The vertebrates also emerged from their invertebrate ancestors via two WGDs, and genomes of diverse gymnosperm trees, unicellular eukaryotes, invertebrates, fishes, amphibians and even a rodent carry evidence of lineage-specific WGDs. Modern polyploidy is common in eukaryotes, and it can be induced, enabling mechanisms and short-term cost-benefit assessments of polyploidy to be studied experimentally. However, the ancient WGDs can be reconstructed only by comparative genomics: these studies are difficult because the DNA duplicates have been through tens or hundreds of millions of years of gene losses, mutations, and chromosomal rearrangements that culminate in resolution of the polyploid genomes back into diploid ones (rediploidisation). Intriguing asymmetries in patterns of post-WGD gene loss and retention between duplicated sets of chromosomes have been discovered recently, and elaborations ofsignal transduction systems are lasting legacies from several WGDs. The data imply that simpler signalling pathways in the pre-WGD ancestors were converted via WGDs into multi-stranded parallelised networks. Genetic and biochemical studies in plants, yeasts and vertebrates suggest a paradigm in which different combinations of sister paralogues in the post-WGD regulatory networks are co-regulated under different conditions. In principle, such networks can respond to a wide array of environmental, sensory and hormonal stimuli and integrate them to generate phenotypic variety in cell types and behaviours. Patterns are also being discerned in how the post-WGD signalling networks are reconfigured in human cancers and neurological conditions. It is fascinating to unpick how ancient genomic events impact on complexity, variety and disease in modern life.Ancient whole-genome duplications (WGDs)— Fast-forward to today’s exciting era of high-throughput genome sequencing, and phylogenomic maps assembled from multiple whole-genome sequences tell the evolutionary story with revised timelines:Darwin was vexed. While natural selection could explain gradual evolutionary transitions, the apparent sudden appearance of diverse flowering plants in the Cretaceous fossil record of approximately 130 million years ago (Mya) was his ‘abominable mystery’2. The nuclear genomes of several gymnosperms have been sequenced recently, a heroic undertaking, given their exceptional size (10 to 40 gigabases) and high density of long terminal repeat (LTR)-retrotransposon repeats5. Within these genomes many non-overlapping duplicated chromosomal regions were identified that display gene synteny, meaning that their gene contents are similar to those of other chromosomal blocks within the same genome and across gymnosperm genomes. These gene synteny patterns and complementary transcriptome data support the hypothesis that the gymnosperms emerged from their common ancestor via a WGD named ζ that occurred an estimated 390 Mya during the Devonian period9. Tell-tale traces of further lineage-specific WGDs have also been discovered in the genomes of Norway spruce, Sequoia and Ginkgo, and in the unusual two-leaved NamibianWelwitschia, suggesting that multiple WGDs contributed to the diversity of these gymnosperms, which include the longest-living and largest organisms on Earth7.From the Carboniferous to early Cretaceous periods (approximately 360 to 130 Mya), the land was dominated by gymnosperms  including the cycads, Ginkgo, and conifers that still flourish in subarctic forests10. Many angiosperm genomes have been selected for sequencing to discover genes for special agronomic traits. The resulting genome assemblies have been used collectively to extrapolate back in time, tracking the genome evolution of the monocot and eudicot angiosperms, as well as more primitive flowering plants, and converging on a reconstructed genome of the most recent common ancestor of all angiosperms6. These new phylogenomic maps solve part of Darwin’s dilemma by confirming that the first flowering plants evolved between 140 and 250 Mya after an unknown gymnosperm went through a WGD (named the ε event) an estimated 300 Mya during the Carboniferous period9. The clearest support for the ε WGD comes from multiple gene synteny blocks in the genome of the primitive angiospermAmborella trichopoda, whose ancestral lineage diverged early on from other flowering plants, experiencing no further post-ε WGDs11.Flowering plants  are the most abundant plant group today, having a rich diversity of some 400,000 species from bananas to water lilies, grasses to beech trees13. In any case, angiosperm evolution was not as sudden an explosion as Darwin thought, and now a comprehensive phylogenomic framework exists to mine for answers about how angiosperm complexity and variety evolved after the ε WGD.The antiquity of the ε WGD means that angiosperms had the entire Triassic and Jurassic periods to evolve and diversify into the species richness reflected in the fossils of approximately 130 Mya that were known to Darwin. Aligning with the recalibrated timelines, older angiosperm microfossils have been discovered, though claims that the earliest ones date to the Triassic are controversial14. Members of gene families generated via WGDs are named ohnologues in his honour15. Another scenario is for the duplicates to each retain subsets of the functions of the ancestral gene 16. Moreover, theen masse diversification of many gene duplicates after a WGD would be expected to create selective pressures between gene families and opportunities for new interactions among them, so that large duplicated gene and protein sets co-evolve as complex systems.In the 1970s, Susumo Ohno had the prescience to propose that evolutionary leaps could occur by WGDs because one of each gene pair may continue to do what it was doing before, giving freedom for the other to either be lost from the genome or to evolve new characteristics Antirrhinum (snapdragon) andArabidopsis. In these seminal studies, homeotic floral mutants were used to deduce an elegant ‘ABC model’, later elaborated into an ‘ABCDE model’ that explains how the four concentric whorls of sepals, petals, male stamens and female carpels develop in these flowers19. A-function genes specify sepals; A, B and E are needed to make petals; B, C and E male stamens; C female carpels; and D for ovules. In homeotic mutants lacking B-function, for example, sepals replace petals and carpels replace stamens18. Most of the A, B, C, D and E functions are performed by combinations of MADS-box transcription factors that operate as homodimers and heterodimers and tetramers with different selectivities for binding to variants of a common motif in the promoters of target genes20. Hence, they target overlapping but distinct sets of floral identity genes many of which are themselves ohnologues21.Floral organs provide a canonical example of how interacting sets of diversified ohnologues can make variant structures. While many gene pairs lost one duplicate after the ε WGD, the retained ohnologue pairs include MADS-box transcription factors. Some of these were characterised more than 20 years ago for their ability to interact with each other in different combinations to specify the floral organs of24. These duplicated and diversified gene sets organised to generate the first now-extinct flowers, and recent reconstructions suggest that these were bisexual with petal-like tepals and pollen-bearing stamens arranged in multiple concentric whorls, and female carpels in a central spiral25. Among living angiosperms, theAmborella lineage evolved a ‘fading borders’ programme, such that the whole flower is a spiral that gradually transitions from bracts to outer then inner tepals (specified by ABc combinations), from inner tepals to stamens (aBC) then carpels (abC), in which upper case indicates functions of greatest influence in the respective organs26. Only in later-evolving flowers such asArabidopsis andAntirrhinum did the tepals subdivide into sepals and petals, by restricting the boundaries of expression of floral identity genes. For example, the transcription of A and C genes became mutually exclusive26. Further evolutionary diversity in flower form occurred by mechanisms that include shifts in the spatial expression of ABC functions across flowers, and by further WGDs that elaborated and extended the ABC regulatory network27. For example, in stylised orchid flowers, subfunctionalisation of duplicated B genes underpins the development of three types of petals: three outer tepals, two inner tepals and a modified lip28. Recent case studies implicate additional ancient WGDs – including one at the base of the eudicots, the γ genome triplication in the Pentapetalae , and ρ, σ and τ polyploidisations for monocots – in evolution of the phenomenal variety of architectural form and size in pollen, fruits and seeds, in diversification of plant defence metabolites, and in the co-evolution of angiosperms with pollinators and symbiotic bacteria34.The recent genome comparisons indicate that male ‘BC’ and female ‘C’ systems already existed to specify reproductive cells in gymnosperm cones, and they were duplicated via the ε WGD, after which the C duplicates diversified into angiosperm C and D genes. An A/E gymnosperm pro-orthologue gave rise to angiosperm A and E genes, and further duplicated A genes were also retained after the ε WGD35. For example, the durum wheat used to make pasta is a tetraploid resulting from hybrid doubling of the genomes of two diploid wild-grass ancestors approximately 0.5 Mya, and was selected for domestication much later by Neolithic farmers, during which time hexaploid bread wheat emerged by hybridisation of the tetraploid with a diploid followed by another WGD35. Like wheat, many well-characterised crop polyploids are allopolyploid36, which means that the genome became polyploid after a hybrid was formed between species, in which case the WGD resolved problems with meiotic pairing by providing each chromosome with a homeologous partner38. However, autopolyploidy events, self-duplication within a species, are suspected in the ancestry of potatoes, bananas, poplar and soybean39.Evolutionarily recent polyploidy events are also prevalent in flowering plants. Many crops including coffee, bananas, peanuts, tobacco, kiwifruit and strawberries were unwittingly selected as polyploids for their exaggerated traits such as large fruits, seeds and leaves43. It makes sense that the odds are stacked against a newly tetraploid plant. Breeding with still-diploid relatives results in triploid progeny that cannot separate evenly into two gametes during meiosis, most often resulting in sterile offspring, and unless self-pollination can occur, chances may be low of finding compatible polyploid mates. Farmers get around such problems by cloning: grafting apple trees or propagating potato tubers instead of seeds, whilst sterility (for example in seedless bananas) is sometimes even preferred44. However, the resulting monocultures may be susceptible to pathogens, as when theFusarium oxysporum fungal pandemic brought the popular triploid Gros Michel banana to the brink of extinction in the 1960s45.Recent statistical comparisons suggest that an individual polyploid plant has a higher risk of extinction than its still-diploid relatives46. Experimental polyploidies show that having extra DNA can produce an immediate phenotypic change, attributable to gene dosage effects. For example, dwarfism in apple plants with colchicine-induced autotetraploidy correlates with increased expression of a microRNA that acts via a gene regulatory network to downregulate synthesis of auxin and brassinosteroid growth regulators47. More generally, polyploid plants are notable for their increased cell and organ size, which is more than a passive consequence of increased nuclear DNA content: in experiments withArabidopsis, increases in cell volume upon tetraploidisation were found to vary in different mutants and according to cell type, indicating a genetic contribution48.Nevertheless, their prevalence suggests that once polyploids have beaten the early survival odds, with or without human intervention, their polyploid traits such as larger organs, stress tolerance and altered flowering time may improve fitness or allow them to adapt to new ecological nichesArabidopsis thaliana (At) andArabidopsis arenosa (Aa), biases in heterologous combinations of components were discovered: the Aa-derived CCA1 hiking expedition (CHE) transcription factor preferentially binds to the promoter of the At circadian clock associated 1 (CCA1) gene, elevating its expression over that of the AaCCA1. Such biased patterns of expression, and of protein–protein and protein–DNA interactions in the circadian regulatory network make the rhythm of the allotetraploid distinct from that of either parent49.For allopolyploids, the relative contributions of the species hybridisations and the WGDs to subsequent evolution are interwoven. In a recent molecular dissection of the circadian clock in allotetraploids formed between diploids51. However, polyploidy is relatively common in ectothermic vertebrates. Also, synthetic fish and shellfish polyploids, generally sterile, have been created to increase food production52. Moreover, helped by technical advances in deep sequencing, genome assembly and pattern-matching software, ancient WGDs have been identified in invertebrate and vertebrate animal lineages, including mammals. The most recent discovery was that the house spiderParasteatoda tepidariorum and bark scorpionCentruroides sculpturatus are common descendants of a WGD that occurred over 450 Mya, and was distinct from an ancestral WGD of horseshoe crabs54.Successful polyploidy is said to be less common in animals than in plants. Based on incidences of chromosomal anomalies in embryos that fail to develop, it appears that when two sperm fertilise one egg or when meiotic cell division fails the result is usually lethal in humans and birdsSaccharomyces cerevisiae and five other fungal genera all stem from the same well-characterised ancestral allopolyploid WGD approximately 100 Mya55, and further fungal WGDs have been identified recently. For example, the opportunistic honeybee fungal pathogenNosema ceranae, which is spreading to beehives worldwide, is a suspected tetraploid56.Although few ancient WGDs have been identified thus far in unicellular eukaryotes, the diploid baker’s yeast57, common principles are emerging that tie disparate WGD events together:In summary, ancient WGDs that were successful in leaving modern descendants have occurred in diverse eukaryotes across eons of time, in terrestrial and aquatic environments. Remarkably, despite their radically different contexts and a sparsity of data on ancient WGDs outside of laboratory models and domesticated speciesSuccessive WGDs have occurred in multiple lineages: As indicated, angiosperms have experienced multiple WGDs, and recursive WGDs among theBrassica crops that include cauliflower, broccoli and cabbages have been precisely mapped recently58. The vertebrate animals emerged from the invertebrates approximately 500 Mya via two sequential rounds of WGD (2R-WGD)59. In fish there was a further teleost-specific WGD  approximately 300 Mya, followed by a salmonid-specific WGD (Ss4R) approximately 95 Mya; and certainXenopus frog species and the red viscacha ratTympanoctomys barrerae also result from lineage-specific WGDs64. The unicellular ciliateParamecium tetraurelia has a history of three successive WGDs65, and WGDs may have contributed to the record number of chromosomes (2n=1260) in the fern-like genusOphioglossum66.Most, though not all, well-characterised WGDs were allopolyploidy events: In common with most polyploid plants, the tetraploid frogXenopus laevis and diploid yeastS. cerevisiae were recently identified to be descendants of allopolyploidies68. In contrast, high similarity between homeologous regions in salmonid genomes indicate that the TSGD/3R and salmonid Ss4R were autopolyploidy events70. The mechanisms of the 2R-WGD at the origin of the vertebrates 500 Mya are unresolved, although early studies argued for two closely spaced autotetraploidies71.Long lag periods may occur between WGDs and subsequent species radiations: A WGD generates a new organism that is immediately distinct from the parental species, especially after inter-species hybridisation allopolyploidies. Intuitively, one would expect this new organism to lead to species radiations due to the availability of new genetic material for evolution to mould in different ways. In practice however, the mechanistic links between WGDs and species radiations are not so clear-cut. In many instances there is a time-lag between WGDs and species radiations, formalised as the WGD Radiation Lag-Time model72. For example, comparisons of post-TSGD fishes, including zebrafish and Japanese medaka, show that post-TSGD genome changes were biphasic. An initial period of bulk losses of chromosomal segments was overlaid by a more extended period of gradual gene losses by pseudogenisation and mutation of the retained ohnologues. The latter phase, after the initial bulk genome reshaping, correlates with radiation of bony fish species62, though how or whether WGD is mechanistically linked to fish diversification is still an open question73. Among the fungi, comparisons of the six genera that share the same ancestral WGD asS. cerevisiae suggest that around 4000 genes still existed in duplicate when these genera were diverging from each other, with subsequent losses of different paralogues in different lineages, such that, for example,S. cerevisiae now has 551 pairs of ohnologues andCandida glabrata has 404 pairs74. The next two bulleted points further discuss how mechanisms of post-WGD genomic evolution, as well as environmental influences, steer the course of speciation and phenotypic diversity after a WGD.Post-WGD chromosomal rearrangements culminate in a return to the diploid state (rediploidisation) as well as lineage divergence: Allopolyploidy results in immediate rediploidisation (diploid pairing of homeologous chromosomes during meiosis/mitosis) if the chromosomes from the two parental species are sufficiently distinct that chromosomes do not form quadrivalents during cell division. In contrast, autopolyploidy leads into a process of gradual rediploidisation37, such that descendants of some WGDs that happened just tens of Mya are still polyploid, whereas species whose last WGD occurred hundreds of Mya (certain angiosperms and most vertebrates) have reverted to diploid. The post-Ss4R salmonids provide interesting snapshots of genomes in transition – some chromosomes are still functionally tetraploid whereas others have become diploid. Examining these genomes in different species reveals that the dynamic interplay of rediploidisation, ohnologue divergence, speciation and post-speciation evolution is complicated75. For example, regions of salmonid genomes have been identified for which rediploidisation and evolutionary diversification of ohnologues occurs after speciation, such that functional divergence of ohnologues occurs in lineage-specific ways . LORe may facilitate adaptations of the distinct species to different ecological contexts76.WGDs and species radiations following WGDs have been linked with major climate change: Many angiosperm WGDs date to the asteroid-triggered Cretaceous-Paleogene boundary events approximately 66 Mya, indicating that polyploid establishment may be favoured during times of environmental stress77. After the Ss4R of approximately 88 Mya, the greatest species radiation occurred in fish that evolved the ability to migrate between fresh water and seawater following the climatic cooling of the Eocene–Oligocene transition 40 to 50 million years later78. Such correlations between polyploidisations and environmental changes strengthen the view that the two are linked, perhaps due to polyploid organisms being more robust to environmental change and stress46.Knowledge of WGDs informs how laboratory animals are used as biomedical models: Zebrafish and polyploidXenopus frogs are valuable models for development and disease. However, they have been through lineage-specific WGDs that humans have not, which means that phenotypes may differ when ohnologues are mutated in zebrafish, polyploid frogs, and humans79. Moreover, families of sister ohnologues from the 2R-WGD may subfunctionalise or neofunctionalise in different ways along different vertebrate lineages, as has been found for the neurogenin and snail/slug genes81. It is therefore important to consider the composite functions of all sister ohnologues when making cross-species comparisons.Asymmetries in the fates of DNA duplicates occur at multiple levels after ancient allopolyploidies: Patterns of gene loss, retention and mutation may differ markedly in the sub-genomes derived from each parent of the original hybrid82. A striking example is the tetraploidX. laevis in which large- and small-scale losses of DNA differ to such an extent that chromosomes derived from one parental species are markedly shorter than chromosomes from the other60. It has been proposed that biases in the fates of DNA duplicates may result from ancient allopolyploidies, but that more evenly balanced post-WGD patterns of gene loss, retention and differentiation may follow on from ancient autopolyploidies39. The proposal is that initial differences in expression levels and in affinities of interactions of ohnologue proteins derived from two parental species49 could propagate into further knock-on biases such as preferential retention of highly-expressed genes66. Another type of post-WGD bias occurred in the shared ancestry ofS. cerevisiae andCandida glabrata. Their ancestral WGD occurred approximately 100 Mya, after one parent from the KLE  clade mated with one of the ZT  clade. However, for various reasons the mixed parentage ofS. cerevisiae was not immediately obvious when its genome was sequenced. One reason is that theS. cerevisiae genome contains more ZT-than KLE-derived sequences, possibly due to biased gene conversion that replaced some KLE-derived sequences with homeologous ZT-derived ones68. Finding thatS. cerevisiae results from an allopolyploidy, rather than an autopolyploidy as originally believed, means that calculations of the relative rates of evolution of its ohnologue pairs82 may need revision.Retained ohnologue gene families are strikingly enriched in signalling and regulatory proteins in plants, fungi and animals85: For example, ancestral WGDs that were identified recently for the fungiMucor circinelloides andPhycomyces blakesleeanus resulted in increased proportions of genes whose transcription is regulated by light86. In humans, while only approximately 25% of genes are ohnologues stemming from the 2R-WGD, ~66% of protein kinases and nearly 90% of well-characterised 14-3-3-binding phosphoproteins are ohnologues; and developmental regulators and post-synaptic density (PSD) brain proteins are also highly enriched in ohnologues92. Signalling in biology ranges from simple signal-response systems to the complex signalling networks of our brains that coordinate complicated actions, create memories and find meaning in patterns. How have WGDs shaped these networks?S. cerevisiae, plants and mammals have shown that regulatory proteins that form oligomers, that interact transiently with multiprotein complexes, and catalyse consecutive steps in metabolic and regulatory pathways are enriched among duplicate pairs that are retained following a WGD94. These findings are interpreted by the gene balance hypothesis, which states that copy numbers of genes encoding multi-protein structures and pathways must be kept in a constant ratio to avoid architectural disruption or metabolic imbalances95, although stoichiometry can also be achieved by other mechanisms such as differential degradation of protein components95.Studies in97.Interestingly, the architectures of sister ohnologue proteins are generally conserved with respect to content and order of their domains, at least in vertebrates. Instead, functional divergence between sisters occurs via small-scale mutations that lead to differences in temporal and spatial patterns of expression, altered sites of regulatory post-translational modifications, and changes in specificities and affinities of catalytic domains and interaction interfacesExamples abound to illustrate how the resulting families of differentiated sister ohnologues contribute to phenotypic robustness, plasticity and complexity. For instance:98.The ‘Gli code’ refers to how different combinations of sister Gli transcription factors—Gli1 and Gli2 that activate transcription and the repressor Gli3—influence tissue shape and size during vertebrate embryonic development. The code can be changed via differential regulation of the sister Gli proteins by multiple signalling pathways99. For example, in mammals the discs large MAGUK scaffold protein 4 (Dlg4) has evolved a role in simple associative learning, whereas its sisters Dlg2 and Dlg3 have distinct and opposing functions in complex cognitive processes87.Genetic and biochemical dissections indicate that sets of sister ohnologues within the PSD ‘supercomplexes’ of mammalian brains can differentially process the signals responsible for individual cognitive abilities and emotionsS. cerevisiae, differential pathways can be created by co-ordinated expression of different combinations of ohnologues that have been largely partitioned into obligate sub-networks100. Switching from one sub-network to another allows the yeast to adapt to different stresses, and to reconfigure fluxes through metabolism to enable growth on different types and quantities of sugars104.InS. cerevisiae however, as mammalian ohnologues have been identified that are convergence points for regulated interactions with 14-3-3 proteins in response to multiple stimuli106.The phosphoprotein-binding 14-3-3 proteins interact with hundreds of (phosphorylated) ohnologue proteins in mammalian cells, suggesting that regulated phosphorylation of 14-3-3 docking sites provides a large-scale mechanism for switching from one set of sister ohnologues to another. Indeed, case studies indicate that one set of ohnologues within a cell may be phosphorylated and consequently bind to 14-3-3s when cells are stimulated by insulin for example, while other combinations of sister ohnologues bind to 14-3-3s in response to phosphorylations that are promoted by growth factors, nutrient stress and adrenalin. Partitioning of ohnologues into obligate sub-networks does not seem to be so clear-cut as inThese few examples—and the aforementioned ABCDE model of floral development—indicate how the parallelised signalling networks of ohnologues generated via WGDs act as multiple-input multiple-output systems. Collectively, these systems generate different cell phenotypes via differential expression and post-translational switching among sets of ohnologues with different kinetic and regulatory properties.108, and there are many examples of heterogeneous patterns of overexpressions and mutations across ohnologue gene families in cancers111. For example, overexpression of insulin receptor substrate 4 (IRS4) drives a subset of breast cancers, while IRS1 and IRS2 are not oncogenic in these cancers, even though all three IRS proteins activate PI 3-kinase–Akt growth signalling. The critical difference is that IRS4 lacks a negative feedback mechanism by which its sisters IRS1 and IRS2 can switch off the pathway via the tyrosine phosphatase SHP2109. This example illustrates how the parallel signalling pathways generated via WGDs can evolve specific regulatory interconnections, which in principle enable these systems to integrate inputs from multiple sensory stimuli, buffer signal noise via responsive feedback loops, and generate a wider repertoire of phenotypic outcomes than would be possible with the original simple circuit113.Deeper understanding of how post-WGD signalling networks operate will underpin advances in understanding polygenic disorders. For instance, mutations in many ohnologue genes are associated with neurological and psychiatric diseases and developmental disorders such as RASopathies114. It will be fascinating to compare the neural network topologies that have been built from gene families generated via WGDs versus those made of multiple small-scale duplications in humans and octopuses.Finally, it should be noted that WGDs do not underpin every evolutionary leap: A WGD was hypothesised to explain why cephalopods  are behaviourally more sophisticated than other molluscs. However, the octopus genome shows no evidence of a WGD. Rather, sensory intelligence in cephalopods is likely to be underpinned by the massively expanded gene families of C2H2 zinc-finger transcription factors and protocadherins that are expressed in their neuronal and sensitive tissues


A growing body of evidence implies that maternal inflammation during pregnancy is associated with increased risk of neurodevelopmental disorders in the offspring. The pathophysiological mechanisms by which maternal inflammation evokes fetal brain injury and contributes to long-term adverse neurological outcomes are not completely understood. In this review, we summarize 10 years of our research experience on maternal inflammation and the implications upon the fetal/offspring brain. We review our findings regarding the underlying mechanisms that connects maternal inflammation and fetal brain injuries ; we discuss our imaging, pathological and behavioral test results which support brain damage following maternal inflammation; and finally we describe some of the therapeutic strategies which might prevent the damage. For many years, intrapartum hypoxia/asphyxia was considered the main etiology for cerebral palsy and fetal brain injuries. In reality, new epidemiological studies have revealed that asphyxia accounts for only 20% of the cases of cerebral palsy, and 70% of the cases arise even before the onset of labor.To understand the mechanisms associated with human fetal brain injury, a model with similar developmental milestones and brain structure proportions should be found. The insult should be delivered at an equivalent stage of intrauterine brain development for an identical period of vulnerability. Measurement of the outcomes of the insult should be feasible by acceptable methods. Clearly, no animal model can fully mimic the development of the human brain, and each species has its own characteristics.Several types of animal models have been suggested for elucidating the mechanisms that link prenatal inflammation and adverse fetal brain development.The development and maturation of the human brain is a complex and continuous process. Primary neurulation occurs during weeks 3–4, neuronal proliferation during months 3–4, migration during months 3–5, and myelination begins during the second trimester and continues into adulthood. Hence, as mentioned above, the timing of an insult is crucial to compare and evaluate the neurodevelopmental response of offspring. While early insult is associated with structural brain abnormalities such as neural tube defects (primary neurulation), later insults may disrupt the migration process of post-mitotic neurons and lead to aberrant cortical development.33Both epidemiological and experimental studies in animals have demonstrated that maternal infection can damage the developing brain.Proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin (IL) 1β, and IL-6 from astrocytes and microglia may directly damage oligodendrocytes and neurons. For example, the injection of IL-1β leads to neuronal death and delayed myelination in neonatal rats.42In light of the potential contribution of cytokines to fetal neurological injury, we first sought to evaluate fetal inflammatory responses to maternal inflammation. One common method of mimicking inflammation associated with infection in animal models is through the exposure of modified proteins. Lipopolysaccharide is a Gram-negative bacterial component that mimics bacterial infections, and LPS exposure was found to induce the production of IL-6, IL-1β, and TNF-α. We used intraperitoneal (i.p.) injections of LPS to induce maternal systemic inflammation in pregnant rats.43In response to maternal i.p. injections of LPS, TNF-α was the first cytokine to peak; this was followed by a quick return to baseline in both maternal circulation and amniotic fluid. Interleukin-1β and IL-6 had delayed responses. Assessment of changes in the mRNA levels of these proinflammatory cytokines revealed that LPS induced increases in TNF-α, IL-6, and IL-10, both in the chorio-amnion and in the placenta. Similar results were demonstrated in the fetal brain .Our results imply that acute maternal inflammation can affect the proinflammatory status of the amniotic fluid, and the chorio-amnion membranes, fetal serum, and brain. All of them are associated with fetal brain injury. Furthermore, we recognize that time does matter. In early gestation, the fetal anti-inflammatory response is decreased due to the immature immune system. Therefore, the earlier maternal inflammation occurs, the worse the prognosis.Increased maternal oxidative stress in pregnancy is associated with an increased risk of poor pregnancy outcome. Several studies have shown that markers of oxidative stress are increased during maternal inflammation. Following maternal LPS injection, elevated oxidative stress has been observed in rat fetus brains several hours and days after delivery.In another study,Our group pioneered the use of magnetic resonance imaging (MRI) to demonstrate the long-term consequences of maternal inflammation on fetal brain development. By using advanced MRI methods,As evident by MRI, offspring of LPS-treated dams exhibited white and gray matter injury. Many of these areas were in regions known to be involved in learning and memory, including the dorsal striatum, medial septal nucleus, entorhinal cortex, and molecular dentate gyrus.23Since maternal infection/inflammation increases offspring brain cytokines and free radicals, both anti-inflammatory substances and free radical scavengers might have therapeutic properties that prevent fetal adverse neurologic outcomes.During the last decade, several prospective studies in pregnant patients have demonstrated the neuroprotective effect of magnesium sulfate (MG) in preventing preterm white matter brain injury.Recently we reportedMaternal LPS at E18 induces brain injury in offspring at 25 days of age, evident by MRI.As described before,Our results support the notion that MG protection is not limited to the white matter, but that it may protect the gray matter and cognitive functions of the offspring as well.N-acetyl cysteine (NAC) is an antioxidant that scavenges free oxygen radicals. In addition, NAC can act indirectly as a stimulant of synthesis of the anti-stressogenic agent glutathione.Following LPS-induced maternal inflammation, NAC administration significantly reduced pro-inflammatory cytokine expression in the maternal circulation, amniotic fluid, fetal blood, and most importantly in the fetal brain .14,16,1720Maternal NAC administration following the i.p. LPS-induced maternal inflammation prevented any visible damage to the offspring neonatal brainsOur findings suggest that NAC might attenuate the short- and long-term sequelae associated with a fetal brain inflammatory response in pregnant women affected by severe inflammation. Although these were encouraging results concerning the influence of NAC upon both fetal and maternal immune responses, further studies regarding this are still needed.Maternal inflammation during pregnancy is associated with an increased risk of neurodevelopmental disorders in the offspring. Activation and up-regulation of inflammatory cytokines and oxidative stress, both systemically and in the fetal brain, are thought to play a key role in altered brain development and may contribute to poor neurodevelopmental outcomes subsequent to maternal inflammation. Possible therapeutic intervention may include antioxidative and anti-inflammatory therapies aimed to reduce the severity and extent of the injury.In order to develop realistic therapeutic options, future research is needed to clarify the mechanisms linking maternal inflammation, fetal brain development, and neurological and behavioral deficits.


The opioid epidemic in the United States carries significant morbidity and mortality and requires a coordinated response among emergency providers, outpatient providers, public health departments, and communities. Anecdotally, providers across the spectrum of care at Massachusetts General Hospital (MGH) in Boston, MA have noticed that Charlestown, a community in northeast Boston, has been particularly impacted by the opioid epidemic and needs both emergency and longer-term resources. We hypothesized that geospatial analysis of the home addresses of patients presenting to the MGH emergency department (ED) with opioid-related emergencies might identify “hot spots” of opioid-related healthcare needs within Charlestown that could then be targeted for further investigation and resource deployment. Here, we present a geospatial analysis at the United States census tract level of the home addresses of all patients who presented to the MGH ED for opioid-related emergency visits between 7/1/2012 and 6/30/2015, including 191 visits from 100 addresses in Charlestown, MA. Among the six census tracts that comprise Charlestown, we find a 9.5-fold difference in opioid-related ED visits, with 45% of all opioid-related visits from Charlestown originating in tract 040401. The signal from this census tract remains strong after adjusting for population differences between census tracts, and while this tract is one of the higher utilizing census tracts in Charlestown of the MGH ED for all cause visits, it also has a 2.9-fold higher rate of opioid-related visits than the remainder of Charlestown. Identifying this hot spot of opioid-related emergency needs within Charlestown may help re-distribute existing resources efficiently, empower community and ED-based physicians to advocate for their patients, and serve as a catalyst for partnerships between MGH and local community groups. More broadly, this analysis demonstrates that EDs can use geospatial analysis to address the emergency and longer-term health needs of the communities they are designed to serve. The United States is facing an opioid epidemic defined by historically high rates of opioid associated overdoses and deaths. More than 2.4 million Americans have a severe opioid-use disorder (OUD) and mortality from opioid use is now the leading cause of accidental death in the United States , 2. In 2Charlestown is a community of approximately 16.5 thousand people in the northeast of the city, which is perennially described as a particularly high-risk community with regards to opioids –10. WithHere, we present a geospatial analysis of the home addresses of patients from Charlestown presenting to the Massachusetts General Hospital (MGH) emergency department (ED) with opioid-related emergencies and identify a hotspot at census tract 040401. This analysis was motivated by an interest in utilizing ED visit data to identify “hot spots” of high opiod-related healthcare utilization within Charlestown to target further investigation, resource deployment, and community-based responses to change the course of the opioid overdose epidemic. Furthermore, while a single site analysis by definition does not consider visits to other EDs or health centers, it does highlight community level risk as viewed from the common model of the dyad of a “mothership” hospital and a “satellite” health center embedded in that community.We performed a geospatial analysis of home address locations based on spatially aggregated patient record data extracted from MGH ED records for all visits to the MGH ED between 2012 and 2015. Self-reported home address, chief complaint, diagnosis on discharge from the emergency department, diagnosis on discharge from the hospital, and basic demographics were collected for all patients who presented to the MGH ED between July 1, 2012 and June 30, 2015. International Statistical Classification of Diseases and Related Health Problem codes were not available in this dataset, so text-based discharge diagnoses were used. If a patient was discharged from the ED, the recorded hospital discharge diagnosis would be the same as the ED discharge diagnosis; if the patient was admitted to the hospital, the hospital discharge diagnosis reflected the diagnosis on discharge from the hospital which may or may not be the same as the ED discharge diagnosis.In July 2014, the MGH ED changed electronic medical record (EMR) providers and diagnosis upon discharge from the ED was no longer recorded distinctly from hospital discharge diagnosis during the remainder of the study period . Where available before the EMR transition, both ED and hospital discharge diagnoses were examined for risk of opioid overdose, as explained below. After the EMR transition, only hospital discharge diagnosis was considered as this was all the available data. All data manipulation and statistical analyses were developed and executed using the R programming language [Opioid-related ED visits were identified using a text search of chief complaint and discharge diagnosis selecting for terms involving narcotic overdose or drug abuse, or involving the name of a specific narcotic medication or naloxone. Specific terms searched for were: "HEROIN", "OVERDOSE", "OPIOID", "OPIATE", "METHADONE", "OPIUM", "NARCOTIC", "NARCAN", "NALOXONE", "FENTANYL", "OXYCODONE", "OXYCONTIN", "PERCOCET", "MORPHINE", "MORPHONE", "DILAUDID", "HYDROMORPHONE", "MS CONTIN", "HYDROCODONE", "CODINE", "TRAMADOL", "DRUG ABUSE", “DRUG”, and “VICODIN”.. Since the chief complaint for each visit was entered as free-text, partial string matching executed using the “grepl” function in R was employed to account for lexical variability. To improve the specificity of this approach, we also searched for matches to the terms “DRUG RASH", "EXPERIMENTAL DRUG", "DRUG RX", "DRUG REACTION", and "DRUGGED" which were structurally similar to the terms of interest but which were thought unlikely to represent opioid-related ED visits and visits with chief complaints or discharge diagnoses matching these second set of terms were excluded from the analysis.Addresses in MA were geocoded using the United States Census Geocoder using address batch geography lookups matched to US Census 2010 data vintage . To de-iBetween July 1, 2012 and June 30, 2015, there were 313,085 patient visits to the MGH ED from 151,250 distinct addresses, including 286,555 patient visits from 132,253 addresses in Massachusetts. From Charlestown, there were 10,577 visits during the study period from 2,822 addresses. Among these 10,577 visits, we identified 210 visits from 111 addresses as opioid-related ED visits; 9,777 visits (92.4%) including 191 opioid-related visits (91.0%) were successfully geocoded. There was no significant association between ability to geocode a visit from Charlestown and whether the visit was opioid-related or not . These 191 geocoded, opioid-related visits from 100 addresses in Charlestown were the basis for further geospatial analysis described below.Census-tract level analysis showed obvious heterogeneity and a 9.5-fold variability in opioid-related ED visits per census tract with the 86 opioid-related ED visits  originating from addresses in census-tract 040401 compared to only 9 (0.5%) opioid-related ED visits from census-tract 040100. There was also a large variability in the number of visits from each address within a census tract, with between 1 and 21 opioid related ED visits per address. To account for the known variability in population across census tracts in Charlestown, we compared the observed distributions of opioid-related ED visits and addresses with opioid-related ED visits to theoretical expected distributions based on population weighting. Since people living in different areas may utilize emergency health resources at different rates, we looked at census-tract level differences in any-cause visit rates to the MGH ED. Our study characterizes geographic variations in opioid-related ED visits among census tracts in Charlestown, Massachusetts. The wide heterogeneity observed was not driven by overall ED usage rates or population size differences between census tracts. These results emphasize that while geographically small, Charlestown is a community exhibiting significant spatial heterogeneity in opioid-related ED utilization with substructure showing a spatial hot spot at census tract 040401. Within Charlestown, these data can be used to facilitate both further investigation into factors effecting opioid use disorder and opioid-related healthcare utilization, as well as efficient distribution of local resources addressing the opioid epidemic. These may include emergency response resources such as bystander naloxone as well as longer term responses such as addiction treatment centers. Additionally, the underlying reason why census tract 040401 is a hotspot of opioid-related ED visits is unclear, and further work is needed to better explore and understand the medical, social, and structural factors in this area.In a broader sense, this study demonstrates the potential value of emergency department data in informing health policy interventions in response to the opioid epidemic and other public health crises. Continuing to collect, and appropriately analyze, emergency visit data and other data that can be geospatially analyzed may serve as a key tool in understanding the potential for place-based patterns of health outcomes and health care utilization . In instFurthermore, this analysis highlights how EDs are uniquely positioned within health systems to provide critical intelligence to other parts of the healthcare system, particularly health clinics and other outpatient facilities, about the risks community facilities may wish to address . This inIn this study, we examined the utility of geospatial analysis of opioid-related ED visits from an a prioi selected community to the ED of a single quaternary care center. Our dataset therefore excludes ED visits to other nearby hospitals, health centers, and urgent cares, so our results should be interpreted more as exploring how an ED can interface with a specific community to guide resource distribution than as providing all-encompassing information about Charlestown specifically. Additionally, people do not necessarily use opioids where they live, so targeting resources to census tracts identified by home address will likely only address part of the opioid-related risk a community carries. Future work should incorporate ED visits to other nearby emergency departments as well as pre-hospital data on emergency medical response to opioid overdoses to further characterize community level distribution of risk.S1 FigStripchart of the number of opioid-related ED visits per address across each census tract in Charlestown, MA. Each circle represents a distinct address within a given census tract. A spatial jitter has been applied to facilitate viewing degenerate points.(TIF)Click here for additional data file.


Auditory input plays an important role in the development of body-related processes. The absence of auditory input in deafness is understood to have a significant, and even irreversible, impact on these processes. The ability to map touch on the body is an important element of body-related processing. In this research, the crossed-arm temporal order judgment (TOJ) task was used to evaluate the spatial mapping of touch. This task elicits a conflict between visual and somatosensory body-related information through a change in posture. We used the crossed-arm TOJ task to evaluate the spatial mapping of touch in deaf participants. Results suggested that a change in posture had a greater impact on congenitally deaf participant TOJ than for hearing participants. This provides the first evidence for the role of early auditory exposure on spatial mapping of touch. More importantly, most deaf participants had auditory prosthetics which provided auditory input. This suggests an important, and possibly irreversible, impact of early auditory deprivation on this body-related process. The representation and control of our body relies on the ability to perceive and distinguish our limbs in space, independent of their posture . This prThe crossed-arm TOJ task , 4] is  is 4] isThe crossed-arm TOJ task is a multisensory task where vision plays an important role along with touch . VisiA recent investigation by Noel & Wallace  on the i13 deaf  and 13 hearing group participants  took part in the study  and hearing group participants . Results from this analysis suggested significant different PCD scores between groups (t(20.146) = -3.373, p = .003). This result indicates that deaf participants had significantly higher TOJ error rates for the crossed posture  = 1.035, p = 0.319, ηp2 = 0.041).Individual scores for proportion of right-hand first answers across different SOA are shown in ture see . Mean grThe objective of this study was to investigate spatial mapping of touch in the deaf using the crossed-arm TOJ task. Results for the task revealed that deaf participants had a higher mean PCD group score, indicating more TOJ errors when crossing the arms, compared to hearing control group members. Results from the non-speeded reaction times for the crossed-arm task revealed no difference between groups. These results suggest that a period of deafness permanently alters spatial mapping of touch, but does not alter the time required to provide that judgment.Many deaf participants provided inverted TOF responses when they crossed their arms see , which cOur results provide an important insight on deafness and the spatial mapping of touch for SOA up to 400 ms. However, future analysis on deaf participants will benefit from longer SOAs to reveal if deaf error rates are along an N-curve , 27], o, o27], oOur findings are consistent with the crossed-arm TOJ results from Noel & Wallace  where paIndeed, two studies have now indicated unimpaired crossed-arm performance in the early blind adults  14]. Th. Th14]. Yamamoto and Kitazawa  proposedThe effects of deafness on tactile abilities are highly variable. Some investigations on body perception in the deaf have reported no differences in tactile perception , whiThese results are the first to investigate the interaction of internal and external frames of references in deaf individuals. The posterior parietal cortex (PPC) has been suggested to play a key role in the interaction of frames of reference in hearing individuals . SeveralParticipants all had similar onset of hearing loss, duration of hearing loss, hearing aids use, and modes of communication. These factors have been revealed to critically impact plasticity and performance in the deaf . FurtheS1 FileIndividual scores for each SOA and individual PCD scores for each hearing (n = 13) and deaf participants (n = 13).(XLSX)Click here for additional data file.


In vitro modeling of complex diseases is now a possibility with the use of patient-derived induced pluripotent stem (iPS) cells. Their stem cell properties, including self-renewal and their potential to virtually differentiate into any cell type, emphasize their importance as a translational tool for modeling disorders that so far have been limited by the unavailability of primary cell lines, animal models, or inaccessible human materials. Around 100 genes with germline mutations have been described to be responsible for cancer predisposition. Familial cancers are usually diagnosed earlier in life since these patients already carry the first transforming hit. Deriving iPS cells from patients suffering from familial cancers provides a valuable tool for understanding the mechanisms underlying pediatric cancer onset and progression since they require less mutation recurrence than adult cancers to develop. At the same time, some familial mutations are found in sporadic cases and are a valuable prognostic tool. Patient-derived iPS cells from germline malignancies can also create new tools in developing specific drugs with more personalized-therapy strategies. Together with John Gurdon’s pioneering experiments during the 1960’s3, it demonstrated that mature and fully differentiated cells can be reprogrammed into a pluripotent state (similar to embryonic stem (ES) cells) with the potential to differentiate into any cellular lineage. Reprogramming of human somatic cells to induced pluripotent stem (iPS) cells overcomes many of the ethical and technical limitations of ES cells, and iPS cells can easily be generated from a tissue biopsy, blood cells, or tumor sample from virtually any person with or without a diagnosis4. iPS cells express similar markers as ES cells, they are capable of self-renewal, and importantly, are able to differentiate in vitro and in vivo into cell types of all three germ layers, thus giving rise to a diverse panel of cells. The unlimited supply of disease-relevant cells have made iPS cells a great tool for studying human diseases, especially for those that previously have been restricted to postmortem samples due to inaccessibility of patient material.The discovery in 2007 by Yamanaka and colleagues that a combination of just four transcription factors (TFs), Oct4, Sox2, Klf4, and c-Myc (OSKM), were able to revert adult human somatic cells back to pluripotency have had a huge impact on basic research, regenerative research, and cancer research5 and Familial dysautonomia6, both monogenic Mendelian diseases, as well as chromosomal diseases such as Down’s syndrome9. However, most diseases are not hereditary but sporadic and are genetically complex with mutations at multiple loci and have a late onset with low penetrance, thus considered much more challenging to model.Until now most disease models using iPS cells have focused on diseases caused by mutations in a single gene, with early disease onset, and often with high penetrance, such as Fragile X syndrome12. However, these are not the only challenges our increasingly aging society face, but also other common pathologies such as cancer and cardiovascular disorders.Nevertheless, complex diseases with sporadic occurrence and late onset have successfully been modeled using disease-specific iPS cells, where vast majority includes neurodegenerative diseases13. This shows the need for developing new models based on disease-relevant human cells to identify the right biomarkers useful for treatment. Most human cancer cell models used to date are based on immortalized cancer cell lines and xenografts studies using cells from established tumors. While these techniques are important for understanding mechanisms operating in late-stage tumor development, they will not model tumor initiation and early progression. Therefore, focusing on end point events of the disease might have given us false positives of what changes are actually driving the disease. Moreover, it may also obscure potential early biomarkers that could be translated into the clinic. Using iPS cells for modeling cancer onset and progression could overcome the disadvantages with current techniques and give us new important insight into tumor initiation and development.Cancer, is among the leading cause of death after cardiovascular diseases in developed countries, firmly linked not just to genetics but also environmental factors and especially due to the increasing age of the population. Since cancer is a multi-step disease and many stages occur before the actual malignancy is developed and detected, there is an urge to understand the genetic mechanisms that are altered from onset to progression of disease. Mouse models of cancer have brought extremely insightful information in terms of understanding cancer progression mechanisms. However, many therapeutic drugs that have shown excellent efficacy in mouse models have failed in human clinical trials14 or by using ectopic expression of TFs to produce iPS cells  cell line, KBM716. Injection of the resulting CML-iPS cells subcutaneously into NOD-SCID mice produced teratomas that contained cells from all three germ layers. The CML-iPS cells were able to differentiate in vitro into cells that express CD43 (pan-T cell marker), CD45 (hematopoietic lineage marker), as well as the stem cell marker CD34, indicating a restoration of differentiation ability into hematopoietic lineages. The parental CML cell lines were dependent on the BCR–ABL pathway; however, the CML-iPS cell lines showed resistance to imatinib, an inhibitor of BCR–ABL signaling, despite expressing the BCR–ABL gene. The CML-iPS cells regained sensitivity to imatinib when differentiated in vitro to hematopoietic lineage cells, suggesting that oncogenic dependency of the CML-iPS cells depended on differentiation status of the cells17.The emerging need of new and improved cancer models has accelerated the optimization of cancer cell line reprogramming. So far, two different ways for reprogramming somatic cells to pluripotency have been described, by somatic cell nuclear transfer ((SCNT) implantation of a somatic nucleus cell into a enucleated oocyte)18, gastric cancer19, glioblastoma20, and pancreatic ductal adenocarcinoma (PDAC)21, among others. Not all reports have described the use of iPS as a model for cancer but solely the success of using this technique to generate pluripotent cells. However, some studies have gone further and showed the use of cancer cell-derived iPS cells to model the disease , and CML27. Even though ectopic expression of pluripotency genes such as reprogramming factors can be achieved using non-integrating techniques28, we have to take in consideration that the OSKM factors are also implicated as potent cancer drivers. In addition, it has been suggested that incomplete reprogramming could lead to cancer development. Using a mouse model with inducible OSKM factors, Ohnishi et al. 29 showed that long-term (4 weeks) in vivo activation of OSKM factors resulted in reprogramming and teratoma formation in vivo, however, a shorter late activation in vivo for 3–9 days resulted in tumor development in various somatic tissues consisting of undifferentiated dysplastic cells and global changes in DNA methylation patterns. The tumor cells could be reprogrammed into iPS cells by OSKM activation for 2 weeks in vitro. When the iPS cells were injected into blastocysts, they gave rise to non-neoplastic normal kidney cells in the chimeric mice29. These findings suggest that epigenetic processes associated with iPS cell reprogramming may also drive cancer development and that this genetic transformation is reversible. Consequently, taking this approach as a potential treatment has to be carefully revised. Due to tumor heterogeneity we might be selecting for clonal subpopulations explaining the discrepancy of reports whether reprogramming cancer cells may alleviate the tumorigenic potential or not.Cancer is not solely driven by genetic defects, extensive research has identified epigenetic changes in cancer cells30. Instead of reprogramming genetically unstable cancer cells with multiple mutations, tumor development could be studied in genetically stable cells carrying only what is thought to be the original driver mutation  database have identified that 43% of cancer predisposition genes with germline mutations overlaps with somatic driver mutationsion Fig. . Using t31. Therefore, iPS cell lines from a BRCA1-carrier with a 5382insC mutation, predicted to be high risk for breast and ovarian cancer, were successfully generated. Although the study did not investigate the cellular phenotype or tumorigenic potential of BRCA1 patient-derived iPS cells32, it encourages further use of this methodology for deeper understanding of BRCA-driven breast cancers. LFS is an autosomal dominant syndrome caused by mutations in the p53 tumor suppressor gene. LFS patients usually develop early onset of a variety of tumors including osteosarcoma (OS), breast cancer, sarcoma, and leukemia33. Non-cancerous fibroblasts from LFS patients with a germline p53 mutation (G245D), also commonly found in sporadic tumors, were reprogrammed into iPS cells. Since one of the major cancers affecting LFS families is OS, they went on to study the effect of this p53 mutation in osteoblasts (OB) differentiation. They found OB derived from LFS-iPS recapitulated OS characteristics, including defective differentiation and tumorigenic potential. Moreover, they found impaired expression of the imprinted H19 gene in LFS-derived OB and more interestingly, restoration of H19 expression improved OB differentiation. LFS mouse models have not been able to fully recapitulate the disease, however by using LFS patient-derived iPS cells they could recapitulate OS features and gene expression signatures and also identify significant dysregulated genes with potential clinical implications34.The tumor suppressors BRCA1/2 play an important role in DNA repair, and mutations in BRCA1/2 are found in familial as well as sporadic breast cancers. Even though mouse models have been informative, differences between mouse and human have to be taken into consideration. As an example, BRCA1 mutation in a single allele has been found to promote genomic instability in human cells but not in murine cells37. Importantly, the majority of these genes are also mutated in sporadic cancers; therefore, generating iPS cells from non-cancerous somatic cells carrying HNPCC-germline mutations would be the perfect alternative for studying not only colon cancer but also the contribution of these genes to other type of cancers. In addition, the differentiation potential of iPS cells into diverse cell lineages might undercover new biological functions of the familial mutated genes and their role in tissue-specific tumor development.In addition, other types of familial cancers could benefit from this type of modeling. For example, lynch syndrome or hereditary non-polyposis colorectal cancer (HNPCC), an autosomal dominant disorder is linked not solely to colon cancer but predisposes to ovarian, endometrium and other cancers that could also be studied using patient-derived lynch syndrome iPS cells. The DNA mismatch repair genes MLH1, MSH2, MSH6, and PMS2, are frequently mutated in HNPCC38. This does not only pose a problem for cancer research but also for other age-related diseases such as neurodegeneration and cardiovascular disorders. For these reasons, several strategies have been developed for increasing the cellular age of iPS cells, including adding chemical compounds that accelerate differentiation and maturation39, transfections of genes that accelerate terminal differentiation40, or promote overall aging by increasing genomic instability (progeria accelerated cellular aging in vitro)41. However, considering that iPS cells and their derivatives in many aspects mimic early stages of human development, they are an attractive model for studying early onset diseases such as childhood cancers.One caveat of using iPS-derived cells to model cancer onset is the immature/young state of the derived cells. The reprogramming process resets the biological clock in the cells, acquiring an early embryonic identity rather than cells of an aging adult tissue42. Childhood cancers generally develop rapidly with fewer genetic aberrations compared to adult cancers, which is thought to be due to defects affecting stem or progenitor cells present during normal embryonic development. The 5-year all-cancer survival in children is around 80%, significantly better than for adults, however, certain cancer types such as pediatric CNS tumors have considerably lower survival rates  is still a hurdle, although some advances have been made. HSCs isolated from iPSC-generated teratomas were capable of restoring the hematopoietic system of immunocompromised mice51. Also direct conversion from somatic cells to HSCs was attempted by using combination of specific TFs such as HoxB453, panmyeloid (ETV2 and GATA2) and erythro-megakaryocytic (GATA2 and TAL1)54, although without the capability of long-term engraftment. However, the addition of strong TFs that directs differentiation towards a specific lineage also raises concerns for its use in a clinical setting. Recently, by direct conversion of iPS cells to hemogenic endothelium (HE)55 in combination with in vivo screening for specific HSCs factors, seven factors were found to be able to convert HE into HSCs that engrafted myeloid, B and T cells in mice and showed multi-lineage potential56, a promising step closer to new treatment options and more reliable models of hematological malignancies.First-line treatment for leukemia often includes whole-body irradiation and transplantation of bone marrow-derived human hematopoietic stem cells (HSCs), peripheral blood, or umbilical cord stem cells. However, limitations arise due to low availability of human leukocyte antigen (HLA) match donors and low yield of HSCs for engraftment, decreasing the chance of successful treatments. The importance of correct donor-host HLA matching is crucial for avoiding graft-vs.-host disease and graft rejections57, so far there has been no attempt of reprogramming cells with these mutations. However, iPS cells have been generated from leukemia-causing somatic mutations. JMLL is initiated by deregulation of cytokine receptor signaling that causes enhanced myelopoiesis. Gandre-Babbe et al. generated iPS cells from malignant cells from two JMLL patients with somatic heterozygous missense mutations in PTPN1126, which encodes SHP-2, a non-receptor tyrosine phosphatase. These JMLL iPS cells could be differentiated into myeloid cells that behaved similarly to primary JMML cells, showing increased proliferative capacity, constitutive activation of granulocyte macrophage colony-stimulating factor (GM-CSF), and enhanced STAT5/ERK phosphorylation. Using iPS cell-derived myeloid cells for drug screening, MEK and JAK1/2 kinases inhibitors have been identified as new treatment options supporting clinical trials for MEK inhibition in JMLL patients.The most common germline mutations in familial leukemia are CEBPA, RUNX1, and GATA258. Recently it was reported that primary blasts from AML patients with MLL rearrangements could be reprogrammed into iPS cells. Patient-derived iPS cells reset epigenetic changes but retained the original genetic rearrangements. Interestingly, only AML-iPS cells that were differentiated into hematopoietic cells gave rise to leukemia in mice, whereas differentiation into other cellular lineages did not. In addition, they generated iPS cells from individual subclones from the AML patients that was used to design therapies targeting each subclone59. Moreover, Kotini et al. were able to show similarities by mimicking hematopoiesis to myelodysplastic syndrome and AML by introducing or correcting mutations using CRISPR–Cas960.Translocations at chromosome 11q23 involving the MLL gene is a frequent event in pediatric ALL and AML, and have been associated with an intermediate to poor outcome61. Medulloblastoma (MB) is one of the most frequent brain tumors, accounting for 20% of all brain tumors62. Despite advances in MB treatments have increased the survival rates in the past years63, post-treatment consequences can lead to severe and permanent neurocognitive defects64. Four sub-classifications can be distinguished within MB; wingless (WNT), sonic hedgehog (SHH) named for the signaling pathways primarily affected during MB pathogenesis, and group 3 and 4, where the pathogenesis of the last two groups is still currently unknown66.Brain tumors and other central nervous system (CNS) cancers are the second most common malignancies in children67. However, their molecular basis can be used to understand the onset and progression of this disease. The SHH and WNT-subtypes of MB constitute about 41% of total MB68. Germline mutations in SHH pathway components, PTCH1, SMO and SUFU predispose to different pathologies that include higher risk of MB69. Around 15% of sporadic MB cases presents any of this mutations70. Gorlin syndrome or nevoid basal cell carcinoma (NBCCS) is a hereditary disease caused by mutations in the PTCH1 gene71. These patients develop different conditions, including basal cell carcinomas (BCC) and congenital skeletal abnormalities, but also other malignancies can be manifested such as MB72. Germline SUFU mutations also lead to infant desmoplastic MB69 and children develop similar symptoms as Gorlin patients73. Mutations in the APC gene are associated with familial adenomatous polyposis and Turcot syndrome. People with Turcot syndrome have an increased risk of developing MB. Modeling human MB using iPS cells from patients with MB-associated syndromes carrying any of these germline mutations could give new insights in how SHH and WNT signaling pathways contribute to MB and as well as initiation of other cancers with the same mutations. Importantly, the understanding of the role of these genes during normal human development is possible by using this type of approach74.Genetic syndromes associated with MB are rare75. Most typical genetic aberrations found in sporadic NB are copy-number alterations; MYCN amplifications, gain of chromosomes 11q, and 17q, and loss of 1p36, as well as different point mutations76. Although rare, germline mutations in anaplastic lymphoma kinase (ALK) and PHOX2B have been found in hereditary NB. PHOX2B, plays an important role during autonomic nervous system development, have been found involved in congenital central hypoventilation syndrome and associated with Hirschsprung disease77 and also found mutated in sporadic tumors78. Similarly, expression patterns of the tyrosine kinase receptor ALK suggest key roles in mammalian nervous system development79. ALK alterations have been described in multiple human cancers, most frequently translocations resulting in ALK fusion proteins in anaplastic large cell lymphoma, inflammatory myofibroblastic tumors and non-small cell lung cancers82. Identification of activating ALK point mutation in familial and sporadic NB opened up new possibilities for using targeted therapy such as Tyrosine kinase inhibitors. Here again, the use of iPS cells derived from patients carrying PHOX2B or ALK germline mutations could be used as models for understanding NB onset and development, other related syndromes and also their role in normal human development.Neuroblastoma (NB) is an embryonic tumor believed to originate from neural crest cells during the development of the sympathetic nervous system. Tumors are often found in sympathetic nervous tissues such as the adrenal gland and paraspinal ganglia of young infants. Although NB incidence is relatively low, the 5-year mortality rate in children with high-risk NB is high and is mostly due to relapse cases83.Pediatric sarcomas is a heterogeneous group of tumors that can be divided into soft tissue sarcomas, mainly rhabdomyosarcomas, and bone sarcomas, most common are Ewing’s sarcoma and osteosarcoma. Although present in young children, sarcomas are mainly occurring in adolescents and young adults. Primary treatment is surgery followed by chemotherapy, and although there has been a tremendous increase in survival the last decades there are still about one third of sarcoma patients that succumb to their disease Fig. . Several84. Interestingly, re-differentiation of EWS-iPS recovered drug sensitivity, suggesting that differentiation status is important for drug responses and the undifferentiated state of iPS cells could be used to study drug resistance mechanisms.In addition to the modeling of sarcoma development using iPS cells derived from Li Fraumeni patients or sarcoma cell lines, iPS cells derived from sarcoma patients have also been used to assess drug resistance mechanisms in Ewing’s sarcoma (EWS). The majority of EWS tumors carry the t chromosomal translocation generating the EWS–FLI1 fusion protein, a promiscuous TF that activates pro-oncogenic programs. Reprogramming of the Ewing’s sarcoma cell line, CHLA-10, into iPS cells (EWS-iPS) showed that although the expression of EWS–FL1 fusion transcript between CHLA-10 and EWS-iPS is similar, EWS-iPS developed resistance to compounds targeting EWS–FL1 driven pathways60. Cancer tissue specificity is an important issue to address. Germline mutations are present in every cell of the body, nevertheless, tumor development is usually restricted to a specific tissue or organ. Due to the differentiation potential of iPS cells, and the unlimited expandable source of material, it can be an important tool for understanding specific genetic mechanisms operating during this specification. In addition, transplanting disease-relevant cell types derived from patient iPS cells into an established microenviroment by using orthotopic xenograft models could generate new in vivo systems for modeling diseases.The discovery of iPS cells opens up a wide spectrum of possible future applications including development of new treatments in regenerative medicine, generation of better and more accurate disease models, and improving drug discovery. Where other tools fail, using patient-specific iPS cells for modeling diseases have an incredible potential to improve our understanding of basic mechanisms operating during healthy and diseased human development and differentiation. With the development of precision editing techniques such as the CRISPR/Cas9 system there is now the possibility to tailor genomic modifications in iPS cell lines, giving new opportunities for disease modeling and for understanding the multi-step tumorigenic process. As proof of principle, with help of CRISPR/Cas9, mutations can be introduced or corrected and their effect on tumor development studiedChildhood cancers and familial cancers develop with shorter latency compared to adult sporadic tumors. It is thought that this is due to the transforming event coincides with mechanisms operating during normal human development. Taking advantage of the immature state of iPS cells would provide us with new potent models to study early events in cancer initiation and progression.


Cumulative evidence suggests that trait rumination can be defined as an abstract information processing mode, which leads people to constantly anticipate the likely impact of present events on future events and experiences. A previous study with remitted depressed patients suggested that enhanced rumination tendencies distort brain mechanisms of anticipatory processes associated with reward and loss cues. In the present study, we explored the impact of trait rumination on neural activity during reward and loss anticipation among never-depressed people. We analyzed the data of 37 healthy controls, who performed the monetary incentive delay (MID) task which was designed for the simultaneous measurement of the anticipation  and consumption (hedonic) phase of reward processing, during functional magnetic resonance imaging (fMRI). Our results show that rumination—after controlling for age, gender, and current mood—significantly influenced neural responses to reward (win) cues compared to loss cues. Blood-oxygenation-level-dependent (BOLD) activity in the left inferior frontal gyrus (IFG) triangularis, left anterior insula, and left rolandic operculum was positively related to Ruminative Response Scale (RRS) scores. We did not detect any significant rumination-related activations associated with win-neutral or loss-neutral cues and with reward or loss consumption. Our results highlight the influence of trait rumination on reward anticipation in a non-depressed sample. They also suggest that for never-depressed ruminators rewarding cues are more salient than loss cues. BOLD response during reward consumption did not relate to rumination, suggesting that rumination mainly relates to processing of the motivational (wanting) aspect of reward rather than the hedonic (liking) aspect, at least in the absence of pathological mood. Rumination, according to the widely used Response Style Theory, is a passive and repetitive thinking process, which focuses one's attention to depressive symptoms, and to reasons and consequences of these symptoms  representations is the emotional charge which, regardless of its valence , will strongly influence the estimation processes and leads to emotional extrapolation. This means that ruminators build their future anticipations on the emotional tone of expected future events  rumination in depressed participants was associated with generally reduced sensitivity to punishment cues compared to reward cues and reduced sensitivity to the probability that a stimulus would be associated with punishment  right-handed volunteers were included in the present study. Participants aged between 18 and 38 years were recruited via newspaper and university advertisements. All volunteers were tested for eligibility. Inclusion criteria were right-handedness assessed with a standardized handedness questionnaire  with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Scientific and Research Ethics Committee of the Medical Research Council (Hungary).Ruminative Response Scale (RRS) of the widely used Response Style Questionnaire . The internal consistency of the RRS was good (Cronbach α = 0.89). The total RRS score was used in the correlational analysis.Rumination was assessed by the Zung Self-Rating Depression Scale , affective (“I feel down-hearted and blue”) and somatic (“I have trouble sleeping at night”) dimensions. Participants were asked to score the items on a 1–4 scale . Internal consistency of the ZSDS proved to be good (Cronbach α = 0.83). The total ZSDS score was used in the correlational analysis. Besides the RRS and ZSDS the test battery contained basic background questions regarding age, sex, ethnicity, family, and personal psychiatric history.Depressive symptoms were measured by the validated Hungarian adaptation of the The self-report measures were taken some days before the scan sessions, in order to avoid any unwanted rumination induction.Subjects performed a variant of the classic monetary incentive delay task  on a laptop out of scanner. To maximize engagement, participants were told that after the experiment they would take part in a prize draw to potentially win the money collected during the MID task.*-weighted echo-planar imaging sequence  with 3 × 3 mm in-plane resolution and contiguous 3-mm slices providing whole brain coverage. A series of high-resolution anatomical images were also acquired during the functional imaging session using a T1-weighted 3D TFE sequence with 1 × 1 × 1 mm resolution.Functional MRI data acquisition was performed on a 3T MRI scanner  using a BOLD-sensitive T2t-tests and correlation analyses as appropriate. All statistical testing used a two-tailed p < 0.05 threshold.Behavioral data including reaction times in response to the target were recorded using E-prime 2.0 software . Demographic and behavioral data were analyzed in SPSS version 22.0  using repeated measures ANOVA, http://www.fil.ion.ucl.ac.uk/spm/software/spm12/; Friston et al., Functional imaging data were analyzed with Statistical Parametric Mapping 12 software  hemodynamic responses were modeled in a mixed design using the general linear model. Each of the three types of incentive cues  were modeled as blocks, while the five types of feedback  were modeled with event-specific regressors. One additional regressor was defined for motion correction using Artifact Detection Tools .To investigate the rumination dependent activations associated with reward and loss anticipation  and consumption  whole brain regression analyses were conducted during the second level analysis. Every contrast served as dependent factor and the individual rumination scores were included in the analysis as covariates along with the age, sex, and Zung depression scores. A whole brain analysis were carried out at a Activated clusters were identified with WFU PickAtlas toolbox, which is based on the Talairach Daemon database , and the mean ZSDS score was 34.76 (SD = 6.52). The ZSDS scores were below the level indicating depression (48 points) and corresponded to the Hungarian healthy average score . Neither of the two constructs showed any sex differences .The mean RRS score was 48.49 . Subjects responded more quickly both to reward cues  and to loss cues  compared to neutral cues . The cue × RRS interaction was not significant , indicating that reaction times of participants did not vary according to rumination between the three incentive conditions.A repeated measures ANOVA was conducted on the reaction times. A main effect of cue was observed , right insula, orbitofrontal cortex and superior frontal gyrus, within the eight significant clusters. Loss consumption contrast [loss outcome-neutral outcome] showed positive activations in two clusters covering the regions of the right insula, bilateral lingual gyrus and orbital part of the inferior frontal gyrus (IFG). The reward vs. loss consumption contrast [win outcome-loss outcome] yielded increased activations in multiple regions within nine clusters such as the ACC, right putamen, and right fusiform gyrus  compared to loss anticipation (loss cues). More precisely, trait rumination -controlled for age, sex, and depressed mood-correlated with one significant cluster of 311 voxels, where the peaks were at the left anterior insula, left inferior frontal gyrus pars triangularis (IFG), and left rolandic operculum. We did not detect any significant rumination-related activations yielded for win-neutral and loss-neutral cues, and reward or loss consumption (win-loss outcome).Our results might indicate elevated sensitivity to reward cues among ruminators, since during reward anticipation they tended to more actively recruit brain areas together, such as anterior insula (AI) and IFG, which are implicated in the salience network  response, and whether reward or punishment (loss) signals are more important for ruminators.Before we interpret the role of rumination on this matter, it is worthwhile to discuss the neuronal activity involved in reward/punishment processing in general. Evidence from animal studies  yielded activations for consumption. However, when inter-individual differences such as depressive mood or rumination were also modeled in our fMRI analyses, AI yielded positive activation for reward anticipation, but not for the consumption phase of reward processing. Our results are in line with the results of Strigo et al.  who inveAccording to some previous results . Although, the exact role of RO in rumination is unclear, evidence suggests that increased RO activation is correlated with some anticipatory processes. For instance, obese adolescents showed greater activations in RO during anticipation of food reward than thin counterparts  we can conclude that participants were indeed motivated. In addition, we are aware that we used a liberal primary threshold during the analysis which can be arguable, but since this is an exploratory study and the significant activated cluster was not too large we think this was a valid decision. Finally, since few studies have previously investigated the neural background of rumination and reward/loss processing  anticipation and consumption in healthy participants. Our results suggest that trait rumination has a significant influence on the anticipation but not on the consumption of rewards. These results might suggest that rumination alters processing of the motivational (wanting) aspect but not the hedonic (liking) aspect of monetary reward, at least in the absence of pathological mood. Further studies will be needed to investigate how rumination affects reward-related prediction error, when there is a difference between the expected and the experienced reward.GJ and GK conceived and designed the study. NK, ES, AÉ, AG, and DP were responsible for subject recruitment and data collection. Data analysis were performed by NK and GK with special assistance from LK and GJ. RE, GB, and GJ contributed to the interpretation to the data. NK, GK wrote the manuscript and all authors provided critical revision to its further development. All authors read and approved the final manuscript.The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: the study was supported by the MTA-SE-NAP B Genetic Brain Imaging Migraine Research Group, Hungarian Academy of Sciences, Semmelweis University (Grant No. KTIA_NAP_13-2-2015-0001); and the Hungarian Academy of Sciences (MTA-SE Neuropsychopharmacology and Neurochemistry Research Group). LK was supported by the Bolyai Research Fellowship Program of the Hungarian Academy of Sciences. RE received consultancy fees from Cambridge Cognition and P1 vital.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.