Type II MADS-box Genes Isolated from the Gymnosperm Cones of Ephedra and Juniper

Type II MADS-box genes determine the organ identity in angiosperm reproductive structures. Current knowledge of Type II MADS-box genes in gymnosperm cones is limited, but it does support the hypothesis that seed plant reproductive structures evolved using a similar genetic toolkit. In this study we focused on isolating and sequencing Type II MADS-box genes from the understudied gymnosperm cones of Ephedra and Juniper. We isolated 11 unique Type II MADS-box gene sequences that belong to four gene lineages. This is the first report of MADS-box genes in Ephedra. Our understanding of the evolutionary history of reproductive structures in gymnosperms and seed plants in general will continue to improve with further work on isolating more sequences and gene expression studies with our increased sampling of gymnosperm Type II MADS-box genes. This knowledge may help determine how cone and flowers are similar at the genetic level and ultimately how flowers evolved from cones

Genetically Modified Organisms: Friend or Foe?

The controversy surrounding genetically modified organisms (GMO\u27s) spans many different aspects of life from food production and medicine to environmental uses. One-hundred ninety-four students at Grand Valley participated in an anonymous voluntary survey in which they expressed their opinions about the impacts of GMO\u27s. Because of the broad span of their uses, our survey divided the applications of GMO\u27s into the categories of crops, animal products, the environment, medicine, fuel, research and household products. We also asked the students about the possible impacts of GMO\u27s in their own lives in the areas of personal health, cost, and moral conflicts. Our results show that if the entirety of GMO\u27s is considered, the student body has a neutral attitude towards them. However, there are distinct results showing opinions of positive impacts in medicine, and negative environmental impacts

Improved internal control for molecular diagnosis assays

The two principal determining steps in molecular diagnosis are the amplification and the identification steps. Accuracy of DNA amplification is primarily determined by the annealing sequence of the PCR primer to the analyte DNA. Accuracy for identification is determined either by the annealing region of a labelled probe for the real time PCR analysis, or the annealing of a sequencing primer for DNA sequencing analysis, that binds to the respective analyte (amplicon). Presently, housekeeping genes (Beta globin, GAPDH) are used in molecular diagnosis to verify that the PCR conditions are optimum, and are thus known as amplification controls [1–4]. Although these genes have been useful as amplification controls, they lack the true definition of an internal control because the primers and annealing conditions are not identical to the analyte being assayed. This may result in a false negative report [5]. The IC-Code platform technology described here provides a true internal control where the internal control and analyte share identical PCR primers annealing sequences for the amplification step and identical sequencing primer annealing sequence for the identification step. • The analyte and internal control have the same PCR and sequencing annealing sequences. • This method assures for little or no false negatives and false positives due to the method’s design of using identical annealing conditions for the internal control and analyte, and by using DNA sequencing analysis for the identification step of the analyte, respectively. • This method also allows for a set lower limit of detection to be used by varying the amount of internal control used in the assay

Detection of BRAF mutations from solid tumors using Tumorplex™ technology

Allele specific multiplex sequencing (Tumorplex™) is a new molecular platform for the detection of single base mutation in tumor biopsies with high sensitivity for clinical testing. Tumorplex™ is a novel modification of Sanger sequencing technology that generates both mutant and wild type nucleotide sequences simultaneously in the same electropherogram. The molecular weight of the two sequencing primers are different such that the two sequences generated are separated, thus eliminating possible suppression of mutant signal by the more abundant wild type signal. Tumorplex™ platform technology was tested using BRAF mutation V600E. These studies were performed with cloned BRAF mutations and genomic DNA extracted from tumor cells carrying 50% mutant allele. The lower limit of detection for BRAF V600E was found to be 20 genome equivalents (GE) using genomic DNA extracted from mutation specific cell lines. Sensitivity of the assay was tested by challenging the mutant allele with wild type allele at 20 GE, and was able to detect BRAF mutant signal at a GE ration of 20:1 × 107 (mutant to wild-type). This level of sensitivity can detect low abundance of clonal mutations in tumor biopsies and eliminate the need for cell enrichment. • Tumorplex™ is a single tube assay that permits the recognition of mutant allele without suppression by wildtype signal. • Tumorplex™ provides a high level of sensitivity. • Tumorplex™ can be used with small sample size with mixed population of cells carrying heterogeneous gDNA

Dysregulation of the histone demethylase KDM6B in alcohol dependence is associated with epigenetic regulation of inflammatory signaling pathways

Epigenetic enzymes oversee long-term changes in gene expression by integrating genetic and environmental cues. While there are hundreds of enzymes that control histone and DNA modifications, their potential roles in substance abuse and alcohol dependence remain underexplored. A few recent studies have suggested that epigenetic processes could underlie transcriptomic and behavioral hallmarks of alcohol addiction. In the present study, we sought to identify epigenetic enzymes in the brain that are dysregulated during protracted abstinence as a consequence of chronic and intermittent alcohol exposure. Through quantitative mRNA expression analysis of over 100 epigenetic enzymes, we identified 11 that are significantly altered in alcohol-dependent rats compared with controls. Follow-up studies of one of these enzymes, the histone demethylase KDM6B, showed that this enzyme exhibits region-specific dysregulation in the prefrontal cortex and nucleus accumbens of alcohol-dependent rats. KDM6B was also upregulated in the human alcoholic brain. Upregulation of KDM6B protein in alcohol-dependent rats was accompanied by a decrease of trimethylation levels at histone H3, lysine 27 (H3K27me3), consistent with the known demethylase specificity of KDM6B. Subsequent epigenetic (chromatin immunoprecipitation [ChIP]-sequencing) analysis showed that alcohol-induced changes in H3K27me3 were significantly enriched at genes in the IL-6 signaling pathway, consistent with the well-characterized role of KDM6B in modulation of inflammatory responses. Knockdown of KDM6B in cultured microglial cells diminished IL-6 induction in response to an inflammatory stimulus. Our findings implicate a novel KDM6B-mediated epigenetic signaling pathway integrated with inflammatory signaling pathways that are known to underlie the development of alcohol addiction.Funding Agencies|National Institute of Mental Health [MH084880]; National Institute on Alcohol Abuse and Alcoholism [R01AA023781]; National Institutes of Health [DA035592, NS071674]; National Institute of Alcohol Abuse and Alcoholism of the National Institutes of Health [R28AA012725]; NIAAA division of Intramural Research; Swedish Research Council; US National Institute of Health [MH084880, DA035592, NS071674]; United States Department of Defense (DoD); NIAAA [1R01AA023781-01A1]; European Union [668863]</p

Dysregulation of the histone demethylase KDM6B in alcohol dependence is associated with epigenetic regulation of inflammatory signaling pathways

Epigenetic enzymes oversee long-term changes in gene expression by integrating genetic and environmental cues. While there are hundreds of enzymes that control histone and DNA modifications, their potential roles in substance abuse and alcohol dependence remain underexplored. A few recent studies have suggested that epigenetic processes could underlie transcriptomic and behavioral hallmarks of alcohol addiction. In the present study, we sought to identify epigenetic enzymes in the brain that are dysregulated during protracted abstinence as a consequence of chronic and intermittent alcohol exposure. Through quantitative mRNA expression analysis of over 100 epigenetic enzymes, we identified 11 that are significantly altered in alcohol-dependent rats compared with controls. Follow-up studies of one of these enzymes, the histone demethylase KDM6B, showed that this enzyme exhibits region-specific dysregulation in the prefrontal cortex and nucleus accumbens of alcohol-dependent rats. KDM6B was also upregulated in the human alcoholic brain. Upregulation of KDM6B protein in alcohol-dependent rats was accompanied by a decrease of trimethylation levels at histone H3, lysine 27 (H3K27me3), consistent with the known demethylase specificity of KDM6B. Subsequent epigenetic (chromatin immunoprecipitation [ChIP]-sequencing) analysis showed that alcohol-induced changes in H3K27me3 were significantly enriched at genes in the IL-6 signaling pathway, consistent with the well-characterized role of KDM6B in modulation of inflammatory responses. Knockdown of KDM6B in cultured microglial cells diminished IL-6 induction in response to an inflammatory stimulus. Our findings implicate a novel KDM6B-mediated epigenetic signaling pathway integrated with inflammatory signaling pathways that are known to underlie the development of alcohol addiction.Funding Agencies|National Institute of Mental Health [MH084880]; National Institute on Alcohol Abuse and Alcoholism [R01AA023781]; National Institutes of Health [DA035592, NS071674]; National Institute of Alcohol Abuse and Alcoholism of the National Institutes of Health [R28AA012725]; NIAAA division of Intramural Research; Swedish Research Council; US National Institute of Health [MH084880, DA035592, NS071674]; United States Department of Defense (DoD); NIAAA [1R01AA023781-01A1]; European Union [668863]</p