miRNA in situ hybridization in circulating tumor cells - MishCTC

irculating tumor cells (CTCs) must be phenotypically and genetically characterized before they can be utilized in clinical applications. Here, we present the first protocol for the detection of miRNAs in CTCs using in situ hybridization (ISH) combined with immunomagnetic selection based on cytokeratin (CK) expression and immunocytochemistry. Locked-Nucleic Acid (LNA) probes associated with an enzyme-labeled fluorescence (ELF) signal amplification approach were used to detect miRNA-21 in CTCs. This protocol was optimized using both epithelial tumor (MDA-MB468) and epithelial non-tumor (MCF-10A) cell lines, and miRNA-21 was selected as the target miRNA because of its known role as an onco-miRNA. Hematopoietic cells do not express miRNA-21; thus, miRNA-21 is an ideal marker for detecting CTCs. Peripheral blood samples were taken from 25 cancer patients and these samples were analyzed using our developed protocol. Of the 25 samples, 11 contained CTCs. For all 11 CTC-positive samples, the isolated CTCs expressed both CK and miRNA-21. Finally, the protocol was applied to monitor miRNA-21 expression in epithelial to mesenchymal transition (EMT)-induced MCF-7 cells, an epithelial tumor cell line. CK expression was lost in these cells, whereas miRNA-21 was still expressed, suggesting that miRNA-21 might be a good marker for detecting CTCs with an EMT phenotype.JJDM thanks the Spanish Ministerio de Economía y Competitividad for a Ramón y Cajal Fellowship (Grant CTQ2012-34778). This research was partially supported by Marie Curie Career Integration Grants within the 7th European Community Framework Program (FP7-PEOPLE-2011-CIG-Project Number 294142 to RMSM and FP7-PEOPLE-2012-CIG-Project Number 322276 toJJDM). Thisresearch wasalso partially supported by Consejeria de Salud de la Junta de Andalucı ́ a (PI0294-2012)

Linear, Deterministic, and Order-Invariant Initialization Methods for the K-Means Clustering Algorithm

Over the past five decades, k-means has become the clustering algorithm of choice in many application domains primarily due to its simplicity, time/space efficiency, and invariance to the ordering of the data points. Unfortunately, the algorithm's sensitivity to the initial selection of the cluster centers remains to be its most serious drawback. Numerous initialization methods have been proposed to address this drawback. Many of these methods, however, have time complexity superlinear in the number of data points, which makes them impractical for large data sets. On the other hand, linear methods are often random and/or sensitive to the ordering of the data points. These methods are generally unreliable in that the quality of their results is unpredictable. Therefore, it is common practice to perform multiple runs of such methods and take the output of the run that produces the best results. Such a practice, however, greatly increases the computational requirements of the otherwise highly efficient k-means algorithm. In this chapter, we investigate the empirical performance of six linear, deterministic (non-random), and order-invariant k-means initialization methods on a large and diverse collection of data sets from the UCI Machine Learning Repository. The results demonstrate that two relatively unknown hierarchical initialization methods due to Su and Dy outperform the remaining four methods with respect to two objective effectiveness criteria. In addition, a recent method due to Erisoglu et al. performs surprisingly poorly.Comment: 21 pages, 2 figures, 5 tables, Partitional Clustering Algorithms (Springer, 2014). arXiv admin note: substantial text overlap with arXiv:1304.7465, arXiv:1209.196

Neuronal Activity Regulates Hippocampal miRNA Expression

Neuronal activity regulates a broad range of processes in the hippocampus, including the precise regulation of translation. Disruptions in proper translational control in the nervous system are associated with a variety of disorders that fall in the autistic spectrum. MicroRNA (miRNA) represent a relatively recently discovered player in the regulation of translation in the nervous system. We have conducted an in depth analysis of how neuronal activity regulates miRNA expression in the hippocampus. Using deep sequencing we exhaustively identify all miRNAs, including 15 novel miRNAs, expressed in hippocampus of the adult mouse. We identified 119 miRNAs documented in miRBase but less than half of these miRNA were expressed at a level greater than 0.1% of total miRNA. Expression profiling following induction of neuronal activity by electroconvulsive shock demonstrates that most miRNA show a biphasic pattern of expression: rapid induction of specific mature miRNA expression followed by a decline in expression. These results have important implications into how miRNAs influence activity-dependent translational control

Genomic and SNP Analyses Demonstrate a Distant Separation of the Hospital and Community-Associated Clades of Enterococcus faecium

Recent studies have pointed to the existence of two subpopulations of Enterococcus faecium, one containing primarily commensal/community-associated (CA) strains and one that contains most clinical or hospital-associated (HA) strains, including those classified by multi-locus sequence typing (MLST) as belonging to the CC17 group. The HA subpopulation more frequently has IS16, pathogenicity island(s), and plasmids or genes associated with antibiotic resistance, colonization, and/or virulence. Supporting the two clades concept, we previously found a 3–10% difference between four genes from HA-clade strains vs. CA-clade strains, including 5% difference between pbp5-R of ampicillin-resistant, HA strains and pbp5-S of ampicillin-sensitive, CA strains. To further investigate the core genome of these subpopulations, we studied 100 genes from 21 E. faecium genome sequences; our analyses of concatenated sequences, SNPs, and individual genes all identified two distinct groups. With the concatenated sequence, HA-clade strains differed by 0–1% from one another while CA clade strains differed from each other by 0–1.1%, with 3.5–4.2% difference between the two clades. While many strains had a few genes that grouped in one clade with most of their genes in the other clade, one strain had 28% of its genes in the CA clade and 72% in the HA clade, consistent with the predicted role of recombination in the evolution of E. faecium. Using estimates for Escherichia coli, molecular clock calculations using sSNP analysis indicate that these two clades may have diverged ≥1 million years ago or, using the higher mutation rate for Bacillus anthracis, ∼300,000 years ago. These data confirm the existence of two clades of E. faecium and show that the differences between the HA and CA clades occur at the core genomic level and long preceded the modern antibiotic era

Downregulation of miR-92a Is Associated with Aggressive Breast Cancer Features and Increased Tumour Macrophage Infiltration

BACKGROUND: MicroRNAs are small non-coding RNAs involved in the regulation of gene expression on a posttranscriptional level. These regulatory RNAs have been implicated in numerous cellular processes and are further deregulated in different cancer types, including breast cancer. MiR-92a is part of the miR-17∼92 cluster, which was first reported to be linked to tumourigenesis. However, little is known about the expression of miR-92a in breast cancer and potential associations to tumour properties. The expression of miR-92a was therefore characterized in 144 invasive breast cancer samples using in situ hybridization and related to clinico-pathological data as well as to selected key properties of the tumour stroma, including the presence of macrophages (CD68) and cancer activated fibroblasts (alpha-SMA). METHODOLOGY/PRINCIPAL FINDINGS: To measure miR-92a levels, an in situ hybridisation protocol was developed and validated using cell lines and miR-92a inhibitors. The expression in the tumour samples was objectively evaluated using digital image analysis program subtracting background activities. We found that the miR-92a expression varied between tumours and was inversely correlated to tumour grade (r = -0.276, p = 0.003) and recurrence-free survival (p = 0.008) and provided independent prognostic information in multivariate Cox analysis (HR: 0.375, CI: 0.145-0.972, p = 0.043). MiR-92a was moreover inversely correlated to the number of infiltrating macrophages in the tumour stroma (r = -0.357, p<0.001), and downregulation of miR-92a promoted cell migration (p<0.01). CONCLUSIONS/SIGNIFICANCE: This study demonstrates that downregulation of miR-92a in breast cancer is linked to key epithelial and stromal properties as well as clinical outcome

Factors associated with influenza vaccination status of residents of a rural community in Japan

<p>Abstract</p> <p>Background</p> <p>The rate of influenza vaccination in Japan has declined over the past several decades. It is essential to identify community-specific factors that affect attitudes toward vaccination, but such parameters have not yet been fully determined in Japan. The present study used the Health Belief Model (HBM) to identify perceptions of influenza vaccination in a rural Japanese community.</p> <p>Methods</p> <p>All subjects were residents of a rural town in the southern part of Kyoto, Japan. An anonymous self-administered questionnaire was mailed to 846 randomly chosen households (containing 2,665 subjects). The survey explored gender, age, history of influenza, and factors associated with obtaining influenza vaccination, based on the HBM.</p> <p>Results</p> <p>A total of 1,182 valid responses (response rate, 44.4%) were received. Sources of information that were associated with vaccination decisions were medical facilities for children (OR = 4.21; 95% CI: 1.17-15.1), workplaces for adults (OR = 2.40; 95% CI: 1.22-4.75), medical facilities, town office and family for elderly subjects (OR = 6.18; 95% CI: 2.42-15.7, OR = 5.59; 95% CI: 2.26-13.8 and OR = 3.29; 95%CI: 1.01-10.6). Subjects, in all age groups, who strongly agreed that the vaccine was effective were significantly more likely to be vaccinated (OR = 10.5; 95%CI: 2.68-41.7 for children; OR = 8.85; 95%CI: 4.61-16.9 for adults; OR = 19.9; 95%CI: 8.28-48.0 for the elderly). The vaccination rate of elderly subjects who expressed concerns regarding adverse vaccine effects (OR = 0.34, 95% CI: 0.15-0.78) or who were worried about practical barriers to the vaccination process (OR = 0.13; 95% CI: 0.05-0.31) was significantly lower than in other populations.</p> <p>Conclusions</p> <p>Our results indicate that vaccination coverage can be increased if accurate information on personal risk, severity of influenza illness, and efficacy of vaccination are provided by responsible information sources that are easily accessible. Such sources include medical facilities and municipal offices. In addition, barriers and inconveniences associated with vaccination should be removed, especially if they impact on elderly people.</p

Factors Influencing Receipt of Iron Supplementation by Young Children and their Mothers in Rural India: Local and National Cross-Sectional Studies

<p>Abstract</p> <p>Background</p> <p>In India, 55% of women and 69.5% of preschool children are anaemic despite national policies recommending routine iron supplementation. Understanding factors associated with receipt of iron in the field could help optimise implementation of anaemia control policies. Thus, we undertook 1) a cross-sectional study to evaluate iron supplementation to children (and mothers) in rural Karnataka, India, and 2) an analysis of all-India rural data from the National Family Health Study 2005-6 (NFHS-3).</p> <p>Methods</p> <p>All children aged 12-23 months and their mothers served by 6 of 8 randomly selected sub-centres managed by 2 rural Primary Health Centres of rural Karnataka were eligible for the Karnataka Study, conducted between August and October 2008. Socioeconomic and demographic data, access to health services and iron receipt were recorded. Secondly, NFHS-3 rural data were analysed. For both studies, logistic regression was used to evaluate factors associated with receipt of iron.</p> <p>Results</p> <p>The Karnataka Study recruited 405 children and 377 of their mothers. 41.5% of children had received iron, and 11.5% received iron through the public system. By multiple logistic regression, factors associated with children's receipt of iron included: wealth (Odds Ratio (OR) 2.63 [95% CI 1.11, 6.24] for top vs bottom wealth quintile), male sex (OR 2.45 [1.47, 4.10]), mother receiving postnatal iron (OR 2.31 [1.25, 4.28]), mother having undergone antenatal blood test (OR 2.10 [1.09, 4.03]); Muslim religion (OR 0.02 [0.00, 0.27]), attendance at Anganwadi centre (OR 0.23 [0.11, 0.49]), fully vaccinated (OR 0.33 [0.15, 0.75]), or children of mothers with more antenatal health visits (8-9 visits OR 0.25 [0.11, 0.55]) were less likely to receive iron. Nationally, 3.7% of rural children were receiving iron; this was associated with wealth (OR 1.12 [1.02, 1.23] per quintile), maternal education (compared with no education: completed secondary education OR 2.15 [1.17, 3.97], maternal antenatal iron (2.24 [1.56, 3.22]), and child attending an Anganwadi (OR 1.47 [1.20, 1.80]).</p> <p>Conclusion</p> <p>In rural India, public distribution of iron to children is inadequate and disparities exist. Measures to optimize receipt of government supplied iron to all children regardless of wealth and ethnic background could help alleviate anaemia in this population.</p

Search for new phenomena in final states with an energetic jet and large missing transverse momentum in pp collisions at √ s = 8 TeV with the ATLAS detector

Results of a search for new phenomena in final states with an energetic jet and large missing transverse momentum are reported. The search uses 20.3 fb−1 of √ s = 8 TeV data collected in 2012 with the ATLAS detector at the LHC. Events are required to have at least one jet with pT > 120 GeV and no leptons. Nine signal regions are considered with increasing missing transverse momentum requirements between Emiss T > 150 GeV and Emiss T > 700 GeV. Good agreement is observed between the number of events in data and Standard Model expectations. The results are translated into exclusion limits on models with either large extra spatial dimensions, pair production of weakly interacting dark matter candidates, or production of very light gravitinos in a gauge-mediated supersymmetric model. In addition, limits on the production of an invisibly decaying Higgs-like boson leading to similar topologies in the final state are presente