NBN gene, which codes for the protein nibrin. Nibrin is an integral member of the MRE11/RAD50/NBN (MRN) complex essential for processing DNA double-strand breaks. Cardinal features of NBS are immunodeficiency and an extremely high incidence of hematological malignancies. Recent studies in conditional null mutant mice have indicated disturbances in redox homeostasis due to impaired DSB processing. Clearly this could contribute to DNA damage, chromosomal instability, and cancer occurrence. Here we show, in the complete absence of nibrin in null mutant mouse cells, high levels of reactive oxygen species several hours after exposure to a mutagen. We show further that NBS patient cells, which unlike mouse null mutant cells have a truncated nibrin protein, also have high levels of reactive oxygen after DNA damage and that this increased oxidative stress is caused by depletion of NAD+ due to hyperactivation of the strand-break sensor, Poly(ADP-ribose) polymerase. Both hyperactivation of Poly(ADP-ribose) polymerase and increased ROS levels were reversed by use of a specific Poly(ADP-ribose) polymerase inhibitor. The extremely high incidence of malignancy among NBS patients is the result of the combination of a primary DSB repair deficiency with secondary oxidative DNA damage.Nijmegen Breakage Syndrome (NBS), an autosomal recessive genetic instability syndrome, is caused by hypomorphic mutation of the  Damage to DNA is extremely dangerous because it can lead to mutations in genes that initiate or accelerate the development of a tumor. Evolution has led to highly complex networks of DNA repair enzymes, which for the majority of individuals are extremely effective in keeping our DNA intact. The devastating consequences of DNA damage are manifested in those individuals in which one or other of the repair pathways is non-functional. Several genetic disorders can be attributed to such DNA repair deficiencies and have the common feature of increased tumor incidence as the major life-threatening symptom. Cancer incidence varies amongst these disorders and is probably highest for the disease Nijmegen Breakage Syndrome, where more than 50% of patients develop a hematological malignancy in childhood. We have sought to understand this extremely high incidence by exploiting cells from a mouse model and cells derived from patients. We find that deficiency in the repair of DNA double-strand breaks leads to disturbances in cellular metabolism, leading ultimately to a loss of antioxidative capacity. The ensuing accumulation of highly reactive oxygen species generates further DNA lesions, thus potentiating the initial damage and increasing the likelihood of malignancy. Genetic cancer susceptibility disorders, such as Xeroderma pigmentosum and Fanconi anemia, generally have deficiencies in DNA repair and cell cycle regulation leading to tumour initiation. The specific mutagen sensitivities underlying these disorders define a set of enzymes and pathways involved in the DNA damage response. Nevertheless, these pathways clearly overlap and components in one pathway can be critically involved in another. For example, the nucleotide excision repair pathway mutated in Xeroderma pigmentosum is also required for the repair of interstrand crosslinks, to which Fanconi anemia patient cells are particularly sensitive Nijmegen Breakage Syndrome (NBS), Nijmegen Breakage Syndrome like disorder (NBSLD), Ataxia telangiectasia (AT) and Ataxia telangiectasia like disorder (ATLD) are clinically and biologically overlapping entities. Whilst the underlying proteins are intimately associated, cancer predisposition is a major life threatening feature of NBS and AT only. The proteins mutated in these four disorders are all involved in the sensing and repair of DNA double-strand breaks (DSB). However, if, as seems likely, the mutation rate in patient cells is increased, this may not be solely due to the primary DNA lesion but, rather, to the cumulative effects of auxiliary cellular disturbances. Thus it has been repeatedly shown that AT patient cells and knockout mice have increased oxidative stress Oxidative stress has not previously been associated with NBS, however, our previous proteomic study of null mutant mice suggested disturbances in the redox homeostasis in the livers of irradiated mice + pool accompanies excessive poly(ADP-ribosyl)ation in NBS cells and this severely compromises the anti-oxidant capacity of the cells. Thus the extremely high incidence of hematological malignancies in NBS may be the result of the combination of a primary DSB repair deficiency and accompanying oxidative damage.We find greatly increased levels of reactive oxygen species in both null mutant mouse cells and NBS patient cells after a DNA damaging exposure. Unlike AT cells, we find a parallel increase in the activity of PARP enzymes as measured by examining poly(ADP-ribosyl)ation of proteins. As we have previously hypothesized, depletion of the cellular NADNbn allele (Nbnins-6), a null mutation, and loxP sites flanking exon six in the other Nbn allele (Nbnlox-6). Treatment of these cells with cre recombinase leads to cells with biallelic Nbnins-6/del6 null mutations Nbn+ and the null mutant Nbnins-6 and Nbndel-6 alleles as Nbn−. As shown in Nbn gene  polymerases and consequent loss of cellular antioxidant capacity Nbn null mutations with bleomycin to induce DSBs and examined the extent and kinetics of poly(ADP-ribosyl)ation by western blot. As shown in We have argued previously that the permanent production of ROS in the absence of nibrin is caused by rapid depletion of NADIn We reasoned that if the increased ROS levels in NBS patient cells are a consequence of increased PARP activity, rather than its cause, inhibition of the enzyme should reduce ROS levels, whilst scavenging of ROS should not affect PARP activity. As shown in +, an important component of the cells antioxidant capacity. Indeed, numerous reports have shown that PARP inhibition prevents the reduction of NAD+ concentrations in cells subject to genotoxins, with a resulting decrease in cellular necrosis + levels in NBS fibroblasts after a bleomycin treatment in comparison to normal fibroblasts. There is very rapid depletion of NAD+ in the patient cells in comparison to the control cells, confirming the hyperactivation of PARP in these cells. Interestingly, the baseline levels of NAD+ in these NBS patient cells were considerably higher than in controls  vs. 875(+/−15) pmol/106 cells) suggesting that even in the absence of exogenous damaging agents, NAD+ requirements are higher in these repair deficient cells. In line with this observation we note that PARP activity in undamaged NBS cells is apparently higher than in controls H and its cellular level is therefore critical for cellular redox homeostasis. NAD+ is also a substrate for the PARP superfamily of enzymes with a common catalytic activity and involved in the DNA damage response + pool Nbn null mutant cells clearly lead to such PARP hyperactivation, as shown here. As previously reported, the −/−Nbn cells attempt to combat the increased ROS levels due to NAD+ depletion by upregulating genes involved in the detoxification of radicals, such as MnSOD. In contrast, enzymes also requiring the NAD+ substrate were downregulated, for example, glyoxylate reductase 6.7-fold NAD−/−Nbn cells and also in NBS patient cells, the loss of full nibrin function leads to a delay in the activation of ATM −/−Nbn mouse livers, transaldolase, a key enzyme of the pentose phosphate pathway, was actually reduced 6-fold In NBN gene are non-viable and NBS patients all have hypomorphic mutations and express a truncated nibrin protein Human cells with null mutation of the + depletion and ROS generation. Patient cells showed approximately 1.5 times the level of ROS after DNA damage in comparison to control cells whilst in null mutant mouse cells the levels were more than two fold increased. We have previously described individual variations in the level of p70-nibrin expression Here we describe increased ROS levels after DNA damage in NBS patient cells. The truncated p70-nibrin is clearly unable to fully prevent the hyperactivation of PARP, NADIn conclusion we present evidence for a further detrimental consequence of NBN mutation. In addition to a DSB repair deficiency and failure in cell cycle checkpoints, lack of fully functional nibrin results in increased ROS levels and oxidative stress. This unique combination would lead to an extremely high mutation rate in cells with an underlying apoptosis deficiency. Oncogene activation and tumour initiation are the consequence.Nbnlox-6/ins-6 mice 2. Environmental oxygen was reduced to 10%. Cells were split 1∶10 at least twice a week.Spontaneous transformed murine fibroblasts were grown from ear explants of NBN657del5/657del5 and a control cell line, LN9, transformed with simian virus 40 (SV40) were cultured using the same conditions described above.The immortalized human NBS cell lines GM7166VA7 and NBS-1LBI homozygous for The cre recombinase fusion protein, HTNC DNA damage was induced by incubating cells for two hours in the radiomimetic drug bleomycin at 10 µg/ml. Cells were then washed with medium and returned to culture for the specified times.Cells were scraped into ice-cold PBS, pelleted and snap frozen in liquid nitrogen. Cell pellets were stored at −80°C. For analysis, pellets were solubilised in LDS-sample buffer (Invitrogen) and sonified for 60 seconds using a model 450 sonifier . Proteins were separated on Tris-Acetate gels (3–8%) and transferred to PVDF membranes .To detect poly(ADP-ribosyl)ated proteins, two different polyclonal antibodies (Abcam) each producing the characteristic smear of PAR modified proteins were used. As a loading control β-actin was detected using a murine polyclonal antibody. Blots were repeated three times using independent lysates. For densitometry, films were scanned using the ScanMaker scanner (Mikrotek) and lanes quantified using ImageQuant software (Molecular Dynamics).The PARP inhibitor KU-0058948 was kindly provided by KuDOS Pharmaceuticals Ltd. (AstraZeneca PLC). The compound was dissolved in 100% dimethylsulfoxid (DMSO) and stored at −20°C. Cells were treated with 1 µM inhibitor in medium containing 0.5% DMSO for 10 hours before induction of DNA damage and then for a further 12 hours. Control cells were incubated in parallel in medium containing 0.5% DMSO.In some experiments, ROS were scavenged by treating cells with the antioxidant vitamin E derivative, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid . Cells were incubated in 500 µM TROLOX for 12 hours after the bleomycin damaging treatment.6 cells were stained in 500 µl PBS with 10 mM 5-(and-6)-chloromethyl-29,79-dichlorodihydrofluorescein diacetate  for 20 min at 37°C in the dark. Samples were subsequently washed using ice-cold PBS and centrifuged for 10 min at 1000 rpm (∼180×g) before being resuspended in FACS dissociation solution  and kept on ice until analysis. Flowcytometry was performed using the FACS-Calibur (Becton Dickinson Bioscience) counting a minimum of 104 cells per sample. The opensource flowcytometry software WinMDI V2.9 was used for data analysis. Gates were placed on dot blots of forward vs. side scatter to exclude apoptotic cells and debris from the fluorescence histograms shown in the figure. In all measurements at least 85% of cells were within this gate. All experiments were repeated independently at least three times. Relative ROS-levels are expressed as [gated mean of bleomycin treated cells]/[gated mean of untreated cells] and were evaluated for statistical significance using the non parametric two-tailed Mann-Whitney U test.The amount of intracellular ROS was monitored before and after the induction of DNA damage in fibroblasts at 50% confluence. Cells were washed and harvested into PBS and 10NBS patient fibroblasts and control fibroblasts were treated in triplicate with bleomycin as indicated above. At the timepoints indicated in + determination by using an enzymatic cycling assay Cells were centrifuged at 1500×g for 10 min and the supernatant was frozen at −20°C before NAD


Rousettus leschenaultii and Hipposideros armiger) have functional L-gulonolactone oxidase (GULO), an enzyme that catalyzes the last step of Vc biosynthesis de novo. Given the uncertainties surrounding when and how bats lost GULO function, exploration of gene evolutionary patterns is needed. We therefore sequenced GULO genes from 16 bat species in 5 families, aiming to establish their evolutionary histories. In five cases we identified pseudogenes for the first time, including two cases in the genus Pteropus (P. pumilus and P. conspicillatus) and three in family Hipposideridae . Evolutionary analysis shows that the Pteropus clade has the highest ω ratio and has been subjected to relaxed selection for less than 3 million years. Purifying selection acting on the pseudogenized GULO genes of roundleaf bats (family Hipposideridae) suggests they have lost the ability to synthesize Vc recently. Limited mutations in the reconstructed GULO sequence of the ancestor of all bats contrasts with the many mutations in the ancestral sequence of recently emerged Pteropus bats. We identified at least five mutational steps that were then related to clade origination times. Together, our results suggest that bats lost the ability to biosynthesize vitamin C recently by exhibiting stepwise mutation patterns during GULO evolution that can ultimately lead to pseudogenization.The traditional assumption that bats cannot synthesize vitamin C (Vc) has been challenged recently. We have previously shown that two Old World bat species ( Moreover, there is an apparent transition of the organs used for the biosynthesis of Vc during evolution, from the kidney of reptiles to the liver of mammals Vitamin C (Vc), or L-ascorbic acid is a water-soluble vitamin that is an essential nutrient impotant in animal metabolism. Vc is involved in tissue growth and repair, and also functions as an antioxidant to block damage caused by free radicals. It is also a cofactor in enzymatic reactions that are catalyzed by CuThe ability to synthesize Vc has been lost independently several times in vertebrates e.g. in some fishes GULO genes in two species (Rousettus leschenaultii and Hipposideros armiger) are still in their intact forms and can produce functional proteins Pteropus and Rousettus belong to the same chiropteran family (Pteropodidae), and although the former has lost the ability to synthesize Vc, the latter retains it Our recent research has challenged the traditional opinion that bats cannot synthesize Vc GULO evolution in bats? Given the uncertainty of when and how bats lost GULO gene function, it is important to sequence GULO genes of more bat to explore patterns of GULO evolution. In this study, we therefore sequenced the GULO genes of 16 bat species and aimed to reconstruct bat GULO evolutionary history. Using ancestral reconstructions, we infer stepwise mutation patterns showing how bats may have lost GULO function.Our previous study on Vc synthesis in bats raises the question-what is the evolutionary pattern that shapes bat Bat wing membrane biopsy specimens were taken from a collection at the Institute of Molecular Ecology and Evolution, East China Normal University. All experiments were conducted under permission to use these specimens granted by the by Animal Care Ethics Committee, East China Normal University . Our screening included 16 bat species from 5 families: Pteropodidae, Hipposideridae, Rhinolophidae, Megadermatidae, and Rhinopomatidae, and sampling locations include China, Cameroon, Australia, India, and Vietnam (supplementary P. vampyrus (GeneScaffold_1205), dog , cow  and pig  that contained GULO genes from the Ensembl database (http://www.ensembl.org/). Polymerase chain reactions (PCR) were performed using Ex TaqTM polymerase (TaKaRa) with the reaction conditions as follows: 94°C for 5 min followed by 30 cycles consisting of 94°C for 30 sec, 57–62°C for 15–30 sec, 72°C for 1 min, and a final extension of 72°C for 10 min. All PCR products were ligated into pGEM-T Easy vectors (Promega) and transformed. The universal T7 (5′-TAA TAC GAC TCA CTA TAG GG-3′) and SP6 (5′-ATT TAG GTG ACA CTA TAG-3′) sequencing primers were used to sequence all positive clones on an ABI 3730 DNA sequencer (Applied Biosystems). All new sequences are submitted to GenBank (supplementary Genomic DNA was extracted by using DNeasy Blood and Tissue Kit (Qiagen) according to the manufacturer's protocols. We aimed to amplify exon-3 to exon-8, the six exons encoding the functional region of the GULO enzyme , of 16 bat species : horse , dog , pig , cow , panda , rabbit , rat , mouse , opossum  and platypus . We then aligned all these sequences with the bat sequences using ClustalW GULO genes are highly conserved in most mammals, evolutionary history was inferred using the Neighbor-joining (NJ) method, which was perfectly used in many studies To reconstruct the bat GULO genes mapped onto the published species tree Pteropus bats) and other branches. To test for the significance of each model used, likelihood ratio tests (LRTs) A widespread method used to inter selection pressures acting on specific genes is to estimate non-synonymous (dN) and synonymous (dS) nucleotide substitution rates and their ratio dN/dS (ω) GULO gene evolution, ancestral reconstruction was employed To trace the amino acid changes during GULO form: Rousettus leschenaultii, R. aegyptiacus, Pteropus rodricensis, P. vampyrus, Eonycteris spelaea (Pteropodidae), Rhinolophus ferrumequinum, Hipposideros armiger, H. ater, H. pratti (Hipposideridae), Megaderma lyra (Megadermatidae), and Rhinopoma hardwickii (Rhinopomatidae) , Coelops frithii, Hipposideros bicolor, and H. speoris (Hipposideridae)  an intact matidae) ; 2) pseuderidae) .GULO  in all main clades of bats, giving a 12–54x higher ω ratio than in the ancestor of Laurasiatheria species (Pteropus clade has the highest ω ratio of 0.648. A pair of two-ratio models (with the Pteropus clade was set as the foreground) was then constructed by having the ω ratio fixed to 1  in one model and a ω ratio not fixing the other. LRT showed no significance when comparing the two models , which suggests that the evolution of Pteropus clade GULO genes is close to being neutral.The phylogenetic gene tree for GULO  closely  species . The PtePteropus suggest their GULO genes have already been subjected to relaxed selection (over a period of less than 3 million years ago (mya), the origination time of this clade) GULO genes of Coelops frithii, Hipposideros armiger, and H. pratti may have been at the early stages of pseudogenization because these genes have relatively low ω ratios (suggesting they are still under purifying selection). Several two-ratio models were also established with different bat clades as the foreground. LRTs (two-ratio model versus one-ratio model) showed significance only in the Pteropus clade (data not shown), supporting the above conclusion that this clade has subjected to relaxed selection.The higher ω ratios of Pteropus bats (around 3 mya) GULO evolution.Interestingly, ancestral sequence reconstruction exhibits a stepwise mutation pattern  that staAs GULO is present in all major vertebrate lineages except some bats (most of these being New World species) GULO genes from 16 species, we carried out detailed evolutionary analyses. Our results show a range of forms of the GULO gene in bats. Combined with our earlier functional studies Pteropus and hipposiderid species, 2) intact genes that functional studies showed loss of function in Vc synthesis (e.g. Pteropus vampyrus), 3) intact genes that maintain some ability to synthesize Vc (Rousettus leschenaultii and Hipposdieros armiger). We found that strong purifying selection has shaped non-Pteropus bat pseudogenes, suggesting these bats are in early stages of loss in their ability to synthesize Vc. In the family Hipposideridae some species possess pseudogenes that show only small changes from the intact and functional genes of their close relatives. Together with the evidence for puryifying selection our results suggest that Vc function has been lost recently in hipposiderid species showing pseudogenized GULO. Relaxed selection acting on Pteropus bat GULO suggests that bats in this genus lost the ability to synthesize Vc within the past 3 mya GULO evolved recently.Having successfully cloned bat GULO evolution. By mapping each mutation step with theorigination times of each clade Click here for additional data file.Table S2GULO genes.Primers used for amplification of exon-3–exon-8 of bat (DOC)Click here for additional data file.


Passer italiae and its parent species, the house sparrow P. domesticus and Spanish sparrow P. hispaniolensis. Reproductive barriers can be difficult to study in hybrid species due to lack of geographical contact between taxa. However, the Italian sparrow lives parapatrically with the house sparrow and both sympatrically and parapatrically with the Spanish sparrow. Through whole-transcriptome sequencing of six individuals of each of the two parent species we identified a set of putatively parent species-diagnostic single nucleotide polymorphism (SNP) markers. After filtering for coverage, genotyping success (>97%) and multiple SNPs per gene, we retained 86 species-informative, genic, nuclear and mitochondrial SNP markers from 84 genes for analysis of 612 male individuals. We show that a disproportionately large number of sex-linked genes, as well as the mitochondria and nuclear genes with mitochondrial function, exhibit sharp clines at the boundaries between the hybrid and the parent species, suggesting a role for mito-nuclear and sex-linked incompatibilities in forming reproductive barriers. We suggest that genomic conflict via interactions between mitochondria and sex-linked genes with mitochondrial function (“mother's curse”) at one boundary and centromeric drive at the other may best explain our findings. Hybrid speciation in the Italian sparrow may therefore be influenced by mechanisms similar to those involved in non-hybrid speciation, but with the formation of two geographically separated species boundaries instead of one. Spanish sparrow alleles at some loci have spread north to form reproductive barriers with house sparrows, while house sparrow alleles at different loci, including some on the same chromosome, have spread in the opposite direction to form barriers against Spanish sparrows.Studies of reproductive isolation between homoploid hybrid species and their parent species have rarely been carried out. Here we investigate reproductive barriers between a recently recognized hybrid bird species, the Italian sparrow  Hybridization between two species has the potential to create a third, hybrid species. However this process, known as hybrid speciation, is thought to be unlikely because it requires reproductive barriers against both parent species to develop despite the barriers between parents being weak enough to allow for the formation of viable, fertile hybrids. The Italian sparrow, which occupies the entire Italian peninsula and some Mediterranean islands, is the product of past hybridization between house and Spanish sparrows and therefore represents one of the few documented cases of vertebrate hybrid speciation in nature. We show that reproductive barriers between Italian sparrows and their parent species exist and that genes on the sex (Z) chromosome and mitochondria are heavily involved. We suggest that speciation in this system may have been driven by dissociation of the sex (Z) chromosome into blocks of different parent species-specific genes, which have shifted alongside mitochondrial genes to form reproductive barriers where the hybrid now meets each of its parent species. Hybridization between divergent populations has diverse impacts on evolution In non-hybrid speciation involving species with chromosomal sex-determination, sex-linked genes have repeatedly been found to strongly influence reproductive isolation (RI) Passer sparrows, however, the distribution of the hybrid Italian sparrow Passer italiaeP. hispaniolensis and the house sparrow P. domesticus  and nearly fixed for the house sparrow allele in the other (PLAA), indicating strong mosaicism on the Z chromosome not paralleled by autosomal markers Previous studies have indicated that Italian sparrows are almost fixed for house sparrow mitochondrial DNA CHD1Z has been shown to be under divergent selection and associated with RI between several non-hybrid bird species pairs [15 and references therein]. For example, CHD1Z shows evidence of divergent selection in sympatric but not allopatric population comparisons of pied and collared flycatchers  CHD1Z prove to be an informative marker in Italian sparrows, it may be particularly likely to represent a functional variant directly involved in RI (or at least a linked marker within the same gene) rather than a linked marker within a neutral gene.The evidence of Z chromosome mosaicism from existing studies Evidence is accumulating that mito-nuclear interactions cause postzygotic isolation and are influential in speciation Here, we analyze species-informative single nucleotide polymorphism (SNP) markers, located within functional transcribed genes, across the breeding range of the Italian sparrow, as well as the contact zones with its parent species. We use a cline analysis approach We identified putatively parent species-diagnostic SNP markers through transcriptome sequencing of six individuals of each of the two parent species. After filtering for coverage, sufficient flanking sequence, genotyping success (>97%) and multiple SNPs per gene, we retained 86 species-informative, genic, nuclear and mitochondrial SNP markers from 84 genes for analysis. Using this marker set, we found the Italian sparrow to exhibit high levels of genomic admixture over the entire study area . We alsoAs gene flow was observed between the Italian sparrow and both parent species, we implemented a cline analysis framework to look for genes exhibiting steep clines, and therefore decreased gene flow at the species boundaries. The SNPs are within functional coding genes and hence any such clines may indicate a direct influence of the gene on RI. They may also, however, be neutral but closely linked to loci under selection. These genes nevertheless represent the most likely candidates to be involved in RI at this inferential stage of analysis.Cline analysis is a method used to measure the steepness, shape and location of changes in allele frequency or locus-specific ancestry, as well as in quantitative traits BGC) BGC analysis revealed 31–35 genes to have excess house sparrow ancestry while 18–22 genes exhibited excess Spanish sparrow ancestry .Combined boundary . These rHSDL2 and MCCC2) P = 0.02; null probability data from The Gene Ontology Project's Gene Association file for Gallus gallus, GOC validation date: 4 December 2012, gaf-version 2.0 and The Gene Ontology Project's Gene Ontology file, date: 4 December 2012, cvs revision version 4708) among the nuclear genes shifting at this boundary. The Z-linked gene HSDL2 exhibited a near-identical pattern of fixation for house sparrow alleles in the Italian sparrow as the mitochondrial marker ND2  with steep clines on range boundaries are Z-linked. This remains a significant overrepresentation of Z-linked markers .The three markers h genome . A conseWhile the steepest genomic clines were found for markers with major geographic clines at the Italian sparrow range boundaries , clines FST between these Spanish sparrows and sympatric Italian sparrows versus Spanish sparrows and nearby allopatric Italian sparrows  and RPS4 (chromosome 4A) at the Italian-house sparrow boundary in the Alps, and HSDL2 (Z chromosome) and ND2 (mitochondria) at the Italian-Spanish sparrow boundary between the Italian mainland and Sardinia. Furthermore, two of the three nuclear markers with steep clines coincident with that of the mitochondria at the Italian-Spanish boundary (HSDL2 and MCCC2) are classified as nuclear-encoded mitochondrial proteins In this study, we utilized the Italian sparrow's gene exchange and geographical overlap with both parent species, house and Spanish sparrows, to investigate hybrid-parent reproductive barriers using a cline analysis framework. Results of geographic and genomic cline analyses reveal that several markers with an excess of Spanish sparrow alleles within Italian sparrows are associated with RI at the house-Italian range boundary, and that several markers with house sparrow excess are associated with RI at the Spanish-Italian range boundary. A disproportionately high number of the markers showing steep clines at one or the other hybrid-parent species boundary are Z-linked, including when potential physical linkage is accounted for. Of these SNPs, which are all within functioning transcribed genes, the strongest candidates to be within genes directly involved in RI are those that exhibit the most extreme cline parameters. This includes Excessive sex linkage of RI supports the hypothesis that HHS in Italian sparrows is facilitated by divergent selection between the parent species. HHS differs from non-hybrid speciation in that an isolating mechanism is required against each parent. This may be aided by transgressive segregation leading to extreme hybrid phenotypes ND2, which is in linkage disequilibrium with all other mitochondrial genes), rather than being directly involved in RI. In the case of RPS4, there is no a priori expectation that it should be involved in RI. However this gene, the sole autosomal representative associated with hybrid-parent isolation, is found on chromosome 4A in zebra finch RPS4 is in fact X-linked in Eutherian mammals SRY, and about one third of this chromosome has even translocated to the sex chromosomes in the whole avian superorder Sylvioidea We recognize that the genes with steep clines may represent neutral markers in linkage disequilibrium with the RI genes under selection  is a chromatin-remodeling factor potentially affecting the expression of many genes CHD1. Centromeric drive has been proposed as an important mechanism in the non-hybrid speciation of pied and collared flycatchers CHD1Z also shows evidence of involvement in RI CHD1Z represents a candidate RI gene, maintaining parapatric differentiation between hybrid Italian sparrows and the house sparrow.We hypothesized that HSDL2, shows a cline almost as steep as mitochondrial ND2. While this may be a linked neutral marker, the fact that HSDL2's protein product is located within mitochondria HSDL2 is thought to be involved in fatty acid metabolism, although its exact functions are unknown We also hypothesized a role for mito-nuclear interactions, and in particular the tracking of spreading mitochondrial variants by nuclear male ‘restorer’ genes, compensating for ‘mother's curse’. The overrepresentation of nuclear genes with a mitochondrial function on the Italian-Spanish sparrow boundary provides some support for this hypothesis. However, only one of these genes, HSDL2 in fact has been shown to have higher expression levels in male than female chickens Our results thus appear consistent with the influence of mito-sex chromosome conflict acting as a reproductive barrier at the Spanish-Italian sparrow boundary. Furthermore, due to the lack of global dosage compensation in birds, Z-linked genes typically have higher expression and – combined with the fact that Z chromosomes spend two thirds of their time in males - stronger fitness effects in males than females. Consequently, genes with male-specific fitness effects are overrepresented on the Z chromosome There is no evidence for hybridization between Spanish and Italian sparrows in the sympatric zone of southeast Italy, supporting previous results In addition to the steep clines at the species range boundaries, some genes exhibited clines steeper than the neutral expectation within the Italian sparrow's range. One possible interpretation of this result is that moving clines of incompatibility genes may have become trapped by environmental transitions or population density troughs before reaching the current hybrid-parent boundaries. In this way, intraspecific incompatibilities within a hybrid species may increase future diversification relative to non-hybrid species, in particular through the effect of divergence hitchhiking in promoting the build-up of novel isolating mechanisms surrounding pre-existing incompatibilities Passer sparrows, and that beneficial alleles often do not sweep across the whole range of a species for a variety of reasons including geographical barriers.The Italian sparrow genome represents a mosaic, particularly on the Z chromosome, in which some Spanish sparrow alleles have spread to form reproductive barriers against house sparrows, while house sparrow alleles at different loci on the same chromosome have spread in the opposite direction to form barriers against Spanish sparrows. We envisage that HHS in the Italian sparrow may match the ‘mosaic genome hybrid speciation’ model Using a cline analysis framework we have identified sets of candidate RI genes and genomic regions between the hybrid Italian sparrow and its parent species. These results support our predictions that mito-nuclear interactions and loci on the Z chromosome strongly influence RI. In this regard, we suggest that HHS in the Italian sparrow resembles non-hybrid speciation, and we would therefore predict that the same loci would be involved in RI between the parent taxa; house and Spanish sparrows. An important next step is therefore to replicate this study in a region of parental sympatry and hybridization n = 612) were taken from 64 locations between 2007 and 2011 (n = 142) from Badajoz, Spain, Sardinia, Italy and a Spanish/Italian sympatric zone in southeast Italy; allopatric house sparrows (n = 85) from Hradec Králové, Czech Republic and Oslo, Norway; Italian sparrows, Italian-house hybrids and parapatric house sparrows from the Italian peninsula and the Alps (n = 385). The sparrows were caught using mist nets. About 25 µl of blood was extracted by venipuncture of a brachial vein and stored in 1 ml of Queens lysis buffer. Appropriate catching and sampling permits were obtained for all sampling locations from the relevant authorities. DNA was isolated using Qiagen DNeasy 96 Blood and Tissue Kits  according to the manufacturer's instructions. For transcriptome sequencing, three house  and three Spanish  sparrows of each sex were sampled in October 2010. Liver, heart and brain tissue samples were taken and stored on RNAlater (100 mg tissue in 1400 µl buffer) according to the manufacturer's protocol.Only males, which are diploid for the Z chromosome, were analyzed to avoid issues related to haplodiploidy of the Z chromosome. Blood samples from the three taxa  including an on-column DNase treatment. From the total RNA samples, poly(A)+ RNA was prepared and fragmented with ultrasound (1 pulse of 30 sec at 4°C). First-strand cDNA was synthesized from the fragmented RNA using a N6 randomized primer and M-MLV RNaseH-reverse transcriptase. 454 adapters A and B were ligated to the 5′ and 3′ ends of the cDNA. The cDNA was amplified with PCR using a proof reading enzyme. Normalization was carried out by one cycle of denaturation and reassociation of the cDNA, resulting in N1-cDNA. Reassociated ds-cDNA was separated from the remaining ss-cDNA  by passing the mixture over a hydroxylapatite column. After hydroxylapatite chromatography, the ss-cDNA was PCR amplified. For GS FLX Titanium sequencing, the cDNA in the size range of 450–700 bp was eluted from preparative agarose gels. The resulting cDNA was double stranded, and had a size of about 450–700 bp. The six samples from each species were pooled in equal amounts and pyrosequenced on a Roche GS FLX Titanium sequencer at the Norwegian Sequencing Center using the manufacturer's protocol.CHD1Z and ND2 genes, which were genotyped using existing primers CHD1Z SNPs in this initial set are within an intron Passer sparrows have so far not been mapped. Genomic locations of the various markers were therefore inferred based on the Zebra finch Taeniopygia guttata genome The house sparrow reads were aligned and mapped against the zebra finch genome, and reads of both species were then mapped against the resulting contigs, providing a list of potential species-informative SNPs. Species-diagnostic SNPs from the twelve samples were chosen and subsequently filtered for those without sufficient flanking sequence for PCR-primer design. Genes were annotated by blasting against the zebra finch and chicken genomes. Two exceptions were SNPs within APC and A2ML1, in which two markers were included due to marked differences in parental allele frequencies).Multiplex sets of PCR primers were designed and all individuals genotyped at each SNP locus using the Sequenom MassARRAY system at CIGENE, Norwegian University of Life Sciences, Ås, Norway. A total of 124 putatively diagnostic SNP markers from 107 different genes were genotyped successfully. Statistical analyses were carried out on a subset of 86 species informative SNPs after further filtering . This inRecent migration between Spanish sparrows on Sardinia and Italian sparrows was identified using the USEPOPINFO model in STRUCTURE BGCND2 locus were coded as diploid homozygote as BGC failed to run with a haploid marker included. Three independent runs with 100,000 iterations each were run with the first 25,000 iterations discarded as burnin, MCMC samples thinned by recording every fifth value, while the rest of the BGC settings were as default. SNPs were identified as significantly deviating from null expectations when the 95% credibility intervals of the cline parameters α and β did not cross zero. Once candidate SNPs were chosen using the genomic clines approach, geographic locations of sharp changes in allele frequency for each SNP were identified in GENELAND BGC indicated a significantly shifted cline center (significant α)  . We furtBGC runs .P value for genotypic disequilibrium between every SNP pair and by employing a Fisher test to combine probabilities across all populations in the Italian peninsula, the Alps and Sardinia  in GENEPOP FST estimates were calculated between i) the Italian sparrows from each population and ii) the Spanish sparrows in Lesina and the Italian sparrows in each of the three populations. A shift in FST towards or away from Spanish sparrows in Italian sparrows from Lesina, relative to the two allopatric populations, would indicate introgression or displacement respectively.To assess if there is introgression between sympatric Italian and Spanish sparrows, FST outlier analysis of molecular markers. We used both softwares to determine which of CHD1Z and CETN3, two highly linked markers, showed stronger evidence for selection in the Alps house-Italian sparrow hybrid zone. Data from all 85 nuclear SNPs  were used and the three transects were pooled, excluding sites with just a single individual sampled. For LOSITAN the options ‘neutral mean FST’ and ‘force mean FST’ were chosen, along with the infinite alleles mutation model and 50 k simulations. For BAYESCAN, default settings were used.BAYESCAN Handling of birds were conducted according to guidelines approved by the relevant authorities in the respective countries , Institute for Environmental Protection and Research – ISPRA , Consejería de Industria, Energía y Medio Ambiente (Spain), Norwegian Food Safety Authority (Norway), Ministrstvo za okolje in proctor, Agencija Republike Slovenije za okolje (Slovenia) and Bundesamt für Umwelt BAFU, Abteilung Artenmanagement (Switzerland)).Figure S1Individuals with intermediate crown color have already been reported in the Alps contact zone (DOC)Click here for additional data file.Figure S2BGC runs. Significant excess house sparrow ancestry is indicated by H's, significant excess Spanish sparrow ancestry is indicated by S's, significantly steep clines are indicated by +'s and significantly narrow clines are indicated by −'s. The number of H's, S's, +'s and −'s indicate for how many runs the parameter estimates for a given marker differed from neutral expectations. When none of the runs differed from neutral expectations, this is indicated by ns. Red panels indicate markers that exhibit significant excess house sparrow ancestry and have clines steeper than neutral expectations, where the difference in allele frequency between the parent species is greater than 0.5 and for which Geneland revealed a shift at the Italian-Spanish sparrow boundary. These are hence candidate RI genes. Blue panels indicate markers that exhibit significant excess Spanish sparrow ancestry, clines steeper than neutral expectations, a difference in allele frequency between the parent species greater than 0.5 and with a shift at the Italian-house sparrow boundary. These are hence candidate RI genes. Green panels indicate markers that have allele frequency differences between the parent species greater than 0.5 and where the clines are steeper than neutral expectations but do not exhibit a major allele frequency cline on either hybrid-parent species boundary. These markers are candidates for being incompatibilities within the Italian sparrow. Only markers with significant ‘+’ on more than one BGC run are highlighted.Bayesian genomic clines for all 86 SNP markers. The legend in each panel indicates genomic location (chromosome number), SNP name and significance of cline center (α) and cline rate (β) parameters for three independent (DOC)Click here for additional data file.Figure S3CHD1Z, CETN3 and RPS4; see main text). Axes represent longitude (x axis) and latitude (y axis) in decimal degrees. Colors refer to posterior likelihood of belonging to the group corresponding to the house sparrow  relative to the Italian sparrow . Black dots denote sampling locations. CHD1Z and RPS4 results for transect 2 are also represented in GENELAND geographic clines for Alps transects 1–3 , for the(DOC)Click here for additional data file.Figure S4P values across all Italian peninsula and Sardinian populations. Dark blue P = 0; medium blue P<0.05; pale blue P<0.1.Heat map of genotypic disequilibrium between Z-linked markers with significantly steep genomic clines. Marker positions in kbp are based on the zebra finch genome. Internal cell values indicate distances between genes in kbp. Cell colors represent genotypic disequilibrium, combining (DOC)Click here for additional data file.Figure S5FST-outlier analyses. (A) LOSITAN graphic of test for evidence of divergent selection in the Alps, using the 85 nuclear SNPs and combining data from all three transects . SNPs in the red area are candidates for positive divergent selection. At a false discovery rate of 0.05 only CHD1Z and RPS4 were significant outliers for divergent selection. (B) BAYESCAN graphic of test for evidence of divergent selection in the Alps, using the 85 nuclear SNPs and combining data from all three transects . The estimated alpha coefficient indicates the strength and direction of selection. A positive value of alpha suggests diversifying selection, whereas negative values suggest balancing or purifying selection. CHD1Z and RPS4 (highlighted in red and named) were the only markers that were significant at a false discovery rate of 0.1.(DOC)Click here for additional data file.Table S1Sample population details.(DOC)Click here for additional data file.Table S2FST between Italian sparrow and its parent species where a steep cline exists on the hybrid-parent boundary, plus estimates of cline shift (α) and steepness (β). Values in shaded boxes are not significant .(DOC)Click here for additional data file.Table S3Details of SNPs used in analyses.(DOC)Click here for additional data file.


The liver is central to most economically important metabolic processes in cattle. However, the changes in expression of genes that drive these processes remain incompletely characterised. RNA-seq is the new gold standard for whole transcriptome analysis but so far there are no reports of its application to analysis of differential gene expression in cattle liver. We used RNA-seq to study differences in expression profiles of hepatic genes and their associated pathways in individual cattle in either mild negative energy balance (MNEB) or severe negative energy balance (SNEB). NEB is an imbalance between energy intake and energy requirements for lactation and body maintenance. This aberrant metabolic state affects high-yielding dairy cows after calving and is of considerable economic importance because of its negative impact on fertility and health in dairy herds. Analysis of changes in hepatic gene expression in SNEB animals will increase our understanding of NEB and contribute to the development of strategies to circumvent it.P <0.05) with 9 KEGG pathways. Seven of these pathways were related to fatty acid metabolism and unexpectedly included ‘Steroid hormone biosynthesis’, a process which mainly occurs in the reproductive organs rather than the liver.RNA-seq analysis was carried out on total RNA from liver from early post partum Holstein Friesian cows in MNEB (n = 5) and SNEB (n = 6). 12,833 genes were deemed to be expressed , 413 of which were shown to be statistically significantly differentially expressed (SDE) at a false discovery rate (FDR) of 0.1% and 200 of which were SDE (FDR of 0.1%) with a ≥2-fold change between MNEB and SNEB animals. GOseq/KEGG pathway analysis showed that SDE genes with ≥2- fold change were associated (RNA-seq analysis showed that the major changes at the level of transcription in the liver of SNEB cows were related to fat metabolism. 'Steroid hormone biosynthesis', a process that normally occurs in reproductive tissue, was significantly associated with changes in gene expression in the liver of SNEB cows. Changes in gene expression were found in this pathway that have not been previously been identified in SNEB cows. Selective breeding for high milk yield in dairy cows has led to breeds in which the nutritional demands of the very high lactation rates following calving are in excess of that which the animal can metabolise from ingested feed . This ab2, or converted to ketone bodies such as beta-hydroxybutyrate (BHB), or esterified into triacylglycerides (TAG) either for delivery into blood as very low density lipoproteins proteins (VLDL) or storage as cytosolic lipid droplets  NEB NEB2, ordroplets .As with the majority of metabolic states, changes in gene expression in the liver are central to NEB. There have been several studies describing such changes at the level of transcription in the liver of NEB cows which have employed qPCR  and boviRNA-seq is a relatively new technique that can be used to analyse changes in gene expression across the entire transcriptome ,14, and Unlike microarrays, the bioinformatic tools for the analysis RNA-seq data are still at the early stages of development ,20. PartHere we describe the use of DEseq  and GOsehttp://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE37544).Twelve polyA RNA-seq single read libraries that had been prepared from total RNA extracted from liver from 6 MNEB animals (libraries 1–6) and 6 SNEB animals (libraries 7–12) were run as 36 bp single reads on 21 lanes randomly distributed across 3 flowcells  and mouse  liver tissue by polyA RNA-seq . RNA-seq2 = 0.9783 NEB vs. SNEB r2 = 0.9905 Lane A vs. Lane B) was predominately due to effect of treatment and not lane effect.To further determine whether the differential expression observed between MNEB and SNEB was due to a real difference between the groups and not simply random variation caused by running samples on different lanes (i.e. lane effect), correlation analysis was performed between FPKM values from the two treatment groups and also the same 9 libraries run as technical replicates on two different lanes Figure . This shDEseq is an R-based software package that was developed specifically for the identification of SDE genes from raw counts of sequence reads generated by RNA-seq analysis that uniquely align to genes. RNA-seq library 1  was not included in these analyses as this animal subsequently became ill and a possible underlying infection may have skewed the gene expression data generated. DEseq analysis showed that 413 genes were SDE (FDR of 0.1%), 201 of which had a fold change of ≥2 (either up or down) between MNEB and SNEB animals  derived from bacterial fermentation in the rumen. VFAs are absorbed across the rumen wall and into the hepatic portal vein which delivers them to the liver. The increase in hepatic gluconeogeneis at the onset of lactation leads to production of very high quantities of glucose. The glucose entry into the blood stream in top performing cows producing 90 kg milk per day has been estimated at 7.4 kg per day of which 4.4 kg will end up as lactose in milk .P <0.05) changes in energy balance (−3.6-fold), blood glucose (−1.5-fold), blood NEFA (+ 4.7-fold), blood BHB (+7.4), blood IGF1(−4.8-fold), liver TAG (+3.9-fold), liver glycogen (−10.4-fold). Blood insulin, oestradiol, urea and liver weight were not different between SNEB and MNEB animals [In our model we were comparing two groups of lactating animals in the early post-calving period . One of these groups was in MNEB and the other was in a state of SNEB which was artificially induced by restricting feed and trebling the milking frequency. From this we hoped to identify novel genes and pathways which are important in SNEB in high-yielding dairy cows. Hormones, metabolites and aspects of liver composition measured at the time of tissue collection showed that, as predicted, relative to MNEB cows, SNEB cows had undergone significant , one of the major rate limiting enzymes in gluconeogenesis [The 1.5-fold drop in blood glucose in SNEB cows would suggest decreased gluconeogenesis in the liver of these animals. However, only a minor decrease ≤2-fold) of pyruvate carboxylase (PYK (KEGG pathway map) = ogenesis  was iden-fold of GHR −1.6-fold, IGFALS −5-fold, IGF1 -5-fold)  as SDE (≤2-fold) in SNEB cow liver.Although pathways related to the GH/IGF1 axis were not identified as overrepresented among the SDE genes identified by RNA-seq, several genes within in this pathway were detected as SDE  which is thought to control fatty acid oxidation was SDE (upregulated 3-fold) in RNA-seq but not picked up in the pathway analysis. KEGG PPAR signalling pathway is also involved in ketogenesis but, although the main gene involved in this (HMGCS2) was detected, it was not identified as SDE. This may be because at the time of slaughter there was little difference in the rate of oxidation and ketogenesis between the two groups, or that there were differences but not at the level of transcription. OLR1 (oxidized low density lipoprotein (lectin-like) receptor 1) is included in the KEGG PPAR signalling pathway and was detected as upregulated . The protein encoded by OLR1 binds, internalizes and degrades oxidized low-density lipoprotein (oxLDL) and is thought to have a role in lowering systemic oxidative stress by removal of oxLDL from blood.Due to the higher amount of NEFAs in the blood of SNEB animals, we were expecting changes in expression of genes to reflect increased oxidation of fatty acids in SNEB animals. However, although eight of the nine fatty acid oxidation genes in the KEGG PPAR signalling pathway were detected, none were SDE  which was down-regulated in SNEB animals  [FADS2, but not FADS1, was identified as SDE. FADS2 catalyses the initial desaturation step of α-linolenic acid (C18:3) and linoleic acid (C18:2) in the enzyme cascade that ultimately leads, via FADS1, to synthesis of the long chain polyunsaturated fatty acids (LC-PUFAs) arachidonic acid (C20:4) and eicosapentaenoic acid (C20:5). Synthesis of LC-PUFAs arachidonic acid and eicosapentaenoic acid mainly occurs in the liver and adipose [FADS1 and FADS2 were associated with reduced serum arachidonic levels in humans [FADS2 gene expression in mice leads to sterility in both male and female mice without affecting their viability [The most SDE gene we identified by RNA-seq was s Figure . FADS1 w partum) . However adipose  and suppn humans . Also, diability .CYP11A1 was up-regulated while UGT2a1, SULTE1 and CYP7A1 were down-regulated. CYP11A1 was also identified as SDE in SNEB cow liver in our microarray study [CYP7A1, UGT2A1 and SULT1E1 were not identified as SDE in either of those studies. CYP7A1 catalyses the rate limiting step of conversion of cholesterol to bile acids and was also included in the KEGG pathway ‘Bile secretion’ which was overrepresented in the ≥2-fold SDE genes identified by RNA-seq . DHEA is the precursor for a number of steroid hormones including oestrogen. Although other tissues, such as brain and foetal rat liver, also express the enzymes for steroid biosynthesis [CYP17A1 was also upregulated and the authors proposed that DHEA (or one of its metabolites) in liver could be involved in a novel hepatic signalling pathway or may serve to protect hepatocytes from the increased oxidative state brought on by fasting. DHEA has been shown to protect endothelial cells from apoptosis. They also suggested that CYP11A1 expression in feed restricted hepatocytes may be involved in regulation of cholesterol homeostasis as CYP11A1 catalyses modifications on cholesterol to produce oxysterols which are known ligands for liver X receptors (LXRs). LXRs regulate cholesterol homeostasis and lipid metabolism. It would therefore be interesting to see if DHEA levels in SNEB cow liver are elevated. In both our microarray and RNA-seq study, SDE genes included in the IPA canonical signalling pathway ‘LXR/RXR activation’ were changed ; ARG, CCL2, IL1RN, RXRG (upregulated)). In our RNA-seq data, many of the genes, including CYP17A1, in the KEGG pathway ‘Steroid hormone biosynthesis’ were not detected as expressed  has an exact copy (LOC784256) at another position in the Bos taurus genome), transcript abundance was too low for detection, or that they are not expressed in the liver thus preventing complete synthesis of steroid hormones in this organ.Although we detected upregulation of ranulosa . Howeverile cows . Up-reguynthesis  these enCYP11A1 was upregulated. In adult cows, this gene is normally only expressed in the adrenal glands and ovaries and not liver. CYP11A1 is a potentially novel gene that may play a key role in metabolic adaptations to NEB in high-yielding dairy cows.RNA-seq analysis showed that the major changes at the level of transcription in the liver of SNEB cows were related to fat metabolism. Unexpectedly, one of the most significantly changed biological pathways was ‘Steroid hormone biosynthesis’ where ad libitum grass silage and 8 kg/day concentrates and milked once daily; SNEB cows were fed 25 kg/day silage and 4 kg/day concentrate and milked three times daily. Measurements of BCS and EB were used to select cows that showed extremes in EB from each group . Cows were slaughtered on days 6–7 of the first follicular wave after calving , based on daily transrectal ultrasonography.The animal model employed in this study has been described previously , and allThe entire liver was removed within 15–30 min after slaughter and weighed. Samples weighing approximately 1 g were dissected, rinsed in RNase-free phosphate buffer, snap-frozen in liquid nitrogen, and stored at −80°C. For triacylglyceride (TAG) analysis, total lipids were extracted from 50 mg samples of liver as previously described .Stabilized (EDTA-treated) whole blood samples were collected on the day of slaughter by jugular venipuncture for haematological analysis. Blood samples were analyzed for glucose, NEFA, β-hydroxybutyrates (BHB), and urea using appropriate kits and an ABX Mira autoanalyser . All metabolite assay coefficients of variation were low and typically <5%.Total RNA was prepared from 100–200 mg of fragmented frozen liver tissue using the TRIzol reagent . Tissue samples were homogenized in 3 ml of TRIzol reagent and chloroform and subsequently precipitated using isopropanol . RNA samples were stored at −80°C. 20 μg of total RNA from each sample for genomic DNA contamination was treated with the RNase-free DNase set  and purified it using the RNeasy mini kit in accordance with guidelines supplied . RNA quality and quantity were assessed using automated capillary gel electrophoresis on a Bioanalyzer 2100 with RNA 6000 Nano Labchips according to manufacturer’s instructions . Samples of RNA had 28 S/18 S ratios ranging from 1.8 to 2.0 and RNA integrity number values of between 8.0 and 10.0., second strand synthesis was performed using Invitrogen DNA Polymerase 1 (Invitrogen). End repair and polyadenylation were performed using NEB Next Tailing Module (New England Biolabs). ® End Repair Module and NEB Next dA- Illumina single read adapters were ligated to blunt ended, polyadenylated fragments with a NEB Quick ligation kit (New England Biolabs). Adapter-ligated cDNA fragment libraries were run on an Illumina GAII using version 3 sequencing kits and version 3 single read cluster generation kits.polyA RNA was isolated from 5–10 ug total RNA with oligo(dT) beads using two rounds of oligo-dT purification. 5–10 ug RNA was fragmented with zinc fragmentase , first strand cDNA synthesis was performed using the Invitrogen random hexamer primers and SuperScript II (Invitrogen)The reads from each of the lanes were aligned separately to the BCM4 genome assembly , using thttp://sourceforge.net/projects/htseq/). The script HTseq-count provided with this package was used to count the number of reads that mapped to each annotated gene, allowing, in some cases, for reads to partially overlap with the exons and still be counted for that gene (i.e. -m union). As our sequencing analysis included technical replicates of individual samples, we summed the counts for all these lanes, resulting in a single count for each gene for each sample. The counts for all the samples were collated into one file and any gene with fewer than 5 reads in all samples was excluded from the subsequent statistical analysis of differential gene expression.The abundance of mRNAs for all annotated genes from the ENSEMBL v59 annotation of the bovine genome , was calWe identified SDE using DEseq (version 1.1.11) . DEseq uGOseq and KEGG pathway analysis tools were used to identify biological pathways that were significantly enriched in the data set of SDE genes Table . To faciThe authors declare that they have no competing interests.MSM generated the RNA-seq libraries, co-ordinated the sequencing runs, helped with retrieval and analysis of data, carried out detailed interpretation of the pathway analysis, and prepared the main manuscript. SW extracted the RNA from NEB liver samples, conceived the idea of performing RNA-seq on the NEB RNA samples and contributed to the biological interpretation of the data and manuscript preparation. DL contributed to the bioinformatics of differential gene expression analysis and pathway analysis and writing and editing of the manuscript. DK, and DM co-ordinated tissue collection and RNA extraction from the NEB model, conducted metabolite studies on the model and contributed to the biological interpretation of the data and writing and editing of the manuscript. CC set up and performed the majority of the bioinformatics on the sequencing runs including data retrieval, read alignments, differential gene expression analysis, KEGG pathway analysis, data interpretation and co-wrote the main manuscript with MSM. All authors read and approved the final manuscript.Table of number and assignment of reads for each flowcell lane.Click here for fileTable SDE genes and their fold-change.Click here for fileComparison of microarray and RNA-seq SDE genes.Click here for fileGraph of effect of SNEB compared to MNEB on blood concentrations of major NEB associated metabolites 1–14 days post-calving.Click here for fileMaps of KEGG pathways (associated with ≥2 fold (FDR 0.1%) SDE genes) overrepresented in SNEB animals.Click here for fileMaps of KEGG pathways associated with all SDE (FDR 0.1%) genes.Click here for file


Prior studies have demonstrated tumor-specific alternative splicing events in various solid tumor types. The role of alternative splicing in the development and progression of head and neck squamous cell carcinoma (HNSCC) is unclear. Our study queried exon-level expression to implicate splice variants in HNSCC tumors.We performed a comparative genome-wide analysis of 44 HNSCC tumors and 25 uvulopalatopharyngoplasty (UPPP) tissue samples at an exon expression level. In our comparison we ranked genes based upon a novel score—the Maximum-Minimum Exon Score (MMES) – designed to predict the likelihood of an alternative splicing event occurring. We validated predicted alternative splicing events using quantitative RT-PCR on an independent cohort.LAMA3, DST, VEGFC, SDHA, RASIP1, and TP63 were selected for further validation studies because of a high frequency of alternative splicing suggested in our graphical analysis, and literature review showing their biological relevance and known splicing patterns. We confirmed TP63 as having dominant expression of the short DeltaNp63 isoform in HNSCC tumor samples, consistent with prior reports. Two of the six genes (LAMA3 and DST) validated by quantitative RT-PCR for tumor-specific alternative splicing events .After MMES scoring of 17,422 genes, the top 900 genes with the highest scores underwent additional manual inspection of expression patterns in a graphical analysis. The genes Alternative splicing events of oncologically relevant proteins occur in HNSCC. The number of genes expressing tumor-specific splice variants needs further elucidation, as does the functional significance of selective isoform expression. Head and neck squamous cell carcinoma (HNSCC) represents the sixth most prevalent solid tumor reported annually TP53 and NOTCH1 genes, found in ∼50% and 15% of HNSCC tumors respectively retinoblastoma (Rb), thereby abrogating a critical tumor suppressor pathway Recent advances in understanding the genetic mutational landscape of HNSCC have shed light upon upregulated oncogenic and disrupted tumor suppressor pathways contributing to this disease. The most prevalent mutations in HNSCC include Another avenue of gene expression regulation in cancer that is relatively under-investigated is alternative splicing—wherein the post-trascriptional modification of mature mRNA greatly increases the functional diversity of gene products. Widespread changes in splicing patterns have been demonstrated when comparing tumor and normal epithelial tissues of the breast and ovaries TP63 is a member of the p53 superfamily from which six major isoforms are transcribed—three short and three long isoforms share common upstream promoters. The short DeltaNp63 isoform is dominant in both normal epithelial cells and HNSCC, and furthermore DeltaNp63 appears to be selectively overexpressed in HNSCC tumor cells p53 superfamily protein, p73, believed to mediate apoptosis in response to DNA damage, may be inactivated by interaction with the DeltaNp63 isoform as was shown previously Similar genome-wide studies of alternative splicing in HNSCC have not yet been reported. There is reason to believe that selective isoform expression may be relevant in HNSCC development. For example Various technologies have been employed in the detection of alternative splicing events. High-throughput quantitative RT-PCR utilizes massive libraries of isoform-specific primers Various approaches to exon array analysis have been utilized in the detection of alternative splicing events. Many studies utilize a variant of the splicing index calculation In this current investigation we employed the Affymetrix Human Exon 1.0ST mRNA expression array to detect alternative splicing events and differential isoform expression specific to HNSCC tumors in comparison to normal upper aerodigestive tract control tissues. We describe our methods for expression microarray analysis at an exon-level and results of validation studies to confirm tumor-specific splice variant expression of genes contributing to the cell-adhesion and cytoskeletal properties of cells.Written consent was obtained from all participants for the procurement and study of human HNSCC tissue samples and normal mucosal tissues. The Johns Hopkins Medical Institutions (JHMI) institutional review board (IRB) approved this study and the consent procedure. Copies of all written consents are maintained by JHMI. Tissues were snap frozen in liquid nitrogen immediately after collection. Microdissection of frozen tissue was performed to assure that more than 75% of tissue contained HNSCC. Microdissection was performed under supervision of Johns Hopkins Hospital head and neck pathologists (W.W and J.B.). For the discovery set, 44 samples of HNSCC and 25 samples of normal oropharyngeal epithelial tissue were used for the Affymetrix Human Exon 1.0 ST mRNA expression array. A separate validation set of 47 microdissected HNSCC tumors and 19 normal oropharyngeal epithelial tissues was used for the validation studies. All normal tissues were derived from patients who underwent uvulopalatopharyngoplasty for obstructive sleep apnea.Total RNA extraction from human tissue samples was performed using Trizol reagent  using standard methods RNA from discovery set samples was labeled using the Whole Transcript Sense Target Labeling protocol described by Affymetrix and reagent from Ambion and Affymetrix. Briefly, 100 ng of total RNA was used to synthesize first strand cDNA using random oligonucleotides with T7 promoter as primer and the SuperScript Choice System . Following the double stranded cDNA synthesis, the double strand cDNA was purified using magnetic beads, and cRNA was generated through in vitro transcription. 10 ug of cRNA was then used to generate sense strand cDNA using random primer, dNTP-dUTP mix, and Superscript II reverse transcriptase . The resulting sense strand cDNA was then purified, fragmented using UDG and APE at 37 C for 60 minutes, and terminal labeled with biotinylated nucleotide and terminal DNA transferase at 37 C for 60 minutes. The labeled sense strand DNA was hybridized to the Affymetrix GeneChip human Exon 1.0 ST arrays for 17 hr at 45 C with constant rotation (60 rpm). Affymetrix Fluidics Station 450 was used to wash and stain the Chips, removing the non-hybridized target and incubating with a streptavidin-phycoerythrin conjugate to stain the biotinilated cDNA. The staining was further amplified using goat IgG as blocking reagent and biotinilated anti-streptavidin antibody (goat), followed by a second staining step with a streptavidin-phycoerythrin conjugate. Fluorescence was detected using the Affymetrix G3000 GeneArray Scanner and image analysis of each GeneChip was perfomed through the Affymetrix GeneChip Command Console version 3.4 (AGCC v3.4) software from Affymetrix. Data from this discovery set is available on the Gene Expression Omnibus, GSE33205.This array interrogates the expression of gene transcripts on a whole-genome level. Over 1.4 million array probesets are designed to target exonic sequences within the human genome based upon a prediction model of exon boundaries described in the Affymetrix GeneChip Technical Note Affymetrix array core probeset data was normalized with the Robust Multiarray Average (RMA) method using the Bioconductor oligo package in R itN  for each tumor.1] After RMA normalization of all core probesets, every probeset signal in each individual tumor is normalized to the mean respective probeset signal in the normal samples, generating a value PitN values for an individual exon is calculated for each tumor, generating a value Exonet .2] Many exons are interrogated by more than one probeset. A mean of all Pet and minimum Exonet is calculated, generating one MMES score per gene, per tumor. A mean tumor MMES score for each gene can be calculated, and a rank list generated with higher mean tumor scores suggesting putative alternative splicing events. Larger differences in the relative expression levels of exons within a single gene are rewarded with higher MMES scores. Greater frequency of high MMES scores within the tumor cohort for a particular gene are reflected by a higher mean MMES score, and consequently ranked more highly.3] In each tumor, for each gene the difference between the maximum ExonDST gene expression data is used as an example  to 3′ (right) end. The vertical axis represents the RMA normalized probeset expression signal on a log2 scale. The flat horizontal tracing represents the mean signal in all probesets across both tumors and normals rounded down to the nearest whole number and merely provides a convenient horizontal reference. The irregular tracing represents for each probeset the difference in the means of the tumor and normal samples relative to the horizontal line indicating zero. This provides a view of the difference shifted to the bulk of the individual data points. The histogram in DST. The vertical axis here represents a select tumor's relative probeset expression compared to the mean for that probeset of the UPPP cohort that is represented by the zero level. By examining individual tumor plots we identify putative alternative splicing events that occur in a significant number of individual samples with less ‘noise’ than may be seen from overall comparisons of tumor and UPPP cohorts.4] From the MMES generated rank list , the top example . All corWe selected 15 tumors from the discovery set for validation of select alternative splicing candidates from our MMES prediction model. For each selected tumor, 2 micrograms of RNA was converted to cDNA using the Quanta Biosciences qScript cDNA SuperMix using standard methods, producing a total of 20 microliters of cDNA Quantitative RT-PCR was performed using custom primer and probe sets specific to each splice variant ., as welTo compare splicing patterns between tumor samples and UPPP samples, splicing indices were calculated for the each gene as:Data was analyzed using Stata v11.0 statistical analyses software . Splicing Indices were compared between tumor and UPPP normal tissue cohorts for significant differences using 2-tailed Student's t test with assumption of unequal variance.The clinicopathologic characteristics of discovery and validation tumors and UPPP normal samples are presented in LAMA3A, DST, RASIP1, SDHA, VEGFC, and TP63. LAMA3 and DST. For the TP63 gene we confirmed a markedly predominant expression of the shorter DeltaNp63 isoform that has been previously reported for epithelia of the upper aerodigestive tract RASIP1, SDHA, VEGFC confirmatory RT-PCR studies did not validate alternative splicing events predicted from microarray analysis and results are summarized in the Supporting Information  share ten consecutive exons from the 5′-end . Three shorter isoforms (DeltaNp63) share eight consecutive exons from the 5′-end . In our exon array analysis, manual review of TP63 gene plots predicted 25 of 44 tumor samples with a splicing pattern predominantly expressing the shorter DeltaNp63 isoforms. DeltaNp63 or TAp63 predominant expression , consistent with prior reports in literature Subsequent quantitative RT-PCR validation of 15 discovery set tumors showed dominant expression of the isoforms . Our indLAMA3 RefSeq annotations demonstrate four major isoforms of this gene, with the two shorter isoforms (LAMA3A NM_001127718.1 and NM_000227.3) excluding the 36 consecutive most 5′ exons, and full-length LAMA3B variants (LAMA3B NM_001127717.1 and NM_198129.1). In our exon array analysis, manual review of LAMA3 gene plots demonstrated 23 of 44 tumor samples with a splicing pattern predominantly expressing the shorter LAMA3A isoforms. LAMA3A or LAMA3B predominant expression —excluding the eight most 3′ exons, and including a long sequence that is intronic and not transcribed in the longer isoforms—or one of four longer isoforms that we probed with a ‘common’ primer/probeset . In our exon array analysis, manual review of DST gene plots demonstrated 14 of 44 tumor samples with a splicing pattern predominantly expressing the shorter 1e isoform. 1e or longer isoform predominant expression  that has not been implicated in squamous cell carcinoma to date. The LAMA3A isoform lacks the N-terminal domain (LN) of the full-length LAMA3 transcript LAMA3B. The LN domain is necessary for the polymerization of laminins and promotion of epithelial basement membrane stability LAMA3A in HNSCC affects tumor invasiveness requires further investigation.LAMA3 expression in HNSCC tumors that were scored as having high versus low hypoxic microenvironments based upon a 99-gene hypoxia expression panel LAMA3A isoform was greater compared to tumors with low hypoxia scores. However the LAMA3B isoform was not differentially expressed. HNSCC cell lines subjected to hypoxic conditions did not replicate the hypoxia-associated increase in LAMA3A expression. This study focused on differential expression of LAMA3 isoforms in biologically different tumors. Our results compare primary HNSCC tumors to normal tissue and confirm a tumor-specific predeliction for LAMA3A isoform expression. The pathophysiological implications of LAMA3A overexpression, and characterization of laminin 332 expression in HNSCC is a new area of interest.A recent study compared DST encodes the sequence for the cytoskeletal bullous pemphigoid antigen 1 (BPAG1) protein that has multiple isoforms variably expressed in different tissue types 1e isoform, with lower expression levels of alternative isoforms. While BPAG1 mutations are implicated in bullous pemphigoid blistering diseases, little is known about a potential role in cancer LAMA3A, isoform 1e interacts with the critical integrin alpha6beta4 that facilitates keratinocyte adhesion to the basement membrane 1e isoform in head and neck squamous carcinoma compared to normal upper aerodigestive tract epithelial cells, and aberrant delocalization of this protein away from the basal membrane, the usual site of hemidesmosomal expression alpha6beta4 integrin and 1e protein expression was distributed along all cell membranes, having lost their polarized expression. In addition, diminished hemidesmosomal formation was seen on electron microscopic analysis of the same tumors, with apparent greater loss in metastatic tumors 1e mRNA expression in a large series of HNSCC tumors compared to normal controls.In conclusion alternative splicing events of oncologically relevant proteins do occur in HNSCC. The number of genes expressing tumor-specific splice variants needs further elucidation, as does the functional significance of selective isoform expression.Figure S1VEGFC.(TIFF)Click here for additional data file.Figure S2SDHA.(TIFF)Click here for additional data file.Figure S3RASIP1.(TIFF)Click here for additional data file.Figure S4Visual screening begins with a graphical comparison of the discovery tumor cohort and UPPP sample expression array data. A,TP63 gene. Comparing the mean expresssion of each probeset in the entire discovery tumor set to the mean in the entire discovery UPPP set. The mean probeset signals for the UPPP sample set have been zeroed , with adjustment of tumor cohort mean probeset signals (oscillating line), 2 scalelog. B, LAMA3 gene. Comparing the mean expresssion of each probeset in the entire discovery tumor set to the mean in the entire discovery UPPP set. The mean probeset signals for the UPPP sample set have been zeroed , with adjustment of tumor cohort mean probeset signals (oscillating line), 2 scalelog. C, DST gene. Comparing the mean expresssion of each probeset in the entire discovery tumor set to the mean in the entire discovery UPPP set. The mean probeset signals for the UPPP sample set have been zeroed , with adjustment of tumor cohort mean probeset signals (oscillating line), 2 scalelog.(TIFF)Click here for additional data file.Table S1Splice variant-specific custom primer and probe sequences.(XLSX)Click here for additional data file.Table S2MMES Gene Rank List.(XLSX)Click here for additional data file.


Many human diseases, arising from mutations of disease susceptibility genes (genetic diseases), are also associated with viral infections , either in a directly causal manner or by indirect associations. Here we examine whether viral perturbations of host interactome may underlie such virally implicated disease relationships. Using as models two different human viruses, Epstein-Barr virus (EBV) and human papillomavirus (HPV), we find that host targets of viral proteins reside in network proximity to products of disease susceptibility genes. Expression changes in virally implicated disease tissues and comorbidity patterns cluster significantly in the network vicinity of viral targets. The topological proximity found between cellular targets of viral proteins and disease genes was exploited to uncover a novel pathway linking HPV to Fanconi anemia. Many “virally implicated human diseases” - diseases for which there is scientific consensus of viral involvement - are associated with genetic alterations in particular disease susceptibility genes. We proposed and demonstrated that for two human viruses, Epstein-Barr virus and human papillomavirus, topological proximity should exist between host targets of viruses and genes associated with virally implicated diseases on host interactome networks . For representative EBV- and HPV16- implicated diseases, genes in the neighborhood of viral targets in the host interactome have significantly shifted expression levels in virally implicated disease tissues, in line with the local impact hypothesis. The viral neighborhoods in the host interactome, along with their disease associations, defined as “viral disease networks”, contain connections known to be informative upon disease mechanisms as well as diseases whose associations with viruses are not yet known. We prioritized these diseases for their candidacy as potential virally implicated diseases based on network topology, and benchmarked this prioritization of candidate diseases using relative risk measurement which depicts population-based clinical associations between candidate diseases and viral infection. Exogenous expression of HPV viral proteins in a human cell line offered evidence for a novel disease pathway that links HPV to Fanconi anemia. We find that the disease susceptibility genes of known virally implicated diseases are in the immediate network vicinity of the host proteins that are targeted by these viruses. We could identify a viral disease module for EBV and HPV, representing a subnetwork of the interactome that contains key mechanistic pathways responsible for the observed virus-disease associations. A computational prioritization procedure, joined by large-scale comorbidity and expression pattern analyses, identified new potential mechanistic disease pathways. To validate several of these pathways, HPV16 E6 and E7 oncogenes were independently expressed in primary human fibroblast (IMR90) and keratinocyte (HFK) cell populations to identify disease-associated genes whose expression levels were significantly altered in these E6/E7-expressing cell populations. We could identify a novel pathway that links HPV to a specific form of Fanconi Anemia. The systematic network-based framework we applied works to decipher the interplay between viruses and disease phenotypes.We define as “virally implicated diseases” those diseases whose association with a particular virus is supported by peer-reviewed publications in the literature. This list includes not only diseases for which there is universally accepted consensus that a virus is causal , but also diseases which have some reproducible evidence of viral association but for which the mechanistic pathways are not worked out. There is significant and legitimate controversy and subjectivity regarding which diseases are virus-associated or virally implicated, so to avoid infusing personal bias in the selection process, we turned to several recently published authoritative review articles Most of the selected virally implicated diseases (13 for EBV and 9 for HPV16) are genetic diseases in that they have been associated with mutations in at least one human gene , as compTo explore the role of macromolecular networks in virus-disease associations we collected four categories of biological connections: 1) lists of previously published experimental virus-human protein-protein P = 2.3×10−6 for EBV and P = 7×10−7 for HPV16, based on empirical calculation). That this shortest path was less than one for both EBV (0.667) and HPV (0.5) indicates that viral proteins preferentially target a disease associated protein directly (hop 0) or a protein that directly interacts with a disease associated protein (hop 1). To mitigate potential investigational biases that accompany literature-curated datasets P = 4.9×10−5 for EBV and average shortest path = 1.0; P = 3×10−4 for HPV16, based on empirical calculation; To test our hypothesis that genes associated with virally implicated diseases are located in the network vicinity of viral targets , we measThe relative shortness of the paths from viral targets to disease genes validates the hypothesis that genes in the “neighborhood” of viral targets are more likely associated with virally implicated diseases, compared to genes in distant regions of the host interactome. But still, given the small world nature of the interactome, large numbers of proteins are within a few hops of the viral targets, potentially implicating hundreds of diseases for which there is no known relationship to HPV or EBV. Accordingly, a procedure is needed to identify the set of host cellular components  that are most likely impacted by the virus, representing the network neighborhood of viral targets. Do the three kinds of interactions used to build the interactome — protein-protein, metabolic and regulatory interactions — play a comparable role in linking viral targets to virally-implicated diseases, and how deep into the interactome should one go, keeping in mind that most proteins are approximately three links from the viral proteins.P = 0.0012 for EBV and P = 0.0005 for HPV based on empirical calculation). We therefore chose this configuration to define the network neighborhood of the viral targets, representing the viral disease modules, leaving aside the host metabolic and protein interactions.To find the optimal neighborhood responsible for the phenotypic impact of a virus, we tested several “configurations” that govern the maximum hops allowed from the viral targets for each type of biological interaction. The simplest configuration includes only viral targets, while the more extended configurations capture increasing number of hops along the links of the interactome network, connecting an increasing number of proteins. The best configuration, as measured by the odds ratio of the enrichment of virally implicated diseases, defined the optimal neighborhood as the viral targets themselves and the genes regulated by them, and was the same for both viruses  associated with virally implicated diseases exceeds 10 viruses . Yet, th viruses : i) EBV The neighborhoods of viral targets in the host interactome, along with their disease associations, represent “viral disease networks” . The vire.g. Burkitt's lymphoma for EBV and bladder cancer for HPV16, Tables S6, S7 in The uncovered viral disease networks contain several diseases that have not been previously associated with infection by the corresponding viruses  To demonstrate the value of the network-based approach to generate new biological hypotheses, we explored whether the cellular perturbations induced by expression of individual viral proteins are similar to those seen in particular disease phenotypes. We generated primary human keratinocyte (HFK) populations with stable expression of the HPV16 E6 or E7 oncoproteins and analyzed the gene expression profiles of multiple independent samples for these cells in concert with expression data from IMR90 cells expressing HPV16 E6 or E7 proteins . Of the FANCC, a gene in the disease network mutated in Fanconi anemia, is up-regulated in the E6 exogenous expression IMR90 cell data, and (iii) FANCC is significantly up-regulated in low density bone marrow cells of Fanconi anemia patients  FANCD2 through an E2F-dependent pathway FANCC pathway, which had not been previously established. FANCC has been reported to be expressed at higher levels upon activation of TP53 Seven out of 39 diseases have high relative risks among HPV patients , of whicThe clinical connection between Fanconi anemia and HPV associated tumors has been subject to debate. Not debatable is that FA patients have a much-increased risk in developing squamous cell carcinomas (SCCs) at anatomical sites infected by HPVs. Our analysis does not necessarily mean that SCCs in Fanconi patients are caused by HPV, but that they arise by similar molecular mechanisms. The well-documented interplay between E7 and FA and our discovery of a possible connection between E6, FANCC and BRCA1 support this hypothesis. Moreover, we observe a relative risk of 3.7 among female HPV patients  toward Fanconi anemia using the US-wide Medicare data, which further supports the identified molecular level relationship between Fanconi anemia and HPV .Given the large number of functional interactions present in human cells and the many possible paths among cellular components, uncovering the precise impact of a virus upon the host interactome is an enormously complicated task. Here we provide evidence that a large proportion of the effect of a virus can be accounted for locally in the network space, which allowed us to develop and test a general methodology designed to elucidate the consequences of viral impacts on the host interactome network, and to prioritize candidate diseases for potential viral implications.A predictive methodology should ideally take into account cell tropism. Tissue-specific gene expression data can be merged with our analysis . We usedThe strategy developed here is not unique to EBV and HPV16. Although the strategy should work better for carcinogenic pathogens, given how well-studied proteins involved in cancer are, it is equally applicable to any pathogen for which protein interactions between the pathogen and the host proteome have been mapped. While still limited by the incompleteness of genome- and proteome-scale datasets Yeast two-hybrid screens (Y2H) between EBV and HPV16 viral proteins and ∼12,200 human proteins encoded by a library of full length human open reading frame (ORFs) clones in Human ORFeome v3.1 Raw data of the gene expression datasets used  was obtained from Gene Expression Omnibus (GEO) To obtain the disease associated genes that are differentially expressed in viral protein induced cell populations, HPV16 E6 and E7 oncogenes were independently transfected into primary human fibroblast (IMR90) and keratinocyte (HFK) cell populations. Affymetrix Human Gene1.0 ST and Human Genome U133 Plus 2.0 arrays, respectively, were used to measure gene expression profiles for five or more replicate samples in each of the cell types. Array data were normalized by RMA, batch effects were removed using ComBat, and the limma package in R/Bioconductor was used to identify differential expression.Relative risk (RR) was calculated as the ratio between the observed co-occurrence and probabilistically-inferred (assuming independence) co-occurrence of two diseases, based on the patient medical history data from United States (U.S.) Medicare, which contains the clinical diagnosis record of each hospital visit (in ICD-9 codes) of 13 million U.S. patients at age 65 or older P values were calculated based on empirical data with 10,000 random configurations. For the analysis of GEO microarray data we used two-tailed t-test statistics.The statistical significance of the average shortest path between viral targets and genes associated with a given virally implicated disease was calculated by randomly sampling human diseases from OMIM (full table of disease in Dataset S1Sheet 1) EBV disease network, (Sheet 2) HPV16 disease network. VH-PPI: virus-host protein-protein interaction, PPI: host protein-protein interaction, PDI: host protein-DNA interaction, MCI: metabolic enzyme-coupled interactions calculated using KEGG, or BIGG databases, or flux coupling analysis.Full list of interactions and gene-disease associations in viral disease networks, including the sources of data. ((XLS)Click here for additional data file.Dataset S2OMIM genes and diseases and their corresponding ICD-9 codes.(XLS)Click here for additional data file.Dataset S3 relative risk between EBV- and HPV-implicated diseases and candidate diseases in the disease network.  relative risk between EBV- and HPV-implicated diseases and all mappable diseases which constitute the random control.Relative risk analysis. (XLS)Click here for additional data file.Dataset S4Sheet 1) EBV (Sheet 2) HPV. Diseases are sorted according to maximum scores.Full list of diseases prioritized by the flow algorithm for ((XLS)Click here for additional data file.Text S1Supporting information, tables and figures are provided in this document.(PDF)Click here for additional data file.