Okay, let's tackle these questions about the pathway enrichment analysis in the given article. The user wants me to go through the Methods section and other relevant parts to answer each question separately. I need to be careful and check each part of the article for details. First question: What tool was used for pathway enrichment analysis? I remember in the Methods section, they mentioned using "nano-LC-MS/MS" for proteomics, but that's for identifying proteins. For pathway analysis, they might have used a different tool. Let me check the Methods again. Wait, under the "Proteomic analysis" subsection, they say, "Functional enrichment analysis of the EV proteome for GO terms..." but they don't specify the tool. The main text in the Results mentions GO terms and biological pathways. Maybe in the supplementary methods or another section. Hmm, the article doesn't explicitly state which software or tool they used for the pathway enrichment. So the answer would be "not described." Second question: Was a tool version number provided? Since the tool isn't mentioned, the version number isn't provided either. Answer: "not described." Third question: What gene set library was queried? The Results mention "GO terms of the 'cellular component' GO domain" and "manually curated biological pathways." So they used GO (Gene Ontology) and possibly other manually curated pathways. The GO terms are part of their analysis. The answer here is GO and manually curated pathways. Wait, the exact wording in the Results is "functional enrichment analysis of the EV proteome for GO terms of the 'cellular component' GO domain" and later "manually curated biological pathways." So the gene set libraries are GO and manually curated ones. But the user asks for the library queried, so the answer would include both. However, the question is phrased as "what gene set library," so maybe they want the specific ones. The answer is GO and possibly others, but the article doesn't specify which manually curated ones. So the answer is "GO" and "manually curated biological pathways." Fourth question: Was a background gene list defined? The article doesn't mention defining a background gene list. They analyzed the proteomic data from EVs, so the background would be the entire proteome, but it's not explicitly stated. So answer: "not described." Fifth question: What statistical test was used? The article mentions "functional enrichment analysis," but doesn't specify the test. For example, hypergeometric test, Fisher's exact test, etc. Since they don't state it, the answer is "not described." Sixth question: Was false discovery rate correction used? The Results mention using "false discovery rate correction" in the proteomic analysis (for identifying differentially expressed proteins), but for pathway analysis, they don't specify. The Results section says, "Using nano-LC-MS/MS... 33% of the proteins were differentially expressed between the two conditions," and "functional enrichment analysis... showed that CSE-exposure caused an up-regulation..." but no mention of FDR for the pathway analysis. So answer: "not described." | **Question** | **Answer** | |--------------|-------------| | 1. What tool was used for pathway enrichment analysis? | **Not described**. The article does not specify the software/tool used for pathway enrichment analysis. | | 2. Was a tool version number provided? | **Not described**. No version number is provided for the tool (if any). | | 3. What gene set library was queried? | **GO (Gene Ontology)** and **manually curated biological pathways** (e.g., "haemostasis," "platelet activation, signalling and aggregation"). | | 4. Was a background gene list defined? | **Not described**. The article does not clarify whether a specific background gene list (e.g., all expressed genes) was used for enrichment analysis. | | 5. What statistical test was used for enrichment analysis? | **Not described**. The article does not specify the statistical test (e.g., hypergeometric, Fisher's exact) used for enrichment analysis. | | 6. Was false discovery rate correction used? | **Not described**. While FDR correction is mentioned for proteomic data analysis (e.g., differential protein expression), it is not explicitly stated for pathway enrichment analysis. | --- ### Key Details from the Article: - **GO enrichment**: The study reports enrichment in GO domains ("cellular component," "molecular function") and biological processes ("immune response," "signal transduction"). - **Manually curated pathways**: Specific pathways like "haemostasis" and "platelet activation" are highlighted but without reference to a standardized library (e.g., KEGG, Reactome). - **Statistical methods**: The article mentions differential expression analysis (e.g., 33% of proteins differentially expressed) but does not detail the statistical framework for pathway enrichment. The analysis appears to rely on in-house or unspecified methods, as no tool, version, or explicit statistical workflow is provided for pathway enrichment.