1,720,976 research outputs found
Allelic discrimination in the diagnosis of somatic BRAF V600E mutation on fine-needle aspiration biopsies
No full textMany studies demonstrated that somatic BRAF gene mutation analysis increases diagnostic accuracy for papillary thyroid carcinoma (PTC), even from very small samples. The gold standard for point mutations research is direct sequencing, that implies DNA extraction, PCR with specific primers, sequencing reaction and run on an automatic sequencer. This is an expensive and time consuming method, and the possible contamination with wild-type DNA not coming from the nodule significantly reduces sensitivity. Allelic discrimination is a real-time PCR application that can discriminate between two alleles differing for the insertion, substitution or deletion of a single base, due to the presence of two real time Taqman probes, each labelled with a different fluorochrome (FAM for the mutated allele and VIC for the wild-type allele).
The aim of our study is to verify whether allelic discrimination can be useful in the diagnosis of BRAF somatic mutation, starting from fine needle aspiration biopsies (FNAB). In each allelic discrimination reactions three positive controls are present, one for each possible genotype; omozygous controls are oligonucleotides containining the target sequence (wild-type or mutated), whereas the eterozygous control is a 4:1 mix of wild-type omozygous control and mutated omozygous control, respectively, in order to mimic wild-type contamination. To evaluate the method sensitivity, mutated DNA has been diluted in wild-type DNA at variable concentrations (1:2, 1:4, 1:10, 1:20, 1:100) and analyzed both by direct sequencing and allelic discrimination. Allelic discrimination was more sensitive since it detected the presence of mutated DNA in all dilutions, while direct sequencing detected the mutation until 1:20 dilution.
Five hundred FNAB have been analyzed with both methods; allelic discrimination identified 55 V600E mutated samples, while direct sequencing identified only 50 V600E mutated samples. Post surgical histological examination confirmed 54 PTC and one anaplastic carcinoma. In conclusion, allelic discrimination is more sensitive (P<0.05) and more accurate (P<0.05) than direct sequencing, and its use in diagnostic procedures is very useful, even when samples (i.e. FNAB) are contaminated with wild-type DNA
Real-time PCR is useful to detect menin gene deletions
No full textFamilial pituitary adenoma is frequently associated with germinal mutations of several genes, including menin gene. MEN1 syndrome is an autosomic dominant disease, characterized by parathyroid adenomas, endocrine gastroenteropancreatic tumors, and pituitary adenomas, due to inactivating mutations of the MenI gene (11q13). MEN1 mutations are scattered within and around the menin open reading frame and are mainly represented by single nucleotide polymorphisms (SNPs), and small deletions/insertions, which can be detected by genomic DNA direct sequencing. However, heterozygous wide deletions in the menin gene cannot be detected by direct sequencing and other techniques have to be employed to characterize the genetic base of some syndromic families. We employed a real-time PCR application, the TaqMan Copy Number Assay, to evaluate a family in which we failed to identify MEN1 SNPs or deletions/insertions by direct sequencing, despite a clear clinical picture of MEN1 syndrome. By directly evaluating the number of genomic copies by quantitative PCR, we identified a wide deletion of the MEN1 gene involving 50% of exon 1 and 100% of exon 2, in three affected family members, but not in the other seven family members, that are, so far, clinically unaffected. We also evaluated the presence of the same genetic alteration in a group of ten unaffected subject, without family history of endocrine tumors, and none of them displayed exon 1 and 2 deletion. Therefore, this new approach allowed us to correctly diagnose three MEN1 patients that were, so far, considered as MEN1 phenocopies. More importantly, we were able to exclude the presence of any MEN1 genetic alteration in the unaffected family members. These results further underline the importance of the new biotechnology approaches in the diagnosis of genetically determined endocrine diseases
mTOR inhibition influences cell viability of medullary thyroid carcinoma primary cultures
No full textEffective medical therapy for persistent/recurrent medullary thyroid carcinoma (MTC) is not available, yet. Everolimus (RAD001) is a Rapamycin derivative, a potent mTOR pathway inhibitor. RAD001 has been employed in several clinical studies demonstrating antiproliferative and apoptotic effects in human tumors, both in vitro and in vivo, also in combination with somatostatin analogs. The aim of our study was to investigate the antiproliferative effects of RAD001 in human MTC primary cultures. Of 10 MTC have been dispersed in primary culture and incubated for 24 h in culture medium without serum. Cells have been then treated without or with increasing concentrations of RAD001 (10 nM – 1 μM) and/or 10 nM SOM230 (a multiligand somatostatin analog) and/or 50 nM IGF-1. Cell viability has been evaluated after 24 h with a colorimetric method. Somatostatin receptor (SSTR) expression has been evaluated by quantitative PCR. We found that RAD001 10 nM slightly (−17%) but significantly reduces cell viability in five MTC, and that this effect is blocked by co-treatment with IGF-1. SOM230 alone did not modify cell viability but enhanced the antiproliferative effects of RAD001 (−23%). In this group, SSTR2 was the mostly expressed SSTR subtype (72×103 molecules × μg total RNA), followed by SSTR1 (59×103 molecules × μg total RNA), while SSTR3 and SSTR5 were not expressed. Both RAD001 and SOM230 did not affect cell viability in the other five MTC, which expressed SSTR1, SSTR2, SSTR3 and SSTR5 (33, 32, 35, and 16×103 molecules × μg total RNA). These results indicate that Everolimus might represent a possible medical therapy aimed at controlling MTC cell growth in some cases, in association with somatostatin analogs
mTOR inhibition hampers cell viability in selected human medullary thyroid carcinoma primary cultures
No full textIt has been demonstrated that everolimus, an mTOR inhibitor, has potent anti-proliferative effect in a human Medullary Thyroid Carcinoma (MTC) cell line, TT, and in two human MTC primary cultures. We aimed at evaluating the possible antiproliferative effects of everolimus in a group of 20 human MTC in primary culture. To this purpose, 20 MTCs were dispersed in primary cultures, treated without or with 1 nM – 1 μM everolimus, 10 nM SOM230, and/or 50 nM IGF1. Cell viability and apoptosis were evaluated after 48 h and Calcitonin (CT) secretion was assessed after a 8 h incubation. Somatostatin receptor expression was investigated by quantitative PCR. We found that in 14 cultures everolimus reduced cell viability (~40%), promoted apoptosis (+30%), inhibited p70S6K activity (−20%) and blocked IGF1 proliferative and anti-apoptotic effects. In selected tissues co-treatment with SOM230 had additive effects. It did not affect CT secretion, but blocked the stimulatory effects of IGF1. In conclusion, everolimus reduced MTCs cell viability by inducing apoptosis, with a mechanism likely involving IGF1 signalling but not CT secretion, suggesting that it might represent a possible medical treatment for persistent/recurrent MTCs
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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