DNA fragments binding CTCF in vitro and in vivo are capable of blocking enhancer activity

<p>Abstract</p> <p>Background</p> <p>Earlier we identified ten 100-300-bp long CTCF-binding DNA fragments selected earlier from a 1-Mb human chromosome 19 region. Here the positive-negative selection technique was used to check the ability of CTCF-binding human genomic fragments to block enhancer-promoter interaction when inserted into the genome.</p> <p>Results</p> <p>Ten CTCF-binding DNA fragments were inserted between the CMV enhancer and CMV minimal promoter driving the herpes simplex virus thymidine kinase (HSV<it>-tk</it>) gene in a vector expressing also the <it>neo</it>^Rgene under a separate promoter. The constructs were then integrated into the genome of CHO cells, and the cells resistant to neomycin and ganciclovir (positive-negative selection) were picked up, and their DNAs were PCR analyzed to confirm the presence of the fragments between the enhancer and promoter in both orientations.</p> <p>Conclusions</p> <p>We demonstrated that all sequences identified by their CTCF binding both <it>in vitro </it>and <it>in vivo </it>had enhancer-blocking activity when inserted between the CMV minimal promoter and enhancer in stably transfected CHO cells.</p

The validation service of the hydrological SAF geostationary and polar satellite precipitation products

Abstract. The development phase (DP) of the EUMETSAT Satellite Application Facility for Support to Operational Hydrology and Water Management (H-SAF) led to the design and implementation of several precipitation products, after 5 yr (2005–2010) of activity. Presently, five precipitation estimation algorithms based on data from passive microwave and infrared sensors, on board geostationary and sun-synchronous platforms, function in operational mode at the H-SAF hosting institute to provide near real-time precipitation products at different spatial and temporal resolutions. In order to evaluate the precipitation product accuracy, a validation activity has been established since the beginning of the project. A Precipitation Product Validation Group (PPVG) works in parallel with the development of the estimation algorithms with two aims: to provide the algorithm developers with indications to refine algorithms and products, and to evaluate the error structure to be associated with the operational products. In this paper, the framework of the PPVG is presented: (a) the characteristics of the ground reference data available to H-SAF (i.e. radar and rain gauge networks), (b) the agreed upon validation strategy settled among the eight European countries participating in the PPVG, and (c) the steps of the validation procedures. The quality of the reference data is discussed, and the efforts for its improvement are outlined, with special emphasis on the definition of a ground radar quality map and on the implementation of a suitable rain gauge interpolation algorithm. The work done during the H-SAF development phase has led the PPVG to converge into a common validation procedure among the members, taking advantage of the experience acquired by each one of them in the validation of H-SAF products. The methodology is presented here, indicating the main steps of the validation procedure (ground data quality control, spatial interpolation, up-scaling of radar data vs. satellite grid, statistical score evaluation, case study analysis). Finally, an overview of the results is presented, focusing on the monthly statistical indicators, referred to the satellite product performances over different seasons and areas

A comprehensive overview of radioguided surgery using gamma detection probe technology

The concept of radioguided surgery, which was first developed some 60 years ago, involves the use of a radiation detection probe system for the intraoperative detection of radionuclides. The use of gamma detection probe technology in radioguided surgery has tremendously expanded and has evolved into what is now considered an established discipline within the practice of surgery, revolutionizing the surgical management of many malignancies, including breast cancer, melanoma, and colorectal cancer, as well as the surgical management of parathyroid disease. The impact of radioguided surgery on the surgical management of cancer patients includes providing vital and real-time information to the surgeon regarding the location and extent of disease, as well as regarding the assessment of surgical resection margins. Additionally, it has allowed the surgeon to minimize the surgical invasiveness of many diagnostic and therapeutic procedures, while still maintaining maximum benefit to the cancer patient. In the current review, we have attempted to comprehensively evaluate the history, technical aspects, and clinical applications of radioguided surgery using gamma detection probe technology

Toward a Rationale for the PTC124 (Ataluren) Promoted Readthrough of Premature Stop Codons: A Computational Approach and GFP-Reporter Cell-Based Assay

The presence in the mRNA of premature stop codons (PTCs) results in protein truncation responsible for several inherited (genetic) diseases. A well-known example of these diseases is cystic fibrosis (CF), where approximately 10% (worldwide) of patients have nonsense mutations in the CF transmembrane regulator (CFTR) gene. PTC124 (3-(5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl)-benzoic acid), also known as Ataluren, is a small molecule that has been suggested to allow PTC readthrough even though its target has yet to be identified. In the lack of a general consensus about its mechanism of action, we experimentally tested the ability of PTC124 to promote the readthrough of premature termination codons by using a new reporter. The reporter vector was based on a plasmid harboring the H2B histone coding sequence fused in frame with the green fluorescent protein (GFP) cDNA, and a TGA stop codon was introduced in the H2B-GFP gene by site-directed mutagenesis. Additionally, an unprecedented computational study on the putative supramolecular interaction between PTC124 and an 11-codon (33-nucleotides) sequence corresponding to a CFTR mRNA fragment containing a central UGA nonsense mutation showed a specific interaction between PTC124 and the UGA codon. Altogether, the H2B-GFP-opal based assay and the molecular dynamics (MD) simulation support the hypothesis that PTC124 is able to promote the specific readthrough of internal TGA premature stop codons