23 research outputs found
Thy-1 interaction with Fas in lipid rafts regulates fibroblast apoptosis and lung injury resolution.
Thy-1-negative lung fibroblasts are resistant to apoptosis. The mechanisms governing this process and its relevance to fibrotic remodeling remain poorly understood. By using either sorted or transfected lung fibroblasts, we found that Thy-1 expression is associated with downregulation of anti-apoptotic molecules Bcl-2 and Bcl-xL, as well as increased levels of cleaved caspase-9. Addition of rhFasL and staurosporine, well-known apoptosis inducers, caused significantly increased cleaved caspase-3, -8, and PARP in Thy-1-transfected cells. Furthermore, rhFasL induced Fas translocation into lipid rafts and its colocalization with Thy-1. These in vitro results indicate that Thy-1, in a manner dependent upon its glycophosphatidylinositol anchor and lipid raft localization, regulates apoptosis in lung fibroblasts via Fas-, Bcl-, and caspase-dependent pathways. In vivo, Thy-1 deficient (Thy1-/-) mice displayed persistence of myofibroblasts in the resolution phase of bleomycin-induced fibrosis, associated with accumulation of collagen and failure of lung fibrosis resolution. Apoptosis of myofibroblasts is decreased in Thy1-/- mice in the resolution phase. Collectively, these findings provide new evidence regarding the role and mechanisms of Thy-1 in initiating myofibroblast apoptosis that heralds the termination of the reparative response to bleomycin-induced lung injury. Understanding the mechanisms regulating fibroblast survival/apoptosis should lead to novel therapeutic interventions for lung fibrosis
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Bento: a toolkit for subcellular analysis of spatial transcriptomics data
The spatial organization of molecules in a cell is essential for their functions. While current methods focus on discerning tissue architecture, cell-cell interactions, and spatial expression patterns, they are limited to the multicellular scale. We present Bento, a Python toolkit that takes advantage of single-molecule information to enable spatial analysis at the subcellular scale. Bento ingests molecular coordinates and segmentation boundaries to perform three analyses: defining subcellular domains, annotating localization patterns, and quantifying gene-gene colocalization. We demonstrate MERFISH, seqFISH + , Molecular Cartography, and Xenium datasets. Bento is part of the open-source Scverse ecosystem, enabling integration with other single-cell analysis tools
Transcriptomic analysis of cutaneous squamous cell carcinoma reveals a multi-gene prognostic signature associated with metastasis
Background: Metastasis of cutaneous squamous cell carcinoma (cSCC) is uncommon. Current staging methods are reported to have sub-optimal performances in metastasis prediction. Accurate identification of patients with tumours at high risk of metastasis would have a significant impact on management.Objective: To develop a robust and validated gene expression profile (GEP) signature for predicting primary cSCC metastatic risk using an unbiased whole transcriptome discovery-driven approach.Methods: Archival formalin-fixed paraffin-embedded primary cSCC with perilesional normal tissue from 237 immunocompetent patients (151 non-metastasising and 86 metastasising) were collected retrospectively from four centres. TempO-seq was used to probe the whole transcriptome and machine learning algorithms were applied to derive predictive signatures, with a 3:1 split for training and testing datasets.Results: A 20-gene prognostic model was developed and validated, with an accuracy of 86.0%, sensitivity of 85.7%, specificity of 86.1%, and positive predictive value of 78.3% in the testing set, providing more stable, accurate prediction than pathological staging systems. A linear predictor was also developed, significantly correlating with metastatic risk.Limitations: This was a retrospective 4-centre study and larger prospective multicentre studies are now required.Conclusion: The 20-gene signature prediction is accurate, with the potential to be incorporated into clinical workflows for cSCC
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Correlation of focal adhesion assembly and disassembly with cell migration on nanotopography.
Selective cell adhesion is desirable to control cell growth and migration on biomedical implants. Mesenchymal cell migration is regulated through focal adhesions (FAs) and can be modulated by their microenvironment, including changes in surface topography. We use the Number and Molecular Brightness (N&B) imaging analysis to provide a unique perspective on FA assembly and disassembly. This imaging analysis generates a map of real-time fluctuations of protein monomers, dimers, and higher order aggregates of FA proteins, such as paxillin during assembly and disassembly. We show a dynamic view of how nanostructured surfaces (nanoline gratings or nanopillars) regulate single molecular dynamics. In particular, we report that the smallest nanopillars (100 nm spacing) gave rise to a low population of disassembling adhesion clusters of ∼2 paxillin proteins whereas the larger nanopillars (380 nm spacing) gave rise to a much larger population of larger disassembling clusters of ∼3-5 paxillin proteins. Cells were more motile on the smaller nanopillars (spaced 100-130 nm apart) compared to all other surfaces studied. Thus, physical nanotopography influences cell motility, adhesion size, and adhesion assembly and disassembly. We report for the first time, with single molecular detection, how nanotopography influences cell motility and protein reorganization in adhesions
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Collagen density modulates triple-negative breast cancer cell metabolism through adhesion-mediated contractility.
Extracellular matrix (ECM) mechanical properties upregulate cancer invasion, cell contractility, and focal adhesion formation. Alteration in energy metabolism is a known characteristic of cancer cells (i.e., Warburg effect) and modulates cell invasion. There is little evidence to show if collagen density can alter cancer cell metabolism. We investigated changes in energy metabolism due to collagen density in five breast cell lines by measuring the fluorescence lifetime of NADH. We found that only triple-negative breast cancer cells, MDA-MB231 and MDA-MB468 cells, had an increased population of bound NADH, indicating an oxidative phosphorylation (OXPHOS) signature, as collagen density decreased. When inhibiting ROCK and cell contractility, MDA-MB231 cells on glass shifted from glycolysis (GLY) to OXPHOS, confirming the intricate relationship between mechanosensing and metabolism. MCF10A cells showed less significant changes in metabolism, shifting towards GLY as collagen density decreased. The MCF-7 and T-47D, less invasive breast cancer cells, compared to the MDA-MB231 and MDA-MB468 cells, showed no changes regardless of substrate. In addition, OXPHOS or GLY inhibitors in MDA-MB231 cells showed dramatic shifts from OXPHOS to GLY or vice versa. These results provide an important link between cellular metabolism, contractility, and collagen density in human breast cancer
Collagen density modulates triple-negative breast cancer cell metabolism through adhesion-mediated contractility.
Extracellular matrix (ECM) mechanical properties upregulate cancer invasion, cell contractility, and focal adhesion formation. Alteration in energy metabolism is a known characteristic of cancer cells (i.e., Warburg effect) and modulates cell invasion. There is little evidence to show if collagen density can alter cancer cell metabolism. We investigated changes in energy metabolism due to collagen density in five breast cell lines by measuring the fluorescence lifetime of NADH. We found that only triple-negative breast cancer cells, MDA-MB231 and MDA-MB468 cells, had an increased population of bound NADH, indicating an oxidative phosphorylation (OXPHOS) signature, as collagen density decreased. When inhibiting ROCK and cell contractility, MDA-MB231 cells on glass shifted from glycolysis (GLY) to OXPHOS, confirming the intricate relationship between mechanosensing and metabolism. MCF10A cells showed less significant changes in metabolism, shifting towards GLY as collagen density decreased. The MCF-7 and T-47D, less invasive breast cancer cells, compared to the MDA-MB231 and MDA-MB468 cells, showed no changes regardless of substrate. In addition, OXPHOS or GLY inhibitors in MDA-MB231 cells showed dramatic shifts from OXPHOS to GLY or vice versa. These results provide an important link between cellular metabolism, contractility, and collagen density in human breast cancer
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Thy-1 interaction with Fas in lipid rafts regulates fibroblast apoptosis and lung injury resolution.
Thy-1-negative lung fibroblasts are resistant to apoptosis. The mechanisms governing this process and its relevance to fibrotic remodeling remain poorly understood. By using either sorted or transfected lung fibroblasts, we found that Thy-1 expression is associated with downregulation of anti-apoptotic molecules Bcl-2 and Bcl-xL, as well as increased levels of cleaved caspase-9. Addition of rhFasL and staurosporine, well-known apoptosis inducers, caused significantly increased cleaved caspase-3, -8, and PARP in Thy-1-transfected cells. Furthermore, rhFasL induced Fas translocation into lipid rafts and its colocalization with Thy-1. These in vitro results indicate that Thy-1, in a manner dependent upon its glycophosphatidylinositol anchor and lipid raft localization, regulates apoptosis in lung fibroblasts via Fas-, Bcl-, and caspase-dependent pathways. In vivo, Thy-1 deficient (Thy1-/-) mice displayed persistence of myofibroblasts in the resolution phase of bleomycin-induced fibrosis, associated with accumulation of collagen and failure of lung fibrosis resolution. Apoptosis of myofibroblasts is decreased in Thy1-/- mice in the resolution phase. Collectively, these findings provide new evidence regarding the role and mechanisms of Thy-1 in initiating myofibroblast apoptosis that heralds the termination of the reparative response to bleomycin-induced lung injury. Understanding the mechanisms regulating fibroblast survival/apoptosis should lead to novel therapeutic interventions for lung fibrosis
Transcriptomic analysis of cutaneous squamous cell carcinoma reveals a multi-gene prognostic signature associated with metastasis
Background: metastasis of cutaneous squamous cell carcinoma (cSCC) is uncommon. Current staging methods are reported to have sub-optimal performances in metastasis prediction. Accurate identification of patients with tumours at high risk of metastasis would have a significant impact on management. Objective: to develop a robust and validated gene expression profile (GEP) signature for predicting primary cSCC metastatic risk using an unbiased whole transcriptome discovery-driven approach.Methods: archival formalin-fixed paraffin-embedded primary cSCC with perilesional normal tissue from 237 immunocompetent patients (151 non-metastasising and 86 metastasising) were collected retrospectively from four centres. TempO-seq was used to probe the whole transcriptome and machine learning algorithms were applied to derive predictive signatures, with a 3:1 split for training and testing datasets. Results: a 20-gene prognostic model was developed and validated, with an accuracy of 86.0%, sensitivity of 85.7%, specificity of 86.1%, and positive predictive value of 78.3% in the testing set, providing more stable, accurate prediction than pathological staging systems. A linear predictor was also developed, significantly correlating with metastatic risk.Limitations: this was a retrospective 4-centre study and larger prospective multicentre studies are now required.Conclusion: the 20-gene signature prediction is accurate, with the potential to be incorporated into clinical workflows for cSCC.<br/
Additional file 1 of Bento: a toolkit for subcellular analysis of spatial transcriptomics data
Additional file 1: Supplementary Figures and Table S1. Supplementary figures and machine learning classifier feature descriptions
Heritage making through community archaeology and the spatial humanities
The archaeology of postindustrial landscapes is still relatively undeveloped. The impact of economic, social, and urban development efforts on both tangible and intangible heritage complicate our attempts to understand these places. Despite this, integrating heritage practice and promotion into the regeneration of a postindustrial landscape continues to grow in popularity. Within this context, genuine public-expert collaboration is the most effective means towards developing a sustainable compromise between protecting community heritage values and fostering economic development and regeneration. In this paper, we suggest three broad categories of challenges for studying and promoting heritage in postindustrial regions – physical, social, and political – and propose a digital data-focused geospatial approach to how community archaeologists and heritage specialists may overcome these challenges. We argue that coupling this data and technology with a robust research agenda and public programming can serve as a crucial two-way link, enabling long-term sustainable heritage-promotion and protection in post-industrial communities