canonical pathways, diseases, and functions and network-building tools of ingenuity pathways analysis (ipa)  (Ingenuity Systems)

Journal: Scientific Reports

Article Title: Next-generation RNA sequencing elucidates transcriptomic signatures of pathophysiologic nerve regeneration

doi: 10.1038/s41598-023-35606-6

Figure Lengend Snippet: Cluster analysis to visualize the broad temporal patterns of genetic expression profiles with similar activity and application to IPA canonical pathways. ( a ) Cluster analysis and heatmap representation identified 5 clusters of genes demonstrating similar expression patterns across experimental timepoints (RStudio 4.2.1: pheatmap 1.0.12. https://cran.r-project.org/web/packages/pheatmap/index.html ). Clusters 1 and 4 reflect immediate changes in activity patterns, with Cluster 1 switching from up- to downregulation by 6 h after injury and Cluster 4 demonstrating the inverse pattern of down- to upregulation. Clusters 2, 3, and 5 depict delayed changes in activity; Clusters 2 and 5 express upregulated activity for control and then transition to downregulation by D2, while the inverse is observed for Cluster 3. Uniquely, Cluster 5 remains the most distinct from control expression levels at the terminal timepoint, while the other clusters return to similar expression profiles as their control counterparts. ( b ) Genes derived from the clustering were then subjected to IPA canonical pathways analysis (QIAGEN Inc., https://digitalinsights.qiagen.com/IPA ) to elucidate representative pathways, with the top 5 significant pathways presented. Down D0 (Cluster 1) genes mostly associate with fibrosis pathways and those implicated in neuronal specific activity. Metabolic regulation paths align with Down D2 (Cluster 2), whereas cell cycle regulation pathways are implicated with Up D2 (Cluster 3). Immune-related signatures dominate Up D0 (Cluster 4) whereas Down D2 + (Cluster 5) implicates pathways with both metabolic and neuronal underpinnings.

Article Snippet: Uniquely, Cluster 5 remains the most distinct from control expression levels at the terminal timepoint, while the other clusters return to similar expression profiles as their control counterparts. ( b ) Genes derived from the clustering were then subjected to IPA canonical pathways analysis (QIAGEN Inc., https://digitalinsights.qiagen.com/IPA ) to elucidate representative pathways, with the top 5 significant pathways presented.

Techniques: Expressing, Activity Assay, Control, Derivative Assay

Journal: Scientific Reports

Article Title: Next-generation RNA sequencing elucidates transcriptomic signatures of pathophysiologic nerve regeneration

doi: 10.1038/s41598-023-35606-6

Figure Lengend Snippet: Temporal genetic signatures of pathologic neuroma-in-continuity formation subject to pathway analysis. ( a ) GSEA (version 4.1.0, Broad Institute, San Diego, CA) presents positive enrichment of pathways involved in the inflammatory response through the terminal timepoint of day 48 (D48). Pathways associated with negative enrichment are profusely metabolic. ( b ) Top 5 statistically significant ( p < .05) IPA canonical pathways (QIAGEN Inc., https://digitalinsights.qiagen.com/IPA ) upregulated at each timepoint. For D0 and D2, a majority of pathways are associated with inflammatory response. Cell cycle and neuronal pathways emerge at D7 and persist through D14. At the terminal timepoint (D48) and neuroma pathology, DEGs align with neurodegenerative pathways while the inflammatory response remains enriched. ( c ) Top 5 statistically significant IPA canonical pathways downregulated at each timepoint, which are predominately metabolic. ( d ) Through IPA diseases and functions analysis, we identified six categories associated with neuroma formation across timepoints. Sub-annotations of specific diseases and functions, out of total number of sub-annotations per day, were used to create a percent representation for each category. Inflammatory response is highly implicated for all timepoints. Both the muscle and neurologic disease categories increase in percent representation overtime. Fibrosis was limited in presentation compared with other categories, yet present for timepoints D0 through D14. Finally, both cell death and cellular growth and proliferation were depicted across all timepoints. ( e ) At the terminal endpoint, the neuroma microenvironment is highly associated with categories involved in cell death, neurologic disease, and the inflammatory response. Representative genes associated with the top functions are indicated.

Article Snippet: Uniquely, Cluster 5 remains the most distinct from control expression levels at the terminal timepoint, while the other clusters return to similar expression profiles as their control counterparts. ( b ) Genes derived from the clustering were then subjected to IPA canonical pathways analysis (QIAGEN Inc., https://digitalinsights.qiagen.com/IPA ) to elucidate representative pathways, with the top 5 significant pathways presented.

Techniques:

Journal: Scientific Reports

Article Title: Next-generation RNA sequencing elucidates transcriptomic signatures of pathophysiologic nerve regeneration

doi: 10.1038/s41598-023-35606-6

Figure Lengend Snippet: Significant genes distinct to the rupture injury state for ( a , b ) day 0 vs. day 2 and ( c , d ) day 2 vs. day 7, subjected to Ingenuity network analysis (QIAGEN Inc., https://digitalinsights.qiagen.com/IPA ) for construction of de novo genetic networks of highly interconnected genes representative of significant biological function. Overlay with diseases and functions pathways permits elucidation of how these networks are associated to the downstream biological functions. Red represents significant upregulation; green, downregulation; orange, predicted activation; blue, predicted inhibition. Grey indicates the gene is not in the isolated dataset, but identified as highly related per network analysis. Five motifs were identified to which pathways repeatedly align: vascularity, Neuronal, Fibrosis, Cell Death, and Inflammation. Only pathways of a p -value < 1 × 10 –5 are shown and annotated to the identified motifs. ( a ) The top network pathways with predicted activation are largely associated with vascularity (Angiogenesis, Vasculogenesis, Proliferation of Endothelial Cells), neuronal processes (Neuritogenesis, Growth of Axons, Cell Movement of Schwann Cells, Cell Viability of Motor Neurons), fibrosis, and inflammation (Activation of Phagocytes, Chemoattraction), while there is conflicting enrichment of cellular death profiles (Activation: Necrosis, Apoptosis; Inhibition: Neuronal Cell Death). ( b ) The second highest network implicates activation of cell death (Necrosis, Apoptosis) and inflammatory pathways (Cellular Infiltration by Lymphocytes, Cellular Infiltration by Mononuclear Leukocytes), inhibition of fibrosis, and predominately inhibition of neuronal pathways (Differentiation of Neural Cells, Myelination, Formation of Nodes of Ranvier). ( c ) Top network pathways are overwhelmingly inhibitory and align with neuronal fibrosis (Scar tissue), and cell death (Necrosis) motifs. ( d ) The second highest network presents pathways that predominately predict activation of inflammatory pathways, yet inhibition of cell death and fibrosis.

Article Snippet: Uniquely, Cluster 5 remains the most distinct from control expression levels at the terminal timepoint, while the other clusters return to similar expression profiles as their control counterparts. ( b ) Genes derived from the clustering were then subjected to IPA canonical pathways analysis (QIAGEN Inc., https://digitalinsights.qiagen.com/IPA ) to elucidate representative pathways, with the top 5 significant pathways presented.

Techniques: Activation Assay, Inhibition, Isolation

Journal: Scientific Reports

Article Title: Next-generation RNA sequencing elucidates transcriptomic signatures of pathophysiologic nerve regeneration

doi: 10.1038/s41598-023-35606-6

Figure Lengend Snippet: Immunofluorescent corroboration of GSEA and IPA analysis. Longitudinal slices of sciatic nerve were obtained at 10 μm and images acquired at × 200. Log2fold change values from the raw data are presented for transcript to protein comparison. Repeating motifs of neurologic disease, fibrosis, and muscular pathways, were identified through multiple pathway analyses. Neurologic disease was corroborated with axonal (anti-NF200) and myelin sheath (anti-MBP) staining. Expression between these markers is complementary: both demonstrate a dramatic decrease in expression over time. MYH4 is represented in multiple muscle categories, yet as a myosin, has functional multiplicity in the cytoskeleton, which is exemplified by apparent overlap with NF200 at day 0 (D0), D2, and D14, along with control specimens. Fibrosis presents with pathological remodeling as early as 2 days post-injury. Col3a has minute representation in the control nerve, with fluctuations in expression and aberrant deposition over the time course of injury progression. Col1a demonstrates decreased protein expression D2 and D7, followed by increased intensity and disordered remodeling. At D48, both collagens depict intense staining and dearth of microtubule delineation. Scale bar, 50 μm.

Article Snippet: Uniquely, Cluster 5 remains the most distinct from control expression levels at the terminal timepoint, while the other clusters return to similar expression profiles as their control counterparts. ( b ) Genes derived from the clustering were then subjected to IPA canonical pathways analysis (QIAGEN Inc., https://digitalinsights.qiagen.com/IPA ) to elucidate representative pathways, with the top 5 significant pathways presented.

Techniques: Comparison, Staining, Expressing, Functional Assay, Control