Automated workflow-based exploitation of pathway databases provides new insights into genetic associations of metabolite profiles

Background: Genome-wide association studies (GWAS) have identified many common single nucleotide polymorphisms (SNPs) that associate with clinical phenotypes, but these SNPs usually explain just a small part of the heritability and have relatively modest effect sizes. In contrast, SNPs that associate with metabolite levels generally explain a higher percentage of the genetic variation and demonstrate larger effect sizes. Still, the discovery of SNPs associated with metabolite levels is challenging since testing all metabolites measured in typical metabolomics studies with all SNPs comes with a severe multiple testing penalty. We have developed an automated workflow approach that utilizes prior knowledge of biochemical pathways present in databases like KEGG and BioCyc to generate a smaller SNP set relevant to the metabolite. This paper explores the opportunities and challenges in the analysis of GWAS of metabolomic phenotypes and provides novel insights into the genetic basis of metabolic variation through the re-analysis of published GWAS datasets. Results: Re-analysis of the published GWAS dataset from Illig et al. (Nature Genetics, 2010) using a pathway-based workflow (http://www.myexperiment.org/packs/319.html), confirmed previously identified hits and identified a new locus of human metabolic individuality, associating Aldehyde dehydrogenase family1 L1 (ALDH1L1) with serine/glycine ratios in blood. Replication in an independent GWAS dataset of phospholipids (Demirkan et al., PLoS Genetics, 2012) identified two novel loci supported by additional literature evidence: GPAM (Glycerol-3 phosphate acyltransferase) and CBS (Cystathionine beta-synthase). In addition, the workflow approach provided novel insight into the affected pathways and relevance of some of these gene-metabolite pairs in disease development and progression. Conclusions: We demonstrate the utility of automated exploitation of background knowledge present in pathway databases for the analysis of GWAS datasets of metabolomic phenotypes. We report novel loci and potential biochemical mechanisms that contribute to our understanding of the genetic basis of metabolic variation and its relationship to disease development and progression

Precision engineering for PRRSV resistance in pigs: Macrophages from genome edited pigs lacking CD163 SRCR5 domain are fully resistant to both PRRSV genotypes while maintaining biological function

Porcine Reproductive and Respiratory Syndrome (PRRS) is a panzootic infectious disease of pigs, causing major economic losses to the world-wide pig industry. PRRS manifests differently in pigs of all ages but primarily causes late-term abortions and stillbirths in sows and respiratory disease in piglets. The causative agent of the disease is the positive-strand RNA PRRS virus (PRRSV). PRRSV has a narrow host cell tropism, limited to cells of the monocyte/macrophage lineage. CD163 has been described as a fusion receptor for PRRSV, whereby the scavenger receptor cysteine-rich domain 5 (SRCR5) region was shown to be an interaction site for the virus in vitro. CD163 is expressed at high levels on the surface of macrophages, particularly in the respiratory system. Here we describe the application of CRISPR/Cas9 to pig zygotes, resulting in the generation of pigs with a deletion of Exon 7 of the CD163 gene, encoding SRCR5. Deletion of SRCR5 showed no adverse effects in pigs maintained under standard husbandry conditions with normal growth rates and complete blood counts observed. Pulmonary alveolar macrophages (PAMs) and peripheral blood monocytes (PBMCs) were isolated from the animals and assessed in vitro. Both PAMs and macrophages obtained from PBMCs by CSF1 stimulation (PMMs) show the characteristic differentiation and cell surface marker expression of macrophages of the respective origin. Expression and correct folding of the SRCR5 deletion CD163 on the surface of macrophages and biological activity of the protein as hemoglobin-haptoglobin scavenger was confirmed. Challenge of both PAMs and PMMs with PRRSV genotype 1, subtypes 1, 2, and 3 and PMMs with PRRSV genotype 2 showed complete resistance to viral infections assessed by replication. Confocal microscopy revealed the absence of replication structures in the SRCR5 CD163 deletion macrophages, indicating an inhibition of infection prior to gene expression, i.e. at entry/fusion or unpacking stages

Pubescenes, jatrophane diterpenes, from Euphorbia pubescens, with multidrug resistance reversing activity on mouse lymphoma cells

The macrocyclic jatrophane diterpene polyesters, pubescenes A-D (1-4) were isolated from the whole dried plant of Euphorbia pubescens, and evaluated for multidrug resistance (MDR) reversing activity on mouse lymphoma cells. All the compounds displayed very strong activity compared with the positive control verapamil. Pubescene D (4) is a new compound, whose structure was established as 3beta9alpha,-diacetoxy-7beta-benzoyloxy-15beta-hydroxy-14-oxo-2betaH-jatropha-5E,12E-diene by spectroscopic methods, including H-1-H-1 COSY, HMQC, HMBC and NOESY

Regulation of Programmed Death Ligand 1 (PD-L1) Expression in Breast Cancer Cell Lines In Vitro and in Immunodeficient and Humanized Tumor Mice

Programmed death ligand 1 (PD-L1) expression is an efficient strategy of tumor cells to escape immunological eradiation. However, only little is known about the factors that affect the cellular expression levels. Here we assessed the PD-L1 expression on different breast cancer cell lines under standard in vitro culture conditions and as a function of Epirubicin or Paclitaxel treatment. Moreover, we evaluated the expression in immunodeficient tumor mice as well as in humanized tumor mice (i.e., in the presence of a human immune system). We found highest PD-L1 levels in JIMT-1 and MDA-MB-231 cells. Epirubicin treatment caused a decrease and Paclitaxel treatment an increased PD-L1 expression in MDA-MB-231 cells. In addition, we identified nuclear PD-L1 in MDA-MB-231 cells. All in vivo transplanted breast cancer cell lines downregulated PD-L1 expression compared to their in vitro counterpart. Neither the gene copy number nor the presence of human immune system in humanized tumor mice had an effect on the PD-L1 content. We demonstrate that the degree of PD-L1 expression amongst breast cancer cell lines varies considerably. In addition, cytotoxic treatments and other extrinsic parameters differentially affect the expression. Hence, further investigations including in vivo evaluations are necessary to understand PD-L1 regulation for advanced breast cancer stratification

Rearranged jatrophane-type diterpenes from Euphorbia species. Evaluation of their effects on the reversal of multidrug resistance

The rearranged jatrophane-type diterpenes (1 - 3), isolated from the Me2CO extracts of Euphorbia portlandica and Euphorbia segetalis, were examined for their effects on multidrug resistance (MDR) in mouse lymphoma cells. Compounds 2 and 3 revealed to be active with the latter being more active than the positive control verapamil, a known resistance modifier. The new compound 1, named portlandicine, was isolated from Euphorbia portlandica and its structure characterised by high-field NMR spectroscopic methods including 2D NMR techniques: COSY, HMQC, HMBC and NOESY. The known diterpene 2, together with aleuritolic acid (4), oleanolic acid (5), and betulin diacetate (6), were also isolated from the same species

Induction of Raf kinase inhibitor protein contributes to macrophage differentiation

Differential gene expression analysis of human blood monocytes has identified the Raf kinase inhibitor protein (RKIP) as a continuously upregulated gene in macrophage and dendritic cell maturation. Using realtime RT-PCR and Western blot analysis we were able to confirm the initial DNA-microarray findings of RKIP induction on mRNA and protein levels. RKIP upregulation in primary cells and overexpression in THP-1 cells did not alter ERK activity but strongly reduced the amount of the NFkappaB subunit p65 in the nucleus. mRNA levels and cell surface expression of maturation markers including the integrin CD11c and the scavenger receptor CD36 were significantly increased in RKIP transfected THP-1 cells. Our data show for the first time that RKIP is upregulated during macrophage and dendritic cell differentiation on mRNA and protein levels and we conclude that RKIP contributes to the monocytic differentiation process via inhibition of the NFkappaB signaling cascade independent from the canonical Ras/Raf/MEK/ERK pathway

Central Cannulation by Seldinger Technique: A Reliable Method in Type A Aortic Dissection Repairs

none11Background: Extensive type A aortic dissections that involve peripheral great vessels can complicate the choice of a cannulation site for cardiopulmonary bypass. We started to employ direct cannulation of the true lumen on the concavity of the aortic arch by Seldinger technique and evaluated the efficacy of this access technique as an alternative arterial inflow target in aortic surgery.Material/Methods: Twenty-four consecutive patients (mean age: 59 +/- 14 years) underwent type A aortic dissection repair using selective antegrade cerebral perfusion. Direct aortic cannulation was used in 14 cases, subclavian access in 6 patients, and femoral entry in 4 patients. Perioperative factors were evaluated to identify the reliability and eventual benefits of direct cannulation method at the aortic arch.Results: There were no operative deaths and cumulative 30-day mortality rate was 25% (6). Permanent neurological deficits were not observed; in 1 patient transient changes occurred (4%). Time to reach circulatory arrest was the shortest in the direct access group, with mean 27 +/- 11 (CI: 20.6-33.3) min vs. 43 +/- 22 (28.0-78.0) min (p=0.058) and 32 +/- 8 (23.6-40.4) min (p=0.34) by femoral cannulation and subclavian entry, respectively. Direct arch cannulation resulted in the best renal function in the first 72 h after surgery and similar characteristics were observed in lactic acid levels.Conclusions: Ultrasound-guided direct cannulation on the concavity of the aortic arch using a Seldinger technique is a reliable method in dissection repairs. Prompt antegrade perfusion provides not only cerebral but also peripheral organ and tissue protection, which is an advantage in this high-risk group of patients.noneGobolos, L; Ugocsai, P; Foltan, M; Philipp, A; Thrum, A; Miskolczi, S; Malvindi, PG; di Gregorio, V; Pousios, D; Navaratnarajah, M; Ohri, SKGobolos, L; Ugocsai, P; Foltan, M; Philipp, A; Thrum, A; Miskolczi, S; Malvindi, Pg; di Gregorio, V; Pousios, D; Navaratnarajah, M; Ohri, S