprotein (Env) mediates viral entry into the host cell. Although the highly dynamic nature of Env intramolecular conformations has been shown with single molecule spectroscopy in vitro, the bona fide Env intra- and intermolecular ... ...

    Abstract The HIV-1 envelope glycoprotein (Env) mediates viral entry into the host cell. Although the highly dynamic nature of Env intramolecular conformations has been shown with single molecule spectroscopy in vitro, the bona fide Env intra- and intermolecular mechanics when engaged with live T cells remains unknown. We used two photon fast fluorescence lifetime imaging detection of single-molecule Förster Resonance Energy Transfer occurring between fluorescent labels on HIV-1 Env on native virions. Our observations reveal Env dynamics at two levels: transitions between different intramolecular conformations and intermolecular interactions between Env within the viral membrane. Furthermore, we show that three broad neutralizing anti-Env antibodies directed to different epitopes restrict Env intramolecular dynamics and interactions between adjacent Env molecules when engaged with living T cells. Importantly, our results show that Env-Env interactions depend on efficient virus maturation, and that is disrupted upon binding of Env to CD4 or by neutralizing antibodies. Thus, this study illuminates how different intramolecular conformations and distribution of Env molecules mediate HIV-1 Env-T cell interactions in real time and therefore might control immune evasion.
    MeSH term(s) HIV-1/physiology ; T-Lymphocytes/virology ; Viral Proteins/metabolism ; Virion/metabolism ; Virus Internalization ; env Gene Products, Human Immunodeficiency Virus/metabolism
    Chemical Substances Viral Proteins ; env Gene Products, Human Immunodeficiency Virus
    Language English
    Publishing date 2021-10-27
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ISSN 2399-3642
    ISSN (online) 2399-3642
    DOI 10.1038/s42003-021-02658-1
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  6. Article: Quantitative FRET-FLIM-BlaM to Assess the Extent of HIV-1 Fusion in Live Cells

    Carlon-Andres, Irene / Padilla-Parra, Sergi

    Viruses. 2020 Feb. 12, v. 12, no. 2

    2020  

    Abstract: The first steps of human immunodeficiency virus (HIV) infection go through the engagement of HIV envelope (Env) with CD4 and coreceptors (CXCR4 or CCR5) to mediate viral membrane fusion between the virus and the host. New approaches are still needed to ... ...

    Abstract The first steps of human immunodeficiency virus (HIV) infection go through the engagement of HIV envelope (Env) with CD4 and coreceptors (CXCR4 or CCR5) to mediate viral membrane fusion between the virus and the host. New approaches are still needed to better define both the molecular mechanistic underpinnings of this process but also the point of fusion and its kinetics. Here, we have developed a new method able to detect and quantify HIV-1 fusion in single live cells. We present a new approach that employs fluorescence lifetime imaging microscopy (FLIM) to detect Förster resonance energy transfer (FRET) when using the β-lactamase (BlaM) assay. This novel approach allows comparing different populations of single cells regardless the concentration of CCF2-AM FRET reporter in each cell, and more importantly, is able to determine the relative amount of viruses internalized per cell. We have applied this approach in both reporter TZM-bl cells and primary T cell lymphocytes.
    Keywords CCR5 receptor ; CXCR4 receptor ; HIV infections ; Human immunodeficiency virus 1 ; T-lymphocytes ; beta-lactamase ; energy transfer ; fluorescence microscopy ; membrane fusion ; viruses
    Language English
    Dates of publication 2020-0212
    Publishing place Multidisciplinary Digital Publishing Institute
    Document type Article
    ZDB-ID 2516098-9
    ISSN 1999-4915
    ISSN 1999-4915
    DOI 10.3390/v12020206
    Database NAL-Catalogue (AGRICOLA)

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  7. Article ; Online: Quantitative FRET-FLIM-BlaM to Assess the Extent of HIV-1 Fusion in Live Cells.

    Carlon-Andres, Irene / Padilla-Parra, Sergi

    Viruses

    2020  Volume 12, Issue 2

    Abstract: The first steps of human immunodeficiency virus (HIV) infection go through the engagement of HIV envelope (Env) with CD4 and coreceptors (CXCR4 or CCR5) to mediate viral membrane fusion between the virus and the host. New approaches are still needed to ... ...

    Abstract The first steps of human immunodeficiency virus (HIV) infection go through the engagement of HIV envelope (Env) with CD4 and coreceptors (CXCR4 or CCR5) to mediate viral membrane fusion between the virus and the host. New approaches are still needed to better define both the molecular mechanistic underpinnings of this process but also the point of fusion and its kinetics. Here, we have developed a new method able to detect and quantify HIV-1 fusion in single live cells. We present a new approach that employs fluorescence lifetime imaging microscopy (FLIM) to detect Förster resonance energy transfer (FRET) when using the β-lactamase (BlaM) assay. This novel approach allows comparing different populations of single cells regardless the concentration of CCF2-AM FRET reporter in each cell, and more importantly, is able to determine the relative amount of viruses internalized per cell. We have applied this approach in both reporter TZM-bl cells and primary T cell lymphocytes.
    MeSH term(s) Cell Line ; Cells, Cultured ; Fluorescence Resonance Energy Transfer/methods ; HEK293 Cells ; HIV-1/physiology ; Humans ; Kinetics ; Optical Imaging/methods ; Single-Cell Analysis/methods ; T-Lymphocytes/physiology ; T-Lymphocytes/virology ; Virus Internalization ; beta-Lactamases/metabolism
    Chemical Substances beta-Lactamases (EC 3.5.2.6)
    Language English
    Publishing date 2020-02-12
    Publishing country Switzerland
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 2516098-9
    ISSN 1999-4915 ; 1999-4915
    ISSN (online) 1999-4915
    ISSN 1999-4915
    DOI 10.3390/v12020206
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  8. Article ; Online: Endogenous Labeling for Light Microscopy during HIV-1 Immune Responses.

    Salmon, Minette / Carlon-Andres, Irene / Padilla-Parra, Sergi

    Trends in immunology

    2020  Volume 41, Issue 12, Page(s) 1056–1059

    Abstract: New approaches in single molecule spectroscopy and microscopy are able to resolve the spatial and temporal resolution of T cell receptor signaling in the context of immune responses to HIV-1 infection. These approaches need to be complemented with novel ... ...

    Abstract New approaches in single molecule spectroscopy and microscopy are able to resolve the spatial and temporal resolution of T cell receptor signaling in the context of immune responses to HIV-1 infection. These approaches need to be complemented with novel techniques that endogenously tag the protein or proteins of interest, yet avoid overexpression, to image protein dynamics under physiological conditions.
    MeSH term(s) HIV-1/immunology ; Humans ; Immunity/immunology ; Microscopy/trends ; Proteins/chemistry ; Receptors, Antigen, T-Cell/metabolism ; Signal Transduction/physiology ; Staining and Labeling/methods ; Staining and Labeling/trends
    Chemical Substances Proteins ; Receptors, Antigen, T-Cell
    Language English
    Publishing date 2020-11-02
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 2036831-8
    ISSN 1471-4981 ; 1471-4906
    ISSN (online) 1471-4981
    ISSN 1471-4906
    DOI 10.1016/j.it.2020.10.009
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  9. Article: exampletestr

    Nolan, Rory / Padilla-Parra, Sergi

    Wellcome open research

    2017  Volume 2, Page(s) 31

    Abstract: In spite of the utility of unit tests, most R package developers do not write them. ...

    Abstract In spite of the utility of unit tests, most R package developers do not write them.
    Language English
    Publishing date 2017-06-21
    Publishing country England
    Document type Journal Article
    ISSN 2398-502X
    ISSN 2398-502X
    DOI 10.12688/wellcomeopenres.11635.2
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  10. Article ; Online: Multicolor lifetime imaging and its application to HIV-1 uptake.

    Starling, Tobias / Carlon-Andres, Irene / Iliopoulou, Maro / Kraemer, Benedikt / Loidolt-Krueger, Maria / Williamson, David J / Padilla-Parra, Sergi

    Nature communications

    2023  Volume 14, Issue 1, Page(s) 4994

    Abstract: Simultaneous imaging of nine fluorescent proteins is demonstrated in a single acquisition using fluorescence lifetime imaging microscopy combined with pulsed interleaved excitation of three laser lines. Multicolor imaging employing genetically encodable ... ...

    Abstract Simultaneous imaging of nine fluorescent proteins is demonstrated in a single acquisition using fluorescence lifetime imaging microscopy combined with pulsed interleaved excitation of three laser lines. Multicolor imaging employing genetically encodable fluorescent proteins permits spatio-temporal live cell imaging of multiple cues. Here, we show that multicolor lifetime imaging allows visualization of quadruple labelled human immunodeficiency viruses on host cells that in turn are also labelled with genetically encodable fluorescent proteins. This strategy permits to simultaneously visualize different sub-cellular organelles (mitochondria, cytoskeleton, and nucleus) during the process of virus entry with the potential of imaging up to nine different spectral channels in living cells.
    MeSH term(s) Humans ; HIV-1/genetics ; Biological Transport ; Cell Nucleus ; Coloring Agents ; Microscopy, Fluorescence
    Chemical Substances Coloring Agents
    Language English
    Publishing date 2023-08-17
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 2553671-0
    ISSN 2041-1723 ; 2041-1723
    ISSN (online) 2041-1723
    ISSN 2041-1723
    DOI 10.1038/s41467-023-40731-x
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