Profiling of circulating microRNAs: from single biomarkers to re-wired networks

The recent discovery that microRNAs (miRNAs) are present in the circulation sparked interest in their use as potential biomarkers. In this review, we will summarize the latest findings on circulating miRNAs and cardiovascular disease but also discuss analytical challenges. While research on circulating miRNAs is still in its infancy, high analytical standards in statistics and study design are a prerequisite to obtain robust data and avoid repeating the mistakes of the early genetic association studies. Otherwise, studies tend to get published because of their novelty despite low numbers, poorly matched cases and controls and no multivariate adjustment for conventional risk factors. Research on circulating miRNAs can only progress by bringing more statistical rigour to bear in this field and by evaluating changes of individual miRNAs in the context of the overall miRNA network. Such miRNA signatures may have better diagnostic and prognostic value

An analytical approach to the β-phase coarsening behaviour in a thermally sprayed CoNiCrAlY bond coat alloy

This paper investigates the β-phase coarsening behaviour during isothermal heat treatment of free-standing CoNiCrAlY (Co-31.7%Ni-20.8%Cr-8.1%Al-0.5%Y, all in wt%) coatings prepared by high velocity oxy-fuel (HVOF) thermal spraying. The microstructure of the coatings was characterised using scanning electron microscopy with energy dispersive X-ray (EDX) analysis and electron backscatter diffraction (EBSD). It comprises a two phase structure of fcc γ-Ni matrix and bcc β-NiAl precipitates. The volume fraction of the γ-Ni and the β-NiAl phases were measured to be around 70% and 30% respectively, with grain sizes varying largely from 0.5 to 2 μm for both phases. Isothermal heat treatments of the free-standing coatings were carried out at 1100 C for times up to 250 h. The β-phase coarsening behaviour during isothermal heat treatments was analysed by quantitative metallography. It is shown that the coarsening behaviour of β phase in the CoNiCrAlY alloy followed the classical Lifshitz-Slyozov-Wagner (LSW) theory of Ostwald ripening. By incorporating a dimensionless factor which correlates with volume fraction of the β phase, a modified LSW model coupled with formulaic interfacial energy and effective diffusion coefficient of the CoNiCrAlY alloy was utilised to interpret the coarsening behaviour of the β phase. The coarsening rate coefficient obtained from the modified LSW model shows good agreement with the corresponding experimental result

Directional Secretory Response of Double Stranded RNA-Induced Thymic Stromal Lymphopoetin (TSLP) and CCL11/Eotaxin-1 in Human Asthmatic Airways

Background Thymic stromal lymphoproetin (TSLP) is a cytokine secreted by the airway epithelium in response to respiratory viruses and it is known to promote allergic Th2 responses in asthma. This study investigated whether virally-induced secretion of TSLP is directional in nature (apical vs. basolateral) and/or if there are TSLP-mediated effects occurring at both sides of the bronchial epithelial barrier in the asthmatic state. Methods Primary human bronchial epithelial cells (HBEC) from control (n = 3) and asthmatic (n = 3) donors were differentiated into polarized respiratory tract epithelium under air-liquid interface (ALI) conditions and treated apically with dsRNA (viral surrogate) or TSLP. Sub-epithelial effects of TSLP were examined in human airway smooth muscle cells (HASMC) from normal (n = 3) and asthmatic (n = 3) donors. Clinical experiments examined nasal airway secretions obtained from asthmatic children during naturally occurring rhinovirus-induced exacerbations (n = 20) vs. non-asthmatic uninfected controls (n = 20). Protein levels of TSLP, CCL11/eotaxin-1, CCL17/TARC, CCL22/MDC, TNF-α and CXCL8 were determined with a multiplex magnetic bead assay. Results Our data demonstrate that: 1) Asthmatic HBEC exhibit an exaggerated apical, but not basal, secretion of TSLP after dsRNA exposure; 2) TSLP exposure induces unidirectional (apical) secretion of CCL11/eotaxin-1 in asthmatic HBEC and enhanced CCL11/eotaxin-1 secretion in asthmatic HASMC; 3) Rhinovirus-induced asthma exacerbations in children are associated with in vivo airway secretion of TSLP and CCL11/eotaxin-1. Conclusions There are virally-induced TSLP-driven secretory immune responses at both sides of the bronchial epithelial barrier characterized by enhanced CCL11/eotaxin-1 secretion in asthmatic airways. These results suggest a new model of TSLP-mediated eosinophilic responses in the asthmatic airway during viral-induced exacerbations

Human papillomavirus in high- and low-risk areas of oesophageal squamous cell carcinoma in China

To examine the potential roles of human papillomavirus (HPV) in oesophageal squamous cell carcinoma (ESCC) development, we examined the presence of HPV DNA in paraffin-embedded ESCC tissues collected from two areas with different ESCC incidence rates in China, that is, Gansu (n=26) and Shandong (n=33), using PCR with SPF10 primers, or PCR with GP5+/GP6+ primers combined with Southern blot hybridisation. HPV genotype was determined by the INNO-LiPA HPV genotyping kit. HPV DNA was detected in 17 cases (65%) in Gansu, where ESCC incidence is much higher than in Shandong, where HPV was positive in two samples (6%). HPV genotypes 16 and 18 were detected in 79 and 16% of HPV-positive samples, respectively. Real-time PCR analysis suggested the presence of integrated form of HPV DNA in all the HPV-16-positive samples, but its viral load was estimated to be only <1–2 copies cell−1. We could not detect HPV 16/18 E6 protein expression by immunostaining in any of the HPV-16-positive samples. Neither p16INK4a nor p53 expression was related to HPV presence in ESCCs. Further studies seem warranted to examine the possible aetiological roles of HPV in ESCC

Association of MMP - 12 polymorphisms with severe and very severe COPD: A case control study of MMPs - 1, 9 and 12 in a European population

BACKGROUND: Genetic factors play a role in chronic obstructive pulmonary disease (COPD) but are poorly understood. A number of candidate genes have been proposed on the basis of the pathogenesis of COPD. These include the matrix metalloproteinase (MMP) genes which play a role in tissue remodelling and fit in with the protease--antiprotease imbalance theory for the cause of COPD. Previous genetic studies of MMPs in COPD have had inadequate coverage of the genes, and have reported conflicting associations of both single nucleotide polymorphisms (SNPs) and SNP haplotypes, plausibly due to under-powered studies. METHODS: To address these issues we genotyped 26 SNPs, providing comprehensive coverage of reported SNP variation, in MMPs- 1, 9 and 12 from 977 COPD patients and 876 non-diseased smokers of European descent and evaluated their association with disease singly and in haplotype combinations. We used logistic regression to adjust for age, gender, centre and smoking history. RESULTS: Haplotypes of two SNPs in MMP-12 (rs652438 and rs2276109), showed an association with severe/very severe disease, corresponding to GOLD Stages III and IV. CONCLUSIONS: Those with the common A-A haplotype for these two SNPs were at greater risk of developing severe/very severe disease (p = 0.0039) while possession of the minor G variants at either SNP locus had a protective effect (adjusted odds ratio of 0.76; 95% CI 0.61 - 0.94). The A-A haplotype was also associated with significantly lower predicted FEV1 (42.62% versus 44.79%; p = 0.0129). This implicates haplotypes of MMP-12 as modifiers of disease severity

MicroRNA-21 Exhibits Antiangiogenic Function by Targeting RhoB Expression in Endothelial Cells

BACKGROUND: MicroRNAs (miRNAs) are endogenously expressed small non-coding RNAs that regulate gene expression at post-transcriptional level. The recent discovery of the involvement of these RNAs in the control of angiogenesis renders them very attractive in the development of new approaches for restoring the angiogenic balance. Whereas miRNA-21 has been demonstrated to be highly expressed in endothelial cells, the potential function of this miRNA in angiogenesis has never been investigated. METHODOLOGY/PRINCIPAL FINDINGS: We first observed in endothelial cells a negative regulation of miR-21 expression by serum and bFGF, two pro-angiogenic factors. Then using in vitro angiogenic assays, we observed that miR-21 acts as a negative modulator of angiogenesis. miR-21 overexpression reduced endothelial cell proliferation, migration and the ability of these cells to form tubes whereas miR-21 inhibition using a LNA-anti-miR led to opposite effects. Expression of miR-21 in endothelial cells also led to a reduction in the organization of actin into stress fibers, which may explain the decrease in cell migration. Further mechanistic studies showed that miR-21 targets RhoB, as revealed by a decrease in RhoB expression and activity in miR-21 overexpressing cells. RhoB silencing impairs endothelial cell migration and tubulogenesis, thus providing a possible mechanism for miR-21 to inhibit angiogenesis. Finally, the therapeutic potential of miR-21 as an angiogenesis inhibitor was demonstrated in vivo in a mouse model of choroidal neovascularization. CONCLUSIONS/SIGNIFICANCE: Our results identify miR-21 as a new angiogenesis inhibitor and suggest that inhibition of cell migration and tubulogenesis is mediated through repression of RhoB

Cluster analysis in severe emphysema subjects using phenotype and genotype data: an exploratory investigation

Background: Numerous studies have demonstrated associations between genetic markers and COPD, but results have been inconsistent. One reason may be heterogeneity in disease definition. Unsupervised learning approaches may assist in understanding disease heterogeneity. Methods: We selected 31 phenotypic variables and 12 SNPs from five candidate genes in 308 subjects in the National Emphysema Treatment Trial (NETT) Genetics Ancillary Study cohort. We used factor analysis to select a subset of phenotypic variables, and then used cluster analysis to identify subtypes of severe emphysema. We examined the phenotypic and genotypic characteristics of each cluster. Results: We identified six factors accounting for 75% of the shared variability among our initial phenotypic variables. We selected four phenotypic variables from these factors for cluster analysis: 1) post-bronchodilator FEV1 percent predicted, 2) percent bronchodilator responsiveness, and quantitative CT measurements of 3) apical emphysema and 4) airway wall thickness. K-means cluster analysis revealed four clusters, though separation between clusters was modest: 1) emphysema predominant, 2) bronchodilator responsive, with higher FEV1; 3) discordant, with a lower FEV1 despite less severe emphysema and lower airway wall thickness, and 4) airway predominant. Of the genotypes examined, membership in cluster 1 (emphysema-predominant) was associated with TGFB1 SNP rs1800470. Conclusions: Cluster analysis may identify meaningful disease subtypes and/or groups of related phenotypic variables even in a highly selected group of severe emphysema subjects, and may be useful for genetic association studies