Adaptive response to stress is a fundamental property of living systems. At the cellular level, many different types of stress elicit an essentially limited repertoire of adaptive responses. Epigenetic changes are the main mechanism for medium- to long-term adaptation to accumulated  stress. We propose the adaptive deregulation of the epigenome in response to stress (ADERS) hypothesis which assumes that the unspecific adaptive stress response grows stronger with the increasing stress level, epigenetically activating response gene clusters while progressively deregulating other cellular processes. The balance between the unspecific adaptive response and the general epigenetic deregulation is critical because a strong response can lead to pathology, particularly to malignant transformation. The main idea of our hypothesis is the continuum traversed by a cell subjected to accumulated stress, which lies between an unspecific adaptive response and pathological deregulation—the two extremes sharing the same underlying cause, which is a manifestation of a unified epigenetically mediated adaptive response to stress. The evolutionary potential of epigenetic regulation in multigenerational adaptation is speculatively discussed in the light of neo-Lamarckism. Finally, an approach to testing the proposed hypothesis is presented, relying on either the publicly available datasets or on conducting new experiments. Any departure from a narrow window of optimal physical, chemical, or biological parameters represents stress, to which all living systems react in an adaptive manner aiming to restore homeostasis, either by influencing their environment or undergoing internal adaptation which enables the new situation to be tolerated. The repertoire of stress responses, especially at the cellular level, is relatively limited when compared with the number of apparently different sources of environmental stress. The idea of unified pathways responding to multiple related or even seemingly unrelated stressors has already been formulated in the context of the gatekeeper hypothesis . The celDamage to the genome, usually in the form of depurination or formation of pyrimidine dimers, elicits a specific repair response mediated by the p53 tumor suppressor and closely connected to other stress-response pathways . This tyAn evolutionary conserved and ancient adaptation pathway is the heat-shock response which, interestingly, activates molecular chaperones not only in response to heat-induced protein unfolding but also to unfolding caused by heavy metal ions, ethanol, or other toxins . The proThe autophagy response, where proteins and defective organelles are degraded by lysosomes, is stimulated by multiple forms of stress: starvation, hypoxia, reactive oxygen species, DNA damage, protein aggregates, and pathogens . This imAnother type of adaptation pathway with a role in cancer is the hypoxia-inducible factor (HIF) response to hypoxia , which pA hub connecting many stress response pathways is the mTOR kinase, itself primarily responsible for nutrient sensing and adaptation to nutrient stress . Its cenPerhaps the best illustration for selective regulation of targeted groups of genes in response to stress is the micro RNA (miRNA) mechanism , based oTranslational reprogramming  is anothMultiple related (or even apparently unrelated) sources of stress elicit unified responses at the cellular level—activation of appropriate “effectors”, which are sets of genes encoding components of the appropriate metabolic pathways responsible for restoring homeostasis. Existence of such functional gene clusters points to the possible role of epigenetic regulation in medium- to long-term adaptation to specific types of stress.Early observations in plants suggest that stress can cause whole genome changes such as large-scale recombination, chromosomal breakage-fusion cycles, and loss of chromosomes that would lead to genome remodeling with likely phenotypic changes . These gOrganisms are able to regulate genome activity in response to stress through specific gene-environment interactions mediated by epigenetic mechanisms, which include DNA methylation, histone modifications, chromatin remodeling activities, and action of various small noncoding RNAs. Abundant evidence suggests that epigenetic pathways are not only targets of stochastic malfunction caused by stress but also parts of the programmed adaptive responses  and TGF-β1 (the major autocrine and paracrine fibrogenic growth factor) [We are, however, interested in the gene-environment interactions mediated by epigenetic mechanisms that result in long-lasting effects. Epigenetic marks are particularly sensitive during the early stages of development, at which point they are influenced by factors such as nutrition, toxins, mother's behavior, and stress. The events form this early life can be strong predictors of adult phenotype, that is, pathological phenotype , 20. Alt factor) . Transge factor) , 26–35.The role of transgenerational epigenetic inheritance in evolution has been discussed by Jablonka and Raz in their extensive review , where tThe Lamarckian concept of evolution assumes the direct influence of the environment on the heritable traits of an organism. This view of evolution and the underlying driving forces has been superseded by the well-known principle of evolution by natural selection. However, considering the role of epigenetics in adaptation—especially the long-term epigenetic adaptation—we can envision a scenario where the environmental factors directly shape heritable traits of an organism.C. elegans that persists over 5 generations, induced by exposure of the animals to a specific, biologically relevant physiological challenge [The notable example of aphids producing a larger proportion of winged offspring when exposed to the alarm pheromone  clearly hallenge , captureYet, we can envision a way to permanently record the ephemeral epigenetic adaptive change in the genome, although the explanation stays speculative . The mosIt is tempting to speculate that an increase in the mutation rate of an epigenetically silenced (hypermethylated) gene might open a window for a permanent record of an epigenetic change in the genome. Assuming that the epigenetic change was an adaptive response to the changing environment, an increase in the mutation rate of the affected gene could be seen as the missing link in the chain of events needed to connect an external influence with a permanent adaptive change in the genome. The probability for such an event would depend on the time the gene (or its promoter) spends in the methylated state, thus linking the mutation rate with the duration of the external stimuli leading to epigenetic adaptation. The idea that epigenetically regulated regions have a higher susceptibility to mutation, which can drive evolution, has been discussed previously , althougThe existence of such proposed “Lamarckian cycle” has yet to be supported by experimental or observational evidence. While confirming its possible role in evolution would be very interesting from the conceptual and the theoretical standpoint, its quantitative contribution as an evolutionary force would in any case be limited in both magnitude and scope, completely overshadowed by—and secondary to—the classical natural selection, to which it would relate as a directed mutation-generating mechanism.Mutations arising by the proposed mechanism would be unique in being guided directly by epigenetic adaptation to produce a more fit phenotype, representing a form of genetic assimilation . The genIn the previous sections, adaptive cellular response to stress has been explained in terms of activation of genes for several metabolic response pathways, each of them responding to several seemingly unrelated but fundamentally connected sources of stress. Arguments for the pivotal role of epigenetics in long-term adaptation and transgenerational inheritance have been presented. At this stage, we can introduce the adaptive deregulation of the epigenome in response to stress (ADERS) hypothesis .It is safe to assume that minor disruptions of cellular processes, brought about by low levels of stress, are quickly dealt with by the appropriate mechanisms, which can restore homeostasis without significant damage to the cell or long-term consequences for cellular function or integrity. However, as the stress level increases or stress becomes constant, more adjustments are necessary to achieve the same goal, which means that activity of more genes (or whole metabolic pathways) needs to be adjusted. We can define accumulated stress as any intense, repeated, or long-term stress—the key being prolonged duration and relatively high intensity—enough to cause a measurable disruption of cellular processes over a time which is long enough to necessitate long-term adaptation.Stress accumulation is mirrored in the accumulation of adaptive responses—it is not the departure from homeostasis  but the long-term activation of stress response pathways that defines the “accumulated” stress.In response to stress accumulation, a cell traverses the adaptive response continuum . At low The apparent epigenetic deregulation can be quantitatively measured as the increase in Shannon entropy (linked to loss of information content) of the epigenome—a feature shared by stress, aging, and cancer, as discussed by Hannum and coworkers in their recent study . In thisAbove a certain level of accumulated stress, epigenetic changes become the primary factor in coordinating adaptive gene expression –44, thusIndeed, epigenetic changes resulting from exposure to accumulated stress  show a striking similarity to changes found in many types of cancer  and seemIn summary, the ADERS hypothesis states that malignant transformation  is facilitated by epigenetically mediated extreme adaptation to accumulated stress. Striking similarities between epigenetic profiles of cells surviving severe stress and cancer cells speak in favor of the hypothesis.Any viable hypothesis needs to be testable in practice; therefore, we propose a plausible way to do this. The main type of experimental data needed is transcription (microarray) and methylation (methylation chip) datasets from different cell types subjected to various types of stress. While such experiments only require common equipment and well-established procedures, many of the needed datasets can be found in publicly available databases of transcription profiling and methylation experiments.A systematic approach to testing the ADERS hypothesis using expression and methylation data would necessarily involve the following steps: (i) demonstrating a global genome response to various types of stress, (ii) showing that accumulated stress triggers epigenetic changes which promote long-term adaptation by activating the global stress response, (iii) with the response increasing as stress accumulates, manifested by epigenetically mediated activation of progressively more stress response gene clusters, and finally, (iv) a link needs to be established between the epigenetic deregulation and pathology, especially malignant transformation.A global response to different types of stress, an idea already supported by some experimental evidence and articulated in the gatekeeper hypothesis , would aDemonstrating long-term epigenetically mediated adaptation to accumulated stress would require a similar approach—gene expression profiling. Long-term adaptation could be demonstrated by retention of a key part of the expression profile acquired under stress .Carefully following the progression and the development of an established transcriptional response to a defined type of stress from essentially zero to a level defined as accumulated stress could give insight into various stages of gene (in)activation. Analyzing DNA methylation at selected time points would give insight into the time point at which epigenetic deregulation becomes operational, thus clarifying the role of epigenetics in adaptation. Discovery of a temporal pattern of gene (or gene ontology class) expression would be in line with the proposed hypothesis, provided it could be demonstrated that progressively more stress-response clusters are activated as stress is accumulated.To link expressional deregulation to pathology, one would need to identify the genes involved in disease—particularly malignant transformation—which are differentially methylated at the later stages of stress accumulation. Alternatively, it might suffice to show that the deregulated gene expression and changed methylation patterns have indeed appeared as a result of stress accumulation. There is already sufficient evidence connecting stem-cell-like expression profile with many types of cancer .We propose the adaptive deregulation of the epigenome in response to stress (ADERS) hypothesis, which considers epigenetically mediated deregulation of transcription under accumulated stress as a means by which cells attempt to restore homeostasis. Finding the right balance between activating many types of adaptive responses and maintaining tight control over gene expression—both factors dependent on the level of transcriptional deregulation—is the key for maximizing adaptability to environmental influences while minimizing the threat of malignant transformation or other pathologies. Proving the hypothesis would give insight into pathological processes—especially malignant transformation—and their relationship to various types of stress, possibly highlighting novel approaches to treatment of disease. Evolutionary aspects of epigenetic inheritance warrant further investigation on their own merit.


Haematopoiesis or blood development has long served as a model system for adult stem cell biology. Moreover, when combined, the various cancers of the blood represent one of the commonest human malignancies. Large numbers of researchers have therefore dedicated their scientific careers to studying haematopoiesis for more than a century. Throughout this period, many new technologies have first been applied towards the study of blood cells, and the research fields of normal and malignant haematopoiesis have also been some of the earliest adopters of genome-scale technologies. This has resulted in significant new insights with implications ranging from basic biological mechanisms to patient diagnosis and prognosis and also produced lessons likely to be relevant for many other areas of biomedical research. This paper discusses the current state of play for a range of genome-scale applications within haemopoiesis research, including gene expression profiling, ChIP-sequencing, genomewide association analysis, and cancer genome sequencing. A concluding outlook section explores likely future areas of progress as well as potential technological and educational bottlenecks. Haematopoiesis represents the process whereby multipotential blood stem and progenitor cells differentiate into more than 10 distinct mature blood cell types. Research over the last 30 years has led to the development of purification protocols that permit the isolation of many of these progenitor and all mature cell types at close to 100% purity. Moreover, biological assays have been developed to validate the functional properties for most of these different cell types including the many progenitors at various stages of maturity. Consequently, differentiation of the blood system is better defined than any other mammalian organ system and has hence become a model system for the wider field of stem cell biology.Since many of the mature blood cell types are short lived, they need to be constantly replenished throughout adult life, with the consequence that the blood system has one of the fastest turnovers of all human organ systems. Production of the various types of mature blood cells is tightly controlled, with transcription factor and signalling proteins playing particularly prominent roles –5. Long-The various types of human leukaemias all share the property of perturbed blood cell production, often with an accumulation of the so-called blast cells that resemble immature blood progenitor cells . With trThe relative ease of accessing blood cells compared with most other human tissues likely is a major reason as to why several advanced approaches for the extraction of new biological knowledge from large-scale gene expression profiling datasets have been pioneered in studies using blood cells. Below I focus in particular on gene expression profiling studies aimed at regulatory network reconstruction and disease classification.With the ever-increasing momentum of genome-scale science driven by, for example, human genome project , 13, gencis-elements were employed to further define the regulatory circuitry, which lead to the definition of densely interconnected cis-regulatory circuits and a number of transcription factors where their differential expression across the different hematopoietic lineages was inferred to be involved in the generation of distinct cellular states. Together with earlier human studies [Coexpression across more extended differentiation hierarchies has more recently been utilised for the definition of haematopoietic gene sets as well as the inference of regulatory networks . Here th studies  as well  studies –30, thisGene expression profiling has also been employed widely across a range of different haematological malignancies, in order to identify new classification schemes with potential diagnostic, prognostic, and/or therapeutic value. One of the early protagonists in the field has been the group of Louis Staudt, who in 2000 reported distinct types of diffuse large B-cell lymphoma through the use of gene expression profiling . DiffuseWith the ever-increasing availability of expression profiling datasets, efforts have been initiated to collate expression data from diverse studies into unified data collections with a view to providing powerful data mining platforms. One example is represented by BloodExpress, an expression profiling resource for mouse haematopoiesis . ThroughWhile BloodExpress demonstrated the feasibility of integrating gene expression profiles from diverse laboratories, cross-dataset normalisation did represent a formidable challenge, so that much of the expression information was eventually discretised in a binary “on/off” fashion, thus giving some confidence to expression states but also losing potentially vital information in quantitative expression changes. Parallel efforts have therefore been initiated to generate unified expression profiling resources generated by a single laboratory or facility. One particularly prominent example here is the hematopoietic fingerprints database from the Goodell lab . The hemComparable expression profiling compendia have more recently also been generated for the human haematopoietic system. The first such study generated a web-accessible resource termed the HaemAtlas . This reA particularly exciting recent development has been an attempt to generate an expression profiling compendia that will allow for an absolute expression quantification . Here moWhile a universally accepted definition of epigenetics as well as epigenomics has this far eluded the field, the current consensus view is that modification to the DNA sequence  as well as posttranslational modification to chromatin proteins  represents key aspects of the epigenetic control of gene regulation. Following the advent of high-throughput sequencing technologies, it was quickly realised that genomewide analysis of histone modification status is particularly amenable to this new technology. To this end, the sheared chromatin fragments are subjected to sequencing following immunoprecipitation with suitable antibodies (chromatin immunoprecipitation or ChIP), with the whole technique commonly referred to as ChIP-Seq. Posttranslational modifications indicating both active and repressed transcriptional status are well recognized and have been mapped at genome scale in a variety of both mouse and human blood cell types –80.It is hoped that genome-scale chromatin maps will eventually provide information complementary to gene expression profiling, with implication for human disease prediction, diagnosis, prognosis, and treatment. Indeed, the European Union invested close to 30 million euro in 2011 into its new BLUEPRINT initiative . BLUEPRIGenome-scale information on DNA methylation can be obtained using a variety of approaches, with direct sequencing of the entire genomic DNA following bisulfite treatment perhaps representing the most comprehensive approach. Indeed, dramatic improvements and falling costs of high throughput sequencing have made bisulfite sequencing (BS-Seq) a viable option for the global analysis of DNA methylation –86. The When using antibodies that specifically recognise transcription factor proteins, the ChIP-Seq technology is also readily amenable to determining genomewide transcription factor binding maps. Indeed, one of the earliest reports using this technology mapped the locations of the NRSF transcriptional regulator in a T-lymphoid cell line . TranscrTranscription factor ChIP-Seq maps have also been utilised to map the genomewide locations of leukaemogenic factors. One recent example mapped the locations of the RUNX1/ETO fusion protein, which is a leukaemia-initiating transcription factor that interferes with RUNX1 function . Global Genomewide association studies (GWASs) examine many common genetic variants in different individuals to see if any variant is associated with a particular trait. GWAS surveys typically focus on associations between single-nucleotide polymorphisms (SNPs) and traits for major diseases, but can also identify variants that are linked to phenotypic variation seen in normal populations. Blood parameters such as the numbers of a particular blood cell type per millilitre of blood vary between different individuals, and much of this variation is thought to be genetic. Since elevated or reduced levels of certain blood cell types can predispose to diseases, GWAS studies have been performed to identify some of the underlying variants. In particular, blood platelets play a pivotal role in cardiovascular disease through their involvement in atherothrombosis following coronary artery plaque rupture. Not only does the platelet response to such an event vary between individuals, but also this variation is largely genetically controlled. A recent integrated systems biology approach performed high density genotyping of 110 genes in a cohort of more than 500 individuals with known levels of platelet response, which was followed by gene expression profiling and proteomics studies on platelets from individuals with the so-called “extreme end” response phenotypes . With thGWAS studies are commonly followed up by meta-analysis of several independent studies. Analysis of 66,867 individuals of European ancestry followed by extensive biological and functional assessment identified 68 loci reliably associated with platelet count and volume . ExpressIdentification of the whole complement of genes that are mutated in human cancers and therefore drive malignant transformation has been a central aim of cancer research ever since the advent of recombinant DNA technology. Following the development of ultra-high-throughput sequencing technologies and the subsequent reductions in the costs of sequencing whole genomes, projects have been initiated in Europe, America, and Asia to decode entire genomes from multiple individuals for a wide range of human cancers. Identification of somatically acquired mutations is thought to provide the most direct route towards delineating genes critical to the development of human cancers, yet it is not entirely clear at this stage what will be the most reliable techniques to distinguish these so-called “driver” mutations from the so-called “passenger” mutations; that are also found in the tumour but occurred by chance without providing a selective advantage to tumour growth and/or survival.Within haematopoiesis research, cancer genome sequencing has now been applied to several different haematological malignancies , 131–145A more recent study investigating cancer genomes in patients with acute myeloid leukaemia (AML) specifically addressed the question of passenger and driver mutations . Of noteThe application of new genome-scale technologies has resulted in an unprecedented increase in the amounts of data available for hypothesis generation in biomedical research. However, the extent to which this explosion in data has contributed to an actual increase in mechanistic understanding has so far been less impressive. To some extent this is to be expected, since the new datasets have been available at most for a few years, and mechanistic studies often have longer timescales. However, there are also cultural, training/educational, and technical issues that need to be overcome to accelerate the exploitation of new datasets.In terms of research culture, it has become apparent that many investigators have found a comfortable niche for themselves, where essentially they end up with repeating the same data collection exercise every couple of years, each time with the next version of whatever genome-scale technology they have become an expert in . This however avoids asking the much harder question whether it is possible to decipher the meaning of any underlying biology from a genome-scale observation and how this might be exploited to deliver actual biomedical advances. Journals and funding agencies should pay more attention to this issue and provide better rewards to those investigators willing to tackle the harder mechanistic questions. Just because an experiment has cost millions of dollars and used the latest technology, this on its own, does not mean that it will have a lasting value.The educational issue is that the exploitation of genome-scale datasets requires biologists with a robust understanding of bioinformatics and statistics and ideally also a significant knowledge of computer programming languages. Universities are increasingly offering relevant courses, yet the demand for bioinformatically trained biologists is still an outstripping supply. Moreover, the longer-term career path for such scientists is not clear either, at least in academic settings, because of the tensions between the need to performing primary, intellectual novel research in order to achieve academic promotions, which does not blend well with carrying out a bioinformatic support function.Finally, there are also scientific conceptual issues that impede interpretation and thus exploitation of genome-scale datasets. For example, it is clear that cell fate decisions are made by individual cells, and that indeed there is substantial heterogeneity within a given biological cell population –152. GenGiven the rapid pace of recent technological innovations particularly in terms of generating descriptive data , the major roadblocks will be to enhance the throughput of downstream functional studies. Importantly, many of the technologies developed to generate the largely descriptive genome-scale maps can also be adapted to multiplex and thus accelerate the analysis of downstream functional assays. For example, high-throughput next generation sequencing can be adapted to many biological assays that require counting. Reporter gene assays can be replaced by measuring transcript abundance rather than luciferase or lacZ enzyme activity, and through the inclusion of sequence tags, many different promoters can be assayed simultaneously . Another


Drosophila eye development, a wave of differentiation follows a morphogenetic furrow progressing across the eye imaginal disc. This is subject to negative regulation attributed to the HLH repressor proteins Hairy and Extramacrochaete. Recent studies identify negative feedback on the bHLH gene daughterless as one of the main functions of extramacrochaete. Here the role of hairy was assessed in relation to daughterless and other HLH genes. Hairy was not found to regulate the expression of Daughterless, Extramacrochaete or Atonal, and Hairy expression was largely unregulated by these other genes. Null alleles of hairy did not alter the rate or pattern of differentiation, either alone or in the absence of Extramacrochaete. These findings question whether hairy is an important regulator of the progression of retinal differentiation in Drosophila, alone or redundantly with extramacrochaete.In  Drosophila neural retina begins at the posterior margin of the third instar larval eye imaginal disc. The specification of the founder R8 photoreceptor precursor cells accompanies the ‘morphogenetic furrow’, a visible groove that moves anteriorly across the eye disc epithelium atonal (ato), the proneural gene responsible for R8 specification, in a band of cells just anterior to the morphogenetic furrow. Notch signaling and lateral inhibition refine Ato expression from this band to the array of single R8 precursor cells that each found one ommatidium. Posterior to the morphogenetic furrow, R8 cells then recruit precursors of other photoreceptor cell types, some of which then express Hh to keep the furrow progressing. The whole retina is differentiating once the morphogenetic furrow has crossed the eye primordium, which takes about two days Neural progenitor cells are specified within proneural regions controlled by members of the helix-loop-helix (HLH) protein family. Differentiation of ∼800 ommatidia in the hairy null mutations nor emc hypomorphic mutations affect morphogenetic furrow progression by themselves, clones of the double mutant combination result in significant faster furrow progression. This observation, along with the expression pattern of the genes, suggested that hairy and emc regulate furrow progression by redundant or overlapping mechanisms In addition to the positive regulation by Hh and Dpp, morphogenetic furrow progression is thought to be regulated negatively by two nuclear HLH proteins, Hairy and Extramacrochaetae . Althougemc has been described as part of a regulatory network of HLH genes emc are in fact mediated by derepressed expression of another HLH protein, Daughterless (Da) Drosophila E-protein, functions as the essential heterodimer partner of Atonal in the eye da expression, emc, the Drosophila homolog of mammalian Inhibitor of DNA-binding (Id) proteins, encodes a HLH protein without the basic DNA-binding domain and so inhibits Ato and Da activity through inactive heterodimer formation Recently, emc was thought to act along with hairy in regulating furrow progression, these recent findings prompted us to examine how hairy fits into the emerging network of HLH protein cross-regulation. Because hairy also encodes a repressor HLH protein, hairy might target da expression, like emc does. Unlike Emc, Hairy is a bHLH protein that acts as a classical transcriptional repressor by sequence specific DNA binding, rather than by heterodimerization with proneural bHLH proteins achaetaeAs emc mutations, clones of emc null mutant cells are now known to show faster morphogenetic furrow progression even in the presence of wild type hairy, and this is due to the elevated Da expression in such clones so that emc da double mutant clones no longer accelerate the furrow emc null alleles compared to 1emc suggests that complete removal of hairy and emc together should have a stronger phenotype still and reveal full extent of negative regulation of differentiation by HLH proteins.Since the original studies of hypomorphic emc, hairy, da and the progression of differentiation. We report that hairy does not seem to be regulated by or a regulator of the Da/Emc network. In addition, we find that hairy null alleles have no effect on morphogenetic furrow movement in the complete absence of emc, challenging the view that these two genes regulate morphogenetic furrow progression together. In fact, our studies of hairy null mutations have yet to identify any specific role for this gene in regulating the morphogenetic furrow.Here we explore regulatory relationships between hairy activity in clones homozygous for the null allele 22h. This allele contains a stop codon within the basic region, so that a truncated protein lacking DNA-binding, dimerization, or repressor domains is predicted 22h cells lacked almost all Hairy antigen, with little consequence for retinal differentiation h clones . These data indicate that da is not directly required to regulate Hairy expression in the same cells, but is responsible for the expression of signals that affect Hairy expression cell-nonautonomously. Hairy downregulation in the furrow requires Notch and Hh signaling cell-autonomously da could affect Hairy expression because both Hh and the Notch ligand Dl are expressed by differentiated cells and depend on ato and da function da clones, but in this case the signals that initiate Hairy expression in the anterior eye are not completely known, except that Dpp signaling contributes Both Emc and Da expression depend on indirect . Specifiundaries . Similaremc regulates Hairy expression, clones of cells null for emc were examined. Hairy expression also changed little in the emc null mutant clones  proteins, which contain similar domains to Hairy, can also repress gene expression when targeted to particular genes by protein-protein interactions Drosophila Hairy is not known to heterodimerize with Emc or any of its proneural gene targets da transcription in a subtle way only revealed in the 1emc backgrounds. For example, Hairy repression of da transcription might be redundant in the presence of wild type emc, and not sufficient to impact da autoregulation in the complete absence of Emc. Detailed information concerning the thresholds of da transcription under different conditions would be required to assess this model.Enhancement of the emc. The possibility remains that hairy may function in a subtle way, perhaps redundantly with other genes, or affect processes other than furrow progression, particularly since many questions remain to be resolved concerning the transcriptional regulation of eye development, such as how ato expression is initiated as the furrow progresses, or all the mechanisms by which the retinal determination genes contribute to eye development hairy gene in eye development, as has been suggested for regulatory pathways that are thought to contribute temperature stability in variable environments The Hairy expression ahead of the morphogenetic furrow certainly seems to provide a marker of an early stage of eye development emc clones and 11 emc h clones.Primary antibodies used were monoclonal mouse anti-Daughterless


In plants, the intercellular communication through the membranous channels called plasmodesmata  plays pivotal roles in the orchestration of development, defence responses, and viral propagation. PD are dynamic structures embedded in the plant cell wall that are defined by specialized domains of the endoplasmic reticulum (ER) and the plasma membrane (PM). PD structure and unique functions are guaranteed by their particular molecular composition. Yet, up to recent years and despite numerous approaches such as mutant screens, immunolocalization, or screening of random cDNAs, only few PD proteins had been conclusively identified and characterized. A clear breakthrough in the search of PD constituents came from mass-spectrometry-based proteomic approaches coupled with subcellular fractionation strategies. Due to their position, firmly anchored in the extracellular matrix, PD are notoriously difficult to isolate for biochemical analysis. Proteomic-based approaches have therefore first relied on the use of cell wall fractions containing embedded PD then on “free” PD fractions whereby PD membranes were released from the walls by enzymatic degradation. To discriminate between likely contaminants and PD protein candidates, bioinformatics tools have often been used in combination with proteomic approaches. GFP fusion proteins of selected candidates have confirmed the PD association of several protein families. Here we review the accomplishments and limitations of the proteomic-based strategies to unravel the functional and structural complexity of PD. We also discuss the role of the identified PD-associated proteins. In plants, intercellular communication must overcome the rigid pectocellulosic wall that encompasses all cells. To achieve that plants have developed membranous pores called plasmodesmata (PD) that perforate the extracellular matrix providing symplastic connections between most cell types Maule, . PD are For a long time, the sparse information available about PD constituents has hindered progress in our understanding as to how these membranous structures function. Over the last 30 years the search for PD proteins has been a constant topic of research and endeavors to identify them have employed a wide diversity of approaches  followed by successive low speed centrifugations to recover and wash wall fragments.Access to PD structures by subcellular fractionation is rendered difficult both by their location, embedded in the extracellular matrix, and by the small physical contribution they make to total plant tissue mass. In fact, PD are not simply inserted into the wall but firmly anchored into it, probably through the action of proteins and/or wall polymers, that would provide stable bridges between the PM and the wall  as a bait, the group of Bill Lucas identified a Non-Cell-Autonomous-Protein-Pathway1  or a Glycosyl Phosphatidyl Inositol (GPI) anchor. A conspicuous drawback of such selection strategy is that it precludes any PD proteins that would associate with PD by other means. A similar strategy was later on also applied to the Arabidopsis PD fraction which despite a major enrichment in PD-derived membranes gave rise a colossal proteomic dataset including likely contaminants (Fernandez-Calvino et al., Arabidopsis wall and PD fractions were searched using bioinformatic prediction programmes, databases, and published work. In each case about 10% of the proteins identified were shown to fulfill the criteria for PD association and were therefore elected for further analysis. Ultimate confirmation of the physical association of selected candidates with PD structures was then achieved through transient expression of GFP fusion products in leaves and eventually by immunolocalization with electron microscopy. So far, this approach resulted in the conclusive identification of several PD-associated proteins including Plasmodesmata Located Proteins (PDLP; Thomas et al., An alternative strategy consists on downstream analysis of the proteomic datasets generated using bioinformatic tools, databases, and literature sources. This approach was employed by the Maule laboratory following the establishment of Arabidopsis cell wall extracts a new family of receptor-like transmembrane proteins named PDLP which were later on shown to act as receptors for viral movement proteins (Amari et al., These proteomic-based studies, combined with functional analysis of identified PD components, have greatly contributed to elucidate PD organization and regulatory principles. For instance, an interesting finding was that PD house receptor-like activities, such as receptor-like kinases (Fernandez-Calvino et al., We must also consider that PD are physically and functionally connected with the endomembrane system. In addition to the continuity of the ER with the desmotubule, the vast majority of PD components identified to date use the secretory pathway for delivery to the channels. For instance, Golgi disrupting treatments prevent both PDLP1 and RGP2 from reaching PD (Sagi et al., The proteomic-based identification of PD components, combined with imaging techniques, pharmacological, and genetic approaches have brought substantial insight into the complexity of PD structure and dynamics. However, our understanding of PD function is still far from comprehensive and much remains to be determined before we fully comprehend the regulatory mechanisms governing symplastic transport. Many of the identified PD proteins still await functional characterization and advances in this area will provide exciting insights. Moreover, current findings concentrate on proteins with a membrane-localized signature, excluding for instance PD-associated soluble proteins or proteins transiently interacting with the channels which are both likely to be lost during PD purification due to extensive washes with salt containing buffer. Finally, many biological processes governed by symplastic transport probably come with a significant remodeling of PD constituents dictating that there are many more analyses to be done before functional PD components are fully described.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.


MicroRNAs (miRNAs) are small non-coding RNAs responsible of post-transcriptional regulation of gene expression through interaction with messenger RNAs (mRNAs). They are involved in important biological processes and are often dysregulated in a variety of diseases, including cancer and infections. Viruses also encode their own sets of miRNAs, which they use to control the expression of either the host’s genes and/or their own. In the past few years evidence of the presence of cellular miRNAs in extracellular human body fluids such as serum, plasma, saliva, and urine has accumulated. They have been found either cofractionate with the Argonaute2 protein or in membrane-bound vesicles such as exosomes. Although little is known about the role of circulating miRNAs, it has been demonstrated that miRNAs secreted by virus-infected cells are transferred to and act in uninfected recipient cells. In this work we summarize the current knowledge on viral circulating miRNAs and provide a few examples of computational prediction of their function. MicroRNAs (miRNAs) are the most studied and best characterized molecules in the class of small regulatory non-coding RNAs . They arViruses encode their own sets of miRNAs. Evidence shows that these miRNAs can act as self-regulators of viral gene expression and/or control host cell pathways through silencing of their nodes . Some viIn this mini review we summarize the current knowledge about circulating miRNAs and their potential regulatory functions, with particular emphasis on extracellular viral miRNAs. We report the promising results of the most recent studies and provide a few examples of computational prediction of viral miRNA function.Extracellular miRNAs have been recently identified stable in most biological fluids, including blood, urine, saliva, semen, cerebrospinal fluid, and breast milk . EvidencMicroRNA profiles of extracellular carriers show distinct sets of miRNAs than their parent cell-type, thus suggesting that some miRNAs might be transcribed only to be exported and not retained in the parent cell . SelectiCirculating miRNAs are highly stable and consistent among individuals of the same species. Specific miRNA expression signatures in extracellular environment have been identified in a variety of human diseases, including cancer and neurological diseases, revealing the diagnostic potential of circulating miRNAs as useful non-invasive biomarkers .in vitro studies have shown that miRNAs transferred by the different types of carriers are functional and can regulate gene expression in recipient cells.Several in vitro and provides atheroprotective effects in vivo  which exploits their intracellular vesicle trafficking pathways to release antigens and viral particles into the extracellular space and  T cells .Generally, viruses implement different strategies during infection essentially consisting in escaping the host immune system and facilitating the invasion and proliferation within the host. Observations suggest that the release of microvesicles containing specific cellular and viral components by infected cells contributes greatly to the preservation of the virus even in a hostile antiviral immune environment .Epstein–Barr virus (EBV), cytomegalovirus (CMV), and hepatitis C virus (HCV) have found means to evade immune responses and increase virus-fusing ability and infectivity by exploiting microvesicles, giving rise to a systematic distribution of viral agents from infected cells able to induce genetic and epigenetic modifications in recipient cells .Tumor-associated viruses, like EBV, may use exosomal transfer to manipulate the growth characteristics of neighboring cells and enhance tumor progression. In particular, exosomes released from nasopharingeal carcinoma (NPC) cells harboring latent EBV were shown to contain the EBV latent membrane protein 1 LMP1; , which iRNA interference (RNAi) most probably was originally selected as a primary mechanism of defense against harmful genetic elements such as viruses. It is of relevant interest that in the evolutionary selection this mechanism was in turn exploited by viruses to their advantage while, as suggested by The first report of viral-encoded miRNAs was published by The majority of natural viruses found to encode miRNAs have thus a DNA component to their replication cycle, can exploit the initiating host miRNA biogenesis machinery in the nucleus where they replicate, and cause long-term persistent infections. DNA viruses such as the ones belonging to the Herpesvirus, Polyomavirus, Ascovirus, Baculovirus, Iridovirus, and Adenovirus families clearly match these characteristics  along wiDespite the established case of BLV, viruses possessing positive or negative sense RNA or double-stranded RNA (dsRNA) genome are not widely accepted to naturally express miRNAs.Nevertheless, HIV-1 has been proven to encode two miRNAs and potentially a third. In fact, hiv1-mir-H1 was proven to be responsible for inducing apoptosis and repressing host gene expression , while hAll viral miRNAs can essentially be grouped into two classes: host analogs and virus-specific. Generally, though, their functions include prolonging longevity of infected cells, evading the immune response, and regulating host or viral genes to limit the lytic cycle. Interestingly, all these functions are essential for infections to be persistent.In fact, miRNAs are likely invisible to the adaptive immune system – a valuable trait for viruses that undergo persistent infection . Thus, iPreventing cell death seems an obvious advantage to viruses that cause persistent or latent infections. Several different viruses including Kaposi’s sarcoma-associated herpesvirus (KSHV), EBV, and Marek’s Disease Virus type 1 (MDV1) encode miRNAs that can play a subtle role in preventing apoptosis by targeting pro-apoptotic host genes and are also associated with tumorigenesis.in vivo. This further supported the proposal that exosomes could most likely serve as deliverers of small RNA due to their specialized biogenesis and presumed entry route  which could explain why exogenous exosomal miRNAs are capable of repressing targets in recipient cells at new subcellular compartments for RNAi activity such as late endosomes . Figure ). The results show that subsets of the targets are significantly involved in cancer pathways, in particular leiomyomatosis, and mesenchymal tumors, for which a connection with EBV had already been described (P < 0.0001), also previously described as related to EBV infections (P < 0.04). Furthermore, although the significance of the P-value is borderline (P < 0.4), it is worth to mention that the top tox functions reported by IPA include increased levels of alkaline phosphatase and LDH, tumour-marker characteristics which have been reported to be significant prognostic factors in metastatic NPC, often associated wih EBV infection (Table 1 summarizes the most significant associations.The correct identification of targets is fundamental to determine miRNA function. Computational miRNA target prediction is still a big challenge, mostly due to the fact that our knowledge about the mechanisms and the molecular rules of miRNA target recognition is still incomplete . Neverthescribed . Other sfections . The prenfection . Table 1These few examples clearly indicate that miRNA functional analysis tools can be of great help in studying the effects of circulating viral miRNAs, allowing the production of plausible hypotheses about their function and involvement in crucial cellular pathways, encouraging the development of more specific tools for computational investigation of cellular and extracellular viral miRNA.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.


Ecological explanations for the success and persistence of invasive species vastly outnumber evolutionary hypotheses, yet evolution is a fundamental process in the success of any species. The Evolution of Increased Competitive Ability (EICA) hypothesis (Blossey and Nötzold 1995) proposes that evolutionary change in response to release from coevolved herbivores is responsible for the success of many invasive plant species. Studies that evaluate this hypothesis have used different approaches to test whether invasive populations allocate fewer resources to defense and more to growth and competitive ability than do source populations, with mixed results. We conducted a meta-analysis of experimental tests of evolutionary change in the context of EICA. In contrast to previous reviews, there was no support across invasive species for EICA's predictions regarding defense or competitive ability, although invasive populations were more productive than conspecific native populations under noncompetitive conditions. We found broad support for genetically based changes in defense and competitive plant traits after introduction into new ranges, but not in the manner suggested by EICA. This review suggests that evolution occurs as a result of plant introduction and population expansion in invasive plant species, and may contribute to the invasiveness and persistence of some introduced species. In the research effort to identify and explain the success of invasive species in their new range, evolutionary explanations for invasiveness are rarely invoked. Only a small proportion of introduced species succeed in their new range, some attaining greater individual size or fitness or establishing populations of greater numbers or densities, than what might be predicted from their native range , or by measuring the growth and fitness of herbivores feeding on the plants, or by quantitating the degree of damage to the plants themselves. This variety of response variables may explain why published reviews explicitly addressing EICA have been qualitative, assessing evidence by comparing the number of significant studies for or against the hypothesis , and “introduced” range as a continent or group of continents where a species was not present preceding European colonialism.We used the search terms “EICA” or “evolution of increased competitive ability” and “ecology” with lemmatization to collect 45 papers from Web of Science in December 2010. In order to be included in the analyses, papers had to meet each of the following criteria: (1) Papers presented data from experiments that tested at least one prediction of the EICA hypothesis using at least one invasive plant species. Thus, studies that presented only the results of models, or in one case applied the EICA model to a fish system, were excluded; (2) Plants from both the introduced and home ranges of each invasive species were grown in a common environment (greenhouse or common garden) so that any variation expressed was due to underlying genetic differences, not to differences in environment or plasticity in response to environment; (3) Each of the introduced and the home ranges were represented by at least two geographically distinct populations. EICA emphasizes the difference between ranges, each of which is comprised of many populations growing under different abiotic and biotic conditions specific to geographically distinct locations. A comparison of only two populations, one from each range, confounds local, population-specific genetic structure with the genetic constraints (stochastic and selective) specific to each range. Including at least two populations from each range ensures that the question of genetic differences between groups is addressed at the scale of range and not population. Following application of these criteria, 27 studies were included in the analysis from which we collected 347 observations of the difference between home and introduced ranges of invasive species in defense, growth, or competitive characteristics .Papers reported comparisons between introduced and home ranges as F-statistics, Chi-squared scores, and t-scores from statistical tests, and in a few cases as mean values and standard deviations for each range. Each observation was converted to a Fischer's Z transformation of the correlation coefficient, except for observations of competitive ability. Competitive ability results were analyzed as natural logarithm-transformed response ratios, as most studies reported comparisons of competitive abilities of home- and introduced-range plants in this form. Positive Z-scores (or response ratios) indicate that the value of the response variable is higher in the introduced range than the home range, and negative Z-scores indicate that the response value is higher in the home range than the introduced range. In the case of response metrics that relate to plant defense, all Z-scores were multiplied by an appropriate weighting variable (−1 or 1) so that negative scores represented higher inferred defenses in the home-range plants and positive scores represented higher inferred defenses in the introduced-range plants.R) were calculated to indicate the number of nonsignificant, unpublished results that would render the summary effect size nonsignificant. If NR exceeded Rosenthal's identified minimum value (5n+10), the result was assumed to be robust against publication bias , indicating that improvements in experimental or analytic techniques were unlikely to explain trends in data.In addition to assessing defense traits, we created models that addressed three components of the hypothesis that there is a difference between ranges in competitive ability. The first model included measures of plant performance related to growth, including height, biomass, and growth rate. These measures were taken from plants from introduced and home ranges when all are grown under noncompetitive conditions, either alone in pots or in common gardens. The second model included measures of plant investment in reproduction, that is, fitness, including floral and seed mass and number, and number of vegetative offspring in plants for which asexual reproduction is important. Although EICA as originally formulated did not make specific predictions for reproductive allocation, reproductive traits have been correlated to abundance of invasive plant species within communities , indicating that there are a few species which support the defense predictions of EICA. We also considered whether expression of defenses would explain variation in leaf chemistry effect sizes, but found no difference in overall effect size between constitutive and induced defenses .We found no general support across invasive plant species for reduced defenses in the introduced range of invaders. There were no overall differences between home and introduced ranges within each species in defense characteristics measured as leaf chemical and physical traits , their eP = 0.86) or plant damage by herbivores (P = 0.76), but classifying herbivores by degree of feeding specialization or geographic range explains some of the heterogeneity in effect sizes. Classifying herbivores by feeding habits—generalist, specialist, or unclassified communities of herbivores—predicted 67% of the variation in effect size of herbivore performance, although these categories were not statistically significant (P = 0.048). There were trends toward a decline in performance of generalists and a rise in performance of specialists when both were placed on introduced plants, indicating that introduced plants tended toward higher defenses against generalists and lower defenses against specialists than home-range plants. However, the degree of herbivore specialization did not significantly explain variation in defenses as inferred from the amount of herbivore damage sustained by plants (P = 0.64). Herbivores were also categorized by geographic range; herbivores collected from the plant species' home range were more negatively impacted by feeding on home-range plants, while herbivores present in both ranges due to universal distribution or human introduction as biocontrol agents were equally impacted by defenses from home- versus introduced-range plants (P = 0.098) or herbivore-induced damage to plants (P = 0.067).Although there were no significant differences overall by plant range for herbivore performance or plant damage inflicted by herbivores, further classification of herbivores revealed significant variation in effect sizes for these metrics. Herbivore family did not explain variation in herbivore performance (e plants . Herbivoe plants . Plant sP = 0.0154). The fail-safe number for this result suggests that this effect is robust against publication biases. However, there was no corresponding difference by range in plant fitness (P = 0.811), although it did explain variation in competitive ability  in which some metric of defense was evaluated, which suggests that release from herbivory is not in general a powerful selective force upon plant defenses in invasive species. We found no support for EICA's prediction that leaf chemical and physical defense traits will in general be reduced in the introduced range. A recent meta-analysis which evaluated putatively generalist-specific defenses did find support for higher levels of leaf toxins in introduced ranges, as suggested by the SDH  and the quantitative genomic approaches (Beaumont and Balding The EICA research has focused primarily upon trait change within species, although the relative importance of stochastic forces as opposed to selective forces in this process (Keller and Taylor Selective factors including climate, resource availability, and biotic interactions may act in concert or in opposition upon ecologically important traits in invasive plant populations. In addition to quantitating the rate and strength of stochastic forces relative to selection, identifying the relative importance of environmental versus biotic selective agents should be a central topic of further research in evolution of invasive plants. Plant populations distributed across a wide geographic range may become locally adapted to climate factors correlated with latitude, both in native species (Macel et al. Ailanthus altissima cultivate soil biota beneficial to their offspring (Pregitzer et al. Recent research on feedbacks suggests that interactions between abiotic and biotic factors, including the environmental impacts of invasive plants, may also serve as selective forces for invasive species. Melaleuca quinquenervia, in which of three leaf terpenoids under selection, cineole and viridiflorol are reduced in correlation with an increase in the terpenoid nerolidol in invasive range plants, despite predictions based on selection skewers analysis of increases in cineole and viridiflorol over time (Franks et al. Alliaria petiolata (Lankau et al. Centaurea maculosa on native grass species (Callaway et al. Further consideration of the possible evolutionary trajectories of plant invasions may inform how researchers determine the risk, impact, and management of invasive plant species. Evidence suggests that the enhancement of traits via evolution may be constrained by negative correlations between phenotypic traits that are subject to selection, for example in


Cocaine addiction is accompanied by a decrease in striatal dopamine signaling, measured as a decrease in dopamine D2 receptor binding as well as blunted dopamine release in the striatum. These alterations in dopamine transmission have clinical relevance, and have been shown to correlate with cocaine-seeking behavior and response to treatment for cocaine dependence. However, the mechanisms contributing to the hypodopaminergic state in cocaine addiction remain unknown. Here we review the positron emission tomography (PET) imaging studies showing alterations in D2 receptor binding potential and dopamine transmission in cocaine abusers and their significance in cocaine-seeking behavior. Based on animal and human studies, we propose that the kappa receptor/dynorphin system, because of its impact on dopamine transmission and upregulation following cocaine exposure, could contribute to the hypodopaminergic state reported in cocaine addiction, and could thus be a relevant target for treatment development. Studies imaging the neurochemistry associated with cocaine addiction in humans have largely focused on dopamine signaling in the striatum. These studies show that pre-synaptic dopamine release, in response to the administration of a stimulant, is reduced in cocaine abusers compared to healthy controls. This has important implications for this disorder, since the reduction in dopamine release has been shown to correlate with increased cocaine-seeking behavior. Importantly, the imaging studies were performed at about 14 days abstinence, which has clinical relevance, since previous studies have shown that cocaine abusers who achieve 2 weeks of abstinence have a better treatment response compared to those who do not  allows imaging of the neurochemistry associated with drug and alcohol addiction in the human brain. This imaging modality uses radionuclide-labeled ligands that bind to a specific receptor, and the radioligands used most frequently in addiction research label the dopamine receptors. Radiotracers that label the dopamine type 2 family of receptors (referred to as D2) can also be used to measure changes in extracellular dopamine. This is performed by imaging with radiotracers that are sensitive to changes in extracellular dopamine, and obtaining scans before and after the administration of a psychostimulant (such as amphetamine or methylphenidate). These stimulants increase extracellular dopamine levels, which results in a reduction of dopamine receptors that are available to bind to the radiotracer, shown in Figure The main outcome measure in radioligand imaging studies is receptor binding to the radiotracer, referred to as BPND, defined as the ratio of specific to non-specific binding  of 15–20%  are reduced in cocaine dependence. This is performed by imaging the D2 receptors before and after acute depletion of endogenous dopamine using alpha-methyl-para-tyrosine (AMPT). Thus, imaging after AMPT administration results in an increase in [11C]raclopride binding, as opposed to the decrease seen after stimulant administration , although dopamine neurons fire in response to the receipt of a reward, and during the expectation of a reward. However, dopamine signaling more likely mediates the reinforcing effects of natural rewards and abused drugs, and makes the behavior required to obtain the reward more likely to be repeated Schultz, . HoweverThe imaging studies in human cocaine abusers show that blunted dopamine release correlates with an increase in cocaine self-administration , which can serve as a marker for the integrity of the dopamine neurons  is the class of peptides cleaved from prodynorphin, which include dynorphin A and B (and others) which have a high affinity for the kappa receptor (KOR)  depending on experimental conditions  can be categorized into at least two subgroups according to their projections sites and the proteins they express Gerfen, . The “dis protein and consequently stimulate the production of adenosine 3′, 5′-monophosphate (cAMP) which leads to the activation of protein kinase A (PKA)-dependent signaling pathways. In contrast, D2 receptor inhibits adenylyl cyclase and cAMP/PKA pathways by recruiting inhibitory Gαi. Accordingly, cocaine activates PKA signaling pathway mainly through activation of D1 receptor and manipulation of this pathway alters behavioral responses to cocaine  function together to increase the negative reinforcing effects of cocaine (Koob et al., Studies in human cocaine abusers have also shown that stress increases the risk of drug abuse and relapse (De La Garza et al., Thus, DYN/KOR signaling appears to play a crucial role in reinstating drug-seeking behavior by mediating the negative effects associated with drug cessation and stress-induced drug taking (Koob and Le Moal, The data presented here suggest that blunted striatal dopamine release measured with imaging in cocaine dependence may be associated with an upregulation of DYN. Acting at the KOR of the dopamine terminals, KOR activation would be expected to produce a decrease in striatal dopamine release. Post-mortem studies in cocaine abusers and animal studies show that both KOR and DYN are upregulated following chronic cocaine exposure, and that this effect is long lasting (Spangler et al., Chronic cocaine exposure induces CREB phosphorylation and changes in gene expression, which increase expression of prodynorphin mRNA in the nucleus accumbens in addition to other factors. As described above, excessive DYN signaling results in a decrease in extracellular dopamine release, which has been shown in the imaging studies of human cocaine abusers. These findings suggest that increasing signaling at the dopamine receptors may be an appropriate treatment approach, but clinical studies using dopamine agonists have not shown efficacy (Amato et al., 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.


Basic science research is not immune to the influence of pharmaceutical and industrial partners. This Perspective by Adriane Fugh-Bermann explains why scientists should be aware of the potential competing interests posed by practices such as selective publication and ghost-writing. In the 1970s, a Baltimore city senator who also owned a tavern backed legislation that helped his business. Accused of having a conflict of interest, Joseph J. Staszak, responded, “What conflict of interest? How does this conflict with my interest?” According to the Institute of Medicine, a conflict of interest is “a set of circumstances that creates a risk that professional judgment or actions regarding a primary interest will be unduly influenced by a secondary influence” In biomedicine, discourse on conflicts of interest  has focused on relationships between industry and physicians or clinical researchers. However, basic scientists are not immune to industry influence on research and publications, and may be important to industry in the production and dissemination of marketing messages.In 2007, industry was the largest funder of biomedical research, paying for nearly twice as much research (58%) as the federal government (33%) Researchers were aware that something was expected in return for the gift. Sponsor expectations that the gift be used for its intended purpose and not be re-gifted, and that the sponsor be acknowledged in publications, are certainly reasonable. Disturbingly, however, about a third (32%) of gift recipients reported that the funder wanted prepublication review of any articles or reports stemming from the use of the gift. This expectation was higher for gifts of biomaterials: 40% of respondents reported that the firm wanted to receive prepublication review of articles or reports. Also, 44% of firms wanted assurances that the biomaterial was not to be used for applications that competed with company products In clinical research, investigators who receive industry funding are more likely to publish results that favor a sponsor's marketing goals than are investigators who do not receive industry funding. The Cochrane Collaboration, renowned for creating and publishing high-quality systematic reviews, analyzed 48 clinical studies, systematic reviews, and meta-analyses that compared results from studies of drugs or medical devices based on sponsorship. This systematic review found that industry-sponsored studies, compared to non-industry-sponsored studies, were more likely to report favorable efficacy results for drugs or medical devices; less likely to find harms; and more likely to conclude that a therapy was beneficial Systematic reviews are important because they utilize a scientifically reliable method that identifies, evaluates, and synthesizes evidence that meets pre-specified criteria and use explicit methods to avoid bias in selecting studies for review. Conventional literature reviews may pick and choose what to include, and while some are useful, non-systematic reviews should be viewed as opinion pieces.The Cochrane review focused on clinical trials, and less is known about the effects of industry funding on basic science research. What information exists, however, is not reassuring.Positive results for therapeutics in animal studies are often not confirmed in clinical studies. Species differences certainly play a role, but faulty experimental design, reporting bias, analytic bias, and publication bias also may be important.A recent analysis of 4,445 animal studies in 160 meta-analyses of neurological drug candidates found that far too many studies  had a “positive” result, when only about half that number (919 studies) would have been expected to be positive Cell culture studies may also suffer from design or reporting bias. Let's take the example of erythropoetin-stimulating agents (ESAs), which were licensed in the early 1990s for treating anemia in cancer patients and became widely used. Early published clinical studies and meta-analyses appeared to show a benefit of ESAs on mortality Long before an increased mortality risk in humans was established, however, questions about potential adverse effects of ESAs had been raised. Basic science studies were conducted to address the question of whether ESAs fostered malignant growth. An analysis of published basic science research found that researchers funded by ESA manufacturers were far less likely than non-industry-funded investigators to report that ESAs promoted tumor growth The majority of researchers (57%) without ties to ESA manufacturers concluded that ESAs were linked to increased malignant potential, but not one researcher funded by ESA makers drew that conclusion. Several industry-paid researchers actually concluded that erythropoietin had potential antitumor effects In other words, industry-supported basic science researchers found industry-friendly results. Although the question of whether ESAs promote tumor progression in humans remains open, the contribution of industry-funded researchers to reassuring clinicians and policymakers regarding risks of ESAs remains concerning.Selective presentations and publications are important tactics for industry. Industry relies on abstracts and posters to convey marketing messages at scientific meetings, because abstracts and posters are usually not peer-reviewed and can be easily altered up to the time of presentation. Posters and abstracts are often used for preclinical studies, case reports, or preliminary results of clinical trials. Promising preliminary results might be presented as a poster, and the results may be publicized, but if the final results of the study do not support commercial goals, the full study may never be published - or may be buried in an obscure, low-impact journal. In either case, scientists may have a positive impression of a therapy from a poster, and never learn that the therapy failed to show efficacy in the final study.The majority of meeting abstracts and posters are never published. Posters and abstracts with positive results are far more likely to be published than negative studies Selective publication of studies that favor a sponsor's drug has obvious commercial benefits. According to the former pharmaceutical executive who shared his perspective on condition of anonymity:“It is to industry's advantage to selectively support particular researchers whose point of view supports marketing goals, and to encourage selective publication of articles.”—Former pharmaceutical executive, personal communicationAlthough many universities frown on researchers signing agreements that give funders the right to suppress the publication of findings, school policies lack uniformity regarding publishing. Researchers cannot be forced to publish studies. Writing up studies requires time and effort, and researchers may be less motivated to publish negative data, especially when it is perceived that negative studies have less impact than positive studies.Anecdotally, researchers say that good journals won't publish negative studies, but journals cannot publish what is not submitted. A study of papers presented at a gastroenterology conference found that 76% (156 of 206) of papers that were never published were never submitted Perhaps because negative outcomes from industry-funded studies are less likely to be submitted for publication, industry-funded clinical trials overall are less likely to be published. An analysis of 546 drug trials listed in ClinicalTrials.gov found that within two years of study completion, about a third of studies that received full (32%) or partial (39%) industry support were published. In contrast, more than half (54%) of trials funded by government, and 56% of trials funded entirely through nonprofit/nonfederal funds, were published Less information is available about basic science studies, but there is reason to be concerned. A key analysis of 16 systematic reviews of experimental animal studies of acute ischemic stroke found that 98% (515 of 525) of unique publications reported significant effects of treatment on infarct volume The authors estimate that publication bias may have accounted for about a third of the efficacy reported in systematic reviews Researchers may not submit papers for publication for many valid reasons. In contrast to clinical research, in which the goal is often to determine whether a specific drug is safe or effective in a specific population, the goal in much of basic science is to define mechanisms and advance understanding of biological processes. Full publication of negative results may be seen as less interesting by basic science researchers. Faculty mentors may encourage students to present work as an abstract as an outlet for disseminating information and recognition of a student's efforts, but without the expectation that a full paper will be written.The question of whether keeping industry sponsors happy affects publishing decisions has important ethical implications. An industry funder provides help, encouragement, and approbation for publishing positive studies. There may be little incentive to preserve the integrity of the medical literature when the unspoken threat of funding withdrawal hangs over a researcher who insists on publishing negative studies.PLOS ONE and numerous other journals have pledged to publish high-quality research with negative results  spoke at many medical meetings, promoting hormones off-label to prevent cardiovascular disease and prevent dementia in menopausal women. No randomized clinical trials with disease endpoints supported this use, so speakers invoked observational studies, studies with surrogate endpoints (i.e. cholesterol-lowering), experimental animal studies, and even cell culture studies.At several medical meetings in the early 2000s, I observed speakers paid by Wyeth showing an industry slide that compared brain cells grown in media with and without estrogen. Brain cells grown in estrogen-free media were shriveled and dying, a pathetic counterpoint to the vigorous confluence of cells grown in estrogen-containing media. In living women's brains, however, estrogen had adverse effects. The Women's Health Initiative Memory Study (WHIMS), which studied 7,479 women over 65, found that hormone therapy increased the risk of dementia and cognitive impairment, decreased global cognitive function, and was associated with more brain atrophy WHIMS was part of the Women's Health Initiative (WHI), a large, long-term, NIH-funded, definitive randomized controlled trial of menopausal hormone therapy in 26,000 women 50–79 years of age, that showed that the risks of menopausal hormone therapy outweighed benefits. Although hormones had been marketed to decrease the risk of cardiovascular disease, the WHI found that hormones were ineffective in preventing heart attacks in healthy women  and increased the risk of stroke and blood clots Wyeth-linked experts fanned out to criticize the WHI, attempting to counter the findings by, among other tactics, touting experimental animal studies that showed that estrogen appeared to prevent manifestations of vascular damage in animals fed a high-fat diet Companies have paid billions of dollars in fines for off-label promotion, often using company-generated research, company-paid speakers, and ghostwritten articles to imply clinical benefits in the absence of clinical trials ; fines have also been imposed for suppressing risks or misleading clinicians about risks Many pharmaceutical companies use medical education and communication companies (MECCs) to recruit academic physicians and scientists to “author” publications crafted by industry Ghostwriting has been used to promote Zyprexa (olanzapine) The extent to which basic scientists participate in ghostwritten articles is unknown. Academic medical centers are starting to pay attention to the fact that academic faculty are putting their names on articles they have not written, but in 2010 only 13 (26%) of the top 50 academic medical centers in the US had policies in place that prohibit participation in medical ghostwriting Even if a researcher does not allow a sponsor to ghostwrite an article, industry review of articles by a sponsor may result in the insertion of subtle marketing messages that researchers may not recognize as advertisements. Marketing messages may not mention the targeted drug; for example, marketing messages may claim that a targeted disease is underdiagnosed, that a mechanism of action is particularly exciting, that a class of drugs has unique benefits, or that a competing drug has significant drawbacks. Marketing messages are disseminated in research studies, case reports, reviews, commentaries, and letters, as well as in presentations and posters at medical meetings Biomedical researchers depend on industry. Accepting a sponsor's gifts or statistical or editorial “assistance” creates an opportunity for results to be suppressed or spun to advantage a targeted drug or disadvantage competing therapies. Participating in ghostwriting or ghost-management of publications or posters is ethically unacceptable, as is accepting sponsor suggestions on whether or not to publish, present, bury, or selectively report specific studies.The underreporting of negative results and misrepresentation of reported results distorts the biomedical literature, makes therapies look better than they are, can mislead both researchers and clinicians, and may have adverse effects on public health. It goes without saying that scientists should accurately report and analyze studies, but publishing negative data should also be considered an obligation to the scientific community. Negative studies are crucial to assessing benefits and risks of therapies and to determining whether further research is indicated.As the former pharmaceutical executive puts it:“Industry is a major funder of basic science research, and academic/industry partnerships and public/private sector partnerships have become common. These partnerships may provide incentive for academic researchers to do work of value to industry partners and to help demonstrate that value over the drug development process. The potential for disruption of the academic mission is obvious.”—Former pharmaceutical executive, personal communicationClinicians and academic medical centers have been grappling with the implications of industry influence on clinical practice, but the issue has not been discussed widely within basic science. It is time for basic scientists to begin a difficult conversation regarding the ethical and scientific hazards of working with industry.


Malignancy is known to be one of the predisposing factors of cerebral venous thrombosis (CVT) due to its hypercoagulable state. CVT is a rare disorder which can lead to frequent misdiagnoses of brain metastases in such cases. We report here the case of a 35-year-old female with metastatic colon adenocarcinoma presenting with sudden neurological symptoms. Brain MRI and magnetic resonance venography confirmed the presence of CVT. She was treated with low molecular weight heparin followed by warfarin. She recovered and is doing well on warfarin after 5 months of diagnosis of CVT. CVT should be strongly suspected as a cause of neurological dysfunction in any case of disseminated malignancy including colon adenocarcinoma. Rapid diagnosis and initiation of therapy should be considered because of its favourable outcome. Cerebral venous thrombosis (CVT), that is, any thrombosis that occurs in intracranial veins or sinuses,  is a rarHerein, we report a case of metastatic colorectal cancer who developed cerebral venous thrombosis while on chemotherapy.A 35-year-old female, a diagnosed case of metastatic colorectal adenocarcinoma,  was on pTo rule out any coexisting prothrombotic condition, homocysteine levels, protein C, protein S, factor V, and antithrombin III were done which were within normal limits. For disease assessment, contrast enhanced CT scan of abdomen and pelvis was done which revealed the disease to be in complete remission. She was started on low molecular weight heparin. There was marked improvement in her symptoms, and she was discharged in stable condition on oral warfarin. She is doing well even after 5 months from the diagnosis of cerebral venous thrombosis.Cerebrovascular events may be the first clinical manifestations in patients with underlying malignancy or may develop subsequently during the course. Systemic thrombosis like deep vein thrombosis or pulmonary embolism is well recognized in cancer patients, although CVTs are uncommon in cancer. Potential mechanisms for an association of cancer with cerebral venous thrombosis (CVT) include direct tumor compression, tumor invasion of cerebral sinuses, the hypercoagulable state associated with cancer, or the chemotherapeutic side effects –5. CerebCerebral venous thrombosis should always be kept in the differential diagnosis of any form of neurological symptoms in a patient with cancer even without cerebral metastases and even if the disease is in complete remission as in our case. As CVT has got a favourable outcome unlike other neurological syndromes, early diagnosis with MRI/MR venography and rapid institution of therapy with heparin should be considered.


Since the time of Darwin, biologists have understood the promise of crop plants and their wild relatives for providing insight into the mechanisms of phenotypic evolution. The intense selection imposed by our ancestors during plant domestication and subsequent crop improvement has generated remarkable transformations of plant phenotypes. Unlike evolution in natural settings, descendent and antecedent conditions for crop plants are often both extant, providing opportunities for direct comparisons through crossing and other experimental approaches. Moreover, since domestication has repeatedly generated a suite of “domestication syndrome” traits that are shared among crops, opportunities exist for gaining insight into the genetic and developmental mechanisms that underlie parallel adaptive evolution. Advances in our understanding of the genetic architecture of domestication-related traits have emerged from combining powerful molecular technologies with advanced experimental designs, including nested association mapping, genome-wide association studies, population genetic screens for signatures of selection, and candidate gene approaches. These studies may be combined with high-throughput evaluations of the various “omics” involved in trait transformation, revealing a diversity of underlying causative mutations affecting phenotypes and their downstream propagation through biological networks. We summarize the state of our knowledge of the mutational spectrum that generates phenotypic novelty in domesticated plant species, and our current understanding of how domestication can reshape gene expression networks and emergent phenotypes. An exploration of traits that have been subject to similar selective pressures across crops  suggests that a diversity of targeted genes and causative mutational changes can underlie parallel adaptation in the context of crop evolution. Origin of Species with a chapter devoted to “Variation under Domestication.” In his introduction, Darwin highlights the value of domesticated organisms for understanding the evolutionary process:At the commencement of my observations, it seemed to me probable that a careful study of domesticated animals and cultivated plants would offer the best chance of making out this obscure problem. Nor have I been disappointed; in this and in all other perplexing cases I have invariably found that our knowledge, imperfect though it be, of variation under domestication, offered the best and safest clue. I may venture to express my conviction of the high value of such studies, although they have been very commonly neglected by naturalists .The recognition that domesticated species serve as excellent models for studying morphological evolution can be traced to Charles Darwin, who famously opens his These comments turned out to be remarkably (but not surprisingly) prescient. The subsequent 150 years of advances in the fields of evolutionary and developmental biology have demonstrated that studies of domestication, particularly in plants, provide a wealth of insights into the genetic and developmental bases of morphological evolution. This is partly due to the central role that crops play in sustaining civilization. Crop species supply the vast majority of humankind's caloric intake, either directly as food or indirectly as livestock feed, and crop species have long been studied by breeders and other plant biologists for purposes of crop improvement. A result has been the development of a rich toolkit in many crops for studying the genetic basis of agronomically-related traits; these resources may include extensive germplasm collections, advanced generation pedigrees for use in genetic manipulations, and ever-expanding “omics” databases such as genome sequences and transcriptome, small RNA and proteome profiles in various tissues. A growing number crop species now feature annotated reference genome sequences [reviewed by Feuillet et al. ], with aAn additional advantage of crop species for evolutionary analysis is the opportunity they provide for studying parallel evolution. The repeated evolution of adaptive traits is a hallmark of evolution; this phenomenon characterizes a wide spectrum of organisms from across the tree of life  and related “omics” approaches. Advances have been particularly evident in research aimed at understanding the genotype–phenotype connection. The dense, genome-wide SNP marker coverage afforded by NGS genotyping [reviewed by Davey et al. ] is now As a complementary approach to genetic mapping, domestication-related changes in transcriptomes and gene expression networks can be explored to assess the genome-scale impacts of domestication on the emergent plant phenotype . Finally, as an exploration of parallel evolution among crop species, we examine the genetic basis of changes in flowering time, a trait that has been subject to selection in many crops, to assess the degree to which parallel adaptation has occurred through shared genetic mechanisms.When our ancestors began to shift from collecting wild plants to actively cultivating them, they imposed intense selective pressures for traits that facilitate human cultivation and harvesting of the crop. The resulting phenotypic changes, shared among many food crops, are collectively referred to as the domestication syndrome Hammer, . DomestiAfter the initial stages of domestication, cultivated crops have been subject to selection for crop improvement traits , and for a diversification in traits that characterize varietal differences . While the distinction between domestication traits and later improvement or diversification traits is not always clear , the latter traits often may be discerned because they remain variable among different varieties or landraces. Table As recently as 2006, the number of crop domestication and improvement traits for which the molecular basis was well-understood was just over two dozen  or hormone synthesis . In contrast, traits that involve specific metabolic pathways, such as carbohydrate or pigment synthesis, may arise either through selection on regulatory genes  locus, which encodes the starch synthase required for amylose synthesis  activity  and gene duplication events. We consider each of these separately below for crop domestication phenotypes.Transposable elements. TE proliferation has played a major role in shaping the evolution of plant genomes, including crop species. A recent review indicates that TEs constitute between 22% and 85% of the genomes of 11 crop species examined  differs from its wild ancestor, teosinte (Zea mays subsp. parviglumis), in lacking axillary branch development; this change is largely due to increased expression of tb1, a transcriptional regulator that represses growth  that is expressed at high levels during fruit development  berry color arises through the absence of anthocyanin synthesis during fruit development. The modern white grape phenotype appears to have arisen through sequential selection for loss-of-function mutations in two adjacent anthocyanin regulatory genes: first, a single-nucleotide non-synonymous mutation in Gene duplication. The tomato fruit shape example above illustrates the potential for TE-mediated gene duplications to alter crop phenotypes. More generally, gene duplication is a prominent feature of plant genome evolution, reflecting a history that includes repeated episodes of whole-genome doubling , which was present in all three ancestral diploid genomes, and which generates the soft wheat phenotype when functional. Deletions of Ha from both of the ancestral genomes of tetraploid wheat created the hard grain phenotype, and the ancestral soft grain phenotype was restored in bread wheat by the contribution of the D-genome Ha locus; subsequent selection for deletions and complex rearrangements in the D-genome locus gave rise to the semihard hexaploid wheats  in sunflower and selection for changes in flowering time during domestication and later crop improvement  in combination with analyses of pathways and networks across various scales  offer new opportunities to reveal the intricacies of domestication and crop improvement, and by extension (echoing the words of Darwin invoked in the introduction to this review), the evolutionary process in general. Much of this review has focused on the mutations responsible for phenotypes found in crop plants, and to be sure, considerable progress has been made in this regard Table . But thecis-regulatory variation. An extension of this work  “fibers”  in both wild and domesticated cotton during five stages representing primary and secondary wall synthesis. They detected significantly altered expression for 9645 genes, or about 25% of the genes in the genome. This is especially remarkable, not just because of the high level of “transcriptomic rewiring” that this reflects, but also because it is observed for a single-celled structure. Other transcriptomic studies in the cotton model system are revealing a comparable, massive rewiring of the transcriptome accompanying domestication  and one diploid (G. herbaceum). In their study of the profilin gene family described above, Bao et al.  photoperiod. The spread of these crops into temperate regions was enabled by selection for reduced or altered SD response, allowing them to be cultivated in the long-day (LD) summer growing seasons of higher latitudes  functions as an activator of downstream floral pathway integrators under LD photoperiod, while its rice ortholog, Hd1, has the opposite effect, repressing downstream genes under LD photoperiod and activating them in SD conditions  genes that mediate photoperiod and related circadian clock functions ; (2) those that mediate vernalization cues ; (3) those involved in other components of floral induction signaling  signaling pathways); and (4) those that function further downstream in the flowering pathway as integrators of the different signaling pathways . Among the domestication-related flowering time genes that have been molecularly characterized in recent years ];  those thinclude:  genes thHd5; Fujino et al., ELF3 have played a role in the emergence of early flowering varieties of peas, lentils, and rice (Matsubara et al., Ghd7 were targets of selection in both rice and maize for earlier flowering under LD photoperiod (Xue et al., FLC (rapeseed BnFLC.A10; Hou et al., FT (sunflower HaFT1 and paralogs; Blackman et al., AP1 (wheat Vrn1; Yan et al., cis-regulatory and protein-coding changes (Table At least seventeen domestication-related flowering time genes have been molecularly characterized in the last 5 years Table , not coues Table .It is an exciting time in evolutionary biology, one characterized by unprecedented experimental power. The application of a suite of advanced technologies to domestication-related traits in multiple crop-wild plant model systems is rapidly increasing our ability to discover the genes affected by human selection during both initial domestication and subsequent phases of crop improvement. This collective effort is providing a rich comparative database of the mutational spectra underlying crop evolution and the manner in which changes at the genetic level propagate through various levels of organization to confer the targeted phenotypes. As the number of molecularly characterized traits continues to grow, it will become increasingly possible to capitalize on these traits and genes to study how the broader developmental context in which they function has shaped the evolutionary process. It is likely that the most exciting and novel insights will derive from the utilization of multiple “omics” tools in an integrative framework, all brought to bear on specific genotype-to-phenotype transformations [reviewed by Ghosh et al. ; Lucas eThe 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.