Beschreibung: Recent advances in genome sequencing technologies provide unprecedented opportunities to characterize individual genomic landscapes and identify mutations relevant for diagnosis and therapy. Specifically, whole-exome sequencing using next-generation sequencing (NGS) technologies is gaining popularity in the human genetics community due to the moderate costs, manageable data amounts and straightforward interpretation of analysis results. While whole-exome and, in the near future, whole-genome sequencing are becoming commodities, data analysis still poses significant challenges and led to the development of a plethora of tools supporting specific parts of the analysis workflow or providing a complete solution. Here, we surveyed 205 tools for whole-genome/whole-exome sequencing data analysis supporting five distinct analytical steps: quality assessment, alignment, variant identification, variant annotation and visualization. We report an overview of the functionality, features and specific requirements of the individual tools. We then selected 32 programs for variant identification, variant annotation and visualization, which were subjected to hands-on evaluation using four data sets: one set of exome data from two patients with a rare disease for testing identification of germline mutations, two cancer data sets for testing variant callers for somatic mutations, copy number variations and structural variations, and one semi-synthetic data set for testing identification of copy number variations. Our comprehensive survey and evaluation of NGS tools provides a valuable guideline for human geneticists working on Mendelian disorders, complex diseases and cancers.

Beschreibung: Assisted reproductive technologies (ART) are associated with several complications including low birth weight, abnormal placentation and increased risk for rare imprinting disorders. Indeed, experimental studies demonstrate ART procedures independent of existing infertility induce epigenetic perturbations in the embryo and extraembryonic tissues. To test the hypothesis that these epigenetic perturbations persist and result in adverse outcomes at term, we assessed placental morphology and methylation profiles in E18.5 mouse concepti generated by in vitro fertilization (IVF) in two different genetic backgrounds. We also examined embryo transfer (ET) and superovulation procedures to ascertain if they contribute to developmental and epigenetic effects. Increased placental weight and reduced fetal-to-placental weight ratio were observed in all ART groups when compared with naturally conceived controls, demonstrating that non-surgical embryo transfer alone can impact placental development. Furthermore, superovulation further induced overgrowth of the placental junctional zone. Embryo transfer and superovulation defects were limited to these morphological changes, as we did not observe any differences in epigenetic profiles. IVF placentae, however, displayed hypomethylation of imprinting control regions of select imprinted genes and a global reduction in DNA methylation levels. Although we did not detect significant differences in DNA methylation in fetal brain or liver samples, rare IVF concepti displayed very low methylation and abnormal gene expression from the normally repressed allele. Our findings suggest that individual ART procedures cumulatively increase placental morphological abnormalities and epigenetic perturbations, potentially causing adverse neonatal and long-term health outcomes in offspring.

Beschreibung: Acute myelogenous leukemia stem cells (AML–LSC) give rise to the leukemic bulk population and maintain disease. Relapse can arise from residual LSCs that have distinct sensitivity and dependencies when compared with the AML bulk. AML–LSCs are driven by genetic and epigenomic changes, and these alterations influence prognosis and clonal selection. Therapies targeting these molecular aberrations have been developed and show promising responses in advanced clinical trials; however, so far success with LSCs has been limited. Besides the genetic diversity, AML–LSCs are critically influenced by the microenvironment, and a third crucial aspect has recently come to the fore: A group of evolutionarily conserved signaling pathways such as canonical Wnt signaling, Notch signaling, or the Hedgehog pathway can be essential for maintenance of AML–LSC but may be redundant for normal hematopoietic stem cells. In addition, early reports suggest also regulators of cell polarity may also influence hematopoietic stem cells and AML biology. Interactions between these pathways have been investigated recently and suggest a network of signaling pathways involved in regulation of self-renewal and response to oncogenic stress. Here, we review how recent discoveries on regulation of AML–LSC-relevant evolutionarily conserved pathways may open opportunities for novel treatment approaches eradicating residual disease. Clin Cancer Res; 21(2); 240–8. ©2015 AACR .

Beschreibung: Plectin, a versatile 500-kDa cytolinker protein, is essential for muscle fiber integrity and function. The most common disease caused by mutations in the human plectin gene, epidermolysis bullosa simplex with muscular dystrophy (EBS-MD), is characterized by severe skin blistering and progressive muscular dystrophy. Besides displaying pathological desmin-positive protein aggregates and degenerative changes in the myofibrillar apparatus, skeletal muscle specimens of EBS-MD patients and plectin-deficient mice are characterized by massive mitochondrial alterations. In this study, we demonstrate that structural and functional alterations of mitochondria are a primary aftermath of plectin deficiency in muscle, contributing to myofiber degeneration. We found that in skeletal muscle of conditional plectin knockout mice (MCK-Cre/cKO), mitochondrial content was reduced, and mitochondria were aggregated in sarcoplasmic and subsarcolemmal regions and were no longer associated with Z-disks. Additionally, decreased mitochondrial citrate synthase activity, respiratory function and altered adenosine diphosphate kinetics were characteristic of plectin-deficient muscles. To analyze a mechanistic link between plectin deficiency and mitochondrial alterations, we comparatively assessed mitochondrial morphology and function in whole muscle and teased muscle fibers of wild-type, MCK-Cre/cKO and plectin isoform-specific knockout mice that were lacking just one isoform (either P1b or P1d) while expressing all others. Monitoring morphological alterations of mitochondria, an isoform P1b-specific phenotype affecting the mitochondrial fusion–fission machinery and manifesting with upregulated mitochondrial fusion-associated protein mitofusin-2 could be identified. Our results show that the depletion of distinct plectin isoforms affects mitochondrial network organization and function in different ways.

Beschreibung: Plumbagin (PL), 5-hydroxy-2-methyl-1,4-naphthoquinone, is a quinoid constituent isolated from the roots of the medicinal plant Plumbago zeylanica L. (also known as chitrak). PL has also been found in Juglans regia (English Walnut), Juglans cinerea (whitenut) and Juglans nigra (blacknut). The roots of P. zeylanica have been used in Indian and Chinese systems of medicine for more than 2500 years for the treatment of various types of ailments. We were the first to report that PL inhibits the growth and invasion of hormone refractory prostate cancer (PCa) cells [Aziz,M.H. et al . (2008) Plumbagin, a medicinal plant-derived naphthoquinone, is a novel inhibitor of the growth and invasion of hormone-refractory prostate cancer. Cancer Res., 68, 9024–9032.]. Now, we present that PL inhibits in vivo PCa development in the transgenic adenocarcinoma of mouse prostate (TRAMP). PL treatment (2mg/kg body weight i.p. in 0.2ml phosphate-buffered saline, 5 days a week) to FVB–TRAMP resulted in a significant ( P 〈 0.01) decrease in prostate tumor size and urogenital apparatus weights at 13 and 20 weeks. Histopathological analysis revealed that PL treatment inhibited progression of prostatic intraepithelial neoplasia (PIN) to poorly differentiated carcinoma (PDC). No animal exhibited diffuse tumor formation in PL-treated group at 13 weeks, whereas 75% of the vehicle-treated mice elicited diffuse PIN and large PDC at this stage. At 20 weeks, 25% of the PL-treated animals demonstrated diffuse PIN and 75% developed small PDC, whereas 100% of the vehicle-treated mice showed large PDC. PL treatment inhibited expression of protein kinase C epsilon (PKC), signal transducers and activators of transcription 3 phosphorylation, proliferating cell nuclear antigen and neuroendocrine markers (synaptophysin and chromogranin-A) in excised prostate tumor tissues. Taken together, these results further suggest PL could be a novel chemopreventive agent against PCa.

Beschreibung: The reconstruction of the history of rearrangements and the reconstruction of ancestral genomes are some of the challenges of bioinformatics today. Many algorithms already exist, treating one or the other question but none treating both. These reconstructions are interdependent and we argue on the interest of treating both problems in parallel to lead to a richer and more complete output. We also argue on the importance of redefining several steps of these algorithms to improve both reconstructions: the identification of synteny blocks has to be as precise as possible, and the treatment of multiple genomes has to be based on pairwise comparisons to ensure the most detailed reconstructions. In this article, we highlight novel solutions to these points and focus on the need of explicitly treating overlapping, included, duplicated and unsigned synteny blocks. To do so, we introduce the new notion of synteny pack , which is a representation of local hypothetical intermediate ancestral genomes. We discuss a number of examples on yeast genomes to illustrate the importance of such a definition.

Beschreibung: The transcription factors Gli2 (glioma-associated factor 2), which is a transactivator of Sonic Hedgehog (Shh) signalling, and myocyte enhancer factor 2C (MEF2C) play important roles in the development of embryonic heart muscle and enhance cardiomyogenesis in stem cells. Although the physiological importance of Shh signalling and MEF2 factors in heart development is well known, the mechanistic understanding of their roles is unclear. Here, we demonstrate that Gli2 and MEF2C activated each other's expression while enhancing cardiomyogenesis in differentiating P19 EC cells. Furthermore, dominant-negative mutant proteins of either Gli2 or MEF2C repressed each other's expression, while impairing cardiomyogenesis in P19 EC cells. In addition, chromatin immunoprecipitation (ChIP) revealed association of Gli2 to the Mef2c gene, and of MEF2C to the Gli2 gene in differentiating P19 cells. Finally, co-immunoprecipitation studies showed that Gli2 and MEF2C proteins formed a complex, capable of synergizing on cardiomyogenesis-related promoters containing both Gli- and MEF2-binding elements. We propose a model whereby Gli2 and MEF2C bind each other's regulatory elements, activate each other's expression and form a protein complex that synergistically activates transcription, enhancing cardiac muscle development. This model links Shh signalling to MEF2C function during cardiomyogenesis and offers mechanistic insight into their in vivo functions.

Beschreibung: Purpose: The phosphoinositide 3-kinase (PI3K) pathway is fundamental for cell proliferation and survival and is frequently altered and activated in neoplasia, including carcinomas of the lung. In this study, we investigated the potential of targeting the catalytic class I A PI3K isoforms in small cell lung cancer (SCLC), which is the most aggressive of all lung cancer types. Experimental Design: The expression of PI3K isoforms in patient specimens was analyzed. The effects on SCLC cell survival and downstream signaling were determined following PI3K isoform inhibition by selective inhibitors or downregulation by siRNA. Results: Overexpression of the PI3K isoforms p110-α and p110-β and the antiapoptotic protein Bcl-2 was shown by immunohistochemistry in primary SCLC tissue samples. Targeting the PI3K p110-α with RNA interference or selective pharmacologic inhibitors resulted in strongly affected cell proliferation of SCLC cells in vitro and in vivo , whereas targeting p110-β was less effective. Inhibition of p110-α also resulted in increased apoptosis and autophagy, which was accompanied by decreased phosphorylation of Akt and components of the mTOR pathway, such as the ribosomal S6 protein, and the eukaryotic translation initiation factor 4E-binding protein 1. A DNA microarray analysis revealed that p110-α inhibition profoundly affected the balance of pro- and antiapoptotic Bcl-2 family proteins. Finally, p110-α inhibition led to impaired SCLC tumor formation and vascularization in vivo . Conclusion: Together our data show the key involvement of the PI3K isoform p110-α in the regulation of multiple tumor-promoting processes in SCLC. Clin Cancer Res; 19(1); 96–105. ©2012 AACR .

Beschreibung: In human tumors, and in mouse models, cyclooxygenase-2 (COX-2) levels are frequently correlated with tumor development/burden. In addition to intrinsic tumor cell expression, COX-2 is often present in fibroblasts, myofibroblasts and endothelial cells of the tumor microenvironment, and in infiltrating immune cells. Intrinsic cancer cell COX-2 expression is postulated as only one of many sources for prostanoids required for tumor promotion/progression. Although both COX-2 inhibition and global Cox-2 gene deletion ameliorate ultraviolet B (UVB)-induced SKH-1 mouse skin tumorigenesis, neither manipulation can elucidate the cell type(s) in which COX-2 expression is required for tumorigenesis; both eliminate COX-2 activity in all cells. To address this question, we created Cox-2 flox/flox mice, in which the Cox-2 gene can be eliminated in a cell-type-specific fashion by targeted Cre recombinase expression. Cox-2 deletion in skin epithelial cells of SKH-1 Cox-2 flox/flox ; K14Cre + mice resulted, following UVB irradiation, in reduced skin hyperplasia and increased apoptosis. Targeted epithelial cell Cox-2 deletion also resulted in reduced tumor incidence, frequency, size and proliferation rate, altered tumor cell differentiation and reduced tumor vascularization. Moreover, Cox-2 flox/flox ; K14Cre + papillomas did not progress to squamous cell carcinomas. In contrast, Cox-2 deletion in SKH-1 Cox-2 flox/flox ; LysMCre + myeloid cells had no effect on UVB tumor induction. We conclude that (i) intrinsic epithelial COX-2 activity plays a major role in UVB-induced skin cancer, (ii) macrophage/myeloid COX-2 plays no role in UVB-induced skin cancer and (iii) either there may be another COX-2-dependent prostanoid source(s) that drives UVB skin tumor induction or there may exist a COX-2-independent pathway(s) to UVB-induced skin cancer.