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Multiplexed, Sequential Secretion Analysis of the Same Single Cells Reveals Distinct Effector Response Dynamics Dependent on the Initial Basal State

ABSTRACT: Multiplexed, Sequential Secretion Analysis of the Same Single Cells Reveals Distinct Effector Response Dynamics Dependent on the Initial Basal State

PROVIDER: PRJNA824066 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR18663661_1.fastq.gz	Fastqsanger.gz
	SRR18663661_2.fastq.gz	Fastqsanger.gz
	SRR18663662_1.fastq.gz	Fastqsanger.gz
	SRR18663662_2.fastq.gz	Fastqsanger.gz
	SRR18663663_1.fastq.gz	Fastqsanger.gz

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Multiplexed, Sequential Secretion Analysis of the Same Single Cells Reveals Distinct Effector Response Dynamics Dependent on the Initial Basal State

Project description:The effector response of immune cells is a highly dynamic process. To explore time course of human macrophage response to toll-like receptor 4 (TLR4) ligand, human U937 cell line-derived macrophages were stimulated with lipopolysaccharide (LPS) and single-cell RNA-Sequencing were conducted at 0, 2, 4 and 6 hours after the initial treatment. The data demonstrated the existence of two major activation states at the transcriptional level, which were enriched for translation versus inflammatory programs, respectively. These results show a cell-intrinsic heterogeneous response in a phenotypically homogeneous cell population, providing dynamic information to better understand how immune cells react to pathogenic challenges over time and how they together constitute a population response.

2022-04-09 | GSE200324 | GEO

SSSMuG: Same Sample Sequential Multi-Glycomics

Project description:The mammalian glycome is structurally complex and diverse, composed of many glycan classes such as N- and O-linked glycans, glycosaminoglycans (GAGs), glycosphingolipids (GSLs) and other distinct glycan features such as polysialic acids (PolySia), sulfation and proteoglycan attachment stubs. Various methods are used to analyse these different components of the glycome, but they require pre-fractionated/partitioned samples to target each glycan class individually. To address this need for a knowledge of the relationship between the different glycan components of a biological system, we have developed a sequential release workflow for analysis of multiple conjugated glycan classes (PolySia, GAGs, GSL glycans, N-glycans O-glycans) from the same tissue lysate, termed SSSMuG: Same Sample Sequential Multi-Glycomics. With this sequential glycan release approach, five glycan classes were characterized (or four glycan classes plus proteomics) from a single sample using enzymatic or chemical release from the same spot immobilized on a polyvinylidene difluoride membrane. The various released glycan classes were then analysed by HPLC and/or MS techniques. Compared to single glycan class release approaches, SSSMuG is able to identify more glycans and more proteins with higher intensity analytical peaks and provides a better comparative normalisation of the different glycan classes of the complex glycome.

2024-02-13 | PXD046012 | Pride

Multiplexed, Sequential Secretion Analysis of the Same Single Cells Reveals Distinct Effector Response Dynamics Dependent on the Initial Basal State.

Project description:The effector response of immune cells dictated by an array of secreted proteins is a highly dynamic process, requiring sequential measurement of all relevant proteins from single cells. Herein, a microchip-based, 10-plexed, sequential secretion assay on the same single cells and at the scale of ≈5000 single cells measured simultaneously over 4 time points are shown. It is applied to investigating the time course of single human macrophage response to toll-like receptor 4 (TLR4) ligand lipopolysaccharide (LPS) and reveals four distinct activation modes for different proteins in single cells. Protein secretion dynamics classifies the cells into two major activation states dependent on the basal state of each cell. Single-cell RNA sequencing performed on the same samples at the matched time points further demonstrates the existence of two major activation states at the transcriptional level, which are enriched for translation versus inflammatory programs, respectively. These results show a cell-intrinsic heterogeneous response in a phenotypically homogeneous cell population. This work demonstrates the longitudinal tracking of protein secretion signature in thousands of single cells at multiple time points, providing dynamic information to better understand how individual immune cells react to pathogenic challenges over time and how they together constitute a population response.

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Single-molecule analysis of RNA:protein interactions reveals sequential binding of different YTHDF proteins to the same mRNA molecules.

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Therapeutic targets and microenvironment in sequential biopsies of classical Hodgkin lymphoma at diagnosis and relapse

Project description:BACKGROUND: Classical Hodgkin lymphoma is dominated by the nonneoplastic microenvironment, while the neoplastic Hodgkin-Reed-Sternberg cells compose only a minority of cells in the lymphoma tissue. Both, the Hodgkin-Reed-Sternberg cells due to their expression of CD30 and PD-L1 and the microenvironment with abundant T-cells and expression of PD1 are specifically targeted by new treatment concepts. AIM: We aimed to understand the dynamics of therapeutic targets in patients treated with conventional chemotherapy. METHODS: We analyzed sequential biopsy specimens obtained at diagnosis and at relapse from the same patient for morphology, immunophenotype and microenvironmental components. RESULTS: The morphological subtype changed between primary and relapse biopsy in 20% of cases. The immunophenotype was stable with respect to CD30, CD3 and LMP1 but variable with respect to CD15 and CD20 expression. Gene expression revealed 8 up- and 20 down-regulated genes at relapse (p≤0.05) with a consistent logarithmic fold change direction in at least 75% of all cases. For PD1, we found discrepant results between gene expression analysis (decrease at relapse) and number of PD1 positive cells assessed by immunohistochemistry (unchanged at relapse). PD-L1 in the neoplastic cells appeared unchanged between primary diagnosis and relapse. CONCLUSION: The expression of the therapeutic targets CD30, PD1 and PD-L1 can reliably be assessed in tumor specimen at first diagnosis and are unchanged under conventional chemotherapy. We used digital multiplexed gene expression (DMGE) with FFPE derived RNA to analyze sequential biopsy specimens obtained at diagnosis and at relapse from the same patient for morphology, immunophenotype and microenvironmental components.

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