scholarly journals Development and validation of a new high-throughput method to investigate the clonality of HTLV-1-infected cells based on provirus integration sites

10.1186/gm568 ◽  
2014 ◽  
Vol 6 (6) ◽  
pp. 46 ◽  
Author(s):  
Sanaz Firouzi ◽  
Yosvany López ◽  
Yutaka Suzuki ◽  
Kenta Nakai ◽  
Sumio Sugano ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Method ◽  
Integration Sites ◽  
Infected Cells ◽  
Development And Validation ◽  
Provirus Integration

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.

scholarly journals A High-Throughput Method for Cloning and Sequencing Human Immunodeficiency Virus Type 1 Integration Sites

2006 ◽  
Vol 80 (22) ◽  
pp. 11313-11321 ◽  
Author(s):  
Sanggu Kim ◽  
Yein Kim ◽  
Teresa Liang ◽  
Janet S. Sinsheimer ◽  
Samson A. Chow
Keyword(s):  
High Throughput ◽  
Integration Site ◽  
Target Site ◽  
Viral Dna ◽  
Target Site Selection ◽  
Immunodeficiency Virus ◽  
High Throughput Method ◽  
Integration Sites ◽  
Hiv 1

ABSTRACT Integration of retroviral DNA is nonspecific and can occur at many sites throughout chromosomes. However, the process is not uniformly distributed, and both hot and cold spots for integration exist. The mechanism that determines target site specificity is not well understood. Because of the nonspecific and widespread nature of integration, studies analyzing the mechanism and factors that control target site selection require the collection and analysis of a large library of human immunodeficiency virus type 1 (HIV-1) proviral clones. Such analyses are time-consuming and labor-intensive using conventional means. We have developed an efficient and high-throughput method of sequencing and mapping a large number of independent integration sites in the absence of any selection or bias. The new assay involves the use of a modified HIV-1 (NL-Mme) containing a type IIS restriction site, MmeI, at the right end of viral DNA. Digestion of genomic DNA from NL-Mme-infected cells generated viral DNA-containing fragments of a discrete size. Subsequent ligation-mediated PCR yielded short integration site fragments termed Int-tags, which were concatemerized for determining multiple integration sites in a single sequencing reaction. Analysis of chromosomal features and sequence preference associated with integration events confirmed the validity of the new high-throughput assay. The assay will aid the effort in understanding the mechanisms of target site selection during HIV-1 DNA integration, and the described methodology can be adapted easily to integration site studies involving other retroviruses and transposons.

scholarly journals Droplet microfluidic sequencing of HIV genomes and integration sites

2020 ◽  
Author(s):  
Chen Sun ◽  
Leqian Liu ◽  
Liliana Pérez ◽  
Xiangpeng Li ◽  
Yifan Liu ◽  
...  
Keyword(s):  
Antiretroviral Therapy ◽  
Genetic Analysis ◽  
Infected Cell ◽  
High Throughput ◽  
Integration Site ◽  
Hiv Cure ◽  
Cellular Junctions ◽  
Integration Sites ◽  
Infected Cells

AbstractSequencing individual HIV-proviruses and their adjacent cellular junctions can elucidate mechanisms of infected cell persistence in vivo. Here, we present a high throughput microfluidic method to sequence entire proviruses in their native integration site context. We used the method to analyze infected cells from people with HIV on suppressive antiretroviral therapy, demonstrating >90% capture and sequencing of paired proviral genomes and integration sites. This method should enable comprehensive genetic analysis of persistent HIV-infected cell reservoirs, providing important insights into the barriers to HIV cure.

scholarly journals Development And Validation of An Eco-Friendly and High-Throughput HPLC Method To Quantify Pyrethroid and Pyriproxyfen in Long-Lasting Insecticide-Treated Nets

2021 ◽  
Author(s):  
Kyle J. Walker ◽  
Christopher T. Williams ◽  
Folasade O. Oladepo ◽  
John Richard Lucas ◽  
David Malone ◽  
...  
Keyword(s):  
High Throughput ◽  
Internal Standard ◽  
Hplc Method ◽  
Insecticide Treated Nets ◽  
High Throughput Method ◽  
Before And After ◽  
Specialized Equipment ◽  
Extraction Volume ◽  
Development And Validation ◽  
Labour Time

Abstract Long-lasting insecticide-treated nets (LLINs) are essential to preventing malaria transmission. The LLINs should last for at least three years, even after repeated washings. Currently, tracking insecticides in LLINs is cumbersome, costly, and requires specialized equipment and hazardous solvents. We therefore developed a low-resource, high-throughput method for detecting insecticides in LLINs. In order to extract insecticides from polyethylene, LLIN samples were heated for 45 minutes at 85oC in 1-propanol-heptane containing dicyclohexylphthalate as an internal standard. Sample size was reduced to ~0.2 g for reduced extraction volume, which is 10% less than what was recommended. We optimized HPLC chromatographic conditions to assess pyrethroid and pyriproxyfen content in polyethylene-based LLINs. The method is capable of quantifying levels ≥ 0.0015% permethrin, 0.00045% alpha-cypermethrin and 0.00025% pyriproxyfen (w/w) in polyethylene, allowing tracking the insecticides before and after LLINs use. A variety of LLINs can be evaluated with this method, including those with 1% pyriproxyfen (pyriproxyfen-LLIN) or 2% permethrin (Olyset® Net), 1% pyriproxyfen and 2% permethrin (Olyset® Duo), or 0.55% pyriproxyfen combined with 0.55% alpha-cypermethrin (Royal Gaurd®). Analysis of 120 samples (40 nets) per run can be done with high precision and accuracy, reducing labour time and costs whilst reducing the environmental impact of organic solvents.

Paraffin-Embedded Cell Line Microarray (PECLIMA): Development and Validation of a High-Throughput Method for Antigen Profiling of Cell Lines

Pathobiology ◽  
2005 ◽  
Vol 72 (5) ◽  
pp. 225-232 ◽  
Author(s):  
Berta Ferrer ◽  
Raquel Bermudo ◽  
Timothy Thomson ◽  
Iracema Nayach ◽  
Marta Soler ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
High Throughput ◽  
High Throughput Method ◽  
Development And Validation
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