Gene set of nuclear-encoded mitochondrial regulators is enriched for common inherited variation in obesity

There are hints of an altered mitochondrial function in obesity. Nuclear-encoded genes are relevant for mitochondrial function (3 gene sets of known relevant pathways: (1) 16 nuclear regulators of mitochondrial genes, (2) 91 genes for oxidative phosphorylation and (3) 966 nuclear-encoded mitochondrial genes). Gene set enrichment analysis (GSEA) showed no association with type 2 diabetes mellitus in these gene sets. Here we performed a GSEA for the same gene sets for obesity. Genome wide association study (GWAS) data from a case-control approach on 453 extremely obese children and adolescents and 435 lean adult controls were used for GSEA. For independent confirmation, we analyzed 705 obesity GWAS trios (extremely obese child and both biological parents) and a population-based GWAS sample (KORA F4, n = 1,743). A meta-analysis was performed on all three samples. In each sample, the distribution of significance levels between the respective gene set and those of all genes was compared using the leading-edge-fraction-comparison test (cut-offs between the 50(th) and 95(th) percentile of the set of all gene-wise corrected p-values) as implemented in the MAGENTA software. In the case-control sample, significant enrichment of associations with obesity was observed above the 50(th) percentile for the set of the 16 nuclear regulators of mitochondrial genes (p(GSEA,50) = 0.0103). This finding was not confirmed in the trios (p(GSEA,50) = 0.5991), but in KORA (p(GSEA,50) = 0.0398). The meta-analysis again indicated a trend for enrichment (p(MAGENTA,50) = 0.1052, p(MAGENTA,75) = 0.0251). The GSEA revealed that weak association signals for obesity might be enriched in the gene set of 16 nuclear regulators of mitochondrial genes

MARIS: Method for Analyzing RNA following Intracellular Sorting

Transcriptional profiling is a key technique in the study of cell biology that is limited by the availability of reagents to uniquely identify specific cell types and isolate high quality RNA from them. We report a Method for Analyzing RNA following Intracellular Sorting (MARIS) that generates high quality RNA for transcriptome profiling following cellular fixation, intracellular immunofluorescent staining and FACS. MARIS can therefore be used to isolate high quality RNA from many otherwise inaccessible cell types simply based on immunofluorescent tagging of unique intracellular proteins. As proof of principle, we isolate RNA from sorted human embryonic stem cell-derived insulin-expressing cells as well as adult human β cells. MARIS is a basic molecular biology technique that could be used across several biological disciplines.Howard Hughes Medical InstituteHarvard Stem Cell InstituteNational Institutes of Health (U.S.) (grant 2U01DK07247307)National Institutes of Health (U.S.) (grant RL1DK081184)National Institutes of Health (U.S.) (grant 1U01HL10040804

An exposure-effect approach for evaluating ecosystem-wide risks from human activities

Ecosystem-based management (EBM) is promoted as the solution for sustainable use. An ecosystem-wide assessment methodology is therefore required. In this paper, we present an approach to assess the risk to ecosystem components from human activities common to marine and coastal ecosystems. We build on: (i) a linkage framework that describes how human activities can impact the ecosystem through pressures, and (ii) a qualitative expert judgement assessment of impact chains describing the exposure and sensitivity of ecological components to those activities. Using case study examples applied at European regional sea scale, we evaluate the risk of an adverse ecological impact from current human activities to a suite of ecological components and, once impacted, the time required for recovery to pre-impact conditions should those activities subside. Grouping impact chains by sectors, pressure type, or ecological components enabled impact risks and recovery times to be identified, supporting resource managers in their efforts to prioritize threats for management, identify most at-risk components, and generate time frames for ecosystem recovery

High efficient differentiation of functional hepatocytes from porcine induced pluripotent stem cells

Hepatocyte transplantation is considered to be a promising therapy for patients with liver diseases. Induced pluripotent stem cells (iPSCs) provide an unlimited source for the generation of functional hepatocytes. In this study, we generated iPSCs from porcine ear fibroblasts (PEFs) by overexpressing Sox2, Klf4, Oct4, and c-Myc (SKOM), and developed a novel strategy for the efficient differentiation of hepatocyte-like cells from porcine iPSCs by following the processes of early liver development. The differentiated cells displayed the phenotypes of hepatocytes, exhibited classic hepatocyte-associated bio-functions, such as LDL uptake, glycogen storage and urea secretion, as well as possessed the metabolic activities of cytochrome P-450 (CYP) 3A and 2C. Furthermore, we compared the hepatocyte differentiation efficacy of our protocol with another published method, and the results demonstrated that our differentiation strategy could significantly improve the generation of morphological and functional hepatocyte-like cells from porcine iPSCs. In conclusion, this study establishes an efficient method for in vitro generation of functional hepatocytes from porcine iPSCs, which could represent a promising cell source for preclinical testing of cell-based therapeutics for liver failure and for pharmacological applications. © 2014 Ao et al

Microbial Functional Capacity Is Preserved Within Engineered Soil Formulations Used In Mine Site Restoration

Mining of mineral resources produces substantial volumes of crushed rock based wastes that are characterised by poor physical structure and hydrology, unstable geochemistry and potentially toxic chemical conditions. Recycling of these substrates is desirable and can be achieved by blending waste with native soil to form a 'novel substrate' which may be used in future landscape restoration. However, these post-mining substrate based 'soils' are likely to contain significant abiotic constraints for both plant and microbial growth. Effective use of these novel substrates for ecosystem restoration will depend on the efficacy of stored topsoil as a potential microbial inoculum as well as the subsequent generation of key microbial soil functions originally apparent in local pristine sites. Here, using both marker gene and shotgun metagenome sequencing, we show that topsoil storage and the blending of soil and waste substrates to form planting substrates gives rise to variable bacterial and archaeal phylogenetic composition but a high degree of metabolic conservation at the community metagenome level. Our data indicates that whilst low phylogenetic conservation is apparent across substrate blends we observe high functional redundancy in relation to key soil microbial pathways, allowing the potential for functional recovery of key belowground pathways under targeted management

Analysis of sequence variations in low-density lipoprotein receptor gene among Malaysian patients with familial hypercholesterolemia

<p>Abstract</p> <p>Background</p> <p>Familial hypercholesterolemia is a genetic disorder mainly caused by defects in the low-density lipoprotein receptor gene. Few and limited analyses of familial hypercholesterolemia have been performed in Malaysia, and the underlying mutations therefore remain largely unknown.</p> <p>We studied a group of 154 unrelated FH patients from a northern area of Malaysia (Kelantan). The promoter region and exons 2-15 of the LDLR gene were screened by denaturing high-performance liquid chromatography to detect short deletions and nucleotide substitutions, and by multiplex ligation-dependent probe amplification to detect large rearrangements.</p> <p>Results</p> <p>A total of 29 gene sequence variants were reported in 117(76.0%) of the studied subjects. Eight different mutations (1 large rearrangement, 1 short deletion, 5 missense mutations, and 1 splice site mutation), and 21 variants. Eight gene sequence variants were reported for the first time and they were noticed in familial hypercholesterolemic patients, but not in controls (p.Asp100Asp, p.Asp139His, p.Arg471Gly, c.1705+117 T>G, c.1186+41T>A, 1705+112C>G, Dup exon 12 and p.Trp666ProfsX45). The incidence of the p.Arg471Gly variant was 11%. Patients with pathogenic mutations were younger, had significantly higher incidences of cardiovascular disease, xanthomas, and family history of hyperlipidemia, together with significantly higher total cholesterol and low density lipoprotein levels than patients with non-pathogenic variants.</p> <p>Conclusions</p> <p>Twenty-nine gene sequence variants occurred among FH patients; those with predicted pathogenicity were associated with higher incidences of cardiovascular diseases, tendon xanthomas, and higher total and low density lipoprotein levels compared to the rest. These results provide preliminary information on the mutation spectrum of this gene among patients with FH in Malaysia.</p

Clinical outcomes in patients treated for coronary in-stent restenosis with drug-eluting balloons: Impact of high platelet reactivity.

BACKGROUND: The impact of high platelet reactivity (HPR) on clinical outcomes after elective percutaneous coronary interventions (PCI) with drug-eluting balloons (DEB) due to in-stent restenosis (ISR) is unknown. OBJECTIVE: We sought to evaluate the prognostic importance of HPR together with conventional risk factors in patients treated with DEB. METHODS: Patients treated with DEB due to ISR were enrolled in a single-centre, prospective registry between October 2009 and March 2015. Only patients with recent myocardial infarction (MI) received prasugrel, others were treated with clopidogrel. HPR was defined as an ADP-test >46U with the Multiplate assay and no adjustments were done based on results. The primary endpoint of the study was a composite of cardiovascular mortality, MI, any revascularization or stroke during one-year follow-up. RESULTS: 194 stable angina patients were recruited of whom 90% were treated with clopidogrel. Clinical characteristics and procedural data were available for all patients; while platelet function testing was performed in 152 subjects of whom 32 (21%) had HPR. Patients with HPR had a higher risk for the primary endpoint (HR: 2.45; CI: 1.01-5.92; p = 0.03). The difference was primarily driven by a higher risk for revascularization and MI. According to the multivariate analysis, HPR remained a significant, independent predictor of the primary endpoint (HR: 2.88; CI: 1.02-8.14; p = 0.04), while total DEB length and statin treatment were other independent correlates of the primary outcome. CONCLUSION: HPR was found to be an independent predictor of repeat revascularization and MI among elective patients with ISR undergoing PCI with DEB

Elevated tumour interleukin-1β is associated with systemic inflammation: a marker of reduced survival in gastro-oesophageal cancer

Systemic inflammation is associated with adverse prognosis cancer but its aetiology remains unclear. We investigated the expression of proinflammatory cytokines within normal mucosa from healthy controls and tumour tissue in cancer patients and related these levels with markers of systemic inflammation and with the presence of a tumour inflammatory infiltrate. Tissue was collected from 56 patients with gastro-oesophageal cancer and from 12 healthy controls. Tissue cytokine mRNA concentrations were measured by real-time PCR and tissue protein concentrations by cytometric bead array. The degree of chronic inflammatory cell infiltrate was recorded. Serum cytokine and acute phase protein concentrations (including C-reactive protein (CRP)) were measured by enzyme-linked immunosorbent assay. Proinflammatory cytokines were significantly overexpressed (interleukin (IL)-1β, IL-6, IL-8 and tumour necrosis factor-α) both at mRNA and protein levels in the cancer specimens compared with mucosa from controls. Interleukin-1β was expressed in greatest (10–100-fold) concentration and protein levels correlated significantly with systemic inflammation (CRP) (P=0.05, r=0.31). A chronic inflammatory infiltrate was observed in 75% of the cancer specimens and was associated with systemic inflammation (CRP: P=0.01). However, the presence of chronic inflammation per se was not associated with altered cytokine expression within the tumour. Both a chronic inflammatory infiltrate and systemic inflammation (CRP) were associated with reduced survival (P=0.05 and P=0.03, respectively). Tumour chronic inflammatory infiltrate and tumour tissue IL-1β overexpression are potential independent factors influencing systemic inflammation in oesophagogastric cancer patients

Cladistic analysis of the apolipoprotein AI-CIH-AIV gene cluster using a healthy French Canadian sample. I. Haploid analysis

A cladistic analysis was carried out to identify haplotypes hypothesized to differ for functional DNA sequence variations within the apolipoprotein (apo) AI-CIII-AIV gene cluster that affect plasma lipid, lipoprotein and apolipoprotein levels. A sample of unrelated healthy French Canadians was studied. First, a cladogram of the observed apo AI-CIII-AIV haplotypes was estimated. Then this cladogram was used to define a statistical analysis of the association between haplotype variation and variation in plasma lipid, lipoprotein and apolipoprotein levels. Three haplotypes were identified which were associated with small (5–12% of the total sum of squares) pleiotropic effects on plasma lipid, lipoprotein and apolipoprotein traits and these effects were context, i.e. gender, dependent.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66070/1/j.1469-1809.1995.tb00742.x.pd

The ter Mutation in the Rat Dnd1 Gene Initiates Gonadal Teratomas and Infertility in Both Genders

A spontaneous mutation leading to the formation of congenital ovarian and testicular tumors was detected in the WKY/Ztm rat strain. The histological evaluation revealed derivatives from all three germ layers, thereby identifying these tumors as teratomas. Teratocarcinogenesis was accompanied by infertility and the underlying mutation was termed ter. Linkage analysis of 58 (WKY-ter×SPRD-Cu3) F2 rats associated the ter mutation with RNO18 (LOD = 3.25). Sequencing of candidate genes detected a point mutation in exon 4 of the dead-end homolog 1 gene (Dnd1), which introduces a premature stop codon assumed to cause a truncation of the Dnd1 protein. Genotyping of the recessive ter mutation revealed a complete penetrance of teratocarcinogenesis and infertility in homozygous ter rats of both genders. Morphologically non-tumorous testes of homozygous ter males were reduced in both size and weight. This testicular malformation was linked to a lack of spermatogenesis using immunohistochemical and histological staining. Our WKY-Dnd1ter/Ztm rat is a novel animal model to investigate gonadal teratocarcinogenesis and the molecular mechanisms involved in germ cell development of both genders