# Gene Networks and Pathways Involved in _Escherichia coli_ Response to Multiple Stressors
## 5 Conclusions  
In summary, this study aimed at using a systems approach, through integrating several data sets, to determine genes that are involved in the response to multiple stresses. Because multiple array platforms were used, we adopted a percentile rank-based approach to pick significantly altered genes, and we gave emphasis to gene sets or pathways rather than individual genes, as gene-level analysis is sensitive to noise, batch effects, and experimental variations.  
The strengths of our approach are that it is unbiased and statistically robust and that it relies on network enrichment to overcome the false positive inclusion or false negative exclusion of individual genes. Most of the statistically significant results were also biologically relevant, or at least biologically explainable. For example, the downregulation of non-essential functions under stress and the upregulation of genes necessary for survival are consistent with an \"emergency response\" in which the cell manages its resources carefully. Meanwhile, a few genes remain unassigned with any functions and will be subject to further experimental validation. Like all computational approaches, the strategy followed here remains hypothesis generating rather than confirmatory and certainly requires future validation via experiments relying, for example, on gene deletion and insertion (loss and gain of function, respectively). In addition, like many high-throughput analyses, the study cannot establish causation, and some of the observed variations might be consequential while some may be collateral. In addition, the approach may overlook genes that may be biologically relevant but fall out of the statistical thresholds for one reason or another. This is exemplified by the absence of some operon members even when most of the operon is selected among stress response genes.  
**Supplementary Materials:** The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/microorganisms10091793/s1](https://www.mdpi.com/article/10.3390/microorganisms10091793/s1)]([https://www.mdpi.com/article/10.3390/microorganisms10091793/s1](https://www.mdpi.com/article/10.3390/microorganisms10091793/s1)), Figure S1: Network of (A) 83 upregulated genes in at least four stressors with medium confidence (cut-off score: 0.4); Figure S2: The main sub-clusters of the upregulated genes in at least four stressors network; Tables S1-S5: Top 250 up- or downregulated genes in each study/sample in heat stress, oxidative stress, cold stress, nitrosative stress, and antibiotic stress, respectively; Table S6: Combined list of upregulated in all samples of each stress; Table S7: Combined list of downregulated genes in all samples of each stress.  
**Author Contributions:** Conceptualization, R.K.A.; experimental design: N.A.H., A.M., R.K.A.; methodology, E.K.A. and A.M.; formal analysis, E.K.A., N.A.H. and A.M.; resources, A.M. and R.K.A.; data curation, E.K.A. and A.M.; writing--original draft preparation, E.K.A. and N.A.H.; writing--review and editing, A.M., N.A.M. and R.K.A.; visualization, E.K.A., N.A.H. and A.M.; supervision, N.A.M. and R.K.A. All authors have read and agreed to the published version of the manuscript.  
**Funding:** E.K.A. was partly supported by Cairo University graduate student funding, which is limited to supplies.  
**Institutional Review Board Statement:** Not applicable.  
**Informed Consent Statement:** Not applicable.  
**Data Availability Statement:** Data tables are available in the Supplementary Material.  
**Conflicts of Interest:** The authors declare no conflict of interest.