{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,7]],"date-time":"2026-10-07T05:50:02Z","timestamp":1791352202346,"version":"4.3.1"},"reference-count":39,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,10,7]],"date-time":"2026-10-07T00:00:00Z","timestamp":1791331200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Physiol."],"abstract":"<jats:sec>\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>Exercise is often described as medicine, but exercise and pharmacological interventions cannot be treated as clinically equivalent. This study developed a Reference Signature Projection Framework (RSPF) to evaluate the directional relationship of external disease and exercise transcriptomic responses relative to a predefined disease-treatment reference axis. The empirical analysis in this study was restricted to the IAV-DI reference axis defined by dimethyl itaconate (DI) treatment in the context of influenza A virus (IAV) infection.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>(1) Reference-signature construction: the IAV-DI reference direction was defined from transcriptional changes between DI-treated and untreated human peripheral blood mononuclear cells in the context of IAV infection. Drug-UP and Drug-DOWN directions were defined according to avg_log2FC, candidate genes were prioritized using random forest Mean Decrease Accuracy (MDA), and the MDA Top 100 was used as the main reference signature. (2) Reference-signature projection: Drug-UP and Drug-DOWN were projected separately onto complete gene-ranking lists for GSE243629 IA versus HC, cardiopulmonary exercise testing (CPX), and Marathon post- versus pre-exercise comparisons, and GSEA was used to calculate NES, nominal p, and FDR. (3) Robustness and supporting evaluation: gene-level similarity, TopN sensitivity, random gene-set negative controls, and pathway-level NES patterns were used to evaluate transcriptomic directionality, robustness, and interpretive boundaries.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      (1) Reference-signature composition: the MDA Top 100 contained 24 Drug-UP and 76 Drug-DOWN genes. (2) External disease-direction evaluation: in GSE243629 IA versus HC, Drug-DOWN was significantly enriched toward the upregulated end (NES\u00a0=\u00a01.724, FDR\u00a0=\u00a04.00 \u00d7 10\n                      <jats:sup>\u22128<\/jats:sup>\n                      ), whereas Drug-UP did not reach FDR&amp;lt; 0.05, indicating a clear opposite relationship of the external disease state relative to the IAV-DI reference axis, with statistical support driven mainly by the Drug-DOWN reference gene set. (3) Overall exercise-direction results: neither Drug-UP nor Drug-DOWN reached FDR&amp;lt; 0.05 in CPX or Marathon; therefore, their direction scores were interpreted only as exploratory directional indicators. Gene-level analysis likewise showed a weak overall directional relationship between CPX and IAV-DI. (4) Pathway-level results: selected functional modules showed the same NES sign for IAV-DI and CPX and the opposite sign for Marathon, but none of the CPX pathways reached FDR &amp;lt; 0.05; these results therefore represent only module-specific exploratory directional patterns.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>RSPF provides a computational framework for evaluating transcriptomic directional relationships relative to a predefined disease-treatment reference axis. In the current IAV-DI proof-of-concept analysis, the external influenza disease comparison showed a clear opposite relationship relative to the IAV-DI reference axis, whereas neither CPX nor Marathon showed statistically supported overall IAV-DI directional concordance; exploratory directional patterns were observed only in selected functional modules. Accordingly, the present findings apply only to the IAV-DI reference axis and cannot be generalized to a universal relationship between exercise and pharmacological interventions or used to infer therapeutic equivalence, drug substitution, or shared pharmacological mechanisms.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.3389\/fphys.2026.1943015","type":"journal-article","created":{"date-parts":[[2026,10,7]],"date-time":"2026-10-07T04:52:49Z","timestamp":1791348769000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Reference-signature projection framework for exercise transcriptomic directionality based on the IAV-DI reference axis"],"prefix":"10.3389","volume":"17","author":[{"given":"Anli","family":"Zhou","sequence":"first","affiliation":[{"name":"Center for Innovation in Sports-Medicine Integration, Capital University of Physical Education and Sports","place":["Beijing, China"]},{"name":"Beijing Key Laboratory of Interdisciplinary Intelligent Technology of Sports, Medicine and Engineering","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Min","family":"Zhang","sequence":"additional","affiliation":[{"name":"Center for Innovation in Sports-Medicine Integration, Capital University of Physical Education and Sports","place":["Beijing, China"]},{"name":"Beijing Key Laboratory of Interdisciplinary Intelligent Technology of Sports, Medicine and Engineering","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cheng","family":"Zhang","sequence":"additional","affiliation":[{"name":"Center for Innovation in Sports-Medicine Integration, Capital University of Physical Education and Sports","place":["Beijing, China"]},{"name":"Beijing Key Laboratory of Interdisciplinary Intelligent Technology of Sports, Medicine and Engineering","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jianjun","family":"Guo","sequence":"additional","affiliation":[{"name":"Center for Innovation in Sports-Medicine Integration, Capital University of Physical Education and Sports","place":["Beijing, China"]},{"name":"Beijing Key Laboratory of Interdisciplinary Intelligent Technology of Sports, Medicine and Engineering","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Min","family":"Zhang","sequence":"additional","affiliation":[{"name":"Department of Nephrology, Beijing Chaoyang Hospital, Capital Medical University","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yan","family":"Zhang","sequence":"additional","affiliation":[{"name":"Center for Innovation in Sports-Medicine Integration, Capital University of Physical Education and Sports","place":["Beijing, China"]},{"name":"Beijing Key Laboratory of Interdisciplinary Intelligent Technology of Sports, Medicine and Engineering","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,10,7]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"783","DOI":"10.1016\/j.cell.2006.02.015","article-title":"Pathogen recognition and innate immunity","volume":"124","author":"Akira","year":"2006","journal-title":"Cell."},{"key":"B2","doi-asserted-by":"publisher","first-page":"1340","DOI":"10.1093\/bioinformatics\/btq134","article-title":"Permutation importance: a corrected feature importance measure","volume":"26","author":"Altmann","year":"2010","journal-title":"Bioinformatics"},{"key":"B3","doi-asserted-by":"publisher","first-page":"459","DOI":"10.1146\/annurev-immunol-020711-074942","article-title":"Neutrophil function: from mechanisms to disease","volume":"30","author":"Amulic","year":"2012","journal-title":"Annu. Rev. Immunol."},{"key":"B4","doi-asserted-by":"publisher","first-page":"1143","DOI":"10.1002\/cphy.c110025","article-title":"Lack of exercise is a major cause of chronic diseases","volume":"2","author":"Booth","year":"2012","journal-title":"Compr. Physiol."},{"key":"B5","doi-asserted-by":"publisher","first-page":"5","DOI":"10.1023\/A:1010933404324","article-title":"Random forests","volume":"45","author":"Breiman","year":"2001","journal-title":"Mach. Learn."},{"key":"B6","doi-asserted-by":"publisher","first-page":"1615","DOI":"10.1096\/fj.14-263699","article-title":"The search for exercise factors in humans","volume":"29","author":"Catoire","year":"2015","journal-title":"FASEB J."},{"key":"B7","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pcbi.1010348","article-title":"Nine quick tips for pathway enrichment analysis","volume":"18","author":"Chicco","year":"2022","journal-title":"PloS Comput. Biol."},{"key":"B8","doi-asserted-by":"publisher","first-page":"607","DOI":"10.1038\/nri3041","article-title":"The anti-inflammatory effects of exercise: mechanisms and implications for the prevention and treatment of disease","volume":"11","author":"Gleeson","year":"2011","journal-title":"Nat. Rev. Immunol."},{"key":"B9","doi-asserted-by":"publisher","DOI":"10.1093\/bib\/bbaf397","article-title":"Single-cell differential expression analysis between conditions within nested settings","volume":"26","author":"Hafner","year":"2025","journal-title":"Brief. Bioinform."},{"key":"B10","doi-asserted-by":"publisher","first-page":"3573","DOI":"10.1016\/j.cell.2021.04.048","article-title":"Integrated analysis of multimodal single-cell data","volume":"184","author":"Hao","year":"2021","journal-title":"Cell."},{"key":"B11","doi-asserted-by":"publisher","first-page":"550","DOI":"10.1038\/s41576-023-00586-w","article-title":"Best practices for single-cell analysis across modalities","volume":"24","author":"Heumos","year":"2023","journal-title":"Nat. Rev. Genet."},{"key":"B12","doi-asserted-by":"publisher","first-page":"54","DOI":"10.22029\/eir.2023.1755","article-title":"A scoping review on the effects of physical exercise and fitness on peripheral leukocyte energy metabolism in humans","volume":"29","author":"Hodgman","year":"2023","journal-title":"Exerc. Immunol. Rev."},{"key":"B13","doi-asserted-by":"publisher","DOI":"10.1093\/bib\/bbac286","article-title":"Benchmarking methods for detecting differential states between conditions from multi-subject single-cell RNA-seq data","volume":"23","author":"Junttila","year":"2022","journal-title":"Brief. Bioinform."},{"key":"B14","doi-asserted-by":"publisher","first-page":"060012","DOI":"10.1101\/060012","article-title":"Fast gene set enrichment analysis","author":"Korotkevich","year":"2016","journal-title":"bioRxiv"},{"key":"B15","doi-asserted-by":"publisher","first-page":"1929","DOI":"10.1126\/science.1132939","article-title":"The Connectivity Map: using gene-expression signatures to connect small molecules, genes, and disease","volume":"313","author":"Lamb","year":"2006","journal-title":"Science"},{"key":"B16","first-page":"18","article-title":"Classification and regression by randomForest","volume":"2","author":"Liaw","year":"2002","journal-title":"R. News"},{"key":"B17","doi-asserted-by":"publisher","first-page":"417","DOI":"10.1016\/j.cels.2015.12.004","article-title":"The Molecular Signatures Database hallmark gene set collection","volume":"1","author":"Liberzon","year":"2015","journal-title":"Cell Syst."},{"key":"B18","doi-asserted-by":"publisher","first-page":"41","DOI":"10.1038\/s41592-021-01336-8","article-title":"Benchmarking atlas-level data integration in single-cell genomics","volume":"19","author":"Luecken","year":"2022","journal-title":"Nat. Methods"},{"key":"B19","doi-asserted-by":"publisher","first-page":"D672","DOI":"10.1093\/nar\/gkad1025","article-title":"The reactome pathway knowledgebase 2024","volume":"52","author":"Milacic","year":"2024","journal-title":"Nucleic Acids Res."},{"key":"B20","doi-asserted-by":"publisher","first-page":"113","DOI":"10.1038\/nature25986","article-title":"Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1","volume":"556","author":"Mills","year":"2018","journal-title":"Nature"},{"key":"B21","doi-asserted-by":"publisher","first-page":"174","DOI":"10.1038\/s41586-023-06877-w","article-title":"Temporal dynamics of the multi-omic response to endurance exercise training","volume":"629","year":"2024","journal-title":"Nature"},{"key":"B22","doi-asserted-by":"publisher","first-page":"7851","DOI":"10.1038\/s41467-022-35519-4","article-title":"A balanced measure shows superior performance of pseudobulk methods in single-cell RNA-sequencing analysis","volume":"13","author":"Murphy","year":"2022","journal-title":"Nat. Commun."},{"key":"B23","doi-asserted-by":"publisher","first-page":"823","DOI":"10.1038\/nri3084","article-title":"Towards a systems understanding of MHC class I and MHC class II antigen presentation","volume":"11","author":"Neefjes","year":"2011","journal-title":"Nat. Rev. Immunol."},{"key":"B24","doi-asserted-by":"publisher","first-page":"1677","DOI":"10.1152\/japplphysiol.00143.2013","article-title":"Transcriptome analysis of neutrophils after endurance exercise reveals novel signaling mechanisms in the immune response to physiological stress","volume":"114","author":"Neubauer","year":"2013","journal-title":"J. Appl. Physiol."},{"key":"B25","doi-asserted-by":"publisher","first-page":"1570","DOI":"10.1038\/s41467-023-37126-3","article-title":"Benchmarking integration of single-cell differential expression","volume":"14","author":"Nguyen","year":"2023","journal-title":"Nat. Commun."},{"key":"B26","doi-asserted-by":"publisher","first-page":"273","DOI":"10.1038\/s41577-019-0128-5","article-title":"Itaconate: the poster child of metabolic reprogramming in macrophage function","volume":"19","author":"O'Neill","year":"2019","journal-title":"Nat. Rev. Immunol."},{"key":"B27","doi-asserted-by":"publisher","first-page":"457","DOI":"10.1038\/nrendo.2012.49","article-title":"Muscles, exercise and obesity: skeletal muscle as a secretory organ","volume":"8","author":"Pedersen","year":"2012","journal-title":"Nat. Rev. Endocrinol."},{"key":"B28","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1111\/sms.12581","article-title":"Exercise as medicine\u2014evidence for prescribing exercise as therapy in 26 different chronic diseases","volume":"25","author":"Pedersen","year":"2015","journal-title":"Scand. J. Med. Sci. Sports"},{"key":"B29","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1010219","article-title":"Itaconate and derivatives reduce interferon responses and inflammation in influenza A virus infection","volume":"18","author":"Sohail","year":"2022","journal-title":"PloS Pathog."},{"key":"B30","doi-asserted-by":"publisher","first-page":"5692","DOI":"10.1038\/s41467-021-25960-2","article-title":"Confronting false discoveries in single-cell differential expression","volume":"12","author":"Squair","year":"2021","journal-title":"Nat. Commun."},{"key":"B31","doi-asserted-by":"publisher","DOI":"10.1186\/1471-2105-8-25","article-title":"Bias in random forest variable importance measures: illustrations, sources and a solution","volume":"8","author":"Strobl","year":"2007","journal-title":"BMC Bioinf."},{"key":"B32","doi-asserted-by":"publisher","first-page":"1437","DOI":"10.1016\/j.cell.2017.10.049","article-title":"A next generation Connectivity Map: L1000 platform and the first 1,000,000 profiles","volume":"171","author":"Subramanian","year":"2017","journal-title":"Cell."},{"key":"B33","doi-asserted-by":"publisher","first-page":"15545","DOI":"10.1073\/pnas.0506580102","article-title":"Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles","volume":"102","author":"Subramanian","year":"2005","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"B34","doi-asserted-by":"publisher","first-page":"805","DOI":"10.1016\/j.cell.2010.01.022","article-title":"Pattern recognition receptors and inflammation","volume":"140","author":"Takeuchi","year":"2010","journal-title":"Cell."},{"key":"B35","doi-asserted-by":"publisher","DOI":"10.1111\/sms.70018","article-title":"Single-cell RNA sequencing analysis reveals exercise-induced transcriptional dynamics in half-marathon runners","volume":"35","author":"Veschetti","year":"2025","journal-title":"Scand. J. Med. Sci. Sports"},{"key":"B36","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pcbi.1009935","article-title":"Urgent need for consistent standards in functional enrichment analysis","volume":"18","author":"Wijesooriya","year":"2022","journal-title":"PloS Comput. Biol."},{"key":"B37","doi-asserted-by":"publisher","DOI":"10.1016\/j.isci.2023.106532","article-title":"Single-cell sequencing of immune cells after marathon and symptom-limited cardiopulmonary exercise","volume":"26","author":"Yu","year":"2023","journal-title":"iScience"},{"key":"B38","doi-asserted-by":"publisher","DOI":"10.1016\/j.isci.2023.108507","article-title":"A single-cell atlas of the peripheral immune response in patients with influenza A virus infection","volume":"26","author":"Zhang","year":"2023","journal-title":"iScience"},{"key":"B39","doi-asserted-by":"publisher","first-page":"738","DOI":"10.1038\/s41467-021-21038-1","article-title":"A practical solution to pseudoreplication bias in single-cell studies","volume":"12","author":"Zimmerman","year":"2021","journal-title":"Nat. Commun."}],"container-title":["Frontiers in Physiology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fphys.2026.1943015\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,10,7]],"date-time":"2026-10-07T04:52:51Z","timestamp":1791348771000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fphys.2026.1943015\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,10,7]]},"references-count":39,"alternative-id":["10.3389\/fphys.2026.1943015"],"URL":"https:\/\/doi.org\/10.3389\/fphys.2026.1943015","relation":{},"ISSN":["1664-042X"],"issn-type":[{"value":"1664-042X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,10,7]]},"article-number":"1943015"}}