Highly conserved gene order and numerous novel repetitive elements in genomic regions linked to wing pattern variation in Heliconius butterflies

This is the final version of the article. Available from the publisher via the DOI in this record.BACKGROUND: With over 20 parapatric races differing in their warningly colored wing patterns, the butterfly Heliconius erato provides a fascinating example of an adaptive radiation. Together with matching races of its co-mimic Heliconius melpomene, H. erato also represents a textbook case of Müllerian mimicry, a phenomenon where common warning signals are shared amongst noxious organisms. It is of great interest to identify the specific genes that control the mimetic wing patterns of H. erato and H. melpomene. To this end we have undertaken comparative mapping and targeted genomic sequencing in both species. This paper reports on a comparative analysis of genomic sequences linked to color pattern mimicry genes in Heliconius. RESULTS: Scoring AFLP polymorphisms in H. erato broods allowed us to survey loci at approximately 362 kb intervals across the genome. With this strategy we were able to identify markers tightly linked to two color pattern genes: D and Cr, which were then used to screen H. erato BAC libraries in order to identify clones for sequencing. Gene density across 600 kb of BAC sequences appeared relatively low, although the number of predicted open reading frames was typical for an insect. We focused analyses on the D- and Cr-linked H. erato BAC sequences and on the Yb-linked H. melpomene BAC sequence. A comparative analysis between homologous regions of H. erato (Cr-linked BAC) and H. melpomene (Yb-linked BAC) revealed high levels of sequence conservation and microsynteny between the two species. We found that repeated elements constitute 26% and 20% of BAC sequences from H. erato and H. melpomene respectively. The majority of these repetitive sequences appear to be novel, as they showed no significant similarity to any other available insect sequences. We also observed signs of fine scale conservation of gene order between Heliconius and the moth Bombyx mori, suggesting that lepidopteran genome architecture may be conserved over very long evolutionary time scales. CONCLUSION: Here we have demonstrated the tractability of progressing from a genetic linkage map to genomic sequence data in Heliconius butterflies. We have also shown that fine-scale gene order is highly conserved between distantly related Heliconius species, and also between Heliconius and B. mori. Together, these findings suggest that genome structure in macrolepidoptera might be very conserved, and show that mapping and positional cloning efforts in different lepidopteran species can be reciprocally informative.The work was funded by U.S. National Science Foundation grants IOB 0344705 and DEB 0715096 to WOM. The H. erato BAC library was constructed by C. Wu, H. Zhang (TAMU), and M. R. Goldsmith (URI) under NSF Grant IBN-0208388. In addition, the Computational Biology Service Unit at Cornell University, which is partially funded by Microsoft Corporation, provided bioinformatics support for our analysis of genomic repeat structure. AFLP analysis and sequencing of PCR products was carried out at the Sequencing and Genotyping Center at the University of Puerto RicoRio Piedras. We thank Nicola Flanagan, Alexandra Tobler, Karla Maldonado, Jenny Acevedo Gonzales, Hector Alejandro Merchan, Yhadi Cotto, Kelitt Santiago and Felix Araujo Perez for help in rearing and maintaining butterfly stocks. Finally, a special thanks to Daniel P. Lindstrom for his support and helpful suggestions during manuscript preparation

Genomic hotspots for adaptation: the population genetics of Müllerian mimicry in Heliconius erato

This is the final version of the article. Available from the publisher via the DOI in this record.Wing pattern evolution in Heliconius butterflies provides some of the most striking examples of adaptation by natural selection. The genes controlling pattern variation are classic examples of Mendelian loci of large effect, where allelic variation causes large and discrete phenotypic changes and is responsible for both convergent and highly divergent wing pattern evolution across the genus. We characterize nucleotide variation, genotype-by-phenotype associations, linkage disequilibrium (LD), and candidate gene expression patterns across two unlinked genomic intervals that control yellow and red wing pattern variation among mimetic forms of Heliconius erato. Despite very strong natural selection on color pattern, we see neither a strong reduction in genetic diversity nor evidence for extended LD across either patterning interval. This observation highlights the extent that recombination can erase the signature of selection in natural populations and is consistent with the hypothesis that either the adaptive radiation or the alleles controlling it are quite old. However, across both patterning intervals we identified SNPs clustered in several coding regions that were strongly associated with color pattern phenotype. Interestingly, coding regions with associated SNPs were widely separated, suggesting that color pattern alleles may be composed of multiple functional sites, conforming to previous descriptions of these loci as "supergenes." Examination of gene expression levels of genes flanking these regions in both H. erato and its co-mimic, H. melpomene, implicate a gene with high sequence similarity to a kinesin as playing a key role in modulating pattern and provides convincing evidence for parallel changes in gene regulation across co-mimetic lineages. The complex genetic architecture at these color pattern loci stands in marked contrast to the single casual mutations often identified in genetic studies of adaptation, but may be more indicative of the type of genetic changes responsible for much of the adaptive variation found in natural populations.Funding: Funding for this study was provided by National Science Foundation grants to WOM (DEB-0715096 and IBN-0344705) and BAC (DEB-0513424). Funding for work on H. melpomene came from a BBSRC grant to CDJ and RHf-C (011845). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

In vitro analysis of the effects on wound healing of high- and low-molecular weight chains of hyaluronan and their hybrid H-HA/L-HA complexes

Abstract Background: Recent studies have reported the roles of Hyaluronic acid (HA) chains of diverse length in wound repair, especially considering the simultaneous occurrence in vivo of both high- (H-HA) and low-molecular weight (L-HA) hyaluronan at an injury site. It has been shown that HA fragments (5 ≤ MW ≤ 20 kDa) usually trigger an inflammatory response that, on one hand, is the first signal in the activation of a repair mechanism but on the other, when it’s overexpressed, it may promote unwanted side effects. The present experimental research has aimed to investigate H-HA, L-HA and of a newly developed complex of the two (H-HA/L-HA) for stability (e.g. hyaluronidases digestion), for their ability to promote wound healing of human keratinocytes in vitro and for their effect on cellular biomarker expression trends. Results: Time-lapse video microscopy studies proved that the diverse HA was capable of restoring the monolayer integrity of HaCat. The H-HA/L-HA complex (0.1 and 1%w/v) proved faster in regeneration also in co-culture scratch test where wound closure was achieved in half the time of H-HA stimulated cells and 2.5-fold faster than the control. Gene expression was evaluated for transformation growth factor beta 1 (TGF-β1) proving that L-HA alone increased its expression at 4 h followed by restoration of similar trends for all the stimuli. Depending on the diverse stimulation (H-HA, L-HA or the complex), metalloproteinases (MMP-2, -9, -13) were also modulated differently. Furthermore, type I collagen expression and production were evaluated. Compared to the others, persistence of a significant higher expression level at 24 h for the H-HA/L-HA complex was found. Conclusions: The outcomes of this research showed that, both at high and low concentrations, hybrid complexes proved to perform better than HA alone thus suggesting their potential as medical devices in aesthetic and regenerative medicine. Keywords: Wound healing, Hyaluronan, MMPs, Hybrid complexe

Jet energy measurement with the ATLAS detector in proton-proton collisions at root s=7 TeV

The jet energy scale and its systematic uncertainty are determined for jets measured with the ATLAS detector at the LHC in proton-proton collision data at a centre-of-mass energy of √s = 7TeV corresponding to an integrated luminosity of 38 pb-1. Jets are reconstructed with the anti-kt algorithm with distance parameters R=0. 4 or R=0. 6. Jet energy and angle corrections are determined from Monte Carlo simulations to calibrate jets with transverse momenta pT≥20 GeV and pseudorapidities {pipe}η{pipe}<4. 5. The jet energy systematic uncertainty is estimated using the single isolated hadron response measured in situ and in test-beams, exploiting the transverse momentum balance between central and forward jets in events with dijet topologies and studying systematic variations in Monte Carlo simulations. The jet energy uncertainty is less than 2. 5 % in the central calorimeter region ({pipe}η{pipe}<0. 8) for jets with 60≤pT<800 GeV, and is maximally 14 % for pT<30 GeV in the most forward region 3. 2≤{pipe}η{pipe}<4. 5. The jet energy is validated for jet transverse momenta up to 1 TeV to the level of a few percent using several in situ techniques by comparing a well-known reference such as the recoiling photon pT, the sum of the transverse momenta of tracks associated to the jet, or a system of low-pT jets recoiling against a high-pT jet. More sophisticated jet calibration schemes are presented based on calorimeter cell energy density weighting or hadronic properties of jets, aiming for an improved jet energy resolution and a reduced flavour dependence of the jet response. The systematic uncertainty of the jet energy determined from a combination of in situ techniques is consistent with the one derived from single hadron response measurements over a wide kinematic range. The nominal corrections and uncertainties are derived for isolated jets in an inclusive sample of high-pT jets. Special cases such as event topologies with close-by jets, or selections of samples with an enhanced content of jets originating from light quarks, heavy quarks or gluons are also discussed and the corresponding uncertainties are determined. © 2013 CERN for the benefit of the ATLAS collaboration

Measurement of the inclusive and dijet cross-sections of b-jets in pp collisions at sqrt(s) = 7 TeV with the ATLAS detector

The inclusive and dijet production cross-sections have been measured for jets containing b-hadrons (b-jets) in proton-proton collisions at a centre-of-mass energy of sqrt(s) = 7 TeV, using the ATLAS detector at the LHC. The measurements use data corresponding to an integrated luminosity of 34 pb^-1. The b-jets are identified using either a lifetime-based method, where secondary decay vertices of b-hadrons in jets are reconstructed using information from the tracking detectors, or a muon-based method where the presence of a muon is used to identify semileptonic decays of b-hadrons inside jets. The inclusive b-jet cross-section is measured as a function of transverse momentum in the range 20 < pT < 400 GeV and rapidity in the range |y| < 2.1. The bbbar-dijet cross-section is measured as a function of the dijet invariant mass in the range 110 < m_jj < 760 GeV, the azimuthal angle difference between the two jets and the angular variable chi in two dijet mass regions. The results are compared with next-to-leading-order QCD predictions. Good agreement is observed between the measured cross-sections and the predictions obtained using POWHEG + Pythia. MC@NLO + Herwig shows good agreement with the measured bbbar-dijet cross-section. However, it does not reproduce the measured inclusive cross-section well, particularly for central b-jets with large transverse momenta.Comment: 10 pages plus author list (21 pages total), 8 figures, 1 table, final version published in European Physical Journal

Altered Expression of Insulin Receptor Isoforms in Breast Cancer

PURPOSE: Insulin-like growth factor (IGF) signaling through human insulin receptor isoform A (IR-A) contributes to tumorigenesis and intrinsic resistance to anti-IGF1R therapy. In the present study, we (a) developed quantitative TaqMan real time-PCR-based assays (qRT-PCR) to measure human insulin receptor isoforms with high specificity, (b) evaluated isoform expression levels in molecularly-defined breast cancer subtypes, and (c) identified the IR-A:IR-B mRNA ratio as a potential biomarker guiding patient stratification for anti-IGF therapies. EXPERIMENTAL DESIGN: mRNA expression levels of IR-A and IR-B were measured in 42 primary breast cancers and 19 matched adjacent normal tissues with TaqMan qRT-PCR assays. The results were further confirmed in 165 breast cancers. The tumor samples were profiled using whole genome microarrays and subsequently subtyped using the PAM50 breast cancer gene signature. The relationship between the IR-A:IR-B ratio and cancer subtype, as well as markers of proliferation were characterized. RESULTS: The mRNA expression levels of IR-A in the breast tumors were similar to those observed in the adjacent normal tissues, while the mRNA levels of IR-B were significantly decreased in tumors. The IR-A:IR-B ratio was significantly higher in luminal B breast cancer than in luminal A. Strong concordance between the IR-A:IR-B ratio and the composite Oncotype DX proliferation score was observed for stratifying the latter two breast cancer subtypes. CONCLUSIONS: The reduction in IR-B expression is the key to the altered IR-A:IR-B ratio observed in breast cancer. The IR-A:IR-B ratio may have biomarker utility in guiding a patient stratification strategy for an anti-IGF therapeutic

Identifying Human Disease Genes through Cross-Species Gene Mapping of Evolutionary Conserved Processes

Understanding complex networks that modulate development in humans is hampered by genetic and phenotypic heterogeneity within and between populations. Here we present a method that exploits natural variation in highly diverse mouse genetic reference panels in which genetic and environmental factors can be tightly controlled. The aim of our study is to test a cross-species genetic mapping strategy, which compares data of gene mapping in human patients with functional data obtained by QTL mapping in recombinant inbred mouse strains in order to prioritize human disease candidate genes.We exploit evolutionary conservation of developmental phenotypes to discover gene variants that influence brain development in humans. We studied corpus callosum volume in a recombinant inbred mouse panel (C57BL/6J×DBA/2J, BXD strains) using high-field strength MRI technology. We aligned mouse mapping results for this neuro-anatomical phenotype with genetic data from patients with abnormal corpus callosum (ACC) development.).This approach that exploits highly diverse mouse strains provides an efficient and effective translational bridge to study the etiology of human developmental disorders, such as autism and schizophrenia

Genomic analysis of Sparus aurata reveals the evolutionary dynamics of sex-biased genes in a sequential hermaphrodite fish

Sexual dimorphism is a fascinating subject in evolutionary biology and mostly results from sex-biased expression of genes, which have been shown to evolve faster in gonochoristic species. We report here genome and sex-specific transcriptome sequencing of Sparus aurata, a sequential hermaphrodite fish. Evolutionary comparative analysis reveals that sex-biased genes in S. aurata are similar in number and function, but evolved following strikingly divergent patterns compared with gonochoristic species, showing overall slower rates because of stronger functional constraints. Fast evolution is observed only for highly ovary-biased genes due to female-specific patterns of selection that are related to the peculiar reproduction mode of S. aurata, first maturing as male, then as female. To our knowledge, these findings represent the first genome-wide analysis on sex-biased loci in a hermaphrodite vertebrate species, demonstrating how having two sexes in the same individual profoundly affects the fate of a large set of evolutionarily relevant genes.European Union KBBE.2013.1.2-10 European Community 311920 Fondazione Cassa di Risparmio Padova e Rovigo FCT - Foundation for Science and Technology research grant SPARCOMP under the Call ARISTEIA I of the National Strategic Reference Framework - by the EU 36 Hellenic Republic through the European Social Fundinfo:eu-repo/semantics/publishedVersio

Type I insulin-like growth factor receptor over-expression induces proliferation and anti-apoptotic signaling in a three-dimensional culture model of breast epithelial cells

INTRODUCTION: Activation of the type I insulin-like growth factor receptor (IGFIR) promotes proliferation and inhibits apoptosis in a variety of cell types. Transgenic mice expressing a constitutively active IGFIR or IGF-I develop mammary tumors and increased levels of IGFIR have been detected in primary breast cancers. However, the contribution of IGFIR activation in promoting breast cancer progression remains unknown. Mammary epithelial cell lines grown in three-dimensional cultures form acinar structures that mimic the round, polarized, hollow and growth-arrested features of mammary alveoli. We used this system to determine how proliferation and survival signaling by IGFIR activation affects breast epithelial cell biology and contributes to breast cancer progression. METHODS: Pooled, stable MCF-10A breast epithelial cells expressing wild-type IGFIR or kinase-dead IGFIR (K1003A) were generated using retroviral-mediated gene transfer. The effects of over-expression of wild-type or kinase-dead IGFIR on breast epithelial cell biology were analyzed by confocal microscopy of three-dimensional cultures. The contribution of signaling pathways downstream of IGFIR activation to proliferation and apoptosis were determined by pharmacological inhibition of phosphatidylinositol 3' kinase (PI3K) with LY294002, MAP kinase kinase (MEK) with UO126 and mammalian target of rapamycin (mTOR) with rapamycin. RESULTS: We found that MCF-10A cells over-expressing the IGFIR formed large, misshapen acinar structures with filled lumina and disrupted apico-basal polarization. This phenotype was ligand-dependent, occurring with IGF-I or supraphysiological doses of insulin, and did not occur in cells over-expressing the kinase-dead receptor. We observed increased proliferation, decreased apoptosis and increased phosphorylation of Ser(473 )of Akt and Ser(2448 )of mTOR throughout IGFIR structures. Inhibition of PI3K with LY294002 or MEK with UO126 prevented the development of acinar structures from IGFIR-expressing but not control cells. The mTOR inhibitor rapamycin failed to prevent IGFIR-induced hyperproliferation and survival signaling. CONCLUSION: Increased proliferation and survival signaling as well as loss of apico-basal polarity by IGFIR activation in mammary epithelial cells may promote early lesions of breast cancer. Three-dimensional cultures of MCF-10A cells over-expressing the IGFIR are a useful model with which to study the role of IGFIR signaling in breast cancer progression and for characterizing the effects of chemotherapeutics targeted to IGFIR signaling