In cancer, dormancy refers to a clinical state in which microscopic residual disease becomes non-proliferative and is largely refractory to chemotherapy. Dormancy was first described in breast cancer where disease can remain undetected for decades, ultimately leading to relapse and clinical presentation of the original malignancy. A long latency period can be explained by withdrawal from cell proliferation (cellular dormancy), or a balance between proliferation and cell death that retains low levels of residual disease (tumor mass dormancy). Research into cellular dormancy has revealed features that define this state. They include arrest of cell proliferation, altered cellular metabolism, and unique cell dependencies and interactions with the microenvironment. These characteristics can be shared by dormant cells derived from disparate primary disease sites, suggesting common features exist between them.High-grade serous ovarian cancer (HGSOC) disseminates to locations throughout the abdominal cavity by means of cellular aggregates called spheroids. These growth-arrested and therapy-resistant cells are a strong contributor to disease relapse. In this review, we discuss the similarities and differences between ovarian cancer cells in spheroids and dormant properties reported for other cancer disease sites. This reveals that elements of dormancy, such as cell cycle control mechanisms and changes to metabolism, may be similar across most forms of cellular dormancy. However, HGSOC-specific aspects of spheroid biology, including the extracellular matrix organization and microenvironment, are obligatorily disease site specific. Collectively, our critical review of current literature highlights places where HGSOC cell dormancy may offer a more tractable experimental approach to understand broad principles of cellular dormancy in cancer. Epithelial ovarian cancer is the 7th most common cancer in women worldwide. It is difficult to treat because it is most often diagnosed at an advanced stage with metastases present . CollectThe metastatic spread of HGSOC is distinct from most forms of human cancer in that cells are shed from the fallopian tubes and ovaries into the peritoneal space where they aggregate and disseminate to other organs both locally in the pelvic region and beyond to destinations higher in the abdominal cavity Fig.  1, 5]. , 5. 1, 5Definitions of cancer cell dormancy vary in the literature with some defining it as minimally as the inhibition of proliferation in cancer cells . In a brPerhaps the biggest challenge in dormancy research is the scarcity of the cells being studied , 18. ThiThe motivation to understand the basic biology of spheroids in HGSOC is to develop better treatment approaches to eliminate this key source of resistance. As detailed in other paradigms of dormancy, the prospect of ‘awakening’ dormant cells to re-sensitize them to chemotherapies that are designed for proliferating cells possesses concerning drawbacks that have been reviewed elsewhere , 21. AltTwo hallmarks of cancer described by Hanahan and Weinberg relate to cell cycle control . Self-suA basic principle of dormant cancer cell signaling in proliferative control that mirrors the hallmarks reasoning above is that pro-growth signals through the Ras-MAPK pathway are often downregulated as evidenced by lowered phospho-ERK. Simultaneously, the stress responsive MAPK, p38 is activated and has been shown to induce growth arrest mechanisms. This core signaling change has been reported in dormancy examples derived from multiple solid tumor types –26, and Reduced cell proliferation implies that dormancy cues block cyclin/CDK activity, reduce Myc levels, and induce other features of quiescence. Experimental cell culture models of dormancy reveal increased levels of CDK inhibitor proteins specifically in growth-arrested conditions , 28, 29.Similar experimental evidence for CDK inhibition of proliferation exists for HGSOC-derived cells and spheroids Fig. A. ReduceDYRK1B gene and dependence on it rather than DYRK1A [Consistent with stress-dependent signals contributing to dormancy, the survival kinases DYRK1A/B are implicated in dormant cell cycle arrest and maintenance of viability . DYRK kin DYRK1A , 37, 39.n DYRK1A , 38. ColMYC oncogene is overexpressed or hyperactivated in numerous cancer types [MYC in a number of experimental models induces dormancy and chemotherapy resistance [The er types , includier types , 42. Itssistance –46. MYC sistance , 47. GivThe widespread importance of negative growth control in dormancy suggests it is an appealing target for inactivation to eradicate residual disease. However, its removal suggests that oncogenic pathways that fuel proliferation may restore cell cycle progression, leading to malignancy. In vitro studies of DYRK1A and DREAM component loss in ovarian cancer cell dormancy indicate that a brief episode of DNA replication is followed by rapid cell death with no evidence of sustained proliferation . FurtherThe PI3K-AKT-mTOR pathway is one of the most widely mutated and activated signaling pathways in human cancers. It has the capacity to promote cell proliferation, mRNA translation, block apoptosis, enhance cell motility and migration, enhance epithelial-mesenchymal transition, and modulate autophagy, to name just a few . HoweverPIK3CA or PTEN deletions, are observed infrequently in HGSOC [Mutations in the PI3K-AKT-mTOR pathway, such as activating missense mutations in in HGSOC . Howeverin HGSOC . PI3K siin HGSOC . Althougin HGSOC , 53, 54.There have been, and continue to be, many clinical trials using PI3K/mTOR inhibitors, including those for epithelial ovarian cancers. This strategy is likely driven by the preponderance of activating driver mutations in this pathway among non-HGSOC ovarian histotypes . HoweverAs malignant cells detach from a primary tumour to spread to distant sites, they commonly trigger stress responses. The inherent cellular mechanisms implicated in HGSOC metastasis may be different from other cancers due to its unique progression within the peritoneal cavity. Metastatic HGSOC cells trigger intracellular stress signaling when they detach into suspension, as well as being deprived of growth factors, ECM components, nutrients, and oxygen. A key pathway that monitors these stressors is the Liver Kinase B1 (LKB1) and AMP-activated protein kinase (AMPK) signaling pathway Fig. A. AMPK aAlthough the stress signaling mediator LKB1 may not be required to control AMPK in HGSOC spheroids, it is still crucial for both spheroid cell viability and metastatic progression , highligPrimary tumors across most disease sites utilize glycolysis to fuel growth and proliferation, a phenomenon known as the Warburg effect . DissemiMacroautophagy, otherwise termed autophagy, is a general intracellular degradation process of organelles, macromolecules and in some cases pathogens. Typically, autophagy is a universal stress-induced phenomenon under nutrient-depleted and starvation-like conditions to facilitate the generation of alternative substrates for energy production . AutophaARHI, a RAS family protein whose signaling can maintain a dormancy-like state of xenografted tumor cells [Autophagy is rapidly induced in HGSOC spheroids Fig. A, and thor cells , 67.When considered together with the section on signaling mechanisms above, a number of important gaps in our knowledge are evident. First, it is unclear if, or to what extent, autophagic mechanisms are controlled through PI3K-AKT and AMPK-ULK1 in cancer dormancy paradigms other than ovarian. Similarly, dependence on fatty acid metabolism and neutralization of reactive oxygen species is suggested by various aspects of HGSOC spheroid biology, but it is not established as it is in other paradigms of residual disease. For example, it is reported that lipid desaturation is critical to the biology of stem-like spheroid cells in HGSOC, which may be implicated in tumor dormancy . Future Given the challenges of PI3K pathway inhibition described above, autophagy potentially offers a more direct consequence of cell signaling in dormancy as a therapeutic target. The majority of clinical trials targeting autophagy in advanced human cancers use hydroxychloroquine, likely because of its known safety profile from use in malaria prophylaxis and rheumatoid arthritis . HydroxyThe transforming growth factor-beta (TGFβ) superfamily of cytokines has widespread implications in human cancers, serving contextual tumour suppressive and pro-metastatic roles . In geneStudies using HGSOC spheroids have observed the reciprocal expression and activity between bone morphogenic protein (BMP) and TGFβ signalling Fig. A, with tRare dormant cancer cells that are capable of initiating new clinically-detectable metastases suggest dormancy and stem cell-like phenotypes go hand-in-hand. Recent work on disseminated cancer cells revealed that negative regulation of WNT signaling suppresses proliferation and prevents immune detection in perivascular niches . This diA theme throughout this review is that common pathways exist to control dormancy that are similar between HGSOC and other disease sites. TGFβ family and WNT signaling pathways contribute to key elements of EMT and stem cell biology in dormancy. Again, there are distinct aspects of how these pathways function in HGSOC spheroids that raise the question of applicability of findings in one disease site paradigm to another. However, tumor dormancy studies across disparate disease sites suggest that TGFβ and WNT signaling pathways are candidates for therapeutic intervention that target dormancy. Thus, appreciating the subtle mechanistic differences in disease site specific aspects of tumor dormancy are likely critical to the successful use of dormancy-directed therapeutics in the future.Research into dormant cancer cell niches highlight three main locations for these cells to reside . The perIt is difficult to see commonalities among these examples as each microenvironment involves different cell-cell contacts and signaling events mediated by distinct cytokines . The comDormant cancer cells in all locations share a common outcome of escaping eradication by the immune system . In someSpheroids from HGSOC patients represent a distinct microenvironment that is mobile and possesses self-contained features essential for survival and dissemination of ovarian cancer Fig. B. SpheroA further distinction between HGSOC dormancy and that of other solid tumors is that native spheroid pathobiology enables metastatic dissemination through distinct interactions with mesothelial surfaces in the abdominal cavity. These interactions involve both cell-cell signaling in which HGSOC cells activate the mesothelium to aid in attachment and infiltration  and cellThe most significant factor affecting both progression-free survival and overall survival rates among women with advanced HGSOC is achieving the lowest level of residual disease following aggressive cytoreductive surgery and combination chemotherapy , 4. It iEpidemiological data suggests the diabetic drug metformin has a protective role for ovarian cancer ; since mMost research implicates tumor dormancy in the context of advanced stages of disease and as a mechanism to evade therapeutic insult or immune-mediated clearance. However, HGSOC tumor cells have the capacity to disseminate from fallopian tubes early in disease progression, long before dormancy mechanisms can impact treatment resistance. The concept of early dissemination in HGSOC is particularly relevant since evidence exists for cells comprising pre-malignant serous tubal intraepithelial carcinoma (STIC) lesions to spread to the ovary, or in rare cases directly into the peritoneum . A critiThe concept of tumor dormancy has wide implications among many human cancers and can impact the ability to attain a durable response to treatment. However, we suggest that reductionist attempts describing it with a single definition, or with a specific set of criteria across all disease sites is counter-productive. In this review, we highlight that there are many common elements of dormancy shared among cancer types, but the underlying molecular mechanisms can often be distinct among different disease sites. Being aware of similarities among dormancy disease site paradigms will likely accelerate our understanding through cross-fertilization of ideas between researchers. At the same time, we conclude from this review that differences among dormancy paradigms are extensive, thus embracing this diversity of discoveries among distinct tumor types is also critical to further advancements in the field. We expect that some knowledge gained through summarizing tumor dormancy in the context of advanced HGSOC will be valuable to investigators studying analogous paradigms in other malignancies and metastatic sites.


The economic and biological implications of plant invasion are overwhelming; however, the processes by which plants become successful invaders are not well understood. Limited genetic resources are available for most invasive and weedy species, making it difficult to study molecular and genetic aspects that may be associated with invasion.Centaurea maculosa, an invasive aster in North America. Seventy-seven percent of the 4423 unique transcripts showed significant similarity to existing proteins in the NCBI database and could be grouped based on gene ontology assignments.As an initial step towards understanding the molecular mechanisms by which plants become invasive, we have generated a normalized Expressed Sequence Tag (EST) library comprising seven invasive populations of C. maculosa EST library represents an initial step towards looking at gene-specific expression in this species, and will pave the way for creation of other resources such as microarray chips that can help provide a view of global gene expression in invasive C. maculosa and its native counterparts. To our knowledge, this is the first published set of ESTs derived from an invasive weed that will be targeted to study invasive behavior. Understanding the genetic basis of evolution for increased invasiveness in exotic plants is critical to understanding the mechanisms through which exotic invasions occur.The  Invasive weeds are regarded as major threats to biodiversity because they can spread through communities, displacing or even eradicating native species. Over 25,000 invasive plant species have been documented in the United States, invading nearly 700,000 hectares per year, with a cost exceeding 34.5 billion dollars per year . MultiplCentaurea maculosa Lam. (spotted knapweed) is a Eurasian native that has become a particularly problematic invasive weed in the northwestern United States, infesting over 4.5 million acres in Montana alone . These studies give partial support to the EICA hypotheses. There is also evidence that Centaurea species (C. maculosa and C. diffusa) produce allelopathic compounds that inhibit growth and germination of North American plant species more severely than their native congneners, lending support to the novel weapons hypothesis [C. maculosa to become invasive in North America.na alone . Spottedna alone . The dipna alone , with a na alone . This trna alone . Both fona alone . Ecologithe weed -17. Alsopothesis ,16, althpothesis . It is nC. maculosa. Here we describe candidate genes that could be utilized in future experiments to correlate plant gene expression and ecological hypotheses proposed for invasive success. To the best of our knowledge, this is the first published set of ESTs derived from an invasive weed that will be targeted to study invasive behavior.As an initial step towards understanding the molecular mechanisms by which plants become invasive, we have generated a normalized Expressed Sequence Tag (EST) library representing seven invasive populations of Centaurea maculosa Lam were grown for one to two months, at which time two to three entire plants per population were harvested. Tissue was frozen in liquid nitrogen and shipped to Agencourt biosciences for normalized cDNA library construction. Single pass directional sequencing was performed on 4969 randomly selected clones from the C. maculosa normalized cDNA library (GenBank accessions EL930664-EL935630). These sequences were assembled into 4423 unique contigs (contiguous consensus sequences) or "unigenes" using Noble Foundation's in-house pipeline based on TIGR Assembler [Seeds from seven invasive populations of ssembler .The library consisted of 894 contig-forming ESTs which created 348 unigenes and 4075 singlet ESTs, each representing a unique sequence. Of the 348 unigene contigs, the majority contained only two EST sequences [see Additional File C. maculosa ESTs, the 4423 unigenes were translated in all frames and searched for similarity against the NCBI non-redundant protein database using BLASTX . Of the entire unigene set, 77% (3392) had significant similarity to genes in the NCBI database, while the remaining 1031 sequences had low similarity and were not able to be annotated. In the group of annotated sequences, 35% (1177 unigenes) had top BLAST hits to transcripts from Arabidopsis thaliana, whereas only 10% (338 unigenes) had top hits to Orzya cultivars. Taxonomically, 64% (2182) of the annotated unigene top hits grouped into the rosids clade (which includes the families Brassicacea and Fabaceae), 20.6% grouped into the asterids clade (which includes the families Asteraceae and Solanacea), and 11.8% grouped into the commelinids clade (which includes members of the Poaceae family and other monocots) [see Additional File To annotate the Centaurea unigene annotations, whereas the molecular function category contained only 2162 annotations. Each category contained approximately 16% unigenes that were annotated as "unknown," but this number does not account for the unigenes that were unable to be annotated in GO format  assignment programs were used to functionally categorize unigenes in the library. Unigene GO terms were counted and grouped in a hierarchical fashion into the major GO functional categories of Cellular Component, Biological Process and Molecular Function. The GO categories of cellular component and biological process contained over 3000 C. maculosa under biotic stress conditions. Only a small percentage of unigene annotations fell into the reproduction category of biological processes (0.3%), but this is not entirely surprising, as reproductive structures such as flowers were not used in the starting material for the library.Approximately 38% of the unigene annotations were grouped into the 'physiological process' category of the Biological Process GO Figure , which iOver 38% (829) of the unigene annotations fell into the 'catalytic activity' category of the molecular function GO Figure . SpecifiCentaurea secondary metabolites and uptake of nutrients, and may aid in understanding the role of transport in competitive ability of Centaurea.Transporters accounted for nearly 8% of all unigene annotations, the majority being ion transporters and transporters with carrier activity (54 and 48 unigenes). Eighteen unigenes fell into the 'ATP-ase coupled transporter' category, which includes transporters of xenobiotics, steroids, sugars, peptides and other small molecules. However, most unigenes could not be subcategorized with a specific transport role. Uptake and translocation of nutrients in plants differ, and regulated expression of specific transporters may allow increased competitive ability in different situations  -25. DepeCentaurea library made up ~6% of the GO annotations for molecular function, whereas ~4% of the Arabidopsis genome sequences are annotated as transcription factors .One hundred twenty-seven unigene annotations were designated as transcriptional regulators, with the majority (118) being subcategorized as having 'transcription factor activity.' Transcription factors are responsible for modulating cellular responses to biotic and abiotic stimuli , and theOver two-thirds of the unigenes were localized by cellular component to either cell or organelle, as shown in Figure Centaurea library. Many of these unigenes could be used as candidates for production of a microarray to visualize changes in global gene expression, or to look at more specific changes in regulation related to plant defense or stress.A wide variety of functional categories were represented in the Centaurea cDNA library potentially involved in genome evolution and plasticity are described below.The EICA hypothesis suggests that when plants are introduced into a new range, they escape their enemies and rapidly evolve to put more resources into growth/reproduction and less into defense . EvolutiCentaurea cDNA library, six transposable element-related unigenes were identified [see Additional File Centaurea cDNA library, have been detected in plants at specific growth stages and under biotic stress conditions such as pathogen attack and wounding [Mobile, or transposable elements (transposons) have the ability to modify DNA sequences by 'jumping' in and out of places in the genome, and certain mobile elements carry gene fragments that, when transposed, lead to repetition or creation of new genetic material . Mobile wounding ,33. Evidwounding , and it Drosophila melanogaster and the cruciferous plant Arabiodpsis [Centaruea cDNA library [see Additional File A. thaliana, suggesting there may be a wide diversity of Hsp90 related sequences in Centaurea. Understanding the mechanism of these proteins in relation to plant genotype, environment and defense response throughout the native and invasive range of C. maculosa may give some clues to the plasticity of the species.Heat shock protein 90 (Hsp90), a stress-induced protein, has been shown to buffer genetic variation in morphogenic pathways in the fruit fly biodpsis . As a chbiodpsis ,35. EighCentaurea spp. are polyacetylenes and related thiophenes, flavonoids (flavones and flavonols and their derivatives in particular) and their glycosides, phenolics and lignans, coumarins, anthocyanins, cyanogenic glycosides (prunasin), mono-, sesqui-, di- and tri-terpenoids (with sesquiterpene lactones particularly diverse), and steroidal compounds [Centaurea unigenes which share sequence similarity with characterized genes involved in plant secondary metabolite biosynthesis. Further study of these candidate genes may aid in understanding the relative influence of AARS in Centaurea invasion.As explained above the AARS hypothesis proposes that novel weapons, often in the form of secondary metabolites, are selected for in invasive plants in the introduced ranges. Members of the aster family are capable of synthesis of a broad spectrum of secondary metabolites that may aid in basal and induced defense response, as well as in competition against other plants ,36. InclC. maculosa is known to accumulate the sesquiterpene lactone, cnicin, at concentrations approaching 2% of dry weight on the leaves of the inflorescence stem [Agapeta zoegana, a biological control agent introduced for the control of C. maculosa in the North America. Additionally, cnicin is phytotoxic to several plant species [C. maculosa [Artemisia annua, Solidago canadensis, Helianthus annuus, Ixeris dentata, Chicorium intybus, and Lactuca sativa. In the Centaurea cDNA library, no unigenes were annotated as a sesquiterpene synthase. However, several unigenes revealed high sequence simlarity to genes involved in the synthesis of the iosprenoid pyrophosphates, metabolic precursors to the terpenoids. BLAST similarity searches revealed two unigenes which closely matched the Antirrhinum majus geranyl diphosphate synthase, which catalyzes the condensation of two isopenyl pyrophosphate units to form a ten-carbon precursor (geranyl pyrophosphate – GPP) of monoterpenoids. GPP can then be extended further to FPP, geranylgeranyl pyrophosphate (GGPP), and ultimately to dolichol phosphate, a polyprenoid involved in the formation of glycoproteins via the endomembrane system. Two unigenes were annotated as dehydrodolichol phosphate synthases by GO annotation.nce stem . This conce stem  includin species , appearsmaculosa ), possesmaculosa , and is maculosa . SesquitC. maculosa. Sesquiterpene lactones commonly occur in the Asteraceae, but the biosynthetic routes for individual metabolites are relatively poorly characterized. One of the most well-characterized of the biosynthetic pathways leading to sesquiterpene lactones is that for artemisinin, a compound of value as an anti-malarial drug [Artemisia annua. Recently the oxidation steps which generate artemisinic acid, a precursor of artemisinin, were characterized and the entire pathway to artemisinic acid reconstructed in yeast [Centaurea library (CENT_UG_03500) demonstrates 93% amino acid sequence identity to this three-step oxidase and a nearly identical gene from the related A. obtusifolia [see Additional File C. maculosa.Following formation of the ring structure, sesquiterpene skeletons can be modified through oxidation, reduction, and glycosylation reactions to form an enormous diversity of secondary products including cnicin, a sesquiterpene lactone found in ial drug  isolatedin yeast . The rea12 oleic acid desaturates, and C. maculosa unigenes annotated as desaturases reveals three C. maculosa ESTs which cluster closely to H. annuus acetylenase, but are distinct from H. annuusΔ12 oleic acid desaturates  are reported in GenBank, and only one Asteraceae sequence is annotated as an FLS gene (from G. hybrida), suggesting that one or more of the unannotated 2-oxoglutarate-dependent dioxygenases may be FLS or FS genes.The basic flavonoid pathway is the best-characterized metabolic pathway of plant secondary metabolism . More thflavones . The earflavones . Many stC. maculosa has been reported to exude catechin, a phytotoxic secondary metabolite, from its root [C. maculosa may help reveal how this compound and other potential 'novel weapons' are synthesized. The AARS hypothesis suggests that plants may evolve to produce more of the effective 'weapon' compounds when in the invaded environment [As its root ,16, the ironment , so it wCentaurea cDNA library contains a variety of unigenes annotated as defense response genes [see Additional File Centaurea sequences similar to three lipoxygenase (LOX) proteins, two phenylalanine ammonia lyase (PAL) proteins, and three calmodulin binding proteins. Also identified was a Centaurea sequence similar to the Arabidopsis activated disease resistance-like (ADR1-like) gene, which contains nucleotide binding site – leucine rich repeat (NBS-LRR) motifs characteristic of defense-response proteins [Centaurea unigenes show high similarity to another LRR containing Arabidopsis transcript (At3g20820) that is suspected to be involved in the defense response signal transduction pathway. The Centaurea library contains seven other unigenes, not annotated as "defense related," that contain LRR motifs or leucine zippers [see Additional File Centaurea unigene shows similarity to the R gene-mediated disease resistance gene (EDS1) from Arabidopsis, which is required for SA accumulation and production of pathogenesis related (PR) proteins [Sambucus nigra, similar to Arabidopsis PR-1-related transcript At4g33720. In addition, twelve of the unigenes annotated as having 'transcription factor activity' in the GO molecular function categorization are similar to WRKY transcription factors [see Additional File C. maculosa sequence information it will be possible to test levels of basal defense responses at the level of gene expression in populations of native and invasive plants. Genes involved only in induced defense response may be under represented in the library, as the plants that were used in library creation did not undergo interspecific competition, pathogen stress, or herbivory and were not grown under field conditions. However, many of the unigenes identified have been implicated in induced defenses in other systems (PR-1 and EDS-like unigenes), and these may be good candidates for studying induced as well as basal defense response. Future experiments can be coupled with more traditional tests of morphology and biochemistry in order to supplement data concerning hypotheses of plant invasion.One of the main predictions of EICA is that plants in their new environment rapidly evolve to put more resources into growth/reproduction and less into defense, as they have escaped their co-evolved pathogens and predators . Thus, nproteins . Four Ceproteins . Two uniCentaurea cDNA library, consisting of 4423 unique transcripts (unigenes), represents an initial step towards looking at gene-specific expression in this species, and will pave the way for creation of other resources such as microarray chips that can help provide a view of global gene expression in invasive C. maculosa and its native counterparts. These technologies can likely be extrapolated to look at other invasive knapweeds  also problematic in North America. By comparing native and invasive C. maculosa plants under different stresses, including herbivory and pathogen infection, it will be possible to test hypotheses such as EICA using molecular resources coupled with classical  techniques.This is the first report of a cDNA library from an invasive weed. The C. maculosa populations, and give insight into the effects of chromosome doubling and polyploidization events in the plant world. Additionally, by looking at secondary metabolite accumulation and the genes responsible for their production in C. maculosa, it may be possible to knock out those genes, create mutants defective in the production of allelochemicals, and to finally determine unequivocally whether allelopathy (novel weapons) is involved in the invasive success of some weeds.This technology will also be useful to help understand differences in gene expression between diploid and tetraploid Centaurea cDNA library provides a unique resource that will be valuable to geneticists, molecular biologists, and ecologists alike.Understanding the genetic basis of evolution for increased invasiveness in exotic plants is critical to understanding the mechanisms through which exotic invasions occur. The Centaurea maculosa Lam were obtained from Ray Callaway . Five populations originated from Montana, one from Washington, and one from Virginia. Six seeds from each of the seven populations were sterilized by heating at 50°C for ten minutes in distilled water. Seeds were cooled to ambient temperature, rinsed with sterile water and placed in Petri dishes containing moist germination paper. Plates were wrapped in parafilm and placed in a growth chamber with a photoperiod of 20 hours light/six hours dark at a constant temperature of 25°C. Upon the emergence of cotyledons, seedlings were planted in 2.5 cm pots in a mix of 70% sand, 10% perlite, and 20% autoclaved potting soil and transported to the greenhouse. Pots were placed in a flat and covered with plastic wrap for approximately one week until seedlings became established. Plants were given sufficient water and fertilized once per week with a dilute solution of Miracle Gro . After approximately two months, two to three plants per population were removed from pots and their roots were washed to remove soil particles. All plants were in the form of small rosettes, lacking stem and floral tissue. Entire plants, including roots, were wrapped in foil, frozen in liquid nitrogen, and stored at -80°C until processing.Seeds from seven invasive populations of R gene was used during construction of the primary library to ensure library quality. Single-stranded DNA was made from a portion of the primary library by phagemid production, and reactions were treated with DNase I to ensure the removal of double-stranded DNA. A second portion of the primary library was linearized and transcribed into anti-sense RNA with biotinylated dNTPs. Oligo dT and primer extension were used to pre-block the poly-A region prior to hybridization. The anti-sense RNA and single-stranded circular DNA were hybridized, and abundant clones were removed using streptavidin. To reduce the amount of empty vectors, a Not1 oligo and Taq polymerase were used to synthesize double stranded DNA from the single stranded normalized library prior to final transfection.Plant tissue was shipped in dry ice to Agencourt Bioscience Corporation (Beverly MA). Total RNA was extracted and optimized first strand cDNA synthesis was performed using a primer adapted with a rare enzyme cut site. cDNA fragments were size-selected by agarose gel electrophoresis, and directionally cloned into a pAGEN-1 vector. A positive control containing the TetThe normalized library was plated and 4969 clones were randomly selected for sequencing. Automated plasmid purification was achieved using the SPRI (SprintPrep™) technique, which harvests plasmid DNA directly from lysed bacterial cultures, trapping both plasmid and genomic DNA to functionalized bead particles and selectively eluting only the plasmid (Beckman Biomek FX robots and CCS Packard DNATraks).® Version 3.1 reactions on ABI3730 instruments at Agencourt Biosciences. Thermal cycling was performed using 384-well Thermal cyclers . Sequencing Reactions were purified using Agencourt's CleanSeq® dye-terminator removal kit. All reads are processed using Phred base calling software and constantly monitored against quality metrics using the Phred Q20. The quality scores for each run were monitored through the Oracle 9i driven Laboratory Information Management System (LIMS). C. maculosa ESTs were trimmed of vector sequence and the data was transferred to a secure site for download.DNA templates were sequenced in 384-well format using BigDye-4 or less) and top hits from GO assignment were deposited in PLAN and can be searched by keyword or unigene accession number . GO annotation was used to categorize unigenes into functional categories by molecular function, cellular component and biological process. A customized in-house program was used to count the number of unigenes being grouped under different GO term categories in a hierarchical fashion [EL930664-EL935630).To determine the number of unique transcripts in the library, an in-house pipeline program was used to cluster and assemble the trimmed EST sequences. The pipeline essentially utilizes TIGR Assembler with its default parameters (overlap of at least 40 bp with 94% identity). The PLAN web system  was used to do a BLASTX search against the non-redundant protein (NR) database for functional annotation, and gene ontology (GO) sequence database for functional categorization . The BLA fashion . The vecAKB: designed and performed research, analyzed data, wrote manuscriptCDB: analyzed data, wrote manuscriptJH, XD, PXZ: provided new analytical techniques (PLAN database)JMV: designed research, edited and approved manuscriptGenBank Accession numbers:EL930664-EL935630Database EST ID (NCBI) numbers:45411770-45416736PLAN database accession numbers for Centaurea EST sequences:CENT_UG_00001 through CENT_UG_04423Centaurea ESTs by cluster sizeDistribution of assembled . The data represent clustering of Centaurea ESTs into unique sequence clusters (unigenes) and show distribution by cluster size. The 4969 Centaurea ESTs were assembled into 4423 unique contigs or 'unigenes' using the PLAN database (Noble foundation). In total, 4075 singlet ESTs were unique (not pictured on graph); 348 could be assembled into clusters containing one or more Centaurea unigene, and were plotted relative to their abundance in the EST library.Click here for fileCentaurea unigenes by sequence lengthTitle: Distribution of . The data represent distribution of Centaurea unigenes by sequence length. The 4423 Centaurea unigenes were plotted by their relative abundance based on sequence length in base pairs.Click here for fileCentaurea unigene top BLAST hitsTaxonomic clades associated with . The data represent taxonomic clades associated with the top similarities of known sequences to Centaurea unigenes. Centaurea unigenes were used to query BLASTX nr database, and the top hit for each unigene was deposited in the PLAN database . These top hits were assembled by taxonomic group. Approximately 35% of the unigenes had top hits to Arabidopsis, which is part of the Rosid clade.Click here for fileCentaurea cDNA libraryEvolution/Plasticity-related sequences in . The table lists sequences identified in the Centaurea cDNA library that may be related to evolution and plasticity, based on similarity to known sequences. Evolution-plasticity related sequences from the Centaurea cDNA library are represented by Centaurea unigene identification number (from the PLAN database). Accession number, organism, functional description, and E value of the top BLAST hit for each unigene is listed.Click here for fileCentaurea unigene (CENT_UG_03500) and related sequencesAlignment of . The data represent predicted amino acid sequence alignment of Centaurea unigene 03500 with related sequences involved in sesquiterpene lactone synthesis. Sesquiterpene lactone synthesis proteins from Artemisia obtusifolia and A. annua were aligned with Centaurea unigene 03500 using . Stars (*) indicate complete sequence conservation;(:) represents amino acids of a similar nature.Click here for fileCentaurea cDNA libraryFlavanoid Pathway- related sequences in . The table lists sequences identified in the Centaurea cDNA library that may be involved in the flavanoid pathway, based on similarity to known sequences. Proposed function (Func) of flavanoid pathway related sequences in the Centaurea cDNA library; PAL , C4H(cinnamate 4-hydroxylase), C4L (4-coumaryl-CoA ligase), CHS , CHI , F3'H (flavanoid 3'-hydroxylase), GT (glycosyl transferase), OMT (O-methyltransferase). The number of unigenes and their identification numbers (PLAN database) are listed for each functional group.Click here for fileCentuarea cDNA libraryDefense-response-related sequences in . The table lists sequences identified in the Centaurea cDNA library that may be involved in defense response, based on similarity to known sequences. Defense-response-related sequences from the Centaurea cDNA library are represented by Centaurea unigene identification number (PLAN database). Accession number, organism, functional description, and E value of the top BLAST hit for each unigene is listed.Click here for file


Congestive heart failure with preserved left ventricular systolic function has emerged as a growing epidemic medical syndrome in developed countries, which is characterized by high morbidity and mortality rates. Rapid and accurate diagnosis of this condition is essential for optimizing the therapeutic management. The diagnosis of congestive heart failure is challenging in patients presenting without obvious left ventricular systolic dysfunction and additional diagnostic information is most commonly required in this setting. Comprehensive Doppler echocardiography is the single most useful diagnostic test recommended by the ESC and ACC/AHA guidelines for assessing left ventricular ejection fraction and cardiac abnormalities in patients with suspected congestive heart failure, and non-invasively determined basal or exercise-induced pulmonary capillary hypertension is likely to become a hallmark of congestive heart failure in symptomatic patients with preserved left ventricular systolic function. The present review will focus on the current clinical applications of spectral tissue Doppler echocardiography used as a reliable noninvasive surrogate for left ventricular diastolic pressures at rest as well as during exercise in the diagnosis of heart failure with preserved left ventricular systolic function. Chronic congestive heart failure, a disease of exercise, and acute heart failure syndromes are characterized by specific pathophysiologic and diagnostic issues, and these two clinical presentations will be discussed separately. Congestive heart failure (HF) has become a highly prevalent medical syndrome in developed countries, which primarily affects older patients with a history of hypertension, coronary artery disease and diabetes mellitus . There iDuring exercise, pulmonary capillary pressure remains unchanged, decreases or slightly increases in healthy subjects ,16, thouAccording to the Starling's principles regarding fluid exchanges through the capillary wall, the fluid movement across the pulmonary capillary membrane is governed by the integrity of the capillary membrane and a balance between opposing filtrative and absorbing forces:Fluid movement = k(CHP - IHP) + k(πif - πc)Where k = filtration coefficient defining the fluid conductance across the capillary membrane, CHP = intra-capillary hydrostatic pressure, IHP = interstitial hydrostatic pressure, πif = colloid osmotic pressure of interstitial fluid and πc = plasma colloid osmotic pressure.Landmark studies have demonstrated experimentally that pulmonary capillary hypertension is the straightforward hemodynamic condition of cardiac pulmonary edema -23, alonGiven the limitations inherent to invasive recordings, Doppler echocardiography has become an invaluable tool for non-invasively determining LV diastolic pressures routinely. Currently, the American Society of Echocardiography recommends the use of color M-mode and spectral tissue Doppler-derived indexes, E/Vp and E/E' respectively, for assessing left atrial pressure in clinical trials . This isThe peak early diastolic mitral E velocity is primarily influenced by left atrial pressure, LV relaxation and LV systolic pressure in order of decreasing significance -43. The Numerous clinical studies using spectral tissue Doppler for recording mitral annular velocities have produced convincing evidence of a positive, linear relation of E/E' with invasively determined mean LV diastolic pressure regardless of LV ejection fraction, rhythm and heart rate ,48-60. SSymptoms of dyspnea and fatigue usually characterize exercise intolerance in chronic congestive HF (stage C of the ACC/AHA classification for chronic HF) , regardlExercise-induced changes in the E/E' ratio are adequately recorded in most patients. In healthy subjects, E/E' remains unchanged during exercise because of a proportional increase in E and E' velocities . It has The clinical application of diastolic stress echocardiography  in the setting of exertional dyspnea is relevant for establishing the cardiac contribution to symptoms. In the study by Skaluba et al in which most patients presented with a normal LV ejection fraction, septal E/E' > 10 at rest was a strong, independent predictor of reduced exercise performance beyond LV systolic and diastolic function, as defined by the LV ejection fraction and the mitral filling pattern respectively . In the Acute, transient exacerbation of symptoms is a common clinical presentation of HFPSF, particularly among elderly patients . The cliAn echocardiographic LV ejection fraction of < 40–45% has been reported to be a powerful predictor of cardiac origin in patients presenting with acute dyspnea ,94. In tNoninvasive assessment of LV diastolic pressures at presentation, before tailored therapy, is clinically relevant particularly for patients with severe symptoms, inconclusive clinical, radiographic and biochemical data and preserved LV ejection fraction at echocardiography (see Table According to the ACC/AHA recommendations, the diagnosis of congestive HF is defined by the association of structural heart disease with symptoms compatible with the diagnosis. In the light of the present review, we can state that tissue Doppler evidence of pulmonary capillary hypertension at rest and unmasked during exercise is likely to become a hallmark for the diagnosis of congestive HF in the setting of preserved LV systolic function, regardless of the clinical presentation.financial competing interestsNoneNon-financial competing interestsNone


A solution to this riddle was that intestinal villi do in fact secrete chloride as a result of rotavirus infection. Also, the overall chloride secretory response is regulated by a phospholipase C-dependent calcium signaling pathway induced by NSP4. However, the overall response is weak, suggesting that NSP4 may exert both secretory and subsequent anti-secretory actions, as did carbachol, hence limiting Cl- secretion. All these characteristics provide the means to make the necessary functional distinction between viral NSP4 and bacterial enterotoxins.Rotavirus is the major cause of infantile gastroenteritis and each year causes 611 000 deaths worldwide. The virus infects the mature enterocytes of the villus tip of the small intestine and induces a watery diarrhea. Diarrhea can occur with no visible tissue damage and, conversely, the histological lesions can be asymptomatic. Rotavirus impairs activities of intestinal disaccharidases and Na+-solute symports coupled with water transport. Maldigestion of carbohydrates and their accumulation in the intestinal lumen as well as malabsorption of nutrients and a concomitant inhibition of water reabsorption can lead to a malabsorption component of diarrhea. Since the discovery of the NSP4 enterotoxin, diverse hypotheses have been proposed in favor of an additional secretion component in the pathogenesis of diarrhea. Rotavirus induces a moderate net chloride secretion at the onset of diarrhea, but the mechanisms appear to be quite different from those used by bacterial enterotoxins that cause pure secretory diarrhea. Rotavirus failed to stimulate Cl Escherichia coli heat-labile and heat-stable toxins.Viral diarrheas are the cause of high mortality among children and animals, including many mammalian and avian species . But desRotavirus infection has always been considered to be confined to the upper two-thirds of the villi of the small intestine, but recent reports suggest that extra-intestinal manifestations may occur . DiarrheRotavirus diarrhea was first considered to be malabsorptive. Since 1996, the prevailing idea in the rotavirus field is that the nonstructural NSP4 protein might play a crucial role in fluid and electrolyte secretion, and hence might represent a novel viral secretory enterotoxin . Being aIn view of current ideas on the subject of rotavirus and NSP4-mediated diarrhea, we feel that a brief but precise description of the pathological mechanisms involved in the disease is necessary at this time. We further discuss how the mechanisms used by the NSP4 enterotoxin produced by rotavirus appear to be quite different from those used by bacterial enterotoxins in leading to mixed type rather than secretory diarrhea. The aim of this review is to clarify the necessary functional distinction between viral NSP4 and bacterial enterotoxins.In figure in vivo in young mice occurred with relatively intact intestinal brush border membrane (BBM) [in vitro in human Caco-2 cells were reduced by infection without any apparent enterocyte destruction, likely caused by a blockade of protein trafficking to BBM [+-D-glucose symport activity present in both villus and crypt cell BBM of rabbit intestine, although higher for villi than for crypt cells, was inhibited by rotavirus in the absence of tissue damage. The glucose uptake value remained much higher for villi BBM in infected rabbits than for crypt cell BBM in control, non-infected rabbits, which would have been improbable if the enterocytes were crypt-like cells [+-L-leucine symport activities [in vivo [It has previously been hypothesized that rotavirus infection kills off most of the mature enterocytes, so that crypt cells invade the villus surface, causing a decrease in the digestive and absorptive capacities of the intestine, and hence generating a malabsorption type of diarrhea . This crne (BBM) . Both thg to BBM . The SGLke cells . Also, tke cells ,16. Becativities  [in vivo .To summarize, rotavirus infection induces maldigestion of carbohydrates and their accumulation in the intestinal lumen as well as malabsorption of nutrients and a concomitant inhibition of water reabsorption, which can lead to a malabsorption component of diarrhea.Since the discovery of the NSP4 enterotoxin, diverse hypotheses have been proposed in favor of an additional secretory component in the pathogenesis of rotavirus diarrhea. Hence, there might be common mechanisms of pathogenesis for bacterial and viral enterotoxins ,11,18.direct experimental evidence of chloride secretion is highly lacking. There is only one instance in the literature in which NSP4 has been shown to directly stimulate iodine influx into distal colon crypts isolated from neonatal mice [ex-vivo on colon crypts, but nothing is known about the direct action of NSP4 on small intestine crypts. Furthermore, the situation in vivo is different. Rotavirus infects the mature enterocytes in the upper two-thirds of the villi of the small intestine, and the question arises – as with most luminal enterotoxins – as to the physical accessibility and binding capacity of the secreted NSP4 to the cells of the crypt region [In reality, tal mice . The expt region . On the t region .indirectly stimulates chloride secretion. In neonatal mouse intestinal mucosal sheets, NSP4 was initially shown to potentiate cAMP-dependent Cl- secretion [- secretory currents induced by NSP4-(114–135) were small compared with those induced by cAMP-mobilizing forskolin, which prompted the same authors soon afterwards to publish findings that NSP4, in contrast to many bacterial enterotoxins, led to a cyclic-nucleotide-independent secretory diarrhea [2+) to intestinal mucosal sheets induced transient, small and almost identical increases in Cl- secretory currents was interpreted as indicating that NSP4 induced a Ca2+-dependent Cl- secretory mechanism. Evidence has been gathered in favor of the mobilization of intracellular calcium associated with NSP4 expressed endogenously or added exogenously [- secretion [Another possibility is that NSP4 ecretion . The Cl-P4-114–13 were smagenously -24. Incrgenously -27. Thisecretion -28.2+ mobilization may trigger the release of amines/peptides from intestinal endocrine cells as well as the release of cytokines, prostaglandins and nitrous oxide from the enterocytes [in vitro and in vivo studies [By analogy to bacterial enterotoxin-induced fluid secretion, it has also been proposed that NSP4-mediated Caerocytes  Fig. 1)2+ mobili studies . All thein vivo.NSP4 has also been found to cause a disruption of tight junctions and a reduction in transepithelial electrical resistance accompanied by an increase in paracellular permeability to macromolecules of 20 kiloDa across MDCK cells , as did 2+ and subsequent ENS stimulation have a role in indirectly regulating chloride secretion might cast doubts as to the secretory agonist action of NSP4. However, the possibility exists that NSP4 might exert positive as well as negative regulation of Cl- secretion, as did carbachol [- secretory currents [-secretion that makes the cells refractory to re-stimulation by a second calcium-dependent agonist [2+ through phospholipase C (PLC) activation. Such PLC activation can lead to transient chloride secretion through inositol  triphosphate (IP3) release. However, NSP4, like carbachol, could promote long-term inhibitory feedback through inositol tetrakisphosphate (IP4) production, preventing Cl- secretion from being sustained [- secretion in rotavirus diarrhea.Taken together, the data show that while NSP4 induces diarrhea in young mice, its importance in chloride secretion remains unresolved. The lack of clear evidence that mobilization of intracellular Caarbachol ,28,32. Fcurrents . Such a  agonist ,28. If N agonist  and NSP4 agonist  had been- secretion during rotavirus diarrhea have revealed discordance between in vitro studies in Ussing short-circuited chambers and in vivo perfusion studies . Davids animals . Conversggestion . Lorrot  rabbits . However- in the stools can be due to decreased absorption in the villus cells and/or increased secretion in the crypt cells [- transport in crypt cell BBM [- reabsorption in villus cell BBM [- secretion at the onset of rotavirus diarrhea. Because rotavirus stimulated both Cl- influx and Cl- efflux in villi, Lorrot et al. proposed that the chloride carrier might function in both normal (absorption) and reversed (secretion) modes, depending on the direction of the chloride electrochemical gradient resulting from rotavirus infection [-/H+ symporter may also explain why rotavirus still results in diarrhea in mice lacking the apical CFTR (Cystic Fibrosis Transmembrane conductance Regulator) chloride channel [While it is widely accepted that loss of Clpt cells ,35-37, tcell BBM , whereascell BBM  Fig. 1)- in the nfection . The pre channel .Escherichia coli, which cause "pure" secretory diarrhea [+ and glucose [+-solute symport activities, hence contributing to massive water loss all along the small intestinal crypt-villus axis [- [- reabsorption in villi [- transport in crypt [- secretion. A solution to this riddle was that intestinal villi do in fact secrete chloride as a result of rotavirus infection [2+-dependent chloride secretory response induced by rotavirus is transient and small, implying that negative signaling events may limit chloride secretion [-into the intestinal lumen at the onset of rotavirus diarrhea. All these considerations support the idea that NSP4 may act as an enterotoxin, but it would act as a viral enterotoxin that would function quite differently from bacterial enterotoxins by inducing mixed type rather than secretory diarrhea.The mechanisms by which rotavirus and the NSP4 enterotoxin cause diarrhea appear to be quite different from those described for bacterial enterotoxins, such as cholera toxin and the heat-labile and heat-stable enterotoxins of diarrhea . To suppdiarrhea ,11,36. Blus axis . Unlike  axis [- , rotavirin villi  without in crypt , questionfection . NSP4 hanfection . Finallyecretion . The pos


There are three peroxisome proliferator-activated receptors (PPARs) subtypes which are commonly designated PPAR alpha, PPAR gamma and PPAR beta/delta. PPAR alpha activation increases high density lipoprotein (HDL) cholesterol synthesis, stimulates "reverse" cholesterol transport and reduces triglycerides. PPAR gamma activation results in insulin sensitization and antidiabetic action. Until recently, the biological role of PPAR beta/delta remained unclear. However, treatment of obese animals by specific PPAR delta agonists results in normalization of metabolic parameters and reduction of adiposity. Combined treatments with PPAR gamma and alpha agonists may potentially improve insulin resistance and alleviate atherogenic dyslipidemia, whereas PPAR delta properties may prevent the development of overweight which typically accompanies "pure" PPAR gamma ligands. The new generation of dual-action PPARs – the glitazars, which target PPAR-gamma and PPAR-alpha (like muraglitazar and tesaglitazar) are on deck in late-stage clinical trials and may be effective in reducing cardiovascular risk, but their long-term clinical effects are still unknown. A number of glitazars have presented problems at a late stage of clinical trials because of serious side-effects (including ragaglitazar and farglitazar). The old and well known lipid-lowering fibric acid derivative bezafibrate is the first clinically tested pan –  PPAR activator. It is the only pan-PPAR activator with more than a quarter of a century of therapeutic experience with a good safety profile. Therefore, bezafibrate could be considered  as an "archetype" of a clinically tested pan-PPAR ligand. Bezafibrate leads to considerable raising of HDL cholesterol and reduces triglycerides, improves insulin sensitivity and reduces blood glucose level, significantly lowering the incidence of cardiovascular events and new diabetes in patients with features of metabolic syndrome. Clinical evidences obtained from bezafibrate-based studies strongly support the concept of pan-PPAR therapeutic approach to conditions which comprise the metabolic syndrome. However, from a biochemical point of view, bezafibrate is a PPAR ligand with a relatively low potency. More powerful new compounds with pan-PPAR activity and proven long-term safety should be highly effective in a clinical setting of patients with coexisting relevant lipid and glucose metabolism disorders. When activated by agonist ligand binding, this heterodimer complex recruits transcription coactivators and regulates the transcription of genes involved in the control of lipid and carbohydrate metabolism [Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors, i.e. ligand-dependent intracellular proteins that stimulate transcription of specific genes by binding to specific DNA sequences following activation by the appropriate ligand. When activated, the transcription factors exert several functions in development and metabolism. There are three PPAR subtypes which are the products of distinct genes and are commonly designated PPAR alpha, PPAR gamma and PPAR beta/delta, or merely delta -4. The Ptabolism -4.PPAR alpha, activated by polyunsaturated fatty acids and fibrates, is implicated in regulation of lipid metabolism, lipoprotein synthesis and metabolism and inflammatory response in liver and other tissues. PPAR alpha is highly expressed in tissues with high fatty acid oxidation , in which it controls a comprehensive set of genes that regulate most aspects of lipid catabolism. Like several other nuclear hormone receptors, it heterodimerizes with RXR alpha to form a transcriptionally competent complex [ complex -3,5. In  complex -3,6.PPAR gamma plays important roles in the regulation of proliferation and differentiation of several cell types, including adipose cells. It has the ability to bind a variety of small lipophilic compounds derived from both metabolism and nutrition. These ligands, in turn, determine cofactor recruitment to PPAR gamma, regulating the transcription of genes in a variety of complex metabolic pathways. PPAR gamma is highly expressed in adipocytes, where it mediates differentiation, promotes lipid storage, and, as a consequence, is thought to indirectly improve insulin sensitivity and enhance glucose disposal in adipose tissue and skeletal muscle [l muscle -9. Its aPPAR delta remained unclear. Animal studies revealed that PPAR delta play an important role in the metabolic adaptation of several tissues to environmental changes. Treatment of obese animals by specific PPAR delta agonists results in normalization of metabolic parameters and reduction of adiposity. PPAR delta appeared to be implicated in the regulation of fatty acid burning capacities of skeletal muscle and adipose tissue by controlling the expression of genes involved in fatty acid uptake, beta-oxidation and energy uncoupling. PPAR delta is also implicated in the adaptive metabolic response of skeletal muscle to endurance exercise by controlling the number of oxidative myofibers, inducing so and enhancing fatty acid catabolism in muscular tissue [Until recently, the biological role of r tissue ,6,10. Mor tissue .Fibric acid derivatives (fibrates) are PPAR alpha ligands. Fibrates have been used in clinical practice for more than four decades as a class of agents known to decrease triglyceride levels while substantially increasing HDL-cholesterol levels, with a limited but significant additional lowering effect on low density lipoprotein (LDL)-cholesterol levels . In addiGlitazones are synthetic PPAR gamma ligands with well recognized effects on glucose and lipid metabolism. The clinical use of these PPARgamma agonists in type 2 diabetic patients leads to an improved glycemic control and an inhanced insulin sensitivity, and – at least in animal models – to a protective effect on pancreatic beta-cell function. Glitazones may also have cardiovascular benefits. Animal models of atherosclerosis have shown that these drugs reduce the extent of atherosclerotic lesions and inhibit macrophage accumulation. Clinical studies have also shown that these drugs improve the lipid profile of patients at risk of developing atherosclerosis and reduce circulating levels of inflammatory markers -18. HoweCurrently, clinical studies regarding PPAR delta ligands are lacking. Given the results obtained with animal models, PPAR delta agonists may have therapeutic usefulness in metabolic syndrome by increasing fatty acid consumption in skeletal muscle and adipose tissue . ProbablCombined treatments with PPAR gamma and alpha agonists may potentially improve insulin resistance and alleviate atherogenic dyslipidemia, whereas PPAR delta properties may prevent the development of overweight which typically accompanies "pure" PPAR gamma ligands like glitazones. With extended use, it is hoped that these effects will reduce the risk of long-term cardiovascular complications. PPAR alpha and gamma stimulation play complementary roles in the prevention of atherosclerosis. Cholesterol accumulation in macrophages located in the endothelium is a crucial step in the formation of atherosclerosis. PPAR gamma activation is necessary for the efflux of cholesterol from macrophage foam cells. Cholesterol taken up by HDL particles containing apolipoportein A-1 is transported to the liver to be disposed of as bile acids ,15,17. PThus, compounds with dual PPAR alpha/PPAR gamma activity appear well-suited for the treatment of diabetic patients with the additional risk factor of dyslipidemia. The finding that PPAR agonists play a role in regulating other processes, such as inflammation, vascular function, and vascular remodeling, has highlighted further potential indications for these agents ,17. So fThe old and well known lipid-lowering fibric acid derivative bezafibrate is the first clinically tested pan –  PPAR activator -33. It iSo, which are the data regarding bezafibrate administration? In a large trial in 1568 men with lower extremity arterial disease, bezafibrate reduced the severity of intermittent claudication for up to three years . In geneOur new data demonstrate that bezafibrate can significantly reduce the incidence of myocardial infarction (MI) in patients with metabolic syndrome . The decPrevious observations have shown beneficial effects of bezafibrate on glucose and insulin metabolism -41. ReceTherefore, the pan –  PPAR activator bezafibrate leads to a considerable raising of HDL cholesterol and a reduction of triglycerides, improves insulin sensitivity and reduces blood glucose level, significantly lowering the incidence of cardiovascular events and new diabetes in patients with features of metabolic syndrome over a long-term follow-up period. We conclude that clinical evidences obtained from bezafibrate-based studies strongly support the concept of a pan-PPAR therapeutic approach to conditions which comprise the metabolic syndrome. However, from a biochemical point of view, bezafibrate is PPAR ligand with a relatively low potency. We believe that more powerful compounds with pan-PPAR activity and proven long-term safety should be highly effective in a clinical setting of patients with coexisting relevant lipid and glucose metabolism disorders.BIP – Bezafibrate Infarction PreventionHDL – high density lipoproteinLDL – low density lipoproteinMI – myocardial infarctionPPAR – peroxisome proliferator-activated receptorRXR – retinoic acid receptorThe author(s) declare that they have no competing interests.All authors have equally contributed in the conception and drafting of the manuscript.


The infiltration of dense non-aqueous-phase liquids (DNAPLs) into the saturated subsurface typically produces a highly contaminated zone that serves as a long-term source of dissolved-phase groundwater contamination. Applications of aggressive physical–chemical technologies to such source zones may remove > 90% of the contaminant mass under favorable conditions. The remaining contaminant mass, however, can create a rebounding of aqueous-phase concentrations within the treated zone. Stimulation of microbial reductive dechlorination within the source zone after aggressive mass removal has recently been proposed as a promising staged-treatment remediation technology for transforming the remaining contaminant mass. This article reviews available laboratory and field evidence that supports the development of a treatment strategy that combines aggressive source-zone removal technologies with subsequent promotion of sustained microbial reductive dechlorination. Physical–chemical source-zone treatment technologies compatible with posttreatment stimulation of microbial activity are identified, and studies examining the requirements and controls  of reductive dechlorination of chlorinated ethenes are investigated. Illustrative calculations are presented to explore the potential effects of source-zone management alternatives. Results suggest that, for the favorable conditions assumed in these calculations , source longevity may be reduced by as much as an order of magnitude when physical–chemical source-zone treatment is coupled with reductive dechlorination. Widespread use of chlorinated solvents in dry cleaning and metal degreasing operations over the last century has resulted in extensive groundwater contamination by compounds such as tetrachloroethene (PCE) and trichloroethene (TCE). When released into the subsurface as dense non-aqueous-phase liquids (DNAPLs), chlorinated solvents tend to migrate downward through the unsaturated zone and can penetrate the water table because of their higher density . During A number of innovative technologies have been developed to enhance contaminant removal from DNAPL source zones . No organisms that grow aerobically with PCE or TCE as a carbon source have been identified. In anoxic environments the metabolic oxidation of chloroethenes is still poorly understood. Although the mineralization of cis-DCE and VC under iron- and manganese-reducing conditions has been demonstrated . Organisms capable of metabolic reductive dechlorination  have been isolated from contaminated and pristine sites  and, in some cases, VC accumulate. cis-DCE and VC, but they identified a minimum threshold chlorinated contaminant concentration below which dechlorination could not be sustained. There is an apparent link between the presence of members of the Dehalococcoides group and complete dechlorination   . Dehalocand slow . A majorcceptors , 2003b.cis-DCE, incomplete dechlorination could still be beneficial for source-zone bioremediation because a) dissolution rates are enhanced 3-fold (b) cis-DCE and VC are more accessible to aerobic degradation in down-gradient aerobic zones  into PCE-DNAPL and decreases in pH due to the release of HCl have also been observed and may affect the dechlorination of the lesser chlorinated ethenes  cic zones , 2002b. ic zones . Competiic zones . Partiti ethenes .cis-DCE dechlorination, complete detoxification requires the presence and activity of Dehalococcoides populations  initiate the reductive dechlorination process in source zones (b) establish bioreactive barriers for treatment of dissolved contaminants down-gradient  must be provided. Chlororespiring populations depend on the activity of fermentative organisms to convert (complex) organic materials into suitable electron donors  . A varieOver the past decade, a number of innovative technologies have been developed that show promise for recovering a large fraction of the DNAPL mass at a given site e.g., . AlthougGiven that innovative source-zone removal technologies have been extensively documented e.g., , 1999, ta) the gas phase will contact the nonaqueous phase, resulting in direct mass transfer from the DNAPL to the vapor phase, and b) the gas phase will strip dissolved contaminants from the aqueous phase (A source-zone remediation technology that has been implemented at many DNAPL-contaminated sites is air sparging . Air is us phase .cis-DCE and VC) are amenable to growth-linked microbial degradation under aerobic conditions, a metabolic process capable of oxidizing PCE and TCE has yet to be identified  was developed to transform contaminants into benign products  [for mechanistic descriptions of ISCO technologies, see •) via Fenton’s chemistry. The hydroxyl radicals are strong oxidants and react rapidly with surrounding molecules. Solutions of hydrogen peroxide, without catalyst, have been introduced into the subsurface  energy demands contribute significantly to project costs (b) the potential for NAPL mobilization  flooding, resistive heating , conductive heating , or some combination thereof [for more detailed descriptions of several thermal technologies, see ct costs  and b) tlization . During lization . Lesser lization , and efflization .In situ rates of cooling are anticipated to be slow enough to allow subsequent microbial rebound  . During in situ flushing technologies that use surfactants to overcome many of the limitations experienced during pump-and-treat remediation of DNAPL source zones  and sodium chloride (as high as 7 g/L) to increase contaminant solubilization capacities > 60 g/L e.g., . Thus, pExisting evidence suggests certain physical–chemical source-zone treatment technologies are more promising for the stimulation of microbial activity as a post-treatment polishing step. Although air sparging, chemical oxidation, and steam flooding may generate an aerobic environment suitable for subsequent metabolic or co-metabolic oxidation, SEAR and co-solvent flushing appear to be the most promising physical–chemical treatments for integration with the microbial reductive dechlorination process. Note that in this assessment, the possibility that DNAPL contaminant distributions resulting from aggressive treatment may be technology specific has not been considered because of the scarcity of data. Residual alcohol or surfactant solutions contribute to oxygen depletion and establishment of anaerobic conditions after aggressive treatment. Further, residual flushing solution may serve as a source of reducing equivalents and stimulate the reductive dechlorination process. Although other technologies may eventually be successfully integrated with posttreatment microbial reductive dechlorination, SEAR seems particularly applicable because of limited toxicity on the microbial community, the establishment of reducing conditions, and the release of reducing equivalents for stimulation of the reductive dechlorination process. Thus, the ultimate fate of the residual surfactant solution and its effect on the dechlorinating population must be considered.Although microbial degradation of surfactants in aerobic environments is well documented , it is uThe residual surfactant concentrations, however, may also alter the bioavailability of a contaminant . Yeh et Although microbial reduction of PCE in DNAPL source zones may be feasible, the relatively low dissolution enhancement factors (3- to 6-fold) reported imply that source longevity would still be measured in multiple decades. Alternatively, if uncertainties in the source zone microbial environment after physical–chemical treatment can be overcome, multiple order-of-magnitude reductions in source-zone mass removal obtained via active physical–chemical treatment might be combined with posttreatment biopolishing to substantially reduce source longevity. Ultimately, it may be possible to devise a posttreatment source-zone strategy that minimizes operations and maintenance efforts while still meeting regulatory standards at down-gradient points of compliance.a) natural gradient dissolution , b) enhanced reductive dechlorination (source-zone bioremediation), and c) physical–chemical treatment followed by source-zone biopolishing (SEAR plus enhanced reductive dechlorination). The four hypothetical field scenarios were selected to span the range of behavior that may be expected in the field and are characterized by a ganglia-to-pool (GTP) ratio, which is a measure of the distribution of mass between low saturation ganglia regions and high saturation pool regions in the source zone. The formation properties, spill scenario, and SEAR characteristics were taken from a recent numerical modeling study that was based on a pilot-scale SEAR demonstration at the Bachman Road site in Oscoda, Michigan (Sn = 1) pools with no ganglia remaining  and the Bachman Road SEAR site  serve as documented case studies where field evidence supports the conclusion that physical–chemical source-zone removal may be coupled with reductive dechlorination. A comparison between observations at the Sages and Bachman sites is shown in Dehalococcoides-targeted primers (cis-DCE production (up to 242 μM) at monitoring points located within the treated zone are indicative of microbial reductive dechlorination.At the Sages site, 34,000 L of a solution consisting of 95% (vol) ethanol and 5% (vol) water were flushed through a DNAPL source zone over a period of 3.5 days followed by a 4.5-day water flood used to recover injected fluids . This co primers . Additio primers . AlthougAt the Bachman Road site, a pilot-scale field demonstration of SEAR was conducted to remove PCE-DNAPL from beneath a former dry cleaning facility. For this source-zone treatment, 68,400 L of an aqueous solution containing 6% (weight) Tween 80 were introduced over a period of 10 days, with 2 additional days of active water flooding . Approxicis-DCE transformation within the treated source zone is consistent with laboratory microcosm studies conducted with aquifer material from the Bachman Road site  they typically occur in proximity to areas of higher population, increasing risk and limiting hydraulic isolation  options, and b) the relatively low NAPL saturations and smaller treated volumes at these sites increase treatment costs as quantified by conventional metrics (dollars per cubic meter of treated soil or dollars per liter of NAPL recovered). Higher costs per volume (treated soil or NAPL) result from a threshold cost associated with establishing a treatment system regardless of site size. Many innovative source-zone technologies offer efficient mass removal at the expense of greater, upfront capital expenditures  the source-zone (dechlorinating) microbial community, before, during, and after the treatment process, and b) contaminant and transformation product concentration distributions after treatment. Indeed, to date, most field observations of enhanced reductive dechlorination in treated source zones have been fortuitous, with little thought devoted to microbial processes in the initial design and implementation of the treatment monitoring scheme. Specific culture-dependent  and culture-independent (nucleic acid-based) tools for assessment of the microbial community are now available for this characterization effort  in source zones is feasible.Future field demonstrations may also be enhanced through exploitation of results obtained from microbial laboratory investigations. Laboratory-scale studies conducted under conditions representative of a source-zone environment  provide heuristic, as well as quantitative, guidance for implementation of posttreatment bioremediation. Substrate amendment strategies that favor chlororespiring populations by maintaining a low concentration of hydrogen may be adapted from the laboratory to the field. However, additional work will be required to explore the effect of unrecovered flushing solutions  typical of a posttreatment source-zone environment on the metabolism of chlorinated NAPLs by chlororespiring organisms. The discovery of numerous dechlorinating populations capable of converting PCE to


Atrial fibrillation (AF) is a very undesirable, but unfortunately a common arrhythmia following coronary artery bypass graft (CABG) surgery, occurring in up to 40% of patients . There iThe pathophysiology of post-operative AF is incompletely understood and appears multi-factorial. There are studies to suggest clinical factors like advanced age, prior history of AF  , post-opIt is crucial to identify who would be at risk of developing AF, post CABG surgery, so as to institute appropriate preventive measures. Pharmacological approach has been widely used wherein preoperative initiation of beta-blocker therapy like oral metoprolol and continuation of the same postoperatively has been very effective in preventing AF . AmiodarBased on the mechanism of postoperative AF, it seems likely that overcoming the slow atrial conduction with reduction in the dispersion of atrial refractoriness and suppression of the atrial ectopy should prevent AF. With these considerations atrial overdrive pacing to prevent post CABG AF has been evaluated with a number of randomized, controlled trials. These trials compared controls with either single site atrial pacing i.e. right atrial pacing (RAP) or left atrial pacing (LAP) and biatrial pacing (BAP) or only RAP and BAP or only LAP and BAP. Bachmann’s bundle pacing as an alternative to two sites, biatrial pacing has also been studied. Different pacing modalities  and different stimulation rates are initiated after the surgery and continued for a period of 3-4 days. Earlier studies with pacing highlighted the limitations including sensing and capture problems, increase in atrial ectopy with demand (AAI) pacing and reported no advantage with pacing. However, most recent studies seem to favor prophylactic biatrial pacing to reduce the incidence of post CABG AF . Two stuThe present study involving 120 patients by Massoud E. et al  in the pIt seems use of oral beta-blockers preoperatively and continuing the same postoperatively definitely prevents AF occurrences in the post-operative patients. Amiodarone therapy is especially rewarding when a hybrid intravenous and oral loading regime is employed as suggested by AFIST II trial. A meta-analysis of smaller pacing studies support the role of biatrial pacing. Few questions remain unanswered. Is pacing a cost-effective strategy; should it be prophylactically used in all patients; is there at all a role of single chamber (RA or LA) pacing and single site biatrial pacing ? A larger, multi-center study with a cost effective analysis, over and above the use of beta-blocker and amiodarone is likely to settle the role of prophylactic pacing. Till such time we need to pre-select the high-risk group and offer biatrial pacing.


Vespula wasps. Furthermore, we compared the patterns found in this species to those found in other taxa in order to investigate how variation in gene expression leads to phenotypic evolution.Organisms are capable of developing different phenotypes by altering the genes they express. This phenotypic plasticity provides a means for species to respond effectively to environmental conditions. One of the most dramatic examples of phenotypic plasticity occurs in the highly social hymenopteran insects , where distinct castes and sexes all arise from the same genes. To elucidate how variation in patterns of gene expression affects phenotypic variation, we conducted a study to simultaneously address the influence of developmental stage, sex, and caste on patterns of gene expression in Vespula squamosa. Comparisons of overall expression patterns indicated that gene-expression differences distinguishing developmental stages were greater than expression differences differentiating sex or caste. Furthermore, we determined that certain sets of genes showed similar patterns of expression in the same phenotypic forms of different species. Specifically, larvae upregulated genes related to metabolism and genes possessing structural activity. Surprisingly, our data indicated that at least a few specific gene functions and at least one specific gene family are important components of caste differentiation across social insect taxa.We constructed 11 different cDNA libraries derived from various developmental stages and castes of Despite research on various aspects of development originating from model systems, growth in understanding how development is related to phenotypic diversity relies on a growing literature of contrasting studies in non-model systems. In this study, we found that comparisons of patterns of gene expression with model systems highlighted areas of conserved and convergent developmental evolution across diverse taxa. Indeed, conserved biological functions across species implicated key functions related to how phenotypes are built. Finally, overall differences between social insect taxa suggest that the independent evolution of caste arose via distinct developmental trajectories. A fundamental goal of the burgeoning field of evolutionary developmental biology is to understand how differences in gene expression contribute to phenotypic diversity. Phenotypic plasticity, the ability of a single genotype to produce alternate forms of morphology, physiology or behavior in response to environmental conditions -3, proviHighly social hymenopteran insects  present one of the most striking examples of polyphenism. Hymenopteran queens, workers, and males all possess the same genes , unlike many other animals, where sex chromosomes play a role in sex determination. Therefore, the phenotypic differences among hymenopteran social insect castes, as well as sexes, are derived from variation in gene expression.Vespula squamosa. Vespula wasps are a particularly good taxon in which to study phenotypic evolution, for several reasons. First, Vespula wasps display distinct female castes; queens differ from workers in size, color, behavior, body proportions, and physiology [Vespula wasps display remarkable similarities to Apis bees, although the two taxa are only distantly related [Vespula and Apis will reveal if analogous environmentally induced phenotypes are generated through similar patterns of gene expression. Finally, Vespula queens and workers are reared in distinct cells; this key feature allows the developmental fate of larvae to be known very early in ontogeny [In this study, we investigated the molecular underpinnings involved in the development of the social wasp ysiology ,8  How do patterns of gene expression differ among developmentally distinct phenotypes?(ii) Are sex-specific developmental patterns similar across insect species?(iii) Do caste-specific developmental patterns display convergent evolution?The most striking result from our data is that developmental stage  plays a much larger role in establishing patterns of gene expression than either caste or sex Figure . In factV. squamosa libraries and thus provided insight into the molecular processes associated with development in this taxon? A general χ2 test  VSQ318 and VSQ031 are both members of the odorant-binding protein family  identifPisum sativum putative senescence-associated protein. Surprisingly, Sharf et al [Reticulitermes flavipes. They suggest that this gene may play a role in ribosomal filtering [R. flavipes (and VSQ709) transcript to 28s rRNA-like sequences.Two other genes show patterns of expression similar to those observed in other species. First, VSQ445, which is homologous to the German cockroach major allergen Bla g 1, is upregulated in adult females, as is the case in cockroaches . Second,rf et al  found thiltering , owing tFinally, several genes of unknown function or those with low or no known homology also showed characteristic expression patterns at particular developmental stages. For example, both VSQ056 and VSQ058 were upregulated during the early and late larval developmental stages, whereas VSQ943 exhibits a striking pattern of upregulation during the female pupal stages Table . The rolV. squamosa support previous studies that have investigated gene-expression patterns among developmental stages. For example, Mathavan et al [Drosophila melanogaster occurred between life stages. Similar to our results, Arbeitman et al found that expression patterns for both male and female adults grouped closely together despite the apparent morphological, physiological and behavioral differences that exist between sexes. In contrast to our results, they found that larval expression was more similar to that of adults, whereas expression of embryos was more similar to that of pupae. The V. squamosa data suggest a more temporal pattern, with EST frequencies more similar between adults and pupae than between adults and larvae.The general expression patterns found in an et al  found clan et al  also fouan et al  discoverV. squamosa further, we used information from the gene ontology (GO) classifications [D. melanogaster [To investigate patterns of gene expression in ications . Use of nogaster . To deteCamponotus festinatus ants and D. melanogaster, and found that some patterns persisted between these species. In particular, the larvae of both species upregulated genes that were involved in protein production and possessed structural activity relative to those in adults. For both of these GO functions, the patterns hold true in V. squamosa when ESTs that show significant similarity to D. melanogaster genes are considered. The mean ± SE number of transcripts associated with protein metabolism is 51.5 ± 15.5 in larvae and 25.3 ± 4.8 in adults, and the mean ± SE number of transcripts possessing structural activity is 16.5 ± 4.5 in larvae and 5.3 ± 0.7 in adults , the male patterns of gene expression are more different and hence diverge before the two female castes . Specifically, the differences in the proportion of genes displaying homology in the adult male and adult female libraries is striking  numbers of homologs  compared with adult males (26%). One possible explanation for these differences is that genes expressed in adult males evolve particularly rapidly relative to those expressed in females and at other developmental stages. This result is consistent with studies in other taxa that have shown that male-specific genes evolve rapidly [de novo gene evolution occurs among male-biased genes. Our data indicate that similar processes may operate in social insects. Whether such putatively rapidly evolving genes are exclusively or primarily expressed in sex-specific tissues, as has been found to be the case in other taxa [Our analysis of sequenced ESTs uncovered another interesting pattern regarding sex in g Figure , with ad rapidly -26. Inde rapidly  deduced her taxa , represeher taxa .The defining feature of social insects is the division of individuals into reproductive and sterile castes . TherefoV. squamosa compare with these other taxa? Four major trends arise from our analyses. First, as might be expected, EST chord distances among castes at earlier life stages are more similar (WLE-QLE = 1.08) than at later life stages (WA-QA distance = 1.23), with intermediate stages at intermediate distances  or early queen larvae  were used in the comparison. Thus, the trends in our data indicate that young larvae are more similar to queen-destined larvae than to worker-destined larvae in V. squamosa. Additionally, the observed differences in chord distances between worker or queen early larvae and worker or queen late larvae suggest that even though young larvae  are potentially bipotent, they may express different genes.Second, our data contrast with a pattern of development identified in honeybees. Evans and Wheeler  suggesteV. squamosa express more genes related to metabolism  than do similarly aged worker larvae  is a methionine-rich member of the hexamerin family that participates in the storage of amino acids accumulated during larval development [V. squamosa exhibit different expression patterns between queen and worker castes. VSQ019 and VSQ233 are upregulated in queen-destined larvae of V. squamosa, whereas VSQ232 and VSQ292 (both hexamerin 70b-like ESTs) are more highly expressed in the late larvae of workers relative to queens. The similarity of this gene-expression pattern among these species suggests that some specific pathways are conserved during social-insect evolution.Fourth, the hexamerin gene family, which plays a significant role in caste differentiation in ellifera , B. terrrrestris  and R. fflavipes ,40,41, aelopment . MoreoveV. squamosa upregulate genes related to metabolism and possessing structural activity. Furthermore, our data provide a provocative example of divergent selection pressures for genes expressed differentially between the sexes. We also discovered that V. squamosa and A. mellifera castes, which arose via independent evolutionary events, may develop through different trajectories. Nevertheless, at least a few specific gene functions and at least one specific gene family appear to be conserved components of caste differentiation. Overall, our results illustrate how the study of phenotypic diversity arising from patterns of gene expression can illuminate evolutionary effects of development in animal taxa.We conducted the first study to simultaneously address the influence of developmental stage, sex, and caste on patterns of gene expression. We found that patterns of expression are more similar across castes for a specific developmental stage than within castes at different stages. Similar to other insect taxa, larvae of our study taxon V. squamosa. The 11 libraries contained copies of transcripts obtained from: (i) eggs collected from queen cells; pooled female larvae from the first three early larval instars sampled from (ii) queen cells and (iii) worker cells; pooled female larvae of the fourth and fifth late larval instars sampled from (iv) queen cells and (v) worker cells; (vi) queen pupae; (vii) worker pupae; (viii) male pupae; (ix) queen adults; (x) worker adults; and (xi) male adults. The cDNA libraries were synthesized using a commercial construction kit . In total, 4224 independent clones were isolated from these libraries, and 3388 single-pass sequences were obtained using the SK primer. After cloned sequences were filtered for vector contamination and quality, we obtained 2144 expressed sequence tags .We constructed 11 directional cDNA libraries from several developmental stages of the wasp 5; Figure These ESTs were grouped into clusters using the BLASTN algorithm . When seWe clustered the 11 libraries using the neighbor-joining method based on chord distances derived from library EST frequencies in order to gain an understanding of how patterns of gene expression were associated with development . FurtherEH and MG conceived the study and participated in its design and coordination. EH carried out the library construction, DNA sequencing, and EST analysis. EH and MG outlined the manuscript together and EH drafted the manuscript. Both authors have read and approved the final manuscript


Both theoretical and applied studies have proven that the utility of single nucleotide polymorphism (SNP) markers in linkage analysis is more powerful and cost-effective than current microsatellite marker assays. Here we performed a whole-genome scan on 115 White, non-Hispanic families segregating for alcohol dependence, using one 10.3-cM microsatellite marker set and two SNP data sets . Two definitions of alcohol dependence (ALDX1 and ALDX2) were used. Our multipoint nonparametric linkage analysis found alcoholism was nominal linked to 12 genomic regions. The linkage peaks obtained by using the microsatellite marker set and the two SNP sets had a high degree of correspondence in general, but the microsatellite marker set was insufficient to detect some nominal linkage peaks. The presence of linkage disequilibrium between markers did not significantly affect the results. Across the entire genome, SNP datasets had a much higher average linkage information content  than did microsatellite marker set (0.57). The linkage peaks obtained through two SNP datasets were very similar with some minor differences. We conclude that genome-wide linkage analysis by using approximately 5,000 SNP markers evenly distributed across the human genome is sufficient and might be more powerful than current 10-cM microsatellite marker assays. In traditional linkage analysis for identifying genomic regions related to disease phenotypes, a whole-genome scan is usually performed using a set of 300–400 microsatellite markers evenly spaced across the genome. To maximize the chances of detecting linkage, the optimal amount of inheritance information is critical. This can be increased by genotyping more families and adding additional markers. With the rapid discovery of SNPs across the genome and the development of large-scale, high-throughput SNP genotyping approaches, high-density SNP assays throughout the genome may be a more rapid, powerful, and cost-effective tool than microsatellite marker assays in linkage analysis . RecentlThe COGA dataset provided to participants in GAW14 was analyzed in this study. Only families with ethnicity self-reported as White, non-Hispanic were kept for analysis. Two diagnostic criteria for alcoholism were used in our analyses. For the first criterion, a diagnosis of alcoholism required positive diagnosis by the DSM-III-R criteria  and defiSNP genetic map positions were interpolated on the deCODE genetic map  through Spairs scoring function [We performed multipoint nonparametric linkage analysis using an affected-only allele-sharing method, which was implemented in the ALLEGRO (version 1.2c) software . We emplfunction , which pfunction  to generfunction .We used 115 White, non-Hispanic families in our analysis. The total number of individuals was 1,245, of which 1,009 were genotyped. Linkage information content for two SNP datasets was very similar except that the less-dense Illumina set had lower linkage information content on the X chromosome due to its poor coverage , we found 12 genomic regions with nominally significant LOD scores  and X (Xp22) in the SNP assays were two examples of signals not detected in the microsatellite analyses. The good concordance between the two SNP marker sets (Affymetrix and Illumina) in both linkage information content and linkage findings suggests that >5,000 SNPs may be excessive for samples with structures similar to the COGA data, and a SNP scan with ~5,000 markers distributed evenly across the human genome is sufficiently dense and powerful in whole-genome linkage analysis. Also, with current technology SNP genotyping is more rapid, requires fewer samples, and is more accurate than microsatellite marker genotyping. High-density SNP marker sets also offer a better localization of linkage peaks, which may save work for fine mapping in regions showing linkage . Since bOur analysis found nominal linkage for alcoholism to 12 genomic regions under both definitions for alcohol dependence (ALDX1 and ALDX2). The results for the two phenotype definitions are somewhat different. It is not clear which criterion is best for identifying genetic susceptibility loci for alcoholism. However, if one genomic region is associated with alcoholism, there should be similar statistical evidence under both criteria. Our finding on chromosome 2 overlaps with that of Reich et al. , who repWe conclude that a high-density SNP scan may offer a more rapid, cost-effective and powerful tool in genome-wide linkage analysis compared to traditional 10-cM microsatellite marker scans. However, further investigation is warranted to explore the effects of genetic map and computational issues on the utility of high density SNP assays in linkage analysis.COGA: Collaborative Study on the Genetics of AlcoholismGAW14: Genetic Analysis Workshop 14LD: Linkage disequilibriumSNP: Single-nucleotide polymorphismQM reconstructed the genetic map, carried out statistical analysis and drafted the manuscript. YY participated in genetic map reconstruction. YM and JF managed the data. LAF supported this study and helped to draft the manuscript. MAW conceived of the study, and participated in its design and helped to draft the manuscript. All authors read and approved the final manuscript.