A simple way to evaluate self-designed probes for tumor specific Multiplex Ligation-dependent Probe Amplification (MLPA)

<p>Abstract</p> <p>Background</p> <p>The Multiplex Ligation-dependent Probe Amplification (MLPA) is widely used for analysis of copy number variations (CNVs) in single or multiple loci. MLPA is a versatile methodology and important tool in cancer research; it provides precise information on increased or decreased copy number at specific loci as opposed to loss of heterozygosity (LOH) studies based upon microsatellite analysis. Pre-designed MLPA kits and software are commercially available to analyze multiple exons, genes, and genomic regions. However, an increasing demand for new gene specific assays makes it necessary to self-design new MLPA probes for which the available software may not be applicable. During evaluation of new self-designed reference probes, we encountered a number of problems, especially when applying the MLPA methodology to tumor samples.</p> <p>Findings</p> <p>DNA samples from 48 unaffected individuals and 145 breast cancer patients were used to evaluate 11 self-designed MLPA probes and determine the cut-off values for CNV, before applying the MLPA probes to normalize the target probes in a cohort of affected individuals. To test the calculation strategy, three probes were designed to cover regions in Regulator of G-protein Signaling 8 (<it>RGS8</it>), which we previously have identified as being affected by allelic imbalance by LOH analysis across <it>RGS8 </it>in the cohort comprising 145 breast tumors. Agreement between the LOH results and the results obtained by each of the three MLPA probes in <it>RGS8 </it>was found for 64%, 73%, and 91%, of the analyzed samples, respectively.</p> <p>Conclusion</p> <p>Here, we present a straightforward method, based upon the normalization pattern in both unaffected and affected individuals, to evaluate self-designed reference probes and to calculate CNV for the MLPA assay with specific focus on the difficulties when analyzing tumor DNA.</p

Дія свинцю (П) в сублетальній концентрації на вміст білкових і кислоторозчинних тіолів та білку в печінці полівки сірої

Induced pluripotent stem cells (iPS) have become crucial in medicine and biology. Several studies indicate their phenotypic similarities with cancer stem cells (CSCs) and a propensity to form tumors. Thus it is desirable to identify a trait which differentiates iPS populations and CSCs. Searching for such a feature, in this work we compare the restriction (R) point-governed regulation of cell cycle progression in different cell types (iPS, cancer, CSC and normal cells) based on the expression profile of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase3 (PFKFB3) and phosphofructokinase (PFK1). Our study reveals that PFKFB3 and PFK1 expression allows discrimination between iPS and CSCs. Moreover, cancer and iPS cells, when cultured under hypoxic conditions, alter their expression level of PFKFB3 and PFK1 to resemble those in CSCs. We also observed cell type-related differences in response to inhibition of PFKFB3. This possibility to distinguish CSC from iPS cells or non-stem cancer cells by PFKB3 and PFK1 expression improves the outlook for clinical application of stem cell-based therapies and for more precise detection of CSCs

Deposition in the inner and outer corners of the JET divertor with carbon wall and metallic ITER-like wall

Rotating collectors and quartz microbalances (QMBs) are used in JET to provide time-dependent measurements of erosion and deposition. Rotation of collector discs behind apertures allows recording of the long term evolution of deposition. QMBs measure mass change via the frequency deviations of vibrating quartz crystals. These diagnostics are used to investigate erosion/deposition during JET-C carbon operation and JET-ILW (ITER-like wall) beryllium/ tungsten operation. A simple geometrical model utilising experimental data is used to model the time-dependent collector deposition profiles, demonstrating good qualitative agreement with experimental results. Overall, the JET-ILW collector deposition is reduced by an order of magnitude relative to JET-C, with beryllium replacing carbon as the dominant deposit. However, contrary to JET-C, in JET-ILW there is more deposition on the outer collector than the inner. This reversal of deposition asymmetry is investigated using an analysis of QMB data and is attributed to the different chemical properties of carbon and beryllium.EURATOM 63305

Overview of the JET ITER-like wall divertor

The work presented draws on new analysis of components removed following the second JET ITER-like wall campaign 2013–14 concentrating on the upper inner divertor, inner and outer divertor corners, life- time issues relating to tungsten coatings on JET carbon fibre composite divertor tiles and dust/particulate generation. The results show that the upper inner divertor remains the region of highest deposition in the JET-ILW. Variations in plasma configurations between the first and second campaign have altered ma- terial migration to the corners of the inner and outer divertor. Net deposition is shown to be beneficial in the sense that it reduces W coating erosion, covers small areas of exposed carbon surfaces and even encapsulates particles.EURATOM 633053RCUK Energy Programme EP/I50104

Cancer-Associated Fibroblasts Modulate Transcriptional Signatures Involved in Proliferation, Differentiation and Metastasis in Head and Neck Squamous Cell Carcinoma

Cancer-associated fibroblasts (CAFs) are known to increase tumor growth and to stimulate invasion and metastasis. Increasing evidence suggests that CAFs mediate response to various treatments. HNSCC cell lines were co-cultured with their patient-matched CAFs in 2D and 3D in vitro models, and the tumor cell gene expression profiles were investigated by cDNA microarray and qRT-PCR. The mRNA expression of eight candidate genes was examined in tumor biopsies from 32 HNSCC patients and in five biopsies from normal oral tissue. Differences in overall survival (OS) were tested with Kaplan–Meier long-rank analysis. Thirteen protein coding genes were found to be differentially expressed in tumor cells co-cultured with CAFs in 2D and 81 in 3D when compared to tumor cells cultured without CAFs. Six of these genes were upregulated both in 2D and 3D (POSTN, GREM1, BGN, COL1A2, COL6A3, and COL1A1). Moreover, two genes upregulated in 3D, MMP9 and FMOD, were significantly associated with the OS. In conclusion, we demonstrated in vitro that CAF-derived signals alter the tumor cell expression of multiple genes, several of which are associated with differentiation, epithelial-to-mesenchymal transition (EMT) phenotype, and metastasis. Moreover, six of the most highly upregulated genes were found to be overexpressed in tumor tissue compared to normal tissue

Cancer-Associated Fibroblasts Modulate Transcriptional Signatures Involved in Proliferation, Differentiation and Metastasis in Head and Neck Squamous Cell Carcinoma

Cancer-associated fibroblasts (CAFs) are known to increase tumor growth and to stimulate invasion and metastasis. Increasing evidence suggests that CAFs mediate response to various treatments. HNSCC cell lines were co-cultured with their patient-matched CAFs in 2D and 3D in vitro models, and the tumor cell gene expression profiles were investigated by cDNA microarray and qRT-PCR. The mRNA expression of eight candidate genes was examined in tumor biopsies from 32 HNSCC patients and in five biopsies from normal oral tissue. Differences in overall survival (OS) were tested with Kaplan–Meier long-rank analysis. Thirteen protein coding genes were found to be differentially expressed in tumor cells co-cultured with CAFs in 2D and 81 in 3D when compared to tumor cells cultured without CAFs. Six of these genes were upregulated both in 2D and 3D (POSTN, GREM1, BGN, COL1A2, COL6A3, and COL1A1). Moreover, two genes upregulated in 3D, MMP9 and FMOD, were significantly associated with the OS. In conclusion, we demonstrated in vitro that CAF-derived signals alter the tumor cell expression of multiple genes, several of which are associated with differentiation, epithelial-to-mesenchymal transition (EMT) phenotype, and metastasis. Moreover, six of the most highly upregulated genes were found to be overexpressed in tumor tissue compared to normal tissue

Beryllium migration in JET ITER-like wall plasmas

JET is used as a test bed for ITER, to investigate beryllium migration which connects the lifetime of first-wall components under erosion with tokamak safety, in relation to long-term fuel retention. The (i) limiter and the (ii) divertor configurations have been studied in JET-ILW (JET with a Be first wall and W divertor), and compared with those for the former JET-C (JET with carbon-based plasma-facing components (PFCs)). (i) For the limiter configuration, the Be gross erosion at the contact point was determined in situ by spectroscopy as between 4% (E-in = 35 eV) and more than 100%, caused by Be self-sputtering (E-in = 200 eV). Chemically assisted physical sputtering via BeD release has been identified to contribute to the effective Be sputtering yield, i.e. at E-in = 75 eV, erosion was enhanced by about 1/3 with respect to the bare physical sputtering case. An effective gross yield of 10% is on average representative for limiter plasma conditions, whereas a factor of 2 difference between the gross erosion and net erosion, determined by post-mortem analysis, was found. The primary impurity source in the limiter configuration in JET-ILW is only 25% higher (in weight) than that for the JET-C case. The main fraction of eroded Be stays within the main chamber. (ii) For the divertor configuration, neutral Be and BeD from physically and chemically assisted physical sputtering by charge exchange neutrals and residual ion flux at the recessed wall enter the plasma, ionize and are transported by scrape-off layer flows towards the inner divertor where significant net deposition takes place. The amount of Be eroded at the first wall (21 g) and the Be amount deposited in the inner divertor (28 g) are in fair agreement, though the balancing is as yet incomplete due to the limited analysis of PFCs. The primary impurity source in the JET-ILW is a factor of 5.3 less in comparison with that for JET-C, resulting in lower divertor material deposition, by more than one order of magnitude. Within the divertor, Be performs far fewer re-erosion and transport steps than C due to an energetic threshold for Be sputtering, and inhibits as a result of this the transport to the divertor floor and the pump duct entrance. The target plates in the JET-ILW inner divertor represent at the strike line a permanent net erosion zone, in contrast to the net deposition zone in JET-C with thick carbon deposits on the CFC (carbon-fibre composite) plates. The Be migration identified is consistent with the observed low long-term fuel retention and dust production with the JET-ILW.EURATOM 63305

Assessment of erosion, deposition and fuel retention in the JET-ILW divertor from ion beam analysis data

Proceedings of the 22nd International Conference on Plasma Surface Interactions 2016, 22nd PSI.Post-mortem analyses of individual components provide relevant information on plasma-surface interactions like tungsten erosion, beryllium deposition and plasma fuel retention with divertor tiles via implantation or co-deposition. Ion Beam techniques are ideal tools for such purposes and have been extensively used for post-mortem analyses of selected tiles from JET following each campaign. In this contribution results from tiles removed from the JET ITER-Like Wall (JET-ILW) divertor following the 2013–2014 campaign are presented. The results summarize erosion, deposition and fuel retention along the poloidal cross section of the divertor surface and provide data for comparison with the first JET-ILW campaign, showing a similar pattern of material migration with the exception of Tile 6 where the strike point time on the tile was ∼ 4 times longer in 2013–2014 than in 2011–2012, which is likely to account for more material migration to this region. The W deposition on top of the Mo marker coating of Tile 4 shows that the enrichment takes place at the strike point location.Peer reviewe

Investigation of deuterium trapping and release in the JET divertor during the third ILW campaign using TDS

Selected set of samples from JET ITER-Like Wall (JET-ILW) divertor tiles exposed in 2015–2016 has been analysed using Thermal Desorption Spectrometry (TDS). The deuterium (D) amounts obtained with TDS were compared with Nuclear Reaction Analysis (NRA). The highest amount of D was found on the top part of inner divertor which has regions with the thickest deposited layers as for divertor tiles removed in 2014. This area resides deep in the scrape-off layer and plasma configurations for the second (ILW-2, 2013–2014) and the third (ILW-3, 2015–2016) JET-ILW campaigns were similar. Agreement between TDS and NRA is good on the apron of Tile 1 and on the upper vertical region whereas on the lower vertical region of Tile 1 the NRA results are clearly smaller than the TDS results. Inner divertor Tile 3 has somewhat less D than Tiles 0 and 1, and the D amount decreases towards the lower part of the tile. The D retention at the divertor inner and outer corner regions is not symmetric as there is more D retention poloidally at the inner than at the outer divertor corner. In most cases the TDS spectra for the ILW-3 samples are different from the corresponding ILW-2 spectra because HD and D2 release occurs at higher temperatures than from the ILW-2 samples indicating that the low energy traps have been emptied during the plasma operations and that D is either in the energetically deep traps or located deeper in the sample.EURATOM 63305

Deposition of impurity metals during campaigns with the JET ITER-like Wall

Post mortem analysis shows that mid and high atomic number metallic impurities are present in deposits on JET plasma facing components with the highest amount of Ni and W, and therefore the largest sink, being found at the top of the inner divertor. Sources are defined as "continuous" or "specific", in that "continuous" sources arise from ongoing erosion from plasma facing surfaces and specific" are linked with specific events which decrease over time until they no longer act as a source. This contribution evaluates the sinks and estimates sources and the balance gives an indication of the dominating processes. Charge exchange neutral erosion is found to be the main source of nickel, whereas erosion of divertor plasma facing components is the main source of tungsten. Specific sources are shown to have little influence over the global mid- and high-Z impurity concentrations in deposits.EURATOM 633053RCUK Energy Programme EP/I50104