scholarly journals Flow Virometry Quantification of Host Proteins on the Surface of HIV-1 Pseudovirus Particles

Viruses ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 1296
Author(s):  
Jonathan Burnie ◽  
Vera A. Tang ◽  
Joshua A. Welsh ◽  
Arvin T. Persaud ◽  
Laxshaginee Thaya ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
High Throughput ◽  
Single Cells ◽  
Surface Current ◽  
High Sensitivity ◽  
Viral Envelope ◽  
High Throughput Analysis ◽  
Associated Proteins ◽  
Hiv 1

The HIV-1 glycoprotein spike (gp120) is typically the first viral antigen that cells encounter before initiating immune responses, and is often the sole target in vaccine designs. Thus, characterizing the presence of cellular antigens on the surfaces of HIV particles may help identify new antiviral targets or impact targeting of gp120. Despite the importance of characterizing proteins on the virion surface, current techniques available for this purpose do not support high-throughput analysis of viruses, and typically only offer a semi-quantitative assessment of virus-associated proteins. Traditional bulk techniques often assess averages of viral preparations, which may mask subtle but important differences in viral subsets. On the other hand, microscopy techniques, which provide detail on individual virions, are difficult to use in a high-throughput manner and have low levels of sensitivity for antigen detection. Flow cytometry is a technique that traditionally has been used for rapid, high-sensitivity characterization of single cells, with limited use in detecting viruses, since the small size of viral particles hinders their detection. Herein, we report the detection and surface antigen characterization of HIV-1 pseudovirus particles by light scattering and fluorescence with flow cytometry, termed flow virometry for its specific application to viruses. We quantified three cellular proteins (integrin α4β7, CD14, and CD162/PSGL-1) in the viral envelope by directly staining virion-containing cell supernatants without the requirement of additional processing steps to distinguish virus particles or specific virus purification techniques. We also show that two antigens can be simultaneously detected on the surface of individual HIV virions, probing for the tetraspanin marker, CD81, in addition to α4β7, CD14, and CD162/PSGL-1. This study demonstrates new advances in calibrated flow virometry as a tool to provide sensitive, high-throughput characterization of the viral envelope in a more efficient, quantitative manner than previously reported techniques.

scholarly journals Plasma Membrane-Associated Restriction Factors and Their Counteraction by HIV-1 Accessory Proteins

Cells ◽  
2019 ◽  
Vol 8 (9) ◽  
pp. 1020 ◽  
Author(s):  
Ramirez ◽  
Sharma ◽  
Singh ◽  
Stoneham ◽  
Vollbrecht ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Immune Surveillance ◽  
Viral Envelope ◽  
Accessory Proteins ◽  
Host Defenses ◽  
Restriction Factors ◽  
Associated Proteins ◽  
Infected Cells ◽  
Hiv 1

The plasma membrane is a site of conflict between host defenses and many viruses. One aspect of this conflict is the host’s attempt to eliminate infected cells using innate and adaptive cell-mediated immune mechanisms that recognize features of the plasma membrane characteristic of viral infection. Another is the expression of plasma membrane-associated proteins, so-called restriction factors, which inhibit enveloped virions directly. HIV-1 encodes two countermeasures to these host defenses: The membrane-associated accessory proteins Vpu and Nef. In addition to inhibiting cell-mediated immune-surveillance, Vpu and Nef counteract membrane-associated restriction factors. These include BST-2, which traps newly formed virions at the plasma membrane unless counteracted by Vpu, and SERINC5, which decreases the infectivity of virions unless counteracted by Nef. Here we review key features of these two antiviral proteins, and we review Vpu and Nef, which deplete them from the plasma membrane by co-opting specific cellular proteins and pathways of membrane trafficking and protein-degradation. We also discuss other plasma membrane proteins modulated by HIV-1, particularly CD4, which, if not opposed in infected cells by Vpu and Nef, inhibits viral infectivity and increases the sensitivity of the viral envelope glycoprotein to host immunity.

scholarly journals Stable integrant-specific differences in bimodal HIV-1 expression patterns revealed by high-throughput analysis

2019 ◽  
Vol 15 (10) ◽  
pp. e1007903
Author(s):  
David F. Read ◽  
Edmond Atindaana ◽  
Kalyani Pyaram ◽  
Feng Yang ◽  
Sarah Emery ◽  
...  
Keyword(s):  
High Throughput ◽  
Expression Patterns ◽  
High Throughput Analysis ◽  
Throughput Analysis ◽  
Hiv 1

Characterization of Microvesicles in Septic Shock Using High-Sensitivity Flow Cytometry

Shock ◽  
2016 ◽  
Vol 46 (4) ◽  
pp. 373-381 ◽  
Author(s):  
Georg Franz Lehner ◽  
Ulrich Harler ◽  
Viktoria Maria Haller ◽  
Clemens Feistritzer ◽  
Julia Hasslacher ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Septic Shock ◽  
High Sensitivity

scholarly journals Virtual-freezing fluorescence imaging flow cytometry

2020 ◽  
Author(s):  
Hideharu Mikami ◽  
Makoto Kawaguchi ◽  
Chun-Jung Huang ◽  
Hiroki Matsumura ◽  
Takeaki Sugimura ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Spatial Resolution ◽  
High Throughput ◽  
Cancer Biology ◽  
Single Cells ◽  
Image Sensor ◽  
Imaging Method ◽  
Microscopy Imaging ◽  
Trade Off ◽  
Imaging Flow Cytometry

ABSTRACTBy virtue of the combined merits of flow cytometry and fluorescence microscopy, imaging flow cytometry (IFC) has become an established tool for cell analysis in diverse biomedical fields such as cancer biology, microbiology, immunology, hematology, and stem cell biology. However, the performance and utility of IFC are severely limited by the fundamental trade-off between throughput, sensitivity, and spatial resolution. For example, at high flow speed (i.e., high throughput), the integration time of the image sensor becomes short, resulting in reduced sensitivity or pixel resolution. Here we present an optomechanical imaging method that overcomes the trade-off by virtually “freezing” the motion of flowing cells on the image sensor to effectively achieve 1,000 times longer exposure time for microscopy-grade fluorescence image acquisition. Consequently, it enables high-throughput IFC of single cells at >10,000 cells/s without sacrificing sensitivity and spatial resolution. The availability of numerous information-rich fluorescence cell images allows high-dimensional statistical analysis and accurate classification with deep learning, as evidenced by our demonstration of unique applications in hematology and microbiology.

scholarly journals MicrobeAnnotator: a user-friendly, comprehensive functional annotation pipeline for microbial genomes

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Carlos A. Ruiz-Perez ◽  
Roth E. Conrad ◽  
Konstantinos T. Konstantinidis
Keyword(s):  
High Throughput ◽  
Functional Annotation ◽  
High Throughput Sequencing ◽  
Gene Annotation ◽  
Single Cells ◽  
Computationally Efficient ◽  
High Throughput Analysis ◽  
Microbial Genomes ◽  
Reference Protein ◽  
User Friendly

Abstract Background High-throughput sequencing has increased the number of available microbial genomes recovered from isolates, single cells, and metagenomes. Accordingly, fast and comprehensive functional gene annotation pipelines are needed to analyze and compare these genomes. Although several approaches exist for genome annotation, these are typically not designed for easy incorporation into analysis pipelines, do not combine results from different annotation databases or offer easy-to-use summaries of metabolic reconstructions, and typically require large amounts of computing power for high-throughput analysis not available to the average user. Results Here, we introduce MicrobeAnnotator, a fully automated, easy-to-use pipeline for the comprehensive functional annotation of microbial genomes that combines results from several reference protein databases and returns the matching annotations together with key metadata such as the interlinked identifiers of matching reference proteins from multiple databases [KEGG Orthology (KO), Enzyme Commission (E.C.), Gene Ontology (GO), Pfam, and InterPro]. Further, the functional annotations are summarized into Kyoto Encyclopedia of Genes and Genomes (KEGG) modules as part of a graphical output (heatmap) that allows the user to quickly detect differences among (multiple) query genomes and cluster the genomes based on their metabolic similarity. MicrobeAnnotator is implemented in Python 3 and is freely available under an open-source Artistic License 2.0 from https://github.com/cruizperez/MicrobeAnnotator. Conclusions We demonstrated the capabilities of MicrobeAnnotator by annotating 100 Escherichia coli and 78 environmental Candidate Phyla Radiation (CPR) bacterial genomes and comparing the results to those of other popular tools. We showed that the use of multiple annotation databases allows MicrobeAnnotator to recover more annotations per genome compared to faster tools that use reduced databases and is computationally efficient for use in personal computers. The output of MicrobeAnnotator can be easily incorporated into other analysis pipelines while the results of other annotation tools can be seemingly incorporated into MicrobeAnnotator to generate summary plots.

scholarly journals Stable integrant-specific differences in bimodal HIV-1 expression patterns revealed by high-throughput analysis

2019 ◽  
Author(s):  
David F. Read ◽  
Edmond Atindaana ◽  
Kalyani Pyaram ◽  
Feng Yang ◽  
Sarah Emery ◽  
...  
Keyword(s):  
Gene Expression ◽  
Infected Cell ◽  
High Throughput ◽  
Expression Patterns ◽  
Clonal Expansion ◽  
High Throughput Analysis ◽  
Integration Sites ◽  
Infected Cells ◽  
Hiv 1 ◽  
Over Time

AbstractHIV-1 gene expression is regulated by host and viral factors that interact with viral motifs and is influenced by proviral integration sites. Here, expression variation among integrants was followed for hundreds of individual proviral clones within polyclonal populations throughout successive rounds of virus and cultured cell replication. Initial findings in immortalized cells were validated using CD4+ cells from donor blood. Tracking clonal behavior by proviral “zip codes” indicated that mutational inactivation during reverse transcription was rare, while clonal expansion and proviral expression states varied widely. By sorting for provirus expression using a GFP reporter in thenefopen reading frame, distinct clone-specific variation in on/off proportions were observed that spanned three orders of magnitude. Tracking GFP phenotypes over time revealed that as cells divided, their progeny alternated between HIV transcriptional activity and non-activity. Despite these phenotypic oscillations, the overall GFP+ population within each clone was remarkably stable, with clones maintaining clone-specific equilibrium mixtures of GFP+ and GFP-cells. Integration sites were analyzed for correlations between genomic features and the epigenetic phenomena described here. Integrants inserted in genes’ sense orientation were more frequently found to be GFP negative than those in the antisense orientation, and clones with high GFP+ proportions were more distal to repressive H3K9me3 peaks than low GFP+ clones. Clones with low frequencies of GFP positivity appeared to expand more rapidly than clones for which most cells were GFP+, even though the tested proviruses were Vpr-. Thus, much of the increase in the GFP-population in these polyclonal pools over time reflected differential clonal expansion. Together, these results underscore the temporal and quantitative variability in HIV-1 gene expression among proviral clones that are conferred in the absence of metabolic or cell-type dependent variability, and shed light on cell-intrinsic layers of regulation that affect HIV-1 population dynamics.SummaryVery few HIV-1 infected cells persist in patients for more than a couple days, but those that do pose life-long health risks. Strategies designed to eliminate these cells have been based on assumptions about what viral properties allow infected cell survival. However, such approaches for HIV-1 eradication have not yet shown therapeutic promise, possibly because much of the research underlying assumptions about virus persistence has been focused on a limited number of infected cell types, the averaged behavior of cells in diverse populations, or snapshot views. Here, we developed a high-throughput approach to study hundreds of distinct HIV-1 infected cells and their progeny over time in an unbiased way. This revealed that each virus established its own pattern of gene expression that, upon infected cell division, was stably transmitted to all progeny cells. Expression patterns consisted of alternating waves of activity and inactivity, with the extent of activity differing among infected cell families over a 1000-fold range. The dynamics and variability among infected cells and within complex populations that the work here revealed has not previously been evident, and may help establish more accurate correlates of persistent HIV-1 infection.

A high-throughput flow cytometry-on-a-CMOS platform for single-cell dielectric spectroscopy at microwave frequencies

Lab on a Chip ◽  
2018 ◽  
Vol 18 (14) ◽  
pp. 2065-2076 ◽  
Author(s):  
Jun-Chau Chien ◽  
Ali Ameri ◽  
Erh-Chia Yeh ◽  
Alison N. Killilea ◽  
Mekhail Anwar ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Dielectric Properties ◽  
Single Cell ◽  
High Throughput ◽  
Dielectric Spectroscopy ◽  
Free Flow ◽  
Label Free ◽  
Microwave Frequencies

This work presents a microfluidics-integrated label-free flow cytometry-on-a-CMOS platform for the characterization of the cytoplasm dielectric properties at microwave frequencies.

scholarly journals An acoustic platform for single-cell, high-throughput measurements of the viscoelastic properties of cells

2020 ◽  
Author(s):  
Valentin Romanov ◽  
Giulia Silvani ◽  
Huiyu Zhu ◽  
Charles D Cox ◽  
Boris Martinac
Keyword(s):  
Mechanical Properties ◽  
Single Cell ◽  
High Throughput ◽  
Single Cells ◽  
Adherent Cells ◽  
Dependent Manner ◽  
Cellular Processes ◽  
Membrane Cytoskeleton ◽  
Change Temperature

ABSTRACTCellular processes including adhesion, migration and differentiation are governed by the distinct mechanical properties of each cell. Importantly, the mechanical properties of individual cells can vary depending on local physical and biochemical cues in a time-dependent manner resulting in significant inter-cell heterogeneity. While several different methods have been developed to interrogate the mechanical properties of single cells, throughput to capture this heterogeneity remains an issue. While new high-throughput techniques are slowly emerging, they are primarily aimed at characterizing cells in suspension, whereas high-throughput measurements of adherent cells have proven to be more challenging. Here, we demonstrate single-cell, high-throughput characterization of adherent cells using acoustic force spectroscopy. We demonstrate that cells undergo marked changes in viscoelasticity as a function of temperature, the measurements of which are facilitated by a closed microfluidic culturing environment that can rapidly change temperature between 21 °C and 37 °C. In addition, we show quantitative differences in cells exposed to different pharmacological treatments specifically targeting the membrane-cytoskeleton interface. Further, we utilize the high-throughput format of the AFS to rapidly probe, in excess of 1000 cells, three different cell-lines expressing different levels of a mechanosensitive protein, Piezo1, demonstrating the ability to differentiate between cells based on protein expression levels.

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