Apparently, these chomokines synergize with the viral envelope protein, triggering cofilin and actin dynamics necessary for the establishment of viral latency. This study opens a new avenue for understanding chemokine interaction with HIV. Traditionally, chemokine control of HIV infection focuses on competitive binding and down-modulation of the corecptors, particularly CCR5. This new study suggests that a diverse group of chemokines may also affect HIV infection through synergistic or antagonistic interaction with the viral coreceptor signaling pathways.A recent paper by Cameron  HIV caner speed ,3. The ver speed ,5; viralin vivo data showing that in HIV-infected patients, enhanced levels of CCL19 and CCL21 correlate with viral load, disease progression and patients' response to HAART.The lack of understanding of viral latency in resting T cells has prompted a search for possible cellular conditions that permit viral integration and latency. In 2007, Lewin's group identified a novel mechanism of HIV latent infection of resting CD4 T cells, in which the CCR7 ligands, CCL19 and CCL21, were found to drastically increase the permissiveness of resting CD4 T cells to HIV infection . SpecifiThese findings open an avenue to examine the role of chemokines in controlling HIV infection, and suggest a potential new way of treating HIV infection. Traditionally, chemokine control of HIV infection focuses on competitive inhibition of viral entry through binding to the chemokine co-receptors, CCR5 in particular. This new result suggests that HIV infection could also be affected with chemokines interacting with multiple receptors such as CCR7, CXCR3, or CCR6 [Chemokines are a group of small proteins with chemoattractant properties, promoting leukocyte movement through binding to G-protein-coupled chemokine receptors (GPCR). Currently there are approximately 50 chemokines and 20 receptors identified Figure . Among tet al., the relationship between HIV infection and multiple chemokines was examined. Several key features emerged: (1) Certain chemokines such as CCL19, CXCL9/CXCL10, and CCL20 promote HIV nuclear migration and integration, whereas others such as CCL1 and CCL13 do not. (2) There are only limited changes in gene expression following chemokine exposure, suggesting that the enhancement on HIV infection may not be at the gene expression level. (3) The chemokine enhancement is not associated with T cell activation, as no changes in surface expression of CD69, HLA-DR, and CD25 were observed. (4) Chemokine enhancement only occurs before or at the time of HIV infection, and it is transit (as little as 3 h after treatment) and reversible (lost if removed for more than 3 h), which is consistent with the plasticity of cellular signal transduction, and suggests that the enhancement likely resulted from rapid changes in signaling pathways rather than from breaking cellular restriction factors.In a recent study by Cameron Although cofilin was identified in this study as the key signaling molecule responsible for the CCL19-mediated enhancement, for the chemokine system as a whole, there are likely multiple mechanisms to affect HIV infection, as chemokines are frequently pleiotropic. The Cameron study also suggested possible new ways of controlling HIV infection. Chemokines may be classified into either "synergizer", "antagonist", or "neutral" based on their relationship with HIV infection. Treatment of target cells with chemokine "synergizers" would enhance HIV infection, whereas treatment with an "antagonist" would do the opposite. "Neutral" chemokines may not affect HIV infection in a significant manner.HIV may also be inhibited through different strategies: (1) through inhibitors that target certain chemokine receptors on the surface. These inhibitors may include either inhibitory antibodies, small-molecule antagonists, nonfunctional chemokines that bind but do not activate viral-dependent pathways, or chemokine antagonists that bind and transduce inhibitory signals for HIV replication; (2) through inhibitors that directly target the intracellular chemokine signaling molecules such as those regulating actin dynamics; (3) through inhibitors that target the down stream effector molecules of chemokine signaling, mainly the cytoskeletal actin that is involved in HIV entry, reverse trancription and nuclear migration ,9,10.et al. calls for an expansion of research scope on chemokine control of HIV infection. It is imperative to initiate a systematical investigation into the chemokine signaling network in relation to HIV infection. This would pave the way for future development of new classes of anti-HIV inhibitors that could potentially act at multiple steps along the chemokine signaling pathways.The recent transformative study by Cameron The author declares that they have no competing interests.


To establish a baseline protein fingerprint of cultured human corneal endothelial cells (HCEC), to determine whether the protein profiles exhibit age-related differences, and to identify proteins differentially expressed in HCEC cultured from young and older donors.Corneas were obtained from five young (<30 years old) and five older donors (>50 years old). HCEC were cultured, and protein was extracted from confluent passage 3 cells. Extracts from each age group were pooled to form two samples. Proteins were separated on two-dimensional (2-D) gels and stained with SyproRuby. Resultant images were compared to identify protein spots that were either similarly expressed or differentially expressed by at least twofold. Protein spots were then identified by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry.Protein spots were well resolved, and patterns were reproducible on 2-D gels using either pH 3–10 or pH 4–7 IPG strips. Two-dimensional gels prepared with pH 4–7 IPG strips were used for differential display analysis, which was reproduced on three separate pairs of gels. MALDI-TOF identified 58 proteins with similar expression; 30 proteins were expressed twofold higher in HCEC from young donors; five proteins were expressed twofold higher in cells from older donors; and 10 proteins were identified in gels from young donors that did not match in gels from older donors. Several proteins expressed at higher levels in younger donors support metabolic activity, protect against oxidative damage, or mediate protein folding or degradation.This is the first proteomic comparison of proteins expressed in HCEC cultured from young and older donors. Although restricted to proteins with isoelectric points between pH 4.0 and pH 7.0, the data obtained represent an initial step in the investigation of molecular mechanisms that underlie physiologically important age-related differences in cultured HCEC, including differences that may affect proliferative capacity. Results indicate that HCEC from older donors exhibit reduced expression of proteins that support important cellular functions such as metabolism, antioxidant protection, protein folding, and protein degradation. These differences may affect the ability to consistently obtain a sufficient number of healthy cultured HCEC for use in preparing bioengineered endothelium as an alternative method for the treatment of endothelial dysfunction. Corneal endothelium is a physiologically important monolayer of cells that functions to maintain corneal transparency. Human corneal endothelial cells (HCEC) in vivo do not normally divide to replace cells lost as the result of disease ,2 or traBecause the aging population requiring corneal transplants is increasing and the donor age requirements and tissue quality limit the availability of donor corneas, there is increased interest in alternative approaches to restore corneal transparency following loss of endothelial function. Several new approaches are taking advantage of the fact that, although HCEC do not divide in vivo, they retain proliferative capacity -14. One Previous studies from this laboratory have used an ex vivo corneal wound healing model to compare the relative proliferative capacity of HCEC from young (<30 years old) and older donors (>50 years old) . ResultsProteomics technology is being widely used in research fields such as aging , biomarkTM rehydration/sample buffer were obtained from Bio-Rad . SyproRuby was purchased from Invitrogen .OptiMEM-I, minimum essential medium (MEM), Dulbecco’s phosphate-buffered saline (PBS), gentamicin, and trypsin/EDTA were purchased from Gibco BRL/Life Technologies . Bovine pituitary extract  was from Biomedical Technologies . Epidermal growth factor  was obtained from Upstate Biotechnologies . Fetal bovine serum (FBS) was from Hyclone . Ascorbic acid, chondroitin sulfate, calcium chloride, 0.02% EDTA solution , and antibiotic/antimycotic solution were purchased from Sigma . Sequential Extraction Reagent III (ER3), Protein Assay Dye Reagent, mineral oil, and ReadyPrep2. Corneas were not accepted for study if the period between time of death and time of preservation was too long (>12 h) or if guttae or other endothelial abnormalities were noted. Corneas were not accepted from donors with glaucoma, sepsis, ocular infection, or on large doses of chemotherapeutic agents. Primary culture and subculture of HCEC followed previously described protocols [Five pairs of human corneas from young donors (<30 years old) and five pairs from older donors (>50 years old) were obtained from the National Disease Research Interchange  and formed two age comparison groups. Donor confidentiality was maintained by the original eye bank, NDRI, and by this laboratory according to the tenets of the Declaration of Helsinki. Donor information is presented in rotocols ,14. PassConfluent passage 3 cells were rinsed with PBS to remove residual culture medium. Cell scrapers were used to remove cells from the culture plates. Harvested cells were centrifuged at 5,000 rpm for 10 min to form firm pellets. Bio-Rad Sequential Extraction Reagent III (ER3) with 1% tributyl phosphine (reducing agent) was added, and the cells were gently pipetted up and down for 1–2 min followed by incubation at room temperature for about 10 min to ensure thorough protein solubilization. Soluble proteins were harvested after centrifugation at 40,000 rpm at room temperature for 1 h and then stored at −80 °C until further analysis.Before gel electrophoresis, equal amounts of protein from five donors per age group were pooled to form two final samples. Protein concentrations of the two, pooled samples were determined by a modified Bio-Rad protein assay. The extracted protein together with De-Streak reagent  and bromophenol blue (Bio-Rad) was loaded onto pH 3–10 or pH 4–7 linear gradient IPG strips of 17 cm (Bio-Rad). Active rehydration was performed for at least 16 h following the Bio-Rad protocol before isoelectric focusing (IEF) in a Protean IEF Cell (Bio-Rad). IEF was run for a total of 60,000 Vhr after reaching maximum 10,000 voltages. The IPG strip was further incubated with equilibration buffers I and II (Bio-Rad) for 15 min each. Proteins were then separated on 8%–16% polyacrylamide pre-cast gels of 19 cm (Bio-Rad) using a Protean II apparatus (Bio-Rad). Electrophoresis was run until bromophenol blue dye just started to disappear from the bottom of the gel (approximately 1440 Vhrs). Gels were fixed for 1 h in 10% methanol and 7% acetic acid solution, stained overnight with SyproRuby protein gel stain, and washed for 1 h in water. Protein spots were then imaged with a ProXPRESS Proteomic Imaging System  using excitation (480/30) and emission (620/30) filters to visualize the SyproRuby.Images from gels prepared using pH 4–7 IPG strips were analyzed using ProFinder version 2005 software . Automatic analysis wizard was used, and spot editing was performed according to the software instructions. After warping to align spots between gels, manual inspection and editing, and automatic background subtraction, the normalized volumes of individual protein spots were compared. For these analyses, the two-dimensional (2-D) gel containing proteins extracted from the HCEC of young donors was used as the reference gel. Density differences between spots were confirmed though the use of various tools such as “montage window” and three-dimensional (3-D) topographic mapping. The software then generated lists indicating protein spots in which the normalized volume was similar (within a twofold range), spots that showed at least a twofold difference in normalized volume between samples, and spots that only appeared in one of the two samples.18 resin of the ZipPlate was then activated with 9 µl acetonitrile for 15 min at 37 °C. Peptides were then washed out of the gel plug with 180 µl 0.1% trifluoroacetic acid (TFA) for 30 min and bound to C18 resin using low vacuum followed by two washings with 100 µl TFA under high vacuum. Peptides were then directly eluted onto a disposable MALDI target plate (Perkin Elmer) by direct vacuum elution with matrix α-cyano-4-hydroxy cinnamic acid  in 50% acetonitrile/50% TFA. The matrix was allowed to air-dry allowing crystals to form. Peptide mass fingerprints were obtained on a Perkin Elmer prOTOF MALDI mass spectrometer, and data was searched on a local copy of the NCBI protein database  using the ProFounder search engine . Identified peptides were grouped into the smallest set of non-redundant proteins possible. Different forms (charge states and modifications) of the same peptide were compressed into a single hit. Protein identifications were reviewed manually to ascertain their accuracy and to ensure that consistent database entries were reported.Gel plugs containing the protein spots of interest were picked from the 2-D gels using a spot-picking robot equipped with a CCD camera  and filter sets for SyproRuby. Plugs were placed in a ZipPlate , dehydrated with 100% acetonitrile for 15 min, rehydrated in 15 µl of 25 mM ammonium bicarbonate, which contained 100 ng Trypsin Gold , and then incubated at 30 °C overnight. The CAge-related differences in morphology and growth characteristics of HCEC observed in this study were very similar to those previously described ,14,18. FProteins were extracted from HCEC cultured from five individual donors per age group  and then pooled to form one sample per age group. We chose to analyze pooled samples to eliminate donor-to-donor variations in relative protein expression so as to detect specific age-related differences more easily. Besides the pooling of five individual samples, we also strictly followed a standardized 2-D separation protocol to eliminate gel-to-gel variation. Protein concentration of the two samples was determined just before the IEF first-dimensional separation. Equal amounts of protein were loaded for both gels. First- and second-dimensional separations of the two pooled samples were performed at the same time. Two-dimensional gel separations were repeated at least three times per sample, and the patterns were compared. Under these strictly controlled conditions, reproducible 2-D protein patterns were obtained using both pH 3–10 and pH 4–7 IPG strips. The ProFinder software compared the normalized volume of individual spots between the two age groups. This form of analysis helped to eliminate any variations caused by protein loading or intra-strip (IPG) protein absorption differences and ensured that the comparison was conducted under well controlled conditions. The software automatically compared the spots based on identical anchoring spots present in both gels. The warping function corrected any distortion caused by changes in gel shape to achieve accurate spot matching. Appendix 1 presents an alphabetical listing of 58 proteins that showed similar expression levels  within a twofold range in HCEC from both young and older donors and that were identifiable by MALDI-TOF analysis. Multiple isoforms of some proteins are reported because their peptide profiles yielded different accession numbers upon search of the NCBI protein database. In a few cases, a single protein was resolved into multiple spots on the 2-D gels and yielded the same protein identification and database accession number upon MALDI-TOF analysis. In each of these cases, the position of the spot within the 2-D gel, the accuracy of the spot-picking, and accuracy of the protein identification were re-checked. Results suggest that these multiple spots represent single proteins that have undergone differential posttranslational modifications that alter the protein’s intrinsic charge and/or relative molecular weight. Appendix 2 presents a list of identified proteins in which the normalized volumes were at least twofold higher in HCEC from young donors compared to older donors, proteins that were expressed at least twofold higher in HCEC from older donors, and proteins that were identified in the gel from young donors that did not have a match in the gel from older donors. The list has been categorized into subgroups based on reported cellular function. Interestingly, several proteins known to function in cellular metabolism, antioxidant protection, protein folding or degradation, and cellular regulation were found to be expressed at least twofold higher in HCEC cultured from young donors.All detectable protein spots were picked and subjected to MALDI-TOF analysis and protein database identification. Bioengineered corneal endothelium is being developed as a new form of treatment to restore visual acuity in patients with critically reduced ECD. Since such bioengineering requires culture of HCEC, its successful development requires exploration of the practicality of inducing proliferation in HCEC to increase cell numbers. In some cases, use of a patient’s own cells as a source for the bioengineered tissue would be optimal to avoid the risk of immune rejection. Because patients with low ECD are usually older, this treatment approach would necessarily involve the culture of cells that exhibit reduced proliferative capacity. Similarly, the majority of corneas available for use as a source of endothelial cells for bioengineered tissue will be obtained from older donors. For these reasons, it is important to explore age-related differences in the protein expression of cultured HCEC to identify differences in metabolism and other functions that may affect the ability of cells to divide and maintain a healthy state within the bioengineered construct. It is clear that donor age negatively affects the proliferative capacity of HCEC ,14,18,19Proteomics technology provides a powerful high throughput screening tool to identify protein expression differences that may underlie age-related changes at the functional level. Protocols similar to those used in the current study have been used to conduct proteomic analyses of other ocular cells ,31,36,37Review of the data obtained in this study provides insight into changes in relative protein expression that suggest an age-related reduction in the ability of cultured HCEC to protect against oxidative stress and to maintain general cellular health. Of particular interest were changes indicating a potential age-related reduction in a subset of proteins involved in protecting against oxidative stress. Redox regulation is required to protect cells from oxidative stress, which is known to contribute to the aging process ,46. ProtA comparison with the available gene expression profiles from human donor corneal endothelium -51 indicThe current studies are an initial effort to use proteomics technology to document age-related differences in the relative protein expression of cultured HCEC and are a first attempt toward understanding the molecular basis for age-related differences that may affect their relative proliferative capacity. Although the results obtained were internally consistent and appeared to reflect age-related differences observed in other cells, it is clear that the study has limitations in that additional, independent studies are needed to confirm the findings from this initial analysis before their relevance can be fully appreciated. Further study is also needed to determine whether the differences in relative protein expression identified in cultured HCEC are also present in HCEC in vivo. Identification of age-related differences in cultured HCEC should aid in the development of methods to optimally culture these cells for use in tissue bioengineering.


Because avian influenza H5N1 infection risks are associated with exposure to infected poultry, we conducted a knowledge, attitudes, and practices survey of poultry-handling behavior among villagers in rural Cambodia. Despite widespread knowledge of avian influenza and personal protection measures, most rural Cambodians still have a high level of at-risk poultry handling. The circulation of the highly pathogenic H5N1 avian influenza (AI) strain throughout Asia since late 2003  and ducks (39%) , althougDuring the previous 6 months, of the 260 households that owned poultry, 162 (62%) experienced poultry deaths; however, only 18 (7%) reported these deaths to local authorities. Half of the respondents (n = 231) believed that it was important to report any poultry deaths because the death may be due to AI (61%) or because the poultry owners may receive management advice from the village veterinarians (39%). Among these 231 respondents, many did not report poultry deaths because they did not know how (41%), were in the habit of not reporting poultry deaths (31%), believed they would have a problem selling poultry if they reported deaths (18%), did not know the risks of AI (7%), or feared poultry culling (5%). Among those respondents who did not believe reporting deaths was important, the reasons provided included the following: “the number of poultry deaths were too few” (62%), “poultry are not as important as cattle” (18%), “no help would be provided from veterinary staff or authorities” (13%), or “because mortality was similar to previous years” (7%). Of respondents that experienced poultry deaths, 62% buried or burned dead poultry, 53% prepared them for food, 22% threw away the dead poultry, 3% used them to feed other animals, and 2% prepared them for sale or gave them to their neighbors.Participants had learned about AI from television (81%) and radio (78%). Thirty-one percent of respondents were able to describe AI symptoms in humans, and 72% believed that AI is a fatal disease among poultry that can be transmitted to humans. Most respondents believed it is unsafe to touch sick or dead poultry with bare hands (67%), eat wild birds (70%), let children touch sick or dead birds with bare hands (83%), and eat meat or eggs that are not fully cooked (86%). Sixty-one percent of respondents mentioned at least 1 of the recommended behavioral practices that protect against AI infection.General media reports about AI through radio and television broadcasts appear to have been effective at reaching rural people. However, despite high awareness and widespread knowledge about AI and personal protection measures, most rural Cambodians still often practice at-risk poultry handling. Anecdotally, we also reported that family members of H5N1-infected patients, who knew about AI risks, still prepared dead or sick poultry for household consumption during massive die-offs, because they observed that neighbors with the same behavior did not become sick . These findings provide evidence that high awareness does not necessary lead to behavior change. Behavior change involves comprehensive and multidisciplinary intervention, which combines risk perception communication and feasible and practical recommendations, including economic considerations. We speculate that it is hardly feasible to sustain good poultry-handling practices if access to personal protective equipment is cost prohibitive, particularly when disease occurrence poultry die-offs are common. Further studies are needed to determine appropriate behavior change strategies in Cambodia.We did find that many of the villagers were willing to report poultry deaths but did not know how. However, this finding should be interpreted in light of some limitations. We observed difficulties and frustrations among farmers whose flocks underwent culling after identification of H5N1 viruses in their flocks because compensation has not yet been approved by the government of Cambodia. In contrast, Thailand and Vietnam have introduced compensation along with the introduction of poultry vaccination in Vietnam and the reduction of backyard poultry ownership in Thailand in an effort to protect the commercial poultry industry. Thus, it is difficult to envision effective control strategies in Cambodia based exclusively on culling. Coincidentally, Vietnam has reported far fewer H5N1 outbreaks in poultry and humans since the introduction of the vaccination program, while Cambodia detected 4 outbreak sites in domestic poultry and 2 unrelated human cases in 2006. The real effect of a no-compensation policy on willingness to report poultry deaths needs to be assessed.Not surprisingly, direct contact with poultry and poultry products was common among household members. Transmission of H5N1 from poultry to humans, even in circumstances in which human–poultry interactions are regular and intense has been limited; however, as the virus continue to circulate and evolve among poultry, bird-to-human transmission may increase. In this context, improvement in risky practices can only be achieved through relentless behavior change efforts. Because lack of knowledge does not appear to be a factor, intervention programs must include feasible options for resource-poor settings that have limited materials for personal protection  and must offer farmers alternative methods to safely work with poultry on a daily basis.


As it becomes clear that mechanical ventilation can exaggerate lung injury, individual titration of ventilator settings is of special interest. Electrical impedance tomography (EIT) has been proposed as a bedside, regional monitoring tool to guide these settings. In the present study we evaluate the use of ventilation distribution change maps (ΔfEIT maps) in intensive care unit (ICU) patients with or without lung disorders during a standardized decremental positive end-expiratory pressure (PEEP) trial.2/FiO2 ratios were obtained at four PEEP levels (15 to 10 to 5 to 0 cm H2O) in 14 ICU patients with or without lung disorders. Patients were pressure-controlled ventilated with constant driving pressure. fEIT images made before each reduction in PEEP were subtracted from those recorded after each PEEP step to evaluate regional increase/decrease in tidal impedance in each EIT pixel (ΔfEIT maps).Functional EIT (fEIT) images and PaOP = 0.002) and from 10 to 5 (P = 0.001) between patients with and without lung disorders. Tidal impedance increased only in the non-dependent parts in patients without lung disorders after decreasing PEEP from 15 to 10 cm H2O, whereas it decreased at the other PEEP steps in both groups.The response of regional tidal impedance to PEEP showed a significant difference from 15 to 10 (During a decremental PEEP trial in ICU patients, EIT measurements performed just above the diaphragm clearly visualize improvement and loss of ventilation in dependent and non-dependent parts, at the bedside in the individual patient. Mechanical ventilation is critical for the survival of most patients with respiratory failure admitted to the ICU, but it has become clear that it can exaggerate lung damage and may even be the primary factor in lung injury . Protectet al. described the use of these ventilation distribution change maps in two ICU patients [et al. composed functional EIT images or ventilation distribution maps by subtracting the EIT images from two PEEP levels to show improvement or loss of regional ventilation between these two PEEP levels [Electrical impedance tomography (EIT) is a noninvasive, real-time imaging method that provides a cross-sectional ventilation image of the lung -6. It ispatients . They inP levels . As the In the present study we evaluate the use of ventilation distribution change maps in ICU patients with two distinct types of lung conditions: with or without lung disorders during a standardized decremental PEEP trial. Furthermore we investigated if the EIT measurements at the bedside may visualize alveolar recruitment and derecruitment in the dependent and non-dependent lung regions.2/FiO2 ratio <300 mmHg and proven pneumonia or abdominal sepsis. All patients were well sedated and ventilated in pressure-controlled mode, without spontaneous breathing activity. All patients were ventilated with constant driving pressures throughout the procedure, mean applied driving pressure was 12 cm H2O (range 9 to 18) in Group N and 16 cm H2O (range 12 to 21) in Group D. Exclusion criteria for participation in the study were: pneumothorax, severe airflow obstruction due to Chronic Obstructive Pulmonary Disease (COPD) , lung transplantation, thoracic deformations and severe cardiovascular instability.Following approval by the local institutional human investigations committee, patients were enrolled after providing informed consent from their legal representatives. The study population consisted of 14 mechanically ventilated patients on a mixed ICU. In eight of these patients, end-expiratory lung volume was also measured and these data have recently been published . For all2O. After a steady state of 15 minutes PEEP was decreased stepwise each 10 minutes to 10, then to 5 and, if clinically acceptable, to 0 cm H2O. The stability of each steady state was evaluated by a stable end-expiratory EIT signal and a stable arterial saturation. Before the end of each PEEP level, EIT was measured during two minutes and hemodynamic and ventilatory parameters were recorded. Dynamic compliance was calculated by dividing expiratory tidal volume by the constant driving pressure set for each patient. In addition, arterial blood gas analysis was performed  in order to calculate the PaO2/FiO2 ratio.In all patients, impedance measurements were performed during two minutes with a silicone belt with 16 integrated electrocardiographic electrodes placed around the thoracic cage at the fifth or sixth intercostal space . The EIT scans consist of images of impedance with a 32 × 32 color-coded matrix relative to the lowest impedance during the PEEP trial (rel. ΔZ). The difference between rel. ΔZ at the end of inspiration and expiration is defined as tidal impedance variation (TIV). This tidal impedance variation is visualized in the functional EIT (fEIT) image, which contains tidal impedance variation per pixel (32 × 32 matrix) averaged over one minute Figure . For ana2/FiO2 ratio and dynamic compliance was evaluated using ANOVA for repeated measurements. The percentage positive of the total tidal impedance change during each PEEP step between both groups was evaluated with the Mann-Whitney U test. For all comparisons, P <0.05 was considered significant.Statistical analysis was performed with Graphpad software package . Due to the small number of patients, results are expressed as median and interquartile range. Regional compliance changes between each PEEP step were evaluated with the Wilcoxon matched pair test. EELV, PaO2O and six patients at a PEEP of 15, 10, 5 and 0 cm H2O. Respiratory data during the PEEP steps are presented in Table 2O and measurements were continued at 10 PEEP cm H2O. All other patients tolerated the PEEP trial well.Table Figure 2O driving pressure is presented for two ventral to dorsal ROIs at all PEEP levels. In both groups, tidal impedance decreased towards 0 cm H2O PEEP. However, this was different for the individual ROIs. In Group N, tidal impedance increased in the non-dependent ROI after decreasing PEEP from 15 to 10 cmH2O, whereas decreased in the dependent ROI during each PEEP step. In Group D, tidal impedance variation was significantly lower compared to Group N in both regions. Further, tidal impedance did not change in the non-dependent region between PEEP steps 15 to 10 and 5 to 0 and in the dependent region between PEEP step 5 to 0. Tidal impedance decreased significantly during the other PEEP changes.In Figure 2/FiO2 ratio and change in dynamic compliance during each PEEP step is presented.The ΔfEIT images between PEEP steps for the individual patient are shown in Figure 2O (P = 0.002) and from 10 to 5 cm H2O (P = 0.001) between patients with and without lung disorders (Figure In Figure This trial shows that EIT is suitable for bedside monitoring of tidal impedance or regional compliance during decremental PEEP steps and can differentiate between dependent and non-dependent lung regions. There was a significant difference in response to a stepwise decrease in PEEP between patients with and without lung disorders, indicating a different PEEP dependency between these two groups.2O; this was due to increased tidal impedance variation in the ventral lung regions, with less loss in the dorsal lung regions (Figures 2O of PEEP alveoli in the ventral part were inflated throughout the ventilatory cycle or overdistended and after lowering the PEEP to 10 cm H2O ventilation increased in the non-dependent part. In this group, after further lowering the PEEP from 10 to 5 cm H2O, tidal impedance increase in the ventral part was exceeded by loss of the tidal impedance in the dorsal part; total impedance change was below 50% during this latter PEEP step (Figure Using EIT, increase or decrease of tidal impedance variation becomes visible in ΔfEIT maps Figure . During  Figures  and 5. Tp Figure  and 5.EIT is able to show the effect of PEEP on tidal impedance change in each pixel of the functional image matrix. If one accepts the proposed linear relationship between impedance and tidal volume -13, the 2O in patients without lung disorders, and at least 15 cm H2O in patients with lung disorders (Figure In the present study, each patient showed an individual response in tidal impedance change during each stepwise decrease in PEEP, and the response differs between dependent and non-dependent lung regions Figure . This iset al. used the same method in an experimental animal study and showed good agreement between CT and regional ventilation [A potential limitation of the present study is that EIT measures an eclipse with a central diameter of 5 to 10 cm. The individual pixel in the EIT matrix contains a vast number of alveoli; during tidal ventilation and/or stepwise change in PEEP, different alveoli might be included in the EIT pixel. However, because the individual pixel contains alveoli from the same lung region, it is unlikely to influence the results of this study. In the present study, we didn't compare our findings to another imaging technique like CT scanning. However, Meier tilation . In addiWe conclude that during a decremental PEEP trial in ICU patients, EIT measurements performed just above the diaphragm clearly visualize improvement or loss of ventilation in dependent and non-dependent parts, at the bedside in the individual patient. There was a significant difference in response to a stepwise decrease in PEEP between patients with and without lung disorders, indicating a different PEEP dependency between these two groups. However, the individual response to the decrease of PEEP within the groups was also different indicating that optimal PEEP should be titrated individually and can not be generalized for a group of patients. In addition, the response to the decrease of PEEP was different between dependent and non-dependent lung regions within a patient, suggesting that optimal PEEP may be defined as an equal balance between increase and decrease in tidal impedance. This definition of the optimal PEEP in order to minimize VILI needs further research to prove its benefit.• EIT is suitable for bedside monitoring of tidal impedance or regional compliance during decremental PEEP steps and can differentiate between dependent and non-dependent lung regions.• During a decremental PEEP trial in ICU patients, EIT measurements performed just above the diaphragm clearly visualize improvement and loss of ventilation in dependent and non-dependent parts, at the bedside in the individual patient.• Differences in response to decremental PEEP steps were found not only between patient groups, but also within groups indicating that the optimal PEEP should be titrated individually and can not be generalized for a group of patients.• A definition of the optimal EIT PEEP is needed if this technique is going to be used in the clinical setting.COPD: Chronic Obstructive Pulmonary Disease; CT: computed tomography; EIT: Electrical Impedance Tomography; fEIT: functional EIT; ΔfEIT: functional EIT images subtracted pixelwise from each other; Group D: group with lung disorders; Group N: group without lung disorders; ICU: intensive care unit; PEEP: positive end-expiratory pressure; ROI: region of interest; TIV: tidal impedance variation; ΔZ: mpedance change.The authors declare that they have no competing interests.IB carried out the data acquisition, analysis, statistical analysis and participated in drafting the manuscript. DRM participated in the statistical analysis and in drafting the manuscript. DG participated in the data acquisition and in drafting the manuscript. SL and JB participated in drafting the manuscript. All authors read and approved the final manuscript.The EIT equipment was supplied by Dräger Medical AG, Lübeck, Germany. The authors thank Laraine Visser-Isles for English language editing.


Genome-wide association scans for genetic loci underlying both Mendelian and complex traits are increasingly common in canine genetics research. However, the demand for high-quality DNA for use on such platforms creates challenges for traditional blood sample ascertainment. Though the use of saliva as a means of collecting DNA is common in human studies, alternate means of DNA collection for canine research have instead been limited to buccal swabs, from which dog DNA is of insufficient quality and yield for use on most high-throughput array-based systems. We thus investigated an animal-based saliva collection method for ease of use and quality of DNA obtained and tested the performance of saliva-extracted canine DNA on genome-wide genotyping arrays.in silico calling of copy number variants was successfully performed and verified by PCR.Overall, we found that saliva sample collection using this method was efficient. Extractions yielded high concentrations (∼125 ng/ul) of high-quality DNA that performed equally well as blood-extracted DNA on the Illumina Infinium canine genotyping platform, with average call rates >99%. Concordance rates between genotype calls of saliva- versus blood-extracted DNA samples from the same individual were also >99%. Additionally, Our findings validate the use of saliva-obtained samples for genome-wide association studies in canines, highlighting an alternative means of collecting samples in a convenient and non-invasive manner. Canis familiaris genome Assemblies of the Because array-based genome-wide genotyping platforms require large quantities of high quality genomic starting material, DNA for such studies has traditionally been obtained from whole blood. However, with increasing demands for large sample sizes to ensure statistical power to detect multiple signals of modest effect as is expected for complex phenotypes, obtaining whole blood samples from large numbers of dogs becomes challenging. In fact, sampling can even become the limiting factor when studying behavioral traits such as severe anxiety disorders where handling by a clinician in itself causes great duress to the animal, and is often only possible with sedation.The utility of dog DNA obtained from buccal sampling is well established for microsatellite marker typing, targeted SNP genotyping and limited resequencing. We have found that use of whole-genome amplification (WGA) provided sufficient quantities of genomic material for use in higher throughput multiplex genotyping assays surveying up to several hundred SNPs Another mode of DNA sampling that has gained increasing utilization is saliva collection, from which DNA has been shown to be of equivalent quality as blood-extracted DNA Saliva-extracted DNA has been demonstrated to be of equivalent quality as blood-extracted DNA in humans http://www.dnagenotek.com/DNA_Genotek_Support_Lit_UI_ANIMAL.html). After sample collection, DNA was preserved by placing the sponges in Oragene·ANIMAL stabilization solution, labeled, and then sent to our laboratory by mail. All saliva samples were stored at room temperature before and after shipping. Blood samples were obtained by 3–5 cc blood draw. All animal work was approved by the local review committee.Saliva and blood samples were collected from four Bearded Collies and one Border Collie in the context of our ongoing genetic studies of canine behavior. Saliva only was also obtained from six additional Bearded Collies recruited for the same study. Saliva samples were collected by owners using the Oragene·ANIMAL  kit as per manufacturer's instructions. Briefly, saliva was collected from dog's mouth using 2–3 absorbent sponges  to remove solution for use in extraction. The solution was collected in original holding container, and then 500 µl was aliquoted via pipette into a 1.5 mL microcentrifuge tube. Absorbent sponges were kept in remaining stabilization solution in the event that additional extractions were required. Because twice the amount of solution was aliquoted for extractions, 20 µl of Purifier was used, and the use of glycogen was omitted. The Animal protocol contains a NaCl step to ensure efficient recovery of DNA; as this step is not in the Human protocol and was added between the two versions of the Animal protocol that we performed (beta testing kit courtesy of DNA Genotek vs. published version PD-PR-095 Issue 2.1), extractions were carried out both with and without the use of NaCl on the same sample (which was not used for the reported genotyping). The single Border Collie sample was extracted without the NaCl step via the beta kit instructions, whereas the ten Bearded Collie samples were all extracted using the NaCl step from the updated protocol. For the final hydration step, 100 µl of Hydration Buffer from the Qiagen kits used for blood extractions  was used to rehydrate DNA, and samples were incubated for at least 24 hours at room temperature prior to final storage at 4°C. Blood sample DNA was extracted in-house using standard methods with the Puregene Blood Kit (Qiagen Inc.).Quantification of all extracted DNA samples was performed on a NanoDrop (ND-1000 v3.3.0) spectrophotometer . Quantification of saliva-extracted samples was not corrected as per notes suggested by DNA Genotek (Laboratory Protocol PD-PR-095 Issue 2.1), but rather were reported as calculated by the NanoDrop for direct comparison with results published by other groups for human saliva and blood. For more details, please see Samples were genotyped on the Infinium Canine SNP20 BeadChip  by the Genomics Core Facility at the University of California, San Francisco. Genotypes were called and quality control was conducted in-house using the GenomeStudio Data Analysis Software package . Clusters of all samples with GenTrain Scores (a measure of reliable SNP detection)<0.60 were visually assessed for quality and either manually reclustered or zeroed due to poor performance . Further exclusion criteria removed SNPs with call rates<95% or minor allele frequency (MAF)<0.02.in silico with the GenomeStudio software  using default criteria. One predicted CNV locus was also evaluated by direct PCR of two genomic segments within the putative deletion region using the following primer pairs: (PLSCR1exon amplicon) forward 5′-TCTAAACCCAGGATTAGCAAGAA-3′, reverse 5′-CCATGTAATTTTGATAGGGTATTTCA-3′ and (CFA23CNV44Mb amplicon) forward 5′-TGTAAACCTCATTTCACTTACATGG-3′, reverse 5′-GGTCCATGGAGGACTCTCTCT-3′. Platinum-Taq was used to amplify segments with a 58°C touchdown protocol in presence of 0.4 µM primer, 100 µM dNTPs, 2.5 mM Mg and 1 mM Betaine.Copy number variation was evaluated All animal work was approved by Institutional Animal Care and Use Program at the University of California, San Francisco (AN079848-02). All dogs were recruited from private owners, who consented to use of de-identified data for research purposes.Twelve sample kits were sent to six Bearded Collie owners who previously consented via written communication to participate in the sample collection. Of those, four owners representing 10 kits (dogs) returned samples to our laboratory, representing a 67% by-owner and 83% by-dog return rate. Surveys sent out with the beta testing version of the Oragene·ANIMAL saliva collection kits reported that owners found the collection to be very easy overall. For all owners, sample collection was successful and took less than 10 minutes.DNA extraction was successful for all saliva samples received from owners. For each sample, 500 µl of saliva-buffer solution was easily extractable from the swabs, with additional volume remaining after the liquid transfer step to the 1.5 mL tube, thus allowing for another extraction for more DNA if necessary. The extraction protocol was very straightforward, and DNA fibers were visible for all extractions performed. When quantified via NanoDrop spectrophotometer, saliva-extracted samples (n = 11) had a mean concentration of 125.5 ng/µl , for an et al. (2005). The array contains 22,362 SNPs with a median of 565 markers per chromosome . The average intermarker spacing is 103.6 kb, with median intermarker spacing of 67.8 kb. Several very large gaps inflate this mean, with the largest gap at 5.6 Mb (on the X chromosome). There are three, four and 27 gaps >3 Mb, >2 Mb and >1 Mb in size, respectively, and 292 SNPs with gaps >500 kb. The average call rate for the 22,362 SNPs surveyed by the Infinium CanineSNP20 array before quality control (QC) of SNP data was 99.2% for saliva samples (n = 11) and 98.5% for comparison blood samples  of the range of GenCall scores across all genotypes called for the individual—were 0.801 and 0.797 for saliva and comparison blood, respectively  was 99.98% . One repin silico were evaluated in all genotyped samples and those appearing to specifically include a subset of the saliva-extracted samples were further assessed for validity via manual inspection of genotype data. A region on chromosome 23 had copy number losses predicted for Sample 2 (homozygous loss) and Sample 5 (heterozygous loss), as well as predicted homozygous loss in three other Bearded Collie blood samples (data not shown). To validate these calls, we investigated this region via direct PCR of genomic samples for two amplicons located within the putative deletion region: PLSCR1exon amplicon designed to span the 8th exon of the PLSCR1 gene, and CFA23CNV44Mb amplicon designed to span a predicted conserved region (annotated in the UCSC Genome Browser) in the middle of a hypothesized minimally deleted region based on no-call genotypes in the three samples predicted to have homozygous deletions . However, because DNA from different tissues may produce source-specific profiles with regard to probe fluorescence —which main silico. Calls are made for putative CNVs and regions of homozygosity based on genotypes across multiple markers. It thus follows that the size of putative copy variable regions relies on the density of the SNP data, and that the size of a reported CNV may be artifactually large due to the requirements of the calling algorithm. Because of the large inter-SNP distances in our data set, direct assessment of genotype calls was therefore also used to hypothesize a minimally deleted region in our samples , which we verified by direct amplification of genomic DNA. Our results demonstrate that saliva samples can also be used reliably in copy variation analysis, although similar requirements for case-control tissue sample consistency still apply.Because they provide high-fidelity SNP genotypes, it appears that saliva-extracted samples can also be used for successful CNV calling We found one saliva sample that demonstrated low concordance (82.6%) with its replicate blood sample. This was likely due to switching samples of dogs within the same breed, as pairwise concordance between known but different dogs of the same breed was similar to that seen in our low-concordance sample, whereas concordance between known dogs of different breeds was much lower . FurtherOne caveat of this work is that the blood samples were genotyped on a separate run several months earlier than the saliva samples, which could introduce artifacts when comparing genotypes and statistics for samples representing the same individual. However, it is more likely that these artifacts would introduce inconsistencies between duplicated samples; this would result in an underestimation of the total concordance seen between duplicates in our study. As our concordances are already greater than 99% (excluding the suspected wrong sample pairing), this suggests that even higher fidelity in genotype calls between blood- versus saliva-extracted DNA samples may be possible if all samples are run in the same genotyping batch. The similarity of clustering data also suggests that samples of diverse provenance can be clustered together using Infinium data. We have recently observed high concordance rates (99.9999%) between blood and saliva replicate pairs on the next generation Illumina canine array with 170,403 QC-filtered SNPs, with a mean call rate for saliva samples of 99.78% .One limitation to this study is the ascertainment bias introduced by our study design. Because we required prior written assent from owners to participate in saliva sample collection prior to kits being sent out, it is likely that our return rates are overestimates of the population at large. However, because saliva collection is so simple and non-invasive, it is probable that return rates would be quite significant, and likely higher than the rate of blood sample collection. Another limitation of this study is the small number of duplicated samples. This limitation highlights the need for replication with larger numbers of dogs from different breeds , and on different genotyping platforms by other groups for further validation of the performance of saliva-extracted DNA for high-throughput assays.In summary, we demonstrate for the first time that saliva sample collection in dogs is a noninvasive means of obtaining high quality DNA for successful use with genome-wide array genotyping, with little danger of loss of information due to the source of data. The dual conveniences of owner sampling in the home and ease of shipping provide alternative means of obtaining samples from rural locales or foreign countries where collection of blood samples may be difficult or impossible. Additionally, ease of sampling allows for collection of large numbers of samples with minimal investment of time and manpower, creating potential for collecting an entire study cohort at a small number of targeted sampling events. Finally, the non-invasive nature of saliva collection makes it particularly appealing when studying dogs whose conditions may otherwise prevent blood collection, such as high levels of anxiety or repeated use of veins for other medical purposes related to disease status. In sum, these factors will lead to increased sample return rates which will increase study sizes and ultimately enhance the ability for geneticists to detect novel genetic loci underlying disease and behavioral traits in a GWAS framework.Table S1Individual saliva and comparison blood sample statistics for DNA extraction and genotyping compared to published data for human saliva and blood, plus manufacturer's report. Individual statistics are given for each saliva and comparison blood sample for DNA concentration (ng/ul), DNA purity (260/280), contamination (260/230), post-QC genotype call rate and post-QC p10 GenCall score (p10 GC). Saliva vs. blood sample concordance rates are also given for every individual represented by both tissue types. Mean values as reported in the main text are provided, as well as the published values for human saliva and blood samples as reported by Hansen et al. and dog saliva statistics as reported by researchers from the manufacturer of the Oragene ANIMAL collection kit, DNA Genotek (Iwasiow 2009).(0.27 MB DOC)Click here for additional data file.Table S2Replicate statistics. Sample identification, tissue source, breed, geographic origin (US vs. foreign) and gender are given for samples (A versus B) that were compared for replicate (concordance) statistics. Sample 2 is suspected to be a switched sample, and demonstrates similar concordance rates as distantly-related dogs of the same breed. Samples 11 & 12 are dogs from a geographically distinct population . BEC = Bearded Collie; BOC = Border Collie. # Correct-total concordant genotype calls; # Errors-total discordant genotype calls; Total-total number of markers with genotype calls in both samples; Rep Freq-replicate frequency (concordance rate); Relation-unrelated refers to dogs that share no grandparents.(0.29 MB DOC)Click here for additional data file.Figure S1Plot of p10 GenCall score versus call rate after quality control for saliva-and blood-extracted DNA samples compared to full set of genotyped samples. Sample 10% GenCall score is plotted against sample call rate as a means of visualizing overall sample performance. Each sample is represented by one data point, with saliva samples represented by open circles, comparison blood samples as filled circles and remaining blood samples also genotyped by our group as grey circles . Overall, saliva samples performed in the same range as all blood samples genotyped. (Please note axes do not start at the origin.) P10 GenCall-10% GenCall score.(0.14 MB DOC)Click here for additional data file.Figure S2Cluster plots for select SNPs. Saliva- versus blood-extracted DNA samples (by columns) are highlighted in cluster plots of genotyped samples  from GenomeStudio.(0.28 MB DOC)Click here for additional data file.


The preferential proliferation of cancer cells in the bone microenvironment is poorly characterised. Expression pattern of bone marrow and other organ microenvironment in contact with osteolytic  and osteoblastic (MatLyLu MLL) metastases were investigated. Fisher and Copenhagen rats received, respectively, W256 and MLL cells injection. Bone and soft tissues were analysed by immunochemistry for DKK1, cathepsin K, RANKL, MCSF or IL6 expression. Tartrate-resistant acid phosphatase (TRAcP)-positive cells were detected by a histoenzymatic technique. In bone, expressions of MCSF and DKK1 were shown in stromal cells of the bone marrow, in contact with metastatic foci of both tumours. Many stromal cells were found RANKL positive in the vicinity of the tumours. Cells expressing cathepsin K and multinucleated TRAcP+ cells were found in direct contact with trabeculae but also in bone marrow spaces near metastatic cells. In extraosseous tumours, cells in contact with malignant cells did not expressed DKK1, MCSF, cathepsin K and IL6. Some RANKL+ cells were found in the periphery of subcutaneous tumours but may represent Langerhans cells. Abnormal presence of TRAcP+ cells was never observed in the vicinity of malignant cells. Interaction between stromal and cancer cells induces the expression on the formers of characteristics leading to osteoclastogenesis only in the bone microenvironment. Bone is a preferential site for metastasis in different types of cancer. Bone metastases induce an increased morbidity  and compromise the long-term survival. They occur in approximately 65–75% of patients with an advanced breast or prostate cancer and 15–40% of patients with a carcinoma of the lung, bladder, skin or kidney. The median survival after first recurrence of breast cancer in bone is 20 months . Once a Interactions between the bone marrow environment and cancer cells were first advocated by Paget, who described them as ‘the seed and the soil’ theory cited by : bone prα4β1, α5β1, αvβ3, αvβ5) (β), vascular endothelium growth factor (VEGF)…)  a release of attractive molecules for these particular types of cancer  inducing specific chemotaxis  ; (3) app(VEGF)…) , growth Bone metastases are commonly characterised as osteolytic or osteoblastic from a radiological point of view. This represents two extremes of a continuum in which dysregulation of the normal bone remodelling process occurs and alters bone mass and bone microarchitecture. Breast cancer is most often associated with osteolytic metastases, whereas osteoblastic metastases are common in prostate cancer. Osteolytic lesions are due to a marked increase in osteoclast number with a reduced osteoblastic activity. Parathyroid hormone-related protein is a major mediator between tumour and bone cells and induces the osteolytic process through the RANKL pathway . On the Most researches have focused on the interactions between tumour cells and osteoclasts or osteoblasts. However, interactions with surroundings cells have been poorly investigated. Because osteoblast precursors are stromal cells (present in the marrow spaces) that interact with haematopoietic osteoclast precursors via the RANK/RANKL/osteoprotegerin system , 100 μg ml−1 of streptomycin sulphate (Eurobio), 1% of non essential amino acids  and 1 mM of sodium pyruvate (Eurobio). Cells were cultured at 37°C in a humidified incubator with 5% CO2. To obtain cells with a bone trophicity, 107 cells were serially passaged intraperitoneally at 7-day intervals in Fisher rats to obtain malignant ascite.The Walker 256/B (W256) cells (issued of a rat mammary carcinoma) are associated with osteolytic metastases , 100 μg ml−1 of streptomycin sulphate (Eurobio) and 250 nM of dexamethasone . Cells were cultured at 37°C in a humidified incubator with 5% CO2.The MatLyLu cell line is an androgen-independent, highly ad libitum.The Animal Care and Use committee at the University of Angers approved all procedures. Nine female Fisher F344/NHsd rats , 3 weeks old were maintained for 6 months in the local vivarium conditions (24°C and a 12 h light/dark cycle). Nine female Copenhagen born from COP/NCrl rats , bred in our University animal facilities were maintained for 6 months in the same conditions. Rats were given a standard laboratory food  and water 7 W256 cells in 0.25 ml saline. They also received intramuscular (quadriceps femoris) and subcutaneous (base of the neck) injection with 107 W256 cells.F344 rats were anaesthetised with isoflurane  and received an intracardiac injection of 104 MLL cells in 0.25 ml saline. They also received intramuscular, intraliver, intraspleen and subcutaneous injections with 3.25 × 104 MLL cells in the same locations than above. For both types of rats, the cell number to be injected was determined as the most appropriate in preliminary experiments. Data were compared with results obtained on control rats.Copenhagen rats were anaesthetised with isoflurane and received an intracardiac injection of 3.25 × 102, 9 days (F344) or 15 days (COP) after the intracardiac injection of tumour cells. These durations have been determined by preliminary studies and were found sufficient to obtain bone metastases before animals die from other tumour localisation. The hindlimbs were carefully dissected; bones and other tissue samples  were placed in a formalin-alcohol-based fixative at +4°C during 24 h.Rats were sacrificed by asphyxiation with COμm) were deparaffinised and stained by Haematoxylin Phloxine Saffron (HPS) as a standard tissue stain. Immunohistochemistry was performed on femur and organs of six F344 and six COP rats. Bone decalcification was done using a ready-to-use decalcifying fluid according to the manufacturer's recommendations . Bone and other tissues were then dehydrated, embedded in paraffin and sectioned at 4 μm. Antigen retrieval was performed by bain-marie heating in a citrate buffer (pH 6). Immunochemistry was performed using DAKOCYTOMATION procedure with the Dako autostainer automat . The following proteins were traced using specific antibodies:Dickkopf-1 (Dkk-1) is an antagonist of the canonical Wnt signalling by binding directly to the LDL receptor-related protein 5/6 (LRP5/6). The interaction between DKK1 ant LRP5/6 induces an inhibition of bone formation (Interleukin 6 (IL-6) is a pleitropic cytokine. Its production by bone marrow stromal cells enhances osteoclastogenesis (with subsequent bone resorption) and tumour cell growth . We usedRANKL is produced by osteoblasts and stromal cells and induces osteoclastogenesis by interacting with its receptor at the osteoclastic surface . We usedCathepsin K is a cysteine protease highly expressed by osteoclasts and is implicated in the bone matrix collagen breakdown (Macrophage-colony stimulating factor (M-CSF) is expressed by stromal cells and osteoblasts. It is required together with RANKL to induce the development of mature osteoclasts (Paraffin sections (4 Control-positive tissues were obtained for each antibody. Sections were counterstained with haematoxylin.μm-thick) were cut dry on a heavy-duty microtome equipped with 50° tungsten carbide knives . Tartrate-resistant acid phosphatase was revealed by a simultaneous coupling reaction using 1-naphtyl phosphate  as substrate and Fast Violet B as the diazonium salt. Sections were counterstained with phosphomolybdic aniline blue (Tartrate-resistant acid phosphatase (TRAcP)-positive cells were detected by a histoenzymatic technique in three F344 and three COP rats. Tartrate-resistant acid phosphatase is a key enzyme found in high level in osteoclasts; it is involved in the intracellular degradation of collagen and noncollagenous proteins of the bone matrix  in the marrow spaces. These cells were mixed with haematopoietic cells and were at a distance from bone surfaces ; they coTwo types of cells exhibited TRAcP positivity: multinucleated cells apposed on trabeculae and either multi- or mononucleated cells encountered in bone marrow spaces were; they were situated far from trabecular surfaces and in the close vicinity of metastatic cells, their density decreased at distance from tumoral foci .Tumour nodules of both types of carcinomas were dense and well delimited by a fibrous capsule. There were composed of a large majority of tumour cells mixed with less numerous stromal cells. Tumour cells showed a different aspect according to the metastatic site: brain metastases were composed of spindle-shaped cells, whereas in other sites, they appeared round or polygonal . For both line cells, tumour cells were large with a high nucleocytoplasmic ratio; prominent nucleoli were often present and the cytoplasms were devoid of vacuoles. Numerous mitoses were observed, but small foci of necrosis were also noted inside nodules.Both tumour models exhibited the same immunohistochemical labelling pattern: tumour cells never exhibited any significant labelling for M-CSF, Dkk-1, RANKL, IL-6 and cathepsin K. Stromal cells in contact with malignant cells did not express Dkk-1, M-CSF and cathepsin K. At a distance from tumour foci, they also failed to express these markers. IL-6 expression was found homogeneous in the liver parenchyma with diffused granular cytoplasmic expression in the hepatocytes. The staining was found in all the liver parenchyma and not restricted to the areas surrounding the tumour. Few stromal cells were located around the tumoral foci and did not exhibit IL-6 expression . Some RATartrate-resistant acid phosphatase-positive cells were found interspersed in the spleen parenchyma whether tumour cells were present or not. These mononucleated TRAcP+ cells appeared round with a large granular cytoplasm; the nucleus was ovoid or reniform . Tartratβ to promote or suppress carcinoma progression and metastasis  that are employed as a paracrine communication system with surrounding cells or as an autocrine regulation system (Interaction between tumour cells and other stromal components also plays a major role in cancer progression. Cancer cells can alter the surrounding connective tissues and modulate the metabolism of fibroblasts, thus resulting in the production of a collagenous stroma that supports the tumoral process. Fibroblast is a key component of tumours, because it acquires a modified phenotype similar to those associated with wound healing. Such ‘activated’ fibroblasts have been coined ‘carcinoma-associated fibroblasts’ . CarcinoThe stromal expression of important cytokines involved in the bone remodelling was found altered only when metastatic cells were developing in a bony microenvironment. No expression of these cytokines could be observed in the tumour stroma when metastastic cells had developed in other organs. The few RANKL+ cells found at the periphery of subcutaneous tumours were probably Langerhans cells. Tartrate-resistant acid phosphatase-positive cells encountered both in invaded and non-invaded spleens probably corresponded to activated macrophages, a condition previously noted by others (Tumour cells do not have the same cytokine pattern of expression according to their location and can modulate it depending on the surrounding stromal cells. For example, breast cancer cells have an increased expression of PTHrP in bone metastases when compared with other sites . As a coIn conclusion, interaction between stromal and cancer cells alters the expression of the former leading to osteoclastogenesis in the bone microenvironment. These modifications allow the growth of the metastases in a convenient microenvironment. It thus explains the preferential localisation observed in our bone metastasis models. The major role of osteoclasts and osteoblasts in bone metastasis formation has led to underestimate the critical function of microenvironment in metastasis growth.


About 85% of the maize genome consists of highly repetitive sequences that are interspersed by low-copy, gene-coding sequences. The maize community has dealt with this genomic complexity by the construction of an integrated genetic and physical map (iMap), but this resource alone was not sufficient for ensuring the quality of the current sequence build. For this purpose, we constructed a genome-wide, high-resolution optical map of the maize inbred line B73 genome containing >91,000 restriction sites (averaging 1 site/∼23 kb) accrued from mapping genomic DNA molecules. Our optical map comprises 66 contigs, averaging 31.88 Mb in size and spanning 91.5%  of the maize genome. A new algorithm was created that considered both optical map and unfinished BAC sequence data for placing 60/66  optical map contigs onto the maize iMap. The alignment of optical maps against numerous data sources yielded comprehensive results that proved revealing and productive. For example, gaps were uncovered and characterized within the iMap, the FPC (fingerprinted contigs) map, and the chromosome-wide pseudomolecules. Such alignments also suggested amended placements of FPC contigs on the maize genetic map and proactively guided the assembly of chromosome-wide pseudomolecules, especially within complex genomic regions. Lastly, we think that the full integration of B73 optical maps with the maize iMap would greatly facilitate maize sequence finishing efforts that would make it a valuable reference for comparative studies among cereals, or other maize inbred lines and cultivars. de novo from individual genomic DNA molecules.The maize genome contains abundant repeats interspersed by low-copy, gene-coding sequences that make it a challenge to sequence; consequently, current BAC sequence assemblies average 11 contigs per clone. The iMap deals with such complexity by the judicious integration of IBM genetic and B73 physical maps, but the B73 genome structure could differ from the IBM population because of genetic recombination and subsequent rearrangements. Accordingly, we report a genome-wide, high-resolution optical map of maize B73 genome that was constructed from the direct analysis of genomic DNA molecules without using genetic markers. The integration of optical and iMap resources with comparisons to FPC maps enabled a uniquely comprehensive and scalable assessment of a given BAC's sequence assembly, its placement within a FPC contig, and the location of this FPC contig within a chromosome-wide pseudomolecule. As such, the overall utility of the maize optical map for the validation of sequence assemblies has been significant and demonstrates the inherent advantages of single molecule platforms. Construction of the maize optical map represents the first physical map of a eukaryotic genome larger than 400 Mb that was created  Zea mays ssp. mays L.) is a pervasive, economically valuable crop supplying the world with food, animal feed, and with biofeedstocks used in the synthesis of a broad range of industrial products. It is also a model system for classical genetics and cytogenetics that has significantly contributed to our understanding of fundamental processes that include reproduction, photosynthesis, biosynthesis of primary metabolites, mobile elements, and chromosome structure-function relationships. Investigators have developed extensive genetic tools over the last two decades dealing with male sterility, QTLs, regeneration of crop species, wide hybridization, marker assisted selection, associative mapping, endosperm mutants, transgenic crops, genetic control of meiosis, transposable elements, chromosome elimination, etc. In addition, diverse germplasms have been accumulated that have leveraged the assessments of genomic modifications during domestication, molecular mechanisms of heterosis, and the roles played by mobile DNA elements affecting genome evolution. Such advances are now being rapidly exploited with paradigm shifting tools and resources that are fostering insights emerging from fully sequenced and annotated genomes. In 2005 three funding agencies – NSF, DOE and USDA – jointly pledged $32 million towards a 4-year program to sequence the maize genome. These agencies' goals were to ensure that cutting-edge genomic resources would be available for maize to accelerate translational research in the agriculture and bioenergy sectors.Maize (The maize genome is estimated to be 2.3–2.5 gigabases (Gb) in size Tekay, Huck, PREM-2, Opie, Ji, etc. The architecture of the maize genome is also heavily punctuated by a complex motif of repetitive elements. About 85% of the genome is made up of a complex mix of repetitive DNA that mainly includes numerous families of retrotransposons such as http://maize-mapping.plantgenomics.iastate.edu/). This augmentation drew new resources from the development of cytological markers based on B-A translocations, molecular markers based on isozymes, restriction fragment length polymorphisms (RFLPs), microsatellite or simple sequence repeat (SSR) markers, single nucleotide polymorphisms (SNPs), and cDNA or expressed sequence tags (EST) markers Given the current need for a broadly informative representation that includes coding sequences and precise physical characterization of gaps between genes, accurate genetic and physical maps are required for guiding the large-scale sequencing of maize genome. The genetic, physical, and integrated maps available for maize are briefly described. The 1935 maize genetic map featured just 62 loci that relied on morphological variants http://www.maizemap.org/iMapDB/iMap.html) has greatly increased the marker density across the entire maize genome and created a framework for directed clone-based sequencing and assembly of chromosome-wide pseudomolecules http://ftp.maizesequence.org/release-3b.50/All Releases/Early physical mapping of maize used a YAC  library constructed from an inbred line UE95 1) The pilot sequencing effort considered large parts of chromosome arms and BAC-end sequence gathered from random clones; this provided an early glimpse into genome structure, organization, and sequence composition 2) The gene-enrichment approaches culled gene-rich templates for side-stepping notoriously complex sequence repeats and high copy number DNA elements present in the maize genome. Enrichment was accomplished by a variety of sequencing approaches that included ESTs, genome filtration (methylation filtration and high-Cot selection), RescueMu (RM), and hypomethylated partial restriction (HMPR) 3) In contrast, clone-by-clone sequencing used a comprehensive, hierarchical, map-based approach that allowed construction of a BAC minimal tiling path across the iMap. Tiled BACs were then individually shotgun-sequenced and assembled Sequencing efforts for the maize genome have progressed through three stages: the pilot, gene enrichment, and clone-by-clone full genome sequencing stages. de novo genome assembly. With this concept in mind, we constructed a high-resolution optical map de novo assembly of a large data set containing ordered restriction maps of individual genomic DNA molecules ∼500 kb in size. This ordered restriction map provides an independent resource that lays out an accurate physical metric across the entire maize genome. Because large molecules were analyzed, we were able to physically map repeat-rich regions and link sequence and map data within complex genomic regions. We show here that our maize optical map identifies gaps within and between sequence contigs and guides the assembly and validation of reference chromosomes.A direct and encompassing way to deal with the formidable architecture of the maize genome is to analyze “chunks” of it, at high-resolution, that are as large as possible. In this way, nests of sequence repeats are largely bridged by chunks that offer a sufficient level of unique sequence information for supporting We constructed a whole-genome shotgun optical map for maize using the CpG methylation insensitive restriction enzyme SwaI. The optical map data set contains 2,116,074 genomic DNA molecules, ranging in size from 300 kb to 3,700 kb, and totaling ∼927,604 Mb, or ∼403× coverage of the maize genome. The maps in this raw data set—one optical restriction map per genomic DNA molecule—have a mean length of 438.4 kb with an average fragment size of 26.1 kb.de novo assembly of maps relied on a divide and conquer strategy that leveraged available cluster computing resources Because of the vast size of the maize optical data set, our The consensus maps were internally validated in an additional iterative assembly step. They were partitioned into a series of overlapping 1 Mb map intervals for use as new seed maps, with the overlaps covering ∼500 kb. Because this diagnostic assembly reproduced the original set of 66 parental contigs, the current optical assembly is apparently free of any chimeric maps. Statistics describing the 66 optical map contigs are shown in Optical contigs terminate to form a gap when the SwaI restriction site density is low, or when a contig reaches the end of a chromosome. Sharply demarcated contig edges may represent telomere associated sequences near chromosome ends. Using these criteria we identified 15 contigs  that have reached the ends of chromosome as evidenced by contig “edges” comprising more than 5 maps that show no significant map “overhangs” . A colleordered SwaI restriction fragments. However, placement of tiled , but unfinished, BAC sequences released by the maize genome sequencing project  on optical maps required development of a new algorithm that considers alignments of FPC clones comprising unordered and unoriented sequence contigs (averaging 11 sequence contigs per BAC). We had developed a new algorithm several years ago to integrate the optical and FPC maps through the alignment of unfinished BAC sequence data  of such clones. This alignment also considers the fragment sizing error model used for alignment of optical and sequence in silico maps vs. FPC maps  onto the 2006 FPC map We assessed the accuracy of BACop by analyzing the expected placement of 15 telomeric optical contigs onto the ends of chromosomes on the iMap. in silico restriction map, and 9 in common between OMcontig_23 and Zm9L_supercontig (Chr 9) covering 3.62 Mb (54.85%). However, the Chr 3 finished supercontigs, corresponding to GenBank EF517601 and EF517600 in silico maps for Chrs 1 and 9 pseudomolecules is not surprising because most of the sequenced BACs are in phase 1 assembly, awaiting the ordering and orienting of their associated sequence contigs. Accordingly, gaps of unknown size remain both within and between these nascent sequence assemblies. Based on these alignments of optical contigs vs. pseudomolecules, we characterized many of these gaps and identified issues with orientation. The assembly of the Zm9L_supercontig appears to be superior to that of the Zm1S_supercontig. This view is further buttressed by the higher proportion of phase 2 BAC sequences (28/56) in the Zm9L_supercontig than in the Zm1S_supercontig (14/60).We evaluated the quality of available and ongoing maize sequence assemblies by comparing optical contigs completely spanning large “supercontigs” (pseudomolecules) from Chrs 1, 3 and 9 vs. sequence contig alignments that tracked, guided, and validated the ongoing sequence finishing efforts of a ∼22 Mb sequence pseudomolecule (FPC ctg182) by the Arizona Genomics Institute (AGI). The process of constructing a large sequence pseudomolecule is an iterative one, drawing support for provisional assembly from many sources that guide the serial generation of hypothetical builds and their subsequent validation. As such, we performed a series of optical The assembly of the accessioned golden path (AGP) involved the merging of 435 correctly ordered FPC pseudomolecules  to span across the entire maize genome, and it is now known as the B73 RefGen_v1 http://www2.genome.arizona.edu/genomes/maize_contig_quality_table). The B73 RefGen_v1 reference chromosomes represent a unified genomic resource showing chromosome-wide placement of sequence and associated gaps. We provided an independent, optical reference map for this important resource via alignments that comprehensively revealed and sized sequence gaps in FPC pseudomolecules and the B73 RefGen_v1, which compose reference chromosomes. Local alignment reveals AGP assembly errors characterized as novel gaps, extra and/or missing cuts, and fragment sizing errors. In total, 1,102 optical contig segments (strings of contiguous restriction fragments) aligned to the B73 RefGen_v1 reference chromosomes (B73 RefGen_v1) from the Arizona Genomics Institute recently released by the maize genome sequencing consortium comprises 10 chromosome-wide “reference chromosomes” (pseudomolecules), and its assembly was guided by several physical maps, including the optical mapping findings presented here  B73 inbred line (PI 550473) genome, which is a parent of the IBM mapping population. 66 optical contigs are included in this map representing 2,103.93 Mb of the maize genome decorated by 91,453 ordered SwaI restriction sites with accurate physical distances between these sites. On average, there is a SwaI site every 23 kb across the genome, and this restriction recognition sequence “marker” density is far greater than those on genetic  and FPC  maps http://maize-mapping.plantgenomics.iastate.edu/). Because the optical data format is a high-resolution ordered restriction map (SwaI), we were able to anchor and orient FPC-sequence contigs  onto this scaffold. While the immediate utility of the maize optical reference map is as an independent reference for sequence finishing and closing gaps, it will also drive comparative studies for unraveling complex patterns of structural variation as additional inbred lines and cultivars are mapped. Here the optical reference map would serve as a scaffold for future map assemblies enabling rapid discernment of genomic architecture. In this regard, optical mapping may be unique since large ∼500 kb molecules are directly mapped, and this advantage supports scalable genome analysis spanning from a restriction site to multi-megabase-sized regions.An optical map was created that spans across ∼91% of the maize  This physical map is free from common cloning and PCR artifacts, since individual genomic molecules are directly analyzed. These advantages are demonstrated by our comprehensive analysis of several pseudomolecules  within the optical reference map, averaging 31.88 Mb in length. In comparison, the May 2006 maize FPC map comprises 721 FPC contigs averaging ∼2.98 Mb in length with a total mass of 2,150 Mb  Maize centromeres have been mapped to regions with flanking molecular markers using many different approaches de novo assembly because of modest read lengths and errors. As such, new sequencing strategies must be developed for wheat and other complex crop genomes that effectively seize the new opportunities enabled by next generation sequencing. To this end, we propose the proactive use of optical mapping data for sequence assembly Although we have demonstrated here that optical mapping offers numerous benefits for physical mapping and genome sequence assembly, its utility would be appreciably extended when combined with next generation sequencing. Genome analysis approaches are now rapidly evolving and tracking the increasingly cost-effective capabilities offered by next generation sequencing. Next generation sequencing approaches using single molecule libraries are now tackling the analysis of complex genomes 2PO4, 155.17 mM NaCl, 2.71 mM Na2HPO4, pH 7.2) to remove glycerol. Rapid DNA concentration assays were conducted by lysing small aliquots of nuclei in TE  with 1 mg/ml proteinase K, and adenovirus DNA added at 25 pg/µl , followed by mounting, restriction digestion, staining and imaging as previously described. Appropriate dilutions for mapping (optimized to minimize molecular crossovers) were made by adjusting the amount of isolated nuclei in the lysing solution , by slowly pipetting up and down several times using a wide-bore pipette tip; samples were incubated at 65°C for 1 hr and at 37°C overnight. Samples were mounted onto optical mapping surfaces and imaged by fluorescence microscopy to assess DNA integrity and concentration of both genomic and reference standard DNA molecules.Maize kernels , obtained from the USDA-Agriculture Research Service North Central Regional Plant Introduction Station , were washed in 10% Clorox bleach for 10 min, rinsed in sterile water , and germinated on moistened brown paper towels in a dark, moist chamber at 30°C for 12 days. Residual ungerminated seeds were removed from maize sprouts prior to nuclei isolation. The procedures for isolation of nuclei and storage have been described previously Surface preparation was done as previously described Genomic DNA molecules (∼400–500 kb) premixed with the adenovirus DNA sizing standards were deposited as stripes on derivatized glass surfaces using a silastic microchannel system Leishmania major genomes With a raw map data set of >2 million maps, a divide and conquer strategy for optical map assembly was needed to deal with the severe computational load through parallel processing. We previously used this approach for the assembly of genome maps spanning the rice as well as the Consensus maps are then stripped from the newly formed contigs as updated seed maps after further processing. The pairwise alignment phase extracted multiple high-scoring alignments based on the efficient linear scaling approach of Huang and Miller BACop” — that utilizes unfinished BAC sequence data. The algorithm for anchoring the optical maps on the maize genetic-physical (FPC) map precedes in four distinct steps: i) matching restriction fragments between the optical map and the in silico restriction fragments from the sequence contigs of the BACs, ii) determining locations of all the BAC sequence contigs along the optical map, iii) anchoring the BACs on the optical maps, and iv) filtering and combining the alignments of BACs in the FPC map and optical map to find the most significant ones  Restriction map view (box = restriction fragment) of FPC contigs aligned to the AGP Chr 6 pseudomolecule; the gap is highlighted. (B) Cartoon showing the alignments of FPC contigs ct280 and ctg281 to Chr6 pseudomolecule. A gap of unknown size is illustrated, with green lines showing alignment to the pseudomolecule. Dashed lines show alignments of ct280 and ctg281 against OMcontig_6 (gold track) and a large gap. (C) Restriction map view of OMcontig_6, locating the large gap within the grayed restriction fragments. FPC gap sizes are calculated as: Gap (kb) = [|−|−|(right sequence coordinate)−(left sequence coordinate)|]/1000.(0.30 MB PDF)Click here for additional data file.Table S1in silico maps of the FPC contig sequence pseudomolecules.Ordering FPC contig sequence pseudomolecules based on the map alignments between optical maps and the (0.10 MB PDF)Click here for additional data file.Table S2in silico maps of the B73 RefGen_v1 reference chromosomes.Discordances in the well-aligned map segments between optical maps and the (0.25 MB PDF)Click here for additional data file.Table S3in silico maps of FPC contig sequence pseudomolecules and B73 RefGen_v1 reference chromosomes.FPC gap estimations based on map alignments between optical maps and the (0.12 MB PDF)Click here for additional data file.


Glycine max) genome has been shown to be composed of approximately four thousand short interspersed homeologous regions with 1, 2 or 4 copies per haploid genome by RFLP analysis, microsatellite anchors to BACs and by contigs formed from BAC fingerprints. Despite these similar regions,, the genome has been sequenced by whole genome shotgun sequence (WGS). Here the aim was to use BAC end sequences (BES) derived from three minimum tile paths (MTP) to examine the extent and homogeneity of polyploid-like regions within contigs and the extent of correlation between the polyploid-like regions inferred from fingerprinting and the polyploid-like sequences inferred from WGS matches.Many of the world's most important food crops have either polyploid genomes or homeologous regions derived from segmental shuffling following polyploid formation. The soybean  were single nucleotide polymorphisms  and single nucleotide indels (SNIs 10%). Larger indels were rare but present (1%). Simulations that had predicted fingerprints of homeologous regions could be separated when divergence exceeded 2% were shown to be false. We show that a 5–10% sequence divergence is necessary to separate homeologs by fingerprinting. BES compared to WGS traces showed polyploid-like regions with less than 1% sequence divergence exist at 2.3% of the locations assayed.The use of HSVs like SNPs and SNIs to characterize BACs wil improve contig building methods. The implications for bioinformatic and functional annotation of polyploid and paleopolyploid genomes show that a combined approach of BAC fingerprint based physical maps, WGS sequence and HSV-based partitioning of BAC clones from homeologous regions to separate contigs will allow reliable de-convolution and positioning of sequence scaffolds (see BES_scaffolds section of SoyGD). This approach will assist genome annotation for paleopolyploid and true polyploid genomes such as soybean and many important cereal and fruit crops. Glycine max) is the second most valuable crop in the U.S., accounting for $12–17 billion in annual revenue . Genomics has had a profound effect on plant biology, but the impact on major crop species such as soybean remains limited to a few marker characterized disease resistant germplasm releases [Arabidopsis thaliana, Medicago truncatulata or Lotus japonicus. Further, the soybean genome shows evidence of a paleopolyploid origin with gene-rich islands that were highly conserved following duplication [Soybean  called the generic model organism databases (e.g. GMOD) , was devArabidopsis thaliana, or 'Mo17' and 'B73' are to Zea mays. The soybean community is committed to advancing both resources, with Williams 82 as the lead for a complete genome sequence. In 2007 there were 7.4 million trace sequences at NCBI. Some preliminary sequence contigs with annotations for about 90% of the genome were available. Sequence contigs can be viewed on the genetic map at a new section of SoyGD [in silico biology that can resolve the physical map and de-convolute the complete genome sequence. Here, these resources were tested for usefulness as tools to determine if the existing contig annotations truly reflect genomic regions that are polyploidy-like, to identify HSVs that can distinguish homeologs within cultivars and to identify HSVs that can distinguish among soybean cultivars.The cultivars Forrest and 'Williams 82' provide a large set of useful genomic tools for soybean genomics ,12,13. TA MegaBlast of Forrest BES Table  to WilliExamination of the second BES from B47P08  to 80 different genetic markers , or about 4% of the markers with sequences in GenBank. About three thousand new microsatellite markers were identified within the whole BES collection.Eighty one homologs of DNA markers found in genetic maps were detected in the BES, i.e. forty two BES's contained sequence highly homologous , there were 7.4 million reads of Williams 82 genome reported in the trace sequence section of NCBI . The totGlycine max WGS database using individual BES [Glycine max-WGS; hits computed 250; all low complexity filter selected; expect was set to 10; word size was set to 32 or 64; percent identity used was normally 90% though 99%, 98%, 95%, and 85% were manually tested in instances noted in the results.MegaBlast enquiries were made of the dual BES . CriteriResults were assembled into groups of 100 by expected copy number and 600 were examined by a manual editor. Distance trees of the results were selected  were filtered out by Blast searching, unlike Shultz et al., [BACs from homeologous regions that assembled into single contigs were picked from BAC library master plates. DNA was extracted as previously described ,17. PrimPolymerase chain reaction (PCR) was performed in a PE 9700 . An initial 95°C denaturation for 5 min was followed by 30 cycles of 95° for 30 s, 55° for 30 s, and 72° for 30 s. After PCR was complete, gel electrophoresis was performed in a 2% (w/v) agarose gel or a 4% (w/v) PAGE stained with ethidium bromide and amplicon documented using a BioRad GelDoc  system. Bands were isolated in pGEM3T. SNP polymorphism was identified by DNA sequencing of PCR amplicons following plasmid isolation using a CEQ2000 .All potential SNPs, SNIs and microsatellites that distinguish either cultivars or homeologs were named with the SIUC_ suffix (at each database entry and first mention in the text) followed by N-, I- or S- prefix. For cultivar polymorphisms this was followed by the motif and BAC of origin. For HSVs the letter H- was suffixed, then followed by the motif and BAC of origin. In contrast, earlier markers were assigned a sequential number -33. The BAC: bacterial artificial chromosome; BES: BAC end sequence; bp: base pair; contig: contiguous set of overlapping clones; EST: expressed sequence tag; HSV: homeolog sequence variant; LIS: legume information system; MTP: minimum tiling path; NCBI: National Center for Biotechnology Information; RFLP: restriction fragment length polymorphism; SNH: single nucleotide polymorphism between homeologs; SNI: single nucleotide insertion; SNP: single nucleotide polymorphism between alleles; SSR: simple sequence repeat.DAL and NS conceived of the study and executed all of the analysis. JS classified the contigs for ploidy based on clones per unique band. DAL drafted the manuscript. NS and JS provided critical review, interpretation of results and funding. All authors read and approved the final manuscript.The WGS trace files homeologous to marker sequence Sat_368. No evidence for multiple homeologs was found.Click here for file


BRCA2 locus at 13q12–q13. The detection of a common haplotype shared by four out of seven families (60%) suggests the presence of a founder BRCA2 mutation. Direct sequencing of BRCA2 coding exons of patients carrying the shared haplotype, allowed the identification of a ‘frame-shift’ mutation at codon 2867 (8765delAG), causing a premature termination-codon. This mutation was found in breast cancer patients as well as one prostate and one bladder cancer patient with shared haplotype. We then investigated the frequency of 8765delAG in the Sardinian breast cancer population by analysing 270 paraffin-embedded normal tissue samples from breast cancer patients. Five patients (1.7%) were found to be positive for the 8765delAG mutation. Discovery of a founder mutation in Sardinia through the identity-by-descent method demonstrates that this approach can be applied successfully to find mutations either for breast cancer or for other types of tumours. © 2000 Cancer Research CampaignSardinian population can be instrumental in defining the molecular basis of cancer, using the identity-by-descent method. We selected seven Sardinian breast cancer families originating from the northern-central part of the island with multiple affected members in different generations. We genotyped 106 members of the seven families and 20 control nuclear families with markers flanking