d-matrix – database exploration, visualization and analysis

BACKGROUND: Motivated by a biomedical database set up by our group, we aimed to develop a generic database front-end with embedded knowledge discovery and analysis features. A major focus was the human-oriented representation of the data and the enabling of a closed circle of data query, exploration, visualization and analysis. RESULTS: We introduce a non-task-specific database front-end with a new visualization strategy and built-in analysis features, so called d-matrix. d-matrix is web-based and compatible with a broad range of database management systems. The graphical outcome consists of boxes whose colors show the quality of the underlying information and, as the name suggests, they are arranged in matrices. The granularity of the data display allows consequent drill-down. Furthermore, d-matrix offers context-sensitive categorization, hierarchical sorting and statistical analysis. CONCLUSIONS: d-matrix enables data mining, with a high level of interactivity between humans and computer as a primary factor. We believe that the presented strategy can be very effective in general and especially useful for the integration of distinct data types such as phenotypical and molecular data

In silico identification of a core regulatory network of OCT4 in human embryonic stem cells using an integrated approach

<p>Abstract</p> <p>Background</p> <p>The transcription factor OCT4 is highly expressed in pluripotent embryonic stem cells which are derived from the inner cell mass of mammalian blastocysts. Pluripotency and self renewal are controlled by a transcription regulatory network governed by the transcription factors OCT4, SOX2 and NANOG. Recent studies on reprogramming somatic cells to induced pluripotent stem cells highlight OCT4 as a key regulator of pluripotency.</p> <p>Results</p> <p>We have carried out an integrated analysis of high-throughput data (ChIP-on-chip and RNAi experiments along with promoter sequence analysis of putative target genes) and identified a core OCT4 regulatory network in human embryonic stem cells consisting of 33 target genes. Enrichment analysis with these target genes revealed that this integrative analysis increases the functional information content by factors of 1.3 – 4.7 compared to the individual studies. In order to identify potential regulatory co-factors of OCT4, we performed a <it>de novo </it>motif analysis. In addition to known validated OCT4 motifs we obtained binding sites similar to motifs recognized by further regulators of pluripotency and development; e.g. the heterodimer of the transcription factors C-MYC and MAX, a prerequisite for C-MYC transcriptional activity that leads to cell growth and proliferation.</p> <p>Conclusion</p> <p>Our analysis shows how heterogeneous functional information can be integrated in order to reconstruct gene regulatory networks. As a test case we identified a core OCT4-regulated network that is important for the analysis of stem cell characteristics and cellular differentiation. Functional information is largely enriched using different experimental results. The <it>de novo </it>motif discovery identified well-known regulators closely connected to the OCT4 network as well as potential new regulators of pluripotency and differentiation. These results provide the basis for further targeted functional studies.</p

Adaptive nanopores: A bioinspired label-free approach for protein sequencing and identification

AbstractSingle molecule protein sequencing would tremendously impact in proteomics and human biology and it would promote the development of novel diagnostic and therapeutic approaches. However, its technological realization can only be envisioned, and huge challenges need to be overcome. Major difficulties are inherent to the structure of proteins, which are composed by several different amino-acids. Despite long standing efforts, only few complex techniques, such as Edman degradation, liquid chromatography and mass spectroscopy, make protein sequencing possible. Unfortunately, these techniques present significant limitations in terms of amount of sample required and dynamic range of measurement. It is known that proteins can distinguish closely similar molecules. Moreover, several proteins can work as biological nanopores in order to perform single molecule detection and sequencing. Unfortunately, while DNA sequencing by means of nanopores is demonstrated, very few examples of nanopores able to perform reliable protein-sequencing have been reported so far. Here, we investigate, by means of molecular dynamics simulations, how a re-engineered protein, acting as biological nanopore, can be used to recognize the sequence of a translocating peptide by sensing the "shape" of individual amino-acids. In our simulations we demonstrate that it is possible to discriminate with high fidelity, 9 different amino-acids in a short peptide translocating through the engineered construct. The method, here shown for fluorescence-based sequencing, does not require any labelling of the peptidic analyte. These results can pave the way for a new and highly sensitive method of sequencing

No evidence for a shift in pyruvate kinase PKM1 to PKM2 expression during tumorigenesis

The Warburg effect describes the circumstance that tumor cells preferentially use glycolysis rather than oxidative phosphorylation for energy production. It has been reported that this metabolic reconfiguration originates from a switch in the expression of alternative splice forms (PKM1 and PKM2) of the glycolytic enzyme pyruvate kinase (PK), which is also important for malignant transformation. However, analytical evidence for this assumption was still lacking. Using mass spectrometry, we performed an absolute quantification of PKM1 and PKM2 splice isoforms in 25 human malignant cancers, 6 benign oncocytomas, tissue matched controls, and several cell lines. PKM2 was the prominent isoform in all analyzed cancer samples and cell lines. However, this PKM2 dominance was not a result of a change in isoform expression, since PKM2 was also the predominant PKM isoform in matched control tissues. In unaffected kidney, lung, liver, and thyroid, PKM2 accounted for a minimum of 93% of total PKM, for 80% - 96% of PKM in colon, and 55% - 61% of PKM in bladder. Similar results were obtained for a panel of tumor and non-transformed cell lines, where PKM2 was the predominant form. Thus, our results reveal that an exchange in PKM1 to PKM2 isoform expression during cancer formation is not occurring, nor do these results support conclusions that PKM2 is specific for proliferating, and PKM1 for non-proliferating tissue

Comparing Three Approaches

Hybridization-based target enrichment protocols require relatively large starting amounts of genomic DNA, which is not always available. Here, we tested three approaches to pre-capture library preparation starting from 10 ng of genomic DNA: (i and ii) whole-genome amplification of DNA samples with REPLI-g (Qiagen) and GenomePlex (Sigma) kits followed by standard library preparation, and (iii) library construction with a low input oriented ThruPLEX kit (Rubicon Genomics). Exome capture with Agilent SureSelectXT2 Human AllExon v4+UTRs capture probes, and HiSeq2000 sequencing were performed for test libraries along with the control library prepared from 1 µg of starting DNA. Tested protocols were characterized in terms of mapping efficiency, enrichment ratio, coverage of the target region, and reliability of SNP genotyping. REPLI-g- and ThruPLEX-FD-based protocols seem to be adequate solutions for exome sequencing of low input sample

Cell array-based intracellular localization screening reveals novel functional features of human chromosome 21 proteins

BACKGROUND: Trisomy of human chromosome 21 (Chr21) results in Down's syndrome, a complex developmental and neurodegenerative disease. Molecular analysis of Down's syndrome, however, poses a particular challenge, because the aneuploid region of Chr21 contains many genes of unknown function. Subcellular localization of human Chr21 proteins may contribute to further understanding of the functions and regulatory mechanisms of the genes that code for these proteins. Following this idea, we used a transfected-cell array technique to perform a rapid and cost-effective analysis of the intracellular distribution of Chr 21 proteins. RESULTS: We chose 89 genes that were distributed over the majority of 21q, ranging from RBM11 (14.5 Mb) to MCM3AP (46.6 Mb), with part of them expressed aberrantly in the Down's syndrome mouse model. Open reading frames of these genes were cloned into a mammalian expression vector with an amino-terminal His(6 )tag. All of the constructs were arrayed on glass slides and reverse transfected into HEK293T cells for protein expression. Co-localization detection using a set of organelle markers was carried out for each Chr21 protein. Here, we report the subcellular localization properties of 52 proteins. For 34 of these proteins, their localization is described for the first time. Furthermore, the alteration in cell morphology and growth as a result of protein over-expression for claudin-8 and claudin-14 genes has been characterized. CONCLUSION: The cell array-based protein expression and detection approach is a cost-effective platform for large-scale functional analyses, including protein subcellular localization and cell phenotype screening. The results from this study reveal novel functional features of human Chr21 proteins, which should contribute to further understanding of the molecular pathology of Down's syndrome

Identification of novel transcriptional regulators involved in macrophage differentiation and activation in U937 cells

Background Monocytes and macrophages play essential role in innate immunity. Understanding the underlying mechanism of macrophage differentiation and the identification of regulatory mechanisms will help to find new strategies to prevent their harmful effects in chronic inflammatory diseases and sepsis. Results Maturation of blood monocytes into tissue macrophages and subsequent inflammatory response was mimicked in U937 cells of human histocytic lymphoma origin. Whole genome array analysis was employed to evaluate gene expression profile to identify underlying transcriptional networks implicated during the processes of differentiation and inflammation. In addition to already known transcription factors (i.e. MAFB, EGR, IRF, BCL6, NFkB, AP1, Nur77), gene expression analysis further revealed novel genes (i.e. MEF2, BRI, HLX, HDAC5, H2AV, TCF7L2, NFIL3) previously uncharacterized to be involved in the differentiation process. A total of 58 selected genes representing cytokines, chemokines, surface antigens, signaling molecules and transcription factors were validated by real time PCR and compared to primary monocyte-derived macrophages. Beside the verification of several new genes, the comparison reveals individual heterogeneity of blood donors. Conclusion Up regulation of MEF2 family, HDACs, and H2AV during cell differentiation and inflammation sheds new lights onto regulation events on transcriptional and epigenetic level controlling these processes. Data generated will serve as a source for further investigation of macrophages differentiation pathways and related biological responses