Sequencing and de novo assembly of 150 genomes from Denmark as a population reference.

Hundreds of thousands of human genomes are now being sequenced to characterize genetic variation and use this information to augment association mapping studies of complex disorders and other phenotypic traits. Genetic variation is identified mainly by mapping short reads to the reference genome or by performing local assembly. However, these approaches are biased against discovery of structural variants and variation in the more complex parts of the genome. Hence, large-scale de novo assembly is needed. Here we show that it is possible to construct excellent de novo assemblies from high-coverage sequencing with mate-pair libraries extending up to 20 kilobases. We report de novo assemblies of 150 individuals (50 trios) from the GenomeDenmark project. The quality of these assemblies is similar to those obtained using the more expensive long-read technology. We use the assemblies to identify a rich set of structural variants including many novel insertions and demonstrate how this variant catalogue enables further deciphering of known association mapping signals. We leverage the assemblies to provide 100 completely resolved major histocompatibility complex haplotypes and to resolve major parts of the Y chromosome. Our study provides a regional reference genome that we expect will improve the power of future association mapping studies and hence pave the way for precision medicine initiatives, which now are being launched in many countries including Denmark.

Discovery of a novel selective PPARγ ligand with partial agonist binding properties by integrated in silico/in vitro work flow.

Full agonists to the peroxisome proliferator-activated receptor (PPAR)γ, such as Rosiglitazone, have been associated with a series of undesired side effects, such as weight gain, fluid retention, cardiac hypertrophy, and hepatotoxicity. Nevertheless, PPARγ is involved in the expression of genes that control glucose and lipid metabolism and is an important target for drugs against type 2 diabetes, dyslipidemia, atherosclerosis, and cardiovascular disease. In an effort to identify novel PPARγ ligands with an improved pharmacological profile, emphasis has shifted to selective ligands with partial agonist binding properties. Toward this end we applied an integrated in silico/in vitro workflow, based on pharmacophore- and structure-based virtual screening of the ZINC library, coupled with competitive binding and transactivation assays, and adipocyte differentiation and gene expression studies. Hit compound 9 was identified as the most potent ligand (IC50 = 0.3 µM) and a relatively poor inducer of adipocyte differentiation. The binding mode of compound 9 was confirmed by molecular dynamics simulation, and the calculated free energy of binding was -8.4 kcal/mol. A novel functional group, the carbonitrile group, was identified to be a key substituent in the ligand-protein interactions. Further studies on the transcriptional regulation properties of compound 9 revealed a gene regulatory profile that was to a large extent unique, however functionally closer to that of a partial agonist.

Inverse regulation of the nuclear factor-kappaB binding to the p53 and interleukin-8 kappaB response elements in lesional psoriatic skin.

Nuclear factor-kappaB (NF-kappaB) is an inducible nuclear transcription factor regulating a range of cellular processes. An imbalance of the DNA binding activity of NF-kappaB may, therefore, be part of the pathophysiological mechanisms in psoriasis. The purpose of this study was to determine the NF-kappaB DNA binding activity in psoriatic skin using three different kappaB sites and to determine how DNA binding activity was modulated by the anti-psoriatic drug calcipotriol. By electrophoretic mobility shift assay, we demonstrated that the NF-kappaB DNA binding to the p53 kappaB site was decreased, whereas the NF-kappaB DNA binding to the interleukin-8 (IL-8) kappaB site was increased in lesional psoriatic skin compared with non-lesional psoriatic skin. No regulation was seen on the NF-kappaB DNA binding to the major histocompatibility complex class I kappaB site. These changes were paralleled by a similar decrease in p53 expression and an increase in IL-8 expression in involved psoriatic skin compared with uninvolved skin as determined by quantitative RT-PCR. The alteration in NF-kappaB DNA binding activity was neither accompanied by any change in the expression of the inhibitor kappaB (IkappaB) kinases, IKKalpha, IKKbeta, and IKKgamma nor in the expression of the NF-kappaB inhibitor proteins, IkappaBalpha and IkappaBbeta. Immunofluorescence analysis revealed that p65 was sequestered in the cytoplasm of keratinocytes, whereas p50 exhibited a cytoplasmic as well as a nuclear localization. Interestingly, this distribution of p50 and p65 was similar in lesional and non-lesional psoriatic skin. Topical application of calcipotriol to lesional psoriatic skin for 4 d resulted in increased NF-kappaB binding to the p53 kappaB site and decreased NF-kappaB binding to the IL-8 kappaB site. Taken together, our data demonstrate that the NF-kappaB DNA binding activity is regulated in a specific manner in psoriatic skin depending on the kappaB sites investigated, and that topical treatment of psoriatic skin normalizes the abnormal NF-kappaB binding activity seen in lesional psoriatic skin.

HDAC activity is required for p65/RelA-dependent repression of PPARdelta-mediated transactivation in human keratinocytes.

Peroxisome proliferator-activated receptors (PPARs) play a key role in differentiation, inflammation, migration, and survival of epidermal keratinocytes. The NF-kappaB has long been known to play pivotal roles in immune and inflammatory responses, and furthermore NF-kappaB has been implicated in the regulation of epidermal homeostasis. Recent studies have established that p65/RelA is a potent repressor of PPARdelta-mediated transactivation in human keratinocytes. In this article we further investigate the molecular mechanisms dictating the NF-kappaB-dependent repression of PPARdelta in human keratinocytes. We demonstrate that repression is unique to p65/RelA, as no other member of the NF-kappaB family had an impact on PPARdelta-mediated transactivation. Interestingly, our results show that p65/RelA only represses PPARdelta-dependent transactivation when PPARdelta is bound to DNA via its DNA-binding domain. We show that repression is sensitive to inhibition of histone deacetylases (HDACs) by tricostatin A (TSA), suggesting that HDAC activity is indispensable for p65/RelA-mediated repression. Accordingly, we demonstrate that a ternary complex consisting of PPARdelta, p65/RelA, and HDAC1 is formed in vivo. Finally, we demonstrate that TSA relieves tumor necrosis factor-alpha (TNFalpha)-induced repression of PPARdelta-mediated transactivation of the PPARdelta target gene adipose differentiation-related protein (ADRP) indicating that cross-talk between PPARdelta and NF-kappaB is of biological significance in human keratinocytes.

Discovery of novel PPAR ligands by a virtual screening approach based on pharmacophore modeling, 3D shape, and electrostatic similarity screening.

Peroxisome proliferator-activated receptors (PPARs) are important targets for drugs used in the treatment of atherosclerosis, dyslipidaemia, obesity, type 2 diabetes, and other diseases caused by abnormal regulation of the glucose and lipid metabolism. We applied a virtual screening workflow based on a combination of pharmacophore modeling with 3D shape and electrostatic similarity screening techniques to discover novel scaffolds for PPAR ligands. From the resulting 10 virtual screening hits, five tested positive in human PPAR ligand-binding domain (hPPAR-LBD) transactivation assays and showed affinities for PPAR in a competitive binding assay. Compounds 5, 7, and 8 were identified as PPAR-alpha agonists, whereas compounds 2 and 9 showed agonistic activity for hPPAR-gamma. Moreover, compound 9 was identified as a PPAR-delta antagonist. These results demonstrate that our virtual screening protocol is able to enrich novel scaffolds for PPAR ligands that could be useful for drug development in the area of atherosclerosis, dyslipidaemia, and type 2 diabetes.

Tetradecylthioacetic acid prevents high fat diet induced adiposity and insulin resistance.

Tetradecylthioacetic acid (TTA) is a non-beta-oxidizable fatty acid analog, which potently regulates lipid homeostasis. Here we evaluate the ability of TTA to prevent diet-induced and genetically determined adiposity and insulin resistance. In Wistar rats fed a high fat diet, TTA administration completely prevented diet-induced insulin resistance and adiposity. In genetically obese Zucker (fa/fa) rats TTA treatment reduced the epididymal adipose tissue mass and improved insulin sensitivity. All three rodent peroxisome proliferator-activated receptor (PPAR) subtypes were activated by TTA in the ranking order PPARalpha > PPARdelta > PPARgamma. Expression of PPARgamma target genes in adipose tissue was unaffected by TTA treatment, whereas the hepatic expression of PPARalpha-responsive genes encoding enzymes involved in fatty acid uptake, transport, and oxidation was induced. This was accompanied by increased hepatic mitochondrial beta-oxidation and a decreased fatty acid/ketone body ratio in plasma. These findings indicate that PPARalpha-dependent mechanisms play a pivotal role, but additionally, the involvement of PPARalpha-independent pathways is conceivable. Taken together, our results suggest that a TTA-induced increase in hepatic fatty acid oxidation and ketogenesis drains fatty acids from blood and extrahepatic tissues and that this contributes significantly to the beneficial effects of TTA on fat mass accumulation and peripheral insulin sensitivity.