The combination of transcriptomics and informatics identifies pathways targeted by miR-204 during neurogenesis and axon guidance

Vertebrate organogenesis is critically sensitive to gene dosage and even subtle variations in the expression levels of key genes may result in a variety of tissue anomalies. MicroRNAs (miRNAs) are fundamental regulators of gene expression and their role in vertebrate tissue patterning is just beginning to be elucidated. To gain further insight into this issue, we analysed the transcriptomic consequences of manipulating the expression of miR-204 in the Medaka fish model system. We used RNA-Seq and an innovative bioinformatics approach, which combines conventional differential expression analysis with the behavior expected by miR-204 targets after its overexpression and knockdown. With this approach combined with a correlative analysis of the putative targets, we identified a wider set of miR-204 target genes belonging to different pathways. Together, these approaches confirmed that miR-204 has a key role in eye development and further highlighted its putative function in neural differentiation processes, including axon guidance as supported by in vivo functional studies. Together, our results demonstrate the advantage of integrating next-generation sequencing and bioinformatics approaches to investigate miRNA biology and provide new important information on the role of miRNAs in the control of axon guidance and more broadly in nervous system development. \uc2\ua9 The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research

Rapamycin inhibits doxorubicin-induced NF-kappaB/Rel nuclear activity and enhances apoptosis in melanoma.

Inhibition of NF-kB/Rel sensitizes many tumor cells to death-inducing stimuli including chemotherapeutic agents and there are findings that disruption of NF-kB may be of therapeutic interest in melanoma. We found that rapamycin sensitized a human melanoma cell line, estabilished by a patient, to the cytolitic effect of doxorubicin. Doxorubicin is a striking NF-kB/Rel-inducer, thus we investigated if rapamycin interfered with the pathway of NF-kB/Rel activation, i.e. IkBa-phosphorylation, –degradation and NF-kB/Rel nuclear translocation, and found that the macrolide agent caused a block of IKK kinase activity responsible for reduced nuclear translocation of the transcription factors. The western blotting analysis of Bcl-2 and c-IAP1 showed increased levels of these anti-apoptotic proteins in cells incubated with doxorubicin, in accord to NF-kB/Rel activation, while rapamycin clearly downmodulated them, in line with the pro-apoptotic ability. The effect of the macrolide on NF-kB/Rel induction appeared to be independent of the block of the PI3k/Akt pathway, because it could not be reproduced by the PI3k inhibitor wortmannin. Very recently the immunophilin FKBP51 has been shown to be essential for the function of IKK kinase. We found high levels of expression of FKBP51 in melanoma cells, moreover we confirmed the involvement of this immunophilin in the control of IKK activity. Indeed apparently IkBa could not be degraded when FKBP51 was downmodulated by siRNAs. These findings provide a possible mechanism of NF-kB downmodulation by rapamycin, since the macrolide agent specifically inhibits FKBP51 isomerase activity. In conclusion our study demonstrated that rapamycin blocked NF-kB/Rel activation in a way independent of PI3k/Akt inhibition suggesting that the macrolide agent could sinergize with NF-kB-inducer anti cancer-drugs also in PTEN positive tumors

Rapamycin stimulates apoptosis of childhood acute lymphoblastic leukemia cells

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Contrast minimization with the new-generation dyevert plus system for contrast reduction and real-time monitoring during coronary and peripheral procedures. First experience

BACKGROUND: Several strategies have been envisioned to reduce the risk of contrast-induced nephropathy, but the most modifiable approach for a treating physician is to minimize contrast administration. To date, there is no report on the use of Osprey Medical's new-generation DyeVert Plus system in coronary or peripheral applications. We aimed to appraise the role of the DyeVert Plus system inclusive of contrast reduction and real-time monitoring in a consecutive series of patients undergoing coronary or peripheral invasive procedures. METHODS: Baseline, procedural, and outcome details for patients undergoing coronary or peripheral invasive procedures were collected from our institutional database. We primarily focused on total and relative amount of contrast saved, as calculated and displayed by the DyeVert Plus system. RESULTS: The DyeVert Plus system was used in 10 patients. All procedures were successfully completed with adequate and high-quality angioscopic and angiographic images. No adverse events occurred up to discharge in any patients, with the notable exception of 1 case of asymptomatic and uneventful contrast-induced nephropathy. Average contrast volume was 79.9 ± 48.8 mL (95% confidence interval [CI], 53.2 to 109.4), thanks to an absolute saving of 55.8 ± 31.9 mL (95% CI, 39.1 to 76.7; P<.05) and a relative saving of 41.8 ± 7.3% (95% CI, 37.5 to 46.4; P<.05). Comparison of contrast volume estimates between DyeVert Plus vs manual measurements showed a minimal difference of 1.6 ± 1.9 mL (95% CI, 2.9 to 0.5; P<.05). CONCLUSION: Use of the new-generation DyeVert Plus system inclusive of contrast reduction and real-time monitoring is feasible in both coronary and peripheral applications while significantly reducing contrast volume

MicroRNA target prediction by expression analysis of host genes

MicroRNAs (miRNAs) are small noncoding RNAs that control gene expression by inducing RNA cleavage or translational inhibition. Most human miRNAs are intragenic and are transcribed as part of their hosting transcription units. We hypothesized that the expression profiles of miRNA host genes and of their targets are inversely correlated and devised a novel procedure, HOCTAR (host gene oppositely correlated targets), which ranks predicted miRNA target genes based on their anti-correlated expression behavior relative to their respective miRNA host genes. HOCTAR is the first tool for systematic miRNA target prediction that utilizes the same set of microarray experiments to monitor the expression of both miRNAs (through their host genes) and candidate targets. We applied the procedure to 178 human intragenic miRNAs and found that it performs better than currently available prediction softwares in pinpointing previously validated miRNA targets. The high-scoring HOCTAR predicted targets were enriched in Gene Ontology categories, which were consistent with previously published data, as in the case of miR-106b and miR-93. By means of overexpression and loss-of-function assays, we also demonstrated that HOCTAR is efficient in predicting novel miRNA targets and we identified, by microarray and qRT-PCR procedures, 34 and 28 novel targets for miR-26b and miR-98, respectively. Overall, we believe that the use of HOCTAR significantly reduces the number of candidate miRNA targets to be tested compared to the procedures based solely on target sequence recognition. Finally, our data further confirm that miRNAs have a significant impact on the mRNA levels of most of their targets

Tirofiban counteracts endothelial cell apoptosis through the VEGF/VEGFR2/pAkt axis

Tirofiban is used in the treatment of patients with acute coronary syndrome submitted to percutaneous coronary intervention (PCI). We have, previously, shown that tirofiban stimulates VEGF expression and promotes proliferation of endothelial cells. VEGF is a well known inhibitor of endothelial cell apoptosis. TNF-α is a pro-apoptotic cytokine released in the site of a vascular injury, including balloon angioplasty. We thought to investigate whether tirofiban was able to protect endothelial cells from cell death induced by TNF-α. For this study, we used human umbilical vein endothelial cells (HUVEC). Analysis of apoptosis was performed by propidium iodide incorporation, annexin V staining and measure of active caspase 3 levels. Western blot served for a semiquantitative measure of Akt activation, VEGF, and the pro-apoptotic Bim and Bak. Our results show that TNF-α was unable to activate caspase 3 and produce cell death in the presence of tirofiban. Activation of apoptosis was preceded by upregulation of Bim and Bak that resulted decreased after addition of tirofiban. The anti-apoptosis effect of tirofiban was reproduced by VEGF and counteracted by VEGFR2 blockade and the cation chelating agent ethylene glycol tetraacetic acid (EGTA). The use of p-Akt inhibitor, BEZ235,and Akt knockdown, suggested that pAkt mediated the prosurvival effect of tirofiban. In conclusion, tirofiban protects endothelial cells from apoptosis stimulated by TNF-α, due to its ability to stimulate VEGF production

Safety and Efficacy of A Single Embolic Protection Device-Stent Combo for Carotid Revascularization

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Safety and Efficacy of A Single Embolic Protection Device-Stent Combo for Carotid Revascularization

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