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The functional engagement between an enhancer and its target promoter ensures precise gene transcription1. Understanding the basis of promoter choice by enhancers has important implications for health and disease. Here we report that functional loss of a preferred promoter can release its partner enhancer to loop to and activate an alternative promoter (or alternative promoters) in the neighbourhood. We refer to this target-switching process as 'enhancer release and retargeting'. Genetic deletion, motif perturbation or mutation, and dCas9-mediated CTCF tethering reveal that promoter choice by an enhancer can be determined by the binding of CTCF at promoters, in a cohesin-dependent manner-consistent with a model of 'enhancer scanning' inside the contact domain. Promoter-associated CTCF shows a lower affinity than that at chromatin domain boundaries and often lacks a preferred motif orientation or a partnering CTCF at the cognate enhancer, suggesting properties distinct from boundary CTCF. Analyses of cancer mutations, data from the GTEx project and risk loci from genome-wide association studies, together with a focused CRISPR interference screen, reveal that enhancer release and retargeting represents an overlooked mechanism that underlies the activation of disease-susceptibility genes, as exemplified by a risk locus for Parkinson's disease (NUCKS1-RAB7L1) and three loci associated with cancer (CLPTM1L-TERT, ZCCHC7-PAX5 and PVT1-MYC).
Assuntos
Fator de Ligação a CCCTC/genética , Elementos Facilitadores Genéticos , Predisposição Genética para Doença , Regiões Promotoras Genéticas , Sistemas CRISPR-Cas , Proteínas de Ciclo Celular/genética , Células Cultivadas , Cromatina , Proteínas Cromossômicas não Histona/genética , Deleção de Genes , Regulação Neoplásica da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Células MCF-7 , Neoplasias/genética , Células-Tronco Neurais , Oncogenes , Doença de Parkinson/genética , CoesinasRESUMO
In glioblastoma (GBM), heterogeneous expression of amplified and mutated epidermal growth factor receptor (EGFR) presents a substantial challenge for the effective use of EGFR-directed therapeutics. Here we demonstrate that heterogeneous expression of the wild-type receptor and its constitutively active mutant form, EGFRvIII, limits sensitivity to these therapies through an interclonal communication mechanism mediated by interleukin-6 (IL-6) cytokine secreted from EGFRvIII-positive tumor cells. IL-6 activates a NF-κB signaling axis in a paracrine and autocrine manner, leading to bromodomain protein 4 (BRD4)-dependent expression of the prosurvival protein survivin (BIRC5) and attenuation of sensitivity to EGFR tyrosine kinase inhibitors (TKIs). NF-κB and survivin are coordinately up-regulated in GBM patient tumors, and functional inhibition of either protein or BRD4 in in vitro and in vivo models restores sensitivity to EGFR TKIs. These results provide a rationale for improving anti-EGFR therapeutic efficacy through pharmacological uncoupling of a convergence point of NF-κB-mediated survival that is leveraged by an interclonal circuitry mechanism established by intratumoral mutational heterogeneity.
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Resistencia a Medicamentos Antineoplásicos/genética , Glioblastoma/fisiopatologia , NF-kappa B/genética , NF-kappa B/metabolismo , Transdução de Sinais/genética , Animais , Comunicação Celular , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Interleucina-6/metabolismo , Camundongos , Camundongos Nus , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
SUMMARY: Leveraging local ancestry and haplotype information in genome-wide association studies and downstream analyses can improve the utility of genomics for individuals from diverse and recently admixed ancestries. However, most existing simulation, visualization and variant analysis frameworks are based on variant-level analysis and do not automatically handle these features. We present haptools, an open-source toolkit for performing local ancestry aware and haplotype-based analysis of complex traits. Haptools supports fast simulation of admixed genomes, visualization of admixture tracks, simulation of haplotype- and local ancestry-specific phenotype effects and a variety of file operations and statistics computed in a haplotype-aware manner. AVAILABILITY AND IMPLEMENTATION: Haptools is freely available at https://github.com/cast-genomics/haptools. DOCUMENTATION: Detailed documentation is available at https://haptools.readthedocs.io. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Estudo de Associação Genômica Ampla , Software , Haplótipos , Genômica , GenomaRESUMO
Reactivation of fetal-specific genes and isoforms occurs during heart failure. However, the underlying molecular mechanisms and the extent to which the fetal program switch occurs remains unclear. Limitations hindering transcriptome-wide analyses of alternative splicing differences (i.e. isoform switching) in cardiovascular system (CVS) tissues between fetal, healthy adult and heart failure have included both cellular heterogeneity across bulk RNA-seq samples and limited availability of fetal tissue for research. To overcome these limitations, we have deconvoluted the cellular compositions of 996 RNA-seq samples representing heart failure, healthy adult (heart and arteria), and fetal-like (iPSC-derived cardiovascular progenitor cells) CVS tissues. Comparison of the expression profiles revealed that reactivation of fetal-specific RNA-binding proteins (RBPs), and the accompanied re-expression of 1,523 fetal-specific isoforms, contribute to the transcriptome differences between heart failure and healthy adult heart. Of note, isoforms for 20 different RBPs were among those that reverted in heart failure to the fetal-like expression pattern. We determined that, compared with adult-specific isoforms, fetal-specific isoforms encode proteins that tend to have more functions, are more likely to harbor RBP binding sites, have canonical sequences at their splice sites, and contain typical upstream polypyrimidine tracts. Our study suggests that compared with healthy adult, fetal cardiac tissue requires stricter transcriptional regulation, and that during heart failure reversion to this stricter transcriptional regulation occurs. Furthermore, we provide a resource of cardiac developmental stage-specific and heart failure-associated genes and isoforms, which are largely unexplored and can be exploited to investigate novel therapeutics for heart failure.
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Insuficiência Cardíaca , Adulto , Processamento Alternativo/genética , Feto/metabolismo , Insuficiência Cardíaca/genética , Humanos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
The identification of a constantly increasing number of genes whose mutations are causally implicated in tumor initiation and progression (cancer genes) requires the development of tools to store and analyze them. The Network of Cancer Genes (NCG 3.0) collects information on 1494 cancer genes that have been found mutated in 16 different cancer types. These genes were collected from the Cancer Gene Census as well as from 18 whole exome and 11 whole-genome screenings of cancer samples. For each cancer gene, NCG 3.0 provides a summary of the gene features and the cross-reference to other databases. In addition, it describes duplicability, evolutionary origin, orthology, network properties, interaction partners, microRNA regulation and functional roles of cancer genes and of all genes that are related to them. This integrated network of information can be used to better characterize cancer genes in the context of the system in which they act. The data can also be used to identify novel candidates that share the same properties of known cancer genes and may therefore play a similar role in cancer. NCG 3.0 is freely available at http://bio.ifom-ieo-campus.it/ncg.
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Bases de Dados Genéticas , Redes Reguladoras de Genes , Genes Neoplásicos , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Humanos , MicroRNAs/metabolismo , Mutação , Neoplasias/genética , Neoplasias/metabolismo , Mapeamento de Interação de Proteínas , Integração de SistemasRESUMO
The regulatory mechanisms underlying the response to pro-inflammatory cytokines during myocarditis are poorly understood. Here, we use iPSC-derived cardiovascular progenitor cells (CVPCs) to model the response to interferon gamma (IFN-γ) during myocarditis. We generate RNA-seq and ATAC-seq for four CVPCs that were treated with IFN-γ and compare them with paired untreated controls. Transcriptional differences after treatment show that IFN-γ initiates an innate immune cell-like response in the vascular cardiac endothelium. IFN-γ treatment also shifts the CVPC transcriptome towards the adult coronary artery and aorta profiles and expands the relative endothelial cell population in all four CVPC lines. Analysis of the accessible chromatin shows that IFN-γ is a potent chromatin remodeler and establishes an IRF-STAT immune-cell like regulatory network. Our findings reveal insights into the endothelial-specific protective mechanisms during myocarditis.
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Stem cells exist in vitro in a spectrum of interconvertible pluripotent states. Analyzing hundreds of hiPSCs derived from different individuals, we show the proportions of these pluripotent states vary considerably across lines. We discover 13 gene network modules (GNMs) and 13 regulatory network modules (RNMs), which are highly correlated with each other suggesting that the coordinated co-accessibility of regulatory elements in the RNMs likely underlie the coordinated expression of genes in the GNMs. Epigenetic analyses reveal that regulatory networks underlying self-renewal and pluripotency are more complex than previously realized. Genetic analyses identify thousands of regulatory variants that overlapped predicted transcription factor binding sites and are associated with chromatin accessibility in the hiPSCs. We show that the master regulator of pluripotency, the NANOG-OCT4 Complex, and its associated network are significantly enriched for regulatory variants with large effects, suggesting that they play a role in the varying cellular proportions of pluripotency states between hiPSCs. Our work bins tens of thousands of regulatory elements in hiPSCs into discrete regulatory networks, shows that pluripotency and self-renewal processes have a surprising level of regulatory complexity, and suggests that genetic factors may contribute to cell state transitions in human iPSC lines.
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Células-Tronco Pluripotentes Induzidas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Redes Reguladoras de Genes , Cromatina/genética , Diferenciação Celular/genética , Fator 3 de Transcrição de Octâmero/genéticaRESUMO
Most GWAS loci are presumed to affect gene regulation, however, only â¼43% colocalize with expression quantitative trait loci (eQTLs). To address this colocalization gap, we identify eQTLs, chromatin accessibility QTLs (caQTLs), and histone acetylation QTLs (haQTLs) using molecular samples from three early developmental (EDev) tissues. Through colocalization, we annotate 586 GWAS loci for 17 traits by QTL complexity, QTL phenotype, and QTL temporal specificity. We show that GWAS loci are highly enriched for colocalization with complex QTL modules that affect multiple elements (genes and/or peaks). We also demonstrate that caQTLs and haQTLs capture regulatory variations not associated with eQTLs and explain â¼49% of the functionally annotated GWAS loci. Additionally, we show that EDev-unique QTLs are strongly depleted for colocalizing with GWAS loci. By conducting one of the largest multi-omic QTL studies to date, we demonstrate that many GWAS loci exhibit phenotypic complexity and therefore, are missed by traditional eQTL analyses.
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Because most DNA-binding transcription factors (dbTFs), including the architectural regulator CTCF, bind RNA and exhibit di-/multimerization, a central conundrum is whether these distinct properties are regulated post-transcriptionally to modulate transcriptional programs. Here, investigating stress-dependent activation of SIRT1, encoding an evolutionarily-conserved protein deacetylase, we show that induced phosphorylation of CTCF acts as a rheostat to permit CTCF occupancy of low-affinity promoter DNA sites to precisely the levels necessary. This CTCF recruitment to the SIRT1 promoter is eliciting a cardioprotective cardiomyocyte transcriptional activation program and provides resilience against the stress of the beating heart in vivo . Mice harboring a mutation in the conserved low-affinity CTCF promoter binding site exhibit an altered, cardiomyocyte-specific transcriptional program and a systolic heart failure phenotype. This transcriptional role for CTCF reveals that a covalent dbTF modification regulating signal-dependent transcription serves as a previously unsuspected component of the oxidative stress response.
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The causal variants and genes underlying thousands of cardiac GWAS signals have yet to be identified. Here, we leverage spatiotemporal information on 966 RNA-seq cardiac samples and perform an expression quantitative trait locus (eQTL) analysis detecting eQTLs considering both eGenes and eIsoforms. We identify 2,578 eQTLs associated with a specific developmental stage-, tissue- and/or cell type. Colocalization between eQTL and GWAS signals of five cardiac traits identified variants with high posterior probabilities for being causal in 210 GWAS loci. Pulse pressure GWAS loci are enriched for colocalization with fetal- and smooth muscle- eQTLs; pulse rate with adult- and cardiac muscle- eQTLs; and atrial fibrillation with cardiac muscle- eQTLs. Fine mapping identifies 79 credible sets with five or fewer SNPs, of which 15 were associated with spatiotemporal eQTLs. Our study shows that many cardiac GWAS variants impact traits and disease in a developmental stage-, tissue- and/or cell type-specific fashion.
Assuntos
Fibrilação Atrial , Coração , Humanos , Miocárdio , Fibrilação Atrial/genética , Pressão Sanguínea , FetoRESUMO
The impact of genetic regulatory variation active in early pancreatic development on adult pancreatic disease and traits is not well understood. Here, we generate a panel of 107 fetal-like iPSC-derived pancreatic progenitor cells (iPSC-PPCs) from whole genome-sequenced individuals and identify 4065 genes and 4016 isoforms whose expression and/or alternative splicing are affected by regulatory variation. We integrate eQTLs identified in adult islets and whole pancreas samples, which reveal 1805 eQTL associations that are unique to the fetal-like iPSC-PPCs and 1043 eQTLs that exhibit regulatory plasticity across the fetal-like and adult pancreas tissues. Colocalization with GWAS risk loci for pancreatic diseases and traits show that some putative causal regulatory variants are active only in the fetal-like iPSC-PPCs and likely influence disease by modulating expression of disease-associated genes in early development, while others with regulatory plasticity likely exert their effects in both the fetal and adult pancreas by modulating expression of different disease genes in the two developmental stages.
Assuntos
Diabetes Mellitus , Locos de Características Quantitativas , Adulto , Humanos , Locos de Características Quantitativas/genética , Estudo de Associação Genômica Ampla , Pâncreas , Sequência de Bases , Diabetes Mellitus/genética , Polimorfismo de Nucleotídeo Único , Predisposição Genética para DoençaRESUMO
Stem cells exist in vitro in a spectrum of interconvertible pluripotent states. Analyzing hundreds of hiPSCs derived from different individuals, we show the proportions of these pluripotent states vary considerably across lines. We discovered 13 gene network modules (GNMs) and 13 regulatory network modules (RNMs), which were highly correlated with each other suggesting that the coordinated co-accessibility of regulatory elements in the RNMs likely underlied the coordinated expression of genes in the GNMs. Epigenetic analyses revealed that regulatory networks underlying self-renewal and pluripotency have a surprising level of complexity. Genetic analyses identified thousands of regulatory variants that overlapped predicted transcription factor binding sites and were associated with chromatin accessibility in the hiPSCs. We show that the master regulator of pluripotency, the NANOG-OCT4 Complex, and its associated network were significantly enriched for regulatory variants with large effects, suggesting that they may play a role in the varying cellular proportions of pluripotency states between hiPSCs. Our work captures the coordinated activity of tens of thousands of regulatory elements in hiPSCs and bins these elements into discrete functionally characterized regulatory networks, shows that regulatory elements in pluripotency networks harbor variants with large effects, and provides a rich resource for future pluripotent stem cell research.
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Duplications of genes encoding highly connected and essential proteins are selected against in several species but not in human, where duplicated genes encode highly connected proteins. To understand when and how gene duplicability changed in evolution, we compare gene and network properties in four species (Escherichia coli, yeast, fly, and human) that are representative of the increase in evolutionary complexity, defined as progressive growth in the number of genes, cells, and cell types. We find that the origin and conservation of a gene significantly correlates with the properties of the encoded protein in the protein-protein interaction network. All four species preserve a core of singleton and central hubs that originated early in evolution, are highly conserved, and accomplish basic biological functions. Another group of hubs appeared in metazoans and duplicated in vertebrates, mostly through vertebrate-specific whole genome duplication. Such recent and duplicated hubs are frequently targets of microRNAs and show tissue-selective expression, suggesting that these are alternative mechanisms to control their dosage. Our study shows how networks modified during evolution and contributes to explaining the occurrence of somatic genetic diseases, such as cancer, in terms of network perturbations.
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Biologia Computacional/métodos , Mapeamento de Interação de Proteínas , Animais , Evolução Biológica , Drosophila melanogaster/genética , Escherichia coli/genética , Evolução Molecular , Duplicação Gênica , Genes Duplicados , Doenças Genéticas Inatas/genética , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Modelos Biológicos , Modelos Genéticos , Saccharomyces cerevisiae/genéticaRESUMO
The Network of Cancer Genes (NCG) collects and integrates data on 736 human genes that are mutated in various types of cancer. For each gene, NCG provides information on duplicability, orthology, evolutionary appearance and topological properties of the encoded protein in a comprehensive version of the human protein-protein interaction network. NCG also stores information on all primary interactors of cancer proteins, thus providing a complete overview of 5357 proteins that constitute direct and indirect determinants of human cancer. With the constant delivery of results from the mutational screenings of cancer genomes, NCG represents a versatile resource for retrieving detailed information on particular cancer genes, as well as for identifying common properties of precompiled lists of cancer genes. NCG is freely available at: http://bio.ifom-ieo-campus.it/ncg.
Assuntos
Biologia Computacional/métodos , Bases de Dados Genéticas , Bases de Dados de Ácidos Nucleicos , Regulação Neoplásica da Expressão Gênica , Genes Neoplásicos , Neoplasias/genética , Animais , Biologia Computacional/tendências , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Genoma de Planta , Humanos , Armazenamento e Recuperação da Informação/métodos , Internet , Mutação , SoftwareRESUMO
Variability in SARS-CoV-2 susceptibility and COVID-19 disease severity between individuals is partly due to genetic factors. Here, we applied colocalization to compare summary statistics for 16 GWASs from the COVID-19 Host Genetics Initiative to investigate similarities and differences in their genetic signals. We identified 9 loci associated with susceptibility (one with two independent GWAS signals; one with an ethnicity-specific signal), 14 associated with severity (one with two independent GWAS signals; two with ethnicity-specific signals) and one harboring two discrepant GWAS signals (one for susceptibility; one for severity). Utilizing colocalization we also identified 45 GTEx tissues that had eQTL(s) for 18 genes strongly associated with GWAS signals in eleven loci (1-4 genes per locus). Some of these genes showed tissue-specific altered expression and others showed altered expression in up to 41 different tissue types. Our study provides insights into the complex molecular mechanisms underlying inherited predispositions to COVID-19-disease phenotypes.
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Variability in SARS-CoV-2 susceptibility and COVID-19 disease severity between individuals is partly due to genetic factors. Here, we identify 4 genomic loci with suggestive associations for SARS-CoV-2 susceptibility and 19 for COVID-19 disease severity. Four of these 23 loci likely have an ethnicity-specific component. Genome-wide association study (GWAS) signals in 11 loci colocalize with expression quantitative trait loci (eQTLs) associated with the expression of 20 genes in 62 tissues/cell types (range: 1:43 tissues/gene), including lung, brain, heart, muscle, and skin as well as the digestive system and immune system. We perform genetic fine mapping to compute 99% credible SNP sets, which identify 10 GWAS loci that have eight or fewer SNPs in the credible set, including three loci with one single likely causal SNP. Our study suggests that the diverse symptoms and disease severity of COVID-19 observed between individuals is associated with variants across the genome, affecting gene expression levels in a wide variety of tissue types.
Assuntos
COVID-19/genética , SARS-CoV-2/genética , Mapeamento Cromossômico/métodos , Biologia Computacional/métodos , Bases de Dados Genéticas , Etnicidade/genética , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Predisposição Genética para Doença/genética , Variação Genética/genética , Estudo de Associação Genômica Ampla/métodos , Humanos , Especificidade de Órgãos/genética , Polimorfismo de Nucleotídeo Único/genética , Locos de Características Quantitativas/genética , SARS-CoV-2/patogenicidade , Índice de Gravidade de Doença , Transcriptoma/genéticaRESUMO
Induced pluripotent stem cells (iPSCs) are an established cellular system to study the impact of genetic variants in derived cell types and developmental contexts. However, in their pluripotent state, the disease impact of genetic variants is less well known. Here, we integrate data from 1,367 human iPSC lines to comprehensively map common and rare regulatory variants in human pluripotent cells. Using this population-scale resource, we report hundreds of new colocalization events for human traits specific to iPSCs, and find increased power to identify rare regulatory variants compared with somatic tissues. Finally, we demonstrate how iPSCs enable the identification of causal genes for rare diseases.
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Variação Genética , Células-Tronco Pluripotentes Induzidas/fisiologia , Locos de Características Quantitativas , Síndrome de Bardet-Biedl/genética , Canais de Cálcio/genética , Linhagem Celular , Ataxia Cerebelar/genética , Metilação de DNA , Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Polimorfismo de Nucleotídeo Único , Proteínas/genética , Doenças Raras/genética , Sequências Reguladoras de Ácido Nucleico , Análise de Sequência de RNA , Sequenciamento Completo do GenomaRESUMO
Induced pluripotent stem cell derived cardiovascular progenitor cells (iPSC-CVPCs) provide an unprecedented platform for examining the molecular underpinnings of cardiac development and disease etiology, but also have great potential to play pivotal roles in the future of regenerative medicine and pharmacogenomic studies. Biobanks like iPSCORE ( Stacey et al., 2013 ; Panopoulos et al., 2017 ), which contain iPSCs generated from hundreds of genetically and ethnically diverse individuals, are an invaluable resource for conducting these studies. Here, we present an optimized, cost-effective and highly standardized protocol for large-scale derivation of human iPSC-CVPCs using small molecules and purification using metabolic selection. We have successfully applied this protocol to derive iPSC-CVPCs from 154 different iPSCORE iPSC lines obtaining large quantities of highly pure cardiac cells. An important component of our protocol is Cell confluency estimates (ccEstimate), an automated methodology for estimating the time when an iPSC monolayer will reach 80% confluency, which is optimal for initiating iPSC-CVPC derivation, and enables the protocol to be readily used across iPSC lines with different growth rates. Moreover, we showed that cellular heterogeneity across iPSC-CVPCs is due to varying proportions of two distinct cardiac cell types: cardiomyocytes (CMs) and epicardium-derived cells (EPDCs), both of which have been shown to have a critical function in heart regeneration. This protocol eliminates the need of iPSC line-to-line optimization and can be easily adapted and scaled to high-throughput studies or to generate large quantities of cells suitable for regenerative medicine applications.
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mESCs can self-renew indefinitely in vitro. However, depending on culture conditions some strains are more unstable than others. In this issue of Cell Stem Cell, Skelly et al. (2020) and Ortmann et al. (2020) shed light into the role genetic variation plays in control of ground state pluripotency.
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Células-Tronco Embrionárias Murinas , Células-Tronco Pluripotentes , Variação Biológica da População , Variação Genética/genéticaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.