scholarly journals Single-cell motile behaviour of $$Trypanosoma brucei $$ in thin-layered fluid collectives

2021 ◽  
Vol 44 (3) ◽  
Author(s):  
Timothy Krüger ◽  
Katharina Maus ◽  
Verena Kreß ◽  
Elisabeth Meyer-Natus ◽  
Markus Engstler
Keyword(s):  
Single Cell ◽  
Trypanosoma Brucei ◽  
Developmental Cycle ◽  
Cell Level ◽  
Unknown Parameters ◽  
Life Cycle Stages ◽  
Cell Velocity ◽  
Cell Groups ◽  
First Time ◽  
Swarming Behaviour

Abstract We describe a system for the analysis of an important unicellular eukaryotic flagellate in a confining and crowded environment. The parasite Trypanosoma brucei is arguably one of the most versatile microswimmers known. It has unique properties as a single microswimmer and shows remarkable adaptations (not only in motility, but prominently so), to its environment during a complex developmental cycle involving two different hosts. Specific life cycle stages show fascinating collective behaviour, as millions of cells can be forced to move together in extreme confinement. Our goal is to examine such motile behaviour directly in the context of the relevant environments. Therefore, for the first time, we analyse the motility behaviour of trypanosomes directly in a widely used assay, which aims to evaluate the parasites behaviour in collectives, in response to as yet unknown parameters. In a step towards understanding whether, or what type of, swarming behaviour of trypanosomes exists, we customised the assay for quantitative tracking analysis of motile behaviour on the single-cell level. We show that the migration speed of cell groups does not directly depend on single-cell velocity and that the system remains to be simplified further, before hypotheses about collective motility can be advanced. Graphic abstract

scholarly journals Single-cell Differential Splicing Analysis Reveals High Heterogeneity of Liver Tumor-infiltrating T Cells

2020 ◽  
Author(s):  
Shang Liu ◽  
Biaofeng Zhou ◽  
Liang Wu ◽  
Yan Sun ◽  
Jie Chen ◽  
...  
Keyword(s):  
T Cells ◽  
Alternative Splicing ◽  
T Cell ◽  
Single Cell ◽  
Regulatory Factor ◽  
Differential Splicing ◽  
Cell Level ◽  
Comprehensive Investigation ◽  
Cancer Initiation ◽  
Cell Groups

Abstract Recent advances in single-cell RNA sequencing (scRNA-seq) have improved our understanding of the association between tumor-infiltrating lymphocyte (TILs) heterogeneity and cancer initiation and progression. However, studies investigating alternative splicing (AS) as an important regulatory factor of heterogeneity remain limited. Here, we developed a new computational tool, DESJ-detection, which accurately detects differentially expressed splicing junctions (DESJs) between cell groups at the single-cell level. We analyzed 5,063 T cells of hepatocellular carcinoma (HCC) and identified 1,176 DESJs across 11 T cell subtypes. Interestingly, DESJs were enriched in UTRs, and have putative effects on heterogeneity. Cell subtypes with a similar function closely clustered together at the AS level. Meanwhile, we identified two novel cell states, pre-exhaustion and pre-activation with the isoform markers CD103-201 and ARHGAP15-205. In summary, we present a comprehensive investigation of alternative splicing differences, which provided novel insights into T cell heterogeneity and can be applied to other full-length scRNA-seq datasets.

scholarly journals scDAPA: detection and visualization of dynamic alternative polyadenylation from single cell RNA-seq data

2019 ◽  
Author(s):  
Congting Ye ◽  
Qian Zhou ◽  
Xiaohui Wu ◽  
Chen Yu ◽  
Guoli Ji ◽  
...  
Keyword(s):  
Single Cell ◽  
Alternative Polyadenylation ◽  
Cellular Heterogeneity ◽  
Supplementary Information ◽  
Rna Seq ◽  
Computational Tool ◽  
Cell Level ◽  
Wilcoxon Rank Sum Test ◽  
Transcriptional Regulatory ◽  
Cell Groups

Abstract Motivation Alternative polyadenylation (APA) plays a key post-transcriptional regulatory role in mRNA stability and functions in eukaryotes. Single cell RNA-seq (scRNA-seq) is a powerful tool to discover cellular heterogeneity at gene expression level. Given 3′ enriched strategy in library construction, the most commonly used scRNA-seq protocol—10× Genomics enables us to improve the study resolution of APA to the single cell level. However, currently there is no computational tool available for investigating APA profiles from scRNA-seq data. Results Here, we present a package scDAPA for detecting and visualizing dynamic APA from scRNA-seq data. Taking bam/sam files and cell cluster labels as inputs, scDAPA detects APA dynamics using a histogram-based method and the Wilcoxon rank-sum test, and visualizes candidate genes with dynamic APA. Benchmarking results demonstrated that scDAPA can effectively identify genes with dynamic APA among different cell groups from scRNA-seq data. Availability and implementation The scDAPA package is implemented in Shell and R, and is freely available at https://scdapa.sourceforge.io. Contact [email protected] Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals Kinetics of HIV-1 Latency Reversal Quantified on the Single-Cell Level Using a Novel Flow-Based Technique

2016 ◽  
Vol 90 (20) ◽  
pp. 9018-9028 ◽  
Author(s):  
G. Martrus ◽  
A. Niehrs ◽  
R. Cornelis ◽  
A. Rechtien ◽  
W. García-Beltran ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Cell Level ◽  
Cell Level ◽  
Gag Protein ◽  
Novel Approach ◽  
Latently Infected ◽  
Infected Cells ◽  
First Time ◽  
Kinetics Of ◽  
Hiv 1

ABSTRACTHIV-1 establishes a pool of latently infected cells early following infection. New therapeutic approaches aiming at diminishing this persisting reservoir by reactivation of latently infected cells are currently being developed and tested. However, the reactivation kinetics of viral mRNA and viral protein production, and their respective consequences for phenotypical changes in infected cells that might enable immune recognition, remain poorly understood. We adapted a novel approach to assess the dynamics of HIV-1 mRNA and protein expression in latently and newly infected cells on the single-cell level by flow cytometry. This technique allowed the simultaneous detection ofgagpolmRNA, intracellular p24 Gag protein, and cell surface markers. Following stimulation of latently HIV-1-infected J89 cells with human tumor necrosis factor alpha (hTNF-α)/romidepsin (RMD) or HIV-1 infection of primary CD4+T cells, four cell populations were detected according to their expression levels of viral mRNA and protein.gagpolmRNA in J89 cells was quantifiable for the first time 3 h after stimulation with hTNF-α and 12 h after stimulation with RMD, while p24 Gag protein was detected for the first time after 18 h poststimulation. HIV-1-infected primary CD4+T cells downregulated CD4, BST-2, and HLA class I expression at early stages of infection, proceeding Gag protein detection. In conclusion, here we describe a novel approach allowing quantification of the kinetics of HIV-1 mRNA and protein synthesis on the single-cell level and phenotypic characterization of HIV-1-infected cells at different stages of the viral life cycle.IMPORTANCEEarly after infection, HIV-1 establishes a pool of latently infected cells, which hide from the immune system. Latency reversal and immune-mediated elimination of these latently infected cells are some of the goals of current HIV-1 cure approaches; however, little is known about the HIV-1 reactivation kinetics following stimulation with latency-reversing agents. Here we describe a novel approach allowing for the first time quantification of the kinetics of HIV-1 mRNA and protein synthesis after latency reactivation orde novoinfection on the single-cell level using flow cytometry. This new technique furthermore enabled the phenotypic characterization of latently infected andde novo-infected cells dependent on the presence of viral RNA or protein.

scholarly journals Single-cell differential splicing analysis reveals high heterogeneity of liver tumor-infiltrating T cells

2020 ◽  
Author(s):  
Shang Liu ◽  
Biaofeng Zhou ◽  
Liang Wu ◽  
Yan Sun ◽  
Jie Chen ◽  
...  
Keyword(s):  
T Cells ◽  
Alternative Splicing ◽  
Single Cell ◽  
Tumor Infiltrating Lymphocytes ◽  
Differential Splicing ◽  
Cell Level ◽  
Comprehensive Investigation ◽  
Cancer Initiation ◽  
Cell Groups ◽  
Infiltrating Lymphocytes

AbstractRecent advances in single-cell RNA sequencing (scRNA-seq), enriched the knowledge of the heterogeneity of the tumor-infiltrating lymphocytes (TIL) for understanding the mechanisms of cancer initiation and progression. However, alternative splicing (AS), as one of the important regulatory factors of heterogeneity, has been poorly investigated. Here, we proposed a computational tool, DESJ-detection, which could fast and accurately detect the differentially expressed splicing junction (DESJ) between cell groups at single-cell level. We analyzed 5,063 T cells of hepatocellular carcinoma (HCC) and identified 1,176 DESJs across 11 T cell subtypes. Cell subtypes with a similar function clustered closer rather than the lineage at the AS level. Meanwhile, we identified two novel cell states, pre-exhaustion and pre-activation with the marker isoform CD103-201 and ARHGAP15-205. In summary, we presented a comprehensive investigation of alternative splicing differences, which provided novel insights for heterogeneity of T cells and can be applied in other full-length scRNA-seq datasets.

scholarly journals Production of proinflammatory and regulatory monokines in hemodialysis patients shown at a single-cell level.

1998 ◽  
Vol 9 (9) ◽  
pp. 1689-1696
Author(s):  
M Girndt ◽  
U Sester ◽  
H Kaul ◽  
H Köhler
Keyword(s):  
Renal Failure ◽  
Single Cell ◽  
Hemodialysis Patients ◽  
Single Cell Level ◽  
Dialysis Patients ◽  
Distinct Population ◽  
Cell Level ◽  
Cytokine Detection ◽  
First Time ◽  
Proinflammatory Factors

Immunologic complications of chronic renal failure are associated with the overproduction of proinflammatory cytokines by monocytes. This is partly due to renal failure itself but is further enhanced by hemodialysis treatment with frequent contact between blood and dialyzer membranes. Previous studies have shown an imbalance of proinflammatory and regulatory monokines in these patients. This study examines monokine production in hemodialysis patients using for the first time a very sensitive method of cytokine detection at a single-cell level by flow cytometry ("cytoflow technique"). Monocytes were stained intracellularly for the production of interleukin-6 (IL-6) and IL-10 after 20 h of culture with lipopolysaccharide. It was shown that high levels of proinflammatory IL-6 in hemodialysis patients are due to an increased number of monocytes producing this cytokine, while IL-6 synthesis per cell remains unchanged. In contrast, elevated levels of regulatory IL-10 are due to an increased synthesis per cell. This study demonstrates that in healthy subjects there is a population of monocytes producing exclusively IL-10 after 20 h of stimulation by lipopolysaccharide. This distinct population of regulatory monocytes is infrequent in dialysis patients, in whom most of the IL-10-positive monocytes also produce IL-6. These findings indicate that overproduction of proinflammatory factors in dialysis patients is at least in part due to a loss of cytokine-specific differentiation in monocytes.

scholarly journals Single-cell differential splicing analysis reveals high heterogeneity of liver tumor-infiltrating T cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shang Liu ◽  
Biaofeng Zhou ◽  
Liang Wu ◽  
Yan Sun ◽  
Jie Chen ◽  
...  
Keyword(s):  
T Cells ◽  
Alternative Splicing ◽  
T Cell ◽  
Single Cell ◽  
Regulatory Factor ◽  
Differential Splicing ◽  
Cell Level ◽  
Comprehensive Investigation ◽  
Cancer Initiation ◽  
Cell Groups

AbstractRecent advances in single-cell RNA sequencing (scRNA-seq) have improved our understanding of the association between tumor-infiltrating lymphocyte (TILs) heterogeneity and cancer initiation and progression. However, studies investigating alternative splicing (AS) as an important regulatory factor of heterogeneity remain limited. Here, we developed a new computational tool, DESJ-detection, which accurately detects differentially expressed splicing junctions (DESJs) between cell groups at the single-cell level. We analyzed 5063 T cells of hepatocellular carcinoma (HCC) and identified 1176 DESJs across 11 T cell subtypes. Interestingly, DESJs were enriched in UTRs, and have putative effects on heterogeneity. Cell subtypes with a similar function closely clustered together at the AS level. Meanwhile, we identified a novel cell state, pre-activation with the isoform markers ARHGAP15-205. In summary, we present a comprehensive investigation of alternative splicing differences, which provided novel insights into T cell heterogeneity and can be applied to other full-length scRNA-seq datasets.

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