The FYDR mouse detects HR-derived sequence rearrangements <i>in situ</i> in intact tissue.

<p>(<b>A</b>) Schematic of the reconstitution of full-length EYFP coding sequence from two truncated copies through replication fork restart by HR. Note that the appearance of fluorescent signal indicates the gain of one repeat unit (a duplication). Arrows represent expression constructs. EYFP coding sequences are in yellow, promoter and polyadenylation signal sequences are in white, and deleted sequences are in black. Drawing is not to scale. (<b>B</b>) Representative image of a FYDR pancreas showing fluorescent foci detectable <i>in situ</i> in intact tissue. Freshly harvested, unfixed whole pancreas was counterstained with Hoechst, compressed to 0.5 mm and imaged under an epifluorescent microscope. Fluorescence is pseudocolored. Original magnification, ×1. Scale bar = 1 cm. (<b>C</b>) Cluster of recombinant cells at ×60 original magnification. Fluorescence is pseudocolored. (<b>D</b>) A recombinant pancreatic acinar cell identified by the overlay of EYFP fluorescence and H&E staining. Fluorescence is pseudocolored. Original magnification, ×40. (<b>E</b>) The model alkylating agent MNU induces HR in the pancreas. Mice received 25 mg/kg MNU i.p., and HR was evaluated 3 to 5 weeks after treatment. Frequencies of recombinant foci per cm² tissue area are significantly greater in MNU-treated mice (n = 15) than in control mice (n = 16). Boxes show 25^th and 75^th percentiles, medians are indicated by horizontal lines. * <i>P</i> < 0.05 (Mann–Whitney <i>U</i>-test).</p

Simultaneous inflammation and cell proliferation induces HR in the pancreas.

<p>(<b>A</b>) Representative images from pancreata of control mice (<i>Top</i>) and mice that experienced combined proliferation and inflammation (<i>Bottom</i>). Freshly harvested whole organs were compressed between glass coverslips and imaged under an epifluorescent microscope. Representative details of composite images are shown, fluorescent foci are apparent <i>in situ</i>. More foci are visible in the pancreas from the proliferation plus inflammation group. Brightness and contrast have been enhanced identically. (<b>B</b>) Numbers of fluorescent foci are higher in mice that experienced combined proliferation and inflammation (n = 18) than in control mice (n = 17). Symbols represent data from individual mice, horizontal bars show medians. **, <i>P</i> < 0.01, (Mann–Whitney <i>U</i>-test). (<b>C</b>) Higher fluorescent cell frequency in the pancreata of mice that experienced combined proliferation and inflammation (n = 18) than in control mice (n = 17). Pancreata were disaggregated into single-cell suspensions and the frequencies of fluorescent cells were determined by flow cytometry. Symbols represent data from individual mice, horizontal bars show median values. *, <i>P</i> < 0.05 (Mann–Whitney <i>U</i>-test).</p

Model for the potentiation of sequence rearrangements induced by endogenous and exogenous DNA damage by inflammation-associated cell proliferation.

<p>Cell proliferation associated with inflammation may be induced by RONS released from inflammatory cells. Regeneration after inflammation also involves cell proliferation to replenish cells lost to inflammation-induced tissue damage. DNA replication is increased in proliferation, and DNA damage during replication can lead to fork breakdown and the formation of DSBs. These DSBs are repaired by HR, but HR can result in LOH, sequence rearrangements, and point mutations. Thus, cell proliferation potentiates the deleterious effect of both endogenous (RONS-induced) and exogenous (exposure-induced) DNA damage, potentially contributing to cancer initiation and recurrence. See text for details.</p

Cerulein treatment induces inflammation in the pancreas, and chronic cerulein pancreatitis induces metaplastic changes.

<p>(<b>A</b>) Tissue sections from pancreata of control mice show normal pancreas architecture. (<b>B</b>) Acute cerulein treatment induces pancreatic inflammation evidenced by edema and an inflammatory infiltrate. (<b>C</b>) Severity of cerulein-induced inflammation as determined by a trained pathologist. Inflammation scores are significantly higher in cerulein-treated mice (n = 30) than in control mice (n = 30). Data are mean ± SEM. *** <i>P</i> < 0.001 (Student’s <i>t</i>-test). (<b>D</b>) Pancreas section from a mouse treated with cerulein for 6 months shows chronic pancreatic inflammation, edema, significant acinar loss, and acinar to ductal metaplasia (arrows). (<b>E</b>) Quantification of metaplastic changes determined by a trained pathologist shows absence of metaplasia in control mice. However, 9 out of 13 mice treated with cerulein for 6 months show metaplastic changes. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004901#sec004" target="_blank"><i>Methods</i></a> for detailed pathological scoring criteria. Statistical testing could not be performed in groups containing only zero values. Panels <b>A,B</b>: Original magnification, ×10. Scale bar = 200 μm. Panel <b>D</b>: Original magnification, ×200. Scale bar = 80 μm.</p

Overlapping bouts of inflammation induce more DSBs than independent bouts of inflammation.

<p>Inflammation, cell proliferation and γH2AX foci formation were quantified in pancreas sections from mice treated with independent bouts of inflammation (blue bars) and with overlapping bouts of inflammation (purple bars). (<b>A,B</b>) Cerulein induces inflammation in both independent (n = 7) and overlapping (n = 8) treatment regimens. Severity of inflammation in control and cerulein-treated mice was quantified by a trained pathologist. (<b>C, D</b>) Quantification of nuclei positive for the proliferation marker Ki-67 shows a moderate increase in independent bouts of inflammation (n = 7), and a large increase in overlapping bouts of inflammation (n = 8). (<b>E,F</b>) Quantification of nuclei positive for the DSB marker γH2AX (nuclei with >5 foci) shows a moderate increase in independent bouts of inflammation (n = 3), and a large increase in overlapping bouts of inflammation (n = 3). Data are mean ± SEM. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004901#sec004" target="_blank"><i>Methods</i></a> for detailed pathological scoring criteria. Statistical testing could not be performed in groups containing only zero values. * <i>P</i> < 0.05; ** <i>P</i> < 0.01, *** <i>P</i> < 0.001 (Student’s <i>t</i>-test).</p

Independent and overlapping bouts of pancreatic inflammation.

<p>(<b>A</b>) For independent bouts of inflammation, three acute cerulein pancreatitis events were induced two weeks apart, and inflammation and proliferation were assessed at the second (analysis time A) and third (analysis time C) bout of inflammation. HR was quantified 10 to 15 days after the last pancreatitis event. (<b>B</b>) For overlapping bouts of inflammation, three acute cerulein pancreatitis events were induced on days 1, 4 and 9. Inflammation and proliferation were assessed at the second (analysis time B) and third (analysis time D) bout of inflammation. HR was quantified 10 to 15 days after the last pancreatitis event. (<b>C</b>) Pancreas section from a control mouse shows healthy tissue. (<b>D,E</b>) Treatment with cerulein (both independent and overlapping) results in edema and an inflammatory infiltrate chiefly of neutrophils, indicating acute inflammation. (<b>F</b>) Ki-67 immunohistochemistry shows low levels of baseline proliferation in control pancreata. (<b>G</b>) Cell proliferation remains low in the pancreas during acute inflammation. (<b>H</b>) During regeneration from acute inflammation, Ki-67 positive nuclei appear, indicating regenerative proliferation. (<b>I</b>) Immunohistochemical detection of γH2AX phosphorylation in pancreas sections show low levels of DSBs in healthy pancreata. (<b>J</b>) During independent bouts of inflammation, nuclei with γH2AX foci (arrowhead) become apparent. (<b>K</b>) During overlapping bouts of inflammation, more γH2AX positive nuclei are visible. (<b>C</b>-<b>E</b>) Original magnification, ×10. Scale bar = 200 μm. (<b>F</b>-<b>H</b>) Original magnification, ×20. Scale bar = 100 μm. (<b>I</b>-<b>K</b>) Original magnification, ×40.</p

PLS-DA and OPLS component contributions for discrimination (R² Y) and variance (Q²) of necrosis at 3 DPI.

<p>Model results are indicated based on using serum or tissue targets alone as well as combined models using the top variables from each independent model.</p

Serum-specific OPLS analysis of <i>C. rodentium</i> infected animals at 3 DPI.

<p>OPLS analysis of <i>C. rodentium</i> infected animals at 3 DPI using serum cytokines and chemistries (X) for prediction of ALT levels (Y). (<b>A</b>) Mice segregated well in the predictive component (principal component 1) with an R²X (1) = 0.35, indicating this component captured ∼35% of the variance present in the X variables. (<b>B</b>) The predictive weight and covariation of serum targets (<b>X variables, </b><b>black triangles</b>) in relation to serum ALT (<b>Y, </b><b>red square</b>). (<b>C</b>) The variables that best separate the two pathological states in relation to the predictive component. (<b>D</b>) Variables in projection (VIPs) for the predictive component where values >1 are have positive influence in determining ALT levels, and VIP <1 have less predictive influence. (<b>E</b>) Observed vs predicted plot for ALT resulted in a R² = 0.9892 indicating a highly predictive model based on serum cytokines. (<b>F</b>) Table representing the VIPs for the predictive component for serum ALT.</p

<i>C. rodentium</i>-induced colonic effects in C57BL/6 mice.

<p>(<b>A</b>) Mock inoculated animals at 0 DPI with normal colonic architecture where epithelial integrity and goblet cells appear intact. (<b>B</b>) Colon at 3 DPI showing epithelial defects at the top of the crypt. (<b>C</b>) Colon at 14 DPI demonstrating hyperplastic crypts and depletion of goblet cells. (<b>D</b>) <i>C. rodentium</i> induced statistically significant histological changes as early as 7 DPI (inflammation, edema, epithelial defects) in colonic sections and found to be most dramatic at 14 DPI. Crypt atrophy and minimal dysplastic changes were only noticeable at 14 DPI. Changes in inflammation, edema, epithelial defects, and hyperplasia as early as 3 DPI were noted, but failed to reach statistical significance (Kruskal-Wallis non-parametric test with Dunn's multiple comparison test: <b>*</b> P<0.05, <b>**</b> P<0.01, <b>***</b> P<0.001). Symbols indicate individual animals and lines indicate group means.</p

Liver-specific OPLS analysis of <i>C. rodentium</i> infected animals at 3 DPI.

<p>OPLS analysis of <i>C. rodentium</i> infected animals at 3 DPI using serum cytokines and chemistries (X) for prediction of ALT levels (Y). (<b>A</b>) Mice segregated well in the predictive component (principal component 1) with an R²X (1) = 0.35, indicating this component captured ∼35% of the variance present in the X variables. (<b>B</b>) The predictive weight and covariation of serum targets (<b>X variables, </b><b>black triangles</b>) in relation to serum ALT (<b>Y, </b><b>red square</b>). (<b>C</b>) Observed vs predicted plot for ALT resulted in a R² = 0.9892 indicating a highly predictive model based on hepatic cytokines. (<b>D</b>) Table representing the VIPs >1 for the predictive component for serum ALT.</p