Decoupling of DNA methylation and activity of intergenic LINE-1 promoters in colorectal cancer

<p>Hypomethylation of LINE-1 repeats in cancer has been proposed as the main mechanism behind their activation; this assumption, however, was based on findings from early studies that were biased toward young and transpositionally active elements. Here, we investigate the relationship between methylation of 2 intergenic, transpositionally inactive LINE-1 elements and expression of the LINE-1 chimeric transcript (LCT) 13 and LCT14 driven by their antisense promoters (L1-ASP). Our data from DNA modification, expression, and 5′RACE analyses suggest that colorectal cancer methylation in the regions analyzed is not always associated with LCT repression. Consistent with this, in HCT116 colorectal cancer cells lacking DNA methyltransferases DNMT1 or DNMT3B, LCT13 expression decreases, while cells lacking both DNMTs or treated with the DNMT inhibitor 5-azacytidine (5-aza) show no change in LCT13 expression. Interestingly, levels of the H4K20me3 histone modification are inversely associated with LCT13 and LCT14 expression. Moreover, at these LINE-1s, H4K20me3 levels rather than DNA methylation seem to be good predictor of their sensitivity to 5-aza treatment. Therefore, by studying individual LINE-1 promoters we have shown that in some cases these promoters can be active without losing methylation; in addition, we provide evidence that other factors (e.g., H4K20me3 levels) play prominent roles in their regulation.</p

The endocannabinoid anandamide causes endothelium-dependent vasorelaxation in human mesenteric arteries

The endocannabinoid anandamide (AEA) causes vasorelaxation in animal studies. Although circulating AEA levels are increased in many pathologies, little is known about its vascular effects in humans. The aim of this work was to characterise the effects of AEA in human arteries. Ethical approval was granted to obtain mesenteric arteries from patients (n = 31) undergoing bowel resection. Wire myography was used to probe the effects and mechanisms of action of AEA. RT‐PCR was used to confirm the presence of receptor mRNA in human aortic endothelial cells (HAECs) and intracellular signalling proteins were measured using multiplex technology. AEA caused vasorelaxation of precontracted human mesenteric arteries with an Rmax of ∼30%. A synthetic CB1 agonist (CP55940) caused greater vasorelaxation (Rmax ∼60%) while a CB2 receptor agonist (HU308) had no effect on vascular tone. AEA-induced vasorelaxation was inhibited by removing the endothelium, inhibition of nitric oxide (NO) synthase, antagonising the CB1 receptor and antagonising the proposed novel endothelial cannabinoid receptor (CBe). AEA‐induced vasorelaxation was not affected by CB2 antagonism, by depleting sensory neurotransmitters, or inhibiting cyclooxygenase activity. RT‐PCR showed CB1 but not CB2 receptors were present in HAECs, and AEA and CP55940 had similar profiles in HAECs (increased phosphorylation of JNK, NFκB, ERK, Akt, p70s6K, STAT3 and STAT5). Post hoc analysis of the data set showed that overweight patients and those taking paracetamol had reduced vasorelaxant responses to AEA. These data show that AEA causes moderate endothelium-dependent, NO-dependent vasorelaxation in human mesenteric arteries via activation of CB1 receptors

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead