responses themselves. We identified patterns of gene expression in the PB of C57Bl6 female mice at 6 hrs, 24 hrs and 7 days post- irradiation which appeared to distinguish the 3 different levels of radiation versus non-irradiated mice (Figure 4A). When the PB metagene profiles of radiation exposure generated from the 6 hr time point were applied against PB samples from mice at the 24 hr and 7 day time points post-irradiation, the profiles appeared less distinct (Figure 4B). A leave-one-out cross-validation analysis demonstrated that the PB metagene profiles from each time point predicted each dose of radiation with 100% accuracy (Figure 4C). Next, a leave-one-out cross-validation analysis was performed using the metagene profiles from the 6 hr time point against each of the PB samples from mice at 24 hr and 7 day time points and the 6 hr metagene profiles demonstrated 100% accuracy in predicting the radiation status of the 24 hr and 7 day time point samples (Figure 4C). Of note, the 7 day time point following 1000 cGy exposure could not be analyzed since we were unable to collect sufficient RNA from these PB samples to allow gene array hybridization to be performed. Although we found that time did not impact the accuracy of PB gene expression profiles in predicting radiation status, the lists of genes which comprised these PB signatures changed significantly over 7 days (Table S4). No genes were found in common between the 6 hr predictors and the 24 hr or 7 day PB signatures of radiation in 50 cGy-, 200 cGy- , or 1000 cGy-treated mice (Table S2). A single gene, Galectin 1 (Lgals1), a carbohydrate binding protein that is involved in the induction of cell death [31], was found in common between the 24 hr and 7 day predictors of 50 cGy. Radiation Signatures Specificity of PB signatures In addition to inter-individual variations [12], human popula- tions are heterogeneous with respect to health status and medical conditions. Therefore, it is critical to determine whether PB gene expression profiles of radiation response are specific to radiation exposure itself or whether these signatures are potentially confounded by other genotoxic stresses. We chose to compare the PB gene expression response to ionizing radiation exposure with that of gram-negative bacterial sepsis, since this syndrome can be expected to induce similar multiorgan toxicity as is observed following radiation injury [15–17,32]. A pattern of gene expression could be identified which effectively distinguished female C57Bl6 mice treated with Escherichia coli-derived lipopolysaccharide (LPS), experiencing sepsis syndrome, from untreated female C57Bl6 mice (Figure 5A). Applying a leave-one-out cross- validation analysis, we found that the PB signature for 50 cGy irradiation in C57Bl6 mice correctly predicted the status of all LPS-treated C57Bl6 mice as non-irradiated, suggesting robust specificity of the signature for low level (50 cGy) irradiation and sepsis syndrome (Figure 5B). The PB signatures for 200 cGy and 1000 cGy also correctly predicted the LPS-treated mice as non- irradiated, although these probabilities were less robust than the application of the 50 cGy signature (Figure 5B). The PB signature of LPS-treatment also correctly predicted the status of all irradiated mice as ‘‘non-LPS treated’’ (Figure 5B, right). These data indicate that the PB gene expression profiles of radiation response and bacterial sepsis are quite specific and able to distinguish one condition from the other with a high level of Figure 4. Impact of time on murine irradiation profiles. (A) Heat map images illustrating expression pattern of genes selected for classifying control, non-irradiated samples versus 50 cGy, 200 cGy, 1000 cGy irradiated samples at time points 6hr, 24hr, and 7days. (B) Heat map images illustrating expression pattern of genes found in the 6hr time point as applied to the dosages 50 cGy, 200 cGy, 1000 cGy at 24 hr and 7 day time points. High expression is depicted as red, and low expression is depicted as blue. (C) A leave-one-out cross-validation analysis of the classification for control (blue) versus 50 cGy (black), 200 cGy (green), and 1000 cGy (red) for the time points 6 hr (circles), 24 hr (squares), and 7 days (triangles) is shown. The control probabilities for each prediction are shown. doi:10.1371/journal.pone.0001912.g004 PLoS ONE | www.plosone.org 5 April 2008 | Volume 3 | Issue 4 | e1912