Analysis of Tyrosine Kinase Inhibitor-Mediated Decline in Contractile Force in Rat Engineered Heart Tissue

Introduction Left ventricular dysfunction is a frequent and potentially severe side effect of many tyrosine kinase inhibitors (TKI). The mode of toxicity is not identified, but may include impairment of mitochondrial or sarcomeric function, autophagy or angiogenesis, either as an on-target or off-target mechanism. Methods and Results We studied concentration-response curves and time courses for nine TKIs in three-dimensional, force generating engineered heart tissue (EHT) from neonatal rat heart cells. We detected a concentration- and time-dependent decline in contractile force for gefitinib, lapatinib, sunitinib, imatinib, sorafenib, vandetanib and lestaurtinib and no decline in contractile force for erlotinib and dasatinib after 96 hours of incubation. The decline in contractile force was associated with an impairment of autophagy (LC3 Western blot) and appearance of autophagolysosomes (transmission electron microscopy). Conclusion This study demonstrates the feasibility to study TKI-mediated force effects in EHTs and identifies an association between a decline in contractility and inhibition of autophagic flux

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

Helium poisoning: new procedure for sampling and analysis.

An increasing number of suicidal asphyxiation with a plastic bag with inert gases, and in particular helium (He), have been reported from numerous countries over the last decade. These cases are differently managed and lead to different and variable interpretations. Based on the 12 last cases analysed in the laboratory and on the review of the most recent literature about this topic, updated autopsy guidelines for sampling have been proposed regarding to the samples choice and analytical challenges required by the gaseous state of this substance. Biological samples from airways (lungs lobe) followed by brain and cardiac blood are the best matrices to take during the autopsy to diagnose He exposure. Gaseous samples from trachea, pulmonary bronchi, gastric and cardiac areas are also recommended as alternative samples. The anatomical site of sampling must be carefully detailed, and to this end, forensic imaging constitutes a beneficial tool. Even if He detection is sufficient to conclude to He exposure, He concentrations in samples may be related to He exposure conditions (duration, breathing rate, etc.). A quantification in biological samples could be helpful to document more precisely the case. He concentrations in gaseous samples are reported up to 6.0 μmol/mL (tracheal gas), 2.4 μmol/mL (pulmonary gas), 0.64 μmol/mL (cardiac gas) and 12 μmol/mL (gastric gas). He concentrations in solid/liquid samples are reported up to 28 μmol/g (lungs) and 0.03 μmol/g (cardiac blood). The other matrices usually sampled during autopsy such as urine, peripheral blood, liver, fat matter and kidney appear as not relevant

Comparison of evolutionary algorithms in gene regulatory network model inference

Background: The evolution of high throughput technologies that measure gene expression levels has created a data base for inferring GRNs (a process also known as reverse engineering of GRNs). However, the nature of these data has made this process very di±cult. At the moment, several methods of discovering qualitative causal relationships between genes with high accuracy from microarray data exist, but large scale quantitative analysis on real biological datasets cannot be performed, to date, as existing approaches are not suitable for real microarray data which are noisy and insu±cient. Results: This paper performs an analysis of several existing evolutionary algorithms for quantitative gene regulatory network modelling. The aim is to present the techniques used and o®er a comprehensive comparison of approaches, under a common framework. Algorithms are applied to both synthetic and real gene expression data from DNA microarrays, and ability to reproduce biological behaviour, scalability and robustness to noise are assessed and compared. Conclusions: Presented is a comparison framework for assessment of evolutionary algorithms, used to infer gene regulatory networks. Promising methods are identi¯ed and a platform for development of appropriate model formalisms is established

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

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

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