These datasets will also facilitate more robust secondary analyses, such as explorations with alternative genetic models, epistasis and gene-environment interactions.A wealth of genetic associations for cardiovascular and metabolic phenotypes in humans has been accumulating over the last decade, in particular a large number of loci derived from recent genome wide association studies (GWAS). True complex disease-associated loci often exert modest effects, so their delineation currently requires integration of diverse phenotypic data from large studies to ensure robust meta-analyses. We have designed a gene-centric 50 K single nucleotide polymorphism (SNP) array to assess potentially relevant loci across a range of cardiovascular, metabolic and inflammatory syndromes. The array utilizes a “cosmopolitan” tagging approach to capture the genetic diversity across ∼2,000 loci in populations represented in the HapMap and SeattleSNPs projects. The array content is informed by GWAS of vascular and inflammatory disease, expression quantitative trait loci implicated in atherosclerosis, pathway based approaches and comprehensive literature searching. The custom flexibility of the array platform facilitated interrogation of loci at differing stringencies, according to a gene prioritization strategy that allows saturation of high priority loci with a greater density of markers than the existing GWAS tools, particularly in African HapMap samples. We also demonstrate that the IBC array can be used to complement GWAS, increasing coverage in high priority CVD-related loci across all major HapMap populations. DNA from over 200,000 extensively phenotyped individuals will be genotyped with this array with a significant portion of the generated data being released into the academic domain facilitating  Cardiovascular disease (CVD), the leading cause of death in the developed world Array-based genotyping technologies that have enabled GWAS also permit flexibility in choosing the scope and density of SNPs for candidate gene studies. For example, they allow improved exploitation of recent deep resequencing data, enabling more accurate capture of genetic diversity across populations We describe here the design and implementation of a custom 50 K SNP genotyping array, primarily aimed at assaying SNPs in candidate genes and pathways for cardiovascular, inflammatory and metabolic phenotypes. Design of this genotyping array was led by investigators from the Institute of Translational Medicine and Therapeutics (ITMAT), the Broad Institute and by the National Heart Lung and Blood Institute (NHLBI) supported Candidate-gene Association Resource (CARe) Consortium. The custom SNP array is hereafter referred to as the “IBC array” (ITMAT-Broad-CARe array). A consortium of international academic and industrial partners have committed to using the IBC array to genotype DNA from more than 200,000 individuals who have been extensively phenotyped for risk factors and clinical evidence of vascular disease. The objectives of forming this consortium were (i) to pool expertise for selection of both loci and SNPs; (ii) to reduce costs by producing a standardized genotyping platform; and (iii) to facilitate cross cohort meta-analyses for a large set of SNPs in high priority candidate loci. Here we formally describe the resource and assess coverage of the genetic variants from prioritized loci generated on the SNP panel with the HapMap populations. We also evaluate the coverage of the array with the major GWAS products.st 2007 for the 2nd version of the IBC array (IBCv2). Key information was collated including: PMID number, publishing journal, size and population examined, loci and SNPs studied  and functional evidence. Over 2,400 published studies were systematically analyzed. Emphasis was placed on sample size, data quality and strength of the described associations. Genes with known of putative association with phenotypes for sleep, lung, and blood diseases were also nominated. Input was also solicited directly from investigators within and outside the consortium.We used the following search string in PubMed ‘ AND  AND ’ covering publications from 1978 to May 2007, for version1 of the IBC array (IBCv1), and to October 1www.pantherdb.org) and BioCarta (www.biocarta.com). These tools were employed to collate additional genes from key pathways including lipid metabolism, thrombogenesis, circulation and gas exchange, insulin resistance, metabolism, and inflammation, oxidative stress and apoptosis.Several pathway-based tools were used to identify additional biologically plausible candidate genes: Kyoto Encyclopedia of Genes and Genomes (KEGG), Early access was provided to a number of unpublished mouse atherosclerosis expression quantitative trait loci (eQTL) datasets. Genes predicted to be causal for atherosclerotic lesion size in genetic crosses of mice with differing susceptibility to atherosclerosis were identified Early access was provided to a number of key findings emerging from five GWAS:The Wellcome Trust Case Control Consortium (WTCCC) Hypertension study The WTCCC type 2 diabetes (T2D) study The WTCCC coronary artery disease study, Finland-United States Investigation of NIDDM Genetics (FUSION) study The Broad-Novartis-Lund Diabetes Genetics Initiative (DGI), A three-way meta-analysis of the WTCCC, Broad-Novartis-Lund and FUSION studies led to the generation of stronger T2D candidate loci for inclusion on the custom array. We also included SNPs reaching genome-wide significance from the WTCCC Rheumatoid Arthritis, Crohn's Disease and Type-1 Diabetes studies http://bmic.upenn.edu/cvdsnp) along with key information displayed for each respective gene: the number of SNPs required to tag the four HapMap representative populations at various minor allele frequencies (MAFs) and r2 thresholds; SymAtlas® expression profiling for over 70 specific human tissues and cell-types http://jaxmice.jax.org) and other phenotypic data. A voting system built into this database facilitated consensus amongst the consortium investigators for ranking genes proposed for inclusion on the IBC array. Over 2,000 of these loci were prioritized into three density criteria for tagging, as described below, based on voting by the participating investigators.Over 2,400 of the collated loci were placed on a database (Group 1 (n = 435 loci); genes and regions with a high likelihood of functional significance, including established mediators of vascular disease, loci derived from GWAS and those shown to be associated with phenotypes of interest. Tag SNPs for these loci were selected to capture known variation with MAF>0.02 and an r2 of at least 0.8 in HapMap populations and SeattleSNPs where available .Group 2 ; candidate loci that are potentially involved in phenotypes of interest or established loci that required very large numbers of tagging SNPs. SNPs for these loci were selected for MAFs>0.05 with an r2 of at least 0.5 in HapMap populations and SeattleSNPs where available.Group 3 (n = 232 loci); comprised mainly of the larger genes (>100 kb) which were of lower interest a priori to the consortium investigators. Only non-synonymous SNPs (nsSNPs) and known functional variants of MAF>0.01 were captured for these loci.Assays for specific SNPs of known or putative functionality and those shown to be highly associated with vascular disease from literature searching were directly ‘forced’ into the array content, with the aim of facilitating more powerful downstream meta-analyses with previously published data. nsSNPs and known functional variants of MAF>0.01 were selected where possible for all genes of interest.http://pga.gs.washington.edu) and Environmental Genome Project (EGP), (http://egp.gs.washington.edu) resequencing data to identify additional tags, not represented in the HapMap populations, using ldSelect SNPs from Group 1 and 2 loci were first chosen using the TAGGER software SNPs were categorized by their assay design scores for the Infinium genomic platform technology , based on a theoretical algorithm and all previously attempted wet-lab Infinium assays. In an attempt to reduce the proportion of failed assays on the final product, we pre-filtered most SNPs for Infinium design scores > = 0.6, finding appropriate proxies where possible.P>0.01). The AIMs panels are listed within the IBC resource site (http://bmic.upenn.edu/cvdsnp/updates/ancestry_informative_markers-ibc-v1.xls). The incorporation of admixture and AIM panels enables admixture mapping in African Americans and adjustment for population stratification in African Americans and European Americans.Two panels of ∼1,500 and ∼400 admixture and Ancestry Informative SNP markers (AIMs) were included for African versus European ancestry, and regional European (e.g. Northern versus Southern) ancestral populations respectively to enable admixture mapping and adjustment for population stratification in studies comprised of individuals from these ancestries. These SNPs were based on panels generated previously Genomic regions demonstrating ultra-high conservation across species were identified as previously described Assays for 49,234 SNPs were attempted using the Infinium technology for IBCv1 2> = 0.8). Suitable proxy assays for the remaining 2,626 SNPs were prioritized for supplementation based on MAFs in the main HapMap populations and their respective r2 value and Infinium design scores;Proxy assays were attempted for a total of 596 SNPs that failed during the manufacture of IBCv1. Some 1,143 of the 3,769 SNPs that failed assay conversion in manufacturing were tagged by at least one passing SNP already on the array  are associated with LDL, two with HDL and five with triglycerides;Combined GWAS analyses of the Diabetes Genetics Initiative −9; FDR∼1%), Schadt and colleagues performed global expression profiling on liver tissue and a GWAS on 427 Caucasian subjects. Genotyping of >750,000 SNPs was performed and SNPs were tested for association to each of the gene expression traits. We included 130 SNPs with MAF>0.04 from this study on IBCv2 that were identified as significantly associated with at least one gene expression trait ; African Americans (n = 384) and South Asians (n = 385). All samples described were genotyped following approval by the relevant institutional review boards. In each respective population, the minor allele frequency for each SNP on the IBCv1 array was determined. Histograms were generated with various allele frequency bins to determine the distribution of allele frequencies in each population.2 values were calculated using the expectation algorithm 2 values were calculated for each SNP list (HapMap release 22) with each SNP on the array being tested. All pair-wise combinations were considered within 200 kb. For chromosome X, only female individuals were used; otherwise, all unrelated individuals were used.We used previously described methods Assays for 49,234 SNPs were attempted during manufacturing with 45,237 SNPs successfully passing the manufacturer's criteria. Reasons for failures included sub-optimal probe synthesis and insufficient resolving of assay traces, potentially due to nearby hidden SNPs or copy number variants (CNVs). http://bmic.upenn.edu/cvdsnp/updates/hapmap_qc_samples-illumina.xls). 37,431 (82.7%) SNPs are evident in HapMap. Approximately 99.5% concordance was observed against respective HapMap data across the 117 samples and 52 Mendelian inconsistencies were observed in 25 HapMap trios ; African Americans (n = 384); and South Asians (n = 385). Some 1415 assays across the complete dataset were monomorphic. 2705 and 2566 assays were, respectively, monomorphic across self-described Caucasians and African Americans. The average number of SNPs across the Group 1 and Group 2 loci of IBCv1 were compared with several GWAS products . The ave2 value was calculated between each HapMap SNP in the region to a SNP in each respective product. 2 thresholds. The coverage using CEUs and CHB+JPT is comparable across all products, although the IBCv1 coverage for YRI is greater. A number of the GWAS products and the IBC array are strongly biased for composition of HapMap SNPs and will obviously have skewed coverage when directly compared. Over 20% of IBCv1 Group1 loci SNPs have not been assayed directly in HapMap with the majority of these additional SNPs derived from SeattleSNPs and the literature. Thus, the IBC array is likely to be more representative of broader population allelic architecture.The coverage of HapMap SNPs was evaluated for all Group 1 loci against the various GWAS products in the HapMap individuals. The maximum rThe combined coverage of IBCv1 with a number of the GWAS products was assessed for Group 1 loci. The coverage of the IBCv1 alone, with both of the 500 K SNP GWAS and with the one million SNP array products across the Group 1 loci is illustrated in We have produced a custom SNP array designed to capture genetic variation in prioritized loci known or postulated to increase risk of cardiovascular, metabolic and inflammatory diseases. Custom SNP selection allowed us to: (a) ensure selective and consistent coverage for a range of prioritized loci across multiple ancestries, (b) provide additional representative coverage to HapMap in loci of major interest, using SNP content from various sources including recent resequencing efforts; and (c) assay directly specific SNPs of interest such as those derived from previously published studies and known non-synonymous SNPs with MAF>0.01. The IBC array reveals greater depth of coverage than GWAS products with respect to information content and haplotype diversity in the high priority regions. This is particularly true of coverage for African HapMap representative samples. A modest fraction of tagging SNPs from the Group 1 loci on the IBC array are derived from SeattleSNPs analyses and were not assayed directly in HapMap, thus it is likely that the cumulative coverage of variation in these regions is actually underestimated in the current results. It is worth noting that as HapMap was predominantly used for the design of the IBC array , then additional densely genotyped or sequenced populations, outside those covered in the original HapMap, would be required for a completely unbiased assessment of coverage.Despite the recent reductions in price of whole genome SNP arrays, GWAS still remain expensive endeavors and power to cost issues are important factors in study design The HapMap project had a bias towards discovery and genotyping of variants with MAFs>0.05, but over 40% of SNPs were observed to have MAFs<0.05 The IBC array is one of the first disease-specific custom arrays with highly focused content to be used on a large scale. We anticipate further generations of the IBC array and that future aggregation of large cohorts and studies with similar disease traits will become commonplace, affording significant cost reductions and increased power to detect effects of modest size.Table S1Bins of SNPs with observed and expected Infinium conversion scores The distribution of SNPs binned according to Infinium score from 0.1 to 1 where a score of 0.8 indicates an 80% likelihood for conversion to a successful assay, 1.0 indicates an assay has ∼100% theoretical score etc. A value of 1.1 indicates that an Infinium assay for SNPs has previously been successful in manufacture and analyses. Percentages are indicated in brackets.(0.05 MB DOC)Click here for additional data file.Table S2Observed IBCv1 Mendelian consistency across 25 HapMap trios. Observed Parent-Parent-Child (PPC) heritability errors across the IBC version1 array using 25 HapMap individuals, where NA number denotes the official HapMap identifier.(0.08 MB DOC)Click here for additional data file.Table S3Observed replicate consistency using six HapMap individuals. Observed IBC version1 array genotyping errors for six replicate HapMap samples, where NA number denotes the official HapMap identifier.(0.05 MB DOC)Click here for additional data file.


In naturally and experimentally occurring scrapie in sheep, prions invade the immune systemand replicate in lymphoid organs. Here we analysed immunohistochemically, in seven spleensof 6-month-old healthy sheep, the nature of the cells expressing prion protein (PrP) potentiallysupporting prion replication, as well as their relationship with autonomic innervation.PrP was identified using either RB1 rabbit antiserum or 4F2 monoclonal antibody directedagainst AA 108–123 portion of the bovine and AA 79–92 of human prion protein respectively.Using double labelling analysis, we demonstrated that PrPc is expressed by folliculardendritic cells using a specific monoclonal antibody (CNA42). We also showed the closevicinity of these PrP expressing cells with noradrenergic fibers, using a polyclonal tyrosinehydroxylase antibody. Our results may help the study of the cellular requirements for the possibleneuroinvasion from the spleen.


Abnormal germline DNA methylation in males has been proposed as a possible mechanism compromising spermatogenesis of some men currently diagnosed with idiopathic infertility. Previous studies have been focused on imprinted genes with DNA methylation in poor quality human sperms. However, recent but limited data have revealed that sperm methylation abnormalities may involve large numbers of genes or shown that genes that are not imprinted are also affected.methylenetetrahydrofolate reductase (MTHFR) gene (NG_013351: 1538–1719) in sperm DNA obtained from 94 idiopathic infertile men and 54 normal fertile controls. Subjects with idiopathic infertility were further divided into groups of normozoospermia and oligozoospermia. Overall, 45% (41/94) of idiopathic infertile males had MTHFR hypermethylation (both hemimethylation and full methylation), compared with 15% of fertile controls (P<0.05). Subjects with higher methylation level of MTHFR were more likely to have idiopathic male infertility . Comparing the two groups of idiopathic infertile subjects with different sperm concentrations, a higher methylation pattern was found in the group with oligozoospermia.Using the methylation-specific polymerase chain reaction and bisulfite sequencing method, we examined methylation patterns of the promoter of MTHFR gene in sperms is associated with idiopathic male infertility. The functional relevance of hypermathylation of MTHFR to male fertility warrants further investigation.Hypermethylation of the promoter of  Infertility is a worldwide reproductive health problem that affects approximately 15% of married couples. Half of these cases are due to male factors Epigenetic alterations in DNA without concomitant changes in underlying genetic codes are now known to occur frequently in various human diseases MTHFR gene may be associated with reduced sperm counts in humans, leading to male infertility in some populations MTHFR promoter hypermethylation was associated with non-obstructive azoospermia in testicular biopsies of patients Methylenetetrahydrofolate reductase (MTHFR) is one of the main regulatory enzymes involved in folate metabolism, DNA synthesis and remethylation reactions. MTHFR catalyses the reduction of methylenetetrahydrofolate  to methyltetrahydrofolate (5-methyl THF). Thus, this enzyme has a key role in balancing the pool of methyl groups between DNA synthesis and DNA methylation. Recently, several association studies have suggested that polymorphic variants in the MTHFR gene is associated with idiopathic male infertility, we utilized carefully selected fertile controls to evaluate the association between the MTHFR promoter hypermethylation and idiopathic male infertility.In this study, to test our hypothesis that hypermethylation of the promoter region of Study subjects were volunteers from the affiliated hospitals of Nanjing Medical University between March 2006 and July 2009 (NJMU Infertile Study). The study was approved by the Institutional Ethics Committee of Nanjing Medical University. All activities involving human subjects were done under full compliance with government policies and the Helsinki Declaration. Consecutive eligible men (with wives not diagnosed as infertile) were recruited to participate, 680 in total were asked. Of those approached, 82.5% consented . Written informed consent was obtained from all study subjects. Infertile men had an infertility history of at least 2 years with their spouses with confirmed normal gynaecological assessment. The controls were healthy men who had fathered at least one healthy child within one year without assisted reproductive measures during the same period as those of the cases recruited in the same hospital. A scheduled interview was arranged for each subject to collect information, including personal background, lifestyle factors, occupational and environmental exposures, sexual and reproduction status, genetic risk factors, medical history and physical activity (e.g. exercise status). After interview, each subject donated a 5-ml peripheral blood sample and a semen sample for genetic testing. These patients and healthy donors were all ethnically Han Chinese from East China.2), testosterone (T), prolactin (PRL), luteinizing hormone (LH), follicle-stimulating hormone (FSH), karyotyping, and molecular tests for Y-chromosome microdeletions Semen samples were obtained in private by masturbation into a sterile widemouth and metal-free glass container after a recommended at least 3-day sexual abstinence. After liquefaction at 37°C for 30 min, conventional semen analysis was conducted in accordance with guidelines of the WHO Laboratory Manual for the Examination of Human Semen The sperms were first washed with phosphate-buffered saline twice and then washed with sperm wash buffer . Motile sperm cells were purified away form lymphocyte contamination, immature germ cells and epithelial cells using swim-up method 5′-TAGATTTAGGTACGTGAAGTAGGGTAGAC-3′, methylated reverse: 5′-GAAAAACTAATAAAAAACCGACGAA-3′, unmethylated forward: 5′-TTTAGGTATGTGAAGTAGGGTAGATGT-3′, unmethylated reverse: 5′-CAAAAAACTAATAAAAAACCAACAAA-3′Genomic DNA  was treated with sodium bisulfite using the EpiTect Bisulfite Kit (QIAGEN) according to the protocol recommended by the manufacturer. Three microliters of the final eluant was used for subsequent PCR amplification. The primers used were: methylated forward: MTHFR, we carried out BSP assay. We examined a total of 18 CpG sites (CpGs) in a 299 bp fragment of the promoter of MTHFR . The primers used were: 5′-TGAGAAAAGATTTTAGATTTAGGTA-3′ (forward), 5′-AATCCAAAATAACAATAAAAAAAA-3′ (reverse). PCR included an initial incubation at 95°C for 10 minutes, followed by 40 cycles of 95°C for 40 seconds, 51.9°C for 40 seconds, and 72°C for 60 seconds, followed by one cycle of 72°C for 10 minutes. The PCR products were purified and cloned into the pCR2.1 vector by TA Cloning kit . To determine the methylation status of the promoter of MTHFR gene, an average of 15 clones (each plate) were sequenced using M13 reverse primer and an automated ABI Prism 3730xl Genetic Analyser .To confirm that the MSP results accurately reflected the methylation pattern of the promoter of MTHFR between case and control groups. To analyze the association between methylation patterns of MTHFR and idiopathic male infertility with normal and abnormal sperm concentrations, chi-square trend tests were performed. Logistic regression analysis was performed to obtain the odds ratios (ORs) for idiopathic male infertility and 95% confidence intervals (95% CI) with adjustment for age, BMI, smoking status and alcohol drinking, wherever it was appropriate. All statistical analyses were carried out using Stata , and P≤0.05 were considered to be significant.Analysis of variance (ANOVA) was used to explore the relationships between fertility status and potentially important covariates, such as age, body mass index (BMI) and ejaculate volume. Differences in sperm concentration and motility between cases and controls were tested using Mann-Whitney test. The chi-squared test was used to evaluate the differences in smoking status, drinking status, duration of sexual abstinence, and the frequency of hypermethylation of MTHFR gene in spermatozoa from 94 idiopathic infertile males and 54 age-matched fertile controls with normal endocrine status and semen parameters. Demographic categories by fertility and sperm concentration are described in We analyzed DNA methylation of the MTHFR gene (NG_013351: 1538–1719). The percentage of hemimethylation  of MTHFR was 29% among idiopathic infertile patients and 13% among fertile controls, while the prevalence of full methylation  of MTHFR in idiopathic infertile males and controls were 16% and 2%, respectively. Overall, 45% (41/94) of idiopathic infertile males had hypermethylation (both hemimethylation and full methylation) in their sperms, compared with 15% of the fertile controls (MTHFR was significantly higher in idiopathic infertile men than in fertile controls (P<0.05). These results indicate that the MTHFR promoter methylation was frequently altered in idiopathic infertile patients.controls . A reprecontrols . The freMTHFR hemimethylation and full methylation was 31% and 14% respectively in Case 2 group (P<0.05). Case 1 and Case 2 groups displayed hypermethylation of 40% and 45%, respectively (P<0.05).For a more detailed analysis, the subjects was stratified by the sperm concentration ≥20 and <20 million spermatozoa/ml into groups of 30 (Case 1) and 64 (Case 2) patients.  2 group , the freectively , while CMTHFR are presented in P-value for trend  = 0.0007]. When we divided the patients into Case 1 and Case 2 group according to a cut-off value of 20 mill./ml of the normal sperm concentration, we found an increased risk for idiopathic infertility among increasing methylation levels for both groups . Comparing the two groups of idiopathic infertile subjects with different sperm concentrations, a higher methylation pattern was found in the group with oligozoospermia.Adjusted ORs and 95% CIs for associations between idiopathic male infertility and methylation patterns of There is a growing body of evidence highlighting the role that aberrant DNA methylation plays in human diseases MTHFR gene is critical in maintaining an adequate methionine pool and is believed to be important in the process of DNA methylation Alterations in DNA methylation patterns impact several critical cellular processes including gene expression, X-inactivation, carcinogenesis, aging and development MTHFR is a common event occurring in sperms of idiopathic infertile men, with 41 of 94 showing such epigenetic aberration. This rate has almost tripled the prevalence found in the fertile control group, which appears to be a frequent abnormality in idiopathic infertile men, playing a significant role in causing infertility. These results were in agreement with the findings of Houshdaran and his colleagues MTHFR promoter hypermethylation was associated with impaired spermatogenesis in infetile men. In the article by Khazamipour et al, hypermethylation of MTHFR was observed in testes of infertile males, suggesting that epigenetic silencing of MTHFR could play a role in male infertility.Our study has unveiled for the first time that the hypermethylation of MTHFR gene is strongly associated with idiopathic male infertility. Therefore, analysis of promoter methylation in specific genes may provide biomarkers valuable for the identification of individuals with an elevated risk of male infertility. However, because our study is relatively small, further large studies of different populations with additional functional analysis of the hypermethylation are needed in order to elucidate the role and the mechanisms of the MTHFR gene in human spermatogenesis and the related pathology.Hypermethylation of the promoter of


MicroRNAs (miRNAs) are small, noncoding RNAs that negatively regulate gene expression by binding to target mRNAs. miRNAs have not been comprehensively studied in recurrent ovarian cancer, yet an incurable disease.Using real-time RT-PCR, we obtained distinct miRNA expression profiles between primary and recurrent serous papillary ovarian adenocarcinomas (n = 6) in a subset of samples previously used in a transcriptome approach. Expression levels of top dysregulated miRNA genes, miR-223 and miR-9, were examined using TaqMan PCR in independent cohorts of fresh frozen (n = 18) and FFPE serous ovarian tumours (n = 22). Concordance was observed on TaqMan analysis for miR-223 and miR-9 between the training cohort and the independent test cohorts. Target prediction analysis for the above miRNA "recurrent metastatic signature" identified genes previously validated in our transcriptome study. Common biological pathways well characterised in ovarian cancer were shared by miR-9 and miR-223 lists of predicted target genes. We provide strong evidence that miR-9 acts as a putative tumour suppressor gene in recurrent ovarian cancer. Components of the miRNA processing machinery, such as Dicer and Drosha are not responsible for miRNA deregulation in recurrent ovarian cancer, as deluded by TaqMan and immunohistochemistry.We propose a miRNA model for the molecular pathogenesis of recurrent ovarian cancer. Some of the differentially deregulated miRNAs identified correlate with our previous transcriptome findings. Based on integrated transcriptome and miRNA analysis, miR-9 and miR-223 can be of potential importance as biomarkers in recurrent ovarian cancer. Ovarian cancer is the leading cause of death from gynaecological malignancy in the western world . OvarianThis new and surprisingly abundant class of RNA regulatory genes has been found to confer a novel layer of genetic regulation in cells. Active, mature miRNAs function as endogenous, highly conserved, small RNA's, 22 nucleotides long that silence gene expression by binding to target mRNAs. Their 5' end binds to its target complementary sequence in the 3'-untranslated region (3'UTR) of mRNA and given the degree of complementarity, miRNA binding appears to result in translational repression, or in some cases, cleavage of cognate mRNAs, causing partial or full silencing of the respective protein-coding genes . An accuFunctional miRNAs are excised from long endogenous transcripts by the sequential action of a pair of endonucleases (Drosha and Dicer) that reside in different compartments of the cell. In the nucleus, the primary microRNA (pri-miRNA) transcript is first cleaved by Drosha, liberating an approximately 60-to-80-nucleotide-long hairpin-shaped precursor miRNA (pre-miRNA). This pre-miRNA is then exported from the nucleus to the cytoplasm, where it undergoes a further processing by the Dicer enzyme and the resulting duplex is then loaded onto the RNA-induced silencing complex (RISC) in order to become more effective. Perfect base pairing between the RISC-bound miRNA and the target mRNA results in cleavage and degradation of the latter, whereas imperfect complementarity generally leads to translational repression of the target . RISC reNot surprisingly, a variety of studies have linked aberrant microRNA expression to carcinogenesis where they act as both oncogenes and tumour suppressor genes . Unique A recent study reported aberrant miRNA expression in ovarian cancers compared to normal ovary . In addiAll samples were obtained at initial cytoreductive procedure from patients treated for ovarian cancer at St James's Hospital, Dublin, Ireland. The study had approval of the hospital ethics committee and informed consent was obtained from each patient by the research team prior to surgery. All tumors were staged according to the International Federation of Gynaecology and Obstetrics standards (FIGO).A pilot study was undertaken to examine the expression of 180 miRNAs with the objective of generating a training set.The initial cohort (training set) consisted of 3 fresh frozen primary serous papillary (stage III and grade III) and 3 fresh frozen recurrent serous papillary ovarian adenocarcinomas of the same grade but from different optimally debulked  patients. The mean age in years for patients in the primary and the recurrent group was 63.3 (range 48–84) and 53.3 (range 45–68). The recurrent group consisted of patients for which primary surgery was performed prior to the commencement of the study. Patients had received no neoadjuvant treatment before surgery and they were mostly treated postoperatively with platinum and/or paclitaxel.A subset of miRNAs selected from the training set was further analyzed in fresh frozen and formalin fixed paraffin embedded (FFPE) cohorts.The additional cohort of fresh frozen tissues, which were analysed with the putative signature miRNA panel comprised 18 serous papillary adenocarcinomas, 11 primary and 7 recurrent cases.This cohort was further expanded to 22 serous papillary adenocarcinomas -14 primary and 8 recurrent- and used to examine the relative expression levels of Dicer and Drosha. An additional 2 primary and 5 recurrent ovarian cancer patients of different histology  were subsequently added to the cohort (n = 29).Specimens were snap frozen on collection within 1 hour of surgery at -80°C. After tissue processing in a cryostat at -20°C, frozen sections were cut and mounted on slides. The slides were stained with H&E and examined by a pathologist to ensure >70% presence of tumour cells.miRNA was extracted from fresh tissues using the Ambion mirVana™ miRNA isolation kit  according to the manufacturer's instructions. Samples were placed in liquid nitrogen, ground thoroughly with a mortar and pestle and homogenised in lysis buffer. In brief, homogenised samples were lysed, followed by an acid-phenol:chloroform extraction. Total RNA samples were collected and small RNAs were purified by precipitation with ethanol. Small RNA species were immobilized on glass-fiber filters, followed by several washes and elution of small RNA with nuclease-free water.An independent set of 22 OSC – 15 primary and 7 recurrent, classified according to the FIGO system: stage (II–III) and grade 2–3, were selected from archival FFPE tissue, between the years 1996–2006 from St James's Hospital, Dublin. H&E slides of all tumors were reviewed by a histopathologist (RF) to confirm original diagnoses.An additional paraffin tissue microarray (TMA) of 56 primary epithelial ovarian carcinomas (EOC) and 15 recurrent ovarian carcinomas (OC) was constructed, composed of tumors arrayed in quadruplicates. Samples were age-matched to 40 normal ovaries containing normal ovarian surface epithelium (NOSE). FFPE blocks were selected that contained over 90% tumour. Slides of archival material were reviewed to determine suitable areas for the TMA construction. 4 representative areas were selected and verified by a second pathologist (CB) for core biopsy.This was performed on the above mentioned independent set of OSC (n = 22) using the Recoverall™ Total Nucleic Acid Isolation Kit . Where tumour cell density was >90% (n = 16), whole sections were taken for miRNA analysis. In cases that tumour stroma component was significantly present (n = 6), Laser Capture Microdissection (LCM) was performed to produce pure cell population for analysis. Up to 3 extractions were performed in parallel with one pellet in each extraction. Following dissection from the blocks, these preparations were deparaffinized, followed by protease digestion for 3 hours at 50°C for RNA isolation, on column DNA digestion and elution as described in the protocol.-7 to 10-6 um3(representing 5000 cells). Following microdissection the Capsures™ were placed in sterile Eppendorf tubes.LCM was employed in six of the recurrent FFPE tumour samples to yield sufficient homogenous populations of malignant epithelial cells. 8 μm sections were cut from each block, dewaxed and stained with haemotoxylin and eosin (H&E). Malignant epithelial cells from the tumour papillae in each case were laser capture microdissected, using the PixCell 11™ System  for subsequent expression analysis. Cells were captured according to a standard protocol as follows: Laser spot size = 30 μm, pulse power = 40 mW, pulse width = 1.5 ms, threshold voltage = 285 mV. The total number of pulses in each case was approximately 700. This yielded a tissue volume in the range of 10® MicroRNA Assay Human Panel Early Access kit  consisting of 180 well characterized miRNAs. The kit uses gene-specific stem-loop reverse transcription primers and TaqMan probes to detect mature miRNA transcripts in a 2-step qRT-PCR assay. Briefly, single stranded cDNA was generated from total RNA sample by reverse transcription using the Applied Biosystems High-Capacity cDNA Archive Kit  following manufacturer's protocol. RT reactions contained 10 ng of total RNA, 50 nM stem-looped RT primer, 1 × RT buffer, 0.25 mM each of dNTPs, 3.33 U/μl Multiscribe reverse transcriptase and 0.25 U/μl RNase Inhibitor. PCR amplification was carried out using sequence specific primers on the Applied Biosystems 7900 Real-Time PCR system. The reactions were incubated in a 96-well optical plate at 95°C for 10 min, following by 40 cycles of 95°C for 15 s and 60°C for 10 min. The assay was run in triplicate for each case to allow for assessment of technical variability. let-7a was used as endogenous control. Four negative controls were also used, ath-mir159a, cel-lin-4, cel-miR-2 and cel-miR-124 as they show no expression in human tissue.miRNA expression levels were examined using the Applied Biosystems TaqManThe Applied Biosystems stemloop RT/PCR kit (AB) and TaqMan microRNA Assays were used to detect and quantify selected mature miRNAs in both fresh frozen and FFPE samples, as described for the Early Access Kit protocol.® Universal PCR master Mix and Assays on demand (Applied Biosystems). Relative quantitation was carried out using the ΔΔCt method for recurrent versus primary with 18S ribosomal RNA as an endogenous control. Transcript quantification was performed in triplicate for each sample.The expanded cohort of fresh frozen samples was used for this analysis. Dicer (Assay ID: Hs00203008_m1) and Drosha (Assay ID: Hs00229023_m1) were analysed using qRT-PCR. Briefly, RNA was initially reverse transcribed using a High Capacity cDNA Archive Kit  and then was amplified in a 10 μL PCR reaction according to the manufacturer's recommended protocol and amplification steps: denaturation at 95°C, followed by 40 cycles of denaturation at 95°C for 15 sec and then annealing at 60°C for 1 min. All reactions were carried out on the ABI Prism 7000 Sequence detection system  using the TaqMan4 μm sections of the OSC tissue array were cut and mounted on glass slides. For antigen unmasking, deparaffinized sections were boiled (eIF6) or microwaved (Dicer) in citrate buffer [10 mmol/L sodium citrate buffer (pH 6.0)] before incubation with primary antibodies. eIF6 (BD Biosciences) and Dicer (Abcam) protein levels were examined using mouse monoclonal antibodies at dilutions of 1:200 and 1:50 respectively. Antibody staining was done using the Biogenex Super Sensitive™ Link-Label IHC Detection system.Intensity of eIF6 and Dicer immunoreactivity was recorded in the neoplastic glands for every specimen. The intensity of staining was graded on a scale from 0 to 3. "0" reflected a lack of immunoreactivity, "1" reflected weak immunoreactivity, "2" reflected moderate immunoreactivity and "3" reflected strong homogenous nucleocytoplasmic (eIF6) or cytoplasmic (Dicer) staining. Scores from all cores were averaged. Two pathologists  scored the stained slides.® software [Relative quantitation using the ΔΔct method in recurrent versus primary was carried out  and foldsoftware  was usedsoftware .Correlations of miRNA expression levels were carried out using student's t-test for age and CA125 and Fischer's test for debulking status and chemosensitivity with significance level set at p < 0.05. Chemosensitivity was defined according to the WHO criteria as complete remission after 6 months post completion of chemotherapy.All statistical analysis of immunohistochemical studies was performed using the Analyse-It™ Software Ltd. Two sample comparisons  were performed with the Mann-Whitney rank sum test.Using the early Access kit distinct expression signatures were identified between primary and recurrent ovarian cancers. 60 miRNAs were greater than 2-fold deregulated between primary and recurrent cases  were selected from the signature panel of 60 miRNAs according to a) their degree of dysregulation (they demonstrated the greatest changes in expression levels) b) their predicted target miRNAs Table .miRNA expression analysis for the 2 selected miRNAs was carried out using real-time PCR in an independent cohort of 15 primary and 7 recurrent FFPE tissues (n = 22). TaqMan data corroborated with the results obtained from the initial cohort in so far as miR-223 was upregulated and miR-9 downregulated . We further expanded our test set to 16 primary and 13 recurrent samples by adding tumours of other than OSC histological subtypes . Fold changes of 1.78 and 1.49 were observed for Drosha and Dicer respectively, but were not significant.We examined the relative expression levels of eIF6 and Dicer on TMAs including 56 primary OSC and 15 recurrent ovarian tumours and determined whether any correlation exists between the two. In this respect, we wanted to examine functionality of the "mature" sector of the miRNA machinery at a protein level. No statistically significant observations were made Figures  and 8.This study demonstrates discrete miRNA signatures between primary and recurrent serous papillary adenocarcinomas on a histologically homogenous set of ovarian tissues from different patients. To our knowledge, this is the first ever study to examine the expression levels of miRNAs in recurrent cancer.Real-time PCR can be employed to quantify miRNA expression profiles and study the potential function of miRNAs as well as miRNA precursors in cancer pathogenesis . We utilWe used a twofold change as a cut-off for miRNA differential expression in our training set. One might argue, much smaller changes could also be significant, reflecting the fact that miRNAs target many genes of even similar function. In this regard, disruption of cooperating genes can be a likely event with subtle miRNA expression . HoweverBecause the expression of some miRNAs is highly tissue specific , it was Real time PCR was further employed to detect expression levels of a subset of miRNAs in an expanded cohort of fresh frozen serous papillary ovarian adenocarcinomas. Selected miRNAs comprised miR-223 (up) and miR-9 (down), which had demonstrated the greatest changes in expression levels in the initial cohort. Similar patterns of altered expression were observed for the top dysregulated genes Figure .We then set out to determine the expression patterns of selected miRNAs across a large panel of FFPE pathological tissues representing primary and recurrent serous ovarian adenocarcinomas, routinely used in hospital pathology laboratories. Unlike mRNAs, miRNAs remain largely intact in these routinely collected clinical samples . "RecurrThis miRNA "recurrent metastatic signature", comprised miR-223 and miR-9 may deserve some special consideration. miR-223 was the most upregulated in recurrent cancers when compared to primary. Recent work by Chen et al. identified among others miR-223 as a murine hematopoietic-specific miRNA and showed functionality of these miRNA through in vitro and in vivo overexpression studies . miR-223miR-9 was the most downregulated in recurrent cancers and appears to play a functional role in the determination of neural fates in embryonic stem (ES) cell differentiation . miR-9 wmiRNAs have been recently implicated in the development of ovarian cancer . SpecifiOur team has previously examined gene expression levels in the training sample and identified over and under represented genes with full functional annotation . In the Common significant biological pathways involving predicted gene targets for miR-9 and miR-223 were identified using PANTHER. It appears that these pathways contain traits that distinguish primary and recurrent ovarian cancers. They have all previously been well characterised in ovarian cancer and chemoresistance. In this respect, if downregulated miRNAs are expected to activate overexpression of mRNA targets, it is fair to suggest that miR-9 downregulation can possibly lead to drug resistance.The target list for miR-223 and miR-9 was interestingly enriched for genes involved in FGF cell signaling (FGFR2 and FGF respectively), which is in concordance with our transcriptome results . This isThe mechanism of miRNA deregulation in recurrent ovarian cancer has not been thoroughly exploited. Fold changes <2 were observed for Dicer and Drosha at a mRNA level in our initial cohort and extended set of tumours with same histology. Inclusion of non serous tumours in the previously homogenous set conferred no statistical significance. To determine whether the observed gene expression pattern was altered at the protein level, primary and recurrent ovarian tumors (n = 71) were analysed immunohistochemically on TMA constructs containing multiple sections, using specific antibodies for Dicer and EIF6. Both proteins are localized in the cytoplasm and have roles in the processing of mature miRNAs.Our results show that Dicer, Drosha and eIF6 may not have a substantive role in recurrent ovarian cancer and may only be involved in early tumorigenesis as shown by Chiosea et al . We are In summary, we propose a miRNA model for the molecular pathogenesis of recurrent ovarian cancer Figure . Some ofThe authors declare that they have no competing interests.AL performed the experiments and drafted the manuscript. SOT performed extensive editing and preparation of manuscript. MR assisted with cutting frozen sections. RF and CB examined the pathological specimens. RF also assisted with LCM and data analysis. LK contributed to RT-PCR analysis of Dicer and Drosha. NG, TDA and EMG provided the tumour samples. SF and ML aided in IHC analysis. CM, OS and BS assisted with critical examination of the manuscript. JOL supervised experimental work and manuscript editing. Final manuscript was read and approved by all authors.miRNA genes differentially expressed between recurrent and primary serous papillary ovarian adenocarcinomas in the training cohort with their numerical fold changes ranging from 2 to 11. Common miRNA genes highlighted in red have been identified as differentially expressed in diverse cancers. This signifies that specific miRNAs are involved in common molecular pathways. miRNAs highlighted in green were the top dysregulated in recurrent vs primary serous papillary adenocarcinomas in the training cohort.Click here for file


To the Editor: The emergence of influenza A pandemic (H1N1) 2009 virus in North America and associated illness and death suggest that humanity faces a dangerous threat. Viruses isolated from a sample of patients with confirmed cases in early phases of the outbreak demonstrated resistance to amantadine and rimantadine. At present, circulating viruses appear to be largely susceptible to the neuraminidase inhibitors oseltamivir and zanamivir, although oseltamivir resistance has been observed in recent cases in Europe, Asia, and North America  that caused few deaths. However, later that year, transmission resurged and was associated in 2 waves with increased illness and deaths. We cannot predict whether the 2009 pathogen will follow a similar temporal pattern and evolve toward increased virulence. Even if vaccine development and delivery could be achieved within 6 months, an aggressive schedule, large supplies of vaccine against pandemic (H1N1) 2009 may not be available until late 2009.Antiviral drugs are used to treat patients with strongly suspected or confirmed influenza. However, until a vaccine is available, specific protection by pharmaceutical products is limited to antiviral drugs. Nonpharmaceutical interventions are also available for prevention. Some governments and organizations are taking steps that would enable mass administration of these drugs (The first viable oseltamivir-resistant human influenza viruses (H1N1) emerged and became prevalent in the United States and Europe in the 2007–08 influenza season, and prevalence of such viruses has continued in 2009. The potential for overuse of antiviral drugs, especially oseltamivir, to select for existing antiviral drug-resistant strains is unknown. Ecologic studies suggest a lack of association between prevalence of oseltamivir use and prevalence of oseltamivir resistance (A similar resistance pattern could occur with pandemic (H1N1) 2009 virus. Regardless of the mutational mechanism for antiviral drug resistance, mass use of antiviral drugs could potentially lead to selection pressure for drug-resistant viruses  and the Centers for Disease Control and Prevention (www.cdc.gov/h1n1flu/recommendations.htm) for prophylaxis should be followed to keep resistance in check and save the lives of patients.What are we to do? Until a vaccine is available, combination antiviral therapy and rapid diagnostic testing may be needed  2009 virus or an evolved variant. Appropriate use of antiviral chemotherapy is complex. Identifying the groups at high risk for serious illness for drug therapy and appropriate antiviral therapy in situations of co-circulation of seasonal and pandemic (H1N1) viruses with various susceptibility patterns needs elucidation. Without clear evidence-based guidance, a global public health disaster could occur if pandemic (H1N1) 2009 reemerges later this year with higher virulence or widespread antiviral drug resistance.


Drosophila melanogaster. Reduced AMPK signaling leads to hypersensitivity to starvation conditions as measured by lifespan and locomotor activity. Locomotor levels in flies with reduced AMPK function were lower during unstressed conditions, but starvation-induced hyperactivity, an adaptive response to encourage foraging, was significantly higher than in wild type. Unexpectedly, total dietary intake was greater in animals with reduced AMPK function yet total triglyceride levels were lower. AMPK mutant animals displayed starvation-like lipid accumulation patterns in metabolically key liver-like cells, oenocytes, even under fed conditions, consistent with a persistent starved state. Measurements of O2 consumption reveal that metabolic rates are greater in animals with reduced AMPK function. Lastly, rapamycin treatment tempers the starvation sensitivity and lethality associated with reduced AMPK function. Collectively, these results are consistent with models that AMPK shifts energy usage away from expenditures into a conservation mode during nutrient-limited conditions at a cellular level. The highly conserved AMPK subunits throughout the Metazoa, suggest such findings may provide significant insight for pharmaceutical strategies to manipulate AMPK function in humans.Organisms must utilize multiple mechanisms to maintain energetic homeostasis in the face of limited nutrient availability. One mechanism involves activation of the heterotrimeric AMP-activated protein kinase (AMPK), a cell-autonomous sensor to energetic changes regulated by ATP to AMP ratios. We examined the phenotypic consequences of reduced AMPK function, both through RNAi knockdown of the gamma subunit (AMPKγ) and through expression of a dominant negative alpha (AMPKα) variant in  Drosophila, a single gene encodes each subunit, and clear homologs of each subunit are present and display 60%, 62%, and 62% identity with human α, β, and γ subunits, respectively. Drosophila AMPK is highly similar to mammalian AMPK, as it is formed via a heterotrimeric complex, is activated by AMP, and has many of the same targets, including acetyl-CoA carboxylase (ACC) The precise mechanisms of how organisms maintain energetic homeostasis in light of dynamic food availability remains largely unknown. A molecule thought to represent a central cellular mechanism mediating energy allocation is the AMP-activated protein kinase, by virtue of its activation by the end product of ATP hydrolysis Genetic modulation of AMPK activity in various model systems suggests that AMPK may represent an underlying mechanism to increase longevity as a consequence of dietary restriction Drosophila leads to a series of behavioral and metabolic phenotypes demonstrating that these animals behave as though they are experiencing chronic starvation. Specifically, animals deficient in AMPK signaling show a hypersensitivity to starvation conditions, altered activity levels, and differential lipid quantities and distributions. These phenotypes further demonstrate a pivotal role of the AMPK molecule in the organization of behavioral and physiological responses to nutrient-limitations, and that such responses appear to be highly conserved throughout the Metazoa.We report here that attenuated AMPK function in K57A transgene was generated by a mutagenic PCR strategy and cloned into the UAS-vector for embryo transformation. The UAS-AMPK-α WT transgene was previously described TED and the UAS-dcr 2. The UAS-dcr2 and UAS-LSD2-GFP lines were kind contributions from Dr. Paul Taghert and Ronald Kuhnlein respectively. All other lines, with the exception of the Ubiq-GAL4 and AMPK alpha transgenes were procured from the Bloomington Stock center . All flies were maintained in an incubator maintained at 25°C and under a 12∶12 LD cycle. Flies were cultured on a standard molasses-malt-cornmeal-agar-yeast medium and housed in uncrowded conditions. All transgenes were backcrossed to the 1118w background. For hsp70-GAL4 lines, we raised and maintained animals at 19°C for all assays (uninduced), and induction was carried out by placing animals at 30°C for 1 hr twice daily for three days, and then assaying animals at 25°C.The AMPKDrosophila as a metric of stress sensitivity e.gMeasurements of lifespan have been widely employed in Locomotor activity was monitored with a TriKinetics Locomotor Monitor  on the aggregate population of thirty 3–5 day old flies 5′- AAAAACAAAACCAAAAGCAACAA and Reverse: 5′AATTATTGGAATTGGAGCTGGAG. Specific primers were also designed to amplify the housekeeping gene, RP49Ninety adult flies were homogenized in 1 mL of TriZol  and total RNA was extracted according to manufacturer's recommendations. RNA was then used for cDNA synthesis using a SuperScript III Reverse Transcription Kit (Invitrogen). Specific primers for the gamma AMPK subunit ORF were designed to flank intervening sequences to distinguish if amplicons originated from cDNA versus contaminating genomic DNA. The primers for the gamma subunit were: Forward: Total triglyceride levels were assessed through use of a Serum Total Triglyceride Kit  according to manufacturer's instructions and as previously described To assess food intake, we employed the CAFE assay Drosophila were put in cages over standard cornmeal-yeast media containing vehicle (ethanol) or 1 µM rapamycin to lay eggs overnight. Larvae were scored  on days 5 and 7 after egg laying and scored for the absence or presence of the green fluorescence of the FM7i balancer. Mutant survival percentages were normalized to their survival at 5 days without rapamycin . For adult rapamycin feeding, adult animals were placed on instant Carolina medium containing 100 µM rapamycin for three days prior to starvation assays.For rapamycin  feeding, 200 adult 2 scavenger, and placed in the bottom of a 15 mL polypropylene tube. Loose cotton was placed over the KOH-saturated filter paper, and 10 flies were loaded into the chamber which was then sealed with wax. Water was delivered to the end of the micromanometer and given ten minutes to equalize. After this time period, the position of the meniscus was marked and re-marked following a ten minute period for three replicates of ten minutes to estimate an average reading. Measurements were taken on two genotypes differing in AMPK function at the same time, since manometric readings may be influenced by external factors such as changes in barometric pressures, and these grouped readings were repeated six times were analyzed for statistical significance using a nested ANOVA .A calibrated 50 µL micropipette was flame bent to fashion a micromanometer. Whatman filter paper was saturated in a KOH solution, to act as a COe.g. To investigate the roles of AMPK in the maintenance of metabolic homeostasis in adults, we relied on two experimental strategies to alter AMPK activity. The first approach was to generate a dominant negative construct (K57A), in which the catalytic domain of the alpha subunit was altered to inactivate the ATP binding domain, as suggested by a number of studies in mammalian systems via the GAL80 element rescued the lethality associated with expression of either the RNAi or the K57A alpha variant. Next, we tested whether it could rescue the lethality caused by a null mutation in the alpha subunit ampk mutant background provided complete restoration of Mendelian segregation ratios  or α (αRNAi) subunits, and observed a significant reduction in the α protein levels in these two genotypes but not in animals possessing the GAL4 alone or in animals expressing an RNAi targeting a different gene  driver for adult survival during starvation, as this driver recapitulates the global expression pattern of the ubiquitin gene, but is not as strong as the Act5C-GAL4 driver Having validated that these genetic strategies were producing phenotypes consistent with reduced AMPK activity, we then asked whether attenuated AMPK function would alter sensitivity to metabolic challenges. We tested the progeny of a UAS-AMPKGiven the developmental defects exhibited in animals lacking functional AMPK, we performed experiments to assess the extent to which potential developmental defects caused by impaired AMPK function contribute to starvation sensitivity in adults. We therefore employed the hsp70-GAL4 element, which allows temporal control through stage-specific induction by heat-shock and thus avoid any developmental disturbances caused by earlier transgene expression. Heat-shock-induced AMPK impairment in adult stages produced a significant reduction in starvation survival , and staDrosophila and other Metazoa, which is believed to be an adaptive response, as elevated activity promotes increased foraging 1118w genetic background controls   .Animals with reduced AMPK function demonstrate hypersensitivity to starvation, despite also displaying hyperphagia, suggesting that these animals are persistently experiencing starvation. Because AMPK null mutants die as larvae and that these larvae are characteristically smaller than wild-type larvae of the same age , we speculated that persistent starvation may be the ultimate cause of reduced larval growth and lethality. We took advantage of previous observations that starvation induces an accumulation of lipid droplets in larval oenocytes DN on total triglyceride stores under fed conditions; with the average amount of triglyceride levels for the dominant negative being significantly lower than AMPK-wild type expressing flies   .Given the observations that animals with reduced AMPK function have higher metabolic rates, we tested the hypothesis that this metabolic activity may stem from failures to reallocate energy during nutritional stress. AMPK signaling can intersect with the target of rapamycin (TOR); AMPK activation would lead to TOR inhibition, which would lead to decreased protein synthesis and decreased energy consumption. We report that animals with reduced AMPK function are highly susceptible to the effects of starvation, and note significant changes in behavioral and physiological responses to starvation caused by altered AMPK function. Demonstrations of increased hyperactive locomotor responses, lower triglyceride levels, increased hunger-driven feeding behaviors, higher metabolic rates, and lipid accumulation phenotypes in liver-like oenocytes indicate that the loss of AMPK leads to a persistent starvation signal both at the organismal and cellular levels.These phenotypes suggest mechanisms underlying reduced survival during starvation in animals with altered AMPK activity. For example, starvation induces hyperactivity in flies with wild-type AMPK function Caenorhabditis elegans. Upon exposure to harsh environmental conditions, including nutrient limitation, C. elegans enter a dormant dauer state to become stress-resistant. C. elegans larvae lacking AMPK activity rapidly consume their energetic stores and die prematurely and are unable to utilize energy efficiently Drosophila. In mammals, a recent report of altered AMPK activity in the mouse also shows some similar phenotypes, specifically higher locomotor activity and reduced triglyceride levels Drosophila does not alter starvation sensitivity We suspect that many of these starvation phenotypes are consequences of a core metabolic defect. While we cannot rule out potential pleitropic effects, we suggest our results are consistent with an idea of compensatory changes in critical physiologies and behaviors stemming from defects in energy allocation, storage, or utilization. Our observations that rapamycin significantly extends lifespan, and that animals with reduced AMPK function consume more oxygen indicate inabilities to appropriately regulate cellular metabolic activities. Our observations are also consistent with multiple observations that AMPK is an important factor regulating cellular activities, and regulate expenditures of energy including protein synthesis Drosophila with compromised AMPK function globally show a similar hyperphagic response. Additionally, AMPK has been shown to inhibit lipolysis i.e., autophagy), which can be independent of AMPK While such observations are consistent with the suspected roles of AMPK as a regulator of cellular activities, our results with an organismal reduction of AMPK function are not entirely expected based on all previous studies and predictions of AMPK function. For example, AMPK is involved in the release of orexigenic peptides from the mammalian hypothalamus during starvation conditions, which acts to stimulate feeding during low energy We also note that these phenotypes likely stem from the acute loss of AMPK function as opposed to developmental defects caused by reduced AMPK signaling. While AMPK is clearly required for normal development, as null mutants are larval lethal hugin – encoding peptides, both of which have been previously shown to modulate larval feeding Drosophila, including muscle function, liver lipolysis, and locomotor behaviors. For example, the neuroendocrine cells that produce the Adipokinetic Hormone (AKH) are critical for the formation of starvation-induced hyperactive behaviors Drosophila with reduced AMPK function through selective introduction of an RNAi element targeting the alpha subunit. Moreover, this report suggests that the selective loss of AMPK signaling in muscle tissue leads to heightened sensitivity to starvation Drosophila.Our results suggest a series of future experiments targeting AMPK function in various central populations that may mediate feeding behaviors, such as neurons that express neuropeptide F or the Drosophila and previously in C. elegansDrosophila (here) and C. elegans suggest loss of AMPK activity leads to difficulty maintaining energy stores and inefficient utilization of them; in essence excessive use of energy stores. If the opposite were true during AMPK activation (for instance with an AMPK-activating drug in humans) one might predict highly-efficient and sparing utilization of energy stores (fat). Since Type 2 Diabetes is associated with obesity and a sedentary lifestyle, such an effect of AMPK activators would be very undesirable and potentially lead to increased energy stores (fat).In addition, if AMPK functions noted here in Figure S1Ubiquitous expression of AMPK alpha K57A and AMPK gamma RNAi elements cause lethality. Adult animals of specific genotypes were counted, as determined by the presence or absence of specific markers. Percent of expected was calculated as determined by expected Mendelian segregation ratios . Numbers above the columns refer to the total number of animals scored. Note that both RNAi elements and the K57A (DN: dominant negative) phenocopy the lethality caused by the AMPKα mutation and is dependent upon GAL4 mediated transcription.(1.11 MB TIF)Click here for additional data file.Figure S2Failure of the K57A transgene to rescue lethality caused by the molecular null ampk allele. We evaluated the extent to which different AMPK transgenes were able to rescue the lethality caused either by the AMPK3 mutation or by expression of the K57A element. Animals were scored with respect to segregation of dominant markers present on balancer chromosomes.(3.70 MB TIF)Click here for additional data file.Figure S3Reduced expression of AMPKα protein caused by expression of different RNAi elements. (A) Western blot for the AMPKα subunit from lysates of animals expressing RNAi for either the alpha (second lane) or the gamma subunit (third lane). Compared to animals possessing the driver alone (first lane) or an RNAi targeting an unrelated gene (ATG8) (fourth lane), there was significantly less expression of the alpha subunit standardized to loading control (tubulin). (B) Semi-quantitative RT-PCR of gamma expression (left) and RP49 (right) derived from animals expressing the gamma RNAi element compared to wild type. Under these PCR conditions, there was no detectable band for the gamma transcript in animals expressing the specific RNAi element.(1.81 MB TIF)Click here for additional data file.Figure S4Median survival time during starvation for animals with reduced AMPK function is decreased. Median survival was determined from three replicate vials of thirty individuals. Non-linear regression was employed to estimate a median survival for each vial; data were pooled, and mean ±SEM is plotted. The addition of a wild-type copy to animals expressing the K57A element leads to intermediate starvation survival times, implicating the K57A as a dominant negative.(5.15 MB TIF)Click here for additional data file.Figure S5Aging is significantly impacted by altered AMPK function. We measured percent survival daily from 300 males and 300 females either expressing the dominant negative or wild type subunit under normal fed conditions, once daily. A non-linear regression was employed to estimate median survival for each vial, and a mean ±SEM is plotted. Overexpression of the wild type alpha subunit leads to increased longevity as compared to w1118 (the genetic background), and in contrast, expression of the dominant negative element leads to decreased longevity .(3.16 MB TIF)Click here for additional data file.


Integrons are genetic platforms that accelerate lateral gene transfer (LGT) among bacteria. They were first detected on plasmids bearing single and multiple drug resistance determinants in human pathogens, and it is abundantly clear that integrons have played a major role in the evolution of this public health menace. Similar genetic elements can be found in nonpathogenic environmental bacteria and in metagenomic environmental DNA samples, and it is reasonable to suppose that integrons have facilitated microbial adaptation through LGT in niches outside infectious disease wards. Here we show that a heavily impacted estuary, exposed for almost a century to products of coal and steel industries, has developed a rich and unique cassette metagenome, containing genes likely to aid in the catabolism of compounds associated with industrial waste found there. In addition, we report that the most abundant cassette recovered in this study is one that encodes a putative LysR protein. This autoregulatory transcriptional regulator is known to activate transcription of linked target genes or unlinked regulons encoding diverse functions including chlorocatechol and dichlorophenol catabolism. Finally, only class 1 integrase genes were amplified in this study despite using different primer sets, and it may be that the cassettes present in the Tar Ponds will prove to be associated with class 1 integrase genes. Nevertheless, our cassette library provides a snapshot of a complex evolutionary process involving integron-meditated LGT likely to be important in natural bioremediation. These arrays are dynamic insofar as closely related organisms show drastically different profiles of cassettes c) after integration Integrons are genetic platforms for lateral gene transfer (LGT) that facilitate adaptation in bacteria, first recognized and named by Ruth Hall and Hatch Stokes in 1989 intI gene  Vibrio species and other proteobacteria, has since revealed much larger chromosome-located cassette arrays, with ORFs of various function Before the late 1990s, most characterized integrons were hospital-derived, transposon-associated and plasmid-borne, carrying between zero and half-a-dozen representatives of a repertoire of about 100 antibiotic-resistance determining cassettes adjacent to a “class 1” Nova Scotia provides an ideal test site for this latter conjecture: the Sydney Tar Ponds. Steel production began at this urban Cape Breton location in the early 1900s and continued for more than 80 years. In that time at least 700,000 tonnes of toxic by-products, including total petroleum hydrocarbons (TPHs), benzene, toluene, ethyl-benzene, xylene (BTEX), polycyclic aromatic hydrocarbons (PAHs) and heavy metals from smelting and associated steel production processes were deposited into a tidal estuary that empties into Sydney Harbour and ultimately the Atlantic Ocean attC sequences in the polymerase chain reaction applied to an environmental DNA sample, in this case a shallow sediment sample from the south Tar Pond. Cassette PCR has shown its effectiveness in recovering single and multiple cassettes (including interspersed attC sites) in the hands of Stokes and colleagues Although the site is now slated to be solidified and stabilized, we were able to obtain Tar Pond samples prior to encapsulation and identify microbial community members using standard 16S rRNA-based phylotyping methods, and to derive some idea of the richness of the pool of gene cassettes that has accumulated there using an environmental polymerase chain reaction (PCR) or specifically, cassette PCR. This entails use of degenerate primers targeted against known Methanosarcina, as were 164 of 199 sequences recovered with archaeal-specific primers. Organisms from this group are known to perform acetoclastic methanogenesis, dismuting acetate to carbon dioxide and methane (CH3COOH→CO2+CH4) in situ methanogenesis (data not shown). Methanogenesis by acetoclastic methanogens is known to occur in anaerobic zones near the source of contaminants Syntrophus aciditrophicusGeobacter species, and Desulfobacterium species as well as Dehalococcoides species. Three ribotypes that identify these organisms were recovered in this study (The taxonomic distribution of ribotypes (phylotypes) recovered in this study is listed in Sequences sharing 97% nucleotide identity or more were assigned to the same Operational Taxonomic Unit (OTU), and the total richness of 16S rRNA genes in the Sydney Tar Ponds (number of OTUs) was calculated using the program DOTUR intI) primers intI sequences obtained with either primer set were of class 1, hitherto most frequently associated with antibiotic resistance  potentially encode proteins with known homologs, consistent with other environmental surveys of integron cassettes Pseudomonas putidaPseudomonas fluorescens Pf-5 The LysR family comprises more than 50 members of autoregulatory transcriptional regulators (LTTRs) and these are found in a diverse range of bacteria Vibrio and Pseudomonas genera, the cassette pool known to facilitate drug-resistance (referred to as class 1 associated cassettes) as well as cassettes from another well-sampled environment . By this measure, it is apparent that the functional profile of cassettes obtained from the Tar Ponds is quite different from any other sampled integron gene cassette pool  have inferred functional roles in the degradation of contaminants present in the Sydney Tar Ponds  and thestte pool . This clThe metabolic atlas of functionally described cassettes highlights the biochemical reactions that cassette-encoded proteins from the Tar Ponds might facilitate, as compared to the entire cassette metagenome previously known . This anThe cassette-encoded catabolic enzymes that potentially degrade compounds associated with industrial processes like catechol 1,4-dioxygenase (EC 1.13.11.1) and 4-carboxymuconolactone decarboxylase (EC 4.1.1.44) are downstream reactions, suggesting that these enzymes likely have a role in degrading catabolic intermediates produced by other enzymes found in contaminated sites. Indeed, it has been suggested that although a complete pathway for a particular substrate may not exist in a single organism, partial and complementary pathway segments may exist in different organisms Shewanella, a genus that has generated much interest in the field of bioremediation given its ability to use a diversity of electron acceptors , an enzyme implicated in 1,4-dichlorobenzene degradation as well as benzoate degradation, a degradation pathway that is downstream to a number of catabolic intermediates present in the Tar Ponds and one that would facilitate funneling these molecules to other biological processes such as phenylalanine biosynthesis and oxidative phosphorylation. Amino acid phylogeny of this cassette-encoded enzyme indicates that while this gene is an out-group to the sampled catechol 1,4-dioxygenases, it is nevertheless most closely related to organisms known to catabolize aromatics . In addicceptors .Five of the catabolic enzymes associated with the degradation of industrial contaminants  are also implicated in other conserved biological functions . Yet givattC in addition to being oriented in the same direction, which is most typical of gene cassette arrays  is part of a cassette array, we used the long walk PCR protocol developed by Katz and colleagues 1999 e arrays . In addiPseudomonas, the most abundant genus and one implicated in contaminant degradation. We used the software package MEGAN, an algorithm based on best BLAST scores Arthrobacter sp. and a Citrobacter sp. Both of these isolates are known to be contaminant degraders intI gene, in addition to several cassettes amplified and sequenced from both organisms. The integron integrase genes of both isolates grouped with the class 1 integron clade illustrated in Escherichia coli, organisms that have not demonstrated the capacity to facilitate bioremediation, their role in syntrophic bioremediation processes should not be discounted. Furthermore, genome studies of the Arthrobacter aurescens strain TC1  revealed that it has likely expanded its metabolic repertoire through processes such as duplication of catabolic genes in addition to funneling metabolic intermediates generated by plasmid-borne genes to chromosomally-encoded pathways Twenty bacterial isolates were cultured from the Sydney Tar Ponds and screened for the presence of integron integrase genes and gene cassettes by PCR. Two bacterial isolates where found to encode integrons by this screen; a putative Collecting and examining cassettes from environments in which particular selection pressures might be inferred is a fruitful approach to assessing the functional diversity of the gene cassette metagenome, which may ultimately prove to be enormous. The Sydney Tar Ponds, with almost a century of exposure to a rich mix of compounds representing both challenges and opportunities to its microbiota, offers an ideal test case. Other surveys of similar environments have been limited in scale: a single potentially niche-relevant cassette was described from a heavy-metal-contaminated mine tailings site Tar Pond sludge was collected from the shoreline of the south Tar Pond located near Muggah Creek, Cape Breton, NS, Canada , a site rich in contaminants associated with coal and steel industrial processes Five grams of sludge collected from the Tar Ponds were added to 50 mL of sterile water , plated on a 2% glucose agar Petri dish 2O. This mixture was subjected to the following PCR protocol using a Peltier thermal cycler-200: 94°C for 3 min, *29 cycles, 68°C for 5 min.Domain-biased archaeal (A751F and U1406R) and universal bacterial (U515F and U1406R) attC regions described in Stokes et al., 2001 TCSGCTKGARCGAMTTGTTAGVC and GCSGCTKANCTCVRRCGTTAGSC, HS287 and HS286 respectively Integron gene cassettes were amplified with degenerate primers targeting conserved intI gene was performed using class 1-specific primers (HS463A: CTGGATTTCGATCACGGCACG and HS464: ACATGCGTGTAAATCATCGTCG)  as well as the more degenerate primer set intI-255F and intI-948R that were designed in Elsaied et al., 2007 PCR using primers specific for the ACCCTTCTTCTAACGGC; GSP2, TTTCGTGCGGCAAACGAAGTC; and GSP3, TGAATTCATCTCAAATCTCG.The long walk PCR protocol was followed according to Katz and colleagues E. coli cells (Invitrogen cat. no. K4750-10). Samples were plated on 1×LB+Kanamycin [50 µg/mL]+X-gal plates [2 mL/L] for blue white colony screening.Each PCR reaction was independently cloned into the pCR-XL-TOPO vector (Invitrogen cat. no. K4750-10) and transformed into TOP10 chemically competent Clones were miniprepped by alkaline lysis with GeneMachine robotics (Genomic Solutions). Sequencing of miniprepped clones was performed using the DYEnamic™ ET Dye Terminator Kit (MegaBACE) and run on a MegaBACE™ 1000 (Amersham). Both forward and reverse sequencing reactions were performed, thus providing two sequencing reads for each clone. These sequence data are publicly available under the EMBL accession numbers: FM866473-FM867587.http://greengenes.lbl.gov/) http://metagenomics.nmpdr.org) built as a modified version of the RAST server http://www.geo.vu.nl/~huik/cluster.htm). Heat map graphics were generated using JavaTreeView DNA sequences were trimmed, edited and assembled with PhredPhrap and Consed 2 = 0.27) for the archaeal-specific primer set suggesting that the Chao1 estimate's uncertainty increases with additional sampling and thus the 95% CI is artificially low for sequence data generated with the archaeal-specific primers. This was not the case for the universal primer set, suggesting that we have sequenced a significant number of clones from this library in order to more accurately calculate this Chao1 richness estimate with 95% CI Click here for additional data file.Figure S2Timeline of events related to steel production in Sydney, Cape Breton, Nova Scotia, Canada.(9.88 MB TIF)Click here for additional data file.Figure S3Rarefaction analysis of 16S rRNA gene clone libraries obtained from the Sydney Tar Ponds. Rarefaction analysis was performed using Distance-based OTU and Richness determination (DOTUR) (8.37 MB TIF)Click here for additional data file.Figure S4intI sequences. This analysis includes all intI sequences amplified from the Tar Ponds as well as those collected from diverse isolates in addition to sequences amplified in a recent environmental survey of class 1 integrases Integron integrase diversity in the Sydney Tar Ponds. (A) General IntI diversity. A diverse selection of IntI protein sequences were retrieved from NCBI for phylogenetic analysis, among these are eight from a deep-sea vent environmental survey of integrons illustrated in bold-type (0.91 MB TIF)Click here for additional data file.Figure S5Partial integron gene cassette array obtained by long-walk PCR on DNA extracted from the Sydney Tar Ponds.(1.95 MB TIF)Click here for additional data file.Figure S6attC recombination sequences embedded in multiple-cassette-amplicons. Primers used for cassette PCR are included at the top of the alignment attC sequences would have been amplified with this primer pair. Asterisks represent positions in the alignment with identical nucleotides. Accession numbers and cassette-encoded functions are listed to the left of the alignment; putative functions are listed relative to their position (left or right) of the given attC. The sequence lengths of the attCs are indicated to the right.Nucleotide alignment of divergent (0.69 MB TIF)Click here for additional data file.Figure S7Dot plot of Chao1 95% CI range obtained from each ordered sampling incidence of 16S rRNA gene sequences amplified by either the archaeal-biased set (A) or the universal primer set (B).(0.59 MB TIF)Click here for additional data file.Table S1(0.03 MB XLS)Click here for additional data file.Table S2(0.02 MB XLS)Click here for additional data file.Table S3(0.01 MB XLS)Click here for additional data file.Table S4(0.02 MB XLS)Click here for additional data file.Table S5(0.02 MB XLS)Click here for additional data file.Table S6(0.02 MB XLS)Click here for additional data file.


FAAH) would identify circulating biomarkers of ECS activation in severe obesity.Obesity is a worldwide epidemic, and severe obesity is a risk factor for many diseases, including diabetes, heart disease, stroke, and some cancers. Endocannabinoid system (ECS) signaling in the brain and peripheral tissues is activated in obesity and plays a role in the regulation of body weight. The main research question here was whether quantitative measurement of plasma endocannabinoids, anandamide, and related N-acylethanolamines (NAEs), combined with genotyping for mutations in fatty acid amide hydrolase  mutation by comparing plasma ECS metabolite levels in the FAAH 385 minor A allele carriers versus wild-type C/C carriers in both groups. The main finding was significantly elevated mean plasma levels of anandamide (15.1±1.4 pmol/ml) and related NAEs in study subjects that carried the FAAH 385 A mutant alleles versus normal subjects (13.3±1.0 pmol/ml) with wild-type FAAH genotype (p = 0.04), and significance was maintained after controlling for BMI.Plasma samples were obtained from 96 severely obese subjects with body mass index (BMI) of ≥40 kg/mFAAH 385 A mutant alleles compared with wild-type FAAH controls. This evidence supports endocannabinoid system activation due to the effect of FAAH 385 mutant A genotype on plasma AEA and related NAE analogs. This is the first study to document that FAAH 385 A mutant alleles have a direct effect on elevated plasma levels of anandamide and related NAEs in humans. These biomarkers may indicate risk for severe obesity and may suggest novel ECS obesity treatment strategies.Significantly increased levels of the endocannabinoid anandamide and related NAEs were found in carriers of the  Endocannabinoids (ECs) are a family of polyunsaturated fatty acid derivatives that function as lipid signaling molecules by acting as endogenous ligands at the two known cannabinoid receptors (CBRs), CB1 and CB2, present in the nervous system and peripheral organs of many living species (FAAH) and monoglyceride lipase (MGLL), which are the principal inactivating enzymes for FAAs and 2-AG. There is an increasing body of data on endocannabinoid control of energy homeostasis and recent evidence for ECS dysregulation in overweight and obesity disorders The complex biosynthetic and enzymatic degradation pathways for these ECS signaling molecules are currently being investigated as potentially selective disease treatment targets Current concepts in this field indicate that body weight and fat metabolism are influenced by complex ECS regulatory mechanisms that modulate energy balance, feeding behavior and peripheral lipid metabolism FAAH expression in adipose tissue and circulating ECs Recent investigations have demonstrated activation of the peripheral ECS in human obesity and type II diabetes FAAH and MGLL. This aim is based on our previous reports that showed an association of the common human FAAH 385 C→A (P129T)non-synonymous mutation with overweight and obesity P129T FAAHFAAH gene ECS biomarkers of severe obesity that have the potential to be clinically useful if confirmed by larger studies.The overall aim of the present study was to investigate circulating biomarkers of ECS activation in severely obese subjects compared to normal weight controls using highly sensitive triple-quadrupole mass spectroscopy plasma quantification methods, combined with genotyping for common mutations in the genes encoding the endocannabinoid and fatty acid amide inactivating enzymes FAAH 385 A alleles compared to normal weight wild-type FAAH carriers. This evidence could further support ECS activation in a sub-type of severely obese individuals and may indicate novel ECS treatment targets in severe obesity.The objective of this study was to investigate readily available circulating biomarkers of ECS activation in severely obese subjects compared to normal weight subjects by combining accurate measurement of plasma endocannabinoid levels with SNP genotyping in critical ECS inactivating enzymes. The main hypothesis herein based on previous studies was that plasma levels of anandamide and related NAEs would be elevated in severely obese carriers of the http://clinicaltrials.gov/ct/show/NCT00263042), a cardiovascular outcomes trial to assess the efficacy of the CB1 receptor antagonist, rimonabant (Acomplia), in reducing the risk of heart attack, stroke, or cardiovascular death in abdominally obese individuals and comprised 18,671 randomized and treated subjects and was conducted by Sanofi-Aventis. Subjects and samples for this study were from a subset of 3101 subjects who volunteered with separate written informed consent for the CRESCENDO Genomic Substudy cohort. A subset of the substudy were selected for this ECS study based on BMI (150 with BMI<30 and 150 with BMI>40). Severely obese subjects for this ECS metabolite and genomics study were selected from the Genomic Substudy cohort based on a body mass index (BMI) of 40 kg/m2 to 60 kg/m2 and waist circumference of greater or equal to 140 cm in men and 131 cm in women. Normal weight controls were included based on a BMI of less than 26 kg/m2 and a waist circumference of less than or equal to 100 cm in men and 90 cm in women. To reduce false-positive findings due to differing genetic backgrounds, and since Whites were highly represented in the patient population sampled, the study was limited to male and female Whites, ranging in age from 55 to 77. All study participants and data were anonymous and biometric data were linked to each plasma and DNA sample by unique study code (see Samples and linked biometric data used in this study were obtained from the rimonabant (Acomplia) CRESCENDO study (code see . The ano2) gas. The lipid layer was then re-solubilized in 100 microliters of 2∶1 CHCl3∶CH3OH.Whole blood samples were collected in evacuated glass tubes containing EDTA. Samples were centrifuged to separate plasma from blood cells and plasma was withdrawn and stored in 1 ml aliquots at −80°C prior to plasma lipid extraction. For each sample, 0.5 ml plasma was added to a glass vial containing 2.0 ml chloroform (CHCl3), 1.0 ml methanol (MeOH) and 0.5 ml (1% v/v) formic acid. To this mixture were added aliquots of 10 pmol D5-2-arachidonlyglycerol (2-AG) and 5 pmol D8-arachidonlyethanolamine (AEA). Vial contents were vortex mixed for 30 seconds and centrifuged at 10°C (1400×g for 10 minutes). The organic layer was carefully removed avoiding the aqueous layer and dried under a stream of nitrogen (NA - 95∶5∶0.1 H20∶methanol∶formic acid and B – 60∶35∶5∶0.1 isopropanol∶methanol∶H2O∶formic acid. Lipids were injected into a 5 microcentimeter C18 column (50×4.6 mm) from Phenomenex  and eluted with a 10 minute solvent B gradient from 60% to 100%. Values for each endocannabinoid and NAE analogue were subsequently calculated using ratios to the deuterated internal standards to calculate absolute concentrations, expressed as picomoles (pmol) of EC metabolite per ml of plasma similar to current methods Quantitative analysis of endocannabinoids and NAE analogues using the deuterated standards for AEA and 2-AG together with calculation of the other EC analogues was based on a ratio to the deuterated standards FAAH (GeneID 2166) and MGLL (GeneID 11343) from dbSNP was performed using the Sequenom platform. The major allele frequencies were determined in all 300 obese and normal subjects, using the Sequenom data for detection of 4 common SNPs in FAAH and 16 in MGLL (data not shown). To reduce false-positive findings due to differing genetic backgrounds, the same patient population used for the EC metabolite analysis was sampled and only male and female Whites were included, as noted above.Genotyping of the SNPs in the apriori or pre-study power calculation to detect a true association in this study was based upon estimations from data reported for measurement of anandamide levels in humans in a clinical trial The FAAH genotype were compared between the severely obese and normal weight groups using the Fisher's exact test. Geometric mean levels of EC analogues were compared between FAAH genotype groups  in both BMI groups combined using the t-test and stratified by BMI group using 2-Way ANOVA. Normal weight wild-type FAAH subjects were compared with severely obese carriers of the FAAH 385 mutant A allele using 1-Way ANOVA with Dunnett's Correction for multiple comparisons. A two-sided p-value <0.05 was considered statistically significant.The distribution of each metabolite was assessed for outliers and normality. Log transformation of data resulted in normal distributions for all EC analogues. Stratification revealed no significant differences in mean metabolite levels by age and sex and these variables were thus not included in further analyses. Geometric means of each metabolite were compared between BMI groups using the t-test. Frequencies of the Participants in this study initially gave broad written informed consent for collection and analysis of plasma and DNA for the Sanofi-Aventis CRESCENDO Genomic Substudy. In this study of EC analogues, all subjects were anonymous and identified only by a code number linked to biometric data. Because informed consent from strictly anonymous CRESCENDO study subjects was not possible, the present study received an exemption for additional written consent from the Scripps IRB.Demographic characteristics of the 96 severely obese subjects and 48 normal weight controls included in the study are shown in The baseline mean endocannabinoid and NAE levels  were notFAAH and MGLL genes for common gene variations or SNPs was completed and there were no common variants of MGLL found in this study. The only coding variant commonly found in the subjects in this obesity study was the FAAH 385 C→A (P129T) as previously reported Analysis of the FAAH C385A mutation on the circulating plasma levels of endocannabinoids and NAE analogues, geometric mean congener levels were compared between carriers of the FAAH 385 mutant A allele and homozygotes for the wild type (WT) C alleles Using the t-test comparing geometric mean plasma metabolite levels in the two groups, there was a significant elevation of mean anandamide (AEA) levels (p = 0.04) and related NAE levels in carriers of the FAAH 385 mutant A alleles versus FAAH-WT carriers ) elevation of AEA and related NAEs  in obese FAAH mutant A allele carriers versus FAAH-WT normal weight subjects using 1-Way ANOVA with Dunnett's correction for multiple comparisons. This amounts to a modest but significant 1.14 fold higher mean AEA plasma level in severely obese mutant FAAH carriers compared to normal FAAH controls. Several other N-acylethanolamines related to AEA that are metabolized by FAAH, including PEA, OEA and LEA, were also significantly elevated in severely obese mutant carriers versus normal controls  of AEA and related NAEs . Because plasma levels of N-acylethanolamine analogues related to AEA, including PEA, OEA and LEA, are significantly elevated in severely obese carriers of the mutant FAAH 385 A alleles, this supports the concept of a functional FAAH enzyme mutation in subjects with FAAH 385 C→A SNPs. Taken together, these findings provide further evidence of activation of the ECS in severely obese subjects with the FAAH 385 C→A mutation. Despite the relatively small subject cohort, tight plasma ranges and statistical significance  in carriers of the FAAH P129T mutation. This is substantiated by demonstration of several of elevated N-acylethanolamine analogues related to AEA that are also metabolized by FAAH. In this study, the FAAH 385 C→A (P129T) mutant genotype, which is the principal catabolic enzyme of many N-acyl ethanolamine ECS signaling molecules, has the same approximately 2-fold prevalence (7.3% vs. 4%) in severely obese subjects compared to normal BMI subjects observed in a previous study (28). Among both obese and normals, carriers of the FAAH 385 minor A alleles had higher levels of AEA and other NAEs, except 2-AG which is not metabolized by FAAH. Likewise, all endocannabinoid levels were positively correlated with each other except 2-AG Click here for additional data file.