scholarly journals Single Cell Label-Free Probing of Chromatin Dynamics During B Lymphocyte Maturation

2021 ◽  
Vol 9 ◽  
Author(s):  
Rikke Morrish ◽  
Kevin Ho Wai Yim ◽  
Stefano Pagliara ◽  
Francesca Palombo ◽  
Richard Chahwan ◽  
...  
Keyword(s):  
Single Cell ◽  
Cell Fate ◽  
B Lymphocytes ◽  
Intracellular Signaling ◽  
Large Scale ◽  
B Lymphocyte ◽  
Biological Effects ◽  
Raman Microscopy ◽  
Phenotypic Characterization ◽  
Label Free

Large-scale intracellular signaling during developmental growth or in response to environmental alterations are largely orchestrated by chromatin within the cell nuclei. Chemical and conformational modifications of the chromatin architecture are critical steps in the regulation of differential gene expression and ultimately cell fate determination. Therefore, establishing chemical properties of the nucleus could provide key markers for phenotypic characterization of cellular processes on a scale of individual cells. Raman microscopy is a sensitive technique that is capable of probing single cell chemical composition—and sub-cellular regions—in a label-free optical manner. As such, it has great potential in both clinical and basic research. However, perceived limitations of Raman spectroscopy such as low signal intensity and the difficulty in linking alterations in vibrational signals directly with ensuing biological effects have hampered advances in the field. Here we use immune B lymphocyte development as a model to assess chromatin and transcriptional changes using confocal Raman microscopy in combination with microfluidic devices and correlative transcriptomics, thereby linking changes in chemical and structural properties to biological outcomes. Live B lymphocytes were assessed before and after maturation. Multivariate analysis was applied to distinguish cellular components within each cell. The spectral differences between non-activated and activated B lymphocytes were then identified, and their correlation with known intracellular biological changes were assessed in comparison to conventional RNA-seq analysis. Our data shows that spectral analysis provides a powerful tool to study gene activation that can complement conventional molecular biology techniques and opens the way for mapping the dynamics in the biochemical makeup of individual cells.

scholarly journals Single cell label-free probing of chromatin dynamics during B lymphocyte maturation

2021 ◽  
Author(s):  
Rikke Morrish ◽  
Kevin Ho Wai Yim ◽  
Stefano Pagliara ◽  
Francesca Palombo ◽  
Richard Chahwan ◽  
...  
Keyword(s):  
Single Cell ◽  
Cell Fate ◽  
B Lymphocytes ◽  
Large Scale ◽  
B Lymphocyte ◽  
Biological Effects ◽  
Raman Microscopy ◽  
Label Free ◽  
Cell Nuclei ◽  
Phenotypic Characterisation

ABSTRACTLarge-scale intracellular signalling during developmental growth or in response to environmental alterations are largely orchestrated by chromatin within the cell nuclei. Chemical and conformational modifications of the chromatin architecture are critical steps in the regulation of differential gene expression and ultimately cell fate determination. Therefore, establishing chemical properties of the nucleus could provide key markers for phenotypic characterisation of cellular processes on a scale of individual cells.Raman microscopy is a sensitive technique that is capable of probing single cell chemical composition - and sub-cellular regions - in a label-free optical manner. As such, it has great potential in both clinical and basic research. However, perceived limitations of Raman spectroscopy such as low signal intensity and the difficulty in linking alterations in vibrational signals directly with ensuing biological effects have hampered advances in the field. Here we use immune B lymphocyte development as a model to assess chromatin and transcriptional changes using confocal Raman microscopy in combination with microfluidic devices and correlative transcriptomics, thereby linking changes in chemical and structural properties to biological outcomes. Live B lymphocytes were assessed before and after maturation. Multivariate analysis was applied to distinguish cellular components within each cell. The spectral differences between non-activated and activated B lymphocytes were then identified, and their correlation with known intracellular biological changes were assessed in comparison to conventional RNA-seq analysis. Our data shows that spectral analysis provides a powerful tool to study gene activation that can complement conventional molecular biology techniques and opens the way for mapping the dynamics in the biochemical makeup of individual cells.

scholarly journals Leveraging high-powered RNA-Seq datasets to improve inference of regulatory activity in single-cell RNA-Seq data

2019 ◽  
Author(s):  
Ning Wang ◽  
Andrew E. Teschendorff
Keyword(s):  
Transcription Factors ◽  
Single Cell ◽  
Cell Fate ◽  
Regulatory Networks ◽  
Large Scale ◽  
Single Cells ◽  
Differential Expression Analysis ◽  
Dropout Rate ◽  
Rna Seq ◽  
Regulatory Activity

AbstractInferring the activity of transcription factors in single cells is a key task to improve our understanding of development and complex genetic diseases. This task is, however, challenging due to the relatively large dropout rate and noisy nature of single-cell RNA-Seq data. Here we present a novel statistical inference framework called SCIRA (Single Cell Inference of Regulatory Activity), which leverages the power of large-scale bulk RNA-Seq datasets to infer high-quality tissue-specific regulatory networks, from which regulatory activity estimates in single cells can be subsequently obtained. We show that SCIRA can correctly infer regulatory activity of transcription factors affected by high technical dropouts. In particular, SCIRA can improve sensitivity by as much as 70% compared to differential expression analysis and current state-of-the-art methods. Importantly, SCIRA can reveal novel regulators of cell-fate in tissue-development, even for cell-types that only make up 5% of the tissue, and can identify key novel tumor suppressor genes in cancer at single cell resolution. In summary, SCIRA will be an invaluable tool for single-cell studies aiming to accurately map activity patterns of key transcription factors during development, and how these are altered in disease.

scholarly journals A single-cell Raman-based platform to identify developmental stages of human pluripotent stem cell-derived neurons

2020 ◽  
Vol 117 (31) ◽  
pp. 18412-18423 ◽  
Author(s):  
Chia-Chen Hsu ◽  
Jiabao Xu ◽  
Bas Brinkhof ◽  
Hui Wang ◽  
Zhanfeng Cui ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Single Cell ◽  
Raman Spectra ◽  
Large Scale ◽  
Developmental Stages ◽  
Single Cells ◽  
Classification Model ◽  
Label Free ◽  
Machine Learning Classification

Stem cells with the capability to self-renew and differentiate into multiple cell derivatives provide platforms for drug screening and promising treatment options for a wide variety of neural diseases. Nevertheless, clinical applications of stem cells have been hindered partly owing to a lack of standardized techniques to characterize cell molecular profiles noninvasively and comprehensively. Here, we demonstrate that a label-free and noninvasive single-cell Raman microspectroscopy (SCRM) platform was able to identify neural cell lineages derived from clinically relevant human induced pluripotent stem cells (hiPSCs). By analyzing the intrinsic biochemical profiles of single cells at a large scale (8,774 Raman spectra in total), iPSCs and iPSC-derived neural cells can be distinguished by their intrinsic phenotypic Raman spectra. We identified a Raman biomarker from glycogen to distinguish iPSCs from their neural derivatives, and the result was verified by the conventional glycogen detection assays. Further analysis with a machine learning classification model, utilizing t-distributed stochastic neighbor embedding (t-SNE)-enhanced ensemble stacking, clearly categorized hiPSCs in different developmental stages with 97.5% accuracy. The present study demonstrates the capability of the SCRM-based platform to monitor cell development using high content screening with a noninvasive and label-free approach. This platform as well as our identified biomarker could be extensible to other cell types and can potentially have a high impact on neural stem cell therapy.

scholarly journals Phosphoproteomic analysis sheds light on intracellular signaling cascades triggered by Formyl-Peptide Receptor 2

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Fabio Cattaneo ◽  
Rosita Russo ◽  
Martina Castaldo ◽  
Angela Chambery ◽  
Cristiana Zollo ◽  
...  
Keyword(s):  
Intracellular Signaling ◽  
Drug Targets ◽  
Large Scale ◽  
Cellular Proliferation ◽  
Biological Effects ◽  
Formyl Peptide Receptor ◽  
Ros Generation ◽  
Systematic Analysis ◽  
Phosphorylated Proteins ◽  
Formyl Peptide

AbstractFormyl peptide receptors (FPRs) belong to the family of seven transmembrane Gi-protein coupled receptors (GPCR). FPR2 is considered the most promiscuous member of this family since it recognizes a wide variety of ligands. It plays a crucial role in several physio-pathological processes and different studies highlighted the correlation between its expression and the higher propensity to invasion and metastasis of some cancers. FPR2 stimulation by its synthetic agonist WKYMVm triggers multiple phosphorylations of intracellular signaling molecules, such as ERKs, PKC, PKB, p38MAPK, PI3K, PLC, and of non-signaling proteins, such as p47phox and p67phox which are involved in NADPH oxidase-dependent ROS generation. Biological effects of FPR2 stimulation include intracellular Ca2+ mobilization, cellular proliferation and migration, and wound healing. A systematic analysis of the phosphoproteome in FPR2-stimulated cells has not been yet reported. Herein, we describe a large-scale phosphoproteomic study in WKYMVm-stimulated CaLu-6 cells. By using high resolution MS/MS we identified 290 differentially phosphorylated proteins and 53 unique phosphopeptides mapping on 40 proteins. Phosphorylations on five selected phospho-proteins were further validated by western blotting, confirming their dependence on FPR2 stimulation. Interconnection between some of the signalling readout identified was also evaluated. Furthermore, we show that FPR2 stimulation with two anti-inflammatory agonists induces the phosphorylation of selected differentially phosphorylated proteins, suggesting their role in the resolution of inflammation. These data provide a promising resource for further studies on new signaling networks triggered by FPR2 and on novel molecular drug targets for human diseases.

scholarly journals Label free, quantitative single-cell fate tracking of time-lapse movies

MethodsX ◽  
2019 ◽  
Vol 6 ◽  
pp. 2468-2475 ◽  
Author(s):  
C. Elizabeth Caldon ◽  
Andrew Burgess
Keyword(s):  
Single Cell ◽  
Cell Fate ◽  
Time Lapse ◽  
Label Free ◽  
Cell Fate Tracking

scholarly journals Shaping development by stochasticity and dynamics in gene regulation

Open Biology ◽  
2017 ◽  
Vol 7 (5) ◽  
pp. 170030 ◽  
Author(s):  
Peng Dong ◽  
Zhe Liu
Keyword(s):  
Gene Expression ◽  
Gene Regulation ◽  
Single Cell ◽  
Cell Fate ◽  
Single Molecule ◽  
Large Scale ◽  
Single Cells ◽  
Individual Gene ◽  
Functional Specification ◽  
Spatio Temporal

Animal development is orchestrated by spatio-temporal gene expression programmes that drive precise lineage commitment, proliferation and migration events at the single-cell level, collectively leading to large-scale morphological change and functional specification in the whole organism. Efforts over decades have uncovered two ‘seemingly contradictory’ mechanisms in gene regulation governing these intricate processes: (i) stochasticity at individual gene regulatory steps in single cells and (ii) highly coordinated gene expression dynamics in the embryo. Here we discuss how these two layers of regulation arise from the molecular and the systems level, and how they might interplay to determine cell fate and to control the complex body plan. We also review recent technological advancements that enable quantitative analysis of gene regulation dynamics at single-cell, single-molecule resolution. These approaches outline next-generation experiments to decipher general principles bridging gaps between molecular dynamics in single cells and robust gene regulations in the embryo.

scholarly journals Raman2RNA: Live-cell label-free prediction of single-cell RNA expression profiles by Raman microscopy

2021 ◽  
Author(s):  
Koseki J. Kobayashi-Kirschvink ◽  
Shreya Gaddam ◽  
Taylor James-Sorenson ◽  
Emanuelle Grody ◽  
Johain R. Ounadjela ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Molecule ◽  
Vibrational Energy ◽  
Temporal Dynamics ◽  
Expression Profiles ◽  
Raman Microscopy ◽  
Live Cells ◽  
Label Free ◽  
Imaging Data

Single cell RNA-Seq (scRNA-seq) and other profiling assays have opened new windows into understanding the properties, regulation, dynamics, and function of cells at unprecedented resolution and scale. However, these assays are inherently destructive, precluding us from tracking the temporal dynamics of live cells, in cell culture or whole organisms. Raman microscopy offers a unique opportunity to comprehensively report on the vibrational energy levels of molecules in a label-free and non-destructive manner at a subcellular spatial resolution, but it lacks in genetic and molecular interpretability. Here, we developed Raman2RNA (R2R), an experimental and computational framework to infer single-cell expression profiles in live cells through label-free hyperspectral Raman microscopy images and multi-modal data integration and domain translation. We used spatially resolved single-molecule RNA-FISH (smFISH) data as anchors to link scRNA-seq profiles to the paired spatial hyperspectral Raman images, and trained machine learning models to infer expression profiles from Raman spectra at the single-cell level. In reprogramming of mouse fibroblasts into induced pluripotent stem cells (iPSCs), R2R accurately (r>0.96) inferred from Raman images the expression profiles of various cell states and fates, including iPSCs, mesenchymal-epithelial transition (MET) cells, stromal cells, epithelial cells, and fibroblasts. R2R outperformed inference from brightfield images, showing the importance of spectroscopic content afforded by Raman microscopy. Raman2RNA lays a foundation for future investigations into exploring single-cell genome-wide molecular dynamics through imaging data, in vitro and in vivo.

scholarly journals Anhydroretinol: a naturally occurring inhibitor of lymphocyte physiology.

1993 ◽  
Vol 178 (2) ◽  
pp. 675-680 ◽  
Author(s):  
J Buck ◽  
F Grün ◽  
F Derguini ◽  
Y Chen ◽  
S Kimura ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
B Lymphocytes ◽  
T Cell Activation ◽  
Intracellular Signaling ◽  
Cell Activation ◽  
B Lymphocyte ◽  
Interleukin 2 ◽  
Fk 506 ◽  
Intracellular Signaling Pathway ◽  
Insect Cell Lines

Vitamin A (retinol) is an essential cofactor for growth of B lymphocytes in culture and for activation of T lymphocytes by antigen receptor-mediated signals. 14-hydroxy-4,14-retro-retinol (14-HRR) a metabolite of retinol, has been implicated as the intracellular mediator of this effect. Anhydroretinol (AR) is a retinol derivative with retro structure produced in activated human B lymphocytes and the insect cell lines SF 21 and Schneider S2. AR reversibly inhibits retinol- and 14-HRR-dependent effects and blocks B lymphocyte proliferation as well as activation of resting T lymphocytes. The intracellular signaling pathway blocked by AR in T cell activation is distinct from the calcineurin/interleukin 2 pathway inhibitable by cyclosporine A or FK-506.

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