Radiation induced genomic instability is a well-studied phenomenon, the underlying mechanisms of which are poorly understood. Persistent oxidative stress, mitochondrial dysfunction, elevated cytokine levels and epigenetic changes are among the mechanisms invoked in the perpetuation of the phenotype. To determine whether epigenetic aberrations affect genomic instability we measured DNA methylation, mRNA and microRNA (miR) levels in well characterized chromosomally stable and unstable clonally expanded single cell survivors of irradiation. While no changes in DNA methylation were observed for the gene promoters evaluated, increased LINE-1 methylation was observed for two unstable clones (LS12 and CS9) and decreased Alu element methylation was observed for the other two unstable clones (115 and Fe5.0–8). These relationships also manifested for mRNA and miR expression. mRNA identified for the LS12 and CS9 clones were most similar to each other (261 mRNA), while the 115 and Fe5.0–8 clones were more similar to each other, and surprisingly also similar to the two stable clones, 114 and 118 (286 mRNA among these four clones). Pathway analysis showed enrichment for pathways involved in mitochondrial function and cellular redox, themes routinely invoked in genomic instability. The commonalities between the two subgroups of clones were also observed for miR. The number of miR for which anti-correlated mRNA were identified suggests that these miR exert functional effects in each clone. The results demonstrate significant genetic and epigenetic changes in unstable cells, but similar changes are almost as equally common in chromosomally stable cells. Possible conclusions might be that the chromosomally stable clones have some other form of instability, or that some of the observed changes represent a sort of radiation signature and that other changes are related to genomic instability. Irrespective, these findings again suggest that a spectrum of changes both drive genomic instability and permit unstable cells to persist and proliferate. Radiation induced genomic instability is a delayed, persistent effect of ionizing radiation exposure that manifests in the unirradiated progeny of irradiated cells as an increased frequency of mitotically heritable genetic alterations. Radiation induced genomic instability is a non-targeted phenomenon that is thought to contribute to radiation carcinogenesis, however the mechanisms underlying this process are poorly understood Epigenetic mechanisms include altered DNA methylation, histone and chromatin modifications, and microRNA (miR) all of which can affect gene expression and cellular phenotype. Epigenetic aberrations have been observed following irradiation and also play a role in carcinogenic processes We hypothesize that epigenetic aberrations are perpetuated in chromosomally unstable cells exhibiting genomic instability and that these epigenetic aberrations play a mechanistic role in the unstable phenotype. To test this hypothesis, DNA methylation, mRNA and miR levels were measured in well characterized clonally expanded single cell survivors of either low linear energy transfer (LET) X-irradiation or high LET iron (Fe) ion irradiation Unless otherwise noted, reagents were obtained from Sigma . All primer sequences and annealing temperatures are listed in 2. Cell cultures were routinely screened to exclude the presence of mycoplasma .The human-Chinese hamster ovary (CHO) hybrid cell line, GM10115, contains a single copy of human chromosome 4 in a background of 20–24 hamster chromosomes . The parental GM10115 cell line and the GM10115-derived stable clones  and unstable clones  were maintained in high glucose Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum , 2 mM L-glutamine , and 0.2 mM L-proline at 34°C with 5% COin situ hybridization (FISH) with chromosome 4 as the probe. A clone was classified as unstable if FISH analysis identified >3 clones showing unique rearrangements of chromosome 4 that make up >5% of the 200 metaphase cells analyzed. In this study each clone was re-characterized cytogenetically to confirm the stable/unstable nature of the clone as described previously These stable and unstable cell lines were established and originally characterized by Limoli and colleagues following irradiations that took place in 1997 in the case of the CS9, LS12, 115, 114, and 118 clones DNA was extracted from all experimental groups in a single batch using the DNeasy kit using standard methods  and stored in TE buffer at −20°C. Bisulfite modification of genomic DNA was also performed in a single batch using the EpiTect bisulfite kit using standard methods (Qiagen). Bisulfite treated DNA was used in specific locus, bisulfite sequencing and repeat element methylation analyses.nuclear factor-kappa B, tumor suppressor in lung cancer 1 and E-cadherin  was evaluated by methylation-specific PCR (MSP) as described previously Promoter DNA methylation for the NFκB was also measured by bisulfite sequencing. Primers were tested for specificity to bisulfite treated DNA and no amplification of genomic DNA occurred. Sequencing was performed at the Institute for Genome Sciences, University of Maryland Biopark. Three biological replicates were performed.Promoter methylation of NFκB sequence were used to determine presence of the genomic sequence. PCR products were resolved on 3% agarose gels, stained using ethidium bromide and images were digitally captured. Two biological replicate experiments were performed for each clone.Primers for genomic T values against the log template amount, and determining the slope of the resulting standard curve −(ΔCt) calculations. Three biological replicate experiments were performed for each clone.Total RNA was extracted from all experimental groups in a single batch using the miRNeasy Mini Kit (Qiagen) following standard kit procedures with a DNAse treatment to eliminate DNA contamination. RNA concentrations and purities were measured using a NanoDrop ND-1000 spectrophotometer . For all RNA, the 260/280 ratios were between 1.97–2.08, and the 260–320 ratios were above 1.5. cDNA was prepared from 1 µg RNA using 5× iScript Reaction Mix kit . Negative control reactions omitting reverse transcriptase were included with all experiments. Quantitative PCR was performed using SsoFast EvaGreen Supermix (Bio-Rad). The efficiency of real-time PCR for each primer was calculated by analyzing a template dilution series, plotting the CLINE-1 and Alu repeat element DNA methylation was evaluated by combined bisulfite restriction analysis (COBRA) Global DNA methylation was evaluated by methylation-sensitive arbitrarily primed PCR (MSAP-PCR) using a single primer, MLG2 The bands observed on the MSAP-PCR gels were excised and the DNA fragments purified using the MinElute Gel Extraction Kit (Qiagen). These PCR products were sequenced at the Biopolymer-Genomics Core  using the MLG2 primer. The sequences were identified using National Center for Biotechnology Information (NCBI) blasts. Bisulfite sequencing was used to validate the methylation status of identified sequences. PCR products were resolved on 3% agarose gels to confirm reaction quality, stained using ethidium bromide, and images were digitally captured. PCR products were cloned into competent bacteria using the TOPO cloning kit (Invitrogen) and subsequent plasmid mini preparation (Invitrogen). Sequencing was performed at the Biopolymer-Genomics Core using the T3 primer (5′-ATT AAC CCT CAC TAA AGG GA-3′). For each sample, ten sub-clones were sequenced from each of the clones used in this study.MiR microarray analysis of total RNA was performed by LC Sciences LLC . Quality control for the integrity of total RNA, enrichment of miR from total RNA, labeling, hybridization to µParaflo microfluidics chip and scanning were performed using miRHuman/Mouse/Chinese Hamster miR array chips, based on Sanger miR-Base Release 12.0. MiR identified as differentially regulated for each clone as compared to the parental GM10115 cell line are provided in the miR Excel in vitro transcription. mRNA identified as differentially regulated for each clone as compared to the parental GM10115 cell line are provided in the mRNA Excel Database for Annotation, Visualization and Integrated Discovery (DAVID) Gene expression microarray analysis was performed by LC Sciences using the Affymetrix GeneChip Mouse Genome 430 2.0 array. The mouse array was used because a hamster genome array was not available, and mouse was deemed the closest match. Quality control for the integrity of total RNA was performed and then the Affymetrix's GeneChip IVT Express kit was used for cDNA synthesis and To determine gene expression pathways affected by altered miR expression in clones exhibiting genomic instability, predicted gene targets of miR were merged with mRNA candidates using the Bioinformatics Resource Manager program version 2.1 Candidate miR with an intensity signal ≥500 and involved in pathways of interest were validated using qRT-PCR. Total RNA used for the microarray analysis was reverse transcribed to cDNA template and TaqMan miR assays were performed in triplicate by LC Sciences. The hsa-miR-16 miR, which was expressed equally in all the samples on the microarray, was used as an internal control.P values <0.05 were considered statistically significant. For the miR expression arrays, preliminary statistical analyses were performed on raw data normalized by the Locally-Weighted Regression (LOWESS) method on the background-subtracted data. ANOVA were then performed to identify differences in miR expression. Two replicate arrays were performed, so the significance threshold was set at P<0.10. For the mRNA expression arrays, three replicate arrays were performed and the data were normalized using the Robust multiarray analysis, and differentially regulated genes were identified with multiple testing and false discovery rate statistics at P<0.05 using the GeneSpring GX software .Unless otherwise noted above, means and standard errors were calculated for all data points from either 3 or 4 replicate experiments. The means were compared between samples by Student's t-test analysis using the StatPlus Mac software package  and NFκB in the unstable phenotype NFκB gene plays a role in genomic instability, a promoter region of NFκB containing 4 CpG was evaluated using methylation sensitive PCR for comparison of the clones to the parental GM10115 cell line  database indicates the presence of a transposable element immediately upstream of the original forward primer's binding site, suggesting a possible hot spot for chromosomal breaks and rearrangement. Analysis of DNA methylation status for two other gene promoters, TSLC1 and CDH1, showed no effects of the instability phenotype or history of irradiation on DNA methylation .Subsequently, qRT-PCR primers based on the human products . Sequencproducts . The forP = 0.02 and 0.01, respectively; P = 0.03 and 0.005, respectively; Both repeat element and global DNA hypomethylation have been linked to genomic instability ▵CGG…3′. The enzyme will not cut if the internal cytosine is methylated. Since the MGL2 PCR primer binds at that cleavage site, presence or increase of a particular PCR product correlates CpG methylation. Changes in DNA methylation at this internal CpG were observed in one or more clones for three out of the eleven bands evaluated (P = 0.01). Band #8 was hypomethylated in the stable clone 118 and the unstable clone 115 . Band #9 was hypomethylated in the stable clone 118 and unstable clones 115 and Fe5.0–8 .Global DNA methylation was evaluated by MSAP-PCR. Genomic DNA was digested using the HpaII restriction enzyme that is sensitive to methylation at the internal cytosine of the recognition site, 5′…Cvaluated . Band #2NFκB experiments regarding the potential for deletion events in those clones  analyses identified significant enrichment of genes involved in signaling pathways affecting neurodegenerative diseases including Parkinson's, Alzheimer's and Huntington's diseases . The AlzP≤0.05, signal ≥100, fold change ≥1.5). These observations again correlated with the repeat element DNA methylation profiles, as well as the gene expression profiles. The highest levels of overlap occurred for the 115 and Fe5.0–8 unstable clones, followed by the stable 114 and 118 clones, with 8 and 6 miR in common, respectively  member A2, clatherin light chain alpha, chromodomain helicase DNA binding protein 2, slingshot homolog 2, caldesmon binding protein 1, and poly(A) polymerase alpha . SSH2, Cald1, Chd2 and PAPOLA are all up regulated for all four clones, Slc35a2 is down-regulated in all four clones, and CTLA is up-regulated for 115 and Fe5.0-8 and down-regulated for 114–118.The 6 anti-correlated mRNA that were common to the 115, Fe5.0–8, 114 and 118 clones are We have tested the hypothesis that clones exhibiting radiation induced genomic instability have unique genetic and epigenetic changes that are different from irradiated, but chromosomally stable cells and as compared to the parental cell line. Further, we hypothesized that these changes play a mechanistic role in the unstable phenotype. These changes were evaluated in two stable clones that had been exposed to 10 Gy of low LET X-rays , three unstable clones that had been exposed to 10 Gy of X-rays  and one clone that had been exposed to 5 Gy of high LET Fe ions (clone Fe5.0–8). Surprisingly, while significant genetic and epigenetic differences were observed in our unstable cells, similar changes were also frequently seen in the chromosomally stable cells. These findings suggest that the observed alterations may be representative of persistent effects of irradiation on that surviving progenitor cell rather than a signature of a mechanism driving genomic instability. Alternatively, while clones 114 and 118 do not exhibit chromosomal instability as measured by FISH analysis, they may have some other form of genomic instability for which they have not been evaluated such as expanded simple tandem repeat (ESTR) instability NFκB plays a role in the unstable phenotype observed in these cell lines NFκB genes in the stable and unstable clones with no consistent trends. Future studies will require follow up work to determine whether the changes in mRNA level are reflective of NFκB protein levels. Similarly, while changes in global and repeat element DNA methylation were observed, there was a lack of consensus among the four unstable clones. However, the common LINE-1 DNA hypermethylation observed for the CS9 and LS12 clones and the common Alu element DNA hypomethylation observed for the 115 and Fe5.0–8 emerged as the first evidence of a compelling story. While it is possible that our data are merely correlative, the persistence of these unique groupings among the clones is also quite clear at the level of mRNA and miR expression.In this study the observed spectrum of changes in our stable and unstable cells included possible deletion events, and altered DNA methylation, mRNA and miR levels. These changes suggest that some cells might compensate for this history of irradiation better than others or at least in different ways. The changes also suggest that there are likely numerous mechanisms or pathways employed to compensate for deficiencies and to maintain cellular homeostasis. Although, it has been suggested previously that We have only evaluated one clonally expanded single cell survivor of high LET irradiation so no conclusions can be drawn, but some observations might be made. The Fe5.0–8 cell line was exposed to 5 Gy of Fe ions as compared to 10 Gy of X-rays for the other clones. This might lead one to expect differences in phenotype based on radiation quality. However, the types of epigenetic and genetic changes that we observed for the Fe5.0–8 clone are not different from the low LET irradiated clones in any obvious way. High LET radiation exposure causes a different spectrum of DNA damage and generally has a higher relative biological effectiveness (RBE) for cell killing than low LET irradiation. In the original study that generated the Fe5.0–8 cell line Limoli and colleagues reported that the RBE of Fe ions for cell killing was 2, while the RBE for inducing chromosomal instability was only 1.3 The results of this study also reinforce the role of oxidative stress and mitochondrial function in the radiation response and genomic instability. Oxidative stress has been clearly shown to persist in these chromosomally unstable cell lines While protein levels or enzyme activities were not evaluated in the current study we can also postulate some potential compensatory or detrimental effects of the epigenetically regulated changes in gene expression for the six miR anti-correlated mRNA that were common to the 115, Fe5.0–8, 114 and 118 clones. There are roles for Ssh2, caldesmon, clatherins, SLC35a2, poly(A) polymerase and Chd2 in everything from actin regulation, endocytosis, galactose transport, pre-mRNA poly adenylation to chromatin structure, DNA damage responses and genomic instability Other studies characterizing these cell lines have not eliminated the possibility that the two clones that are cytogenetically stable exhibit other forms of genomic instability Table S1PCR and qRT-PCR primer pairs. LS, Low Stringency; HS High Stringency.(DOCX)Click here for additional data file.Table S2miR with anti-correlated mRNA for 115-Fe5.(DOCX)Click here for additional data file.Table S3miR with anti-correlated mRNA for 114-118.(DOCX)Click here for additional data file.Workbook S1MiR identified as differentially regulated for each clone as compared to the parental GM10115 cell line.(XLSX)Click here for additional data file.Workbook S2mRNA identified as differentially regulated for each clone as compared to the parental GM10115 cell line.(XLSX)Click here for additional data file.


The introduction of next-generation sequencing (NGS) into clinics has revealed the important role of somatic NLRP3 mosaicism in these syndromes by means of its detection in a high proportion of patients who were apparently mutation-negative by Sanger sequencing. Thus, NGS technologies are becoming essential for routinely identifying the genetic cause of the suspected autoinflammatory disease.Cryopyrin-associated periodic syndromes (CAPS) usually present in early childhood as an urticaria-like skin rash associated with an increased inflammatory response, with additional manifestations  typically restricted to certain phenotypes. CAPS are caused by dominantly inherited or NLRP3 mutation as a somatic mutation.To describe a Spanish patient with CAPS that start in adulthood in whom molecular analyses detected a novel MEFV, TNFRSF1A, MVK, NLRP3, NOD2 and PSTPIP1) was performed by NGS. Additional molecular studies of somatic NLRP3 mosaicism were performed by targeted deep sequencing (TDS).Genomic DNA was extracted from peripheral blood. The analysis of the six most common genes associated with autoinflammatory diseases  with an allele frequency of 12.2% (coverage: 738x). We hypothesized that this variant could be a somatic NLRP3 mutation. TDS confirmed the somatic NLRP3 mosaicism at 18.4 % . Additional analyses showed that this variant has never been reported in public databases, that is located on a highly evolutionary conserved amino acid residue and that is predicted to be possibly damaging by PolyPhen-2 algorithm. Additional functional and genetic studies are currently ongoing.The patient is a 63 year-old Spanish male who presented with a generalized urticarial rash, a gradually worsening oligoarthritis at wrists, elbows and knees, and a moderate bilateral sensorineural hypoacusia starting in his 50s. Laboratory results showed a marked leucocytosis, neutrophilia and increased inflammatory markers without evidence of circulating autoantibodies. Multiple therapeutic approaches including NSAID, corticosteroids, methotrexate and colchicine result in poor or partial responses. Screening of autoinflammatory-associated genes identified a novel NLRP3 mosaicism. Our findings highlight the diagnostic utility of NGS technologies in detecting low-level somatic gene mosaicism and support its use as a routine genetic screening tool.We herein describe the case of a patient with a clinical picture compatible with MWS, with the exception of a late onset of the disease, who carries a somatic Written informed consent for publication of their clinical details was obtained from the patient/parent/guardian/relative of the patient.


Arnt (aryl hydrocarbon receptor nuclear translocator) and Gdi2 (GDP dissociation inhibitor 2). Here we show that ectopic expression of Arnt and Gdi2 promoted LMD in mice bearing Sonic hedgehog (Shh)-induced medulloblastomas. We overexpressed Arnt and Gdi2 in a human medulloblastoma cell line (DAOY) and an immortalized, nontransformed cell line derived from mouse granule neuron precursors (SHH-NPD) and quantified migration, invasiveness, and anchorage-independent growth, cell traits that are associated with metastatic competence in carcinomas. In SHH-NPD cells. Arnt and Gdi2 stimulated all three traits. In DAOY cells, Arnt had the same effects, but Gdi2 stimulated invasiveness only. These results support a mechanism whereby Arnt and Gdi2 cause cells to detach from the primary tumor mass by increasing cell motility and invasiveness. By conferring to tumor cells the ability to proliferate without surface attachment, Arnt and Gdi2 favor the formation of stable colonies of cells capable of seeding the leptomeninges.Leptomeningeal dissemination (LMD), the metastatic spread of tumor cells via the cerebrospinal fluid to the brain and spinal cord, is an ominous prognostic sign for patients with the pediatric brain tumor medulloblastoma. The need to reduce the risk of LMD has driven the development of aggressive treatment regimens, which cause disabling neurotoxic side effects in long-term survivors. Transposon-mediated mutagenesis studies in mice have revealed numerous candidate metastasis genes. Understanding how these genes drive LMD will require functional assessment using in vivo and cell culture models of medulloblastoma. We analyzed two genes that were sites of frequent transposon insertion and highly expressed in human medulloblastomas: The online version of this article (doi:10.1186/s40478-014-0085-y) contains supplementary material, which is available to authorized users. An important goal in designing therapies for patients with the malignant brain tumor medulloblastoma is to reduce the risk of metastasis. A defining characteristic of metastasis in medulloblastoma is leptomeningeal dissemination (LMD), the spread of tumor cells via the cerebrospinal fluid (CSF) to the leptomeningeal spaces of the brain and spinal cord. The need to minimize metastasis risk is critical because survival times are very low once LMD has occurred. Guided by this principle, pediatric oncologists have designed multimodality treatments, which combine surgery, chemotherapy, and craniospinal radiation ,2. TheseBecause the prospect for long-term survival is so poor for patients with LMD, radiation to the entire neuraxis remains an indispensable part of medulloblastoma treatment. Unfortunately, intellectual capacity and academic achievement decline even in children treated with protocols that use reduced radiation doses . Hope foAnalysis of the medulloblastoma transcriptome in large cohorts of patients has shown that medulloblastomas do not comprise a uniform disease entity, but rather a diverse set of tumors, which have different gene expression profiles, rates of metastatic dissemination, and patient survival times -9. DespiWe reported previously that ectopic expression of genes encoding Eras (embryonic stem cell–expressed Ras), Lhx1 (LIM-class homeobox gene 1), Ccrk (cell cycle–related kinase), and the phosphatidyl inositol 3-kinase signaling protein Akt shifted the growth characteristics of Shh-induced medulloblastoma from a localized pattern to one in which tumor cells seeded the leptomeninges of the brain and spinal cord . The ideExtensive evidence from the field of epithelial cancer metastasis indicates that a large number of genes choreograph the multistep process whereby tumor cells breach the basement membrane in the originating tissue, intravasate into the bloodstream, and colonize distant organs ,21. AlthArnt (aryl hydrocarbon receptor nuclear translocator) and Gdi2 (GDP dissociation inhibitor 2), which had been identified previously as common insertion sites for the Sleeping Beauty (SB) transposon, in cerebellar neural progenitor cells in mice by retroviral transfer in combination with Shh. Here we show that ectopic expression of Arnt and Gdi2 promotes spinal LMD in mice bearing Shh-induced medulloblastomas and demonstrate the effects of these genes on the motility, invasiveness, and anchorage-independent growth of medulloblastoma tumor cells and precursor cells in culture.To expand the set of LMD-driving genes, we transferred and expressed Arnt and Gdi2 were obtained from the American Type Culture Collection , where they were deposited by the Integrated Molecular Analysis of Genomes and their Expression (IMAGE) consortium (http://www.imageconsortium.org). RCAS vectors were prepared by ligating a PCR-generated cDNA corresponding to the complete coding sequence into the parent retroviral vector RCASBP(A)  ÷ 20. The denominator of 20 is used to account for the ten hours of observation and two advancing cell fronts. Each experiment was repeated on a separate day. Standard deviation (SD) for the mean migration rate was derived from 30 data points.SHH-NPD or DAOY cells, previously infected with HIV-ZsGreen lentivirus and expressing 5), previously infected with HIV-ZsGreen lentivirus and expressing Arnt or Gdi2, were plated into the upper chamber of an 8-μm, 6-well BD Biocoat Matrigel invasion chamber  in 500 μl of serum-free DMEM. Prior to plating, the cells were treated with 10 μg/ml mitomycin C to suppress proliferation. Each bottom chamber was filled with 2 ml of DMEM containing 10% fetal bovine serum and then incubated for 24 hours at 37°C under 10% CO2. After incubation, the noninvading cells are removed from the upper chamber with a cotton swab. The invading cells on the lower surface of the membrane were stained with Hema 3  and counted under a microscope. Five nonoverlapping microscope fields (10× objective) were counted in each well. Each assay was repeated, and the mean number of invading cells per well was calculated. SD was derived from 20 data points.SHH-NPD or DAOY cells (2 × 104) were suspended in culture medium (2 ml) containing 0.3% agar  and plated over a layer of 0.5% agar in the same culture medium in tissue culture plates (35 mm in diameter). Triplicate cultures were incubated at 37°C under 5% CO2. Colonies were counted 14 days after plating by viewing under a microscope through a 10× objective and a graduated eyepiece reticle. Any colonies larger than 38 μm in diameter (>10 cells) were scored as positive. SD was derived from three data points.Cells  served as internal controls for sample variation in mRNA degradation and gel loading. Reaction products were separated by electrophoresis through agarose gels and visualized by UV illumination after immersion in ethidium bromide solution.Cerebella were removed from euthanized mice and frozen immediately in liquid nitrogen. Tissue specimens were homogenized in Trizol, and total RNA was extracted using RNeasy Mini Kit  according to the manufacturer’s instructions. RT-PCR was carried out using the SuperScript III One-Step RT-PCR kit  as described previously . In briewww.tcag.ca). Expression analysis was performed using Affymetrix Expression Console (Version 1.1) as previously described [Human primary medulloblastomas were profiled on Affymetrix Genechip Human Exon 1.0ST arrays at The Centre for Applied Genomics in Toronto, Canada . CandidArnt and four in Gdi2 . These were genes in which (a) the SB transposon had inserted into the 5’ untranslated region or intron I and (b) the murine stem cell virus promoter of the SB transposon pointed in the direction of gCIS-associated gene transcription. Two such insertions occurred in Arnt and Gdi2 indicated that activated transcription of these genes plays a causal role in the LMD program. To test this hypothesis, we used an established mouse model system, in which retroviral transfer of Shh to Nestin+ neural progenitor cells in the cerebella of mice induces medulloblastomas that have a very low incidence of metastatic dissemination [Arnt and Gdi2, in combination with Shh, in the cerebella of newborn mice and examined histological sections of brain and spinal cord in a 4-month observation period. Our results demonstrated that Arnt and Gdi2 increased the incidence of spinal LMD  to 67% (Shh + Arnt) and 53% (Shh + Gdi2)  Table . The enhmparable .Figure 2Shh, Shh + Arnt, and Shh + Gdi2 groups  excluded the possibility of constructing an Arnt-IRES-GFP bicistronic vector. Therefore, we prepared an epitope-tagged version by appending six repeats of a human c-Myc sequence (EQKLISEEDL), which is recognized by monoclonal antibody 9E10, to the carboxy terminus of Arnt, in-frame with the coding sequence. The presence of nuclear immunoreactivity in tumor cells indicated that RCAS-transferred Arnt was being expressed and transported to the nucleus, as expected for this transcription factor . Gdi2 increased the migration of SHH-NPD cells 1.2-fold and paradoxically decreased that of DAOY cells (P < 0.0001 by ANOVA for both cell lines).To assess cell migration, we used the in vitro scratch assay, which measures the rate of cell movement across a gap created in a confluent monolayer of cells growing on the surface of a tissue culture plate . We overn system . The aveArnt and Gdi2 enhanced the ability of both SHH-NPD and DAOY cells to penetrate the matrigel barrier .To measure invasiveness, we used the matrigel chemoinvasion assay, in which the percentage of cells invading through a gel matrix is determined as a function of time. Because matrigel contains the principal macromolecular components of epithelial basement membranes, the ability of cells to invade through this barrier has long been considered a reliable experimental model of the initial step in metastasis—transgression of the basement membrane . Resultsr Figure B. For exArnt and Gdi2 , whereas Gdi2 had no significant effect. The ability of Arnt and Gdi2 to promote anchorage-independent growth is not derived from a general mitogenic effect because neither gene stimulated the proliferation of adherent cells when tested in an MTT colorimetry assay (data not shown).A general feature of transformed cells is the ability to proliferate without attachment to a solid surface (anchorage-independent growth). This would seem to be an essential attribute of disseminating medulloblastoma cells, which must survive passage through the CSF before implanting on the pial surfaces of the spinal cord. To assess anchorage-independent growth, we measured the efficiency with which DAOY and SHH-NPD cells formed colonies in soft agar before and after lentivirus-mediated expression of 2 Figure C. The abARNT and GDI2 was associated with an increased risk of metastasis in medulloblastoma patients. To answer this question, we analyzed two publicly accessible sets of gene expression data on human medulloblastomas, which had been assigned to one of four molecular subgroups . Previously published work showed that metastasis  was significantly more common in Group 3 (46.5%) and Group 4 (29.7%) than in the WNT (17.9%) and SHH (19.1%) subgroups [We asked whether increased expression of ubgroups . The patubgroups  and the oup 4 29.% than inubgroups .ARNT mRNA in two sets of patients from the MAGIC cohort: a 103-patient discovery set, which was divided into four molecular subgroups  in tumors showing high ARNT expression (top quartile) than low expression (bottom quartile) (χ2P = 0.02)  Figure C. We didGDI2 expression in the human tumor data sets revealed that GDI2 mRNA levels were higher in medulloblastomas than in normal adult and fetal cerebellum , and GDI2 in six (75%). Although this observation is consistent with a selective growth advantage conferred to cells by ARNT and GDI2, larger sets of primary-metastatic tumor pairs will be needed to verify this trend.An unanswered question is whether expression of LMD-driving genes increases over time in a subpopulation of tumor cells, leading to metastatic dissemination at a later time point. Answering this question will require analyzing medulloblastoma cells that have already disseminated, a project that is hindered by the fact that surgical excision of metastatic tumors is rarely indicated in patients. We profiled matched pairs of primary and metastatic medulloblastomas from eight patients on Affymetrix exon arrays . Arnt oA substantial body of evidence indicates that the role of Arnt/AhR signaling in cell biology is not restricted to the xenobiotic response but rather extends to normal developmental processes and tumorigenesis (reviewed in ). DuringGdi2 prevents the dissociation of GDP from Rab proteins, a family of membrane-localized GTPases, which switch between active GTP-bound and inactive GDP-bound states. There is extensive literature to support the general concept that Rab proteins coordinate the intracellular movement of membrane vesicles (reviewed in ). The fuArnt and Gdi2 cause shedding of cells from the primary tumor mass into the CSF by increasing cell motility and invasiveness. By making tumor cells competent to proliferate without surface attachment, expression of Arnt and Gdi2 would also favor the formation of suspended colonies of cells capable of surviving in the CSF before reimplanting on a leptomeningeal surface. The ability of medulloblastoma cells to circumvent apoptotic programs normally triggered by detachment from a solid surface (anoikis) is most likely a prerequisite for LMD. This adaptive response is analogous to that of metastasizing carcinoma cells, which must survive the passage through the bloodstream before colonizing a distant organ.Extrapolation of our cell culture results to the in vivo growth of medulloblastoma supports a mechanistic scheme whereby We could not determine in all cases whether the presence of tumor cells in the brain stem and forebrain resulted from direct extension from the primary tumor or dispersal through the CSF. Nevertheless, the cell traits that empower cells to disseminate to the spinal column  could also promote extension from the cerebellum to the fourth ventricle or remote sites in the forebrain.Arnt and Gdi2 in Shh-induced medulloblastomas in vivo and validated these effects in culture using cell lines in which the Shh signaling pathway is active. Therefore, we cannot conclude that Arnt and Gdi2 are LMD drivers in Shh-independent medulloblastomas even though Arnt and Gdi2 are expressed in all subgroups. Nevertheless, the fact that medulloblastomas of any molecular subgroup can metastasize, either at initial presentation or relapse, broadens the clinical significance of LMD driver genes discovered in different genetic backgrounds [We demonstrated the LMD-inducing effects of kgrounds .The multiple cellular transitions connecting tumor stromal detachment, CSF passage, and finally leptomeningeal spreading indicate that LMD might approach the complexity of the invasion–metastasis model used to describe hematogenous metastasis of epithelial cancers. Transposon mutagenesis revealed hundreds of candidate genes, although we do not know how many of these genes are drivers as opposed to passengers in the LMD process. Through the functional validation studies we have reported here and previously , we know


Cell lines recapitulate cancer heterogeneity without the presence of interfering tissue found in primary tumor. Their heterogeneous characteristics are reflected in their multiple genetic abnormalities and variable responsiveness to drug treatments. In order to understand the heterogeneity observed in Asian gastric cancers, we have performed array comparative genomic hybridization (aCGH) on 18 Asian gastric cell lines. Hierarchical clustering and single-sample Gene Set Enrichment Analysis were performed on the aCGH data together with public gene expression data of the same cell lines obtained from the Cancer Cell Line Encyclopedia. We found a large amount of genetic aberrations, with some cell lines having 13 fold more aberrations than others. Frequently mutated genes and cellular pathways are identified in these Asian gastric cell lines. The combined analyses of aCGH and expression data demonstrate correlation of gene copy number variations and expression profiles in human gastric cancer cells. The gastric cell lines can be grouped into 2 integrative clusters (ICs). Gastric cells in IC1 are enriched with gene associated with mitochondrial activities and oxidative phosphorylation while cells in IC2 are enriched with genes associated with cell signaling and transcription regulations. The two clusters of cell lines were shown to have distinct responsiveness towards several chemotherapeutics agents such as PI3 K and proteosome inhibitors. Our molecular integrative clustering provides insight into critical genes and pathways that may be responsible for the differences in survival in response to chemotherapy. Gastric cancer is the second leading cause of cancer death worldwide, and is particularly common in East Asia Traditionally, classification of gastric cancers is based on histopathological findings. The widely used Lauren’s classification divides gastric cancers into two major histological types, namely intestinal type or diffuse type Cell lines formed the foundation of cancer biology and the quest for drug treatments. Common alterations in cell lines, which include gains and losses of entire arms of chromosomes, are often the same ones found in primary tissue Many Asian gastric cell lines are available commercially and they have contributed to the progress in gastric cancer biology and treatment. Despite being relatively homogenous and devoid of tissue complexity, these Asian gastric cell lines are found to have heterogeneous response or susceptibility to drug treatment . We reasoned that subtle genomic variations may contribute to the underlying differences observed among these gastric cell lines. Unlike tumor tissue, these differences would be intrinsic and a signature to the biology of the particular gastric cell line since there is no interference from other cell types.We analyzed the gene copy number and LOH in 18 Asian gastric cell lines. Coupling our results to gene expression profiles of these cell lines available from CCLE, we identified two distinct genomic signatures based on genetic aberrations among these Asian gastric cancer cell lines. The clustering was further validated by in vitro chemotherapy sensitivity study done in our lab and with data publicly available from CCLE and Sanger COSMIC databases. A molecular classification on gastric cancer patients would have great clinical impact as it leads to more accurate prediction of prognosis, allowing targeted therapy based on the underlying biology of each subgroup.Asian gastric cell lines SNU-1, SNU-5, SNU-16 and Kato-III, were purchased from the American Type Culture Collection. MKN1, MKN7, MKN45, MMKN74, Fu97, AZ-521, SCH, OCUM-1, NUGC-3, NUGC-4, IM95 and IM95 m were purchased from Japan Health Sciences Foundation. SNU-216 was purchased from Korea Cell Line Bank. YCC-3 6 cells were used for the aCGH hybridization. The genomic DNA quality, hybridization signal strengths and internal controls satisfied Affymetrix required standard at each step before proceeding to the next. Data from the 18 gastric cell lines on Affymetrix Cytoscan HD array were pre-processed using Affymetrix Chromosome Suite 1.2.2.271 using the single sample analysis workflow and default settings. Hidden Markov model segmentation was applied to call DNA copy number gain, loss or loss of heterozygosity (LOH) status. For matching purposes, the Entrez Gene ID was assigned to each gene name in this data set using the NCBI’s Entrez Gene Database CGH array was performed by Origen Labs (Singapore) using the Affymetrix CytoScan HD array platform. Cell pellets containing 1×10Affymetrix U133 Plus2 DNA microarray gene expressions of 27 gastric cancer cell lines  were downloaded from Cancer Cell Line Encyclopedia (CCLE) Unsupervised hierarchical clustering  was performed on the cell lines using the aCGH data. Putative driver genes of which copy number aberrations correlated to mRNA gene expression were identified to determine subtypes or clusters that are driven by different mechanisms. This was done using Mann Whitney U-test with p<0.05, and Spearman Correlation Coefficient test with Rho >0.6. We then performed consensus clustering ssGSEA was used to estimate pathway activities of the gastric cancer cell line in the Molecular Signature Database v3.1 (Msigdb v3.1) 50). P-values were computed from Mann Whitney U-test.A panel of 39 compounds against various cellular targets was obtained from Selleck Chemicals  and reduced secretion of gastric acid due to atrophic mucosa is observed in gastric cancer.We found that 72% of the Asian gastric cell lines have gene copy number gain in  kinases . AmplifiThe NCI-Nature Pathway Interaction Database Additionally, we investigated the correlation between copy number aberrations and gene expression data of these cell lines in the public domain. The integrated analysis of DNA copy number variations and corresponding gene expression data would allow identification of significant genes and cellular pathways critical to the gastric cancer pathophysiology. Of the 18 gastric cell lines that we have performed aCGH, only 14 cell lines have corresponding mRNA expression data in CCLE. CCLE has a total of 27 Asian gastric cell lines at the time of analysis (March 2013). We used Mann Whitney U-test and Spearman Correlation Coefficient test to identify 1762 putative driver genes of which copy number aberrations correlate to mRNA gene expression  were obtained from CCLE  . Similar50 ∼100 µM) towards XAV939, a tankyrase inhibitor Since we observed gene loss and LOH in the nuclear beta-catenin pathway, we postulate that targeting this pathway may have a therapeutic effect in gastric cancer Cells in IC1 have enrichment of genes associated with oxidative phosphorylation and mitochondria functions. We found that cells in IC1 are more resistant to proteosome inhibitors bortezomib and MG132 . ProteosInterestingly, we found that a subset of gastric cells within the IC1 are more sensitive towards the MEK/ERK inhibitor PD0325901  (p = 0.027), GSK269962A (Rho kinase inhibitor) (p = 0.019), and midostaurin (Flt3 and multiple kinase inhibitor) (p = 0.012).In support of our findings that the gastric cells in IC2 are enriched for genes involved in cell signalling, we found that cells in IC2 are generally more sensitive to kinase inhibitors than cells in IC1. Cell lines in IC2 are more sensitive to treatment with PI3 K inhibitors BEZ235, ZSTK424, PI-103 and PIK-75  . This reThe absolute magnitude of the differential drug sensitivities ranges from 2–10 fold in the gastric cell lines based on our clustering. The modest differences may still be clinically meaningful given the small therapeutic windows associated with cytotoxicity even in targeted chemotherapy. A large patient cohort study will be needed to confirm the value of the molecular clustering strategies by us or others in predicting chemosensitivity and prognosis.In conclusion, combination of aCGH and gene expression analysis to identify potential candidate oncogenes or tumor suppressor genes is a powerful and proven approach that has been reported in other cancer studies. This study provides insight into DNA copy number variations and their correlation to gene expression profiles in Asian gastric cell lines. A schematic diagram of the overall workflow is shown in Fig. S1 located in File S1Table S1: A panel of target-specific compounds for chemosensitivity study in the Asian gastric cell lines. Table S2: Putative driver genes selected using correlated genes between copy number aberration and mRNA gene expression . Table S3: Integrative cluster signature (Copy number and mRNA correlated genes). Table S4: Integrative cluster-specific pathway. Table S5: Compounds showing significant differences in sensitivity between the two integrative clusters of Asian gastric cell lines. Figure S1: A schematic diagram of the analysis workflow.(XLSX)Click here for additional data file.


Dear Editor,We thank Dr. Kiani for highlighting again the importance of risk stratification in patients with non-valvular atrial fibrillation (AF). We do agree with him as stated also in our review  that the CHA2DS2-VASc score should be currently applied to patients with CHADS2-Score < 2 for further risk stratification as also recommended by current guidelines . As it iFinally, although clinically used in Europe, there is no conclusive evidence that left atrial appendage (LAA) occlusion reduces the risk of stroke in patients with non-valvular AF . Althoug


Copy number variation (CNV) represents an important source of genetic divergence that can produce drastic phenotypic differences and may therefore be subject to selection during domestication and environmental adaptation. To investigate the evolutionary dynamics of CNV in the yak genome, we used a read depth approach to detect CNV based on genome resequencing data from 14 wild and 65 domestic yaks and determined CNV regions related to domestication and adaptations to high-altitude.We identified 2,634 CNV regions (CNVRs) comprising a total of 153 megabases (5.7 % of the yak genome) and 3,879 overlapping annotated genes. Comparison between domestic and wild yak populations identified 121 potentially selected CNVRs, harboring genes related to neuronal development, reproduction, nutrition and energy metabolism. In addition, we found 85 CNVRs that are significantly different between domestic yak living in high- and low-altitude areas, including three genes related to hypoxia response and six related to immune defense. This analysis shows that genic CNVs may play an important role in phenotypic changes during yak domestication and adaptation to life at high-altitude.We present the first refined CNV map for yak along with comprehensive genomic analysis of yak CNV. Our results provide new insights into the genetic basis of yak domestication and adaptation to living in a high-altitude environment, as well as a valuable genetic resource that will facilitate future CNV association studies of important traits in yak and other bovid species.The online version of this article (doi:10.1186/s12864-016-2702-6) contains supplementary material, which is available to authorized users. Copy number variations (CNVs), a form of genomic structural variation, are defined as duplications or deletions of DNA fragments that range in size from at least 50 base-pairs (bp) to more than several megabase-pairs (Mb), causing a different copy number of specific genomic regions among individuals of a species –4. CNV rAMY2B gene probably allowed dogs to thrive on a relatively starch-rich diet during their domestication [ASIP CNV allele is almost entirely associated with different coat colors in different goat breeds [SOX5 gene [AKR1C gene is considered possibly responsible for testicular androgen synthesis and sexual development in horse [Ntn1 gene may influence meat quality in pigs [HSFY and ZNF280BY genes are associated with male reproductive traits in Holstein bulls [PLA2G2D located in a CNV region is associated with aspects of body size in Chinese bulls such as heart girth and hucklebone width [HSPA1B gene [APOL3 gene involved in lipid transport is highly duplicated in beef breeds [As a common feature of vertebrate genomes, the foundational studies of CNV have been conducted in humans. Around 2,057,368 CNVs that correspond to over 24,032 CNV regions have been identified in humans and these may account for >9.5 % of the human genome, with over 12 % of gene sequences involved in CNV regions , horse , sheep , dog 1414 and chtication . The ASIt breeds  and in Tt breeds . The mosOX5 gene . A CNV i in pigs . In boviin bulls ; PLA2G2Dne width ; hereditA1B gene ; and theBos grunniens) is the only major livestock that can survive the extremely cold, harsh and oxygen-poor conditions and take full advantage of the limited grassland resources on the Qinghai-Tibetan Plateau (QTP) [The yak (au (QTP) . Yak werau (QTP) . Nowadayau (QTP) . It shouau (QTP) , 28. Theau (QTP) . Indeed,au (QTP) .Although comparative genomic hybridization (CGH) and SNP arrays are routinely used for CNV identification, the performance of these methods are heavily depending on the marker density and the specially designed non-polymorphic probes –32. The Here we describe the first genome-wide and systematic analysis of CNVs in yak using NGS genome re-sequencing data from wild and domestic yak. This CNV map was constructed for three main reasons. First, to develop and enrich new genomic variations in order to facilitate further research on yak and other bovid species and make it available to the scientific community. Second, to determine genes affected by CNV that differ between wild and domestic yak, and evaluate whether extensive CNV is related to domestication. Third, to compare the CNVs in individuals from different altitudes and assess the evolutionary impact of CNVs in adaptations to life at high altitude.We used whole genome re-sequenced data from our previous study, based on 14 wild and 65 domestic yaks from widely spaced locations across the QTP  by merging all overlapping calls across multiple individuals into unique regions and filtering the ones that were present in fewer than four individuals. These CNVRs occupied a total of 153 Mb or 5.7 % of the yak genome, substantially more than the recent study in two yak individuals based on a CGH array approach  . The lenWe evaluated the accuracy of individual copy number variations predicted by performing a series of real time-polymerase chain reactions (qPCR). The experimental validation showed that 88 % of the CNVs 56/64) had an accurate copy number and 100 % were in agreement with the predicted patterns  were shown to be one of the catalysts and hotspots for CNV formation in many species , 24, 41,Among the 2,634 CNVRs described above, 961 (36.5 %) harbored a total of 3,879 protein-coding genes according to the current yak genome  relatedTaken together, our results suggest that some CNVRs may have been under selection pressure during yak domestication and these are associated with behavior, physical characteristics and economically significant traits. Consequently, additional functional experiments are needed to verify the contributions of the identified genes located in CNVRs to domestication.MRP4 [DCC [DEXI [MRP4 gene encodes ATP-binding cassette (ABC) transporters, regulating intracellular levels of cAMP and cGMP in arterial smooth muscle cells; inhibition of MRP4 expression can prevent and reverse hypoxia-induced pulmonary arterial hypertension [DCC gene encodes the Netrin 1 receptor, which plays an important mediating function in protecting against hypoxia-induced mitochondrial apoptosis [DEXI expression was increased in emphysema tissues and is associated with disease progression [ULBP17 encodes the immune system-activating receptor on T-cells and is related to pathogen- and parasite-resistance [CIITA is a positive regulator of class II major histocompatibility and mutations in this gene are associated with diseases of the immune system [CATHL1 encodes cathelicidin, which is important in innate immune defense against bacterial infection  and low-altitude <2,500 m) areas. We found 85 CNVRs that are significantly different between these two groups: 29 of them harbor 41 protein-coding genes  with default parameters. SAMTOOLS [Picard software .Using a custom Perl script, we filtered out the low quality reads based on the following criteria: (i) reads with ≥10 % unidentified nucleotides (N); (ii) reads for which more than 65 % of the read length had a Phred quality value ≤7; (iii) reads with more than 10 bp aligned to the adapter, allowing 2 bp mismatches; and (iv) duplicate reads. Reads were also trimmed if they had three consecutive base pairs with a Phred quality value of 13 or below, and discarded if they were shorter than 45 bp. The pair-end sequence reads were mapped to the SAMTOOLS  was usedSAMTOOLS  was usedSAMTOOLS  after asp < 0.05). Previous studies have shown that RD based analysis have reduced power and reliability to detect small CNVs events [CNVs were detected through read-depth analysis, in which the number of copies presented is inferred from sequence depth of whole genome sequencing data . By combs events , 88. To s events , 39. In The copy number variation region (CNVR) is defined as a combined region of overlapping CNVs on the genome. CNVRs are merged from different samples with any amount of overlap by extending the boundaries of the overlapping CNVs . Here, aBTF3) gene was chosen as a reference location for all qPCR experiments, because neither CNVs nor SDs overlapping this gene were found in our dataset nor in any previous studies of bovid species [http://frodo.wi.mit.edu/). The amplicon length was set to 100–200 bp, and the regions with GC percentage were between 30 and 60 % . A segment of bovine basic transcription factor 3  re-sequencing dataset based on three domestic and three wild yaks to estimate the accuracy of our original identified CNV events. We calculated the coefficient of variation (cv%) between the high-depth-sequencing predicted and our original predicted copy numbers. The difference was considered to be acceptable if cv % was <0.25.Using the yak_1.1 yak genome assembly, we applied two well-established computational approaches, whole-genome shotgun sequence detection (WSSD) and whole-genome assembly comparison (WGAC) to detect putative segmental duplications. Briefly, WGAC identifies paralogous sequences >1 kb in length with >90 % sequence identity, and WSSD identifies genomic regions that exhibit significant depth of coverage by aligning whole-genome shotgun sequencing reads to the reference genome sequence. The final SD dataset was obtained by merging these two results. Association between CNVs and SDs was tested by random simulations using a custom Perl script as previously published .http://wego.genomics.org.cn/cgi-bin/wego/index.pl).By searching for the coordinate of each CNVR in the yak genome assembly and gene annotation, we assessed the gene contents of each CNVR. Only the genes that were primarily comprised of CNVRs (>50 %) were retained. Functional classification of GO categories was performed using the Blast2GO program . EnrichmFST (the fixation index) named VST [VST was calculated using the formula VST = (VT–VS)/VT as previously published. Here, VT is the total variance in CN between the two populations and VS is the average of the variance within each population, weighted for its sample size [VST values and P-value < 0.05) were considered to be population-differentiated CNVs, representing a rigorous criteria to identify differential CNVRs between populations. All these statistics were calculated using the R package.We carried out two comparative studies to identify CNV genes with high differentiation between populations. One was domestication related, including 65 domestic and 14 wild samples; the other was high-altitude adaptation related, only including domestic yak living at high  and low-altitude  areas. With the aim of identifying population-differentiated CNVRs more accurately, two statistical measures were used during comparisons: a metric analogous to amed VST  and the ple size . The disWe performed heatmap analyses based on the depth of each sample. For each individual, the depth of every base was computed using the “depth” command in SAMTOOLS . As the All animal specimens were collected legally. Animal collection and utility protocols were approved by the Ethics Committee of Animal Experiments at Lanzhou University and in accordance with guidelines from the China Council on Animal Care.Not applicable.The datasets supporting the conclusions of this article are available in the European Molecular Biology Laboratory (EMBL/EBI) Nucleotide Sequence Database repository (accession code PRJNA285834). Other supporting data are included as Additional files:


In addition, a significant reduction in long chain-CoA (LC-CoA) was observed in INS-1 cells at both basal and stimulatory glucose following inhibition of MGL. Our data implicate an important role for MGL in insulin secretion.Lipid signals derived from lipolysis and membrane phospholipids play an important role in glucose-stimulated insulin secretion (GSIS), though the exact secondary signals remain unclear. Previous reports have documented a stimulatory role of exogenously added mono-acyl-glycerol (MAG) on insulin secretion from cultured β-cells and islets. In this report we have determined effects of increasing intracellular MAG in the β-cell by inhibiting mono-acyl-glycerol lipase (MGL) activity, which catalyzes the final step in triacylglycerol breakdown, namely the hydrolysis of MAG to glycerol and free fatty acid (FA). To determine the role of MGL in GSIS, we used three different pharmacological agents . All three inhibited GSIS and depolarization-induced insulin secretion in INS-1 (832/13). JZL184 significantly inhibited both GSIS and depolarization-induced insulin secretion in rat islets. JZL184 significantly decreased lipolysis and increased both mono- and diacyglycerol species in INS-1 cells. Analysis of the kinetics of GSIS showed that inhibition was greater during the sustained phase of secretion. A similar pattern was observed in the response of Ca Decades of research have demonstrated a critical role for insulin resistance in the manifestation of Type 2 Diabetes (T2D). However, a decline in β-cell function characterized by an inability to adequately respond to an acute glucose challenge, clearly distinguishes those who go on to develop for T2D from those who can maintain glucose homeostasis . Our und2+]i) that promotes the ‘triggering’ of insulin granule fusion with the plasma membrane via SNARE proteins i, resulted in a sustained increase in control islets while only a transient spike was observed in the islets exposed to JZL184.While INS-1 cells retain many of the characteristics of normal β-cells, the fact that they are a tumor cell line necessitates confirmation in primary β-cells. Isolated rat islets showed an approximate 35-fold increase in insulin secretion when stimulated with 15 mM glucose . JZL184 + (KATP)-channel agonist diazoxide (DZ). Insulin secretion is increased under these conditions with or without stimulatory glucose via a sharp peak and mildly elevated sustained phase of cytosolic [Ca2+]. Additionally, it is known that glucose can stimulate insulin secretion in a concentration dependent manner under these conditions -Glucose was incubated with INS-1 cells overnight to label the lipid pool. Stimulation with 8 mM glucose for 30 min resulted in a 50% increase in total MAGs (Fig To further support this concept, we used thin layer chromatography to determine the affect of JZL184 on lipid species at both basal and stimulatory glucose. i, which directly promotes the fusion of insulin granules with the plasma membrane [2+]i was reduced in response to these stimuli. We tested this concept using the cytosolic [Ca2+]i indicator fura-2. Rat islets were perifused with basal glucose followed by an increase to 15 mM with and without JZL184, similar to what was done in the insulin secretion perifusions. A clear sustained rise in cytosolic [Ca2+]i was observed in response to 15 mM glucose (2+]i induced by KCl/DZ was slightly inhibited by JZL184 and the sustained phase was decreased by more than half in INS-1 cells (The final common step in the triggering pathway of GSIS and depolarization is a rise in cytosolic i in response to glucose. Consistent with this, depolarization of the plasma membrane with KCl/DZ resulted in a similar but slightly smaller peak rise in [Ca2+]i with concurrent JZL184 incubation and decreased the sustained phase [Ca2+]i rise. Inhibition of MGL also caused a reduction in LC-CoA, an important coupling factor derived endogenously from lipid stores during lipolysis..Herein we have demonstrated a role for MGL activity in GSIS. Inhibition of MGL resulted in a pronounced defect in the sustained phase of GSIS. Importantly, we were able to demonstrate that this effect was partially a result of reduced [CaThe role of LC-CoA in GSIS has been debated in the literature . The wel2+]i influx. Our data suggest that the triggering of insulin secretion requires signals in addition to [Ca2+]i, which we have previously suggested i, which goes in the opposite direction and suggests that the MGL inhibitors are not affecting KATP channels. Additionally, because an affect is still observed with KCl and diazoxide, which takes offline the KATP channel, an affect on the KATP channel is unlikely. A role for LC-CoA in mediating Ca2+ influx through L-type Ca2+ channels has been documented [2+]i via an affect on Ca2+ stores. These data differed from other published studies showing that ABHD6 is the primary monoglyceride lipase in β-cells and is essential for GSIS i influx, which is not sustained. In contrast, we observed a marked inhibition of both glucose and KCl-induced insulin secretion using the same inhibitor, URB602. Several factors may explain the apparent discrepancy between this result and our own data. Our experiments were conducted over a longer time frame and therefore a transient increase in insulin secretion may have been masked by a later prolonged decrease, which Li et al. also observed. While we cannot conclude that the pharmacological inhibitors used in our study were specific, we were able to measure both a reduction in glycerol secretion and a definitive increase in total MAG accumulation using thin layer chromatography, thereby demonstrating that indeed the drugs used did inhibit MAG hydrolysis, as expected.Several previous papers have characterized the role of MAGs and MGL in the regulation of GSIS. Li et al. used the MGL inhibitor URB602 and observed an enhancement of insulin secretion at both basal and stimulatory glucose concentrations in MIN6 cells and human islets . This inA recent report by Zhao et al. clearly demonstrates that pharmacological or genetic inhibition of the plasma membrane hydrolase, ABHD6, stimulates GSIS possibly by action of increased MAGs binding to the exocytotic protein Munc13-1 . While i2+-dependent pathway via which glucose and KCl induce insulin secretion as well as a failure to increase cytosolic LC-CoA. We cannot rule out the possibility that the build-up of specific MAG species in a specific location may be responsible for the observed inhibition of GSIS. Alternatively, lack of specificity of the lipase inhibitors may alter other lipid species not measured. Further research will be needed to determine the role of specific MAG species on GSIS.In summary, we have demonstrated that pharmacological inhibition of MGL reduces both KCl and glucose induced insulin secretion. This reduction is the result of processes that may partially involve the classical Ca


Cultured primary cells and various immortalized cells have been frequently used as skin models but their comparability to intact skin has been questioned. Moreover, when analyzing KC transcriptomes, fluctuation of polyA+ RNA content during the KCs’ lifecycle has been omitted.Keratinocytes (KCs) are the most frequent cells in the epidermis, and they are often isolated and cultured We performed STRT RNA sequencing on 10 ng samples of total RNA from three different sample types: i) epidermal tissue (split-thickness skin grafts), ii) cultured primary KCs, and iii) HaCaT cell line. We observed significant variation in cellular polyA+ RNA content between tissue and cell culture samples of KCs. The use of synthetic RNAs and SAMstrt in normalization enabled comparison of gene expression levels in the highly heterogenous samples and facilitated discovery of differences between the tissue samples and cultured cells. The transcriptome analysis sensitively revealed genes involved in KC differentiation in skin grafts and cell cycle regulation related genes in cultured KCs and emphasized the fluctuation of transcription factors and non-coding RNAs associated to sample types.The epidermal keratinocytes derived from tissue and cell culture samples showed highly different polyA+ RNA contents. The use of SAMstrt and synthetic RNA based normalization allowed the comparison between tissue and cell culture samples and thus proved to be valuable tools for RNA-seq analysis with translational approach. Transciptomics revealed clear difference both between tissue and cell culture samples and between primary KCs and immortalized HaCaT cells.The online version of this article (doi:10.1186/s12864-015-1671-5) contains supplementary material, which is available to authorized users. Skin is a multi-layered tissue that is composed of continuously renewing epidermis – with keratinocytes (KCs) as a predominant cell type – and underlying dermis populated mostly by fibroblasts. The life span of epidermal keratinocytes is controlled by two alternative pathways: differentiation as their normal function or activation as an altered function in wound healing or skin diseases . EpidermTP53 alleles [The relatively noninvasive sampling together with the methods that allow culturing of pure KCs have greatly facilitated research on skin and KCs. In cell culture, KCs are uncoupled from their tissue environment that naturally provides a network of homeostatic control signals; they are induced to either retain an active proliferative state or to differentiate. However, the prolonged KC culturing leads to the induction of cellular senescence  and ther alleles , 7 they  alleles . The comGenome-wide expression profiling is an useful approach to screen key genes with respect to different cellular statuses and to further model the regulatory networks . MicroarIn this study, we revisit the skin and KC transcriptome with respect to fluctuation of polyA+ RNA content by the keratinocyte statuses; differentiated, activated, senescent and immortalized. Four types of human keratinocyte samples represented these cell statuses: epidermal tissue , cultured primary KCs in early and late passages, and HaCaT cell line. To reduce the sample size and sequencing costs and to control the fluctuation of mRNA concentration, we applied single-cell tagged reverse transcription (STRT) sequencing method for expression profiling using 10 ng of total RNA per sample which is ten times less than required for a conventional RNA-seq method . For accThe protocol for sample preparation is depicted in Fig. RPLP0 and RPL13A, as predicted by the spike-in-based normalization, both of which were predicted unchanged or downregulated by the gene-based normalization , whereas the KCs contained cytokeratins typical of cells that maintain their proliferative capacity (KRT5 and 14) or that are activated by wound healing, hyperproliferative skin diseases or in vitro culturing  [KRT8 and KRT18 that are the developmentally first keratins absent from normal skin and rather characterizing simple epithelia are in our data expressed by both cultured KCs and HaCaTs supporting their proliferative and undifferentiated nature [When we assessed the transcriptome profiles in the different samples, we found 11,908 differentially expressed genes  . KRT8 and nature .Fig. 3DiKRT4, 13, 15, 19 and 81). Furthermore, several cytokeratins  characterising HaCaT by the spike-in normalization  and late (LKC) passage KCs in the cytokeratin levels. In contrast, HaCaT was remarkably different from both SGs, EKC and LKC expressing higher levels of cytokeratins 4, 13, 15, 19 and 81 . Our results showed that 136 out of 7,801 gene sets correlated positively, being upregulated in the SGs  is a known transactivator of the suprabasal layer marker KRT10 [−4), and is also a known repressor for the basal layer marker KRT14 [Since the PC scores representing the functions and phenotypes were calculated by linear combination of the expression profile and the loading coefficients, genes with large loading coefficients on each PC would be the key regulators for the functional contrast. For example, in case of PC1, genes with high positive loading coefficients contribute to the characteristic phenotypes and functions of SGs, and conversely, genes with high negative loading coefficients contribute to common characteristic phenotypes and functions of HaCaTs and KCs. When we extracted genes contributing to PC1, 223 genes showed positive loading and 104 genes showed negative loading  [PC2 captured the second largest variation which mostly shows the contrast between HaCaTs and EKCs, with intermediate LKCs Fig. . Three t × 10−3) .Table 4TAPBB1 [ESPL1 [TCF19 [ZNF300 [UCA1 was a negatively loading lncRNA, known as essential for bladder cancer cell proliferation via CREB-dependent pathway [Then we proceeded to find genes that are the key regulators for the functional contrast on PC2. 137 genes showed high positive loading to PC2, and 111 genes showed negative loading  [LMNA was consistently negatively correlated . It’s well known that the mutations of LMNA lead to Hutchinson–Gilford progeria syndrome characterized by a premature aging [We next attempted to find supporting genes for this interpretation. A total of 117 genes showed high positive loading to PC3, and 144 genes showed high negative loading  , and expre aging . TherefoSTRT RNA-seq method complemented with synthetic RNAs revealed a variation of polyA+ RNA content per total RNA in different cell types, SGs, EKCs, LKCs and HaCaTs, reflecting the activity of the cell type. Even though the STRT reads are concentrated towards the 5’end of the polyA+ transcripts and the method has limited resolution at 2nd and more downstream exons, the advantages of the method include the small amount of starting material needed for library preparation, early multiplexing of up to 92 samples reducing the cost and time of library preparation, and the inclusion of external spike-in RNAs as a standard procedure. The spike-in normalization has been shown to be a valuable tool when comparing samples with fluctuating polyA+ RNA contents , 44. We We applied PCA to elucidate dissimilarity between the samples, and also to decompose the differences. The three first PCs represented differentiation and the mitochondrial phenotypes between SGs and cultured cells, G1/S-transition between HaCaTs and EKCs, and senescence and cellular aging responces between HaCaTs and LKCs. All cultured cells differed from tissue samples and HaCaT cells differed remarkably from other cultured cells based on both PCA and the comparison of previously known KC markers, cytokeratins. Our results thus suggest that great caution should be payed when using cultured primary KCs and cell models like HaCaTs as models for skin, especially when focusing on the pathways revealed by PCA. The transcriptomes of cultured primary KCs and HaCaTs resemble that of acivated skin rather than normal skin as shown also by others , 11.In this study, we present an approach to compare highly varying cell types by applying synthetic RNA based normalization. For further studies or applications of other biological events, the key is to find the hidden associations between genes and phenotypes which are contrasted on PCs. We also note that there are still many poorly annotated genes in the genome that might be revealed by our approach. Moreover, PCA without sample pre-classification might be applied in studying gene expression in complex disorders using a large enough cohort.All subjects involved in this study provided written informed consent under a protocol adherent to the Helsinki Guidelines and approved by the Institutional Review Board of the Helsinki University Central Hospital.Split-thickness skin grafts (SGs) were harvested as outlined previously . BrieflyEpidermal cells were isolated from the full thickness skin with dispase digestion followed by trypsinization to enable collection of all primary cell types and phenotypes of the epidermis in the initial harvest. Human primary keratinocytes were cultured in Keratinocyte Growth Medium 2 (PromoCell # C-20011) with calcium (0.06 mM) and PromoCell supplements (#C-39016). Cell culture dishes were coated with collagen I . Cells were routinely passaged, and samples were collected from early  and late passages . The human immortalized keratinocyte HaCaT cell line was grown in low glucose DMEM (Lonza) supplemented with 5 % FBS, 2 mM L-glutamine, 1 mM sodium pyruvate solution, 0.1 mM non-essential amino acids, 100 U/ml penicillin and 100 μg/ml streptomycin at 37 °C and 5 % CO2. HaCaT cells were collected at passage 43 for RNA isolation.Total RNA was extracted by miRNeasy kit (Qiagen) from both tissue samples and cells. RNA concentrations were measured by Nanodrop and Qubit and the quality was controlled by Bioanalyzer  were used for RNA sequencing library preparation according to the STRT protocol , which wThe qualified total RNA samples 10kD, 10k1 and 10k6, which were used for sequencing with STRT method, were subjected to cDNA synthesis. Equal amount of the SPIKE-in RNA mix  was added to each cDNA synthesis reaction according to the STRT-library preparation protocol described above. cDNA synthesis was carried out using oligo dT-primers and SuperScript III First-Strand synthesis system for RT-PCR  according to manufacturer’s instructions. 5 ng of cDNA was applied to each qPCR assay and each sample was run in three technical replicates. qPCR was carried out using an ABI PRISM 7500 Fast Real-Time PCR System with Fast SYBR® Green Master mix  according to manufacturer’s instructions. The primer sequences are shown in Additional file http://www.ebi.ac.uk/ena/data/view/PRJEB8997).The processed STRT reads supporting the results of this article are available in the European Nucleotide Archive (


Nature, the Cancer Cell Line Encyclopedia and the Cancer Genome Project, provide a wealth of data regarding the drug sensitivity and genomic profiles of many different types of cancer cells. In the present study we have mined these two studies for new therapeutic agents for SCLC and identified heat shock proteins, cyclin-dependent kinases and polo-like kinases (PLK) as attractive molecular targets with little current clinical trial activity in SCLC. Remarkably, our analyses demonstrated that most SCLC cell lines clustered into a single, predominant subgroup by either gene expression or CNV analyses, leading us to take a pharmacogenomic approach to identify subgroups of drug-sensitive SCLC cells. Using PLK inhibitors as an example, we identified and validated a gene signature for drug sensitivity in SCLC cell lines. This gene signature could distinguish subpopulations among human SCLC tumors, suggesting its potential clinical utility. Finally, circos plots were constructed to yield a comprehensive view of how transcriptional, copy number and mutational elements affect PLK sensitivity in SCLC cell lines. Taken together, this study outlines an approach to predict drug sensitivity in SCLC to novel targeted therapeutics.There are currently no molecular targeted approaches to treat small-cell lung cancer (SCLC) similar to those used successfully against non-small-cell lung cancer. This failure is attributable to our inability to identify clinically-relevant subtypes of this disease. Thus, a more systematic approach to drug discovery for SCLC is needed. In this regard, two comprehensive studies recently published in  Small cell lung cancer (SCLC) represents 15% of all lung carcinomas and is typically diagnosed when the disease has metastasized Nature, the Cancer Cell Line Encyclopedia (CCLE) A different approach to drug discovery for SCLC is needed and may lie in mining available databases on the drug sensitivities of SCLC cell lines. That is, as most SCLC cells are derived from metastatic sites or pleural effusions, they may be representative of extensive disease SCLC and its associated drug vulnerabilities. In this regard, two comprehensive drug-screening studies recently published in In the present study we have specifically extracted the data on SCLC cell lines from these two studies and outline a bioinformatic approach to identify new therapeutics for SCLC using polo-like kinase (PLK) inhibitors as an example.50≤1 µM), represented by paclitaxel. ‘Ineffective’ drugs, represented by erlotinib and sunitinib, produced no growth inhibition in most SCLC cells (IC50≥8 µM), although ‘outliers’ may be present. ‘Selective’ drugs, represented by rapamycin, demonstrated a long boxplot and can be considered effective for only a subset of SCLC cell lines.Initially we sought a global view of SCLC drug sensitivity in the CCLE 50 just outside 1 µM include AZD-7762, a CHK inhibitor; and JW-7-52-1, an MTOR inhibitor. HSP90 (17-AAG) and HDAC (panobinostat) inhibitors may also represent ‘effective’ drugs, although their efficacy varied among the two studies. ‘Effective’ drugs likely carry the best translational potential.As shown in Gene array data has been used extensively in cancer research to identify expression ‘signatures’ that may be either prognostic or predictive of tumor behavior. Therefore, we examined if gene expression clustering could be used to identify subgroups of drug-sensitive SCLC cell lines, particularly for drugs that demonstrated a broad efficacy range against SCLC cells in We next determined if CNV clustering could be used to identify subgroups of drug-sensitive SCLC cell lines. Three clusters were again identified using all 426 interrogated genes ; however50 values for the PLK inhibitors in most cell lines is between 10-100 nM, supporting our hypothesis that SCLC cells are broadly sensitive to PLK inhibitors.Our analyses identified PLK inhibition to have promising efficacy and little clinical trial activity in SCLC. Therefore, we used PLK inhibition as an example of how to use the CGP datasets to develop a genomic profile of SCLC drug sensitivity. First we sought to validate the efficacy of PLK inhibitors in SCLC. In these validation experiments we used SCLC cell lines, as well as inhibitors, that were different from those used in the original studies to highlight the efficacy of these new therapeutic agents. The SCLC cell lines used were H1048, H1688, SW1271 and DMS454. PLK inhibitors included BI-6727 (volasertib) and ON-01910 (rigosertib). In these experiments irinotecan served as a positive control while erlotinib served as a negative control. The results are shown in 50 values. We identified a list of 185 genes that were significantly differentially expressed between these two groups  with that for the five least sensitive SCLC cell lines ; as defined in the CGP by their BI-2536 ICTo validate that the PLK gene signature does, in fact, predict sensitivity to PLK inhibitors, we determined the efficacy of the PLK inhibitor BI-6727 in a cell line, H1092, which had gene expression data but no PLK sensitivity data in the CGP study, represented by a grey box at the top of It was of interest to determine whether the PLK gene signature was present in patient tumors and could potentially be used to predict tumor sensitivity to PLK inhibitors. The largest study of SCLC tumor gene expression is that of Rudin et al. TP53 or RB1, and sometimes both genes, whereas all sensitive cells displayed mutations of unknown protein significance (intronic and missense), typically in only one of these genes. All but one of the gene mutations was homozygous, indicating that resistant cells likely have no functional RB1 or TP53 protein. All sensitive cells display MYC , MYCN  or MYCL (CORL88) amplification, whereas only one resistant cell line displays MYC amplification (H2171). There also seems to be little or no CNV on the X chromosome in sensitive cells relative to resistant cells, which typically display some CNV loss. Genes on chromosome 13 are generally upregulated in PLK sensitive cells and downregulated in PLK resistant cells, whereas genes located on chromosome 19 are generally downregulated in PLK sensitive cells and upregulated in PLK resistant cells. Taken together, these results suggest that gene expression, CNV and mutational status may all contribute to the sensitivity of SCLC cells to PLK inhibition.Finally, we used circos plots Small-cell lung cancer is a disease in urgent need of new drug therapies. Unfortunately, the limited availability of tumor tissue hinders the acquisition of complete genomic analyses required for the identification and validation of new drugable targets in this cancer. Thus, alternative drug discovery strategies need to be developed until our understanding of the genomics driving SCLC can begin to approximate that of NSCLC, which benefits from comprehensive analyses of large cohorts of patient tumors, such as the The Cancer Genome Atlas (TCGA).In this report we have taken a bioinformatic approach to drug discovery for SCLC by data mining two large drug screening studies in cultured cell lines, the CCLE The CGP study also identified drug sensitivities that tended to cluster across all cell lines  and 53 SCLC cell lines examined. None of these PLK mutated cell lines were included in our study. Our PLK gene signature does, however, include genes on chromosomes typically deleted  and amplified (19p) in SCLC It was recently reported that mutations in the MYC-amplified cell lines, they also found the PLK inhibitor BI-2536 to be active, similar to our results, but did not pursue it further. They also showed that the majority of cells sensitive to the Aurora kinase inhibitor VX-680 demonstrated MYC-amplification. Interestingly, the CGP also included two Aurora kinase inhibitors (VX-680 and ZM-447439) in their study; however, it did not find any significant clustering of PLK with Aurora kinase inhibitors, indicating no link between these two classes of inhibitors when analyzed in the general population of cancer cell lines.During our study a report by Sos et al. In the present study we consistently identified three subtypes of SCLC cell lines using unsupervised clustering of either gene expression or CNV datasets from the CGP or CCLE  studies.Other unique approaches have been taken to identify new and effective therapies for SCLC. Jahchan et al. identified a surprising sensitivity of SCLC cells to tricyclic antidepressants in a drug repositioning study, which used bioinformatics to find drugs that induced changes in gene expression opposite to the gene expression profile of SCLC cells All cells were obtained from the ATCC and grown in their recommended medium. Cell proliferation was determined quantitatively by fluorescent DNA assay 50), gene expression, CNV, and mutation data was downloaded from http://broadinstitute.org/ccle (CCLE) and http://cancerrxgene.org (CGP) http://www.r-project.org/).The publically available CCLE and CGP drug sensitivity (IC50 greater than 8 µM in the CGP were thresholded at 8 µM. Boxplots were drawn for the IC50 from the two datasets respectively using R (http://www.r-project.org/). Mosaic plots were drawn using proc sgrender in SAS version 9.3 .For SCLC only, 24 drugs and 53 cell lines were included in the CCLE, while 92 drugs and 31 cell lines were included in the CGP. All ICNormalization of gene expression data  involved four steps: 1) raw data were normalized via the robust multi-array average (RMA) method; 2) probes without a gene name were removed; 3) gene level data was obtained by averaging the probe value within each gene and 4) the gene level data was then standard normalized by gene. For the gene expression data, the CCLE included 51 cell lines, while the CGP included 30 cell lines, among which three cell lines had duplicate measurements. Averaging the duplicates generated 27 unique cell lines for the CGP. Unsupervised consensus clustering was performed on the normalized gene level data with the R package “Consensus Cluster Plus”; parameters were set as default except maxK was set at 10. The Kruskal-Wallis test was used to assess expression differences between subtypes based on the consensus clustering results. Unsupervised hierarchical clustering was then performed using the significant genes only  and visualized using heatmaps via the R package “gplots”.Copy number data was obtained for the CGP data only for 426 genes The fastq data was aligned with tophat 2.0.9 Figure S1Boxplot of drug sensitivity in SCLC cells using the CCLE dataset. There are 53 cell lines for small cell lung cancer. The boxplots show drugs listed on the x-axis and the corresponding IC50 values (in µM) listed on the y-axis, similar to (PDF)Click here for additional data file.Figure S2Mosaic plot of drug sensitivity using CNV clustering of SCLC cells. Drug sensitivity was color-coded according to the legend at the bottom. Drugs are grouped along the y-axis according to their target molecule. Cells are arranged along the x-axis identical to their gene expression clustering identified in (TIF)Click here for additional data file.Figure S3Full-size circos plots of SCLC cells. Circos plots are shown full-size as individual panels for all the SCLC cell lines shown in collectively (PPTX)Click here for additional data file.Figure S4Unsupervised clustering of SCLC cells by gene expression using the CCLE dataset. Unsupervised consensus clustering was performed using the all 53 cell lines (only 51 gene expression available) and showed that 3 clusters was optimal for this dataset. With this assignment, we performed non-parametric one way ANOVA  test on those 3 clusters and obtained 4749 significant genes. We then generated the heatmap with those significant genes.(TIF)Click here for additional data file.Table S1Numerical data for drug efficacy determined in the CGP study. The 25%, 50% and 75% quantiles for all 92 drugs used to construct the boxplot in (DOC)Click here for additional data file.Table S2Numerical data for drug efficacy determined in the CCLE study. The 25%, 50% and 75% quantiles for all 24 drugs used to construct the boxplot in (DOC)Click here for additional data file.Table S3Significant genes used in CGP and CCLE gene expression clustering. The 1006 and 4749 significant genes used to cluster SCLC cell lines in the CGP ( the CGP  and CCLE the CGP  datasets(XLS)Click here for additional data file.Table S4Significant genes used in PLK gene expression clustering. The 185 significant genes used to cluster SCLC cell lines based upon their sensitivity to BI-2536 ( BI-2536  using th(XLS)Click here for additional data file.


Calidris mauri) utilized biofilm as a food resource across a large and variable stopover location during northward (spring) migration. We investigated the spatial heterogeneity in diet composition, to determine whether shorebirds were consistently feeding on biofilm or whether diet varied between naturally and anthropogenically delineated sites. We used stable isotope analysis to estimate that biofilm conservatively comprised 22% to 53% of Western Sandpiper droppings across all sampling sites and that prey composition differed significantly between areas within the stopover location. Widespread biofilm consumption demonstrates the importance of biofilm as a dietary component. Variable diet composition suggests that habitat heterogeneity may be an important component of high quality stopover locations in the context of “state-dependant trade-offs” of Western Sandpiper population sub-groups. Future management decisions must consider and address potential impacts on the biofilm community throughout a stopover location, as single site studies of diet composition may not be adequate to develop effective management strategies for entire stopover sites.Many shorebird species undertake long-distance migrations punctuated by brief stays at food-rich, estuarine stopover locations. Understanding use of these food resources helps guide conservation and responsible development decisions. We determined the extent and degree to which Western Sandpiper ( High-quality stopover locations are integral in the life cycle of many migratory birds –3. SucceIntertidal mud and sandflats often host matrix-enclosed microorganism communities generically known as biofilm –9. BiofiMajor estuarine stopover locations supporting biofilm are often strategic places for transport, industrial and residential development. Such development can significantly change the extent and quality of estuarine ecosystems by altering hydrology and sedimentation, converting habitats, and introducing pollutants or invasive species. Each of these factors could potentially affect biofilm quality, abundance and distribution. An understanding of the extent and degree to which migratory shorebirds use biofilm is therefore necessary, to assess the possible impacts of development, and to help guide design and management.Calidris mauri) population and the entire population of the Pacific subspecies of Dunlin  migrate along North America’s Pacific flyway, regularly stopping during spring northward migration at a few, food-rich locations that are separated by hundreds or thousands of kilometres [A globally significant portion of the world’s Western Sandpiper  and nitrogen (δ15N) in shorebird droppings and prey items to determine the relative contribution of each prey type to the overall diet [We sampled the diet of Western Sandpipers across the entire stopover location. We followed the approach used by Kuwae et al. , and modall diet , 40. Droall diet –36 and titrogen δN in shorall diet –42.2, in order to gather a sufficient amount of material for isotope analysis. Surface sediment samples (n = 118) were collected from the same locations, using a toothbrush to scrape off the uppermost 1 mm of surface sediment [We focused on spring rather than fall migration in the Fraser Estuary—Boundary Bay system because the density of Western Sandpipers is higher and the period of migration shorter  , 23, presediment . Macroinsediment . SedimenDropping samples were treated prior to stable isotope analysis to remove potential metabolites such as urea and ammonium. A 5 mg subsample of each powdered dropping sample was mixed with a 1.4 ml 2:1 chloroform:methanol solution. Particulate matter was allowed to settle and the solution was removed using a pipette. This procedure was repeated a minimum of three times per sample followed by oven drying.Due to the high sediment content in samples collected from the intertidal zone, all samples were treated to remove non-dietary carbonates prior to isotope analysis using an acid wash . A few dδ13C) and nitrogen (δ15N), percent carbon content, and percent nitrogen content were measured using either a Thermo-Finnigan Delta Plus mass spectrometer (droppings and invertebrates) or a Delta XP isotope-ratio mass spectrometer (sediment and microphytobenthos) interfaced to an Elemental Analyzer via the Conflo II or Conflo III, respectively. Isotope values are presented relative to international measurement standards Vienna Peedee Belemnite (VPDB) and atmospheric nitrogen for δ13C and δ15N, respectively. Stable-isotope values are expressed in δ notation as deviation from the appropriate international measurement standard in parts per thousand (‰).All samples were weighed into tin capsules and combusted in either a Carlo Erba NC2500 (dropping and invertebrate samples) or a Costech 4010 Elemental Analyzer (sediment and microphytobenthos samples) and delivered to mass spectrometers via continuous flow systems (Conflo II or Conflo III) using helium as a carrier gas. Stable-isotope values of carbon  were used to ensure that values used in calibration spanned the range of expected values, and composition of unknowns. These showed that instrument precision was 0.2 for or δ15N . In addiδ13C and δ15N values of surface sediments and microphytobenthos were similar in both magnitude and direction from dropping samples  were compared to those of Kuwae et al. [δ13C = -15.43 ± 1.57, δ15N = 12.01 ± 1.07) we found a difference of only 0.23 δ13C and 0.5 δ15N, less than 4%; suggesting that polychaete sample size did not significantly impact the interpretation of our results. Variation in small invertebrate isotopic values was comparatively low among sites and sample sizes were higher. We therefore pooled invertebrates within the three naturally delineated Wildlife Management Areas 1: Boundary Bay, 2: Roberts Bank and 3: Sturgeon Bank.We used the Bayesian mixing model SIAR  in R 2.1To measure the similarity of the estimates of biofilm consumption by Western Sandpipers between sites, we used the approach of Bond and Diamond , and calDuring daily sample collection for isotope analysis, numbers of Western Sandpiper and Dunlin in each site were estimated within three hours of the high tide. Experienced observers estimated the size of flocks, by tallying the number of groups of 100 or 500 birds within each flock. This method has an estimated error of approximately 20% . The speThe proportion of biofilm in Western Sandpiper diet varied spatially with the highest proportions observed in Site 1:a Mud Bay, and Site 2:e Westham Island, and lowest proportions in Area 3: Sturgeon Bank , Fig 3. Diet composition of Western Sandpipers varied significantly between Areas 1: Boundary Bay, 2: Roberts Bank and 3: Sturgeon Bank, but not between sites within each of these three areas . Diet coWestern Sandpiper and Dunlin were widely distributed in large numbers across the Fraser Estuary and Boundary Bay during the study period. The highest counts were recorded at Site 2:d Brunswick Point , where 5Biofilm was estimated to comprise 22.8%  to 53%  of Western Sandpiper droppings, suggesting that it is an important dietary component throughout the stopover location. Given that droppings provide a conservative estimate of biofilm grazing  and thatRegular movements of Western Sandpiper and Dunlin between sites and areas suggest that birds have knowledge of food and habitat variation and make site selection decisions accordingly. The proportion of biofilm, small invertebrates and polychaete worms in the diet differed significantly between Site 1:a Mud Bay, Site 1:b Boundary Bay East, Area 2: Roberts Bank and Area 3: Sturgeon Bank. Two non-mutually exclusive hypotheses exist that may explain this diet heterogeneity: i) the diet composition in areas supporting the highest numbers of Western Sandpipers is optimal and the population is operating under an ideal free distribution , and ii)The sites with the highest proportion of biofilm in the diet, Area 2: Roberts Bank and Site 1:a Mud Bay either supported the greatest numbers of Western Sandpipers, or exhibited the highest proportions of Western Sandpipers to Dunlin, suggesting that site use was related to biofilm consumption. Analysis of bird density in comparison to biofilm abundance was not possible here, as density per unit area does not necessarily correlate to density per unit biofilm. The extent and density of biofilm varies spatially and temporally  yet effiIdeal free distribution theory assumes that all individuals have the same foraging preferences. Given that fattening rates of Western Sandpipers neither correlate to feeding strategy, nor vary within stopover sites  and WestAlthough we focused on spring migration, similar studies during the extended fall migratory period are needed in order to understand the implications of diet heterogeneity and biofilm feeding throughout the full life cycle of this species. Shorebirds have a generally high flexibility of invertebrate prey species choice, and previous management regimes have focused on maintaining wetland habitats and diverse invertebrate populations . HoweverCalidris pusilla) in the Bay of Fundy [Biofilm feeding by vertebrates is a relatively recent discovery , 13. Ourof Fundy  and has of Fundy . The potS1 Table(DOCX)Click here for additional data file.S2 TableDropping samples represent 10 droppings from different individuals pooled as one sample. Small invertebrates and polychaete samples represent a pool of all individuals captured at each excavation location.(DOCX)Click here for additional data file.