Miniature pigs are useful model animals for humans because they have similar anatomy and digestive physiology to humans and are easy to breed and handle. In this study, whole blood microarray analyses were conducted to evaluate variations of correlation among individuals and ages using specific pathogen-free (SPF) Clawn miniature pigs. Whole blood RNA is easy to handle compared to isolated white blood cell RNA and can be used for health and disease monitoring and animal control. In addition, whole blood is a heterogeneous mixture of subpopulation cells. Once a great change occurs in composition and expressing condition of subpopulations, their associated change will be reflected on whole blood RNA. From 12 to 30 weeks of age, fractions of lymphocytes, monocytes, neutrophils, eosinophils, and basophils in white blood cells showed insignificant differences with age as a result of ANOVA analysis. This study attempted to identify characteristics of age-related gene expression by taking into account the change in the number of expressed genes by age and similarities of gene expression intensity between individuals. As a result, the number of expressed genes was less in fetal stage and infancy period but increased with age, reaching a steady state of gene expression after 20 weeks of age. Variation in gene expression intensity within the same age was great in fetal stage and infancy period, but converged with age. The variation between 20 and 30 weeks of age was comparable to that among 30 weeks individuals. These results indicate that uniformity of laboratory animals is expected for miniature pigs after 20 weeks of age. Furthermore, a possibility was shown that whole blood RNA analysis is applicable to evaluation of physiological state. Pigs are a useful model animals of humans because they have similar anatomy and digestive physiology to human Microarray techniques allow to detect genome-wide perturbations during various treatments and to measure various responses by multitude of gene probes. Toxicogenomics, in which microarray techniques are specifically used in toxicology test, has been widely recognized as one of standard safety procedures for chemicals Use of whole blood was intended on two accounts. First, we are aware that RNA expression and degradation are susceptible to artificial manipulation such as cell separation and extraction. Whole blood manipulation avoids this risk by using a RNA blood collection tube. Second, whole blood is a heterogeneous population of lymphocytes , granulocytes , and platelets. It is expected that representative subpopulations in white blood cells may change depending on the condition of an individual. When a great alteration occurs in some subpopulations, whole blood will depart from the normal state of its age, because whole blood is a heterogeneous mixture of such subpopulations. We consider that it is particularly important to identify gene expression characteristics and variation of heterogeneous population of cells with age in whole blood.The present microarray analysis was conducted from the following aspects. First analysis addresses the number and kind of expressed genes. Quantity and characteristics of expressed genes by age were examined. Second analysis deals with similarity among individuals based on the correlation coefficient. Variations among individuals of the same age group and that between different age groups were examined. These results offer age-related gene expression characteristics of miniature pigs when whole blood is employed.Body weight change and hematological variation during breeding period are shown To characterize the age-related gene expression in whole blood from miniature pigs, RNA analysis was conducted on bloods sampled from fetal stage, 12, 20, and 30 weeks subjects. Each RNA sample was analyzed by an Agilent #G2519F#20109 Porcine Gene Expression Microarray (44K) consisting of 43603 oligonucleotide probes.Initially, we examined the change in the number of expressed genes to identify age-related characteristics. Microarray gene expressions were divided into two groups; “absent” and “present”, using flag indicators given by the scanner. Background level was determined from spot intensities outside the gene probing area. “Absent” was assigned to the spots whose intensities were less than the background level, while the rests were marked as “present.” Then each gene was judged as either “expressed” or “unexpressed” based on the number of “present” events. We defined a certain gene as “expressed” when “present” exceeds 75% out of replicated events. “A threshold of 75% was chosen by considering experimental deviation. Detailed explanation is given in the following part of Section “The number of expressed genes increased with age for both male and female subjects, as shown in We evaluated variations in correlation coefficients among individuals of the same age and different age groups. Pearson correlation coefficient was used for correlation analysis. Correlation coefficients for a total of 31 microarrays were obtained in normalized signals log-scale after excluding “absent” spots. A color-coded pairwise correlation matrix is shown in The average correlation coefficient within the same age group were 0.87±0.04 ,0.93±0.03, 0.93±0.03, 0.88±0.04, 0.98±0.01, 0.98±0.00 ,0.96±0.02, 0.94±0.05 for fetal sage males, fetal stage females, 12 weeks males, 12 weeks females, 20 weeks males, 20 weeks females, 30 weeks males, and 30 weeks females, respectively. Variations in gene expression were greater for younger subjects, but it diminished with age while generating resembling expression patterns . CorrelaNext, we analyzed correlation between different age groups. In All spots on the microarray were divided into 16 categories as shown To estimate the cause of variation in gene expression during infancy period, correlation coefficients between same-aged individuals were calculated for all spots and those belonging to Categories 1, 2, 4, and 8 . CorrelaTo characterize gene expression in each category, TC Annotator List (Porcine version 14.0 3-11-10) was downloaded from the TIGR gene Indices. TC Annotator List includes the gene number and the GO terms. Out of 43,603 probes in the Agilent porcine microarray (#G2519F#20109), 6,019 genes bear GO annotation. Microarray cDNA probes were classified by GO terms of “biological processes”. Out of all genes, fraction in Categories 1, 2, 4, 8, and 16 were 31%, 20%, 8%, 2%, and 38% respectively.Then the difference in gene expression between all spots and those in 4 categories  was examined. Among more than 20 genes classified under “biological processes” in GO term, dominant ones in Category 1 and 2 are shown in Next, expression intensity of immunity gene was examined. “Biological Process” contains immune system process (GO accession 0002736) attaching 11 subsets below . AntigenAntigen processing and presentation (GO:0019882) and T cell selection (GO0045058) include the major histocompatibility complex (MHC) genes. By presenting antigens, MHC is involved in elimination of bacterial or viral pathogen, rejection of cancer cells, and rejective response on organ transplantation. Also MHC is indispensable in the immune system. Swine leukocyte antigens (SLA) are important immunogens for humoral responses and important mediators of the cellular immune responses through both direct and indirect presentation of peptides to T-cells The Agilent porcine microarray had 7 probes with 7 types of interferon and 7 probes for 4 types of interferon receptors. All of 7 interferon genes fell under Category 16. Normally these genes remain unexpressed but expressed upon necessity. In contrast, 1 type of interferon receptor gene fell under Category 1, 3 fell under Category 2, and were expressed until 12 weeks of age. Their signal intensities stayed at constant levels after 12 weeks .Toll-like receptors (TLRs) are the principal pattern recognition receptors. With this innate immunity, the first immune response is mediated into reserved foreign patterns on recognition. TLRs recognize reserved molecular patterns, start rapid response to protect the host upon infection, and produce signals, such as cytokines and co-stimulatory molecules to activate the adaptive immune system This study aimed to evaluate the “healthy state” gene expression by whole blood microarray analysis of miniature pigs at different age groups up to 30 weeks. Results of our gene expression analysis can be applied to health management of laboratory animals, and eventually, to health monitoring of humans.Use of whole blood was intended on two accounts. First, we are aware that RNA expression and degradation is susceptible to artificial manipulation such as cell separation and extraction. The whole blood manipulation avoids this risk, unlike dealing with extracted white blood cells. In addition, whole-blood RNA can be stabilized immediately by using RNA blood sampling tube such as PAXgene. This avoids the cell separation process after sampling and minimizes the possibility of RNA denaturation. Usually, peripheral blood mononuclear cells (PBMCs) separation employs the difference of specific gravity between other blood components, which should be followed immediately after the blood sampling. Such manipulation requires a skilled operator to reduce the influence of separation procedures on gene expression. Second, the whole blood is a heterogeneous population of cells. One can expect that representative subpopulations in white blood cells may vary depending on the health condition of an individual. When a great alteration occurs in some subpopulations, the whole blood may also depart from the normal state of its age, because whole blood is a heterogeneous mixture of such subpopulations. Therefore, identification of gene expression characteristics and age-related variation in subpopulations in whole blood are essential issues.A major drawback in whole blood RNA manipulation includes a decrease in detection sensitivity caused by excessive globin mRNA. A previous study showed that globin elimination from whole blood RNA lead to alteration in original gene expression profiles Staphylococcus enterotoxin B-stimulated and unstimulated blood from healthy subjects was significantly higher in leukocyte-derived samples the in whole blood, suggest that the method of RNA isolation of from whole blood is critical variable in blood RNA assay 2 = 0.85) for 8273 genes expressed in both whole blood RNA and PBMC RNA samples Whole blood contains a variety of cell types as red blood cells, granulocytes, lymphocytes, and platelets. Most of the nucleated cells in blood are white blood cells such as neutrophils, T-cells, B-cells, and monocytes. The intraclass correlation, a measure of signal to noise ratio, of the difference between As a result of gene expression analysis based on whole blood RNA, the number of expressed genes was less in fetal stage and infancy period but increased with age, presenting a decreased rate of gene expression after 20 weeks of age. Variation in gene expression intensity within the same age was great in fetal stage and infancy period, but decreased with age. The variation between 20 and 30 weeks of age was comparable to that among individuals. If we assume that the whole blood RNA is originated from blood cells, variation of gene expression in infancy period is attributed to that of blood cells themselves. Correlation coefficient analysis of genes expressed constantly from fetal stage to 30 weeks (Category 1) also agrees with this assumption. These results indicate that uniformity of laboratory animals is expected for miniature pigs after 20 weeks of age. The organs and reproductive function of the Clawn miniature pigs become mature at about 26 weeks. Physiological state will become stable from 20 weeks and gene expression will reach a steady state.The expressed genes were classified into those expressed constantly and those emerged from certain age. Their functions were analyzed by Gene Ontology. As a result, we found that mitosis and immunity genes were distinctively expressed throughout the period from fetus to 30 weeks old, while defense and control genes began to express from 12 weeks old. The number of expressed genes converged after 20 weeks old. Functions for those expressed at 20 and 30 weeks old were unavailable due to limited number of porcine gene annotations.Then, we examined variation of correlation coefficients for genes bearing GO terms such as neutrophil, monocyte, and platelet. Variation under “neutrophil” and “platelet” within the same age group was small from fetal stage to 30 weeks. On the other hand, that under “monocyte” became greater than the former, and even greater under “metabolic process.” These results suggest that variation of gene expression during fatal stage and infant period is caused by cell activity, rather than by blood cell subpopulation functions. Thus, whole blood RNAs is useful to monitor health condition of an individual since they reflect the status of white blood cell subpopulations.In clinical use of whole blood RNA analysis, we need to validate that the state of immune-related gene expression in whole blood reflects the state of the immune system. Our Gene Ontology analysis showed that immune-related genes accounted for 87% of total expressed genes at 12 weeks, and 94% at 20 weeks. Our results indicated that MHC class I and class II genes were constantly expressed from fetal stage to 7 weeks and that the gene expression rate increased with age until 20 weeks, reaching a steady state afterwards. This tendency agrees with that noted in previous studies In this study, we proved that whole blood RNA analysis can be used in practical applications by considering variations of gene expression by individual specificity and age in miniature pigs. Further study on gene expression profiles for the early stages of disease and physiological state under stress will contribute to the promotion of xenotransplantation in miniture pigs, as well as to standardization of their breeding method. The blood RNA diagnostics under development can eventually be applied to human health monitoring.2 at the SPF facility of the breeder  for 18 weeks. Mean body weights of males and females at the beginning of the experiment were 7.0 kg and 6.9 kg respectively. During this period, all animals were fed with 450 g/day standard dry feed  with free access to water. Fetuses were taken out from their mothers on days 77 to the 84 days of the pregnancy by a Caesarean section. The unborn baby's sex was determined based on the shape of the vulva.Five males and five females of 12 week old Clawn miniature pigs were housed individually in cages of 1.5 mhttp://www.patho.co.jp/index.html)  using standard clinical methods.All blood samples were collected from the superior vena cava at 12, 16, 20, 24, and 30 weeks of age. Blood (EDTA), plasma (EDTA) and serum samples for hematology and biochemical tests were collected 24 hours after fasting. Hematology and biochemical tests were conducted by Clinical Pathology Laboratory, Inc. (The microarray experiments described in this manuscript are MIAME compliant and the raw data has been deposited in the Gene Expression Omnibus (GEO) database (Accession number GSE25393).Blood samples for microarray analysis were collected from each subject in PAXgene™ tubes , incubated at room temperature for 4 hours for RNA stabilization, and then stored at −80°C. RNA was extracted from whole blood using the PAXgene™ Blood RNA System Kit  following the manufacturer's guidelines. Quality of the purified RNA was verified on an Agilent® 2100 Bioanalyzer . RNA concentrations were determined using a NanoDrop® ND-1000 spectrophotometer . cRNA labeled with fluorescent Cyanine 3-CTP was used for hybridization onto porcine oligo microarray slides  containing 43,603 oligonucleotide probes at 65°C for 17 h. Hybridized microarray slides were washed according to the manufacturer's instructions and were scanned with an Agilent DNA Microarray Scanner  at 5-µm resolution. The scanned images were analyzed numerically using Agilent Feature Extraction Software version 9.5.3.1. (Agilent Technologies)http://www.geneontology.org/) was used to functionally categorize gene expression profiles. GO terms were obtained from the TIGR pig gene indices, Porcine version 14.0 3-11-10 (http://compbio.dfci.harvard.edu/cgi-bin/tgi/gimain.pl?gudb=pig).Normalized data using quantile normalization were analyzed using GeneSpring GX software version 10.0.1 (Agilent Technologies). The Gene Ontology Database .


Cardiac vein arterialization is seldom applied for treating right coronary artery disease. This study aimed to improve outcomes of cardiac vein arterialization in a porcine model using intramammary artery anastomosis.A chronic, stenotic coronary artery model was established in 12 of 14 Chinese experimental miniature pigs of either sex, which were randomly divided into equal control (n = 6) and experimental (n = 6) groups. In experimental animals, blood flow was reconstructed in the right coronary artery using intramammary artery. Arterialization involved dissection of right internal mammary artery from bifurcation to apex of thorax followed by end-to-side anastomosis of internal mammary artery and middle cardiac vein plus posterior descending branch of right coronary artery. Intraoperative heart rate was maintained at 110 beats/min. Graft flow assessment and echocardiography were performed when blood pressure and heart rate normalized.The experimental group had significantly higher mean endocardial and epicardial blood flow postoperatively than control group , p<0.001; mean epicardial blood flow: 0.29 vs. 0.22, p = 0.014). Transmural blood flow was also higher in experimental group than in control group ; ejection fraction increased from 0.46% at baseline to 0.51% (p = 0.0038) at 6 hours postoperatively, and mean blood flow of internal mammary artery was 44.50, perfusion index 0.73 at postoperative 6 months, 43.33 and 0.80 at 3 months.Successful cardiac vein arterialization via intramammary artery in a porcine model suggests that this may be a viable method for reconstructing blood flow in chronic, severe coronary artery disease. Currently, most patients with chronic or acute  coronary artery disease (CAD) are treated with coronary artery bypass grafting (CABG) or percutaneous coronary intervention (PCI). Long-term outcomes of these procedures are generally favorable. The advent of antiplatelet therapy and drug-eluting stents have increased the success rates of PCIIn the 1970s, based on the idea that atherosclerosis does not affect the venous system, the concept of selective coronary vein arterialization was proposed for patients not able to receive CABG or PCICurrently, however, cardiac vein arterialization is not a major treatment for CAD because procedural aspects, graft materials and outcomes are still being questioned. For example, common use of the great saphenous vein as the bypass graft is complicated by the inability of the coronary vein to tolerate high arterial pressure for a long period of time, which may result in hemorrhage, edema, endometrial hyperplasia or even occlusion of the coronary venous systemAll animal procedures were approved by the Ethics Committee of Beijing An Zhen Hospital, Capital Medical University, and the protocols followed the ethical guidelines of this committee. All animals were allowed free access to water and diet.Experimental animals: Chinese experimental miniature pigs were purchased from Beijing Agriculture University (n = 14) and were of either sex with an average body weight of 40±5 kg. They were fed a high cholesterol diet for 4 weeks  and then underwent endarterectomy of the right coronary artery. Aspirin 325 mg was administered one day before surgery and the animals were fasted 8 hours before surgery. Anesthesia involved intramuscular injection of diazepam (1.5 mg/kg) and ketamine (3.5 mg/kg) 15 min before surgery. Endotracheal intubation was then performed and the ventilator  was set at 15–20 times/min to assist breathing as close as possible to normal physiological status. Intraoperative anesthesia was maintained with diazepam (0.5 mg/kg), and continuous ECG monitoring was carried out. During intervention, fentanyl  was used as an analgesic and 1.5 mg/kg atracurium besylate intravenous bolus was administered to induce muscular relaxation. Marginal ear vein was punctured to establish intravenous access. Right femoral artery puncture was performed, and the femoral artery sheath was rinsed with heparin and inserted via guide wire . Heparin 1000 U/kg was injected, and heparin 2000 U was injected intravenously once every hour. Coronary angiography was performed and 6F JR catheter  was sent to the middle section of the ascending aorta along the arterial sheath under digital subtraction angiography (DSA). The catheter was pushed slowly to the opening of the right coronary artery and 76% diatrizoate was injected. A 6F JR catheter was used for right coronary angiography. Balloon  expansion and stretching were done first to send the 7F JR guiding catheter to the opening of the right coronary artery through the femoral artery sheath, and then the guide wire was sent to the distal segment of the right coronary artery through the guiding catheter. The 2.5 mm or 3.0 mm balloon catheter (selected according to right coronary artery diameter) was inserted along the guidewire into the distal, middle-distal and middle segments of the right coronary artery in succession to perform balloon expansion. The ratio of the balloon diameter to the coronary artery diameter was 1.3∶1, and the balloon inflation pressure was 4–6 atm. In order to cause mechanical exfoliation of the arterial intima, each segment of the right coronary artery was expanded by the balloon and stretched 3 times, for 30 seconds each time. The interval was 60 seconds. The contrast agent was aspirated, creating negative pressure in the balloon before it was withdrawn. The pigs were then raised in individual cages and continued to receive the high-cholesterol diet. Intramuscular injection of 4 million U penicillin was administered for 3 days to prevent infection. After 8 weeks, coronary angiography was performed via the femoral artery on the opposite side (left) to observe the coronary artery stenosis. Animals with severe diffuse right coronary artery stenosis  were suitable for undergoing subsequent experiments.Of 14 animals, severe diffuse right coronary artery stenosis was successfully modeled in 12 animals. They were randomly and equally divided into an experimental group that would receive cardiac vein arterialization and a control group that would receive exploratory thoracotomy only. Postoperative evaluation was conducted in both groups and results were compared.After anesthesia and median sternotomy and thoracotomy, the right internal mammary artery was dissected for later use. In order to ensure sufficient length, the right internal mammary artery was dissected from the bifurcation to the apex of the thorax . The proGraft flow assessment was performed using the Medistim Veri-Q Flow Meter System with QuickFit TTFM probe . Echocardiography (HP SONOS 5500) was used to calculate the pre-operative and postoperative ejection fraction (EF), E/A ratio . The ultrasound section was a parasternal left ventricular long axis view.Non-radioactive colored microsphere method  was used to detect the blood flow of various myocardial layers, including subendocardial, subepicardial, and transmural blood flow. The animals received injections of 2 ml yellow non-radioactive colored microspheres  through the aortic root and the internal mammary artery graft, respectively. At the same time, a fixed speed pump was used to draw the reference blood samples via the common carotid artery catheter at a rate of 7.64 mL/min for a total of two minutes. The animals were sacrificed with an intravenous overdose infusion of potassium chloride solution and 4 g samples of the right ventricular myocardium were collected from the two groups. Samples were divided into equal-sized endocardial and epicardial myocardial tissue blocks and sent for pathologic examination. Each tissue block weighed about 2 g. The microsphere dye extraction reagents (Triton Technology) were used to extract the dye. A spectrophotometer was used, and a filter with a 10 nm bandwidth and center absorption wavelength of 450 nm was selected to measure the optical density of the dye extract of yellow microspheres. The unit myocardial blood flow (MBF) was calculated. The MBF ml/(g.min)  =  (ODm×Qr)/(ODr×Wm), of which MBF is the unit myocardial blood flow, Qr is the drawing velocity of the reference blood samples, and ODr and ODM are the optical density of the reference blood samples and the microsphere dye extract of the myocardial tissue, respectively. Wm is the weight of the myocardial tissue block.Data are presented as mean with standard deviation (mean±SD). Comparisons between control and SCVBG groups were performed by the independent two samples t-test. Comparisons for the changes of the three time points from baseline to post-surgery within groups were performed by repeated-measures analysis of variance, with Bonferroni post-hoc tests. Statistical hypothesis tests were two-sided. A p-value of <0.05 was considered as statistically significant. Statistical analyses were performed using SPSS15.0 .A porcine model of severe diffuse coronary artery stenosis was successfully established in 12 of 14 animals. One animal died due to refractory bradycardia induced by balloon expansion and one animal failed due to a stenosis rate of <90%. The 12 animals were equally and randomly divided into control and selective coronary vein bypass graft (SCVBG) groups. Control group animals received exploratory thoracotomy and experimental animals received arterialization of the middle cardiac vein via the internal mammary artery while maintaining heart rate. At baseline, animals' weights and diffuse coronary artery stenosis rates were comparable in control and SCVBG groups. After surgery, significantly higher mean blood flow was observed in the endocardium and epicardium of the SCVBG group compared to that in the control group , p<0.001; mean epicardial blood flow: 0.29 vs. 0.22, p = 0.014). The transmural blood flow was also higher in the SCVBG group than in the control group . In addition, the SCVBG animals had mean internal mammary artery blood flow of 44.50 and 43.33 and PI of 0.73 and 0.80 at 6 hours and at 3 months after surgery. No significant change was observed in mean blood flow of internal mammary artery and PI between 6 hours and 3 months after surgery. No significant differences were found in baseline EF and E/A between the two groups. At 6 hours and at 3 months postoperatively, significantly higher EF, E/A ratio, and FS were observed in the experimental group than in the control group . Also, significantly lower postoperative LVEDD and LVESD were observed in the experimental group than in the control group . In addition, a significant increase of EF from 0.46% at baseline to 0.51% at post-operative 6 hours (p = 0.0038), significant increases of E/A ratio from 1.5 at baseline to 1.64 at post-surgery 6 hours and 3 months (p = 0.0067 and p = 0.009), and significant increases of FS from 37.33 at baseline to 46.67 and 47.0 at post-surgery 6 hours and 3 months were observed in the experimental group (p = 0.013 and p = 0.011). Significant decreases of LVEDD from 64.67 mm at baseline to 55.0 and 51.33 mm at post-operative 6 hours and 3 months were observed (p<0.001 and p = 0.001). Significant decreases of LVESD from 15.0 mm at baseline to 9.5 and 9.67 mm at post-operative 6 hours and 3 months were observed (p = 0.03 and p = 0.04). Additional images are provided showing 3-month follow-up of experimental and control groups, including the ligation site fibrosis status, cardiac wall and myocyte arrangement. The author successfully established a chronic, stenotic coronary artery porcine model focused on the right coronary artery, and blood flow was reconstructed using the intramammary artery for arterialization. Evaluation of this modified SCVBG procedure revealed that the SCVBG group had significantly higher mean blood flow in the endocardium and epicardium compared to controls and the transmural blood flow was also higher. At two time points, 6 hours and 3 months postoperatively, the mean blood flow of the internal mammary artery was 44.50 and 43.33, respectively, and PI was 0.73 and 0.80, respectively. Significantly higher EF and E/A ratios were also observed in the experimental group at these time points. Our satisfactory results in a porcine model show that revascularization is not only possible but that, as we had hypothesized, replacing the saphenous vein with the internal mammary artery as the graft material allows effective reconstruction of blood flow in severe, diffuse coronary artery disease, avoiding potential complications commonly associated with bypass grafting.We are not the only investigators to construct an animal model of CAD and to substitute the mammary artery for SCVBG. Møller et al. constructed a porcine model of CAD, focusing on the left coronary vein, and conducted selective retrograde coronary vein bypass grafting using an off-pump technique, applying various modern methods of monitoring the physiological effects of this procedureGiven the encouraging results from our study and that of Møller et al., we must look at the clinical importance of a chronic CAD model, and the possible reasons for unsatisfactory results of previous studies of cardiac vein arterialization. In previous studies of selective retrograde coronary venous perfusion, animal models of acute myocardial infarction were established by ligating the initial segment of the anterior descending branchModels of chronic myocardial ischemia are mainly established through chronic mechanical compression of the coronary artery, which requires complex open chest surgery and causes great trauma to the animals, who cannot be raised after surgery. This study focused on the right coronary artery for these reasons. In clinical practice, sequential coronary artery bypass grafting is usually adopted, but since the right coronary artery is the terminal branch of the sequential graft, the patency of the right coronary artery bypass graft will directly affect the patency of the entire sequential graft. We applied balloon expansion and stretching to establish a chronic diffuse right coronary artery stenosis model in pigs. A great number of choices are available for experimental animals for model building. Rabbits fed with a high cholesterol diet can develop atherosclerosis, and balloon expansion can cause restenosis lesions, although rabbits are small for difficult heart surgeries. A dog model usually has poor stability because collateral circulation forms too easily in dogs after myocardial ischemia. Nevertheless, Ido et al. (2001) showed that coronary sinus occlusion enhanced coronary collateral flow and reduced subendocardial ischemia in a dog modelA recent report by Yu et al. (2011) described performance of off-pump coronary artery bypass (OPCAB) surgery in 17 patients as a less invasive way to achieve myocardial revascularization. The procedure allows performance of sequential bilateral mammary artery grafting combined with selective arterialization of the coronary venous system; ischemia was reduced in all patients and substantial improvement was seen in cardiac function and quality of life, suggesting a better long-term prognosis and the likelihood of extensive application of the techniqueThe limitations of coronary angiography must be considered when evaluating results of the present study. In depth studies on the hemodynamics and pathophysiology of the coronary artery have shown that coronary angiography as a single modality is not a unique standard able to guide the performance of vascular reconstructive surgery. Coronary angiography fails to completely evaluate the physiological functions of the stenotic coronary artery and therefore we cannot determine whether the stenotic coronary artery may cause evident myocardial ischemia by applying coronary angiography alone. Fractional flow reserve (FFR), however, may provide additional evidence on functional changes in the stenotic coronary artery based on evaluation by coronary angiography and also provide evidence for therapeutic strategies The present study has certain other limitations. First, the sample was small with only a limited number of animals in each group, which may affect analysis of results. Further, because controls were not subject to revascularization, results cannot be compared to other forms of arterialization in the myocardial venous system. We also did not have data to indicate whether angiogenesis was improved in the ischemic myocardium. However, our results indicate the potential benefit of direct revascularization and suggest that further study is needed to confirm this benefit in clinical practice.In conclusion, successful cardiac vein arterialization via intramammary artery anastomosis in a porcine model suggests that this may be a viable method for reconstructing blood flow in chronic, severe coronary artery disease. The methods we have employed were shown to be effective and to avoid complications associated with bypass grafting in CAD; they may be suitable for chronic CAD patients who require compensation of collateral circulation, which may have a synergistic effect on retrograde flow from the arterialized coronary vein. This alternate technique could be especially relevant for patients with severe, diffuse coronary artery disease whose arteries are inaccessible for CABG and PCI or who have had previous failed surgeries or restenosis.


Reported cases of “pure” duplication of the entire short arm of chromosome 16 (16p) are rare, with only 7 patients described in the literature. We report on a female infant with de novo 16p duplication localized to the short arm of chromosome 6, detected by chromosomal analysis and characterized by array CGH and fluorescence in situ hybridization. This baby girl presented with clinical symptoms characteristic of patients with duplications of the short arm of chromosome 16: psychomotor retardation, constitutional growth delay and specific dysmorphic features, including proximally placed hypoplastic thumbs. In addition, she exhibited evidence of neonatal hemochromatosis as shown by direct hyperbilirubinemia, iron overload and elevated liver enzyme levels. To our knowledge, this is the first report of signs of neonatal hemochromatosis in a patient with 16p duplication. Duplication of the short arm of chromosome 16 is a well described condition with severe prenatal and postnatal growth retardation, dolichocephalus, facial dysmorphisms, clinodactyly and hypoplastic thumbs -8. TheseHFE gene) [Neonatal hemochromatosis (NH) is a severe clinical and pathological entity that affects newborns and is characterized by liver failure associated with hepatic and especially extrahepatic iron deposition . There iFE gene) . In contFE gene) ,11.de novo duplication of the entire short arm of chromosome 16.Here, we report the case of a female infant with neonatal liver failure suggestive of NH together with psychomotor retardation, external malformations and facial dysmorphisms. Cytogenetic analysis revealed the presence of additional chromosomal material at the terminal short arm of chromosome 6. Characterization by both aCGH and FISH analyses defined that additional chromosomal region as a th week of gestation. A fetal ultrasound at that time demonstrated a female singleton fetus with intrauterine growth retardation and anomalies of the central nervous system, heart and midface. A caesarean section was performed at the end of the 34th gestational week due to pathologic cardiotocograph changes.The patient is the second child of healthy non-consanguineous parents. Her father was 46 years old and her mother 29 years old when she was born. She had one healthy older brother, and there was no family history of congenital anomalies, intellectual disability (ID) or miscarriages. Following an otherwise uneventful pregnancy, the mother had premature rupture of her membranes at the beginning of the 34At birth, the baby weighed 1860 g (10-25th percentile) and measured 39 cm in length (<3rd percentile), and her head circumference measured 30.5 cm (25-50th percentile). Her Apgar scores were 5/7/9, and her cord blood pH was 7.35. The newborn screening was normal. Due to respiratory problems, she was transferred to the neonatal intensive care unit. As the respiratory situation did not improve and weaning from CPAP-support was not possible a tracheostomy was performed.At three days of age, the patient’s facial appearance Figure a-b was cPostnatal echocardiography demonstrated a complex cardiac defect with ectasia of the root of the pulmonary artery, a dysplasic aortic valve, a persistent left vena cava, an atrial and subaortic ventricular septal defect. A persistent Ductus arteriosus (PDA) required surgical ligation.Cranial ultrasonography revealed agenesis of the corpus callosum, enlargement of the posterior ventricles, a large cisterna magna and sparse gyration. Her electroencephalogram was normal. Due to the presence of anal atresia with a subvaginal fistula, the baby required a colostomy on her third day of life. Ultrasound of the abdomen was normal.Direct hyperbilirubinemia was found, and it lasted for three months. Serum transaminases, lactate dehydrogenase and γ-glutamyl transferase also showed prolonged elevation. Beginning at 4 weeks of life, her ferritin levels were markedly elevated and fluctuated between 1984 and 4254 μg/L (reference range: 10–204 μg/L). Her transferrin saturation peaked at 92% (reference range: 11–52%) at 7 weeks of life. Laboratory tests for toxoplasmosis, syphilis, coxsackie virus and echo virus showed normal results.Unexpectedly, at 5 months of age, her physical condition deteriorated, and she died within a few hours from cardiovascular failure.Blood samples were collected from the patient and her parents after obtaining the parents’ signed informed consent. The manuscript is a retrospective case report that does not require ethics committee approval at the University of Göttingen. For rapid karyotyping in the newborn, FISH analysis was performed using multicolor DNA probes for chromosomes 13, 18, 21, X and Y . Metaphase chromosome spreads were prepared from phytohemagglutinin (PHA)-stimulated peripheral blood cultures using standard protocols, and 10 GTG-banded metaphases were analyzed.Genome-wide copy number scans were performed with the patient’s and her parents’ lymphocyte DNA using an Agilent SurePrint G3 Human CGH Microarray Kit 4×180K and was read using an Agilent Microarray Scanner G256BA along with Agilent Feature Extraction Software V9.1  according to the manufacturer’s instructions. The results were analyzed using Agilent Cytogenomics 2.0 software. To confirm the aCGH results of the newborn FISH was performed using the DNA probes 16pter and 22q11.2 .FISH analysis of the newborn’s interphase chromosome spreads was performed immediately after birth with probes for chromosome 13, 18, 21, X and Y but yielded normal results. GTG-banded metaphases prepared from lymphocyte cultures demonstrated distal elongation of the short arm of chromosome 6, preliminary karyotype 46,XX,add(6)(p25). Because cytogenetic analysis alone could not identify the origin of this chromosomal region, aCGH was performed, which demonstrated duplication of the entire short arm of chromosome 16 (16pter to 16p11.2) with a size of approximately 33.83 Mb  of NH and the availability of prophylaxis of GALD-related NH by prenatal treatment with intravenous immunoglobulin ,11 make Consequently, a definite recommendation regarding maternal prenatal treatment with intravenous immunoglobulin during future pregnancy could not be made, and the mother’s prognosis for the recurrence of a neonatal liver failure in a subsequent offspring could not be established. The risk of recurrence of 16p duplication in future pregnancy is very low because this chromosomal aberration has occurred de novo. However, a low probability of germ cell mosaicism in one of the parents remains.Written informed consent was obtained from the parents of the patient for publication of this Case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.EMCS evaluated the aCGH analysis, participated in the genetic counseling of the patient’s parents and drafted the manuscript. IB was responsible for the cytogenetic analysis and helped with the final version of the manuscript. HK helped with the final version of the manuscript. HK and ML were responsible for the medical treatment of the patient. PB was responsible for the aCGH analysis and helped with the final version of the manuscript. RA participated in the evaluation of the aCGH analysis. BZ did the genetic counseling of the patient’s parents. All authors read and approved the final manuscript.


Hurricanes Katrina, Rita, Gustav, and Ike deposited large quantities of sediment on coastal wetlands after making landfall in the northern Gulf of Mexico. We sampled sediments deposited on the wetland surface throughout the entire Louisiana and Texas depositional surfaces of Hurricanes Katrina, Rita, Gustav, and the Louisiana portion of Hurricane Ike. We used spatial interpolation to model the total amount and spatial distribution of inorganic sediment deposition from each storm. The sediment deposition on coastal wetlands was an estimated 68, 48, and 21 million metric tons from Hurricanes Katrina, Rita, and Gustav, respectively. The spatial distribution decreased in a similar manner with distance from the coast for all hurricanes, but the relationship with distance from the storm track was more variable between events. The southeast-facing Breton Sound estuary had significant storm-derived sediment deposition west of the storm track, whereas sediment deposition along the south-facing coastline occurred primarily east of the storm track. Sediment organic content, bulk density, and grain size also decreased significantly with distance from the coast, but were also more variable with respect to distance from the track. On average, eighty percent of the mineral deposition occurred within 20 km from the coast, and 58% was within 50 km of the track. These results highlight an important link between tropical cyclone events and coastal wetland sedimentation, and are useful in identifying a more complete sediment budget for coastal wetland soils. The infrequent, but intense, storm surge events regularly punctuating the microtidal Louisiana coastal zone can reach heights exceeding normal tidal cycles by several orders of magnitude. Data from offshore buoys during Hurricane Katrina, for example, show that the maximum wave height 100 km east of the hurricane path was about 17 meters, with a wave period exceeding 14 seconds The suspended sediment is carried in waves whose fate depends on the bathymetry encountered as they propagate shoreward. Massive hurricane depositional events have been observed offshore of coastal Louisiana. Following hurricanes Katrina and Rita, the deposition on the inner continental shelf was estimated to be 1160 MMT (million metric tons) - 10 times the average annual deposition rate for that area 234Th activity, which was interpreted to indicate an offshore sediment origin Sediment deposition in inshore waters has also been reported following hurricane events. Sediment accumulations up to 2 m thick were found in tidal creeks of the Florida Everglades following Hurricane Andrew in 1992 Sediment deposition on the wetland platform has been more widely studied than subaqueous deposition, possibly because it is more readily distinguished from the existing soil surface. Early reports noted storm surge deposition on wetlands as far back as Hurricane Audrey in 1957 The deposition of these sediments is influenced by a variety of factors related to various physical and biological conditions, but there is little understanding about the spatial distribution of the sediment deposited, and how it varies from storm to storm. The focus of this analysis is to investigate the spatial distribution of sediments on coastal wetlands following hurricanes at an event or coast-wide scale. We conducted a large-scale spatial analysis, and incorporated an analysis of smaller scale variations by examining the residuals within a landscape-scale model. The objective of this research is to determine how the quality and quantity of sediments deposited during 4 hurricanes in the last 10 years varies spatially within a Louisiana coastal landscape.We sampled sediments deposited within four months of landfall for Hurricanes Katrina (2005), Rita (2005), Gustav (2008) and Ike (2008) . These dNo specific permits were required for the described field studies. We did not sample in any areas marked as private property or that displayed “posted” or “no trespassing” signs.The field sampling was designed to encompass the entire depositional area following Hurricanes Katrina, Rita, Gustav, and Ike. The entire Louisiana depositional areas were sampled , but theEach observation consisted of measuring sediment thickness, percent mineral content, and bulk density. In order to accurately quantify sediment deposition, several preliminary samples were taken before the final sample was collected using the method discussed by Turner and others −2. Bulk density and mineral content were compared at increasing distances from storm path and the coastline.Sediment samples were dried and analyzed for inorganic content by loss on ignition. Mineral accretion was used to model sediment deposition, and derived by multiplying deposition depth by sample bulk density and percent mineral content to yield mineral accretion in g cm−2 near the bay to 0.6 g cm−2 19 km inland. Samples were prepared for analysis using standard procedures 2O2. The sediment samples were then dispersed with 0.05% sodium hexametaphosphate. Sediment grain size distributions were determined using a laser diffraction particle size analyzer (Beckmann Coulter LS 13 320).We used a subsample from 24 locations impacted by Hurricane Gustav to investigate grain size distributions within the event. The subsample was drawn from an area 30 km east of the track at the head of Terrebonne Bay, where sampling density was greatest, and included a gradient of inorganic sediment from 6.6 g cmAll spatial analyses were conducted using ArcInfo 10.0 . Two distinct interpolation methods were tested to estimate depositional patterns on a coastwide scale. A kriging analysis was not a suitable method when observed deposition lacked significant spatial autocorrelation, as was the case in several locations within the sampling areas. Inverse distance weighting (IDW), which does not assume input data are spatially autocorrelated, was applied to these datasets and provided the most statistically viable results. Inverse distance weighting is considered an exact interpolator, in that the output surface passes through each observation. The areas between sampled locations are estimated based on nearby samples, and are weighted to favor closer samples over more distant observations. We selected model parameters for neighborhood and power that minimized interpolation error. We could only estimate the Louisiana portion of the deposition during Hurricane Ike because the depositional area in Texas was not sampled.2. About 15% of the study area is in Universal Transverse Mercator zone 16-north, but the difference in area computed in its native zone compared to zone 15-north at this latitude was 0.1% in one area tested.The method used to bound the depositional area was imperfect because the observed areas with zero deposition did not always completely encircle the areas with deposition. Based on decay relationships within areas that were more densely sampled, we applied a 20 kilometer buffer to each sample area, and assumed that no deposition occurred beyond this boundary. This served as the outer bound of spatial interpolation. To analyze Hurricanes Katrina and Gustav we divided the study areas east and west of the Mississippi River, and recombined them following interpolation. All data were processed in North American Datum 1983 Universal Transverse Mercator zone 15 north and a pixel size of 1 km2) for each event was multiplied by the area of wetlands in each area to produce an estimate of total deposition. These estimates were formulated for all Louisiana coastal wetlands, and all Texas coastal wetlands to Galveston Bay. The root mean square error, also in units of mineral sediment deposition per cm2, from each interpolation was used to calculate interpolation error terms for each estimate.We used United States Fish and Wildlife Service National Wetlands Inventory data to calculate wetland areas because it is consistent in coverage and source data throughout the study area. The mean pixel value  and directly along marshes that extend to the Gulf. Land-to-water ratios within each area were also calculated to test if the results reflected differing wetland coverage rather than deposition.The storm surges of Hurricanes Katrina, Rita, and Gustav deposited 67.8±8.6, 48.2±8.3, and 20.6±3.9 (estimate ± root mean square error) million metric tons (MMT) of inorganic sediment on coastal wetlands, respectively, when calculated using the IDW method , Table 1The storm surge from Hurricane Ike resulted in sedimentation farthest from the storm path (214 km) when compared to the other three storms . SedimenHurricane Katrina deposited 80% of the total amount within 60 km of the hurricane track, which is more than from Hurricanes Rita or Gustav, . By compThe patterns of deposition relative to distance from the coast were more consistent than distance from storm track, and nearly identical for the two deltaic plain events, Katrina and Gustav, as a percent of their total deposition . The inl−2, 28 km from the storm path and 5 km from Lake Borgne, while the maximum for the remainder of the study area was 9.92 g cm−2, 8 km from the path and 3 km from Breton Sound. The percent mineral sediment in this area was as much as 98%, which was much greater than the 66% observed in wetlands 10 km south of this area. This high mineral content was more similar to that of areas 0 to 15 km from the storm path (µ = 93%). In contrast to Hurricane Katrina, the maximum sedimentation in the other storm events occurred nearest coastal bays and the Gulf of Mexico, and decreased inland.The sediment characteristics in wetlands near Bayou Bienvenue, the area bounded by the Mississippi River on the west and the Mississippi River Gulf Outlet on the east, were similar to that of the surrounding areas following Hurricane Katrina. The maximum observed mineral accretion in this area was 20.81 g cm−3 ±0.04 SE .There were significant trends in organic content and bulk density with distance from the coast, but the relationships were noisy . There wr2 = 0.35, F = 13.68, p = 0.001) and bulk density  = 5.86, p = 0.023). The peak bulk density was observed 31 km east of the storm path, which is also where the greatest deposition occurred. The bulk density of Hurricane Katrina sediment samples decreased until 100 km from the hurricane track, and then there was a sharp increase, with the maximum bulk density observed at 148 km from the track. The mineral content of samples collected immediately after Hurricane Gustav decreased slightly with increasing distance from the hurricane track  = 4.44, p = 0.039), but there was no significant relationship between bulk density and distance from the hurricane track, which initially increased with distance, but then decreased in samples closer to Mississippi River levees. The peak bulk density from Hurricane Gustav was observed 35 km to the east of the hurricane track. Hurricane Ike sediments exhibited similar patterns to the other Chenier Plain event, Rita. Sediment mineral content  = 10.74, p = 0.007) and bulk density  = 7.89, p = 0.017) both decreased significantly with distance from the storm track, but there were fewer samples than the other events, and no samples closer than 75 km from the storm track.These results demonstrated that sediment characteristics with respect to distance from the storm track were more variable between depositional events. For this reason, each event was analyzed separately. With increasing distance from track, sediments deposited following Hurricane Rita decreased in mineral content  = 7.19, p = 0.013; r2 = 0.26, F = 7.88, p = 0.010). Overall, the mean grain size was 34.8 µm, which is classified as silt. However, 17.3% of the total sample volume was comprised of grains larger than very fine sand (63.4 µm), and the range extended to fine sand (256.9 µm).The mode grain size within the subsampled area of Hurricane Gustav decreased with increasing distance from shoreline (8 t yr−1 (100 MMT yr−1), but the spatial and temporal distribution of these events can vary widely. The amount of sediment deposited on coastal wetlands ranged considerably between the hurricanes, with Hurricane Katrina depositing nearly three times the total sediment of Hurricane Gustav. Hurricane Rita deposited about 70% of the amount left by Hurricane Katrina. Our combined estimate of 116 MMT for Hurricanes Katrina and Rita is 89% of the 131 MMT reported by Turner and others using the same dataset The quantity of sediment left on the marsh surface following tropical cyclone events can exceed 10A previously published estimate of the total deposition from Hurricane Ike is 16.2 MMT, but the sampling did not include the entire area impacted by the storm Sediment deposition tended to be greatest just east of the storm path, and nearer the coastline, for the two storms making landfall west of the Mississippi River. The areas of high deposition generally coincided with the highest storm surges in previously reported models, but modeled surges covered a broader area The majority of sedimentation (80%±5 SE) occurred within the first 20 km inland, and declined exponentially. With distance from the storm track, about half (48%±10 SE) of the total occurred within the first 50 km, but the distribution was more variable. Comparing between the three events, there are remarkably consistent depositional patterns along a shoreline-to-inland gradient. These patterns were more variable with distance from the path, because the high sedimentation during Hurricane Katrina was closest to its storm track, whereas the other two events peaked 50 km east of the storm tracks. A similar relationship with distance from landfall was reported for Hurricane Ike, where deposition was greatest about 40 km east of landfall, and then decreased with distance The high deposition we observed in the Bayou Bienvenue area following Hurricane Katrina was anomalous compared to nearby sample locations and the otherwise decreasing inland trend, but is consistent with high storm surge energy reported for that area The reduction in storm surge as it propagates shoreward is reflected in observations of deposition ranging from offshore to inshore ponds and wetlands. Offshore deposition in 2005 was at least 10 times greater than our estimate for wetlands 2 was observed Landscape-scale wetland sedimentation events have been observed in other areas following tropical cyclones, such as the Florida coast. In southwest Florida following Hurricane Andrew in 1992, an average deposition of 7 cm (range: 0–20 cm) across an area of 110 kmThe areas of highest mineral content and bulk density were near the coast, which is where storm surge energies were greatest, and decreased inland. Similar trends in organic content and bulk density were observed following Hurricane Andrew in Louisiana Although grain size measurements were more spatially limited, an inland trend of decreasing grain size was also observed in Florida following Hurricane Andrew, but the Florida sediments were predominantly carbonate-rich sand 2 of new water area was observed, but a portion of this may be due to the removal of floating aquatic vegetation rather than wetland Some of the sediment deposited during storms from inshore sources may include newly eroded wetland soils. The amounts of this lateral erosion of coastal wetlands during tropical cyclones can vary widely; some effects recover within months, while others may remain for decades The contribution of these powerful, but infrequent, events to the long-term sedimentary record of wetlands may vary widely −1Tropical cyclone events represent both potential agents of land loss, but also a significant input of sediment to the wetland soil profile. In a deltaic system with rates of isostatic sea level rise approaching one cm yr


TERT) and cleft lip and palate transmembrane 1-like (CLPTM1L) genes, which have been implicated in carcinogenesis. A common sequence variant, rs401681, located in an intronic region of CLPTM1L, has been reported to be associated with lung cancer risk based on genome-wide association study. However, subsequent replication studies in diverse populations have yielded inconsistent results. In addition, genetic variants at 5p15.33, including rs401681, have been shown to be involved in the susceptibility to multiple malignancies. Nevertheless, the role of these TERT-CLPTM1L variants in the etiology of esophageal squamous cell carcinoma (ESCC) remains unknown.The human 5p15.33 locus contains two well-known genes, the telomerase reverse transcriptase  and 860 healthy individuals.P = 0.031; CT/TT vs. CC: adjusted OR = 0.786; 95% CI = 0.635–0.972, P = 0.026). Stratification analysis by histology type indicated that rs401681 T genotypes were associated with a significantly reduced risk of both adenocarcinoma and squamous cell carcinoma. Furthermore, no significant association was observed between rs401681 and the risk of ESCC .Logistic regression analyses revealed that rs401681 T genotypes were associated with a significantly decreased risk of lung cancer (CT vs. CC: adjusted OR = 0.782, 95% CI = 0.625–0.978, Our findings provide further evidence supporting rs401681 as a genetic variant associated with the risk of lung cancer. In addition, we investigated the correlation between the rs401681 variant and the risk of ESCC in a Han Chinese population, and our results suggest that this genetic variant may not be involved in ESCC risk. Lung cancer has been the most common cancer in the world for several decades as well as the most common cause of death from cancer. In addition, esophageal cancer is the eighth most common cancer worldwide, with 482,000 new cases estimated in 2008, and represents the sixth most common cause of death from cancer, being responsible for 407,000 deaths TERT-CLPTM1L variants at 5p15.33 may influence the risk of developing lung cancer as well as other types of cancer TERT and CLPTM1L are attractive candidate genes, as they have both been implicated in carcinogenesis. TERT encodes the catalytic subunit of telomerase, an enzyme that maintains telomere ends by adding the telomere repeat TTAGGG. Telomeres are the protein-bound DNA repeat structures at the ends of chromosomes and are important in maintaining genomic stability CLPTM1L, which has been reported to be involved in the cellular response to genotoxic stress and cisplatin resistance CLPTM1L has been observed in several types of cancer, including lung cancer Recently, genome-wide association studies (GWASs) have shown that common CLPTM1L, has been reported to be associated with lung cancer risk based on genome-wide association study The rs401681 polymorphism, located in an intronic region of TERT-CLPTM1L region, including rs401681, have shown possible associations in multiple cancers In most cases, the common genetic variants that have been associated with cancer risk seem to be specific to a particular cancer type. However, single-nucleotide polymorphisms (SNPs) in the This case-control study included 726 patients with lung cancer, 753 patients with esophageal cancer and 860 healthy controls. All of the subjects in this study were genetically unrelated ethnic Han Chinese individuals from Shandong Province in North China. The cases with histologically confirmed primary lung or esophageal cancer were recruited from 2011 to 2013 at Qilu Hospital and the Provincial Hospital Affiliated with Shandong University . The histological type of the tumors was diagnosed on the basis of biopsies or resected specimens. The esophageal carcinomas were all squamous cell carcinomas. Patients with primary cancer outside the lung and the esophagus and with cancer of unknown primary origin were excluded. The healthy participants, who presented no history or diagnosis of cancer or genetic disease, were recruited from individuals who visited the same hospitals for a routine check-up during the same period. Patients or controls who had recently (in the last 6 months) received blood transfusions were excluded. Cases and controls were frequency matched by age (±5 years) and sex. All participants were given an explanation of the study, and written informed consent was obtained from each subject. This study was approved by the Ethics Committees of the Provincial Hospital Affiliated with Shandong University.A structured questionnaire was completed for each case and control by a trained interviewer to obtain demographic data  and information on related risk factors . Individuals who had smoked one cigarette per day for over 1 year were considered smokers. Subjects were considered alcohol drinkers if they drank at least once per week for over 1 year.A venous blood sample was collected from each subject, and genomic DNA was extracted within 1 week after sampling using the RelaxGene Blood DNA System (Tiangen Biotech (Beijing) Co., Ltd.) according to the manufacturer’s protocol. The quality and quantity of DNA were checked using GeneQuant Pro (Amersham Biosciences). The rs401681 polymorphism was genotyped using TaqMan methodology in 96-well plates and read with Sequence Detection Software  using the Applied Biosystems (ABI) 7500 Real-Time PCR System. Genotyping was performed without knowledge of the subjects’ case or control status. The genotyping assays were randomly repeated for 12% of the samples, and the results were 100% concordant.2 test was used to examine the differences in the distributions of category variables, including demographic characteristics, selected variables, and genotype/allele frequencies, between the cases and controls. Hardy–Weinberg equilibrium was tested using a goodness-of-fit χ2 test to compare the observed genotype frequencies to the expected frequencies among the control subjects. The associations between the rs401681 polymorphism and cancer risk were estimated by computing the odds ratios (ORs) and their 95% confidence intervals (CIs) through logistic regression analyses for crude ORs and adjusted ORs when adjusting for age, sex and smoking and drinking status. Statistical analyses were carried out using SPSS (version 19) and Stata (version 12.0), and a P-value of <0.05 was set as the criterion for statistical significance.Pearson’s χP<0.001 for both). In addition, the proportion of alcohol drinkers in both the lung cancer and ESCC patient groups was significantly different from that in healthy controls. Therefore, these variables were further adjusted for in subsequent multivariate logistic regression analyses. Within the lung cancer group, patients with squamous cell carcinoma, adenocarcinoma, small-cell carcinoma, and other carcinomas accounted for 34.6%, 37.7%, 19.4%, and 8.3% of the subjects, respectively. The esophageal carcinomas were all squamous cell carcinomas.A total of 1,479 cancer patients (726 with lung cancer and 753 with ESCC) and 860 healthy controls were included in the analysis. The demographic distributions of the lung cancer and ESCC patients and healthy controls are shown in P = 0.121). However, a significant difference in the combined genotype (CT+TT) frequency was observed between patients with lung cancer and control subjects (P = 0.040).The genotype and allele frequencies of the rs401681 polymorphism in the lung cancer and ESCC patients and healthy controls are presented in P = 0.031; CT/TT vs. CC: adjusted OR = 0.786; 95% CI = 0.635–0.972, P = 0.026). Moreover, the rs401681 T allele was borderline significantly associated with a lower risk of lung cancer. We further evaluated the association between rs401681 and lung cancer risk stratified by histology type. As shown in As shown in For the patients with esophageal carcinoma, the genotype frequencies of the rs401681 variant were not significantly different between the cases and controls. In addition, the rs401681 T allele was less frequent among cases than among controls, though this difference was not statistically significant . FollowiCLPTM1L rs402710, which is in strong LD with rs401681, has been reported to be associated with significantly higher levels of bulky aromatic/hydrophobic DNA adducts in lung tissue adjacent to tumors TERT rs2736100 and CLPTM1L rs401681) act as determinants of lung cancer risk, differentially impacting lung cancer histology. The risk associated with rs2736100 is largely confined to adenocarcinoma. However, rs401681 influences the risk of all lung cancer histologies, but its strongest effect is on squamous cell carcinoma Telomere biology has been implicated in the pathogenesis of a variety of lung diseases A recent meta-analysis by cancer type demonstrated that rs401681 [T] carriers show a modestly increased risk of pancreatic carcinoma and skin melanoma. In contrast, a modest reduction of risk was observed for bladder, lung and prostate cancer as well as basal cell and skin squamous cell carcinomas TERT-CLPTM1L that we did not include in the present study of ESCC risk. Finally, we incorporated the major confounding variables in our analysis. However, other detailed risk factors were not included in our logistic regression models because of incomplete or missing information.Several limitations of our study should be discussed. First, selection bias may not be avoidable. However, the cases and healthy control subjects were recruited from the same area and matched for age and sex. Second, the sample size in this study was not sufficiently large to draw a firm conclusion, and the statistical power of the study may be limited. Third, we cannot exclude the potential roles of other functional SNPs or tagging SNPs for TERT-CLPTM1L locus need to be investigated to further evaluate the role of this genomic region in esophageal tumorigenesis.In summary, our findings suggested that rs401681 may modify susceptibility to lung cancer but not ESCC. Although no significant association was found between this genetic variant and susceptibility to ESCC, more variants at the


Recent studies have shown that urinary excretion of podocyte proteins is an indicator of podocyte injury, and that podocyte abnormalities and elevated concentrations of Amadori-modified glycated albumin (AGA) are linked to the development of diabetic nephropathy and to each other. We evaluated relationships between urinary markers of podocyte damage, increased AGA and filtration function in rats made diabetic by streptozotocin injection and treated for 8 weeks with a compound that inhibits the formation of AGA, with age-matched nondiabetic and diabetic rats serving as controls. Blood and urine were collected for measurement of glycated albumin, creatinine, albumin, nephrin, podocalyxin, and βig-h3 protein. The elevated circulating concentrations of glycated albumin and higher urinary levels of these podocyte markers as well as of albumin that were observed in diabetic rats compared with nondiabetic controls were significantly reduced in animals receiving test compound, and decrease in urinary biomarkers correlated with reduction in AGA. The results provide evidence that lowering the concentration of AGA, independent of filtration status and hyperglycemia, reduces urinary nephrin, podocalyxin, and βig-h3 protein, linking the increased glycated albumin associated with diabetes to podocyte abnormalities and shedding of podocyte proteins into the urine. Although the pathogenesis of diabetic nephropathy remains incompletely delineated, results of recent studies support the hypothesis that damage to glomerular podocytes and abnormalities in podocyte function are intimately involved in the development of diabetic renal disease and in the increased albumin excretion that heralds its onset  associated with diabetes was established with the demonstration that lowering the circulating concentration of AGA without change in blood glucose levels restores the reduced nephrin that is observed in the kidneys of diabetic mice  served as nondiabetic controls. The diabetic rats were divided into two groups, one of which (n = 10) served as the diabetic control and the other (n = 11) received the test compound as the potassium salt. The drug was administered by gavage at a dose of 10 mg kg−1 day−1 in two equally divided portions for eight consecutive weeks commencing 3 days after induction of diabetes. This dosage corresponded to that employed in previous studies in diabetic rodents in which 23CPPA has been shown to reduce glycated albumin concentrations without affecting hyperglycemia  aged 6 weeks and weighing between 120 and 140 g were made diabetic by intravenous injection (50 mg/kg) of streptozotocin  into the tail vein. All diabetic animals had blood glucose levels, measured using a One Touch Glucometer , greater than 250 mg/dL at 48 h after streptozotocin injection. Age-, weight-, and gender-matched Wistar rats . The albumin and nephrin assays are competitive immunoassays with specificities for the rat proteins with respective sensitivities to 0.1 and 0.02 μg/mL, and have intra- and interassay coefficients of variation <5%. The podocalyxin assay is a competitive enzyme-linked immunoassay (ELISA) immunospecific for the rat protein with a sensitivity to 0.5 ng/mL and intra- and interassay coefficients of variation of <7%. The βig-h3 assay is a sandwich ELISA immunospecific for the rodent protein with a sensitivity to 10 pg/mL and intra- and interassay coefficients of variation of <7%. For measurement of plasma creatinine, the method was modified by including a deproteination step with acid tungstate, and the alkaline picric reagent was added after absorption with Fuller's earth, which eliminates interference by confounding chromogens that may falsely elevate plasma creatinine , and correlations were calculated by Pearson's correlation analysis. A P = 0.08 and P = 0.06). Creatinine clearance values were approximately 50% higher in diabetic compared to nondiabetic controls, and were not significantly different from diabetic controls in diabetic rats receiving 23CPPA.General characteristics of the animals conformed to those typically found in this model of experimental diabetes Table . Body wer = 0.98) between the 24-h urinary albumin excretion and the albumin:creatinine ratio (ACR) for all animals. The incomplete normalization of albumin excretion in the diabetic rats receiving test compound is consistent with the persistent and marked hyperglycemia in these animals.Urine albumin excretion was markedly increased in diabetic compared to nondiabetic animals and was significantly reduced in diabetic rats receiving 23CPPA compared to the diabetic controls, with values ∼50% less than those of diabetic controls whether expressed as mg/24 h Fig. A or as mr = 0.94) between the 24-h urine nephrin excretion and the nephrin:creatinine ratio (NCR) for all animals. There was also a significant positive linear correlation (r = 0.63) between the NCR and ACR  and between the βCR and the ACR (r = 0.77) , and with each other (r = 0.93) as shown in Figure Figure 77) Fig. C and D. r r = 0.9 as shownr = 0.58; Fig. r = 0.70; Fig. r = 0.50, Fig. Figure This study employed ELISA methodology to quantify urinary excretion of nephrin, podocalyxin, and βig-h3 in rats with streptozotocin diabetes to explore relationships of these analytes with each other and with albumin excretion, and to determine if administration of a compound that inhibits the formation of AGA affects these markers of podocyte and glomerular dysfunction. The experimental protocol, initiated at ∼6 weeks of age and continued for eight consecutive weeks, was consistent with that employed in other studies using the streptozotocin diabetic rat to assess abnormalities in early diabetic nephropathy. For example, glomerular expression of fibronectin, collagen IV and TGF-β1,smad2/3, and ERK1/2 signaling, urinary TGF-β1 and renal glucose transporter (GLUT 1) have been found to be increased after 6–8 weeks of diabetes, and these parameters are improved with various interventions of 2–3 months duration  but, because this study employed the same methodology in all animals, the higher creatinine clearance values in diabetic compared to nondiabetic rats after 8 weeks of diabetes are viewed as corresponding with the hyperfiltration of early onset and long duration that has been observed in this model of insulin deficient diabetes (O'Donnell et al. In summary, we provide evidence that, 8 weeks after the induction of diabetes, streptozotocin-diabetic rats manifest increased urine levels of nephrin, podocalyxin, and βig-h3 that show positive correlations with each other as well as with albuminuria, and that administration of a compound that lowers the concentration of AGA significantly decreases urinary levels of these analytes, linking the elevated concentrations of AGA associated with diabetes to the abnormalities resulting in podocyte dysfunction and shedding of podocyte proteins in the urine.


Aplysina aerophoba is a common Mediterranean sponge inhabiting shallow waters in the Mediterranean and its area of influence in Atlantic Ocean. This species contains large concentrations of brominated alkaloids (BAs) that play a number of ecological roles in nature. Our research investigates the ecological variation in BAs of A. aerophoba from a scale of hundred of meters to thousand kilometers. We used a nested design to sample sponges from two geographically distinct regions , with two zones within each region (less than 50 km), two locations within each zone (less than 5 km), and two sites within each location (less than 500 m). We used high-performance liquid chromatography to quantify multiple BAs and a spectrophotometer to quantify chlorophyll a (Chl a). Our results show a striking degree of variation in both natural products and Chl a content. Significant variation in Chl a content occurred at the largest and smallest geographic scales. The variation patterns of BAs also occurred at the largest and smallest scales, but varied depending on which BA was analyzed. Concentrations of Chl a and isofistularin-3 were negatively correlated, suggesting that symbionts may impact the concentration of some of these compounds. Our results underline the complex control of the production of secondary metabolites, with factors acting at both small and large geographic scales affecting the production of multiple secondary metabolites.Understanding the scale at which natural products vary the most is critical because it sheds light on the type of factors that regulate their production. The sponge  Conducting research at multiple spatial or temporal scales can substantially increase our understanding of numerous ecological processes, making scale a central problem in ecology . EcologiThe production of marine natural products is an area that could benefit tremendously from a multi scale approach. We know that marine natural products play multiple roles in nature ,3,4 and Aplysina. There are multiple bromotyrosine alkaloids (BAs) described for Aplysina spp. [The production of natural products is widespread in the benthic realm . Spongesina spp. ,30,31,32ina spp. . These cina spp. ,35,36,37ina spp. ,39,40. CAplysina species [Aplysina aerophoba vary between cells [Aplysina aerophoba a perfect organism to investigate natural product variation across multiple spatial scales ranging from a few meters to thousands of kilometers apart, and thus provide a great opportunity to assess the spatial scale at which chemical variation varies the most.BAs may vary remarkably within the same  species , which cen cells , tissuesen cells ,42,43, sen cells , and geoen cells . Moreoveen cells ,42. ThesA. aerophoba [a (Chl a) can be used as a traditional proxy of the abundance of photosynthetic symbionts in sponges [a varied at the same spatial scale as BAs and further investigated whether both Chl a and secondary metabolites were related. It is known that photosynthetic symbionts can contribute to the production of secondary metabolites ,45,46 anerophoba ,47. Sinc sponges ,42,48,49a? In this study we tested these hypotheses by looking at the spatial variation in BAs and Chl a of the sponge Aplysina aerophoba in neighboring bays to locations over 2500 km apart. We followed a nested design with two distant geographic regions, two zones within each region, two locations within each zone, and two sites within each location. This design calculates the magnitude of the variance attributable to the four spatial scales and will suggest the most relevant scale at which chemical variation should be addressed in this species. We found a spatial scale where BAs and Chl a varied the most and shed light on the complex mechanisms behind the production of natural products within a single species. This is the first report to take a broad spatial approach in marine chemical ecology, but further research will clarify whether this complex trend in the production of natural products is common among benthic organisms. Is there a spatial pattern of variation in BAs? Do BAs vary the most between, near, medium, or far away locations? Do individual BAs follow the same pattern of variation? Do these patterns match the pattern of variation in Chl a concentration (Chl a) of Aplysina aerophoba. We identified and quantified the four major brominated alkaloids in our samples: aerophobin-1 , aerophobin-2 , aplysinamisin-1 , and isofistularin-3 , by comparing their retention times and UV profiles to those of purified, characterized standard compounds. We quantified a total of 126 samples , between 49% and 77% remained unexplained . Experimental errors, however, would either amplify or reduce our perception of the existing natural variation. Since our methods minimized compound alteration in this species and were consistent across all samples, we believe that our study accurately describes the natural variation in the production of BAs in xplained ; thus ita slightly decreased with increasing depth, which explained over 15.29% of the variation in Chl a in our samples , we a  was collected by scuba diving at different sampling sites of Tenerife (Canary Islands) and Cap de Creus (Northwestern Mediterranean)  . We coll−1 and the injection volume was 100 µL. Peaks were detected at 320 nm and each BA compound detected was identified and carefully isolated. Dry pure BAs were recovered using rotatory evaporation for use as standards. Then, series of dilution on pure compounds coupled to peak area calculation in HPLC (at 245 nm) allowed tracing calibration-curves. The major compounds observed in the HPLC chromatograms were characterized by classic spectrometric techniques  and by their retention times. For BA isolation, 50 g of freeze-dried sponge were extracted three times  with methanol . The crude extract (CE) was first fractionated by Flash-Chromatography. High Performance Liquid Chromatography (HPLC) was performed on a Waters HPLC with an Alliance separation module 2695, column heater, and 2998 photodiode array detector. Separation was achieved using a hydrophobic column . Mobile phase consisted of 70% of MeOH, 30% of MilliQ water; the flow rate was 3 mL·min−1. Samples were injected in 10 µL volumes and column temperature was maintained at 30 °C. Peaks were identified at 245 nm and integrated by applying the detector response based on peak areas to calibration curves. Concentrations of brominated compounds were expressed as mg·g−1 of dry mass of sponge tissue. For BA quantification, approximately 50 mg of freeze-dried sponge tissue was extracted three times with 1.5 mL of MeOH in an ultrasonic tank for 15 min each time. The CE was filtered through a 20 μm polytetrafluoroethylene filter (PTFE) and added in a 5 mL beaker. The final volume was adjusted to 5 mL with MeOH and an aliquot of 1.5 mL was passed through a 13 mm, 0.2 μm PTFE syringe-filter before HPLC injection. Separation was achieved using a hydrophobic column  with a mobile phase of buffered (0.1% trifluoroacetic acid) water and acetonitrile. We used a linear gradient from 30% to 80% acetonitrile over 18 min, with an additional 10 min at 100% acetonitrile at the end of the run, with a flow rate of 0.4 mL·mina (Chl a) as an indication on the density of autotrophic symbionts within A. aerophoba. Approximately 100 mg of freeze-dried sponge tissue was extracted with 5 mL of 90% acetone for 12 h in the dark at 1 °C. 2 mL of the extract solution were passed through a 0.2 μm PTFE syringe-filter. The 8452A diode array spectrophotometer (Hewlett Packard) was used to perform the absorbance measurements. We measured absorbance at four wavelength  according to the trichromatic equation of Jeffrey and Humphrey [a concentration (1). Chl a was expressed as ng·mg−1 of dry mass of sponge tissue.We used the concentration of Chlorophyll Humphrey  to calcua content. Nested analyses of covariance (ANCOVAs) were performed on ranked compound abundances and Chl a concentrations with region (2 regions), zone (4 zones), location (8 locations), and sampling site (16 sites) as factors  of the variation in BAs. This minor effect was however overshadowed by the larger influence of spatial scale in the concentrations of BAs. Depth played a trivial role, if any, regulating the concentration of BAs in A. aerophoba remains an open question. There is indirect evidence that supports the role of symbionts, including the correlation between Isofistularin-3 and chlorophyll a detected in this study. The circumstantial evidence available provides a number of testable hypotheses that, with the use of new molecular techniques, should contribute to the development of this particularly elusive research area.The putative role of symbionts on the production of BAs in


Genomic structure in a global collection of domesticated sheep reveals a history of artificial selection for horn loss and traits relating to pigmentation, reproduction, and body size. Through their domestication and subsequent selection, sheep have been adapted to thrive in a diverse range of environments. To characterise the genetic consequence of both domestication and selection, we genotyped 49,034 SNP in 2,819 animals from a diverse collection of 74 sheep breeds. We find the majority of sheep populations contain high SNP diversity and have retained an effective population size much higher than most cattle or dog breeds, suggesting domestication occurred from a broad genetic base. Extensive haplotype sharing and generally low divergence time between breeds reveal frequent genetic exchange has occurred during the development of modern breeds. A scan of the genome for selection signals revealed 31 regions containing genes for coat pigmentation, skeletal morphology, body size, growth, and reproduction. We demonstrate the strongest selection signal has occurred in response to breeding for the absence of horns. The high density map of genetic variability provides an in-depth view of the genetic history for this important livestock species. During the process of domestication, mankind recruited animals from the wild into a captive environment, changing their morphology, behaviour, and genetics. In the case of sheep, domestication and subsequent selection by their animal handlers over thousands of years has produced a spectrum of breeds specialised for the production of wool, milk, and meat. We sought to use this population history to search for the genes that directly underpin phenotypic variation. We collected DNA from 2,819 sheep, belonging to 74 breeds sampled from around the world, and assessed the genotype of each animal at nearly 50,000 locations across the genome. Our results show that sheep breeds have maintained high levels of genetic diversity, in contrast to other domestic animals such as dogs. We also show that particular regions of the genome contain strong evidence for accelerated change in response to artificial selection. The most prominent example was identified in response to breeding for the absence of horns, a trait now common across many modern breeds. Furthermore, we demonstrate that other genomic regions under selection in sheep contain genes controlling pigmentation, reproduction, and body size. Man's earliest agricultural systems were based on the captive management of sheep and goats. The transition from hunting to animal husbandry involved human control over the reproduction, diet, and protection of animals. The process of domestication was initiated approximately 11,000 years ago in the Fertile Crescent ovine SNP50 Beadchip, an array consisting of approximately 50,000 evenly spaced SNP. We present the relationship between breeds in terms of divergence time, estimated from the extent of haplotype sharing. Secondly, we sought to characterise the genetic legacy that selection and adaptation have imparted on the sheep genome. By performing a genome-wide scan for the signatures of selection, 31 genomic regions were identified that contain genes for coat pigmentation, skeletal morphology, body size, growth, and reproduction. By combining the collection of a global sample of ovine breeds with the ability to interrogate 50,000 genetic loci, the results provide unprecedented insight into the phylogeographic structure of sheep populations and the results of centuries of breeding practices.Patterns of genetic variation have long proven insightful for the study of domestication, breed formation, population structure, and the consequences of selection. Variation within the mitochondrial genome has documented the global dispersal of two major haplogroups in modern sheep ovine SNP50 Beadchip, an array consisting of SNP derived from three separate sequencing experiments  within breeds and between regions . This appears much less pronounced in sheep compared with human migration out of Africa Ne, Ne of 150 or less Ne exceeding 500 and only two sheep populations showed evidence of a comparatively narrow genetic base (Ne<150).Analysis of genetic variation was performed for 2,819 animals in the global sheep diversity panel. Breeds were sampled from each continent across the species range , includi regions . Allele  methods . Followi methods . This liK) K = 2), a clear division was observed between Northern European and Asian breeds  was located at Mb position 29.54 near the Relaxin/insulin-like family peptide receptor 2 (RXFP2), which was recently linked with the absence of horns (poll) in sheep FST between breeds defined as either being horned or polled. This recapitulated a single strong and striking selection signal at RXFP2  and their neighbouring SNP. Global osome 10 . The higat RXFP2 . ImportaFST  and bone resorption (p = 4.0×10−5). Given it is unlikely all 181 genes have undergone selection but each contributed to the GO analysis, caution is required during interpretation. Nonetheless, the content of the differentiated regions strongly suggests enrichment for genes under selection given their roles in pigmentation, body size, reproduction, animal production, and domestication. Selection for specialised coat pigmentation represents breed-defining characteristics across domestic animals including sheep. Selection signals were detected spanning KIT, ASIP, and MITF  is a key regulator of mammalian reproduction that is critical for the onset of lactation and is associated with milk traits in dairy cattle TSHR) in chicken, which given its pivotal role in metabolic regulation and the control of reproduction, was postulated to be a domestication gene FST peak on Chromosome 6 spanned the FGF5 gene, recently shown to contain mutations in dog responsible for variation in hair type KIT, FGF5, MITF, and RXFP2 are targets for selection across multiple mammalian lineages . Conversely, sheep from the Americas (Brazil and the Caribbean) had high Ne and clustered separately from European, African, or Asian populations. Decomposing the genome into two or more components  gives the fraction of total SNP that displayed both alleles within each population. Expected heterozygosity (He) and the inbreeding coefficient (F) were estimated using PLINK Ar) and private allele richness (pAr) were estimated by ADZE Five metrics were used to estimate levels of within-breed genetic diversity . The proAnalysis of allele frequency distributions, plotted separately for SNP identified by Roche 454 and Illumina GA sequencing, indicated the presence of ascertainment bias . To deteK (number of subpopulations). For supervised clustering, prior population information was introduced from six meta-populations consisting of regional pool of breeds considered to represent ancestral populations. The same meta-populations were used for updating the allele frequencies during the simulations. NeighborNet graphs were constructed from a matrix of Reynolds' distances using Splitstree In order to understand the relationship within and between breeds across each major geographic group, Principal Components Analysis (PCA) was performed using EigenStrat 2r between all SNP pairs where MAF for each SNP in the pair was >0.10. 2r values were grouped into bins based on the distance between SNP from the physical map. tN was then calculated as (1−2r)/(42cr), where c is the distance between the SNPs in Morgans (we assumed 100 Mb = 1 Morgan) and tN is the effective population size t generations ago, where t = 1/2c. The most recent estimate of effective size was taken as tN when c = 1 Mb. We performed simulations to assess the sensitivity of the estimates of effective population size over generations based on LD, in populations with and without admixture events (2) was calculated between all marker papers and Ne estimated at different times in the past as described for the real data. Five replicate simulations were performed for each scenario. The extent of haplotype sharing among populations was characterised with the r statistic, where r is a signed 2rr values for all locus pairs separated by the same physical distance among two breeds requires that the same haplotypes are found within both breeds. This means the sign of the r statistic is preserved across breeds only if the phase relationship among alleles is the same in both populations . The correlation of r between breeds was calculated for SNP separated by <10 kb, 10–25 kb, 25–50 kb, 50–100 kb, and 100–250 kb . To determine the extent of this error, we calculated the correlation of the r values at these different lengths of haplotypes for the Merino and Industry Merino samples, which are samples from the same breed. This gave a correlation between the r values for each bin size of 0.6. All correlations of r values for all breed comparisons were then divided by 0.6 to correct for sampling. Only corrected values are presented. As detailed in r between two breeds with increasing marker distance can be used to estimate generations since divergence from a common ancestral population. From r after T generations of divergence is E(rT) = ecT−2. The natural logarithm of the expected correlation of r then follows a linear decrease as a function of distance with slope −2T, and this was used to calculate divergence time between all breeds  was calculated as e events . A mutat0–250 kb , S8, S9.l breeds .FST was calculated as described by FST values were smoothed using a local variable bandwidth estimator as described in FST within each population was smoothed into 500 genomic divisions (98 SNP per region). The number of breeds with smoothed FST in excess of one standard deviation of the mean was plotted for values at each tail of the distribution. Analysis was performed to identify gene ontology (GO) terms that were significantly overrepresented in 181 genes residing within the 31 regions under selection  based on SNP type. MAF was estimated for each of five geographically defined breed groupings separately using either 33,115 SNP derived using Roche 454 (top panel) or 15,427 SNP derived using Illumina GAII (bottom panel). The breed membership of each group is given in (TIF)Click here for additional data file.Figure S2Diversity between breeds compared using different SNP panels. Expected heterozygosity was calculated using four SNP panels: “49034” contains all SNP passing quality control ; “20279”(TIF)Click here for additional data file.Figure S3Principal components analysis (PCA) of European-derived sheep. To visualise the complex relationships between European-derived populations, PCA was performed separately using northern European breeds  and central and southern European breeds . The inbred Soay, Boreray, and Macarthur Merino have been omitted. In the left panel, PC1 and PC2 resolve all British and most central European breeds and show the intermediate position of the Swiss crossbred Swiss Alpine, Swiss Mirror, and Swiss Brown-Black Mountain sheep. Individuals from three geographically distinct populations of Texel formed a single cluster Click here for additional data file.Figure S4K components, which may correspond to the genomes of ancestral populations. Representative results are shown for unsupervised clustering using K = 2, 5, and 8. At K = 2, a marked division is observed between European and Asian breeds, which corresponded to the first principal component in PCA , South-West Asian (Middle Eastern), Asian, and African breeds. Separate clusters were assigned to the Soay and Boreray (at K = 5), Dorset (at K = 6), Friesian (K = 7), and Wiltshire breeds (K = 9). The admixed nature of Brazilian and Caribbean animals can be seen at higher values of K. Essentially the same patterns were obtained with the Admixture program K (unpublished data). Supervised clustering  was performed using six predefined regional genomic components reconstructed by pooling breeds as indicated by the coloured horizontal line.Model-based clustering of sheep. The diverse origin of the sheep genome was examined by model-based clustering using STRUCTURE t in PCA . For inc(TIF)Click here for additional data file.Figure S5Proportion of variance explained in principal component analysis. Genetic distance between each pair of animals in the global sheep diversity panel was analysed using PCA. The proportion of variance explained by each of the first 50 PCs is given at left (blue diamonds) and the cumulative proportion is shown at right (red triangles). The first PC explained approximately 3% of the variance, while the first 20 PCs together explain 16.3% of the total variation.(TIF)Click here for additional data file.Figure S6Ne was estimated at different times in the past as described for the real data. This shows the admixture event did affect the inferred Ne, with a higher estimate for the generations in which the admixture event is occurring. However, impact on estimates of Ne for generations that were not within 100 generations of the admixture event were minimal. Further, the pattern of inferred Ne for the admixed population suggests that there is information in the pattern of LD to pick up such events.Effective population size inferred from linkage disequilibrium (r2) with and without an admixture event in a simulated sheep population. The decline in effective population size was simulated to be similar to that observed in the real HapMap data for many breeds. The admixture event occurred 220 to 200 generations ago and lasted 20 generations before the breeds diverged for a further 200 generations without further admixture. LD (r2) was calculated between SNP and (TIF)Click here for additional data file.Figure S7r statistic r was calculated using SNP pairs separately by short physical distances (0–10 Kb). The values are given as heat map, where each cell represents haplotype sharing between a breed pair. This revealed a high degree of conservation in LD phase between sheep populations.Extensive sharing of short-length haplotypes between breeds. The persistence of haplotype phase between breeds was evaluated using the signed (TIF)Click here for additional data file.Figure S8r statistic r was calculated using SNP pairs separately by short physical distances (10–25 Kb). The values are given as heat map, where each cell represents haplotype sharing between a breed pair. The values were used to plot haplotype sharing in Haplotypes sharing between breeds at intermediate physical distances. The persistence of haplotype phase between breeds was evaluated using the signed (TIF)Click here for additional data file.Figure S9Long-distance haplotype sharing between breeds. The persistence of haplotype phase between breeds was calculated in the same way as in (TIF)Click here for additional data file.Figure S10Divergence time between breeds. Divergence time (in generations) between breeds was calculated from the extent of haplotype sharing that persists at increasing physical distance between SNP pairs (TIF)Click here for additional data file.Figure S11The effect of SNP ascertainment on breed relationships. To evaluate the effect of SNP ascertainment on visualised relationships between sheep breeds, NeighborNet graphs were constructed and compared using five different SNP sets. Graphs were constructed using all 49,034 SNP (A); 33,115 SNP identified using Roche 454 (B); 15,427 SNP identified using Illumina GA (C); 22,678 SNP identified by application of LD pruning of the full SNP set (D); and 20,279 SNP polymorphic in non-domestic sheep that displayed LD<0.05 (E). The topology of each graph is very similar, indicating SNP ascertainment doe not have a strong impact on interpretation of genetic relationships.(TIF)Click here for additional data file.Table S1Global Sheep Diversity Panel.(DOC)Click here for additional data file.Table S2ovine SNP50 BeadChip.SNP discovery for the (DOC)Click here for additional data file.Table S3Quality control filters used to remove SNP.(DOC)Click here for additional data file.Table S4Genetic diversity and recent effective population size.(DOC)Click here for additional data file.Table S5Enrichment analysis of gene ontology (GO) terms for genes in regions under selection.(DOC)Click here for additional data file.Table S6Selection signals identified in both sheep and cattle.(DOC)Click here for additional data file.