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Heidelberg, Germany

Debbache J.,U.S. National Institutes of Health | Zaidi M.R.,U.S. National Cancer Institute | Davis S.,NCI Inc | Guo T.,U.S. National Cancer Institute | And 8 more authors.
Genetics | Year: 2012

The microphthalmia-associated transcription factor (MITF) is a basic helix-loop-helix leucine zipper protein that plays major roles in the development and physiology of vertebrate melanocytes and melanoma cells. It is regulated by post-translational modifications, including phosphorylation at serine 73, which based on in vitro experiments imparts on MITF an increased transcriptional activity paired with a decreased stability. Serine 73 is encoded by the alternatively spliced exon 2B, which is preferentially skipped in mice carrying a targeted serine-73-to-alanine mutation. Here, we measured the relative abundance of exon 2B+ and exon 2B- RNAs in freshly isolated and FACS-sorted wild-type melanoblasts and melanocytes and generated a series of knock-in mice allowing forced incorporation of either alanine, aspartate, or wild-type serine at position 73. None of these knock-in alleles, however, creates a striking pigmentation phenotype on its own, but differences between them can be revealed either by a general reduction of Mitf transcript levels or in heteroallelic combinations with extant Mitf mutations. In fact, compared with straight serine-73 knock-in mice with their relative reduction of 2B+ Mitf, forced incorporation of alanine 73 leads to greater increases in MITF protein levels, melanoblast and melanocyte numbers, and extent of pigmentation in particular allelic combinations. These results underscore, in vivo, the importance of the link between alternative splicing and post-translational modifications and may bear on the recent observation that exon 2B skipping can be found in metastatic melanoma. © 2012 by the Genetics Society of America.

Furnrohr B.G.,Friedrich - Alexander - University, Erlangen - Nuremberg | Furnrohr B.G.,Kings College London | Furnrohr B.G.,Innsbruck Medical University | Stein M.,Friedrich - Alexander - University, Erlangen - Nuremberg | And 7 more authors.
Journal of Immunology | Year: 2015

Clustering of surface receptors is often required to initiate signal transduction, receptor internalization, and cellular activation. To study the kinetics of clustering, we developed an economic high-throughput method using flow cytometry. The quantification of receptor clustering by flow cytometry is based on the following two observations: first, the fluorescence signal length (FL time-of-flight [ToF]) decreases relative to the forward scatter signal length (FSc-ToF), and second, the peak FL (FL-peak) increases relative to the integral FL (FL-integral) upon clustering of FL-labeled surface receptors. Receptor macroclustering can therefore be quantified using the ratios FL-ToF/FSc-ToF (method ToF) or FL-peak/FL-integral (method Peak). We have used these methods to analyze clustering of two immune receptors known to undergo different conformational and oligomeric states: the BCR and the complement receptor 3 (CR3), on murine splenocytes, purified B cells, and human neutrophils. Engagement of both the BCR and CR3, on immortalized as well as primary murine B cells and human neutrophil, respectively, resulted in decreased FL-ToF/FSc-ToF and increased FL-peak/FLintegral ratios. Manipulation of the actin-myosin cytoskeleton altered BCR clustering which could be measured using the established parameters. To confirm clustering of CR3 on neutrophils, we applied imaging flow cytometry. Because receptor engagement is as a biological process dependent on cell viability, energy metabolism, and temperature, receptor clustering can only be quantified by gating on viable cells under physiological conditions. In summary, with this novel method, receptor clustering on nonadherent cells can easily be monitored by high-throughput conventional flow cytometry. © 2015 by The American Association of Immunologists, Inc.

Murtas D.,U.S. National Institutes of Health | Murtas D.,University of Cagliari | Maric D.,Flow Cytometry Core Facility | De Giorgi V.,U.S. National Institutes of Health | And 19 more authors.
British Journal of Cancer | Year: 2013

Background:Several lines of evidence suggest a dichotomy between immune active and quiescent cancers, with the former associated with a good prognostic phenotype and better responsiveness to immunotherapy. Central to such dichotomy is the master regulator of the acute inflammatory process interferon regulatory factor (IRF)-1. However, it remains unknown whether the responsiveness of IRF-1 to cytokines is able to differentiate cancer immune phenotypes.Methods:IRF-1 activation was measured in 15 melanoma cell lines at basal level and after treatment with IFN-γ, TNF-α and a combination of both. Microarray analysis was used to compare transcriptional patterns between cell lines characterised by high or low IRF-1 activation.Results:We observed a strong positive correlation between IRF-1 activation at basal level and after IFN-γ and TNF-α treatment. Microarray demonstrated that three cell lines with low and three with high IRF-1 inducible translocation scores differed in the expression of 597 transcripts. Functional interpretation analysis showed mTOR and Wnt/β-cathenin as the top downregulated pathways in the cell lines with low inducible IRF-1 activation, suggesting that a low IRF-1 inducibility recapitulates a cancer phenotype already described in literature characterised by poor prognosis.Conclusion:Our findings support the central role of IRF-1 in influencing different tumour phenotypes. © 2013 Cancer Research UK. All rights reserved.

Riddell A.,Flow Cytometry Core Facility | Riddell A.,Cambridge Stem Cell Institute | Gardner R.,Instituto Gulbenkian Of Ciencia | Perez-Gonzalez A.,Flow Cytometry Core Facility | And 3 more authors.
Methods | Year: 2015

Sorting performance can be evaluated with regard to Purity, Yield and/or Recovery of the sorted fraction. Purity is a check on the quality of the sample and the sort decisions made by the instrument. Recovery and Yield definitions vary with some authors regarding both as how efficient the instrument is at sorting the target particles from the original sample, others distinguishing Recovery from Yield, where the former is used to describe the accuracy of the instrument's sort count. Yield and Recovery are often neglected, mostly due to difficulties in their measurement. Purity of the sort product is often cited alone but is not sufficient to evaluate sorting performance. All of these three performance metrics require re-sampling of the sorted fraction. But, unlike Purity, calculating Yield and/or Recovery calls for the absolute counting of particles in the sorted fraction, which may not be feasible, particularly when dealing with rare populations and precious samples. In addition, the counting process itself involves large errors.Here we describe a new metric for evaluating instrument sort Recovery, defined as the number of particles sorted relative to the number of original particles to be sorted. This calculation requires only measuring the ratios of target and non-target populations in the original pre-sort sample and in the waste stream or center stream catch (CSC), avoiding re-sampling the sorted fraction and absolute counting. We called this new metric Rmax, since it corresponds to the maximum expected Recovery for a particular set of instrument parameters. Rmax is ideal to evaluate and troubleshoot the optimum drop-charge delay of the sorter, or any instrument related failures that will affect sort performance. It can be used as a daily quality control check but can be particularly useful to assess instrument performance before single-cell sorting experiments. Because we do not perturb the sort fraction we can calculate Rmax during the sort process, being especially valuable to check instrument performance during rare population sorts. © 2015 The Authors.

Blake J.,Genomics Core Facility | Riddell A.,Flow Cytometry Core Facility | Riddell A.,University of Cambridge | Theiss S.,University of Heidelberg | And 10 more authors.
PLoS ONE | Year: 2014

Balanced chromosome abnormalities (BCAs) occur at a high frequency in healthy and diseased individuals, but cost-efficient strategies to identify BCAs and evaluate whether they contribute to a phenotype have not yet become widespread. Here we apply genome-wide mate-pair library sequencing to characterize structural variation in a patient with unclear neurodevelopmental disease (NDD) and complex de novo BCAs at the karyotype level. Nucleotide-level characterization of the clinically described BCA breakpoints revealed disruption of at least three NDD candidate genes (LINC00299, NUP205, PSMD14) that gave rise to abnormal mRNAs and could be assumed as disease-causing. However, unbiased genome-wide analysis of the sequencing data for cryptic structural variation was key to reveal an additional submicroscopic inversion that truncates the schizophrenia- and bipolar disorder-associated brain transcription factor ZNF804A as an equally likely NDD-driving gene. Deep sequencing of fluorescent-sorted wild-type and derivative chromosomes confirmed the clinically undetected BCA. Moreover, deep sequencing further validated a high accuracy of mate-pair library sequencing to detect structural variants larger than 10 kB, proposing that this approach is powerful for clinical-grade genome-wide structural variant detection. Our study supports previous evidence for a role of ZNF804A in NDD and highlights the need for a more comprehensive assessment of structural variation in karyotypically abnormal individuals and patients with neurocognitive disease to avoid diagnostic deception. © 2014 Blake et al.

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