An emerging body of evidence indicates that early-life arsenic (As) exposure may influence the trajectory of health outcomes later in life. However, the mechanisms underlying these observations are unknown.The objective of this study was to investigate the influence of prenatal As exposure on global methylation of cord blood DNA in a study of mother/newborn pairs in Matlab, Bangladesh.Maternal and cord blood DNA were available from a convenience sample of 101 mother/newborn pairs. Measures of As exposure included maternal urinary As (uAs), maternal blood As (mbAs) and cord blood As (cbAs). Several measures of global DNA methylation were assessed, including the [3H]-methyl-incorporation assay and three Pyrosequencing assays: Alu, LINE-1 and LUMA.In the total sample, increasing quartiles of maternal uAs were associated with an increase in covariate-adjusted means of newborn global DNA methylation as measured by the [3H]-methyl-incorporation assay (quartile 1 (Q1) and Q2 vs. Q4; p = 0.06 and 0.04, respectively). Sex-specific linear regression analyses, while not reaching significance level of 0.05, indicated that the associations between As exposures and Alu, LINE-1 and LUMA were positive among male newborns (N = 58) but negative among female newborns (N = 43); tests for sex differences were borderline significant for the association of cbAs and mbAs with Alu  and for the association between maternal uAs and LINE-1 (p = 0.07). Sex-specific correlations between maternal urinary creatinine and newborn methyl-incorporation, Alu and LINE-1 were also evident (p<0.05).in utero may potentially influence disease outcomes later in life. Additional studies are needed to confirm these findings and to examine the persistence of DNA methylation marks over time.These results suggest that prenatal As exposure is associated with global DNA methylation in cord blood DNA, possibly in a sex-specific manner. Arsenic-induced epigenetic modifications  Naturally occurring arsenic (As) is a ubiquitous element in the environment. Through processes that are incompletely understood, As in the solid phase can become solubilized and released into groundwater. Numerous aquifers around the world are enriched with this metalloid, posing a tremendous public health challenge in populations whose drinking water is obtained from contaminated aquifers in utero exposure to As induced sex-specific tumor formation in adulthood. Male offspring developed liver and adrenal tumors and female offspring developed ovarian and lung tumors in utero As exposure Arsenic is classified as a human carcinogen by the International Agency for Research on Cancer and the U.S. Environmental Protection Agency in vitro and animal studies have demonstrated that As exposure leads to reductions in global DNA methylation in vitro studies have further indicated that As exposure can induce alterations in post-translational modifications of histone tails The collective evidence supporting the role of As exposure in the dysregulation of DNA methylation is provided in two excellent reviews In our study of mother/newborn pairs from 101 pregnancies in Bangladesh, we previously demonstrated strong positive correlations between maternal blood As (mbAs) and cord blood As (cbAs), indicating that As readily crosses the placenta This study was approved by the Institutional Review Board of the Columbia University Medical Center and the Ethical Review Committee of the ICDDR-B.Between December 2004 and April 2005, pregnant women who presented at the main hospital in Matlab, Bangladesh, were asked by an attending physician to participate in a study designed to characterize the As metabolic profiles among mother/newborn pairs Near the time of delivery, women were asked to provide written informed consent, to answer a brief questionnaire, to provide a blood and urine sample prior to delivery, and to allow for a blood sample to be obtained from the discarded umbilical cord after the birth of the baby. The questionnaire was administered by a trained nurse, who also obtained the blood and urine samples. The length, weight and head circumference of the baby were also recorded.12 and homocysteine analyses. All samples were then frozen at −80°C. Urine samples were also collected in acid-washed tubes and frozen at −80°C. All of the samples were ultimately shipped to New York on dry ice and analyzed at Columbia University.Samples were collected in EDTA vacutainers and immediately processed in the hospital laboratory. Whole blood for blood As measurements was aliquoted into acid-washed tubes. Serum was separated for folate, BFor 100 of the 101 women, a tube well water sample was obtained from the primary well shortly after delivery, usually within a day, into 60-mL acid cleaned polyethylene bottles. These were shipped to Columbia University's Lamont Doherty Earth Observatory where they were acidified prior to analysis. Analytical procedures have previously been described in detail Urinary As (uAs) concentrations were assayed by graphite furnace atomic absorption spectrophotometry (GFAA) using a Perkin-Elmer AAnalyst 600 system  as described Maternal and cord blood samples were analyzed for total blood As concentration using a Perkin-Elmer Elan DRC II ICP-MS equipped with an AS 93+ autosampler as described Genomic DNA was isolated at Columbia University from 300 µl of frozen whole blood samples using the FlexiGene DNA kit (Qiagen) following the manufacturer's protocol, except for an additional centrifugation step at 10,000× g for 5 min immediately after protease digestion to pellet any remaining proteins or lipids. The supernatant was then transferred into a new microcentrifuge tube containing 150 µl of isopropanol. The remainder of the procedures were conducted according to the manufacture's protocol. All extractions yielded ≥2 µg of DNA with a 260/280 ratio ≥1.7.3H]-SAM in the presence of the SssI prokaryotic methylase enzyme, which indiscriminately methylates all unmethylated cytosines in CpG sequences. The ability of DNA to incorporate [3H] methyl groups in vitro is inversely related to endogenous DNA methylation. Briefly, 250 ng of DNA was incubated for 1 hr at 37°C with 3 U of SssI methylase , 3.8 uM (1.1 µCi) 3H labeled SAM , and EDTA, DTT and Tris-HCL (pH 8.2). The reaction mixtures were applied onto Whatman DE81 filter papers and washed on a vacuum filtration apparatus and air dried. Dried filters were analyzed by a Packard Tri-Carb 2100TR Liquid Scintillation Analyzer. Each DNA sample was processed in duplicate and each processing run included samples for background , a hypomethylation control (HeLa cell DNA) and a quality control sample (DNA extracted from a whole-blood sample). The intra- and inter-day coefficients of variation (CVs) were 1.8% and 5.3%, respectively. To quantify the amount of double-stranded input DNA in each reaction, an aliquot of the assayed DNA was used to determine DNA concentrations using PicoGreen dsDNA Quantitation Reagent (Molecular Probes). All disintegrations per minute (DPM) values were expressed per µg DNA.The methyl-incorporation assay was performed by the method of Balaghi and Wagner DNA samples (500 ng at 20 ng/µl) were bisulfite-treated using the EZ-96 DNA Methylation-Gold Kit™ . Bisulfite conversion of DNA changes unmethylated cytosine to uracil and subsequently to thymidine after PCR whereas methylated cytosines are protected from bisulfite conversion, resulting in methylation-dependent differences in DNA sequences. Bisulfite-converted DNA was stored at −20°C until further use. LINE-1 and Alu methylation was measured by quantitative Pyrosequencing (Qiagen) using primers and conditions as described previously 5′- CCGG -3′ sequences by the methylation sensitive restriction enzyme (HpaII) and its methylation insensitive isoschizomer (MspI) in parallel reactions. Additionally, EcoRI is included in all reactions to normalize the amount of DNA input. The extent of restriction cleavage is then measured by bioluminometic polymerase extension via Pyrosequencing.The Luminometric Methylation Assay (LUMA) has been described in detail elsewhere GTGTCACATGTGTGBriefly, genomic DNA (300 ng) was digested with HpaII+EcoRI and MspI+EcoRI in parallel reactions containing 2 µl of Tango buffer , 5 U of HpaII or MspI, 5 U of EcoRI and dH2O to a final volume of 20 µl. Samples were incubated at 37°C for 4 hours. After the incubation, 15 µl of Pyrosequencing annealing buffer was added to samples. After mixing, 30 µl of the sample reaction containing the annealing buffer was transferred to 96 well Pyrosequencing plates and run with the following nucleotide dispensation order: Plasma folate and total cobalamin from maternal and cord blood were analyzed by radioimmunoassay  as described previously Descriptive statistics were calculated for sample characteristics of mothers and their newborns and by newborns' sex. Sex differences for continuous variables were assessed using the Wilcoxon rank-sum test. Pyrosequencing data that did not pass Pyro Q-CpG Software quality scores or were not reproducible were excluded from analyses; these included three samples . For the methyl-incorporation assay, quality data was available for 98 samples. Spearman correlation coefficients were used to assess bivariate associations between maternal and newborn DNA methylation and sex specific correlations between uCrn and DNA methylation measures. Wald tests were used to evaluate differences in correlations by sex. Linear regression models were used to examine associations between As exposures and DNA methylation measures controlling for uCrn, including a uCrn by sex interaction term as appropriate. An As by sex interaction term was added to test for sex differences in the effect of As on DNA methylation. We also categorized uAs adjusted for uCrn, defined as the residual from regressing log uAs on sex-specific log uCrn, into quartiles, and computed mean levels of global DNA methylation for each quartile. All analyses were performed using SAS .The demographic and clinical data of the study population are presented in Global DNA methylation levels for the methyl-incorporation, Alu, LINE-1 and LUMA assays for mothers and newborns are also provided in Table S1). However, in linear regression models adjusting for sex-specific effects of maternal uCrn levels, maternal uAs was negatively related with newborn methyl-incorporation levels  showed a positive association In secondary analyses, we found that newborn sex modified the association between As exposure and global methylation of cord blood DNA. Although the associations did not reach statistical significance, arsenic exposure and DNA methylation were positively associated in males and inversely associated in females. These results were unanticipated and were not part of our a priori hypothesis. However, a growing body of evidence in humans suggests that sex differences may arise in epigenetic responses to prenatal environmental exposures Our findings for sex-specific associations between maternal uCrn and global DNA methylation in cord blood were unanticipated. Numerous studies from our group have showed that uCrn is a strong predictor of As methylation An advantage of this study is the use of multiple assays to estimate global DNA methylation levels. Although the four methods have been used widely as indicators of global DNA methylation Alu and LINE-1 are highly repetitive elements. Approximately 1.5 million copies of Alu and 0.5 million copies of LINE-1 are dispersed throughout the genome. Together they comprise around 25% of the genome and > 40% of methylated CpG domains An unanticipated finding was that the associations between maternal DNA methylation and that of her newborn as measured by Pyrosequencing differed by newborn sex, such that LINE-1 methylation was correlated only among male newborn/maternal pairs, whereas Alu methylation was correlated among female newborn/maternal pairs. These inconsistencies complicate the interpretation of Alu and LINE-1 as surrogates for global DNA methylation. A potential explanation for these differences may be related to the fact that Alu and LINE-1 use different types of internal RNA polymerase promoters, suggesting that these repetitive elements may be under different selective pressures A limitation to this study is our use of a convenience sample of participants drawn from a study that was originally designed to characterize the As metabolite profiles among only 101 mother/newborn pairs In conclusion, the results of this study suggest that newborn sex may modify the association between prenatal As exposure and global methylation of cord blood DNA. Our data offer a plausible epigenetic mechanism by which prenatal As exposure may contribute to observed sex differences in As-induced health outcomes later in life. Additional studies are needed to confirm these results and to examine the persistence of DNA methylation marks across the lifecourse.Table S1Spearman correlation coefficients between As exposures and maternal and newborn DNA methylation.(DOCX)Click here for additional data file.


Drosophila is an outstanding model system to study the mechanisms involved in large-scale tissue rearrangements. Ventral cells accumulate myosin at their apical sides and, while being tightly coupled to each other via apical adherens junctions, execute actomyosin contractions that lead to reduction of their apical cell surface. Thereby, a band of constricted cells along the ventral epithelium emerges which will form a tissue indentation along the ventral midline . Here we adopt a 2D vertex model to simulate ventral furrow formation in a surface view allowing easy comparison with confocal live-recordings. We show that in order to reproduce furrow morphology seen in vivo, a gradient of contractility must be assumed in the ventral epithelium which renders cells more contractile the closer they lie to the ventral midline. The model predicts previous experimental findings, such as the gain of eccentric morphology of constricting cells and an incremental fashion of apical cell area reduction. Analysis of the model suggests that this incremental area reduction is caused by the dynamical interplay of cell elasticity and stochastic contractility as well as by the opposing forces from contracting neighbour cells. We underpin results from the model through in vivo analysis of ventral furrow formation in wildtype and twi mutant embryos. Our results show that ventral furrow formation can be accomplished as a “tug-of-war” between stochastically contracting, mechanically coupled cells and may require less rigorous regulation than previously thought.Ventral furrow formation in For the developmental biologist it is a fascinating question how cells can coordinate major tissue movements during embryonic development. The so-called ventral furrow of the Drosophila embryo is a well-studied example of such a process when cells from a ventral band, spanning nearly the entire length of the embryo, undergo dramatic shape change by contracting their tips and then fold inwards into the interior of the embryo. Although numerous genes have been identified that are critical for ventral furrow formation, it is an open question how cells work together to elicit this tissue rearrangement. We use a computational model to mimic the physical properties of cells in the ventral epithelium and simulate the formation of the furrow. We find that the ventral furrow can form through stochastic self-organisation and that previous experimental observations can be readily explained in our model by considering forces that arise when cells execute contractions while being coupled to each other in a mechanically coherent epithelium. The model highlights the importance of a physical perspective when studying tissue morphogenesis and shows that only a minimal genetic regulation may be required to drive complex processes in embryonic development. Drosophila embryogenesis and an outstanding model system to address the mechanisms by which cell shape changes evoke a large-scale tissue rearrangement. During a remarkably fast time-span of about 10 minutes ventral cells constrict their apices and form an indentation in the ventral epithelium  which subsequently invaginates into the interior of the embryo to commence the development of mesodermal structures  take place at the apical side of ventral cells, so imaging of surface sections was frequently used as the method of choice to investigate the cell biological mechanisms of ventral furrow formation e.g.: . We applDrosophila embryo in surface view is modelled as a sheet of hexagonal cells. We use a vertex model to describe the components of potential energy relevant to the cellular events during furrow formation (0). Line tension refers to tension arising along cell-cell boundaries. In order to account for previous findings revealing asymmetrical tension across the tissue during ventral furrow formation The ventral epithelium of the ormation . Similarormation . Area elStarting from a relaxed condition, i.e. cells having their preferred area and preferred boundary lengths, contractility makes cells constrict their area until contractile and elastic forces balance each other and a local energy minimum is reached. The degree of constriction largely depends on the ratio of contractility and area elasticity . We prefIn gastrulating embryos, constriction only occurs in ventral cells . However, among these cells different constriction levels are achieved since cells lying closer to the ventral midline yield higher constriction than those lying further lateral before they become invaginated e ( = 0) . In the e ( = 0) . Experime ( = 0) , consiste ( = 0) , Fog [4]With temporarily constant contractility enabled, both the cutoff and the gradient model result in the emergence of a band of constricted cells with lateral cells moving towards the midline, as seen in live-imaging data –S3. The in vivo data for which a slow rate of constriction at the onset of furrow formation has been documented  will certainly not occur independently from adjacent cells since the cells are part of a mechanically coherent epithelium. Therefore, one has to take the possibility into account that contractions of adjacent cells may have a considerable effect on each other’s shape change. For instance, temporary dilations (i.e. increase of cell area), seen both in live-imaging analysis and in the model -1. Howevin vivo. As cells are tightly coupled via adherens junctions and execute asynchronous stochastic actomyosin contractions, cell constriction will be constrained by contraction activity of neighbouring cells. Consequently, we hypothesize that stagnations in area reduction could be explained through the counterbalancing effects of elastic forces within the cell and the contractile action of neighbouring cells.Based on these finding we assume that analogous mechanisms work during apical constriction in ventral furrow formation twi mutants with only a subset of ventral cells undergoing area reduction which is also considerably slowed down , the graded contractility in our model is a qualitative concept whose numerical parameters must be chosen to match the experimental data. Lower overall contractility, for instance, will only yield low constriction until force balance is reached, so only an inconspicuous furrow is formed . Possibly, Sna equips all cells within its expression domain  with a minute basic contractility . Line tension In order to model cell shape change during ventral furrow formation we set-up a variant of a commonly used vertex model (compare e.g.: Z) ( = 15), i the cell row  and j the cell column (antero-posterior coordinate) and  = 0.15). Time-dependence in the gradient model was achieved by using  = 25 to avoid unlimited increase of contractility. Finally, for the stochastic gradient model autonomous stochastic dynamics were implemented by adding Brownian motion to the contractility function viatwi PE::sna mutants, contractility in the formula above was modified via U being a uniform random number between 0.0 and 0.5, drawn independently for each cell.For the cutoff model, contractility (eps (eps = 0.1), they undergo a swapping process (“T1 transition”) by changing connections with adjacent vertices (in vivo. All data from a simulation run (including the stochastic contractility dynamics) are saved to disk so simulation runs can be identically replicated or replicated after directed manual modifications like in Each vertex in the model moves following the directive to minimize the energy in the sheet. To derive the equation of motion for each vertex, we assume balance between frictional force and potential force as described previously in vertices . Real-tiSpider:GFPsqh:mCherryspaghetti-squash (sqh) encoding myosin regulatory light chain); EY53twistg]P[snaPE::sna, this construct drives sna expression using a twi-independent enhancer element, so a twi loss-of-function will not be superimposed by additional partial loss of sna). rabbit anti-RhoGEF2 , mouse anti-Nrt , GAM-Cy5 , GAR-Cy3 .1∶10000, , rabbit For antibody staining and cross-section imaging, embryos were heat-fixed using standard procedures and manually cut with a needle in AquaPolymount (Polysciences). For live-imaging, dechorionated embryos were glued ventral side up onto a slide and covered in water-saturated 3 S Voltalef oil. Image series were recorded on a Leica SP2 IRBE at 8 or 12 sec./frame starting from the onset of gastrulation (cellularization front has reached the yolk). Scanning depth was 2 µm below the apical surface. Automated image analysis (segmentation and cell tracking) was performed like in Figure S1Variations of the gradient model. A: Stochastic gradient model with standard parameters. B: Stochastic gradient model with reduced overall contractility . This way, the cell’s contractility will increase the further it approaches the midline. The model shows similar performance as the stochastic gradient model with cells gaining eccentricity and undergoing constriction with or without stagnation periods, but with a slightly wider furrow.(TIF)Click here for additional data file.Movie S1Ventral Furrow Formation. Confocal live-recording of the ventral epithelium of a Drosophila embryo expressing Spider:GFP to mark cell membranes. Imaging plane 2 µm below the apical surface. Movie accelerated 120×, spanning 10 minutes real-time.(MOV)Click here for additional data file.Movie S2Cutoff Model. Model of ventral furrow formation using a cutoff contractility function and standard parameters ((MOV)Click here for additional data file.Movie S3Gradient Model. Model of ventral furrow formation using a gradient contractility function and standard parameters ((MOV)Click here for additional data file.Movie S4Time-dependent Gradient Model. Model of ventral furrow formation using a time-dependent gradient contractility function (without stochastic fluctuation) and standard parameters ((MOV)Click here for additional data file.Movie S5Stochastic Gradient Model. Model of ventral furrow formation using a time-dependent gradient contractility function with stochastic fluctuations ((MOV)Click here for additional data file.Movie S6Stochastic Gradient Model, with contractility coded by colour. Same model as in (MOV)Click here for additional data file.Movie S7Phenotype of twi mutants. Confocal live-recording of the ventral epithelium of a twi mutant embryo (twi PE::sna) expressing Spider:GFP to mark cell membranes. Imaging plane 2 µm below the apical surface. Movie accelerated 120×, spanning 20 minutes real-time.(MOV)Click here for additional data file.Movie S8twi Model. Model of ventral furrow formation in twi mutants using random reduction of contractility across the cell sheet and standard parameters otherwise (see wise see .(MOV)Click here for additional data file.Movie S9Modified twi Model. Model of ventral furrow formation in twi mutants using random reduction of contractility across the cell sheet and a widened contractility gradient ((MOV)Click here for additional data file.


IR increased ATC intra-cellular ROS levels resulting in a corresponding decrease in reducing equivalent levels. Exogenous manipulation of cellular ROS and reducing equivalent levels altered ATC radiosensitivity in a predictable manner. Irradiation of ATC xenografts resulted in an acute drop in reducing potential measured using HP-MRS, reflecting the shunting of reducing equivalents towards ROS neutralization. Residual tumor tissue post irradiation demonstrated heterogeneous viability. We have adapted HP-MRS/MRSI to non-invasively measure IR mediated changes in tumor reducing potential in real time. Continued development of this technology could facilitate the development of an adaptive clinical algorithm based on real-time adjustments in IR dose and dose mapping.Ionizing radiation (IR) cytotoxicity is primarily mediated through reactive oxygen species (ROS). Since tumor cells neutralize ROS by utilizing reducing equivalents, we hypothesized that measurements of reducing potential using real-time hyperpolarized (HP) magnetic resonance spectroscopy (MRS) and spectroscopic imaging (MRSI) can serve as a surrogate marker of IR induced ROS. This hypothesis was tested in a pre-clinical model of anaplastic thyroid carcinoma (ATC), an aggressive head and neck malignancy. Human ATC cell lines were utilized to test IR effects on ROS and reducing potential  Therefore, total dose, dose fractionation and dose painting decisions are largely based on empirical data derived from previous prospective and retrospective trials Ionizing radiation (IR) is commonly employed in the treatment of most solid head and neck tumors as definitive therapy or in the adjuvant setting Over the last 20 years, multiple investigators have demonstrated that generation of ROS plays a crucial role in radiation cytotoxicity by inducing DNA damage and triggering tumor cell death 13C]-pyruvate allow measurement of in vivo processes that were previously impossible Recent advances in hyperpolarized MR tracers such as  pyruvic acid was prepared to a final solution containing 80 mM pyruvate with a nominal pH of 7.6 and temperature of 37°C as previously described 13C-Pyruvate, 40 mM Lactate, and 3.92 U/mL Lactate Dehydrogenase  in a Tris buffer  . Conversion of HP pyruvate to lactate was measured in 3 separate scans using 0.0, 1.0 and 4.0 mM NADH .Hyperpolarized . Ph13C labeled pyruvate into 13C lactate 13C pyruvate to 13C lactate reaction on reducing potential is further demonstrated using a synthetic phantom, consisting of substrate (13C pyruvate), enzyme and NADH. As shown in We have previously demonstrated that it is possible to indirectly measure tumor reducing potential using HP-MRS by measuring the rate of conversion of exogenous in vitro data demonstrate a reciprocal relationship between tumor cell reducing potential and tumor ROS levels, we hypothesized that exposure to IR will result in a decrease in tumor reducing potential, which could be measured in decreased conversion of exogenous pyruvate into lactate . It is important to note that our experimental design controls for several experimental pitfalls. First, by normalizing post-IR measurements to the pre-IR measurements we account for inter-animal variability. Second, by using repeated imaging in sham irradiated animals, we account for the potential physiologic effects of the imaging tracer described above. Using this dual control, we were able to repeatedly demonstrate a measurable effect of IR on tumor HP-MRS signal. Because repeated measurements were performed within 2 hours of each other, tumor growth, or changes in the rate of growth following irradiation are not likely to be a significant contributor to the changes measured in this experiment.To determine whether these effects can be measured urements . We suspControl (n = 3) and irradiated tumors (n = 3) were also imaged at 2 weeks post treatment in order to evaluate chronic effects of IR on ATC tumor reducing potential. Anatomic measurements were made using T2-weighted MRI images . ControlUsing previously established imaging sequences, we measured the relative levels of HP pyruvate and lactate in a representative slice through the large control ATC tumor in vitro and in vivo approaches.One clinically relevant aspect of the data contained in in vitro and animal model data. The ability to adjust total dose and dose painting based on real-time, tumor-specific information would represent a paradigm shift in clinical practice and potentially lead to improved clinical effectiveness.Successful locoregional control of aggressive solid tumors, such as ATC, requires improved ability to monitor radiation effects in real time in order to adjust total dose, dose painting and timing of radiosensitizing agents. Currently, design and implementation of clinical IR protocols are largely empirically driven processes, guided in part by extrapolation of IR cytotoxicity is largely a function of ROS generation in vitro conditions. Based on these data, we hypothesized that IR induced ROS increases would result in reciprocal decreases in tumor reducing potential which could be monitored in real time using HP- MRS measurements of pyruvate to lactate conversion. We first demonstrated a rapid, transient, dose dependent decrease in lactate production measured biochemically under in vitro conditions. The transient nature of this change is completely consistent with the transient reciprocal perturbations we had previously measured in ROS and reducing potential in ATC cells, and served as additional confirmation that HP-MRS using labeled pyruvate should be able to detect perturbations in tumor reducing potential immediately following irradiation.We and others have demonstrated an inverse relationship between tumor reducing potential and ROS levels Data from two recent studies indicate that HP-MRS can be used to ascertain IR effects on tumor cell death and surrounding tissue toxicity HP-MRS of irradiated tumors demonstrated a second important phenomenon. Although IR exposed ATC tumors exhibited significantly slower growth rates, growth was not completely arrested. Despite smaller tumor volume, irradiated ATC tumors exhibited high reducing potential. This may be a result of two interacting biological phenomena: 1) selection of tumor cells which exhibit increased reducing potential and 2) increasing metabolic heterogeneity associated with large solid tumor volumes . It is possible that residual tumor tissue following irradiation exhibits a metabolic phenotype required to survive the initial cytotoxic event. This would be consistent with data presented by Albers et al., which demonstrated that increased conversion corresponded to more aggressive solid tumor histology in vitro with exquisite detail, in vivo studies have been limited by an inability to monitor acute IR effects on tumors in real-time. For the first time, we provide evidence that such measurements are in fact possible, by monitoring changes in tumor reducing potential using HP-MRS/MRSI. This is the first study to demonstrate acute perturbations in tumor reducing potential following irradiation using a non-invasive means suitable for clinical translation predictably monitor IR effects in real time. Overcoming these challenges will require the continued development of acquisition, reconstruction, and signal analysis strategies in order to more precisely characterize tumor metabolic changes.Improved IR effectiveness in controlling the growth of ATC and other aggressive solid tumors is an important clinical goal. Although investigators have been able to characterize IR effects


CTEN/TNS4 is an oncogene in colorectal cancer (CRC) which enhances cell motility although the mechanism of Cten regulation is unknown. We found an association between high Cten expression and KRAS/BRAF mutation in a series of CRC cell lines (p = 0.03) and hypothesised that Kras may regulate Cten. To test this, Kras was knocked-down RNA) in CRC cell lines SW620 and DLD1 (high Cten expressors and mutant for KRAS). In each cell line, Kras knockdown was mirrored by down-regulation of Cten Since Kras signals through Braf, we tested the effect of Kras knockdown in CRC cell line Colo205 (which shows high Cten expression and is mutant for BRAF but wild type for KRAS). Cten levels were unaffected by Kras knockdown whilst Braf knockdown resulted in reduced Cten expression suggesting that Kras signals via Braf to regulate Cten. Quantification of Cten mRNA and protein analysis following proteasome inhibition suggested that regulation was of Cten transcription. Kras knockdown inhibited cell motility. To test whether this could be mediated through Cten, SW620 cells were co-transfected with Kras specific siRNAs and a Cten expression vector. Restoring Cten expression was able to restore cell motility despite Kras knockdown . Since KRAS is mutated in many cancers, we investigated whether this relationship could be demonstrated in other tumour models. The experiments were repeated in the pancreatic cancer cell lines Colo357 & PSN-1(both high Cten expressors and mutant for KRAS). In both cell lines, Kras was shown to regulate Cten and forced expression of Cten was able to rescue loss of cell motility following Kras knockdown in PSN-1 . We conclude that, in the colon and pancreas, Cten is a downstream target of Kras and may be a mechanism through which Kras regulates of cell motility. Cten, TNS4) is a member of the Tensin gene family. This gene family comprises four members  and their products are localised to the cytoplasmic tails of integrins at focal adhesions. Tensins play an important role in various biological processes such as cell adhesion, migration, proliferation, differentiation, apoptosis and invasion C-terminal tensin-like , phosphatidylinositol-3-kinase (PI3-K), and Crk-associated substrate p130KRAS. All cell lines were cultured in Dulbecco's modified Eagle's medium  supplemented with 10% fetal calf serum (Invitrogen) and 1% penicillin/streptomycin (Invitrogen) in 5% CO2 in a humidified atmosphere.The CRC cell lines used in this study  were kindly donated by Prof I Tomlinson and have been previously described sequence scrambled control (ssc) duplexes (i.e. duplexes with the same base composition arranged in a random order). Two different duplexes were used for Kras knockdown and the sequences of the gene-specific duplexes and scrambled controls are as follows: (1) Kras specific 5′CAGGGUGUUGAUGAUGCCUUCUAUA′3 and scrambled control 5′CAGUGUAGUAGUCGUUUCUCGGAUA′3 (2) Kras specific 5′UAUAGAAGGCAUCAUCAACACCCUG′3 and scrambled control 5′UAUCCGAGAAACGACUACUACACUG′3.In order to knock down Cten, Braf and Kras, cells were transfected with small interfering RNA (siRNA) duplexes using Lipofectamine 2000 (Invitrogen) as previously described In order to induce Cten expression, cells were transfected with a CMV promoter driven expression construct containing Green Fluorescent Protein (GFP) tagged Cten (GFP-Cten) using Lipofectamine 2000 (Invitrogen) as previously described In order to functionally evaluate the functional relationship between Kras and Cten, a specific cellular condition was created whereby Kras protein was depleted and Cten protein was restored. This was achieved by co-transfection of Kras-specific siRNA and GFP-Cten and, for this purpose, the protocol for siRNA transfection was used with appropriate amounts of plasmid mixed in with the siRNA prior to adding to Lipofectamine 2000. Cells were analysed 72 hours later by Western blot or functional assays.In order to ascertain the role of proteasomal degradation in the Kras mediated regulation of Cten, the proteasomal inhibitor Z-Leu-Leu-Phe-CHO, , was added to SW620 cells 24 hours after transfection with siRNA duplexes (either Kras specific or scrambled control).Total RNA was extracted from cells using the RNeasy Mini Kit (Qiagen) following the manufacturer's protocols and quantified on a NanoDrop ND-1000 UV-Vis Spectrophotometer  and 250 nM primers. PCR was performed on an MX3005P Real-time PCR machine  and cycling conditions were 5 minutes denaturation at 95°C followed by 40 cycles of: 30 seconds denaturation at 95°C/30 seconds annealing [60°C for Cten/59°C for HPRT/50°C for Kras/30 seconds extension at 72°C and a single final extension for 10 minutes. The data for Q-PCR were analyzed using the MxPro-QPCR software for Mx3005P QPCR system. PCR primer sequences are available from the authors.Quantification of Cten and Kras was performed using the standard curve method. All experiments were conducted in triplicate and test gene values were normalized to the housekeeping gene Whole cell extracts were prepared using lysis buffer ). 30 µg protein was loaded on a 10% SDS–PAGE gel and transferred onto PVDF membranes by semi-dry transfer. After blocking, membranes were incubated overnight at room temperature with the indicated primary antibody. Antibodies were used at the following dilution: anti-Cten , anti-E-Cadherin , mouse anti-Kras , mouse anti-β-actin  and mouse anti-CD24 . After three washes in TBS/Tween-20 (0.05%), blots were incubated for 1 hour at room temperature with the appropriate horseradish peroxidase-linked secondary antibody. After three further washes, detection was performed using the Enhanced Chemiluminescence Kit (Pierce). Bands were visualised using X-Ray films (Kodak) and quantified using ImageJ software.In order to test the transfection efficiency of GFP-Cten into SW620, cells were initially transfected with GFP-CTEN as above. After 48 hours, the cells were non-enzymatically detached by incubating with TE buffer (10 mM Tris-HCl (pH 8), 1 mM EDTA, Sigma) for 5–10 minutes. These then underwent anlaysis in an Epics Altra Flow cytometer (Beckman Coulter). 600,000 events were analysed using a GFP filter with the appropriate gate parameter.5 cells were incubated with PE-labelled anti-CD24 antibody (BD Biosciences) diluted 1∶100 in FACS wash  for 15 minutes at 4°C. An isotype and concentration matched PE labelled control antibody  was used and samples labelled with this antibody were used to set the gating levels. After three 5 minute washes with FACS wash, the cells were re-suspended and fixed in solution containing FACS wash with 2% formaldehyde. Determination of percentage of CD24+ cells was performed on an Epics Altra flow cytometry machine (Beckman Coulter). The results were analyzed using WinMDi 2.9 computer software.Confirmation of the efficacy of the DN-TCF expression vector was tested by the effect on CD24 expression (a well known Wnt target gene) 4 cells were seeded and cell migration was assessed after 48 hr by fixing the cells attached to the lower surface in 10% methanol for 30 minutes. These were then stained for 30 minutes with methylene blue and the stained cells manually counted. Cell invasion was measured in the same way as described for migration, except that prior to cell seeding the upper chamber was prepared by coating the filter with 100 µl Matrigel  and the cells attached to the lower surface were fixed, stained and counted. Cell wounding migration assays were performed in six-well plates. Cells were grown to confluence and then serum-deprived for 24 hrs. A sterile 200 µl pipette tip was used to create three separate parallel wounds and migration of the cells across the wound line was assessed at 0, 24, 48 and 72 hr. Photographs were taken using a charge-coupled device (CCD) camera  attached to the inverted phase-contrast microscope at a power of 40×. In order to statistically analyse cell migration in the wounding assay, images were taken from three separate wounds All images were taken at the same magnification and converted into binary images using the ImageJ image analysis program (http://rsb.info.nih.gov/ij/index.html) and the area of the wound was measured. All assays were performed in triplicate on at least two separate occasions.Transwell cell migration was measured using a Boyden chamber containing a polycarbonate filter with an 8 µm pore size (Costar). Five ×10Statistical analysis was undertaken using SPSS 13. 0 Software. All evaluations were done using unpaired two tailed Student's T-test. For cell counting studies cell numbers (as quantified by the methylene blue assay) were analysed. For the cell wounding studies, total pixel numbers with an intensity value of 0 were analysed as previously described We have previously shown that forced expression of Cten induces cell motility in cell lines not expressing Cten KRAS/BRAF mutation in a series of CRC cell lines (KRAS mutation and is a high expressor of Cten. Knockdown of Kras in SW620 (annotated as SW620Kras-) resulted in down-regulation of Cten (ssc). This effect was validated in the cell line DLD1 which also contains a KRAS mutation and is a high expressor of Cten (DLD1Kras- versus DLD1ssc) both of which produced Kras knockdown and both of which resulted in down-regulation of Cten.Having found an association between Cten expression and ll lines , we soug of Cten  when comBRAF but wild type for KRAS). In this case the level of Cten was unaffected  and compared with cells co-transfected with Kras specific siRNA and GFP expressing empty vector controls (SW620Kras-/evc). Experimental controls were cells co-transfected with scrambled siRNA duplexes and GFP empty vector (SW620ssc/evc) creating a condition in which neither Kras nor Cten were altered  tagged Cten . However, ectopic expression of GFP-Cten restored the cell motility . Cell wounding assays using the same transfection protocol demonstrated the same effect and validated the data , Kras was knocked and Cten restored (PSN1Kras-/GFP-Cten) and neither was altered (PSN1ssc/evc). The data paralleled those in the CRC cell lines and Kras knockdown was demonstrated to inhibit cell motility  whilst this could be rescued by restoration of Cten expression  repressed cell motility and caused up-regulation of E-cadherin thereby validating our previous data and firmly establishing the role of Cten in regulating cell motility.KRAS/BRAF mutation in CRC cell lines. We have demonstrated Cten is a true target of Kras signalling since (i) knockdown of Kras results in down-regulation of Cten in two cell lines which are mutant for KRAS and (ii) knockdown of Kras in a cell line mutant for BRAF has no effect on Cten expression whilst knockdown of Braf in this cell line does result in down-regulation of Cten. Furthermore, quantification of Cten mRNA and use of proteasomal inhibitors to prevent protein degradation suggested that the level of control lay at Cten transcription.Little is known of the mechanism of Cten regulation and, in this study, we have found an association between high Cten expression and Taking cognizance of the fact that there are a large number of reported targets of Kras and it is unlikely that they will all be functionally relevant KRAS which show low levels of Cten. Conversely, there are cell lines which are wild type for KRAS/BRAF but which have high levels of Cten. Recently Stat3 has been reported as a modulator of Cten expression KRAS/BRAF mutation, the association is likely to be causal.This is the first report of Cten as a target of Kras signalling although it is clear that there are other mechanisms controlling Cten expression since we have identified occasional cell lines mutant for Our data suggest that a Kras-Cten signalling pathway exists which regulates cell motility. Taken together with studies in breast cancer showing that EGFR signalling can regulate Cten expression, a wider EGFR-Kras-Cten signalling pathway can be inferred. Circumstantial support for this comes from studies showing high levels of Cten expression in lung cancer KRAS/BRAF mutation are refractory to the therapeutic effects of anti-EGFR antibodies. If future studies confirm that Cten is a part of the EGFR-Kras signalling pathway, it may represent a new therapeutic option for the significant number of CRCs (approximately 60%) containing KRAS/BRAF mutation and therefore ineligible for anti-EGFR therapy.As well as raising some fascinating questions about the role of Cten in regulating cell motility, our data also have some therapeutic implications. The use of biologics targeted to the EGFR can have a dramatic effect on CRC resulting in marked reduction in tumour size and clinical down-staging Figure S1DN-TCF4) or empty vector (SW620evc). Gating levels were ascertained using an isotype PE-labelled control antibody and it was shown that DN-TCF4 resulted in a 53% reduction in cells expressing CD24.Validation of the functional activity of DN-TCF4 was shown by flow cytometry. The expression of cell surface CD24 was tested following transfection into SW620 of either an expression construct expressing DN-TCF4 (SW620(TIF)Click here for additional data file.Figure S2To confirm that the proteasomal inhibitor (PI) was effective, RKO cells were incubated either with the inhibitor or with DMSO and levels of β-catenin were quantified. Exposure to the proteasomal inhibitor resulted in inhibition of β-catenin degradation making it detectable by Western blotting.(TIF)Click here for additional data file.Figure S3High efficiency of transfection of GFP-Cten into SW620 was confirmed by flow cytometry. To ensure that exposure to lipofectamine (the transfection reagent) did not influence fluorescence, control cells were transfected with a scrambled siRNA control. In comparison with control cells, there was approximately 60% transfection efficiency.(TIF)Click here for additional data file.Table S1CTEN message expression compared with the mean value for normal mucosa . The column alongside indicates whether there is a mutation in the hotspots of KRAS or BRAF.This is a table of the cell lines showing fold change in (DOC)Click here for additional data file.


Epigenetic control is essential for maintenance of tissue hierarchy and correct differentiation. In cancer, this hierarchical structure is altered and epigenetic control deregulated, but the relationship between these two phenomena is still unclear. CD133 is a marker for adult stem cells in various tissues and tumour types. Stem cell specificity is maintained by tight regulation of CD133 expression at both transcriptional and post-translational levels. In this study we investigated the role of epigenetic regulation of CD133 in epithelial differentiation and cancer.DNA methylation analysis of the CD133 promoter was done by pyrosequencing and methylation specific PCR; qRT-PCR was used to measure CD133 expression and chromatin structure was determined by ChIP. Cells were treated with DNA demethylating agents and HDAC inhibitors. All the experiments were carried out in both cell lines and primary samples.+/α2β1integrinhi prostate (stem) cells, when compared to CD133-/α2β1integrinhi (transit amplifying) cells or CD133-/α2β1integrinlow  cells, selected from primary epithelial cultures. Condensed chromatin was associated with CD133 downregulation in all of the cell lines, and treatment with HDAC inhibitors resulted in CD133 re-expression in both cell lines and primary samples.We found that CD133 expression is repressed by DNA methylation in the majority of prostate epithelial cell lines examined, where the promoter is heavily CpG hypermethylated, whereas in primary prostate cancer and benign prostatic hyperplasia, low levels of DNA methylation, accompanied by low levels of mRNA, were found. Moreover, differential methylation of CD133 was absent from both benign or malignant CD133CD133 is tightly regulated by DNA methylation only in cell lines, where promoter methylation and gene expression inversely correlate. This highlights the crucial choice of cell model systems when studying epigenetic control in cancer biology and stem cell biology. Significantly, in both benign and malignant prostate primary tissues, regulation of CD133 is independent of DNA methylation, but is under the dynamic control of chromatin condensation. This indicates that CD133 expression is not altered in prostate cancer and it is consistent with an important role for CD133 in the maintenance of the hierarchical cell differentiation patterns in cancer. Epigenetic regulation of gene expression is a dynamic mechanism, which permits precise regulation throughout differentiation . It playThere is mounting evidence to support the hypothesis that cancers can retain the hierarchical structure present in normal tissues, but that homeostasis is disrupted, leading to aberrant replication and differentiation. As in normal tissues, a small percentage of cancer cells can persist and initiate new tumour growth (tumour-initiating cells or cancer stem cells - CSCs), while most cells proceed to terminal differentiation .2β1integrin are two markers that, when used in combination, have been demonstrated to enrich for a cell population with stem cell features [+ cells from human primary tissues represent a very small subpopulation  located in the basal compartment. Tissue stem cells are restricted to the α2β1integrinhi population, have a high colony-forming ability and proliferative potential, and are able to regenerate fully differentiated prostate epithelium in vivo. The stem cells express basal cell markers, such as CD44 and CK5/14, but do not express luminal markers AR, PSA or depend on androgens for their survival [CD133 is a pentaspan membrane glycoprotein first identified in humans as a hematopoietic stem cell marker  and is cfeatures . Indeed,survival -11. Othesurvival  and CD11+ cells from prostate cancer biopsies (PCSCs) are similar in phenotype to normal prostate stem cells. They are rare  and represent the clonogenic population with highest proliferative potential. Moreover, they express basal cell cytokeratins, but do not express AR [CD133press AR .Disruption of epigenetic mechanisms is found in all cancers and, together with genetic changes, plays a key role in cancer initiation and progression . EpigeneHowever, these studies do not take into account the hierarchical structure of cancer, since they describe epigenetic alterations that occur in the bulk population of cancer cells. It has been proposed that disruption of epigenetic control may result in formation of aberrant self-renewing cells , culminaCD133 expression is controlled at multiple steps including transcriptional regulation, alternative transcription initiation sites, alternative splicing and post-translational modifications ,19. ThisHere, we show that CD133 expression is efficiently repressed by promoter methylation in prostate cell lines. However, in primary cultured cells from prostate epithelium and tumour xenografts, regulation of CD133 expression is independent of DNA methylation, indicating that, for this gene, cell lines are not indicative of DNA methylation status in primary tissues. In addition, the CD133 promoter is not hypermethylated in prostate cancer tissues, highlighting the important role for CD133 in the maintenance of the hierarchical structure of cancer.CD133 promoter methylation was quantified by pyrosequencing in prostate cell lines Figure . Using tTo assess whether the levels of methylation were consistent along the entire CpG island, PYRO 2 and 3 assays were carried out, and both showed comparable patterns of methylation with the PYRO 1 assay . CD133 expression (measured by qRT-PCR) was induced in 2 out of 4 benign cell lines (BPH-1 and PNT1A) and all of the CaP cell lines analysed  with the exception of VCaP Figure . The lacNext, 3 cell lines representative of the panel used were treated with 1 μM 5-Aza-2'-deoxycytidine for 96 h and analysed by FACs for the expression of the glycosylated form of CD133 Figure  and 2C. Taken together, these results show that DNA demethylation induces CD133 upregulation, indicating that promoter methylation suppresses CD133 expression in prostate cell lines.A direct comparison between CD133 expression, measured by qRT-PCR, and DNA methylation (PYRO 1 assay), confirmed that hypermethylation of the CD133 promoter results in downregulation of gene expression Figure . Cell li+ cells and had detectable levels of mRNA. In contrast, CD133+ cells were not found in PC3 and DU145 cells, although detectable levels of mRNA were found; while in LnCaP and VCaP cells, CD133 was not detectable at either the mRNA or protein level. Finally, in SerBob cells, although CD133 was undetectable at the mRNA level, we detected a very small subpopulation of cells expressing very low levels of CD133 protein.Moreover, mRNA abundance and expression of the glycosylated isoform of CD133 (detected by the 293C3 antibody) directly correlated in prostate cell lines , TA (α2β1hi/CD133-) and CB (α2β1low/CD133-) cell populations isolated from low passage (<10) primary prostate epithelial cultures (1 BPH and 2 CaP)  Figure . In BPH We then wanted to analyse whether this differential regulation was sustained by DNA methylation. Pyrosequencing  or inactive (trimethylation of lysine 27 of histone H3 - H3K27me3) chromatin around the CD133 promoter was determined by chromatin immunoprecipitation (ChIP) in RC-165N/hTERT, PNT2-C2, P4E6 and PC3 cells Figure . H3K4 diP4E6 cells showed a very similar expression and DNA methylation pattern to primary epithelial cultures and primary tissues, containing low levels of CD133 mRNA and promoter methylation. In this cell line CD133 was maintained in a repressed state by a highly condensed chromatin structure. In line with these results, CD133 mRNA was highly overexpressed after treatment of P4E6 cells with trichostatin A  Figure , a well Treatment of BPH and CaP derived primary epithelial cultures with 0.6 μM TSA and 10 mM NaBu (a less potent and specific HDAC inhibitor) also resulted in overexpression of CD133 mRNA Figure  after 48CD133 is widely used as a marker for CSCs in many different solid tumours including: colon ,25, braiThe data presented here indicated that DNA methylation mediated suppression of CD133 expression in prostate epithelial cell lines, where an inverse correlation between expression and DNA methylation was clearly seen, together with re-expression of both the mRNA and the glycosylated protein after gene demethylation by 5-Aza-2'-deoxycytidine treatment.However, data obtained from prostate tissue and primary epithelial cultures displayed a stark contrast to that obtained in cell lines. A huge variation  in CD133 expression was found across the cell lines analysed, while CD133 was undetectable or expressed at very low levels in prostate primary epithelial cultures. These results provide strong evidence that regulation of CD133 expression can be disrupted during long-term culture in vitro.Moreover, the low levels of CD133 mRNA detected in primary epithelial cultures suggested that the CD133 gene was repressed in the vast majority of prostate basal epithelial cells (with the exception of the stem cell population). The very low levels of promoter methylation found in these samples indicated that repression of CD133 expression was independent of promoter methylation and implied that other mechanisms were required to control CD133 expression in prostate tissues and primary cultures. Interestingly, CD133 expression was also not deregulated in primary prostate cancer, suggesting that a tight regulation of CD133 expression was important in the hierarchical structure of both normal and cancerous prostate.+), TA and CB (CD133-) cell populations from primary epithelial cultures were analysed separately, the results obtained showed no differential methylation of the CD133 promoter in individual populations. Therefore mechanisms other than DNA methylation must regulate CD133 expression in the prostate epithelial cell hierarchy.Prostate epithelium is heterogeneous and composed of various cell populations at different differentiation stages. When SC . This is in line with the concept of crosstalk between different epigenetic mechanisms  and withIn prostate tissues, the glyscosylated form of CD133 was expressed only in a very small subpopulation of basal epithelial cells ,35. HoweAlthough CD133 is widely used as a stem cell marker in various types of cancer, very little is known about its molecular function and its functional involvement in tumour and metastasis formation. Even if there is evidence that CD133- positive cells from various cancers are more resistant to anti-cancer therapies -38, it ide novo methylation is a common event during cell line establishment [The results obtained show two different mechanisms for regulation of CD133 expression in cell lines (DNA methylation dependent) and primary tissues and recently established prostate cancer xenografts (DNA methylation independent). Although discrepancies between cell lines and primary samples have been discussed in the literature for at least 20 years , cell lilishment ,43 as palishment . The resin vitro adaptation.Thus, the choice of the correct cell model system is of paramount importance when studying epigenetic regulation of gene expression in cancer biology and stem cell biology. The results with CD133 highlight the need for verification (using primary tissues) of the results obtained with cell lines. For example, in the last few years, several high-throughput epigenetic studies compared commercially available normal epithelial cultures  with established cancer cell lines in prostate ,45 and oin vitro culture affects the fine gene regulation that is essential for the maintenance of prostate epithelial hierarchy. Moreover, the data presented by Pfeiffer and Schalken (2010) [Finally, in recent studies designed to isolate cells with stem cell features from prostate cancer cell lines a great discordance was reported regarding the expression of CD133 on the surface of several CaP cell lines. The data presented here, reveal the limitations of the use of cell lines in such studies, and indicate that prolonged n (2010) , is in aWe present here a comprehensive study of the epigenetic regulation of CD133 promoter in cell lines, primary epithelial cultures, tissue and tumour xenografts from the human prostate. We conclude that CD133 expression is regulated by different mechanisms in cell lines relative to the other samples, and that regulation in primary cultures is independent of methylation, where this gene is maintained in a repressed state by condensed chromatin structure. These results also have implications in the choice of models that are chosen to analyse epigenetic changes in cancer cells, and highlight the complexity of regulation of this common stem cell marker.A list of the cell lines used, origin, culture conditions and identification is provided in Additional File Human prostatic tissue was obtained from patients undergoing transurethral and retropubic prostatectomy for BPH or undergoing radical prostatectomy for CaP . BPH or CaP diagnosis was confirmed by histological examination of representative adjacent fragments.+ cells were selected from cells that adhered within 20 minutes using MACS microbeads linked to anti-human CD133, according to the manufacturer's instruction (Miltenyi Biotec) [Tissues were disaggregated and cultured as described previously ,10. Basa Biotec) .-/- gamma C-/- mice. Tumours generated were serially passaged in vivo and routinely genotyped to confirm the original patient's genotype. Early passages (1-3) were used for DNA methylation studies.Xenografts were generated by subcutaneous grafting of CaP tissue in RAG2PC3 xenografts were generated by injecting subcutaneously 106 PC3 cells embedded in 100 μl of Matrigel (BD Biosciences) in Balb/c Nude mice. Tumours were harvested after 29 days from the injection.DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen) and the QIAamp DNA micro Kit (Qiagen) for small samples. 0% and 100% methylated controls were purchased from Qiagen (EpiTect PCR Control DNA). 50 ng/1 μg of DNA was bisulfite converted using the EpiTect Bisulfite Kit (Qiagen). Converted DNA from SCs was amplified using the EpiTect Whole Bisulfitome Amplification Kit (Qiagen).The CD133 promoter sequences were amplified by PCR using specific primers for 3 regions of the CpG island  and reverse transcribed using random hexamers and reverse transcriptase . Real time PCR was carried out using either Power mix SYBR Green and specific primers -APC antibody (1:11) (Miltenyi Biotec) and analysed on a CyAn ADP flow cytometer . Doublet cells were gated out with pulse width. Dead cells were gated out by DAPI exclusion. More than 250,000 events were analysed.ChIP assays were performed as previously described . The antThe authors declare that they have no competing interests.DP: Conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing. RJP: Collection and assembly of data, data analysis and interpretation. FMF: Data analysis and interpretation, manuscript writing, final approval of the manuscript. EEO, PAB, MCL, MJS and MSS: Provision of study material or patient samples, collection and assembly of data. ATC and NJM: Conception and design, manuscript writing, final approval of the manuscript. All authors read and approved the final manuscript.Additional analysis of CD133 promoter methylation in prostate cell lines. Figure S1: Methylation specific PCR (MSP) analysis of the CD133 promoter with two different primer sets (MSP1 and MSP2) in a panel of prostate cell lines (A). PCR products recognizing methylated (M) and unmethylated (U) CpG sites were analyzed on 2% agarose gels . Pyrosequencing methylation assay of CD133 CpG island in a panel of prostate cell lines -APC antibody (Miltenyi Biotec) (CD133-APC) or without any antibody (No Antibody) and analysed by FACs. For each dot plot, X axes: FITC channel fluorescence (not stained); Y axes: APC channel fluorescence (CD133 or No Antibody control); Percent of CD133+ cells is indicated.Click here for fileAdditional analysis of CD133 promoter methylation in prostate primary epithelial cultures and prostate tissues. Figure S3: MSP analysis of the CD133 promoter with two different primer sets (MSP1 and MSP2) in prostate primary epithelial cultures (A) and in selected populations from primary epithelial cultures (B) . Pyrosequencing methylation assay of CD133 promoter in prostate tissues  and in prostate primary epithelial cultures  Diagram depicting a typical histogram generated by pyrosequencing methylation analysis of CD133 promoter with PYRO 1 assay and the position of each CpG site on the genomic sequence (Exon 1 A is shown in yellow). (B-C) Example of typical pyrograms generated with PYRO 1 assay using human (B) or mouse (C) bisulfite converted DNA as a template for PCR. No PCR product or sequence was generated with mouse DNAClick here for file


We describe the genetic diversity of sapovirus (SaV) in children in Nicaragua and investigate the role of host genetic factors and susceptibility to SaV infections. Our results indicate that neither ABO blood group, Lewis phenotype, nor secretor status affects susceptibility to SaV infection in Nicaragua. Caliciviridae, is a causative agent of gastroenteritis in children and adults. Symptoms of SaV infection seem to be milder than symptoms of rotavirus and norovirus (NoV) infections; thus, SaV infection prevalence is higher in nonhospitalized children than in hospitalized children.Human sapovirus (SaV), family FUT2), which gives rise to the nonsecretor phenotype, has been found to provide almost complete protection from experimental and natural NoV infection  expressed on cells in the gastrointestinal tract  were randomly selected among 694 symptomatic and 158 asymptomatic children who participated in a community- and hospital-based study of sporadic acute diarrhea in León, Nicaragua, during 2005–2006. In brief, clinical cases were evaluated according to the World Health Organization strategy for diarrhea management , according to manufacturer’s instructions. RNA underwent reverse transcription (RT) to produce cDNA by using 50 pmol of random hexadeoxynucleotides (pd[N]6)  and RT-PCR beads (Amersham Biosciences), according to manufacturer´s instructions.The RT products were amplified by SaV PCR using forward JV33 and reverse SR80 primers  , or a nonsecretor (se428se428).A single nucleotide polymorphism at position 428 in the 2 or Fisher exact tests. GraphPad  was used for statistical analysis.SaV-positive specimens were compared with specimens from a control group of children who tested negative for SaV, which was matched in terms of sex, age group, clinical status, and HBGAs using 2-tailed significance with χOf 292 samples analyzed, 33  (11%) were SaV positive. This study found that diarrhea is as common in SaV-infected children as it is in NoV-infected children in Nicaragua . Why theResults of a previous seroepidemiologic study of SaV infections suggested that SaV infections are frequent in children during the first 2 years of life because >90% of these children had antibodies against SaV .SaV infections were most frequently detected during June–July 2005, in the rainy period , and werTo investigate whether certain SaV genogroups  were associated with clinical severity, we examined 25 SaV-positive symptomatic and asymptomatic children. All human SaV genogroups were found in study participants as observed .FUT2 mutation were infected with NoV (428) and nonsecretors (se428se428 and Lea+b-) were susceptible to SaV infections. Our data suggest that SaV can infect secretors and nonsecretors, those who are Lewis phenotype positive as well as Lewis phenotype negative, and persons of all ABO blood groups. However, it must be noted that these data represent susceptibility to symptomatic GI SaV infection. To our knowledge, only 1 report in the literature has explored binding properties of recombinant SaV GI/1 (Mc114) and GV/1 (NK24) to HBGAs and revealed that the recombinant SaV strains investigated showed no specific binding activity to HBGAs from or to synthetic carbohydrates (We have previously investigated whether HBGA were susceptibility markers for NoV infections in children in Nicaragua and observed that secretors of blood type O were highly susceptible to infections with different NoV genotypes (with NoV . ChildreOur results demonstrate that SaV, similar to NoV, frequently causes acute gastroenteritis in children in Nicaragua (


Maximum-likelihood phylogenetic analysis of those sequences revealed that most (>70%) strains from east Africa segregated in a single regional-specific monophyletic group, here called CEA. A second major Ethiopian subtype C lineage and a large collection of minor Kenyan and Tanzanian subtype C clades of southern African origin were also detected. A Bayesian coalescent-based method was then used to reconstruct evolutionary parameters and migration pathways of the CEA African lineage. This analysis indicates that the CEA clade most probably originated in Burundi around the early 1960s, and later spread to Ethiopia, Kenya, Tanzania and Uganda, giving rise to major country-specific monophyletic sub-clusters between the early 1970s and early 1980s. The results presented here demonstrate that a substantial proportion of subtype C infections in east Africa resulted from dissemination of a single HIV local variant, probably originated in Burundi during the 1960s. Burundi was the most important hub of dissemination of that subtype C clade in east Africa, fueling the origin of new local epidemics in Ethiopia, Kenya, Tanzania and Uganda. Subtype C lineages of southern African origin have also been introduced in east Africa, but seem to have had a much more restricted spread.The HIV-1 subtype C accounts for an important fraction of HIV infections in east Africa, but little is known about the genetic characteristics and evolutionary history of this epidemic. Here we reconstruct the origin and spatiotemporal dynamics of the major HIV-1 subtype C clades circulating in east Africa. A large number ( Subtype C is the most prevalent variant, accounting for nearly half (48%) of all global infections Human immunodeficiency virus type 1 (HIV-1) sequences belonging to the pandemic group M are classified into nine subtypes , six sub-subtypes , and a variety of inter-subtype recombinant forms  Little is known about the genetic characteristics of HIV-1 subtype C strains circulating in east Africa. Previous studies showed that two genetically different subtype C strains designated C and C′, have been co-circulating in roughly similar prevalence and among the same risk groups and geographical areas in Ethiopia pol sequences sampled from the east , southern  and central (Angola and Democratic Republic of Congo) African regions over a time period of 25 years (1986–2010).To obtain a more comprehensive picture of the spatiotemporal dynamics of the HIV-1 subtype C epidemic in east Africa, we analyzed a large number of subtype C pol sequences from east, southern, and central African countries, that matched the selected genomic region (nt 2253–3272 relative to HXB2 clone) were retrieved from the Los Alamos HIV Database (http://hiv.lanl.gov). Countries were grouped in geographical regions according to the classification proposed by Hemelaar et alpol sequences sampled from 12 different African countries  ML phylogenetic trees were inferred under the GTR+I+Γet alPutative intrasubtype C/C′ recombinant sequences in Ethiopia were identified by Bootscanning using Simplot version 3.5.1 μ, units are nucleotide substitutions per site per year, subst./site/year), the age of the most recent common ancestor , and the spatial dynamics of major subtype C clades from east Africa were jointly estimated using the Bayesian Markov Chain Monte Carlo (MCMC) approach implemented in the BEAST software package v1.6.2 4 nucleotide substitution model, an uncorrelated Lognormal relaxed molecular clock model 8 generations and adequate chain mixing was checked by calculating the effective sample size (ESS) after excluding an initial 10% for each run using program TRACER v1.4 The evolutionary rate  downloaded from the Los Alamos HIV Database (http://hiv.lanl.gov) was used to characterize the relationship between subtype C sequences sampled from east, central and southern African countries. The transition/transversion vs divergence graphics showed that both type of nucleotide substitutions increase linearly with the genetic distance, with transitions being higher than transversions  monophyletic cluster, here called CEA, that contains sequences from all five east African countries analyzed (EA clade comprises a minor proportion (9%) of the 54 sequences from central Africa, but none of the 1,576 sequences from southern Africa here included. A minor fraction (11%) of subtype C sequences from east Africa branched in a second well supported (aLRT = 0.94) monophyletic cluster that comprises sequences from Ethiopia, and corresponds to the so called Ethiopian-C′ (C′ET) clade (n≤5 sequences) that were intermixed among strains from southern African countries  subtype C sequences from east Africa branched within a highly supported  clade . The remountries .EA. Subtype C strains from Ethiopia, by contrast, were mainly distributed into clades CEA (61%) and C′ET (37%). Finally, about 64% of subtype C sequences from Kenya and 49% from Tanzania branched within the major clade CEA, while the remaining sequences were distributed in the multiple minor clades of southern African origin. Such geographical variation in the prevalence of different subtype C clades could be also observed at a more local scale in Tanzania (EA clade. In the Kilimanjaro region (northeast), sequences from both the CEA and “southern African” clades reach a roughly similar prevalence. In the Mbeya region (southwest), only “southern African” clades were detected.The analysis of sequence distribution among clades by country of origin revealed three different patterns within east Africa represented by Burundi/Uganda, Ethiopia and Kenya/Tanzania . All or Tanzania . In the EA clade showed that sequences from Burundi occupies the most basal position in the clade , sequences with unexpectedly long branches in the phylogenetic analysis (n = 10), and Ethiopian sequences with evidence of intra-subtype recombination  were excluded from this analysis. This resulted in a final dataset of 236 sequences  sampled between 1990 and 2010.A closer inspection of the HIV-1 Che clade , thus suEA clade originated in Burundi (PP = 1) and was later exported to the other east African countries where it further spread, establishing new local epidemics (EA from Burundi to Ethiopia (n = 4), Kenya (n = 5), Tanzania (n = 8) and Uganda (n = 8) were detected, as well as from Tanzania to both Kenya (n = 3) and Uganda (n = 7). Importation of the CEA lineage into Burundi from other east African countries, and viral exchanges between Ethiopia, Kenya and Uganda were seldom detected in our dataset.The Bayesian MCC tree supports the hypothesis that the Cpidemics  and 4. Epidemics . SeveralEA lineage. Consistent with the ML topology (PP≥0.93) had a Burundian origin  and this observation should be interpreted with caution.The Bayesian analysis also supports an important phylogeographic subdivision within the Ctopology , most sun origin . The CETpol region of the CEA clade was 1.8×10−3 (95% highest posterior density [HPD]: 1.1×10−3–2.4×10−3) subst/site/year, similar to that previously estimated for HIV-1 subtype C lineages circulating in South America EA clade and supporting the use of a relaxed molecular clock model to reconstruct the time-scale of this lineage. According to this analysis the CEA clade started to spread in Burundi at 1962 (95% HPD: 1942–1975), while major sub-clades CET1/CET2, CKE, CTZ and CUG began to expand in Ethiopia, Kenya, Tanzania and Uganda, respectively, by the early 1970s (The median estimated evolutionary rate for the ly 1970s .ET and C′ET) was also estimated by combining all sequences from this country in a single dataset and incorporating the posterior distribution of the substitution rate previously estimated for the CEA lineage as an informative prior. This analysis resulted in a Bayesian MCC tree in which clades CET and C′ET were poorly supported (PP<0.5) and several strains branched outside those major clades (ET (n = 8) or C′ET (n = 10) and those strains that branched outside major Ethiopian clades (n = 2) are putative C/C′ intrasubtype recombinant viruses (ET and C′ET segregate in two highly supported (PP>0.9) reciprocally monophyletic groups  of sequences from central Africa, but no sequence from southern Africa, thus indicating that its circulation is mainly restricted to the east African region. Of note, the genealogies previously inferred for HIV-1 subtypes A and D also support a model of limited introduction of each subtype into east Africa, followed by a subsequent local expansion This study demonstrates a significant phylogeographic subdivision of HIV-1 subtype C strains circulating in the east respect to those circulating in the central and southern African regions, consistent with a recent study EA clade most probably originated in Burundi and after a period of local expansion, this viral lineage was disseminated at multiple times to Ethiopia, Kenya, Tanzania and Uganda, where it generated new local epidemics. Several introductions of the CEA lineage from Tanzania into both Kenya and Uganda were also detected, while viral exchanges between Ethiopia, Kenya and Uganda were less frequent. Five major country-specific monophyletic sub-clusters were detected within the CEA clade that comprise 44%, 33%, 37%, and 39% of all sequences from Ethiopia, Kenya, Uganda and Tanzania here included, respectively. Thus, despite frequent viral movement among east African countries, a significant proportion of subtype C infections in Ethiopia, Kenya, Tanzania and Uganda most likely resulted from the expansion of a few ancestral CEA strains.Our phylogeographic study suggests that the CEA lineage in southern African countries is consistent with this model, considering the relative inaccessibility between the principal population centers of eastern and southern African regions. This model, however, is not consistent with the proposed role of Burundi as the main hub of dissemination of the CEA clade in the region, as this small country is poorly interconnected to other east African countries. Previous studies have also shown a strongly supported phylogenetic relationship between subtype C sequences from Brazil, the UK, Burundi and Kenya; thus indicating that the CEA clade has also been disseminated to South America and Europe EA clade at both local and global scale.It has been suggested that interconnectivity between population centers was a critical factor in the spread of HIV-1 subtypes A and D across Africa EA lineage in Burundi to the early 1960s, while the onset date of the major sub-clades circulating in Ethiopia, Kenya, Tanzania and Uganda was estimated at around the early 1970s, coinciding with the first large Burundian migration flow. These analyses support the notion that the Burundian migration flow occurring in 1972 may have played a fundamental role in the regional and international dissemination of the CEA clade.Burundi has known many violent ethnic conflicts mainly since the 1960s that resulted in large migration flows. Two major civil conflicts that took place in Burundi in 1972 and 1993 generated especially large human movements with the former producing around 300,000 refugees and the latter producing about 687,000 EA clade, a second major subtype C lineage is also circulating in Ethiopia. Our results showed that the two Ethiopian lineages previously designated C and C′ While subtype C epidemic in Burundi and Uganda is largely dominated by the CEA clade, although they were particularly prevalent (100%) in southwest Tanzania (Mbeya region), close to Zambia and Malawi. The co-circulation of subtype C sequences from both east and southern African origin in Tanzania is consistent with its intermediate geographical position between eastern and southern countries. It is unclear whether subtype C clades of southern African origin detected in Kenya were introduced from Tanzania and/or directly from southern Africa.A large collection of minor subtype C lineages of southern African origin were detected in Kenya and Tanzania, which together represent 36% and 51% of sequences from those countries here analyzed, respectively. These lineages seem to have a more restricted expansion than the CET is associated with initially lower HIV-1 RNA plasma loads but more rapid onset of disease than infections with clade C′ETET may be less efficiently transmitted than clade C′ET, which is consistent with epidemiological evidence that show that the strain C′ET has gained ground and surpassed the clade CET over time It is also unclear the relevance of these findings for HIV-1 vaccine design. Possible correlations of distinct HIV-1 subtype C clades with differential susceptibility to neutralizing antibody and/or cellular immune responses should be explored to justify the selection of vaccines incorporating one or multiple immunogens derived from major African subtype C clades In conclusion, the results presented here point to the existence of a HIV-1 subtype C lineage characteristic of east Africa, which accounts for >70% of subtype C infections in this African region. This lineage probably emerged in Burundi in the 1960s and about 10 years later spread to Ethiopia, Kenya, Uganda and Tanzania, where it disseminated establishing new local epidemics. The subtype C epidemics in Ethiopia, Kenya and Tanzania also resulted from the introduction and dissemination of additional lineages of southern African origin. The explanation for the pattern of spread of the HIV-1 subtype C epidemic in east Africa is probably multifactorial and includes founder effects, massive migration between countries as a consequence of ethnic conflicts and geographical proximity.Figure S1Substitution saturation and likelihood mapping analyses. a) Transition (blue line) and transversion (green line) versus divergence plot for the HIV-1 subtype C pol dataset. b) Likelihood mapping of 10,000 random quarters selected from the HIV-1 subtype C pol dataset. Distribution (left triangle) and percentage (right triangle) of dots plotted in each region of the map. Each dot represents the likelihoods of the three possible tree topologies for a set of four sequences (quartets) selected randomly from the dataset. The dots localized on the vertices, in the centre and on the laterals represent the tree-like, the star-like and the network-like phylogenetic signals, respectively.(PPT)Click here for additional data file.Figure S2EA lineage despited in Close view of the HIV-1 C. The color of branches represents the country from where the sequence originated, according to the legend shown on the left. The boxes highlight the position of the major country-specific sub-clades detected in our study. The aLRT support values are indicated only at key nodes.(PPTX)Click here for additional data file.Figure S3Time-scaled Bayesian MCC tree of HIV-1 subtype C pol sequences from Ethiopia. Branches are colored according to the initial clade assignment of each sequence based on ML analysis: CET (blue), C′ET (red), other clades (green). The PP support is indicated only at key nodes. Positions of the putative interclade C/C′ recombinant sequences are marked with asterisks. Horizontal branch lengths are drawn to scale with the scale at the bottom indicating years. The tree was automatically rooted under the assumption of a relaxed molecular clock. Representative bootscanning plots of some putative C/C′ intrasubtype recombinant sequences are depicted on the right. Query sequences were compared to reference sequences of HIV-1 clades A1 (AB253429), D (AY371157), CET (AY242589), and C′ET (AY242581).(PPTX)Click here for additional data file.


E. coli (ETEC).The clinical onset and severity of intestinal disorders in humans and animals can be profoundly impacted by early life stress. Here we investigated the impact of early weaning stress in pigs on intestinal physiology, clinical disease, and immune response to subsequent challenge with enterotoxigenic F18 Pigs weaned from their dam at 16 d, 18 d, and 20 d of age were given a direct oral challenge of F18 ETEC at 26 d of age. Pigs were monitored from days 0 to 4 post-infection for clinical signs of disease. On Day 4 post-ETEC challenge, ileal barrier function, histopathologic and inflammatory cytokine analysis were performed on ileal mucosa.Early weaned pigs (16 d and 18 d weaning age) exhibited a more rapid onset and severity of diarrhea and reductions in weight gain in response to ETEC challenge compared with late weaned pigs (20 d weaning age). ETEC challenge induced intestinal barrier injury in early weaned pigs, indicated by reductions in ileal transepithelial electrical resistance (TER) and elevated FD4 flux rates, in early weaned pig ileum but not in late weaned pigs. ETEC-induced marked elevations in IL-6 and IL-8, neutrophil recruitment, and mast cell activation in late-weaned pigs; these responses were attenuated in early weaned pigs. TNF levels elevated in ETEC challenged ileal mucosa from early weaned pigs but not in other weaning age groups.These data demonstrate the early weaning stress can profoundly alter subsequent immune and physiology responses and clinical outcomes to subsequent infectious pathogen challenge. Given the link between early life stress and gastrointestinal diseases of animals and humans, a more fundamental understanding of the mechanisms by which early life stress impacts subsequent pathophysiologic intestinal responses has implications for the prevention and management of important GI disorders in humans and animals. E. coli (ETEC) are a major cause of these infections and are responsible for an estimated 300,000-5000 deaths in children under the age of 5E. coli heat-stable enterotoxin-1 (EAST1) enterotoxins that trigger intracellular signaling pathways inducing massive fluid secretion that contribute to diarrhea. The LT toxin of ETEC binds to GM1 ganglioside receptors on the enterocyte brush border leading to translocation, activation of adenylate cylase, and increases in cAMP-PKA pathways that induce phosphorylation-dependent increases in apical CFTR channel activity, resulting in Cl-, HC03- and fluid movement into the intestinal lumenEnteric infections resulting in diarrhea are a major cause of morbidity and mortality among both humans and agriculturally relevant animal populations worldwide. Enterotoxigenic It has become increasingly clear that environmental factors, including stress and diet, play a major role in gastrointestinal health and defense against important intestinal diseases in both humans and animals. Multiple lines of evidence derived from epidemiological and basic animal research studies show the important role of stress in the development and clinical onset of variety of debilitating gastrointestinal disorders including irritable bowel syndrome (IBS), the inflammatory bowel diseases (IBDs), and chronic intestinal infectionsad libitum access to water and standard nursery diet that met or exceeded NRC recommendations .All studies were approved by the North Carolina State University Institutional Animal Care and Use Committee. A total of 48 Large White × Landrace barrows, obtained from a commercial swine farm in North Carolina, were used in this study. Piglets selected for this study were genotyped at 1 week of age to confirm genetic susceptibility to F18 E coli based on the restriction fragment length polymorphism test described by Frydendahl et al.E coli receptor protein in the intestineSeparate rooms were used to house control (non-infected) and infected pigs for the duration of the study. Pigs were challenged when they reached 26 days of age to ensure that all pigs were expressing F18 Our rationale for studying F18 ETEC in this model is because of (1) our previous experience with this infection modelE. coli. Challenge (Day 0) and at the end of the study . Fecal scores were recorded daily throughout the experiment and were conducted by trained personnel that were blinded to experimental groups. Fecal scores were based on a standard scoring system . Fecal scoring began on the day of arrival  and continued for the duration of the study (Day 4). Body weights were recorded on Day 0 and Day 4 of the experiment to calculate the % body weight change over the 4 day experimental period.All pigs were weighed prior to On Day 4 post infection , all pigs were euthanized by penetrating captive bolt, exsanguinated and a complete necropsy was performed to assess gross pathology including presence of fluid-filled intestines, edema, and hyperemia of abdominal organs.2aperture Ussing chambers . Ileal mucosa was bathed on the serosal and mucosal sides with 10 ml Ringer solution. The serosal bathing solution contained 10 mM glucose, which was osmotically balanced on the mucosal side with 10 mMmannitol. Bathing solutions were oxygenated (95% O2- 5% CO2) and circulated in water-jacketed reservoirs maintained at 37°C. The spontaneous potential difference (PD) was measured using Ringer-agar bridges connected to calomel electrodes, and the PD was short-circuited through Ag-AgCl electrodes using a voltage clamp that corrected for fluid resistance. Tissues were maintained in the short-circuited state, except for brief intervals to record the open-circuit PD. Transepithelial electrical resistance (Ω⋅cm2) was calculated from the spontaneous PD and short-circuit current (Isc), as previously describedIscwas recorded at 15-minute intervals over a 1-hour period and then averaged to derive the basal TER and Isc values for a given animal.Distal small intestine (ileum) was harvested from each pig immediately after euthanasia and opened along the anti-mesenteric border. The intestinal mucosa was stripped from the seromuscular layer in oxygenated (95% O2- 5% CO2) Ringer solution  and mounted in 1.13 cmAfter a 30-min equilibration period on Ussing chambers, FITC-albumin  was added to the mucosal bathing reservoir of Ussing chambers. After a 15 minute equilibration period, standards were taken from the serosal side of each chamberand a 60 minute flux period was established by taking 0.5 ml samples from the mucosal compartment. The quantity of FITC-albumin was established by measuring the fluorescence in mucosal reservoir fluid samples in a fluorescence plate reader at 540 nm. Data was presented as the rate of FD4 flux in mg FD4 flux.min.Ileum was fixed in 10% neutral buffered formalin and processed for paraffin-embedding. Paraffin blocks were sectioned (5 µm thick) and stained with hematoxylin and eosin for histological analysis. Photomicrographs were acquired with 20×, 40× and 100× magnifications at a resolution using imaging software  running a high resolution digital camera  equipped to a clinical light microscope . Measurements for crypt depth and villous height were taken utilizing the calibrated measurement caliper option in the Villus measurements were taken from three well-oriented villi.Villi chosen for measurement were based on the criteria that 1) the entire crypt and villi were captured in cross section and 2) the central lacteal was present. Villi overlying gut associated lymphoid tissue was excluded from measurement.The number of neutrophils was counted in histologic sections based on nuclear and cytoplasmic morphology. Neutrophil counts were performed at 40× magnification and counts were expressed as number of neutrophils/hpf.The number of mast cells and their activation status was performed as described previously Ileal mucosa was homogenized in PBS containing protease inhibitors and the supernatant was collected and analyzed for protein content us a BCA assayData were reported as means±SE based on the experimental number (n). Time course data  were analyzed using a 2-way ANOVA on repeated measures with time and experimental group as the main factors. The remaining data were analyzed by using a standard one-way ANOVA . A post hoc Tukey’s test was used to determine differences between treatments following ANOVA. Statistical significance was set at a level of p<0.05.Prior to ETEC challenge, there was no indication of diarrhea among any of the pigs in this study . CompareIn control  pigs, ileal mucosal FD4 flux rates, an index of intestinal paracellular permeability, were elevated in the 16d and 18d weaning age pigs compared with pigs in the 20 d weaning age groupp<0.05; . ETEC chIsc, an index of electrogenic ion transport, compared with pigs weaned at 18 d or 20 d of age  relative to group-matched, unchallenged controls  pigs weaned at 16 d of age exhibited greater baseline d of age . In respe groups . Given tcontrols  and demoIn response to ETEC challenge, pigs in the 16 d and 18 d weaning age groups exhibited marked villus atrophy as indicated by reductions in villus height and morphological appearance of villi whereas no measureable changes in villus morphology were observed in ileum from challenged 20 d weaned pigs . There wIncreased numbers of mast cells were observed in the lamina propria of pigs challenged with ETEC however; this response was significant greater in pigs weaned at 20 d of age . HistoloThe concentration of inflammatory cytokines IL-6, IL-8, and TNF-α were determined in ileal mucosal samples . Ileal TEmerging evidence indicates that the the clinical onset and severity of a number of acute and chronic intestinal diseases, is profoundly and negatively influenced by early life stress E. coliKlebsiella pneumoniaStreptococcus pneumoniain vitro stimulation of whole blood with LPS from rats subjected to maternal separation stress induced an increase in TNF release compared with control In response to an infectious challenge, the primary goal of the innate immune response is to rapidly clear or contain offending pathogens to prevent prolonged inflammation and sepsis Intestinal mast cells play a central role in innate immune response to bacterial, parasitic, and viral infections by releasing pro-inflammatory cytokines  that mediate neutrophil recruitment into infected tissues and bacterial clearance -, HCO3- secretion and fluid loss that is responsible for the clinical signs of diarrhea In the present study, early weaning led to marked differences in the intestinal pathophysiologic response to ETEC challenge. While late weaned pigs exhibited no measurable intestinal barrier injury in response to ETEC challenge (based on TER and FD4 flux measurements), intestinal barrier function was in challenged, early weaned pigs was significant. The differential responses to ETEC challenge were independent of age and feed intake as all piglets were challenged at 26 d of age and feed intake was similar between weaning age group during the experiment. The diminished ETEC-mediated inflammatory response observed in early weaned pigs does not support a major role for inflammatory signaling pathways in mediating intestinal barrier injury in this study. It is well-established that ETEC mediates its pathogenesis via the binding to receptors expressed on the intestinal epithelium. Once bound, ETEC elaborates heat stable (STa and STb) and heat labile enterotoxins (LT) that stimulate epithelial secretory signaling pathways resulting in massive ClIsc responses to ETEC challenge (by 3.7 and 2.1-fold compared with late-weaned pigs), yet exhibited increased diarrheal scores. This finding was surprising given that Isc is reflective of net electrogenic ion transport, associated with Cl- and HCO3- secretion, which drives fluid movement into the intestinal lumen resulting in diarrhea. The enhanced secretory response to ETEC challenge, along with the robust innate immune response observed in the late-weaned pig ileum, could represent an enhanced capacity of the late-weaned pig to rapidly “flush-out’ and clear the offending pathogen and thus reduce clinical disease. However, it remains unclear why enhanced electrogenic Isc responses in the late weaned ileum, was associated with less severe clinical diarrhea. One potential explanation could be due to enhanced colonic reabsorption of ETEC-stimulated ileal fluid in the late-weaned pig. In previous studies in pigs infected with TGE, it was shown that survival and reduced clinical diarrhea in older pigs (compared with neonates) was due to the more developed colonic microbiota and short chain fatty acid production that was responsible for compensatory fluid reabsorption in the colon Compared with late-weaned pigs, early weaned pigs exhibited diminished Overall these data demonstrate that early weaning stress in pigs has a profound impact on subsequent clinical severity and intestinal injury in response to ETEC challenge. The exacerbated clinical and pathophysiological consequences of early weaning stress in pigs coincided with defects in intestinal epithelial barrier function and suppressed mucosal innate innate immune responses that were possibly mediated via alterations in mast cell function. Given the emerging relationship between early life stress and subsequent gastrointestinal disease susceptibility in humans, and the central role of impaired intestinal epithelial and innate immune defense barriers in the onset of such diseases, a more fundamental understanding of the precise mechanisms by which early life stress compromises long-term intestinal defense mechanisms could lead to innovative management and therapeutic strategies for a number of important GI diseases of humans and animals.