Mutation positive breast cancer sample analyzed using a tiling 32 K BAC array and an Agilent 244 K oligonucleotide CGH array

<p><b>Copyright information:</b></p><p>Taken from "Normalization of array-CGH data: influence of copy number imbalances"</p><p>http://www.biomedcentral.com/1471-2164/8/382</p><p>BMC Genomics 2007;8():382-382.</p><p>Published online 22 Oct 2007</p><p>PMCID:PMC2190775.</p><p></p> Correction lines for Median (orange), Lowess (green), and popLowess (red) normalization are superimposed in panels (e) and (f). Identified copy number populations are differentially colored in panels (g) and (h) according to size where yellow corresponds to the largest identified copy number population, red to the second largest, and green to the smallest. Data in panels (g) and (h) are centered on the middle population Genome plot of un-normalized BAC data. Genome plot of un-normalized Agilent data. M-A plot of un-normalized BAC data. M-A plot of un-normalized Agilent data. Contour plot of copy number population enriched BAC data. Contour plot of copy number population enriched Agilent data. Genome plot of BAC data after popLowess. Genome plot of Agilent data after popLowess

Example of segmentation of data after alternative normalization methods

<p><b>Copyright information:</b></p><p>Taken from "Normalization of array-CGH data: influence of copy number imbalances"</p><p>http://www.biomedcentral.com/1471-2164/8/382</p><p>BMC Genomics 2007;8():382-382.</p><p>Published online 22 Oct 2007</p><p>PMCID:PMC2190775.</p><p></p> Segmented copy number profile of chromosome 4 from smoothed (50 kBp) Agilent data for the sample from figure 7 with superimposed adaptive thresholds (± 0.171). Three different segments are highlighted with colored arrows in each panel to exemplify regions with different copy number level Segmentation applied after Lowess. M values for selected segments; blue arrow 0.16, red arrow 1.02, and green arrow -0.36. Segmentation applied after popLowess. M values for selected segments; blue arrow -0.07, red arrow 0.94, and green arrow -0.64.Segmentation applied after Lowess normalization subsequently followed by centralization of data on median M of an individual population. M values for selected segments; blue arrow -0.05, red arrow 0.81, and green arrow -0.57

Cluster analysis of C (blue), N (red) and CN (grey) expression profiles where all samples were median-centred and treated as they were independent experiments

<p><b>Copyright information:</b></p><p>Taken from "Non-coding antisense transcription detected by conventional and single-stranded cDNA microarray"</p><p>http://www.biomedcentral.com/1471-2164/8/295</p><p>BMC Genomics 2007;8():295-295.</p><p>Published online 29 Aug 2007</p><p>PMCID:PMC2020490.</p><p></p> Minimum presence per cDNA clone was set to 7 out of 9 in each data set. Histogram of pair-wise Euclidean distances over all samples for IMAGE clones. For each IMAGE in 9 dimensions, i.e. hybridization 1–9, the Euclidean distance between C and N is √(Σi = 1 (C-N)) where Cand Nis the log2ratio for C and N probe respectively for hybridization i. Histogram of distances between C and N probes for all IMAGE for each hybridization

Strand-specific end modifications (amino-linkers) are incorporated into DNA by two parallel PCR reactions

<p><b>Copyright information:</b></p><p>Taken from "Non-coding antisense transcription detected by conventional and single-stranded cDNA microarray"</p><p>http://www.biomedcentral.com/1471-2164/8/295</p><p>BMC Genomics 2007;8():295-295.</p><p>Published online 29 Aug 2007</p><p>PMCID:PMC2020490.</p><p></p> After clean-up, the PCR product for each end-modified strand is printed separately onto the same microarray glass slide. The amino terminal groups are further coupled to the glass. The probes are digested with 5'-3' T7 gene 6 exonuclease and then immersed in boiling water. As a result of this treatment, only end-modifed strands remain attached to the surface. Hybridization of labelled transcribed RNA from the β-lactamase gene with β-lactamase single-stranded sense and antisense DNA capture probes. Control spots containing ds-DNA probes or amino-modified PCR primers alone were also included on the array (not shown). Both probes and controls were spotted in 10 × 10 replicates. Scatter plot showing the distribution of raw median pixel signal intensities from the hybridization performed in (b). Signals from all replicates are clearly discriminated according to the strand from which they originated (sense or antisense). Control spots for background intensities (printing buffer, PCR and amplification primers) demonstrate the specificity of the single stranded probes

Table_1_FGF/FGFR1 system in paired breast tumor-adjacent and tumor tissues, associations with mammographic breast density and tumor characteristics.docx

IntroductionMammographic breast density (MBD) is an established breast cancer risk factor, yet the underlying molecular mechanisms remain to be deciphered. Fibroblast growth factor receptor 1 (FGFR1) amplification is associated with breast cancer development and aberrant FGF signaling found in the biological processes related to both high mammographic density and breast cancer microenvironment. The aim of this study was to investigate the FGF/FGFR1 expression in-between paired tumor-adjacent and tumor tissues from the same patient, and its associations with MBD and tumor characteristics.MethodsFGFR1 expression in paired tissues from 426 breast cancer patients participating in the Karolinska Mammography Project for Risk Prediction of Breast Cancer (KARMA) cohort study was analyzed by immunohistochemistry. FGF ligand expression was obtained from RNA-sequencing data for 327 of the included patients.ResultsFGFR1 levels were differently expressed in tumor-adjacent and tumor tissues, with increased FGFR1 levels detected in 58% of the tumors. High FGFR1 expression in tumor tissues was associated with less favorable tumor characteristics; high histological grade (OR=1.86, 95% CI 1.00–3.44), high Ki67 proliferative index (OR=2.18, 95% CI 1.18–4.02) as well as tumors of Luminal B-like subtype (OR=2.56, 95%CI 1.29–5.06). While no clear association between FGFR1 expression and MBD was found, FGF ligand (FGF1, FGF11, FGF18) expression was positively correlated with MBD.DiscussionTaken together, these findings support a role of the FGF/FGFR1 system in early breast cancer which warrants further investigation in the MBD–breast cancer context.</p

Robotic table football - position detection of opponent

The aim of this thesis is design of the sensing device to detect the positions of the four independent human axes robotic table soccer and create an output program applicable to game strategy. In the introduction is comparison of previously developed solutions. Next part is the comparison and selection of appropriate sensors applicable to the design table. For specific solutions then select a suitable module for integration into the control system and then test the functionality of the system. Implementation part of the proposal includes the design of construction for detection and next includes all necessary upgrades in the construction of the machine ware to commissioning

IGV view (Integrative Genomics Viewer) showing the enrichment of AGO-CLIP sequencing reads in miR-4728-3p transfected vs non-transfected control around the IS target site on the 3′UTR of the Ubiquitin carboxyl-terminal hydrolase 1 (USP1) gene.

<p>The positions of the 7- and 6-mer IS target sites are highlighted below the USP1 gene.</p

miR-4728-3p IS regulates ESR1.

<p><b>A.</b> qRT-PCR analysis of ESR1 and HER2 transcripts and miR-4728-3p among a panel of 38 breast cancer tumors (19 HER2+, 19 HER2-). Calibrated Normalized Relative Quantity (CNRQ) of miR-4728-3p (left) and HER2 (right) is plotted against expression levels of ESR1. Tumors classified as HER2+ by ISH are shown in red, HER2- in grey. Expression was normalized to a panel of reference genes. For details see text and material and methods. <b>B.</b> Luciferase assay in BT-474 with ESR1 3′UTR constructs carrying either wild type target site of miR-4728-3p internal seed (WT) or mutated internal seed site (MUT). Firefly luciferase activity was normalized against Renilla luciferase. Reporter activity is given as % of WT in respective experiment. Repression of WT ESR1 construct by endogenous miR-4728-3p (left) is alleviated by an antisense oligo (AS) against endogenous miRNA (right) but not by a non-targeting control (middle). <b>C.</b> Western blot (left) and protein quantification (right) of ESR1 in MCF7. The two main isoforms of ESR1 (47 and 66 kDa), plotted as percentage of control signal of matching size, are down regulated upon transfection of miR-4728-3p mimics. Levels of HER2, (p)MAPK and (p)AKT remain largely unchanged. <b>D.</b> MCF7 cells were transfected with indicated concentrations of miR-4728-3p mimic. ESR1 levels show a concentration-dependent down-regulation that is most pronounced at highest tested concentration (25 nM). <b>E.</b> Western blot (left) and protein quantification (right) of ESR1 in BT474. ESR1 is up regulated when blocking endogenous miR-4728-3p with AS-oligonucleotides, while pMAPK and pAKT remain largely unchanged. <b>F.</b> Western blot (left) and protein quantification (right) of ESR1 in HCC1954 cells. ESR1 isoform of 47 kDa is up regulated under miR-4728-3p blocking. The main 66 kDa isoform is not detectable in this ER- cell line. Signals were quantified with ImageJ and normalized to total protein by Coomassie stain. Tubulin was used as a loading control. Asterisks denote p-values of <0.05 (*), and <0.005 (**) in Student′s t-test.</p

IS targeting is not restricted to miR-4728-3p.

<p>SylArray enrichment landscape from a microarray experiment of miR-30a overexpression in HCT116 cells. Data generated by Baraniskin <i>et al.</i> was analyzed by SylArray. Word designations and graph details as in Fig. 1a, with CS and IS referring to corresponding miR-30a positions.</p

The main isoform of miR-4728-3p is 24-26nt long and interacts with its targets through an internal seed.

<p><b>A.</b> SylArray enrichment landscape plots for 6-, 7- and 8-mer words (from top to bottom) for ranked genes from a microarray experiment of miR-4728-3p overexpression in MCF 10A. 3′ UTRs are sorted on the x-axis from most down (left) to up regulated (right). Significance of the words is given as log-transformed P-values, where over- and underrepresentation are shown on the positive and negative y axis, respectively. Highlighted are targets corresponding to canonical seed (CS, red), CS shifted by one base towards the miRNA'3′ end (CS+1, green), internal seed (IS, blue) and IS +1 (purple). CS+1 is below significance cut-off and it is not highlighted for 8-mer. <b>B.</b> Northern blot of total RNA from cells A) untransfected and B) transfected with miR-4728-3p mimics with a radiolabeled miR-4728-3p probe. Control lane shows signal from synthetic miR-4728-3p RNA and the ethidium bromide staining in the lower panel shows tRNA bands as loading control. <b>C.</b> Alignment of small RNA sequencing reads to miR-4728-3p genomic context. Positions of CS, CS+1, IS, and IS+1 are highlighted in color as in panel A above the alignment. Sequencing reads of mir-4728 in cell lines with endogenous expression (BT-474, JIMT1 and SKBR3), mimic-transfected MCF 10A (Mimic) and tumors are given to the right as percentages of total miR-4728-3p reads. <b>D.</b> Empirical cumulative distribution functions (ECDF) show effectiveness of CS and IS target sites. mRNA abundance after miRNA transfection in MCF 10A was monitored with microarrays. Distributions of changes for 3′ UTRs of mRNAs containing CS, IS, both, or neither are colored as denoted on the right. Each class contains the seed and the respective shifted seed (+1). <b>E.</b> Conservation plot. Top 250 down regulated genes from a microarray experiment of miR-4728-3p overexpression were filtered for IS and CS target sites respectively. Target sequence context of 10 bases on either side of the seeds was extracted and analyzed with multiple sequence alignment. The IS conservation plot shows conservation of the target site but not of surrounding nucleotides. <b>F.</b> Distribution of duplex energies of 3' UTRs containing IS target sites comparing genes down regulated by miR-4728-3p overexpression (t-statistics of <-4, TRUE) with unaffected genes (t-statistics ≥4, FALSE). Thermodynamic stability of hybrid formation between targets and miR-4728-3p was calculated using RNAduplex from the Vienna package. RNA duplex score is shown on the x-axis while target density is expressed on the y-axis. Folding stability is higher in a small number of regulated targets but otherwise similar to non-regulated targets, and only one motif with high duplex stability was part of a longer motif (10nt).</p