Linking crowdsourced observations with INSPIRE

Ponencias, comunicaciones y pósters presentados en el 17th AGILE Conference on Geographic Information Science "Connecting a Digital Europe through Location and Place", celebrado en la Universitat Jaume I del 3 al 6 de junio de 2014.The combination of spatial data from the variety of sources on the web, being either legislative, commercially or voluntarily driven, is a major requirement for the establishment of a fully integrated geospatial web. Therefore, spatial data fusion techniques need to be linked to current web-developments, in particular on Spatial Data Infrastructures and the Semantic Web, to allow for standardized and effective use of combined spatial data for information retrieval. In this paper, crowdsourced environmental observations, representing the rapidly increasing amount of voluntarily collected data on the web, and INSPIRE, acting as the legal framework for spatial environmental information in Europe, are chosen to design and develop capabilities for spatial data fusion on the web. Possible use cases show mutual benefits for both volunteers and INSPIRE data providers, and might thus facilitate further collaboration. A prototypical implementation based on common SDI and Linked Data standards demonstrates the feasibility of the proposed approach and offers a starting point for further exploration

Integrating systems biology with clinical research

A report on the conference 'Systems Genomics 2008', Heidelberg, Germany, 2-3 May 2008

Alternative pre-mRNA processing regulates cell-type specific expression of the IL4l1 and NUP62 genes

BACKGROUND: Given the complexity of higher organisms, the number of genes encoded by their genomes is surprisingly small. Tissue specific regulation of expression and splicing are major factors enhancing the number of the encoded products. Commonly these mechanisms are intragenic and affect only one gene. RESULTS: Here we provide evidence that the IL4I1 gene is specifically transcribed from the apparent promoter of the upstream NUP62 gene, and that the first two exons of NUP62 are also contained in the novel IL4I1_2 variant. While expression of IL4I1 driven from its previously described promoter is found mostly in B cells, the expression driven by the NUP62 promoter is restricted to cells in testis (Sertoli cells) and in the brain (e.g., Purkinje cells). Since NUP62 is itself ubiquitously expressed, the IL4I1_2 variant likely derives from cell type specific alternative pre-mRNA processing. CONCLUSION: Comparative genomics suggest that the promoter upstream of the NUP62 gene originally belonged to the IL4I1 gene and was later acquired by NUP62 via insertion of a retroposon. Since both genes are apparently essential, the promoter had to serve two genes afterwards. Expression of the IL4I1 gene from the "NUP62" promoter and the tissue specific involvement of the pre-mRNA processing machinery to regulate expression of two unrelated proteins indicate a novel mechanism of gene regulation

SMART amplification combined with cDNA size fractionation in order to obtain large full-length clones

BACKGROUND: cDNA libraries are widely used to identify genes and splice variants, and as a physical resource for full-length clones. Conventionally-generated cDNA libraries contain a high percentage of 5'-truncated clones. Current library construction methods that enrich for full-length mRNA are laborious, and involve several enzymatic steps performed on mRNA, which renders them sensitive to RNA degradation. The SMART technique for full-length enrichment is robust but results in limited cDNA insert size of the library. RESULTS: We describe a method to construct SMART full-length enriched cDNA libraries with large insert sizes. Sub-libraries were generated from size-fractionated cDNA with an average insert size of up to seven kb. The percentage of full-length clones was calculated for different size ranges from BLAST results of over 12,000 5'ESTs. CONCLUSIONS: The presented technique is suitable to generate full-length enriched cDNA libraries with large average insert sizes in a straightforward and robust way. The representation of full-coding clones is high also for large cDNAs (70%, 4–10 kb), when high-quality starting mRNA is used

The 3of5 web application for complex and comprehensive pattern matching in protein sequences

BACKGROUND: The identification of patterns in biological sequences is a key challenge in genome analysis and in proteomics. Frequently such patterns are complex and highly variable, especially in protein sequences. They are frequently described using terms of regular expressions (RegEx) because of the user-friendly terminology. Limitations arise for queries with the increasing complexity of patterns and are accompanied by requirements for enhanced capabilities. This is especially true for patterns containing ambiguous characters and positions and/or length ambiguities. RESULTS: We have implemented the 3of5 web application in order to enable complex pattern matching in protein sequences. 3of5 is named after a special use of its main feature, the novel n-of-m pattern type. This feature allows for an extensive specification of variable patterns where the individual elements may vary in their position, order, and content within a defined stretch of sequence. The number of distinct elements can be constrained by operators, and individual characters may be excluded. The n-of-m pattern type can be combined with common regular expression terms and thus also allows for a comprehensive description of complex patterns. 3of5 increases the fidelity of pattern matching and finds ALL possible solutions in protein sequences in cases of length-ambiguous patterns instead of simply reporting the longest or shortest hits. Grouping and combined search for patterns provides a hierarchical arrangement of larger patterns sets. The algorithm is implemented as internet application and freely accessible. The application is available at . CONCLUSION: The 3of5 application offers an extended vocabulary for the definition of search patterns and thus allows the user to comprehensively specify and identify peptide patterns with variable elements. The n-of-m pattern type offers an improved accuracy for pattern matching in combination with the ability to find all solutions, without compromising the user friendliness of regular expression terms

Automated production of recombinant human proteins as resource for proteome research

<p>Abstract</p> <p>Background</p> <p>An arbitrary set of 96 human proteins was selected and tested to set-up a fully automated protein production strategy, covering all steps from DNA preparation to protein purification and analysis. The target proteins are encoded by functionally uncharacterized open reading frames (ORF) identified by the German cDNA consortium. Fusion proteins were produced in <it>E. coli </it>with four different fusion tags and tested in five different purification strategies depending on the respective fusion tag. The automated strategy relies on standard liquid handling and clone picking equipment.</p> <p>Results</p> <p>A robust automated strategy for the production of recombinant human proteins in <it>E. coli </it>was established based on a set of four different protein expression vectors resulting in NusA/His, MBP/His, GST and His-tagged proteins. The yield of soluble fusion protein was correlated with the induction temperature and the respective fusion tag. NusA/His and MBP/His fusion proteins are best expressed at low temperature (25°C), whereas the yield of soluble GST fusion proteins was higher when protein expression was induced at elevated temperature. In contrast, the induction of soluble His-tagged fusion proteins was independent of the temperature. Amylose was not found useful for affinity-purification of MBP/His fusion proteins in a high-throughput setting, and metal chelating chromatography is recommended instead.</p> <p>Conclusion</p> <p>Soluble fusion proteins can be produced in <it>E. coli </it>in sufficient qualities and μg/ml culture quantities for downstream applications like microarray-based assays, and studies on protein-protein interactions employing a fully automated protein expression and purification strategy. Future applications might include the optimization of experimental conditions for the large-scale production of soluble recombinant proteins from libraries of open reading frames.</p

Liquid-phase electron microscopy of molecular drug response in breast cancer cells reveals irresponsive cell subpopulations related to lack of HER2 homodimers

The development of drug resistance in cancer poses a major clinical problem. An example is human epidermal growth factor receptor 2 (HER2) overexpressing breast cancer often treated with anti-HER2 antibody therapies, such as trastuzumab. Because drug resistance is rooted mainly in tumor cell heterogeneity, we examined the drug effect in different subpopulations of SKBR3 breast cancer cells and compared the results with those of a drugresistant cell line, HCC1954. Correlative light microscopy and liquid-phase scanning transmission electron microscopy were used to quantitatively analyze HER2 responses upon drug binding, whereby many tens of whole cells were imaged. Trastuzumab was found to selectively cross-link and down-regulate HER2 homodimers from the plasma membranes of bulk cancer cells. In contrast, HER2 resided mainly as monomers in rare subpopulations of resting and cancer stem cells (CSCs), and these monomers were not internalized after drug binding. The HER2 distribution was hardly influenced by trastuzumab for the HCC1954 cells. These findings show that resting cells and CSCs are irresponsive to the drug and thus point toward a molecular explanation behind the origin of drug resistance. This analytical method is broadly applicable to study membrane protein interactions in the intact plasma membrane, while accounting for cell heterogeneity

Spatially resolved observation of uniform precession modes in spin-valve systems

Using time-resolved photoemission electron microscopy the excitation of uniform precession modes in individual domains of a weakly coupled spin-valve system has been studied. A coupling dependence of the precession frequencies has been found that can be reasonably well understood on the basis of a macrospin model. By tuning the frequency of the excitation source the uniform precession modes are excited in a resonant way.Comment: This article has been accepted by Journal of Applied Physics. After it is published, it will be found at http://jap.aip.or

RNAi-based validation of antibodies for reverse phase protein arrays

<p>Abstract</p> <p>Background</p> <p>Reverse phase protein arrays (RPPA) have been demonstrated to be a useful experimental platform for quantitative protein profiling in a high-throughput format. Target protein detection relies on the readout obtained from a single detection antibody. For this reason, antibody specificity is a key factor for RPPA. RNAi allows the specific knockdown of a target protein in complex samples and was therefore examined for its utility to assess antibody performance for RPPA applications.</p> <p>Results</p> <p>To proof the feasibility of our strategy, two different anti-EGFR antibodies were compared by RPPA. Both detected the knockdown of EGFR but at a different rate. Western blot data were used to identify the most reliable antibody. The RNAi approach was also used to characterize commercial anti-STAT3 antibodies. Out of ten tested anti-STAT3 antibodies, four antibodies detected the STAT3-knockdown at 80-85%, and the most sensitive anti-STAT3 antibody was identified by comparing detection limits. Thus, the use of RNAi for RPPA antibody validation was demonstrated to be a stringent approach to identify highly specific and highly sensitive antibodies. Furthermore, the RNAi/RPPA strategy is also useful for the validation of isoform-specific antibodies as shown for the identification of AKT1/AKT2 and CCND1/CCND3-specific antibodies.</p> <p>Conclusions</p> <p>RNAi is a valuable tool for the identification of very specific and highly sensitive antibodies, and is therefore especially useful for the validation of RPPA-suitable detection antibodies. On the other hand, when a set of well-characterized RPPA-antibodies is available, large-scale RNAi experiments analyzed by RPPA might deliver useful information for network reconstruction.</p

Extending pathways based on gene lists using InterPro domain signatures

<p>Abstract</p> <p>Background</p> <p>High-throughput technologies like functional screens and gene expression analysis produce extended lists of candidate genes. Gene-Set Enrichment Analysis is a commonly used and well established technique to test for the statistically significant over-representation of particular pathways. A shortcoming of this method is however, that most genes that are investigated in the experiments have very sparse functional or pathway annotation and therefore cannot be the target of such an analysis. The approach presented here aims to assign lists of genes with limited annotation to previously described functional gene collections or pathways. This works by comparing InterPro domain signatures of the candidate gene lists with domain signatures of gene sets derived from known classifications, e.g. KEGG pathways.</p> <p>Results</p> <p>In order to validate our approach, we designed a simulation study. Based on all pathways available in the KEGG database, we create test gene lists by randomly selecting pathway genes, removing these genes from the known pathways and adding variable amounts of noise in the form of genes not annotated to the pathway. We show that we can recover pathway memberships based on the simulated gene lists with high accuracy. We further demonstrate the applicability of our approach on a biological example.</p> <p>Conclusion</p> <p>Results based on simulation and data analysis show that domain based pathway enrichment analysis is a very sensitive method to test for enrichment of pathways in sparsely annotated lists of genes. An R based software package <it>domainsignatures</it>, to routinely perform this analysis on the results of high-throughput screening, is available via Bioconductor.</p