Cellular sheddases are induced by Merkel Cell Polyomavirus Small Tumour Antigen to mediate cell dissociation and invasiveness

Merkel cell carcinoma (MCC) is an aggressive skin cancer with a high propensity for recurrence and metastasis. Merkel cell polyomavirus (MCPyV) is recognised as the causative factor in the majority of MCC cases. The MCPyV small tumour antigen (ST) is considered to be the main viral transforming factor, however potential mechanisms linking ST expression to the highly metastatic nature of MCC are yet to be fully elucidated. Metastasis is a complex process, with several discrete steps required for the formation of secondary tumour sites. One essential trait that underpins the ability of cancer cells to metastasise is how they interact with adjoining tumour cells and the surrounding extracellular matrix. Here we demonstrate that MCPyV ST expression disrupts the integrity of cell-cell junctions, thereby enhancing cell dissociation and implicate the cellular sheddases, A disintegrin and metalloproteinase (ADAM) 10 and 17 proteins in this process. Inhibition of ADAM 10 and 17 activity reduced MCPyV ST-induced cell dissociation and motility, attributing their function as critical to the MCPyV-induced metastatic processes. Consistent with these data, we confirm that ADAM 10 and 17 are upregulated in MCPyV-positive primary MCC tumours. These novel findings implicate cellular sheddases as key host cell factors contributing to virus-mediated cellular transformation and metastasis. Notably, ADAM protein expression may be a novel biomarker of MCC prognosis and given the current interest in cellular sheddase inhibitors for cancer therapeutics, it highlights ADAM 10 and 17 activity as a novel opportunity for targeted interventions for disseminated MCC

Interplay of microRNA-21 and SATB1 in epidermal keratinocytes during skin aging

YesNottingham Trent University, United Kingdom, UoA03 QR and Capital Funds (MIA), as well as by the grant from Amway, USA to VAB and NVB

The Galaxy platform for accessible, reproducible, and collaborative data analyses: 2024 update

YesGalaxy (https://galaxyproject.org) is deployed globally, predominantly through free-to-use services, supporting user-driven research that broadens in scope each year. Users are attracted to public Galaxy services by platform stability, tool and reference dataset diversity, training, support and integration, which enables complex, reproducible, shareable data analysis. Applying the principles of user experience design (UXD), has driven improvements in accessibility, tool discoverability through Galaxy Labs/subdomains, and a redesigned Galaxy ToolShed. Galaxy tool capabilities are progressing in two strategic directions: integrating general purpose graphical processing units (GPGPU) access for cutting-edge methods, and licensed tool support. Engagement with global research consortia is being increased by developing more workflows in Galaxy and by resourcing the public Galaxy services to run them. The Galaxy Training Network (GTN) portfolio has grown in both size, and accessibility, through learning paths and direct integration with Galaxy tools that feature in training courses. Code development continues in line with the Galaxy Project roadmap, with improvements to job scheduling and the user interface. Environmental impact assessment is also helping engage users and developers, reminding them of their role in sustainability, by displaying estimated CO2 emissions generated by each Galaxy job.NIH [U41 HG006620, U24 HG010263, U24 CA231877, U01 CA253481]; US National Science Foundation [1661497, 1758800, 2216612]; computational resources are provided by the Advanced Cyberinfrastructure Coordination Ecosystem (ACCESS-CI), Texas Advanced Computing Center, and the JetStream2 scientific cloud. Funding for open access charge: NIH. ELIXIR IS and Travel grants; EU Horizon Europe [HORIZON-INFRA-2021-EOSC-01-04, 101057388]; EU Horizon Europe under the Biodiversity, Circular Economy and Environment program (REA.B.3, BGE 101059492); German Federal Ministry of Education and Research, BMBF [031 A538A de.NBI-RBC]; Ministry of Science, Research and the Arts Baden-Württemberg (MWK) within the framework of LIBIS/de.NBI Freiburg. Galaxy Australia is supported by the Australian BioCommons which is funded through Australian Government NCRIS investments from Bioplatforms Australia and the Australian Research Data Commons, as well as investment from the Queensland Government RICF program

The Galaxy platform for accessible, reproducible, and collaborative data analyses: 2024 update

YesGalaxy (https://galaxyproject.org) is deployed globally, predominantly through free-to-use services, supporting user-driven research that broadens in scope each year. Users are attracted to public Galaxy services by platform stability, tool and reference dataset diversity, training, support and integration, which enables complex, reproducible, shareable data analysis. Applying the principles of user experience design (UXD), has driven improvements in accessibility, tool discoverability through Galaxy Labs/subdomains, and a redesigned Galaxy ToolShed. Galaxy tool capabilities are progressing in two strategic directions: integrating general purpose graphical processing units (GPGPU) access for cutting-edge methods, and licensed tool support. Engagement with global research consortia is being increased by developing more workflows in Galaxy and by resourcing the public Galaxy services to run them. The Galaxy Training Network (GTN) portfolio has grown in both size, and accessibility, through learning paths and direct integration with Galaxy tools that feature in training courses. Code development continues in line with the Galaxy Project roadmap, with improvements to job scheduling and the user interface. Environmental impact assessment is also helping engage users and developers, reminding them of their role in sustainability, by displaying estimated CO2 emissions generated by each Galaxy job.NIH [U41 HG006620, U24 HG010263, U24 CA231877, U01 CA253481]; US National Science Foundation [1661497, 1758800, 2216612]; computational resources are provided by the Advanced Cyberinfrastructure Coordination Ecosystem (ACCESS-CI), Texas Advanced Computing Center, and the JetStream2 scientific cloud. Funding for open access charge: NIH. ELIXIR IS and Travel grants; EU Horizon Europe [HORIZON-INFRA-2021-EOSC-01-04, 101057388]; EU Horizon Europe under the Biodiversity, Circular Economy and Environment program (REA.B.3, BGE 101059492); German Federal Ministry of Education and Research, BMBF [031 A538A de.NBI-RBC]; Ministry of Science, Research and the Arts Baden-Württemberg (MWK) within the framework of LIBIS/de.NBI Freiburg. Galaxy Australia is supported by the Australian BioCommons which is funded through Australian Government NCRIS investments from Bioplatforms Australia and the Australian Research Data Commons, as well as investment from the Queensland Government RICF program.Please note, contributors are listed in alphabetical order

3D-FISH analysis of the spatial genome organization in skin cells in situ

NoSpatial genome organization in the cell nucleus plays a crucial role in the control of genome functions. Our knowledge about spatial genome organization is relying on the advances in gene imaging technologies and the biochemical approaches based on the spatial dependent ligation of the genomic regions. Fluorescent in situ hybridization using specific fluorescent DNA and RNA probes in cells and tissues with the spatially preserved nuclear and genome architecture (3D-FISH) provides a powerful tool for the further advancement of our knowledge about genome structure and functions. Here we describe the 3D-FISH protocols allowing for such an analysis in mammalian tissue in situ including in the skin. These protocols include DNA probe amplification and labeling; tissue fixation; preservation and preparation for hybridization; hybridization of the DNA probes with genomic DNA in the tissue; and post-hybridization tissue sample processing

Characterization of Changes in the Proteome in Different Regions of 3D Multicell Tumor Spheroids

Three dimensional multicell tumor spheroids (MCTS) provide an experimental model where the influence of microenvironmental conditions on protein expression can be determined. Sequential trypsin digestion of HT29 colon carcinoma MCTS enabled segregation into four populations comprising proliferating cells from the surface (SL), an intermediate region (IR), nonproliferating hypoxic cells from the perinecrotic region (PN), and a necrotic core (NC). Total protein was extracted from each population and subjected to iTRAQ-based quantitative proteomics analysis. From a total of 887 proteins identified, 209 were observed to be up-regulated and 114 were down-regulated in the PN and NC regions relative to the SL. Among the up-regulated proteins, components of glycolysis, TCA cycle, lipid metabolism, and steroid biosynthesis increased progressively toward the PN and NC regions. Western blotting, immunohistochemistry, and enzyme assays confirmed that significant changes in the expression of proteins involved in cellular metabolism occur in the nonproliferating fraction of cells within the viable rim. The presence of full length, functional proteins within the NC was unexpected, and further analysis demonstrated that this region contains cells that are undergoing autophagy. This study has identified possible targets that may be suitable for therapeutic intervention, and further studies to validate these are required

Characterization of Changes in the Proteome in Different Regions of 3D Multicell Tumor Spheroids

Three dimensional multicell tumor spheroids (MCTS) provide an experimental model where the influence of microenvironmental conditions on protein expression can be determined. Sequential trypsin digestion of HT29 colon carcinoma MCTS enabled segregation into four populations comprising proliferating cells from the surface (SL), an intermediate region (IR), nonproliferating hypoxic cells from the perinecrotic region (PN), and a necrotic core (NC). Total protein was extracted from each population and subjected to iTRAQ-based quantitative proteomics analysis. From a total of 887 proteins identified, 209 were observed to be up-regulated and 114 were down-regulated in the PN and NC regions relative to the SL. Among the up-regulated proteins, components of glycolysis, TCA cycle, lipid metabolism, and steroid biosynthesis increased progressively toward the PN and NC regions. Western blotting, immunohistochemistry, and enzyme assays confirmed that significant changes in the expression of proteins involved in cellular metabolism occur in the nonproliferating fraction of cells within the viable rim. The presence of full length, functional proteins within the NC was unexpected, and further analysis demonstrated that this region contains cells that are undergoing autophagy. This study has identified possible targets that may be suitable for therapeutic intervention, and further studies to validate these are required

Characterization of Changes in the Proteome in Different Regions of 3D Multicell Tumor Spheroids

Three dimensional multicell tumor spheroids (MCTS) provide an experimental model where the influence of microenvironmental conditions on protein expression can be determined. Sequential trypsin digestion of HT29 colon carcinoma MCTS enabled segregation into four populations comprising proliferating cells from the surface (SL), an intermediate region (IR), nonproliferating hypoxic cells from the perinecrotic region (PN), and a necrotic core (NC). Total protein was extracted from each population and subjected to iTRAQ-based quantitative proteomics analysis. From a total of 887 proteins identified, 209 were observed to be up-regulated and 114 were down-regulated in the PN and NC regions relative to the SL. Among the up-regulated proteins, components of glycolysis, TCA cycle, lipid metabolism, and steroid biosynthesis increased progressively toward the PN and NC regions. Western blotting, immunohistochemistry, and enzyme assays confirmed that significant changes in the expression of proteins involved in cellular metabolism occur in the nonproliferating fraction of cells within the viable rim. The presence of full length, functional proteins within the NC was unexpected, and further analysis demonstrated that this region contains cells that are undergoing autophagy. This study has identified possible targets that may be suitable for therapeutic intervention, and further studies to validate these are required

Characterization of Changes in the Proteome in Different Regions of 3D Multicell Tumor Spheroids

Three dimensional multicell tumor spheroids (MCTS) provide an experimental model where the influence of microenvironmental conditions on protein expression can be determined. Sequential trypsin digestion of HT29 colon carcinoma MCTS enabled segregation into four populations comprising proliferating cells from the surface (SL), an intermediate region (IR), nonproliferating hypoxic cells from the perinecrotic region (PN), and a necrotic core (NC). Total protein was extracted from each population and subjected to iTRAQ-based quantitative proteomics analysis. From a total of 887 proteins identified, 209 were observed to be up-regulated and 114 were down-regulated in the PN and NC regions relative to the SL. Among the up-regulated proteins, components of glycolysis, TCA cycle, lipid metabolism, and steroid biosynthesis increased progressively toward the PN and NC regions. Western blotting, immunohistochemistry, and enzyme assays confirmed that significant changes in the expression of proteins involved in cellular metabolism occur in the nonproliferating fraction of cells within the viable rim. The presence of full length, functional proteins within the NC was unexpected, and further analysis demonstrated that this region contains cells that are undergoing autophagy. This study has identified possible targets that may be suitable for therapeutic intervention, and further studies to validate these are required

Characterization of Changes in the Proteome in Different Regions of 3D Multicell Tumor Spheroids

Three dimensional multicell tumor spheroids (MCTS) provide an experimental model where the influence of microenvironmental conditions on protein expression can be determined. Sequential trypsin digestion of HT29 colon carcinoma MCTS enabled segregation into four populations comprising proliferating cells from the surface (SL), an intermediate region (IR), nonproliferating hypoxic cells from the perinecrotic region (PN), and a necrotic core (NC). Total protein was extracted from each population and subjected to iTRAQ-based quantitative proteomics analysis. From a total of 887 proteins identified, 209 were observed to be up-regulated and 114 were down-regulated in the PN and NC regions relative to the SL. Among the up-regulated proteins, components of glycolysis, TCA cycle, lipid metabolism, and steroid biosynthesis increased progressively toward the PN and NC regions. Western blotting, immunohistochemistry, and enzyme assays confirmed that significant changes in the expression of proteins involved in cellular metabolism occur in the nonproliferating fraction of cells within the viable rim. The presence of full length, functional proteins within the NC was unexpected, and further analysis demonstrated that this region contains cells that are undergoing autophagy. This study has identified possible targets that may be suitable for therapeutic intervention, and further studies to validate these are required