scholarly journals Single cell sequencing of the small and AT-skewed genome of malaria parasites

2020 ◽  
Author(s):  
Shiwei Liu ◽  
Adam C. Huckaby ◽  
Audrey C. Brown ◽  
Christopher C. Moore ◽  
Ian Burbulis ◽  
...  
Keyword(s):  
Single Cell ◽  
Copy Number ◽  
Mammalian Cells ◽  
Copy Number Variations ◽  
Single Cell Level ◽  
Cell Level ◽  
Single Cell Sequencing ◽  
Base Content ◽  
Amplification Method ◽  
Adaptive Mechanisms

AbstractSingle cell genomics is a rapidly advancing field; however, most techniques are designed for mammalian cells. Here, we present a single cell sequencing pipeline for the intracellular parasite, Plasmodium falciparum, which harbors a relatively small genome with an extremely skewed base content. Through optimization of a quasi-linear genome amplification method, we achieve better targeting of the parasite genome over contaminants and generate coverage levels that allow detection of relatively small copy number variations on a single cell level. These improvements are important for expanding accessibility of single cell approaches to new organisms and for improving the study of adaptive mechanisms.

Accurate and sensitive single-cell-level detection of copy number variations by micro-channel multiple displacement amplification (μcMDA)

Nanoscale ◽  
2018 ◽  
Vol 10 (37) ◽  
pp. 17933-17941 ◽  
Author(s):  
Junji Li ◽  
Na Lu ◽  
Yuhan Tao ◽  
Mengqin Duan ◽  
Yi Qiao ◽  
...  
Keyword(s):  
Single Cell ◽  
Copy Number ◽  
High Performance ◽  
Multiple Displacement Amplification ◽  
Copy Number Variations ◽  
Reaction Vessel ◽  
Single Cell Level ◽  
Micro Channel ◽  
Cell Level ◽  
Cnv Detection

An improved multiple displacement amplification (MDA) approach realized by compressing the geometry of the reaction vessel exhibits high performance for single-cell-level CNV detection.

scholarly journals Single-cell sequencing of the small and AT-skewed genome of malaria parasites

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Shiwei Liu ◽  
Adam C. Huckaby ◽  
Audrey C. Brown ◽  
Christopher C. Moore ◽  
Ian Burbulis ◽  
...  
Keyword(s):  
Single Cell ◽  
Genetic Variants ◽  
Mammalian Cells ◽  
Intracellular Parasite ◽  
Linear Amplification ◽  
Small Genome ◽  
Single Cell Sequencing ◽  
Base Content ◽  
Amplification Method ◽  
Variant Analysis

AbstractSingle-cell genomics is a rapidly advancing field; however, most techniques are designed for mammalian cells. We present a single-cell sequencing pipeline for an intracellular parasite, Plasmodium falciparum, with a small genome of extreme base content. Through optimization of a quasi-linear amplification method, we target the parasite genome over contaminants and generate coverage levels allowing detection of minor genetic variants. This work, as well as efforts that build on these findings, will enable detection of parasite heterogeneity contributing to P. falciparum adaptation. Furthermore, this study provides a framework for optimizing single-cell amplification and variant analysis in challenging genomes.

Measurement of Molecular Mobility in Mammalian Cells

2004 ◽  
Author(s):  
Alptekin Aksan ◽  
Mehmet Toner
Keyword(s):  
Single Cell ◽  
Real Time ◽  
Chemical Reaction ◽  
Molecular Mobility ◽  
Mammalian Cells ◽  
Free Water ◽  
Single Cell Level ◽  
Extracellular Water ◽  
Cell Level

Preservation of mammalian cells requires establishing a reversible stasis condition by reducing the intra/extracellular molecular mobility ensuring reduced chemical reaction and deterioration rates. Molecular mobility may be reduced by various techniques. For example, in cryopreservation, mobility within and surrounding the cell is reduced through freezing the free water that constitutes 70–90% of the cell’s composition. In dried-state preservation applied successfully to preserve seeds, pharmacological materials and foodstuff (mimicking the anhydrobiosis phenomenon seen in nature), reduction in molecular mobility is established by removing intra/extracellular water. Certain carbohydrates (such as trehalose and sucrose) can be artificially uploaded into mammalian cells to replace the removed water and to form an intra/extracellular glass. In this research, a fluorescent rotor is utilized to determine the changes in intracellular molecular mobility during carbohydrate uploading of mammalian cells. It was shown that using this technique, it is feasible to make real-time mobility measurements at a single cell level.

scholarly journals Single-Cell Sequencing Confirms Transcripts and VHDJH Rearrangements of Immunoglobulin Genes in Human Podocytes

Genes ◽  
2021 ◽  
Vol 12 (4) ◽  
pp. 472
Author(s):  
Zhenling Deng ◽  
Huige Yan ◽  
Zhan Shi ◽  
Xinyu Tian ◽  
Zhuan Cui ◽  
...  
Keyword(s):  
Single Cell ◽  
Sanger Sequencing ◽  
Single Cell Level ◽  
Normal Kidney ◽  
Cell Level ◽  
Glomerular Diseases ◽  
Single Cell Sequencing ◽  
Heavy Chains ◽  
Single Cell Rna Sequencing ◽  
Healthy Control

Most glomerular diseases are associated with inflammation caused by deposited pathogenic immunoglobulins (Igs), which are believed to be produced by B cells. However, our previous study indicated that the human podocyte cell line can produce IgG. In this study, we aimed to confirm the transcripts and characterize the repertoires of Igs in primary podocytes at single cell level. First, single-cell RNA sequencing of cell suspensions from “normal” kidney cortexes by a 10xGenomics Chromium system detected Ig transcripts in 7/360 podocytes and Ig gene segments in 106/360 podocytes. Then, we combined nested PCR with Sanger sequencing to detect the transcripts and characterize the repertoires of Igs in 48 single podocytes and found that five classes of Ig heavy chains were amplified in podocytes. Four-hundred and twenty-nine VHDJH rearrangement sequences were analyzed; podocyte-derived Igs exhibited classic VHDJH rearrangements with nucleotide additions and somatic hypermutations, biased VH1 usage and restricted diversity. Moreover, compared with the podocytes from healthy control that usually expressed one class of Ig and one VHDJH pattern, podocytes from patients expressed more classes of Ig, VHDJH patterns and somatic hypermutations. These findings suggested that podocytes can express Igs in normal condition and increase diversity in pathological situations.

scholarly journals LINE-1 Retrotransposon expression in cancerous, epithelial and neuronal cells revealed by 5’-single cell RNA-Seq

2021 ◽  
Author(s):  
Wilson McKerrow ◽  
Shane A. Evans ◽  
Azucena Rocha ◽  
John Sedivy ◽  
Nicola Neretti ◽  
...  
Keyword(s):  
Single Cell ◽  
Cancer Cells ◽  
Early Development ◽  
Immune Cells ◽  
Copy Number ◽  
Single Cells ◽  
Single Cell Level ◽  
Rna Seq ◽  
Cell Level ◽  
Mouse Hippocampus

AbstractLINE-1 retrotransposons are known to be expressed in early development, in tumors and in the germline. Less is known about LINE-1 expression at the single cell level, especially outside the context of cancer. Because LINE-1 elements are present at a high copy number, many transcripts that are not driven by the LINE-1 promoter nevertheless terminate at the LINE-1 3’ UTR. Thus, 3’ targeted single cell RNA-seq datasets are not appropriate for studying LINE-1. However, 5’ targeted single cell datasets provide an opportunity to analyze LINE-1 expression at the single cell level. Most LINE-1 copies are 5’ truncated, and a transcript that contains the LINE-1 5’ UTR as its 5’ end is likely to have been transcribed from its promoter. We developed a method, L1-sc (LINE-1 expression for single cells), to quantify LINE-1 expression in 5’ targeted 10x genomics single cell RNA-seq datasets. Our method confirms that LINE-1 expression is high in cancer cells, but low or absent from immune cells. We also find that LINE-1 expression is elevated in epithelial compared to immune cells outside of the context of cancer and that it is also elevated in neurons compared to glia in the mouse hippocampus.

scholarly journals Accumulation of Amphotericin B in Human Macrophages Enhances Activity against Aspergillus fumigatus Conidia: Quantification of Conidial Kill at the Single-Cell Level

1998 ◽  
Vol 42 (10) ◽  
pp. 2569-2575 ◽  
Author(s):  
Bernhard Jahn ◽  
Albert Rampp ◽  
Christian Dick ◽  
Andreas Jahn ◽  
Michael Palmer ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Single Cell ◽  
Amphotericin B ◽  
Mammalian Cells ◽  
Single Cell Level ◽  
Cell Level ◽  
Streptolysin O ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony

ABSTRACT A cytofluorometric assay that allowed assessment of damage to phagocytosed Aspergillus fumigatus conidia at the single-cell level was developed. After ingestion by monocyte-derived macrophages (MDMs), conidia were reisolated by treatment of the cells with streptolysin O, a pore-forming toxin with lytic properties on mammalian cells but not on fungi. The counts obtained by staining of damaged conidia with propidium iodide and quantification by cytofluorometry correlated with colony counts. By the use of this method, we demonstrate that MDMs differentiated in vitro by low-dose granulocyte-macrophage colony-stimulating factor and gamma interferon have only a limited capacity to damageAspergillus conidia in vitro. The killing rate 12 h after phagocytosis was found to be only 10 to 15%. However, intracellular loading of the phagocytes with amphotericin B (AmB) dose dependently enhanced the anticonidial activity. Preincubation of macrophages with only 1 μg of AmB per ml resulted in an uptake of 18 fg of AmB/cell, leading to killing rates of 50 to 60%. The experimental protocol provides a new tool for the rapid quantification of anticonidial activity against A. fumigatus in vitro. Intracellular accumulation of AmB may represent an important factor underlying the efficacy of this antifungal drug in the prophylaxis and treatment ofAspergillus infections.

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