DBA/2J.Thy1(YFP)) and DiOlistically labelled retinal ganglion cells in DBA/2J mice. Here we show retinal ganglion cell dendritic degeneration in DiOlistically labelled DBA/2J retinal ganglion cells but not in the DBA/2J.Thy1(YFP) retinal ganglion cells suggesting that a potential downregulation of Thy1 allows only ‘healthy’ retinal ganglion cells to express YFP. These data may highlight alternative pathways to retinal ganglion cell loss in DBA/2J glaucoma.Glaucoma is a complex disease affecting an estimated 70 million people worldwide, characterised by the progressive degeneration of retinal ganglion cells and accompanying visual field loss. The common site of damage to retinal ganglion cells is thought to be at the optic nerve head, however evidence from other optic neuropathies and neurodegenerative disorders suggests that dendritic structures undergo a prolonged period of atrophy that may accompany or even precede soma loss and neuronal cell death. Using the DBA/2J mouse model of glaucoma this investigation aims to elucidate the impact of increasing intraocular pressure on retinal ganglion cell dendrites using DBA/2J mice that express YFP throughout the retinal ganglion cells driven by Thy1 ( Glaucoma is a complex multifactorial disease, which affects an estimated 70 million people worldwide. It is characterised by the selective and progressive loss of retinal ganglion cells (RGCs) and associated reduction in visual field in vivo imaging in mice expressing Thy1-YFP in RGCs Several lines of experimental enquiry have confirmed that the optic nerve head is a site of early damage. These have been confirmed in human glaucoma, in animals with a scleral lamina  and those with a glial lamina Thy1(YFP) to generate mice that develop spontaneous pigmentary glaucoma a subset of RGCs that express YFP in the cell soma and dendritic tree. We also labelled RGCs DiOlistically using the carbocyanine dyes, DiI and DiO The DBA/2J strain of mice is one of the most widely used in glaucoma research Thy1(YFP) mice were analysed as a control group. All eyes were categorised into 3 cohorts on the basis of optic nerve damage Both eyes from DBA2J.YFP mice at 9.5–11 months were assessed by which time they would be expected to manifest retinal damage. Retinas from 1–3 month DBA/2J.Retinal ganglion cell identity was confirmed by the presence of an axon running toward the optic disc in the retinal nerve fibre layer. Typically we observed that YFP filled the terminal parts of the dendritic tree; regardless of the intensity of YFP fluorescence, we did not observe points in the dendritic tree that suggested a barrier to the distribution of YFP. In total we analysed 68 YFP positive cells from DBA/2J mice at 9.5–11 months and 35 cells from 1–3 month old mice. The mean (SD) number of cells imaged per retina was 2.59(2.33).P>0.05) for all parameters tested ). There was also no significant difference in retinal ganglion cell dendritic morphology between retinal ganglion cells in the NOE group verses MOD or SEV, or between MOD and SEV groups  (Representative YFP positive RGCs from glaucomatous and control eyes are shown in  tested) , 4.Thy1 promoter in the transgenic mice and endogenous Thy1 has been shown to be down-regulated in animal models of RGC damage or glaucoma Thy1-driven YFP expression would mask early dendrite changes in DBA/2J glaucoma. Therefore, in a second cohort of mice, we assessed dendrite morphology in RGCs that were labelled DiOlistically with the carbocyanine dyes DiI and DiO. These dyes distribute in the plasma membrane and can be used to label cells regardless of their physiological status Gpnmb+ (D2-+Gpnmb) mice (and their 4 month old counterparts to serve as a control) and 4 month C57BL/6J . A representative panel of labelled cells is shown in +Gpnmb strains and we used this as a check against the possible of labelling bias. Using the Sun classification YFP is driven by the +Gpnmb age and sex match controls as assessed by total dendritic field area , total dendritic length  and Sholl analysis area under the curve (AUC)  . No significant difference  was between any of the control groups; 4 month C57BL/6J vs 4 and 11–12 month D2-+Gpnmb. As sectorial damage in the retina during glaucoma has been shown independently by different groups There was a significant decrease in dendritic architecture between DBA/2J eyes with no significant optic damage (NOE) and the D2- P<0.05) , 7. TherThy1(YFP) mice. The differences in dendrite mapping are highlighted by comparison of the Sholl plots for YFP and Diolistics . Although axon damage was not assessed in all regions of the optic nerve, we have shown previously that earliest axonal changes occur near or close to the optic nerve head Our data are consistent with a pathophysiological model in which glaucoma induced dendritic atrophy occurs in RGCs with intact axons. In all labelled cells, axons could be traced to the optic nerve head. Also, these changes occurred in eyes with no significant optic nerve damage  using a CFP fluorescent protein transgene that expresses in RGCsThe maintenance and degeneration of dendritic arbours is a complex process that shows considerable variation throughout the CNS. Current evidence suggests limited scope for remodelling of dendrites and that dendritic pruning is complex. The underlying mechanism appears to depend on whether this is driven by injury of as part of development. The mechanisms underling dendritic atrophy remain unclear in glaucoma. Studies in experimental glaucoma have indicated that the retrograde transport of BDNF Atrophic changes in RGCs are likely to influence the response characteristics of RGCs. This has been explored in the primate glaucoma model using explant preparations which showed reduced contrast sensitivity e.g. in Opa1 mutants) in which dendritic atrophy and mitochondrial beading is a hallmark of early disease Several factors are likely to conspire in dendritic degeneration. Histological studies in human glaucoma have confirmed the presence of varicosities within the axons of retinal ganglion cells These considerations do not rule out the role of exogenous (non-RGC related factors). Macrophage activity, in the form of microglial activation and migration, is an early feature of both experimental and human glaucoma ad libitum. All breeding and experimental procedures were undertaken in accordance with the Association for Research for Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Research. The Institutional Biosafety Committee (IBC) and the Animal Care and Use Committee (ACUC) at the Jackson Laboratory approved this study. The DBA/2J, DBA/2J-Gpnmb+, and DBA/2J.Thy1(YFP) strains have been described in detail elsewhere. C57BL/6J mice from The Jackson Lab facility  were used as an alternative age matched control. All mice were female. We used D2-+Gpnmb mice, a non glaucomatous substrain of DBA/2J that carry a wild type version of the Gpnmb gene responsible for the development of the glaucomatous phenotype. Although these animals develop mild pigment dispersion from the iris as a result of a mutation in the Tyrp1b gene this is not associated with elevated IOP Mice were housed in a 14 h light/10 h dark cycle as previously described with food and water available Gpnmb+, and DBA/2J were injected with 1–2 µl (1 mg/ml) Alexa Fluor 594-Cholera Toxin subunit B conjugate (Invitrogen) into the vitreous using a Hamilton syringe (35-gauge needle). After 48 to 72 hours they were anesthetized and euthanized via 4% PFA cardiac perfusion. Brains were submersion fixed for 24 hours after perfusion, cryoprotected in 30% sucrose overnight, OCT cryoembedded, and sectioned at 50 µm. Alexa Fluor 594 was visualized using an SP5 confocal microscope (Leica). The entire SC was assessed.Thirty 10–12 months old DBA/2J-Gpnmb+, and DBA/2J mice  were killed by cervical dislocation and the eyes quickly enucleated and placed in Neurobasal -A media (Invitrogen) at RT. The eyes were punctured at the limbus and a slit cut in the sclera to remove the cornea and sclera anterior to the ora serrata, along with the lens and vitreous. Three cuts were made in the retina before it was flat-mounted ganglion cell layer up on a cell culture insert (Millipore) and submerged Neurobasal –A media. Retinas were incubated at 37°C and 4% CO2 ready for DiOlistic labelling using a gene gun. The total time between death and DiOlistic labelling was less than 10 minutes.4 and 12 month female C57BL/6J, DBA/2J-n = 7) and 9.5–11 month female DBA/2J.Thy1(YFP) mice (n = 16) were killed by cervical dislocation, the eyes quickly enucleated and placed into 4% PFA at RT for 1 h. Retina were removed and dissected as above, mounted onto slides, coverslipped and sealed under Floromount (Sigma Aldrich) ready for imaging.1–3 month  from 5 cm with a 3.0 µm pore size, high pore density, cell culture insert (Becton Dickinson) to block the passage of aggregated tungsten particles. Retinas were then incubated for 30 min to facilitate dye diffusion before being placed in 4% PFA at RT for a further 30 min. Retinal preparations were then mounted retinal ganglion cell side up and coverslipped under Floromount and sealed with nail polish. Images were taken within 48 hours.n = 28; DBA/2J-Gpnmb+, 4 months n = 38, 12 months n = 86; DBA/2J, 4 months n = 53, 12 months n = 92; DBA/2J.Thy1(YFP), 1–3 month n = 35, 9.5–11 month n = 68) were obtained with a Zeiss LSM 510 confocal microscope (Carl Zeiss) or Leica TCS SP5 confocal microscope (Lieca) using a 20× objective to allow the capture of the entire dendritic tree within a single image. Dendritic morphologies were analysed using ImageJ to measure dendritic field area (measured using the convex polygon tool to join the outer most points of the dendritic tree), an ImageJ plugin, NeuronJ to measure total dendritic length, and a custom Matlab macro to run a Sholl analysis Image stacks (0.1 µm slice width) of 400 RGCs  and stained with PPD. Typically 30–50 sections are taken from each nerve. Homology between sections is considered during grading. Optic nerves were analysed and given 1 of 3 gradings The processing of optic nerves and staining with paraphenylenediamine (PPD) which darkly stains the axoplasm and myelin sheath of dying axons has been reported previously No or early damage (NOE) – less than 5% axons damaged. This level of damage is seen in age and sex matched non-glaucomatous mice,Moderate damage (MOD) – average of 30% axon loss,Severe (SEV) – >50% axonal loss and damage.


The aim of the study was determination of serum Se concentration and identification of genetic variations in genes related to metabolism of selenium as markers of cancer risks for carriers of BRCA1 gene mutation and individuals with susceptibility to other common unselected cancers .Eight genotypes of 4 most common SNPs localised in GPX1, GPX4, TXNRD2 and SEP15 were selected. Genotyping was performed in 93 affected and 186 unaffected matched BRCA1 carriers as well as on pairs matched 1:1 consisting of 108 breast, 50 ovarian and 105 prostate consecutive cancer patients and healthy controls.The following techniques for laboratory analyses have been applied: a) sequencing on ABI310, b) SimpleProbe or TaqMan analysis (a melting-curve genotyping with fluorescence-labeled probes based on the LightCycler 480 System (Roche Applied Science), c) determination of selenium concentration in plasma using atomic absorption spectrometer AAnalyst600 (Perkin Elmer).In none of studied groups statistically significant differences on cancer risk could be found when serum selenium concentration was assessed as a single factor. However, when selenium level data were combined with some selenoprotein genotypes some strong associations with cancer risk have been identified.The strongest association was found for carriers of SEP15 nGG genotype  and GPX1 nCC/TXNRD2 nGG/SEP15 GG .Results were not statistically significant however tendencies similar to those found in BRCA1 carriers and unselected breast cancer were observed.For four genotypes: GPX1 CC, GPX4 nCC, TXNRD2 GG and SEP15 nGG lower risk (~3 times) was found for selenium level ~90µg/l.


TET2 or EZH2 that are implicated in epigenetic mechanisms. We have performed genome-wide DNA methylation arrays and mutational analysis of TET2, IDH1, IDH2, EZH2 and JAK2 in a group of 24 patients with CMML. 249 genes were differentially methylated between CMML patients and controls. Using Ingenuity pathway analysis, we identified enrichment in a gene network centered around PLC, JNK and ERK suggesting that these pathways, whose deregulation has beenrecently described in CMML, are affected by epigenetic mechanisms. Mutations of TET2, JAK2 and EZH2 were found in 15 patients (65%), 4 patients (17%) and 1 patient (4%) respectively while no mutations in the IDH1 and IDH2 genes were identified. Interestingly, patients with wild type TET2 clustered separately from patients with TET2 mutations, showed a higher degree of hypermethylation and were associated with higher risk karyotypes. Our results demonstrate the presence of aberrant DNA methylation in CMML and identifies TET2 mutant CMML as a biologically distinct disease subtype with a different epigenetic profile.Chronic myelomonocytic leukemia (CMML) has recently been associated with a high incidence of diverse mutations in genes such as  Chronic myelomonocytic leukemia (CMML) is a rare clonal hematological disorder, characterized by the neoplastic transformation of the hematopoietic stem cell RUNX1, JAK2, EZH2, CBL, TET2, ASXL1 and FLT3 genes TET2 mutations are by far the most frequently event in CMML patients TET2 protein plays an important role in the pathogenesis of CMML. However, data regarding the impact of TET2 mutations on the prognosis of CMML patients is still controversial Several mutations have been found in CMML patients including TET2 mutations as a plausible cause for aberrant epigenetic regulation of gene expression in CMML. Clinical studies further support this hypothesis, as clinical responses in CMML patients to treatment with Decitabine, a de-methylating agent, have been shown TET2 mutations has not been addressed.The TET family proteins  have been shown to catalyze the conversion of 5-methyl-cytosine (mC) to 5-hydroxymethyl-cytosine (hmC), a recently identified epigenetic mark, and participate in the epigenetic regulation of gene expression during embryogenesis and cancer TET2 as well as other mutations with potential epigenetic impact, namely IDH1, IDH2, JAK2 and EZH2.Genome-wide strategies offer a unique and systematic approach to adequately establish functional and biological impact of single gene function alterations. Therefore, the present study was designed to establish a global methylation profiling of CMML and to analyze the association between such DNA methylome and the presence of JAK2V617F mutations was performed by the ARMS technique and mutations of TET2, EZH2, exon 4 of IDH1  and exon 4 of IDH2  by direct sequencing as previously described Bone marrow (BM) aspirates and peripheral blood (PB) mononuclear cells (MNC) (N = 24) were collected from patients with chronic myelomonocytic leukemia (CMML). Diagnosis of CMML was made according to the WHO) classification system of hematological malignancies Microarray-based DNA methylation profiling was performed with CMML and control samples using the Human Methylation27 Beadchip  according to the instructions of the manufacturer Before proceeding with methylation data analysis, 1092 CpGs located on chromosomes X and Y were excluded to avoid biological biases, due to the known methylation-mediated inactivation of one of the X chromosomes in female individuals. Additionally, we evaluated the detection probabilities  for all CpGs and excluded those with values of p>0.05 in more than 10% of cases. From a total of 26,486 CpGs in autosomal chromosomes, 89 CpGs showed a poor detection p value (i.e. above 0.05) in more than 10% of cases and consequently were eliminated from the study. Finally, 26,397 autosomal CpGs were investigated.T test: significantly differentially methylated CpGs (dMCPG) or differentially unmethylated CpGs (dUMCPG) were identified using a difference of at least 0.34 between mean β values of each analysis group  and a false discovery rate (FDR) below 0.05, calculated using permutated t tests or analysis of variance if more than two groups were compared.Initially, a global view on the DNA methylation profile of CMML and healthy PB/BM control samples was obtained using an unsupervised hierarchical cluster analysis including only CpGs with standard deviation (SD) >0.25 among samples  A second bioinformatic analysis approach was carried out using R and Bioconductor A third analysis defined differential methylation according to the following criteria: Genes in CMML samples versus control BM/PB samples were considered as hypermethylated when the mean β values of controls were <0.25 and at least a 20% of the CMML samples had a β value >0.5. Hypomethylated genes in CMML samples versus control BM/PB samples were considered when the mean β values of controls were >0.75 and at least a 20% of the CMML samples had a β value<0.5. Raw array data files were deposited in a MIAME compliant database Gene Expression Omnibus (GEO) and are available under the accession number GSE31600.http://www.ensembl.org). Ingenuity Pathway Analysis software was also used to identify deregulated gene networks containing differentially methylated genes between CMML patients and healthy controls . Both GO and Ingenuity pathway analyses were performed integrating the differentially methylated genes between CMML patients and healthy control samples coming from the three strategies utilized .Functional enrichment analysis of Gene Ontology (GO) was performed using differentially methylated genes between CMML and control samples using standard hypergeometric tests http://www.mirbase.org/; and snoRABase, http://www-snorna.biotoul.fr/index.php). Subsequently array genes were annotated as including miRNA or snoRNA using gene symbol ID.Analyzed genes were classified as polycomb repressive complex 2 (PRC2) targets according to the genome-wide mapping of Polycomb target genes in embryonic stem cells (ESCs) provided by Lee et al The methylation level of LAX1, SLC22A12 and VHL genes was analyzed by pyrosequencing technique as previously described Quantification of 5 hmC and 5 mC content in specific regions was performed using the EpiMark 5-hmC and 5-mC analysis kit , according to the instructions of the manufacturer, followed by qPCR. The kit distinguishes 5 mC from 5 hmC by the addition of glucose to the hydroxyl group of 5 hmC utilizing T4 B-glucosyltransferase. Briefly, 1 µg of genomic DNA was treated with 30 units of T4 B-glucosyltransferase. Glucosylated DNA was digested with 100 units of MspI or 50 units of HpaII or no enzyme (mock digestion) at 37°C overnight. The MspI and HpaII resistant fraction (in the context of CCGG) was quantified by qPCR using specific primers that covered at least one MspI/HpaII site. Resistance to digestion with the enzyme MspI, which is blocked by glucosilated 5 hmC, translates directly into the level of methylation in 5 hmC while 5 mC levels were obtained by subtracting the percentage of 5 hmC from the resistance to digestion with the enzyme HpaII, which is blocked by 5 mC, 5 hmC and glucosilated 5 hmC. Primers and conditions used to qPCR are described in the Before analyzing the methylation profile of samples from patients with CMML, we compared the methylation profile of bone marrow and peripheral blood samples from healthy donors in order to know whether there were differences between these two control groups. This analysis revealed no significant differences in methylation for any of CpG analyzed in the array, suggesting that there are both samples are equivalent in terms of methylation profile and can be combined.AIM2, CDKN2A, POU4F2 or WT1) and from these CpGs, 156 (79%) where located at CpG islands. In contrast, 62 CpG probes (24%) where hypomethylated in CMML , being 38 (61%) of them located outside of CpG islands.Unsupervised analysis performed on 8 samples obtained from healthy donors and 24 samples from patients with CMML including all probes on the array except the probes located on chromosome X, show that CMML samples cluster together and separated from healthy controls, while all the control samples were grouped together . The comThe 260 differentially methylated CpGs were validated in a second set of samples from a recently published study in which samples from 18 CMML and 9 controls were analyzed using the same methylation array hsa-mir-204 and hsa-mir-153-1) were found to be located in differentially hypermethylated regions and one mirtron (hsa-miR-1231) in a hypomethylated region in CMML samples when compared to healthy donor samples  and 94 snoRNAs (177 probes). The majority of these CpG were found to be located in regions unmethylated in CMML as well as in healthy donor samples (76% miRNA/mirtrons and 86% snoRNAs). Two miRNAs  indicating an enrichment in PRC2 targets among differentially hypermethylated genes in CMML.Previous reports have shown that genes acquiring de novo hypermethylation in cancer are frequently targeted by the polycomb repressor complex 2 (PRC2) in embryonic stem cells (ESCs) As hypermethylation frequently targets genes with dense CpG islands JAK2, UTX, DNMT3a, EZH2 and TET2 are a frequent events in patients with CMML JAK2, EZH2, TET2, IDH1 or IDH2 could be implicated in the epigenetic regulation in CMML. In order to validate our hypothesis, we analyzed the mutations in these genes and their association with the DNA methylation patterns observed in CMML patients.Mutations in epigenetic genes such as JAK2, EZH2, TET2, IDH1 or IDH2 genes in CMML patients are represented in TET2 gene mutations. In 4 patients, a JAK2V617F gene mutation was found while only 1 patient showed a mutation of EZH2. No mutations in IDH1 and IDH2 genes were identified (TET2 (TET2-wt) clustered together and separately from mutated TET2 (TET2-mut), with TET2-wt samples showing a higher number of differentially hypermethylated genes than TET2-mut samples  between TET2-wt and TET2-mut CMML samples, with 13 CpGs being hypermethylated in TET2-mut CMML and 50 hypermethylated in TET2-wt samples  . Indeed CMML patients with TET2-wt belonged to the recently described high or intermediate cytogenetic risks groups TET2-wt CMML patients belong to high or intermediate risk groups while only one of 13 TET2-mut patients) (Comparing DNA methylation profiles between control samples and CMML R = 2.4) . The levhe array . Interesatients) .EZH2 was found to be mutated only in one patient precluding any further meaningful analysis of the relation between EZH2 and methylation. EZH2 was found not to be methylated in CMML or in healthy controls according to the results of the methylation arrays with β values <0.3, indicating that the methylation of the EZH2 promoter region itself was not responsible for the differences in methylation in CMML. JAK2V617F mutations were found in 16.6% of our CMML cohort (4 JAK2V617F and 20 JAK2 wild type) , suggesting that at least in CMML, the presence of JAK2V617F is not related with a specific epigenetic profile. As it was the case for EZH2, differences in methylation of the JAK2 promoter were not related to the different methylation patterns observed in patients with CMML. A β value between 0.3 and 0.7 was found in 7 healthy donor samples and 14 CMML samples (2 of them with JAK2V617F), a β value >0.7 in 1 CMML and 9 CMML samples with a methylation β value <0.3, indicating a hypomethylation of JAK2 in these 9 cases (2 of them with JAK2V617F) .ld type) . Cluster profile . HoweverK2V617F) . The anaIDH1 and IDH2 were found not to be methylated in CMML or in healthy controls according to the results of the methylation arrays with β values <0.2 and <0.1 respectively, indicating that the methylation of these two genes were not responsible for the differences in methylation in CMML.TET2 is mutated, has not been analyzed. To determine the 5 hmC content, we specifically analyzed the promoter regions of 3 of the 13 genes hypermethylated in TET2-mut CMML patients. This analysis was performed in PB samples obtained from 2 healthy donors, 8 TET2-mut and 5 TET2-wt CMML patients samples. These genes were chosen based on the fact that only CpG dinucleotides associated to CCGG motifs can be analyzed using MspI-HpaII enzymes. For the LAX1 gene, we analyzed two CpG located 5′upstream (−38 and −244 bp respectively) to the CpG analyzed in the methylation array  and another one downstream (58 bp) to the CpG analyzed in the methylation array  the content of 5 hmC varies among different genes regardless of the l status ; 2) chanCDKN2B in 58% of cases with CMML and hypomethylation of C-MYC in 2 patients has been previously reported Despite the use of epigenetic drugs as decitabine, 5-azacitidine or the histone deacetylase inhibitor valproic acid for the treatment of patients with CMML Previous studies have shown that genes frequently targeted by the polycomb repressor complex 2 (PRC2) in ESCs are predisposed for cancer-specific hypermethylation BCR-ABL1 oncoprotein JAK2 gene Ingenuity Pathway Analysis software using genes differentially methylated between CMML and controls identified a specific gene network centered in PLC, JNK and ERK pathways. Mice deficient for PLC-beta3 and Lyn develop a myelodysplastic/myeloproliferative neoplasm similar to human CMML TET2, CBL, RUNX1, RAS, IDH1, IDH2, NPM1, ASXL1, NPM1 or EZH2EZH2 or TET2 have been associated with the prognosis of the disease their role is currently unclear TET2 mutations More than 80% of CMML patients harbor mutations in diverse genes including TET2 induces a decrease in the levels of 5 hmC in myeloid progenitors and a dysregulation of hematopoietic stem cells leading to the development of myeloid malignancies similar to CMML TET2-mut patients would be expected to show an increase in hypermethylation in comparison with TET2-wt (due to the lack of capacity to convert 5 hmC) TET2-wt CMML patients. Hypermethylation could be related to the statistically significant association between TET2-wt patients and the presence of abnormal karyotypes (p<0.01). Indeed, TET2-wt and abnormal karyotype have been described as poor prognostic factors in patients with CMML TET2 participates in the conversion of 5 mC to 5 hmC and thus is required for DNA de-methylation TET2-mut patients has not been analyzed. Our results indicate that hypermethylated genes in TET2-mut CMML patients have a high content of 5 mC as expected from the lack of function of TET2. However, the decrease of 5 hmC and increase of 5 mC do not occur equally in every hypermethylated gene or even in different CpGs of the same gene. On the other hand, genes differentially methylated in TET2-wt could be enriched both in 5 mC or 5 hmC. Some of the hypermethylated genes in TET2-wt CMML have been directly or indirectly related to cell cycle arrest, tumor suppression or myeloid differentiation . Whether these genes are in fact silenced by epigenetic mechanisms should be explored in order to determine their role in the pathogenesis of CMML.Although recent studies have demonstrated that silencing of TET2 protein induces a decrease in the content of 5 hmC in normal and malignant myeloid cells TET2 showed a different methylation profile than patients wild type TET2, and these differences segregate CMML patients in 2 groups with an increase in hypermethylated genes in non-mutated patients; 3) although TET2 mutations induced a decrease in the content of 5 hmC, the analysis of specific genes and CpGs demonstrate a heterogeneous behavior in terms of 5 mC and 5 hmC content. These results along with recent studies that demonstrate the role TET2 in hematopoiesis and the development of myeloid malignancies, suggest that epigenetic changes are not likely the mechanism by which inactivation of the TET2 protein induces myeloid malignancies.From our results, we may conclude: 1) CMML is associated with an abnormal epigenetic profile with 249 genes differentially hypermethylated; 2) CMML patients with mutations in Figure S1Unsupervised analysis including all probes on the array except the probes located on chromosome X. Samples are color coded. The top bar beneath the dendrogram shows CMML and healthy donor samples.(TIF)Click here for additional data file.Figure S2Analysis of 260 differentially methylated CpGs in CMML samples from our study and the study from Ko et al. Hierarchical cluster analysis based on abnormally methylated genes identified using 24 CMML patients and 8 controls, validated in another 18 CMML 9 healthy donor samples from the study of Ko M et al. β values are depicted using a pseudocolor scale . Samples are color coded. The top bar beneath the dendrogram refers CMML or healthy donor samples, second bar indicates CMML samples and healthy donor samples of our series and series of Ko et al.(TIF)Click here for additional data file.Figure S3Methylation results obtained by pyrosequencing in CMML patients samples with TET2-mut or TET2-wt. Pyrosequencing results of analyzed CpG loci on the array and hipermethylated in CMML TET2-mut patient samples, corresponding to LAX1, SLC22A12 and VHL genes. In addition one CpG located 5′upstream (−38) to the CpG analyzed in the methylation array in the case of LAX1; one downstream CpG (58 bp) in the case of SLC22A12 gene and one CpG downstream (5 bp) in the case of VHL gene were analyzed. The values are expressed as percentage of methylation. Median values of percentage of DNA methylation are indicated and P values were obtained using the 2-tailed T test or U Mann Whitney test.(TIF)Click here for additional data file.Table S1Specific primers and probes corresponding to pyrosequencing analysis.(XLS)Click here for additional data file.Table S2Primers, probes and conditions used to quantification of 5 hmC methylation levels by qPCR. The CCGG location refers to the position of this motif with respect to the analyzed CpG dinucleotide in the methylation array. All primers and probes are in 5′ to 3′ and probes are labbeled with FAM in 5′and TAMRA in 3′. In all of PCR the Tm was 60°C.(XLS)Click here for additional data file.Table S3Differentially methylathed genes between CMML patient samples and healthy donor samples. Gene Name; Red: Genes hypermethylated in CMML samples; Green: genes hypomethylated in CMML samples. CpG ISLAND; TRUE: probe inside in CpG island; FALSE: probe outside CpG island. POLYCOMB; NO: no target of Polycomb group; YES: target of Polycomb group; UD: unknown. PROMOTER CLASS: HCP: high CpG content; ICP: intermediate CpG content; LCP: low CpG content; UD: unknown. miRNA; NO: no annotated miRNA in gene sequence. snoRNA; NO: no annotated snoRNA in gene sequence.(XLS)Click here for additional data file.Table S4Description of Cariotype and JAK2, TET2 and EZH2 gene mutations of CMML patients. *: High risk Caryotype. **: Intermediate risk Caryotype. WT: wild type sequence. UD: undetermined.(XLS)Click here for additional data file.Table S5Differentially methylathed genes between TET2-wt and TET2-mut CMML patient samples. Gene Name; Red: Genes hypermethylated in TET2-mut respect to TET2-wt CMML samples; Green: Genes hypermethylated in TET2-wt respect to TET2-mut CMML samples. CpG ISLAND; TRUE: probe inside in CpG island; FALSE: probe outside CpG island. POLYCOMB; NO: no taget of Polycomb group; YES: target of Polycomb group; UD: unknown. PROMOTER CLASS: HCP: high CpG content; ICP: intermediate CpG content; LCP: low CpG content; UD: unknown. miRNA; NO: no annotated miRNA in gene sequence. snoRNA; NO: no annotated snoRNA in gene sequence.(XLS)Click here for additional data file.


Oral lichen planus (OLP) is a common chronic inflammatory mucocutaneous disease. Clinical diagnosis of OLP requires clinical work-up and histologic examination to rule out possible dysplasia and carcinoma. It is possible that oral mucosal viral infections including HPV infection may have a causative role in OLP pathogenesis. The aim of this study was to examine the coincidence of human papilloma virus type 18 and oral lichen planus.This study was a case-control study. Twenty nine paraffinized specimens of previously diagnosed oral lichen planus and 14 paraffinized specimens of nonpathogenic mucosa were studied. Polymerase Chain Reaction (PCR) analyze used for detection of DNA HPV 18. The data were analyzed with SPSS software and Fisher’s exact test was used to find the possible relation between HPV18 infection and oral lichen planus.Nine out of 29 (31.0%) lichen planus samples and one out of 14 (7.1%) controls were HPV 18 positive. No significant correlation (P = 0.128) was observed between HPV18 infection and oral lichen planus.According to the findings there might be a co-incidence of human papilloma virus type 18 and oral lichen planus. Oral lichen planus (OLP) is a chronic immunologic inflammatory mucocutaneous disease.2472, 100 μM deoxynucleotide triphosphate (dNTP), 200 pmoles of primer and 2.5 U of Taq polymerase (invitrogen). A total reaction of 50 μl containing 1 μl template DNA was amplified by incubating at 94°C for 5 min followed by 40 cycles  and 72°C for 10 min. After PCR reaction, the electrophoresis was performed on PCR products and the bands were observed using a UV eliminating Transilluminator ,10uminator . Those sThis study included 29 OLP cases with minimum age of 22 years and maximum age of 84 years (mean age of 49.55 ± 13.12) and 14 healthy con-trols with minimum age of 29 and maximum age of 70 years (mean age of 48.5 ± 10.18); 12 of cas-es and 7 of controls were female. Nine out of 29 (31.0%) lichen planus cases and one out of 14 (7.1%) controls were HPV 18 positive . Given tA lot of researchers have found that racial and geographical features may affect viral factors which involve in human diseases. Some viral factors have been studied and proposed as etiologic factors in oral lichen planus. Researchers showed that CD8+ cells cause apoptosis in virally infected cells. So it may be possible to consider a role for viral infections in OLP pathogenesis.125However in the present study, no significant difference was observed between the case and the control groups regarding HPV 18 infection. In 2006, Giovannelli reported that HPV infection can be affected by keratinization, so that keratinized tissue is more resistant to HPV infection.In the current study, no significant relation was observed between HPV /18 infection and OLP. According to the findings, there might be a coincidence of human papilloma virus type 18 and oral lichen planus. Given the fact that HPV 16 is a high risk type, it is suggested that another study be conducted to investigate the relation between HPV16 and other subtypes of HPV and OLP lesions.


HHIP), 4q22 (FAM13A), and 19q13 . Significant eQTLs were replicated in two independent datasets (n = 363 and 339). SNPs previously associated with COPD and lung function on 4q31  were associated with the mRNA expression of HHIP. An association between mRNA expression level of FAM13A and SNP rs2045517 was detected at 4q22, but did not reach statistical significance. At 19q13, significant eQTLs were detected with EGLN2. In summary, this study supports HHIP, FAM13A, and EGLN2 as the most likely causal COPD genes on 4q31, 4q22, and 19q13, respectively. Strong lung eQTL SNPs identified in this study will need to be tested for association with COPD in case-control studies. Further functional studies will also be needed to understand the role of genes regulated by disease-related variants in COPD.Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of mortality worldwide. Recent genome-wide association studies (GWAS) have identified robust susceptibility loci associated with COPD. However, the mechanisms mediating the risk conferred by these loci remain to be found. The goal of this study was to identify causal genes/variants within susceptibility loci associated with COPD. In the discovery cohort, genome-wide gene expression profiles of 500 non-tumor lung specimens were obtained from patients undergoing lung surgery. Blood-DNA from the same patients were genotyped for 1,2 million SNPs. Following genotyping and gene expression quality control filters, 409 samples were analyzed. Lung expression quantitative trait loci (eQTLs) were identified and overlaid onto three COPD susceptibility loci derived from GWAS; 4q31 ( Chronic obstructive pulmonary disease (COPD) is the fourth most common cause of death worldwide and is predicted to be the third leading cause of mortality in the world by the year 2030 FAM13A), 4q31 (HHIP), 15q25 (CHRNA3/CHRNA5/IREB2) and 19q13  The number of susceptibility genes for COPD is expanding rapidly with lists tabulated at 57 genes in 2009 The goal of this study is to identify lung expression quantitative trait loci (eQTL) within COPD susceptibility loci identified by GWAS. As part of the lung eQTL consortium, we have recently performed a genome-wide search for eQTLs in 1,111 human lung samples http://www.federa.org).All lung tissue samples were obtained in accordance with Institutional Review Board guidelines at the three sites: Laval University , University of British-Columbia  and Groningen University . All patients provided written informed consent and the study was approved by the ethics committees of the Institut universitaire de cardiologie et de pneumologie de Québec (IUCPQ) and the UBC-Providence Health Care Research Institute Ethics Board for Laval and UBC, respectively. The study protocol was consistent with the Research Code of the University Medical Center Groningen and Dutch national ethical and professional guidelines ; at Groningen, the lung specimens were provided by the local tissue bank of the Department of Pathology, and at UBC, the lung specimens were provided by the James Hogg Research Center Biobank at St Paul's Hospital. COPD diagnosis and severity were determined according to the GOLD recommendations Study subjects and lung specimens were described previously Genome-wide gene expression and genotyping profiles were obtained using a custom Affymetrix array (see GEO platform GPL10379) and the Illumina Human1M-Duo BeadChip array, respectively. Gene expression data are available through the Gene Expression Omnibus (GEO) repository with the accession number GSE23546. Standard quality controls were performed as described previously and only subjects that passed genotyping and expression quality controls were included in this study with 409, 363, and 339 subjects from Laval, Groningen, and UBC, respectively CHRNA3/CHRNA5/IREB2 locus that we have reported on previously FAM13A), 4q31 (HHIP) and 19q13 . SNPs associated with COPD from previous GWAS were tabulated for the three loci , 9 genes on 4q31  and 45 genes on 19q13 .Lung eQTLs were overlaid onto COPD susceptibility loci identified by previous GWAS except for the 15q25-ree loci . SNPs gecis-eQTLs were those passing Bonferroni correction considering the effective number of independent SNPs and genes tested at each locus. The number of independent variables was determined by using the definition of Li and Ji −6 for 4q22 (0.05/(279.64 SNPs×35 probesets), 1.50×10−5 for 4q31 (0.05/(128.26 SNPs×26 probesets) and 3.43×10−6 for 19q13 (0.05/(246.73 SNPs×59 probesets). Significant eQTLs in Laval dataset were then validated in the UBC and Groningen datasets. P values lower than 0.05 were considered significant in the replication sets.Lung eQTLs were identified using a different model than in our previous genome-wide lung eQTL mapping study PPM1K, GPRIN3, SNCA, MMRN1). Significant linkage disequilibrium (LD) was observed among the 64 SNPs (PPM1K (PPM1K and the direction of the effect was the same in the three cohorts. None of the SNPs previously associated with COPD on 4q22 (2>0.5)  and in modest LD (r2 = 0.53–0.69) with COPD SNPs were nominally associated with the expression levels of FAM13A (p = 4.1×10−5). The FAM13A-rs2045517 eQTL was replicated in UBC, but not in Groningen  . These froningen .FREM3, BC029578, HHIP, OTUD4) were involved in the significant eQTLs. Only eQTLs associated with BC029578 (35) and OTUD4 (1) were replicated in the two replication sets. eQTL-SNPs on chromosome 4q31 are subdivided in two strong LD blocks . Two SNPs  previously associated with COPD were found to affect the expression of HHIP. Rs1828591 was the most significant SNP associated with HHIP in the Laval dataset. This eQTL was replicated in UBC, but not in Groningen . 210 eQTLs were validated in both replication cohorts. SNPs associated with gene expression were distributed across four LD blocks  and rs7937 was genotyped in our lung eQTL dataset. Rs7937 was not associated with expression of genes located in this predefined 19q13 locus. The gene most significantly regulated by rs7937 was NUMBL (p = 0.0187). Three SNPs were regulating the expression of EGLN2, a gene previously associated with COPD. The most significant association with EGLN2 was with rs4803369 (p = 8.9×10−7). Rs4803369 is located at 13,274 bp away from rs7937 and is in modest LD (r2 = 0.33) with the latter. The eQTL results for rs4803369-EGLN2 from the three cohorts are illustrated in On 19q13, 739 SNPs and 95 probesets covering 45 different genes were tested. The expression levels of detected . 174 SNPD blocks . 26 SNPsA/CYP2A6 . These eFAM13A), 4q31 (HHIP) and 19q13 . We identified genetic variants influencing gene expression at each locus and replicated findings in two independent cohorts.The goal of this study was to identify causal variants and genes within susceptibility loci associated with COPD. GWAS have indicated four loci associated with this disease as defined by lung function measures MMRN1. Lung eQTLs on 4q22 were found and validated with four genes: PPM1K, SNCA, PPM1 and GPRIN3 genes. A SNP located in SNCA (rs2035268) has been associated with accelerated FEV1/FVC decline 2 between one of our significant eQTL-SNP and rs2035268 was 0.047. No SNP previously associated with COPD within and near FAM13A2>0.5). The latter were nominally associated with the expression of FAM13A and validated in one replication set. Accordingly, our results provide some support that FAM13A is the COPD causal gene on 4q22.The first study to identify an association between the 4q22 locus and COPD was published in 2010 BC029578 transcript and another associated with OTUD4. This transcript is located between the GYPE and GYPB genes. SNPs regulating BC029578 are distributed across a 400 kb region. Further studies are needed to understand the function of BC029578. eQTLs were also associated with FREM3 and HHIP, a member of the hedgehog-interacting protein family. HHIP has been associated with COPD in three GWAS The 4q31 locus was first associated with COPD and lung function in two studies in 2009 CEACAM21 was associated with COPD susceptibility in a sputum eQTLs study on COPD patients AXL, NUMBL, SERTAD3, B3GNT8, CEACAM4, CYP2G1P, LOC100505495 or ZNF780A to the development of COPD or related phenotypes. Rs7937, a SNP located in RAB4B, EGLN2 and MIA-RAB4B and identified in previous GWAS, was genotyped in our datasets. However, no association was detected between this SNP and the expression level of any gene. The strongest association with a suspected COPD gene is EGLN2-rs4803369. This gene is known to be involved in regulating hypoxia tolerance and apoptosis in cardiac and skeletal muscle. These results support that EGLN2 is a potential causal COPD gene on 19q13.There are many genes present in the 19q13 locus. This locus was recently associated with COPD and so far no replication study has been published eQTLs obtained in this study are derived from non-tumor lung parenchymal samples. As all organs, the lung is multicellular. The cellular heterogeneity constitutes an inherent limitation of our study and will inevitably reduce the power to detect eQTLs. It is known that many eQTLs will be missed by studying heterogeneous tissues FAM13A gene were indirectly (in LD) associated with the mRNA expression levels of FAM13A. On 4q31, the suspected candidate in this region, HHIP, was found to be regulated by SNPs previously associated with COPD. On 19q13, SNPs associated with COPD were consistently associated with the expression level of EGLN2. Further functional studies will be needed to verify the contribution of susceptibility genes in COPD. Strong lung eQTL SNPs will also need to be tested for association with COPD in case-control studies and in additional eQTL mapping studies in other disease-relevant tissues and cell types. This study is an important step forward to better understanding the underlying biology of the COPD susceptibility loci. It also shows the potential of eQTLs in a relevant tissue to leverage the results of previous GWAS and extend their functional meaning to gene expression.In conclusion, we used a large collection of human lung specimens from patients with and without COPD to identify SNPs that regulated gene expression in three COPD susceptibility loci derived from GWAS. Strong lung eQTLs were detected in the three COPD loci. However, the eQTL-SNPs were not necessarily the SNPs associated with COPD. On 4q22, SNPs associated with COPD near the Figure S1Linkage disequilibrium plot of significant SNPs on the 4q22 locus in the 1000 Genome Project. The white horizontal bar on the upper part of the figure illustrates the location of SNPs on a physical scale. LD values (r2) are indicated in each box. The color of the squares illustrate the strength of pairwise r2 values on a black and white scale where black indicates perfect LD (r2 = 1) and white indicates perfect equilibrium (r2 = 0). Red rectangles are SNPs previously associated with COPD (ith COPD . The gen(TIFF)Click here for additional data file.Figure S2Linkage disequilibrium plot of significant SNPs on the 4q31 locus in the 1000 Genome Project. The white horizontal bar on the upper part of the figure illustrates the location of SNPs on a physical scale. LD values (r2) are indicated in each box. The color of the squares illustrate the strength of pairwise r2 values on a black and white scale where black indicates perfect LD (r2 = 1) and white indicates perfect equilibrium (r2 = 0). Red rectangles are SNPs previously associated with COPD (ith COPD . The gen(TIFF)Click here for additional data file.Figure S3Linkage disequilibrium plot of significant SNPs on the 19q13 locus in the 1000 Genome Project. The white horizontal bar on the upper part of the figure illustrates the location of SNPs on a physical scale. LD values (r2) are indicated in each box. The color of the squares illustrate the strength of pairwise r2 values on a black and white scale where black indicates perfect LD (r2 = 1) and white indicates perfect equilibrium (r2 = 0). Red rectangles are SNPs previously associated with COPD (ith COPD . The gen(TIFF)Click here for additional data file.Table S1Significant eQTLs at the 4q22 locus in the Laval dataset and replication in UBC and Groningen datasets.(DOCX)Click here for additional data file.Table S2Significant eQTLs at the 4q31 locus in the Laval dataset and replication in UBC and Groningen datasets.(DOCX)Click here for additional data file.Table S3Significant eQTLs at the 19q13 locus in the Laval dataset and replication in UBC and Groningen datasets.(DOCX)Click here for additional data file.


Shigella sonnei, we performed multilocus variable number tandem repeat analysis of 1,672 isolates obtained since 1943 from 50 countries on 5 continents and the Pacific region. Three major clonal groups were identified; 2 were globally spread. Type 18 and its derivatives have circulated worldwide in recent decades.To investigate global epidemiology of  Shigella sonnei is the most commonly isolated species among the 4 Shigella species in industrialized countries  and the Pacific region by multilocus VNTR analysis (MLVA) to investigate the global epidemiology of S. sonnei.A total of 26 variable number tandem repeats (VNTRs) have been used to type Isolates were obtained from 50 countries on 5 continents and the Pacific region . Of thes<7 loci among the 26 loci. The 3 large clusters displayed distinct allelic diversity features. Eight loci  had Simpson diversity values >0.5 for >1 of the 3 large clusters (Table). Differences in diversity values >0.3 among the 3 clusters were observed for 9 of the 26 VNTRs. The largest difference was in 2 hypervariable VNTRs (SS1 and SS6). These 2 VNTRs displayed a high degree of allelic diversity in cluster A, but were invariant in cluster B. SS1 was invariant but SS6 displayed a high degree of diversity in cluster C.The high resolving power of MLVA is primarily caused by highly diverse VNTRs  were obtained from patients who acquired infections in Taiwan. Most isolates belonged to an insertion element IS1 interspacer 1 clone  A and C were globally spread, and clonal group B was found in countries in Africa, Asia and Europe only.Cluster D contained 2 isolates obtained in French Guiana and Senegal in 2003. The isolate for singleton E was obtained in Malaysia in 1999.S. sonnei isolates obtained since 1943 from 50 countries on 5 continents and the Pacific region. Three large clonal groups were identified; they displayed distinct allelic diversity features, particularly for 2 hypervariable VNTRs (SS1 and SS6). Clonal groups A and C were globally spread. One MLVA18 type (SS18.2) and several of its SLVs were widely distributed over 5 continents in the past 10 years.Genetic analysis using MLVA presented a simple clonal structure for 1,672


Angiopoietin-1 (Ang1) signals via the receptor tyrosine kinase Tie2 which exists in complex with the related protein Tie1 at the endothelial cell surface. Tie1 undergoes regulated ectodomain cleavage in response to phorbol esters, vascular endothelial growth factor (VEGF) and tumour necrosis factor-α (TNFα). Recently phorbol esters and VEGF were found also to stimulate ectodomain cleavage of Tie2. Here we investigate for the first time the effects of factors activating ectodomain cleavage on both Tie1 and Tie2 within the same population of cells, and their impact on angiopoietin signalling. We find that phorbol ester and VEGF activated Tie1 cleavage within minutes followed by restoration to control levels by 24 h. However, several hours of PMA and VEGF treatment were needed to elicit a detectable decrease in cellular Tie2, with complete loss seen at 24 h of PMA treatment. TNFα stimulated Tie1 cleavage, and induced a sustained decrease in cellular Tie1 over 24 h whilst increasing cellular Tie2. These differential effects of agonists on Tie1 and Tie2 result in dynamic modulation of the cellular Tie2∶Tie1 ratio. To assess the impact of this on Ang1 signalling cells were stimulated with VEGF and TNFα for differing times and Ang1-induced Tie2 phosphorylation examined. Elevated Tie2∶Tie1, in response to acute VEGF treatment or chronic TNFα, was associated with increased Ang1-activated Tie2 in cells. These data demonstrate cellular levels of Tie1 and Tie2 are differentially regulated by pathophysiologically relevant agonists resulting in dynamic control of the cellular Tie2∶Tie1 balance and modulation of Ang1 signalling. These findings highlight the importance of regulation of signalling at the level of the receptor. Such control may be an important adaptation to allow modulation of cellular signalling responses in systems in which the activating ligand is normally present in excess or where the ligand provides a constitutive maintenance signal. Induction of Tie1 cleavage by VEGF would be expected to enhance Ang1 activation of Tie2, and this has indeed been demonstrated Cleavage of both Tie1 and Tie2 has been reported to be strongly activated by PMA in endothelial cells VEGF is a key physiological activator of both Tie1 and Tie2 cleavage with similar, though less marked, effects as PMA TNFα is also an important activator of Tie1 cleavage in endothelial cells The data presented in We also examined levels of released Tie1 and Tie2 ectodomain at early (30 min) and late (24 h) time points in cells treated with VEGF and TNFα. As shown in Taking the findings on full-length cellular Tie1 and release of ectodomain together these data indicate that VEGF activates Tie1 cleavage acutely but not chronically, whereas TNFα causes Tie1 cleavage acutely and further loss of Tie1 chronically, via effects nt involving cleavage. Furthermore, chronic, but not acute, VEGF treatment stimulates loss of Tie2 whereas chronic TNFα treatment elevates cellular Tie2. The net result of these ligands therefore is for VEGF to decrease full-length Tie1∶Tie2 ratio acutely and increase the ratio chronically whereas TNFα causes a clear decrease in Tie1∶Tie2 ratio with increasing time of treatment.The cell-associated fragment of Tie1 remaining after ectodomain cleavage encompasses transmembrane and intracellular domains and is clearly observed as an approximately 45 kDa protein that accumulates in endothelial cells following activation with PMA, VEGF and other stimuli As a physiological activator, VEGF induces a more modest Tie2 cleavage than PMA and it was important to test whether this physiologically relevant stimulus is sufficient to induce appearance of the Tie2 endodomain. Therefore HUVEC were stimulated for 30 min and 24 h with VEGF before analysis for the presence of Tie2 endodomain by immunoblotting . As witht-Butyl-Ester), treated with or without PMA and Tie1 and Tie2 detected in cell lysates by immunoblotting. As shown in It has previously been shown that once formed by ectodomain proteolysis Tie1 endodomain is degraded by a two step process involving release of the intracellular domain from the membrane tethered transmembrane sequence, by γ-secretase activity, followed by proteosomal degradation of the 42 kDa intracellular kinase domain Previously we reported the presence of complexes comprising full-length Tie2 physically associated with Tie1 endodomain in endothelial cells treated with VEGF We and others have shown that loss of full-length Tie1 or cleavage of Tie1 ectodomain enhances Ang1 signalling through Tie2 As shown in In addition to signalling from full-length Tie2 it is possible that the Tie2 intracellular domain fragment detected in cells following ectodomain cleavage could have signalling activity. This could occur in the absence of Ang1 as a result of loss of the Tie2 regulatory ectodomain. Indeed, some other intracellular kinase domains released following ectodomain cleavage of full-length receptor tyrosine kinases, such as ErbB4 and TrkA, appear to be active as judged by their phosphorylation status As shown in In this study we have examined the effect different physiological activators have on relative levels of Tie receptors and Ang1 signalling. Our data shows that cleavage of cellular Tie1 and Tie2 are both regulated by VEGF but with different kinetics. This results in an increase in cellular Tie2∶Tie1 ratio early after VEGF stimulation and a decrease in the ratio at later time points. In contrast, TNFα differentially affects the receptors by enhancing Tie1 loss whilst increasing Tie2 levels leading to a progressive increase in Tie2∶Tie1 ratio in cells treated with this ligand. Furthermore, this modulation of Tie1 and Tie2 by VEGF and TNFα affects Ang1 signalling, with a decrease in Tie1 relative to Tie2 enhancing Ang1-induced cellular Tie2 activation The mechanisms responsible for the different time courses of Tie1 and Tie2 cleavage in response to PMA and VEGF are not known. However, it is possible that this reflects involvement of different proteases in cleavage of each of the receptors. Recently matrix metalloprotease-14 (MMP14) has been implicated in mediating Tie2 cleavage and the increase in PMA-activated Tie2 cleavage correlates with induction of increased MMP14 expression The ability of TNFα to cause a time dependent increase in Tie2∶Tie1 ratio in endothelial cells was found to involve both effects on Tie1 cleavage and effects on expression of Tie1 and Tie2. As shown in Together the findings of the present study show that the molecular balance between cellular Tie2∶Tie1 is dynamically regulated by pathophysiologically relevant factors and this balance influences the ability of Ang1 to signal in endothelial cells. These findings identify the Tie2∶Tie1 ratio as a key determinant of angiopoietin signalling in endothelial cells. Interestingly, Ang1 is considered to provide a fairly constant maintenance signal to endothelial cells, is present bound to extracellular matrix and is produced by perivascular cells at a relatively constant rate in vivo. A prediction from the cellular findings is that in vivo Ang1 signalling under basal conditions is limited by the responsiveness of the Tie2 receptor, which is determined by the Tie2∶Tie1 ratio in the cell, to the pool of perivascular Ang1. Furthermore, short-term increases in local VEGF concentration would enhanced Tie2 responsiveness and increase Ang1 signalling. This would allow the endothelium to maintain a quiescent state due to the presence of perivascular Ang1 without requiring maximal signalling through Tie2 under normal basal conditions whilst having the ability to enhance this pro-quiescent signal by increasing Tie2 responsiveness to Ang1 in the presence of transient increases in VEGF in vivo. Such a mechanism of local regulation of Tie2 responsiveness by VEGF would prevent short-term fluctuations in VEGF from causing vascular destabilization without having to maintain constant maximal levels of Ang1 signalling in the endothelium. The endothelium would retain the ability to respond acutely to VEGF if there was a coincident increase in Ang2. In addition, chronic VEGF elevation would also be expected to result in vessel destabilization as the enhancement of Tie2 responsiveness to Ang1 does not occur under conditions of long-term VEGF treatment. Whilst it is possible to speculate on the functional significance of the cellular effects in vivo it will be important in future work to test these hypotheses and examine the significance of the cellular changes in receptors and signalling in pathophysiological situations in vivo such as during angiogenesis.The focus of the present study has been to examine the effects of VEGF and TNFα on the relative levels of Tie1 and Tie2 within the same population of endothelial cells and the effects on Ang1 signalling. Clearly it will be important to determine the functional significance of the effects of VEGF and TNFα on Ang1 signalling In conclusion this study has shown the levels of intact Tie2 and Tie1 in endothelial cells are differentially controlled in both the short- and long-term by factors regulating ectodomain cleavage of the two receptors. This results in modulation of the molecular Tie2∶Tie1 balance in cells by various stimuli. Furthermore, the molecular balance between these two receptors determines the level of Ang1-induced Tie2 activation in the cell. These findings highlight the importance of regulation of signalling at the level of the receptor. Such control may be an important adaptation to allow modulation of cellular signalling responses in systems in which the activating ligand is normally present in excess or where the ligand provides a constitutive maintenance signal.Human umbilical vein endothelial cells (HUVEC) were from Promocell and were maintained in Medium 199 containing 20% foetal calf serum, 5 units/ml heparin, and 50 µg/ml endothelial cell growth supplement. As indicated in Results, cells were treated with the following concentrations of reagents: PMA, 10 ng/ml ; VEGF, 100 ng/ml ; TNFα, 100 ng/ml ; Ang1 200 ng/ml , unless otherwise indicated. The γ-secretase inhibitor  was added prior to addition of the PMA and remained in the media throughout treatment. Annealed, purified and de-salted double-stranded siRNA oligonucleotides have been previously detailed Cells were lysed with ice-cold lysis buffer  and lysates centrifuged at 13,000× g for 5 min to remove particulate matter.For analysis of whole cell lysates, Laemmli sample buffer containing dithiothreitol, to produce a final concentration of 100 mM, was mixed with cleared lysates and boiled for 5 min before loading equal amounts of protein onto SDS-PAGE and resolving. For immunoprecipitates, supernatants cleared of particulate material by centrifugation at 13,000× g for 5 min were immunoprecipitated by the addition of 2 µg of the indicated antibody for2–3 h in the presence of protein-A beads. In some experiments Tie2 and Tie1 extracellular domain was immunoprecipitated from conditioned medium, in these cases medium was conditioned for 24 h and cleared by centrifugation before immunoprecipitation as for cell lysates. In all cases immunoprecipitated proteins were recovered by centrifugation at 13,000× g for 5 min and washed 3 times with wash buffer (as lysis buffer but with 0.1% Triton X-100). Proteins were eluted from beads by the addition of Laemmli sample buffer containing 100 mM dithiothreitol and boiling for 5 min before SDS-PAGE. For immunoblotting proteins were transferred to nitrocellulose membranes electrophoretically and membranes probed with Tie1, Tie2 or phospho-Tie2 antibodies as appropriate. Blots were stripped and re-probed with anti-tubulin. Immunoreactive proteins were visualized with peroxidase-conjugated secondary antibodies and chemiluminescent detection Bands on Western blots were quantified by densitometric scanning of films. Graphs were derived from 3 or more independent experiments and data is plotted as means and standard error. Statistical analysis was performed using Student's ‘t’ test and differences between means judged statistically significant for p<0.05.


Escherichia coli (ETEC) F4ac is a major determinant of diarrhea and mortality in neonatal and young pigs. Susceptibility to ETEC F4ac is governed by the intestinal receptor specific for the bacterium and is inherited as a monogenic dominant trait. To identify the receptor gene (F4acR), we first mapped the locus to a 7.8-cM region on pig chromosome 13 using a genome scan with 194 microsatellite markers. A further scan with high density markers on chromosome 13 refined the locus to a 5.7-cM interval. Recombination breakpoint analysis defined the locus within a 2.3-Mb region. Further genome-wide mapping using 39,720 informative SNPs revealed that the most significant markers were proximal to the MUC13 gene in the 2.3-Mb region. Association studies in a collection of diverse outbred populations strongly supported that MUC13 is the most likely responsible gene. We characterized the porcine MUC13 gene that encodes two transcripts: MUC13A and MUC13B. Both transcripts have the characteristic PTS regions of mucins that are enriched in distinct tandem repeats. MUC13B is predicated to be heavily O-glycosylated, forming the binding site of the bacterium; while MUC13A does not have the O-glycosylation binding site. Concordantly, 127 independent pigs homozygous for MUC13A across diverse breeds are all resistant to ETEC F4ac, and all 718 susceptible animals from the broad breed panel carry at least one MUC13B allele. Altogether, we conclude that susceptibility towards ETEC F4ac is governed by the MUC13 gene in pigs. The finding has an immediate translation into breeding practice, as it allows us to establish an efficient and accurate diagnostic test for selecting against susceptible animals. Moreover, the finding improves our understanding of mucins that play crucial roles in defense against enteric pathogens. It revealed, for the first time, the direct interaction between MUC13 and enteric bacteria, which is poorly understood in mammals.Enterotoxigenic  Escherichia coli (ETEC) expressing the F4 (previously known as K88) fimbriae is a major cause of diarrhea in neonatal and pre-weaned piglets Enterotoxigenic S  and s . It is assumed that susceptibility towards ETEC F4ac is determined by the intestinal receptor that allows the bacterium to adhere to the intestinal tract or not. The identification of the receptor locus is thus desirable for the pig industry as it would enable us to accurately and efficiently eliminate the susceptible allele from nucleus breeding populations, leading to decreased mortalities caused by ETEC F4ac infection.Three antigenic variants of F4 have been described: F4ab, F4ac and F4ad, of which F4ac is the most prevalent LMLN-S0283 region by recombination breakpoint analysis MUC4MUC13MUC20TFRCin vitro F4ac adhesion phenotypes in specific pig populations have been described MUC13 is the responsible gene for the intestinal receptor conferring susceptibility to ETEC F4ac infection in pigs. We further identified MUC13 markers that are in complete linkage disequilibrium with the resistant causal allele in a broad panel of Western pig populations. The finding allowed us to select for the F4ac resistant animals and would greatly benefit the worldwide pig industry.The locus encoding the intestinal receptor for ETEC F4ac, denoted as F4acR, has been initially mapped to the q41 region on pig chromosome 13 (SSC13) by two independent linkage analyses 3 intercross population 2 and 461 F3 animals were recorded for in vitro F4ac adhesion phenotypes by a microscopic enterocyte adhesion assay as described previously 2 pedigree for 194 microsatellite markers covering the pig genome and performed a whole genome scan. The linkage analysis mapped F4acR to a region of 7.8 cM flanked by SW207 and S0075 in the q41 region on SSC13, which confirmed the previous reports of other investigators To identify loci affecting economically important traits in pigs, we constructed a large scale White Duroc × Erhualian FSW207 - S0075 interval on SSC13. A panel of 50 informative markers including 32 microsatellite and 18 SNPs on SSC13 were genotyped on all animals in the White Duroc × Erhualian F2 cross. A multipoint linkage analysis showed that the UMNp997– S0283 interval of 5.7 cM was defined as the most likely region harboring F4acR as the association of this region was 100-fold stronger than that for any other region in the genome . We identified susceptible and resistant haplotypes of founder animals by their complete association with adhesion and non-adhesion phenotypes in the cross, respectively. Recombination events in the candidate region of F4acR were observed in one F2  and one F3 animals . Individual 3314 was a non-adhesive animal and should be homozygous for the resistant allele. In the F4acR region, this animal carried a non-recombinant resistant chromosome from Erhualian founder sows and a recombinant chromosome from White Duroc founder boars. The recombinant SWR2054– UMNp595 interval was identical to the susceptible haplotype, which thus positioned F4acR downstream of UMNp595  on SSC13  at 140.93 Mb on SSC13  as the SNP. We further performed LDLA analysis for F4acR using the 60K chip data and adhesion phenotype data of the F2 population. The analysis detected the most significant marker (MARC0096736) in the 2.3-Mb region on SSC13  that has also been proposed as a candidate of F4acR by other investigators MUC13 g.28784 T>C was the most significantly associated marker in both Chinese and Western pigs. Especially, this SNP had an accuracy of more than 97% (144 out of 148) distinguishing susceptible and resistant animals in the 148 independent Western pigs  that was highly expressed in the jejunum. As the deduced MUC13 protein lacked the typical PTS region of mammalian mucins in the N-terminus that is enriched in proline, threonine and/or serine, we speculated that pig MUC13 could have another much longer transcript containing the PTS region MUC13 transcripts compared with our previous finding MUC13A (JN613414) and MUC13B (JN613417), share the same 5′UTR of 35 bp, transcription start site and 3′UTR of 1497 bp, but have distinct PTS regions that are rich in tandem repeats spanning approximate 3–5 kb (MUC13B is a string of 8 amino acid residues comprising threonine and proline (TPTPTTTP or TPTPTTTL). It is noteworthy that we failed to characterize the exact number and length of repeats of both transcripts, as the repetitive sequences were unsuccessfully amplified or sequenced using the current available technologies possibly due to the complex second structures of the sequences. Nevertheless, Southern blot analysis revealed that the length of the tandem repeat region was approximate 3–5 kb long (data not shown).We have previously isolated a 2679-bp cDNA of pig e 3–5 kb Figure 4MUC13, we screened 4 pig genomic DNA libraries and identified positive BAC/PAC clones encompassing the MUC13 gene from the libraries. By using the Solexa deep sequencing technology, we obtained the DNA sequences of these clones  and characterized the genomic structure of the porcine MUC13 gene. Each BAC clone contained a single MUC13 gene, corresponding to one of the above-mentioned two transcripts of MUC13  exhibit a high degree of sequence identity at the nucleotide level (>95%), and both consist of 12 exons and 11 introns . The Indel was used as a diagnostic marker for MUC13A and MUC13B alleles for the following analysis. Like the cDNA analysis, we unsuccessfully determined the complete DNA sequence of the PTS regions as the Solexa sequencing technology generated short pair-end reads of 148 bp that can not reveal the definite number of tandem repeats. Sequencing mucin genes has been shown to be technically difficult due to the large size and the repetitive structure of these molecules. For example, the missing sequence information for the PTS region is also encountered for MUC3A, MUC6, MUC7, MUC12 and MUC13 in cattle To determine the complete genomic DNA sequence of pig of MUC13 Figure 4MUC13A and MUC13B transcripts are encoded by a single gene or two separate loci, we developed a genomic qPCR assay to quantify copy numbers of MUC13 in the pig genome. The copy number assay measured the relative copy ratio between MUC13 and the reference GAPDH gene. We performed the assay on 60 representative pigs from Chinese and Western diverse breeds. The assay showed that all tested animals had a single MUC13 gene with the copy number ratio of 1.0 to GAPDH from diverse breeds for the diagnostic Indel marker, and analyzed association of the two MUC13 alleles with F4ac adhesion phenotypes in these pigs. We found that all 124 pigs homozygous for the MUC13A allele from the broad breed panel were resistant (non-adhesive) to ETEC F4ac. Moreover, all 594 susceptible animals carried at least one MUC13B allele  showed the complete (100%) association with the adhesion phenotypes in Western MUC13B homozygous pigs (Table S1). It further supported the MUC13 gene as F4acR.To examine the effect of B allele Table 1.MUC13B is associated with both susceptibility and resistance while MUC13A confers only resistance to ETEC F4ac, we analyzed the O-glycosylated site of the two MUC13 transcripts as the site is presumed to be the binding site of the bacteria O-glycosylated, i.e. addition of many short O-linked glycans, such as N-acetyl-galatosamine , to the peptides of mucins. The O-glycosylation is essential for the function of mucins as it is required to maintain an extended conformation to create a long, filamentous structure. The highly elaborate structures allow mucins to mediate the interactions between epithelia and their surroundings. The abnormal interactions have been implicated in many disease processes including infectious and inflammatory diseases, cancer and metastasis To elucidate why O-glycosylation sites . This indicates that the peptide of MUC13A can not form the proper filamentous structure by the O-glycosylation for the attachment of ETEC F4 fimbriae. It hence explains why MUC13A homozygous animals are all resistant to ETEC F4ac. For MUC13B, it has potential O-glycosylation sites predominantly in the PTS region . Therefore, MUC13B could be heavily or lightly O-glycosylated depending on the variable tandem repeat sequences of the PTS region. This is concordant with the observation that MUC13B is associated with both susceptibility and resistance towards ETEC F4ac.Protein motif/domain analysis showed that MUC13A does not have n sites ,Figure S2O-linked mucin-type sialoglycoproteins of 210–240 kDa MUC13B PTS sequence. These findings give additional strong supporting evidence for the MUC13 gene determining susceptibility/resistance to ETEC F4ac.It has been reported that the intestinal receptor for F4ac is MUC13 did not differ significantly in the small intestine of susceptible and resistant animals . The finding is consistent with the recent report that the expression of MUC13 is not related to susceptibility towards ETEC F4ac MUC13 causative mutation(s), we screened variants in the complete coding region except for the PTS repetitive sequences using RNA of susceptible and resistant animals from both White Duroc and Erhualian breeds. We detected 14 nonsynonymous mutations out of 24 cSNPs. All cSNPs along with 55 intronic SNPs of MUC13 were included in the data set of 188 SNPs that were genotyped on the 292 outbred pigs. To test if these mutations of interest contribute to susceptibility towards ETEC F4ac, we analyzed their association with the adhesion phenotypes in the 292 animals. The protein-altered SNPs occurred in both susceptible and resistance animals, thereby excluding them as the causative mutation.Quantitative RT-PCR analysis showed that the expression level of O-glycolysation sites that are essential for the biological functions of mucins. Hence, variance in the number, length and sequence of the tandem repeats can impact the extent and type of glycosylation and consequently the functions of mucins. For example, the variable tandem repeats in a variety of mucins have been associated with disease susceptibility in humans  MUC13 maps to the 2.3-Mb critical region containing F4acR; (2) MUC13 is proximal to the most significant markers in both GWAS and LDLA analyses based on large scale SNPs scan across the pig genome; (3) Of the 188 SNPs around the critical region, the six most significant SNPs that had 1000-fold stronger association than any other SNP in diverse outbred populations were all located in MUC13 (4) MUC13A allele was completely associated with the F4ac non-adhesion phenotype across diverse pig populations; (5) All susceptible animals from the broad breed panel carried at least one MUC13B allele; (6) MUC13 is perfectly consistent with the known biochemical properties of F4acR, as MUC13B has the unique O-glycosylation region that forms the binding site of bacterium and is rich in threonine and proline while MUC13A does not. Altogether, these data allow us to conclude that the MUC13 gene confer susceptibility/resistance to ETEC F4ac in pigs.We herein described the causality of the MUC13 PTS region that can not be amplified and sequenced using the current technologies.Overall, our findings have important practical consequences and will have immediate impact on pig breeding programs, as they allow the rapid elimination of the susceptible allele and consequently greatly benefit animal welfare and the pig industry. The findings also provide novel insights into the functions of mammalian mucins, as it establishes, for the first time, the direct interaction between MUC13 and enteric bacteria. Further endeavors will be directed to identify causative mutations in the All animal work was conducted according to the guidelines for the care and use of experimental animals established by the Ministry of Agriculture of China. The ethics committee of Jiangxi Agricultural University specifically approved this study.3 intercross population, one Western commercial population, one Chinese cultivated population (Sutai) and 15 outbred populations. The intercross population was constructed with two divergent founder breeds: White Duroc and Chinese Erhualian. Two White Duroc boars were mated to 17 Erhualian sows, and 9 F1 boars were then intercrossed with 59 F1 sows avoiding full-sib mating to generate 1912 F2 animals, of which 87 boars and 299 sows were intercrossed to produce 5311 F3 animals. In this study, 755 F2 and 461 F3 animals at day 240 were slaughtered for ETEC F4ac adhesion phenotype recording. The management of the experimental population has been described previously www.iciba.com/strain/, animals of each Chinese breed except for Lantang pigs were collected from at least 3 unrelated sire families (no common ancestry for 3 generations) each with 2 to 4 animals. For the three Western breeds, piglets of each breed were collected from 5 nucleus populations representing 18 (Duroc), 8 (Landrace) and 21 (Large White) sire families. Genomic DNA was extracted from ear tissues using a routine phenol/chloroform way and diluted to a final concentration of 20 ng/µl.Experimental animals were from one White Duroc × Erhualian Fin vitro ETEC F4ac adhesion phenotypes with slight modification as described previously −1) at 37°C for 30 min with gentle shaking. Each brush border was subsequently tested for its adhesion with F4ac by a phase contrast microscopy (Leica). A total of 20 well-separated and intact brush borders were examined in each specimen. In cases where less than four brush borders bound more than two bacteria, an additional 20 brush borders were scored. According to the classification standard proposed by Baker et al. A microscopic enterocyte adhesion assay developed by Baker et al. 2 population as described in Guo et al. 1 boars was amplified with primers listed in Table S2 and amplicons were directly sequenced in a 3130xl Genetic Analyzer (Applied Biosystem) using original primers. Additional microsatellite markers in the critical region were mined from the pig genome assembly (Sscrofa10 at http://www.ncbi.nlm.nih.gov/mapview/map_search.cgi?taxid=9823). The newly developed microsatellite and SNP markers were genotyped for all animals in the intercross F2 pedigree with primers given in Table S3 by using the fluoresce dye labeled primers (for microsatellite), SNapshot (Applied Biosystem) and PCR-RFLP technologies. A multipoint linkage analysis was performed on SSC13 with Allegro version 2 A panel of 194 informative microsatellite markers covering the pig genome was genotyped across the White Duroc × Erhualian F2 and F3 animals that carried a recombinant susceptible haplotype from the founder animals in the F4acR region were explored to define the genomic location of F4acR by recombination breakpoints.Haplotypes in the candidate region of F4acR were reconstructed for all tested animals in the White Duroc × Erhualian cross using SimWalk2 software 2 intercross on an Illumina iScan System following the manufacture’s protocol. The bead arrays with call rate <85% were excluded for further analyses. Genome-wide association studies were performed on all SNPs with a minor allele frequency (MAF) >0.05 and call rate >95% by GenABEL The PorcineSNP60 BeadChips (Illumina) were used to genotype all animals across the White Duroc × Erhualian FLDLA was performed in a haplotype-based approach with the assumption that each current founder population was originated from a history population (K = 20) after recombination and drift of N generations. The haplotypes of the history population can be reconstructed by a Hidden Marcov model Table S4. A final panel of 188 informative SNP markers (Table S4) in 24 genes was genotyped on the 292 purebred animals with the adhesion phenotypes by iPLEX SEQUENOM MassARRAY platform. SNP genotype calls were filtered and checked manually, and aggressive calls were omitted from the dataset. Associations of SNP markers with F4ac adhesion phenotypes were evaluated with the standard χ-test implanted in BEAGLE Polymorphisms in the responsible region of F4acR defined by recombinant breakpoint analysis were identified by comparative sequencing of genomic DNA of two adhesive White Duroc and two resistant Chinese Erhualian animals using primers given in MUC13 specific primers F1/R1 and F2/R2 (Table S5) were designed from the 5′- and 3′- regions of the previously isolated MUC13 mRNA sequence (NM_001105293). Three BAC and one PAC clones harboring the complete porcine MUC13 gene were identified by PCR screening of 4 genomic DNA libraries constructed from Western MUC13 specific primers. These clones were sequenced at 300×coverage by the Solexa (Illumina) technology at Beijing Genomic Institute, Shenzhen.2 animals using the Rneasy Fibrous Tissue Mini Kit (Qiagen). The first strand-complementary DNA was synthesized with the SMART RACE cDNA synthesis Kit (Clontech) and the 5′-Full RACE Kit (TaKaRa). To obtain the extended 5′-end of MUC13 cDNA, the first strand cDNA (Clontech) was first amplified with MUC13 nested primers F3/NF3 (Table S5) and universal primers UPM/NUP . To isolate the further 5′-end sequence of cDNA, primers F4/NF4 and F5/NF5 (Table S5) were designed from a conserved region of the first exon of mammalian MUC13 and the extended 5′ cDNA sequence by Clontech RACE. The primers together with 5′RACE Outer and Inner Primers  were used to amplify the first strand cDNA (TaKaRa). Primers F6/R6 (Table S5) were designed to amplify a fragment filling the gap of MUC13A transcript. All RACE PCR products were cloned to pGEM-T Easy Vector (Promega) for sequencing analysis using M13 universal primer. The full-length MUC13 cDNA sequence was obtained by joining the 5′RACE amplicon sequences with the previously isolated cDNA sequence MUC13 genomic DNA sequence was determined by the alignment of the obtained cDNA sequence with the BAC/PAC sequences.Total RNA was extracted from the jejunum of both adhesive and non-adhesive FMUC13 and GAPDH specific amplicons of 437 bp and 368 bp were generated by routine PCR with primers MUC13-FP1/RP1 and GAPDH-FP1/RP1 (Table S6), respectively. The two amplicons were connected to form an 805-bp fragment by bridge PCR using primers MUC13-FP1 and GAPDH-RP1 (Table S6). The fused fragment was cloned into a pGEM-T Easy vector (Promega). Sequence analysis confirmed that the recombinant plasmid clone contained a single copy of MUC13 and GAPDH fragments. The plasmid DNA was used as the reference sample in subsequent genomic qPCR assays, which determined copy numbers of MUC13 in the pig genome.Table S6) were designed for target (MUC13) and reference (GAPDH) genes. The target and reference probes were 5′ labeled with 6-FAM and VIC, respectively. Both probes were 3′ labeled with the minor groove binder non-fluorescent quencher (ABI). The amplification efficiencies of MUC13 and GAPDH were measured and validated by the CT slope method over a fivefold range dilution of the reference DNA. Standard curves were created by plotting the CT values against the logarithm amount of DNA. Genomic qPCR assays were performed using 60 independent animals from Chinese and Western diverse breeds. The 60 animals were classified into 10 groups according to their adhesion phenotypes and genotypes at MUC13A and MUC13B alleles. The target/reference ratios of all samples are normalized by the target/reference ratio of the calibrator sample (the plasmid DNA) using the method described in TaqMan probes and primers . The MUC13A allele was indicated by amplicons of 151 bp, and the MUC13B allele was represented by amplicons of 83 bp. The diagnostic test for the most significant MUC13 marker (g.28784 T>C) in outbred populations was performed using the ABI SNapshot protocol. A 280-bp DNA fragment was amplified with the F8/R8 primer pairs . SNapshot reactions were performed with Multiplex Ready Reaction Mix (Applied Biosystem) and an extension primer (5′-TTT TTT TTT TTT TTT CCA TGT ACA TTT CAG AGT CTG AGG GAT-3′) using an ABI 3130XL Genetic Analyzer (Applied Biosystem).The Indel in intron 2 distinguishing O-glycosylation sites and N-Glycosylation sites were predicted using the DictyOGlyc and NetNGlyc server, respectively Computational analyses were performed to identify the protein domains of MUC13A and MUC13B. Since the exact number of tandem repeat in the PTS region was not known, we initially assumed this repeat number as 10 for the following analyses. To make sure the analyses to be robust, we compared the results from the sequences with repeat number varying from 10 to 100. The protein domains were identified using Pfam Figure S1Detection of the diagnostic Indel marker for MUC13A and MUC13B alleles by PCR analysis. Genomic DNA was amplified with forward (5′-TTC TAC TCT GAT TCC ACA TCA CG-3′) and reverse (5′-TGG TCA TGT CTA GGA CTC TTT GAG-3′) primers. Amplicons of 151 bp and 83 bp indicate the MUC13A and MUC13B alleles, respectively. Lanes 1–3, 5, 8, 11 and 12: AB; lane 10: AA; lanes 4, 6, 7 and 9: BB; M: 50 bp marker.(TIF)Click here for additional data file.Figure S2Plots of probabilities indicating the potential O-glycosylation sites in the deduced peptides of MUC13A (upper panel) and MUC13B (lower panel). The positions of amino acids are given on the x-axis. Vertical green lines indicate the probabilities for the O-glycosylation at each residue. The red line indicates the threshold for the predicted O-glycosylation site.(TIF)Click here for additional data file.Figure S3Real-time RT-PCR analysis of MUC13B expression in the small intestine of susceptible and resistant animals from White Duroc and Erhualian breeds. Tissue samples were collected from piglets at the age of 6–8 weeks for RNA extraction. Three susceptible and three resistant animals homozygous for MUC13B were sampled from each breed. Real-time PCR was performed in triplicate. MUC13B expression levels normalized with β-actin are given (mean ± s.e.). No significant difference was observed in MUC13B expression levels between susceptible and resistant pigs. EHL+: Erhualian adhesive pigs; EHL-: Erhualian non-adhesive pigs; WD+: White Duroc adhesive pigs; WD-: White Duroc non-adhesive pigs.(TIF)Click here for additional data file.Table S1The complete association of MUC13B SNPs with F4ac adhesion phenotypes in Western purebred pigs.(DOC)Click here for additional data file.Table S2Primers for identification of SNP markers in the region of F4acR that were genotyped in the intercross population.(DOC)Click here for additional data file.Table S3The microsatellite and SNP markers in the region of F4acR that were genotyped in the intercross population.(DOC)Click here for additional data file.Table S4Primers for identification of SNP markers in the region of F4acR that were genotyped in outbred populations.(DOC)Click here for additional data file.Table S5Primers for isolation of the full-length cDNA and genomic DNA sequence of the porcine MUC13 gene.(DOC)Click here for additional data file.Table S6Primers for the copy number assay of the porcine MUC13 gene.(DOC)Click here for additional data file.