Molecular Changes in Pre-Metastatic Lymph Nodes of Esophageal Cancer Patients

Lymph node metastasis indicates poor prognosis in esophageal cancer. To understand the underlying mechanisms, most studies so far focused on investigating the tumors themselves and/or invaded lymph nodes. However they neglected the potential events within the metastatic niche, which precede invasion. Here we report the first description of these regulations in patients on transcription level. We determined transcriptomic profiles of still metastasis-free regional lymph nodes for two patient groups: patients classified as pN1 (n = 9, metastatic nodes exist) or pN0 (n = 5, no metastatic nodes exist). All investigated lymph nodes, also those from pN1 patients, were still metastasis-free. The results show that regional lymph nodes of pN1 patients differ decisively from those of pN0 patients--even before metastasis has taken place. In the pN0 group distinct immune response patterns were observed. In contrast, lymph nodes of the pN1 group exhibited a clear profile of reduced immune response and reduced proliferation, but increased apoptosis, enhanced hypoplasia and morphological conversion processes. DKK1 was the most significant gene associated with the molecular mechanisms taking place in lymph nodes of patients suffering from metastasis (pN1). We assume that the two molecular profiles observed constitute different stages of a progressive disease. Finally we suggest that DKK1 might play an important role within the mechanisms leading to lymph node metastasis

Normalization and Statistical Analysis of Multiplexed Bead-Based Immunoassay Data Using Mixed-Effects Modeling

Multiplexed bead-based flow cytometric immunoassays are a powerful experimental tool for investigating cellular communication networks, yet their widespread adoption is limited in part by challenges in robust quantitative analysis of the measurements. Here we report our application of mixed-effects modeling for the normalization and statistical analysis of bead-based immunoassay data. Our data set consisted of bead-based immunoassay measurements of 16 phospho-proteins in lysates of HepG2 cells treated with ligands that regulate acute-phase protein secretion. Mixed-effects modeling provided estimates for the effects of both the technical and biological sources of variance, and normalization was achieved by subtracting the technical effects from the measured values. This approach allowed us to detect ligand effects on signaling with greater precision and sensitivity and to more accurately characterize the HepG2 cell signaling network using constrained fuzzy logic. Mixed-effects modeling analysis of our data was vital for ascertaining that IL-1α and TGF-α treatment increased the activities of more pathways than IL-6 and TNF-α and that TGF-α and TNF-α increased p38 MAPK and c-Jun N-terminal kinase (JNK) phospho-protein levels in a synergistic manner. Moreover, we used mixed-effects modeling-based technical effect estimates to reveal the substantial variance contributed by batch effects along with the absence of loading order and assay plate position effects. We conclude that mixed-effects modeling enabled additional insights to be gained from our data than would otherwise be possible and we discuss how this methodology can play an important role in enhancing the value of experiments employing multiplexed bead-based immunoassays.United States. Army Research Office (Contract W911NF-09-D-0001)National Institutes of Health (U.S.) (NIH P50-GM68762

Identification of differentially expressed microRNAs in human male breast cancer

<p>Abstract</p> <p>Background</p> <p>The discovery of small non-coding RNAs and the subsequent analysis of microRNA expression patterns in human cancer specimens have provided completely new insights into cancer biology. Genetic and epigenetic data indicate oncogenic or tumor suppressor function of these pleiotropic regulators. Therefore, many studies analyzed the expression and function of microRNA in human breast cancer, the most frequent malignancy in females. However, nothing is known so far about microRNA expression in male breast cancer, accounting for approximately 1% of all breast cancer cases.</p> <p>Methods</p> <p>The expression of 319 microRNAs was analyzed in 9 primary human male breast tumors and in epithelial cells from 15 male gynecomastia specimens using fluorescence-labeled bead technology. For identification of differentially expressed microRNAs data were analyzed by cluster analysis and selected statistical methods.</p> <p>Expression levels were validated for the most up- or down-regulated microRNAs in this training cohort using real-time PCR methodology as well as in an independent test cohort comprising 12 cases of human male breast cancer.</p> <p>Results</p> <p>Unsupervised cluster analysis separated very well male breast cancer samples and control specimens according to their microRNA expression pattern indicating cancer-specific alterations of microRNA expression in human male breast cancer. miR-21, miR519d, miR-183, miR-197, and miR-493-5p were identified as most prominently up-regulated, miR-145 and miR-497 as most prominently down-regulated in male breast cancer.</p> <p>Conclusions</p> <p>Male breast cancer displays several differentially expressed microRNAs. Not all of them are shared with breast cancer biopsies from female patients indicating male breast cancer specific alterations of microRNA expression.</p

Organotypic Culture of Physiologically Functional Adult Mammalian Retinas

BACKGROUND: The adult mammalian retina is an important model in research on the central nervous system. Many experiments require the combined use of genetic manipulation, imaging, and electrophysiological recording, which make it desirable to use an in vitro preparation. Unfortunately, the tissue culture of the adult mammalian retina is difficult, mainly because of the high energy consumption of photoreceptors. METHODS AND FINDINGS: We describe an interphase culture system for adult mammalian retina that allows for the expression of genes delivered to retinal neurons by particle-mediated transfer. The retinas retain their morphology and function for up to six days— long enough for the expression of many genes of interest—so that effects upon responses to light and receptive fields could be measured by patch recording or multielectrode array recording. We show that a variety of genes encoding pre- and post-synaptic marker proteins are localized correctly in ganglion and amacrine cells. CONCLUSIONS: In this system the effects on neuronal function of one or several introduced exogenous genes can be studied within intact neural circuitry of adult mammalian retina. This system is flexible enough to be compatible with genetic manipulation, imaging, cell transfection, pharmacological assay, and electrophysiological recordings

Genome-Wide Gene Expression Analysis in Cancer Cells Reveals 3D Growth to Affect ECM and Processes Associated with Cell Adhesion but Not DNA Repair

Cell morphology determines cell behavior, signal transduction, protein-protein interaction, and responsiveness to external stimuli. In cancer, these functions profoundly contribute to resistance mechanisms to radio- and chemotherapy. With regard to this aspect, this study compared the genome wide gene expression in exponentially growing cell lines from different tumor entities, lung carcinoma and squamous cell carcinoma, under more physiological three-dimensional (3D) versus monolayer cell culture conditions. Whole genome cDNA microarray analysis was accomplished using the Affymetrix HG U133 Plus 2.0 gene chip. Significance analysis of microarray (SAM) and t-test analysis revealed significant changes in gene expression profiles of 3D relative to 2D cell culture conditions. These changes affected the extracellular matrix and were mainly associated with biological processes like tissue development, cell adhesion, immune system and defense response in contrast to terms related to DNA repair, which lacked significant alterations. Selected genes were verified by semi-quantitative RT-PCR and Western blotting. Additionally, we show that 3D growth mediates a significant increase in tumor cell radio- and chemoresistance relative to 2D. Our findings show significant gene expression differences between 3D and 2D cell culture systems and indicate that cellular responsiveness to external stress such as ionizing radiation and chemotherapeutics is essentially influenced by differential expression of genes involved in the regulation of integrin signaling, cell shape and cell-cell contact

Effective DNA/RNA Co-Extraction for Analysis of MicroRNAs, mRNAs, and Genomic DNA from Formalin-Fixed Paraffin-Embedded Specimens

Background: Retrospective studies of archived human specimens, with known clinical follow-up, are used to identify predictive and prognostic molecular markers of disease. Due to biochemical differences, however, formalin-fixed paraffinembedded (FFPE) DNA and RNA have generally been extracted separately from either different tissue sections or from the same section by dividing the digested tissue. The former limits accurate correlation whilst the latter is impractical when utilizing rare or limited archived specimens. Principal Findings: For effective recovery of genomic DNA and total RNA from a single FFPE specimen, without splitting the proteinase-K digested tissue solution, we optimized a co-extraction method by using TRIzol and purifying DNA from the lower aqueous and RNA from the upper organic phases. Using a series of seven different archived specimens, we evaluated the total amounts of genomic DNA and total RNA recovered by our TRIzol-based co-extraction method and compared our results with those from two commercial kits, the Qiagen AllPrep DNA/RNA FFPE kit, for co-extraction, and the Ambion RecoverAll TM Total Nucleic Acid Isolation kit, for separate extraction of FFPE-DNA and-RNA. Then, to accurately assess the quality of DNA and RNA co-extracted from a single FFPE specimen, we used qRT-PCR, gene expression profiling and methylation assays to analyze microRNAs, mRNAs, and genomic DNA recovered from matched fresh and FFPE MCF10A cells. These experiments show that the TRIzol-based co-extraction method provides larger amounts of FFPE-DNA and –RNA tha

Establishment and characterization of a new human pancreatic adenocarcinoma cell line with high metastatic potential to the lung

<p>Abstract</p> <p>Background</p> <p>Pancreatic cancer is still associated with devastating prognosis. Real progress in treatment options has still not been achieved. Therefore new models are urgently needed to investigate this deadly disease. As a part of this process we have established and characterized a new human pancreatic cancer cell line.</p> <p>Methods</p> <p>The newly established pancreatic cancer cell line PaCa 5061 was characterized for its morphology, growth rate, chromosomal analysis and mutational analysis of the K-<it>ras</it>, EGFR and p53 genes. Gene-amplification and RNA expression profiles were obtained using an Affymetrix microarray, and overexpression was validated by IHC analysis. Tumorigenicity and spontaneous metastasis formation of PaCa 5061 cells were analyzed in pfp^-/-/rag2^-/-mice. Sensitivity towards chemotherapy was analysed by MTT assay.</p> <p>Results</p> <p>PaCa 5061 cells grew as an adhering monolayer with a doubling time ranging from 30 to 48 hours. M-FISH analyses showed a hypertriploid complex karyotype with multiple numerical and unbalanced structural aberrations. Numerous genes were overexpressed, some of which have previously been implicated in pancreatic adenocarcinoma (GATA6, IGFBP3, IGFBP6), while others were detected for the first time (MEMO1, RIOK3). Specifically highly overexpressed genes (fold change > 10) were identified as EGFR, MUC4, CEACAM1, CEACAM5 and CEACAM6. Subcutaneous transplantation of PaCa 5061 into pfp^-/-/rag2^-/-mice resulted in formation of primary tumors and spontaneous lung metastasis.</p> <p>Conclusion</p> <p>The established PaCa 5061 cell line and its injection into pfp^-/-/rag2^-/-mice can be used as a new model for studying various aspects of the biology of human pancreatic cancer and potential treatment approaches for the disease.</p

Determination and verification of reference interval limits in clinical chemistry. Recommendations for laboratories on behalf of the Working Group Guide Limits of the DGKL with respect to ISO Standard 15189 and the Guideline of the German Medical Association on Quality Assurance in Medical Laboratory Examinations (Rili-BAEK)

Laboratory measurement values require interpretative assistance e.g. so-called guide limits (GL), as an interpretative aid. Legal and normative requirements for medical laboratories do not provide specific information for their implementation and verification. A German Society for Clinical Chemistry and Laboratory Medicine (DGKL) Working Group GL (WG-GL) has, therefore, developed recommendations to support medical laboratories in the management of GL. A specific objective was to create a framework that mainly takes into account those aspects that can be realistically implemented by routine laboratories and that should improve the management of GL of frequently requested quantitative measurement procedures in clinical chemistry. Thus, the focus of these recommendations is on the distinction between reference interval limits and clinical decision limits as well as the determination and verification of reference interval limits. Indirect approaches are highlighted, as they enable routine laboratories with a broad analytical spectrum but limited resources to evaluate or to establish reference limits

Effective local evolutionary searches distributed on an island model solving bi-objective optimization problems

Using multiple local evolutionary searches, instead of single and overall search, has been an effective technique to solve multi-objective optimization problems (MOPs). With this technique, many parallel and distributed multi-objective evolutionary algorithms (dMOEAs) on different island models have been proposed to search for optimal solutions, efficiently and effectively. These algorithms often use local MOEAs on their islands in which each local search is considered to find a part of optimal solutions. The islands (and the local MOEAs), however, need to communicate to each other to preclude the possibility of converging to local optimal solutions. The existing dMOEAs rely on the central and iterative process of subdividing a large-scale population into multiple subpopulations; and it negatively affects the dMOEAs performance. In this paper, a new version of dMOEA with new local MOEAs and migration strategy is proposed. The respective objective space is first subdivided into the predefined number of polar-based regions assigned to the local MOEAs to be explored and exploited. In addition, the central and iterative process is eliminated using a new proposed migration strategy. The algorithms are tested on the standard bi-objective optimization test cases of ZDTs, and the result shows that these new dMOEAs outperform the existing distributed and parallel MOEAs in most cases