Injury to the tunica media initiates atherogenesis in the presence of hyperlipidemia

Background and aimsFatty streaks initiating the formation of atheromatous plaque appear in the tunica intima. The tunica media is not known to be a nidus for lipid accumulation initiating atherogenesis. We assessed changes to the tunica media in response to a micro-injury produced in the pig aorta. In addition, we assessed human carotid endarterectomy plaques for indication of atheroma initiation in the tunica media.MethodsThree healthy landrace female pigs underwent laparotomy to inject autologous blood and create micro-hematomas at 6 sites within the tunica media of the infrarenal abdominal aorta. These pigs were fed a high-fat diet (HFD) for 4–12 weeks. Post-mortem aortas from all pigs, including a control group of healthy pigs, were serially stained to detect lipid deposits, vasa vasora (VV), immune cell infiltration and inflammatory markers, as well as changes to the vascular smooth muscle cell (vSMC) compartment. Moreover, 25 human carotid endarterectomy (CEA) specimens were evaluated for their lipid composition in the tunica media and intima.ResultsHigh lipid clusters, VV density, and immune cell infiltrates were consistently observed at 5 out of 6 injection sites under prolonged hyperlipidemia. The hyperlipidemic diet also affected the vSMC compartment in the tunica media adjacent to the tunica adventitia, which correlated with VV invasion and immune cell infiltration. Analysis of human carotid specimens post-CEA indicated that 32% of patients had significantly greater atheroma in the tunica media than in the arterial intima.ConclusionThe arterial intima is not the only site for atherosclerosis initiation. We show that injury to the media can trigger atherogenesis

A non-synonymous coding change in the CYP19A1 gene Arg264Cys (rs700519) does not affect circulating estradiol, bone structure or fracture

Background The biosynthesis of estrogens from androgens is catalyzed by aromatase P450 enzyme, coded by the CYP19A1 gene on chromosome 15q21.2. Genetic variation within the CYP19A1 gene sequence has been shown to alter the function of the enzyme. The aim of this study is to investigate whether a non-synonymous Arg264Cys (rs700519) single nucleotide polymorphism (SNP) is associated with altered levels of circulating estradiol, areal bone mineral density or fracture. Methods This population- based study of 1,022 elderly Caucasian women (mean age 74.95 ± 2.60 years) was genotyped for the rs700519 SNP were analyzed to detect any association with endocrine and bone phenotypes. Results The genotype frequencies were 997 wildtype (97.6%), 24 heterozygous (2.3%) and 1 homozygous (0.1%). When individuals were grouped by genotype, there was no association between the polymorphism and serum estradiol (wildtype 27.5 ± 16.0; variants 31.2 ± 18.4, P = 0.27). There was also no association seen on hip bone mineral density (wildtype 0.81 ± 0.12; 0.84 ± 0.14 for variants, P = 0.48) or femoral neck bone mineral density (0.69 ± 0.10 for wildtype; 0.70 ± 0.12 for variants, P = 0.54) before or after correction of the data with age, height, weight and calcium therapy. There were also no associations with quantitative ultrasound measures of bone structure (broadband ultrasound attenuation, speed of sound and average stiffness). Conclusions In a cohort of 1,022 elderly Western Australian women, the presence of Arg264Cys (rs700519) polymorphism was not found to be associated with serum estradiol, bone structure or phenotypes

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

14-3-3 proteins in calcium-sensing receptor signalling

The Calcium-sensing receptor (CaR) belongs to Family C of G protein-coupled receptors. The receptor’s main role is to maintain calcium homeostasis. In addition, the CaR plays an important functional role in processes such as cell proliferation and apoptosis. These processes are mediated by various intracellular signalling pathways and the CaR tail is believed to play a major role in receptor signal transduction. To provide insight into mechanisms that control CaR signalling, recent yeast two-hybrid (Y2H) studies in our laboratory, using the CaR tail as bait, identified a number of interacting proteins including two isoforms of 14-3-3 (theta and zeta). 14-3-3 proteins are ubiquitously expressed and highly conserved, and are emerging as a group of multifunctional adapter proteins with a recognised role as chaperones. 14-3-3 proteins bind to numerous partner proteins and have a preference for targets containing phosphorylated motifs. Y2H deletion mapping studies performed in our laboratory have delineated the interaction region for 14-3-3 theta to residues 865-923 in the CaR tail. This region contains a consensus 14-3-3 binding motif that includes serine 895, a known protein kinase C substrate. The in vivo interaction between the CaR and 14-3-3 theta has been demonstrated by co-immunoprecipitation in mammalian cells. In addition, a direct in vitro interaction has been confirmed between 14-3-3 theta and the CaR tail using pulldown assays. An immediate aim of our studies is to establish the significance of the consensus 14-3-3 interaction motif in CaR signalling

14-3-3 proteins in calcium-sensing receptor cell signalling

The calcium-sensing receptor (CaR) maintains calcium homeostasis, but also influences processes such as cell proliferation, differentiation and apoptosis through activation of signalling pathways such as Rho and ERK1/2. To provide insight into mechanisms controlling CaR signalling, a yeast two-hybrid (Y2H) screen was performed using the CaR intracellular tail as bait. Several interacting proteins were identified including the 14-3-3 isoforms theta and zeta. 14-3-3 proteins are chaperones which bind to numerous partner proteins, preferring targets containing phosphorylated motifs. They influence a multitude of cellular processes, including ERK1/2 signalling. Co-immunoprecipitation (co-IP) and pulldown assays have confirmed CaR-14-3-3 in vivo and in vitro interaction, respectively, for 14-3-3 theta. Confocal microscopy has demonstrated that both 14-3-3 isoforms co-localise with the CaR in the endoplasmic reticulum. Y2H deletion mapping delineated the interaction site for 14-3-3 zeta to residues 965-980 in the CaR tail. By contrast, the 14-3-3 theta interaction site is confined to residues 865-923. A 14-3-3 consensus binding motif, Rx1-2Sx2-3S, exists within this region of the CaR tail where serine 895 is putatively phosphorylated. Site-directed mutagenesis and co-IP assays have shown that the serine 895 is not primarily responsible for mediating CaR-14-3-3 theta interaction. Further studies aim to establish the significance of the entire consensus motif for CaR-14-3-3 theta interaction. To determine the role of 14-3-3 proteins in CaR-mediated ERK1/2 activity, HEK-293 cells stably expressing the CaR were transfected with 14-3-3 theta or zeta, stimulated with extracellular calcium and analysed for ERK1/2 activity by Western blot analysis. Neither 14-3-3 isoform modulated ERK1/2 activity through the CaR. In addition, the possible role of 14-3-3 theta and zeta in CaR-mediated modulation of Rho-dependent stimulation of serum-response element transcription was examined in HEK293 cells using a luciferase assay. Preliminary results suggest that both 14-3-3 theta and zeta inhibit Rho signalling to a similar extent. Differential binding of the two 14-3-3 isoforms to the CaR tail initially suggested differences in the way these isoforms may influence CaR-mediated signalling but experiments to date have not exposed such differences. The functional significance of the CaR-14-3-3 interaction will be further examined with other CaR-mediated signalling pathways

The role of 14-3-3 proteins in calcium-sensing receptor-mediated Rho signalling

The calcium-sensing receptor (CaR) is pivotal in maintaining calcium homeostasis, but also regulates a number of other cellular processes. To achieve this, the CaR activates a number of different cell signalling pathways including Rho-dependent serum-response element (SRE) activation, which is enhanced by the partner binding protein, filamin. To provide further insight into mechanisms controlling CaR signalling, a yeast two-hybrid screen was performed using the CaR intracellular tail as bait. A number of interacting proteins were identified including the 14-3-3 isoforms theta and zeta. 14-3-3 proteins are chaperones which bind to numerous partner proteins, influencing a multitude of cellular processes, including cell signalling. Yeast two-hybrid mapping studies delineated the interaction site for both 14-3-3 isoforms to residues 865-922 on the CaR tail, and co-immunoprecipitation studies confirmed CaR/14-3-3 theta and zeta in vivo interaction in mammalian cells. To investigate the possible role of 14-3-3 theta and zeta in CaR-mediated Rho signalling, HEK-293 cells stably expressing the CaR (HEK-293/CaR) were transfected with either 14-3-3 theta or zeta and an SRE luciferase reporter, which allowed for the measurement of SRE activation. Results demonstrated that over-expression of both 14-3-3 theta and zeta inhibited CaR-mediated SRE activation in these cells. This effect was not seen in CaR-transfected M2 cells unable to express filamin but was not restored in CaR-transfected M2 cells stably expressing filamin. We propose a mechanism whereby 14-3-3 theta and zeta either competitively bind with or sequester filamin leading to the inhibition of CaR-mediated SRE activation in HEK-293/CaR cells