Postupalenko V.,CNRS Laboratory of Design and Application of Bioactive Molecules |
Desplancq D.,CNRS Biotechnology and Cell Signaling Laboratory |
Orlov I.,Center for Integrative Biology |
Arntz Y.,CNRS Laboratory of Design and Application of Bioactive Molecules |
And 6 more authors.
Angewandte Chemie - International Edition | Year: 2015
Recombinant proteins with cytosolic or nuclear activities are emerging as tools for interfering with cellular functions. Because such tools rely on vehicles for crossing the plasma membrane we developed a protein delivery system consisting in the assembly of pyridylthiourea-grafted polyethylenimine (πPEI) with affinity-purified His-tagged proteins pre-organized onto a nickel-immobilized polymeric guide. The guide was prepared by functionalization of an ornithine polymer with nitrilotriacetic acid groups and shown to bind several His-tagged proteins. Superstructures were visualized by electron and atomic force microscopy using 2 nm His-tagged gold nanoparticles as probes. The whole system efficiently carried the green fluorescent protein, single-chain antibodies or caspase 3, into the cytosol of living cells. Transduction of the protease caspase 3 induced apoptosis in two cancer cell lines, demonstrating that this new protein delivery method could be used to interfere with cellular functions. © 2015 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim.
Ellen A.F.,Center for Integrative Biology |
Ellen A.F.,University of Groningen |
Zolghadr B.,University of Groningen |
Zolghadr B.,Max Planck Institute for Terrestrial Microbiology |
And 2 more authors.
Archaea | Year: 2010
Although archaea have a similar cellular organization as other prokaryotes, the lipid composition of their membranes and their cell surface is unique. Here we discuss recent developments in our understanding of the archaeal protein secretion mechanisms, the assembly of macromolecular cell surface structures, and the release of S-layer-coated vesicles from the archaeal membrane. © 2010 Albert F. Ellen et al.
PubMed | CNRS Laboratory of Design and Application of Bioactive Molecules, Center for Integrative Biology and CNRS Biotechnology and Cell Signaling Laboratory
Type: Journal Article | Journal: Angewandte Chemie (International ed. in English) | Year: 2015
Recombinant proteins with cytosolic or nuclear activities are emerging as tools for interfering with cellular functions. Because such tools rely on vehicles for crossing the plasma membrane we developed a protein delivery system consisting in the assembly of pyridylthiourea-grafted polyethylenimine (PEI) with affinity-purified His-tagged proteins pre-organized onto a nickel-immobilized polymeric guide. The guide was prepared by functionalization of an ornithine polymer with nitrilotriacetic acid groups and shown to bind several His-tagged proteins. Superstructures were visualized by electron and atomic force microscopy using 2nm His-tagged gold nanoparticles as probes. The whole system efficiently carried the green fluorescent protein, single-chain antibodies or caspase3, into the cytosol of living cells. Transduction of the protease caspase3 induced apoptosis in two cancer cell lines, demonstrating that this new protein delivery method could be used to interfere with cellular functions.
Cereseto A.,Center for Integrative Biology |
Giacca M.,International Center for Genetic Engineering and Biotechnology
Methods in Molecular Biology | Year: 2014
Advancements in fluorescent microscopy techniques now permit investigation of HIV-1 biology exploiting tools alternative to conventional molecular biology. Here we describe a novel, fluorescence-based method to visualize HIV-1 viral particles within intact nuclei of infected cells. This method allows investigating the localization of pre-integration complexes within the nuclear compartment with respect to the nuclear envelope and the chromatin territories. © 2014 Springer Science+Business Media, LLC.
Andreotti V.,Italian National Cancer Institute |
Ciribilli Y.,Center for Integrative Biology |
Monti P.,Italian National Cancer Institute |
Bisio A.,Center for Integrative Biology |
And 6 more authors.
PLoS ONE | Year: 2011
Background: The p53 tumor suppressor, which is altered in most cancers, is a sequence-specific transcription factor that is able to modulate the expression of many target genes and influence a variety of cellular pathways. Inactivation of the p53 pathway in cancer frequently occurs through the expression of mutant p53 protein. In tumors that retain wild type p53, the pathway can be altered by upstream modulators, particularly the p53 negative regulators MDM2 and MDM4. Methodology/Principal Findings: Given the many factors that might influence p53 function, including expression levels, mutations, cofactor proteins and small molecules, we expanded our previously described yeast-based system to provide the opportunity for efficient investigation of their individual and combined impacts in a miniaturized format. The system integrates i) variable expression of p53 proteins under the finely tunable GAL1,10 promoter, ii) single copy, chromosomally located p53-responsive and control luminescence reporters, iii) enhanced chemical uptake using modified ABC-transporters, iv) small-volume formats for treatment and dual-luciferase assays, and v) opportunities to co-express p53 with other cofactor proteins. This robust system can distinguish different levels of expression of WT and mutant p53 as well as interactions with MDM2 or 53BP1. Conclusions/Significance: We found that the small molecules Nutlin and RITA could both relieve the MDM2-dependent inhibition of WT p53 transactivation function, while only RITA could impact p53/53BP1 functional interactions. PRIMA-1 was ineffective in modifying the transactivation capacity of WT p53 and missense p53 mutations. This dual-luciferase assay can, therefore, provide a high-throughput assessment tool for investigating a matrix of factors that can influence the p53 network, including the effectiveness of newly developed small molecules, on WT and tumor-associated p53 mutants as well as interacting proteins.
Lecca P.,University of Trento |
Lecca P.,Association for Computing Machinery |
Re A.,Center for Integrative Biology
Lecture Notes in Computer Science (including subseries Lecture Notes in Artificial Intelligence and Lecture Notes in Bioinformatics) | Year: 2016
While computational systems biology provides a rich array of methods for network clustering, most of them are not suitable to capture cellular network dynamics. In the most common setting, computational algorithms seek to integrate the static information embedded in near-global interaction networks with the temporal information provided by time series experiments. We present a novel technique for temporally informed network module detection, named TD-WGcluster (Time Delay Weighted Graph CLUSTERing). TD-WGcluster utilizes four steps: (i) time-lagged correlations are calculated between any couple of interacting nodes in the network; (ii) an unsupervised version of k-means algorithm detects sub-graphs with similar time-lagged correlation; (iii) a fast-greedy optimization algorithm identify connected components by sub-graph; (iv) a geometric entropy is computed for each connected component as a measure of its complexity. TD-WGcluster notable feature is the attempt to account for temporal delays in the formation of regulatory modules during signal propagation in a network. © Springer International Publishing Switzerland 2016.
Mazza T.,Mendel Institute |
Ballarini P.,École Centrale Paris |
Guido R.,University of Calabria |
Prandi D.,Center for Integrative Biology
IEEE/ACM Transactions on Computational Biology and Bioinformatics | Year: 2012
Important achievements in traditional biology have deepened the knowledge about living systems leading to an extensive identification of parts-list of the cell as well as of the interactions among biochemical species responsible for cell's regulation. Such an expanding knowledge also introduces new issues. For example, the increasing comprehension of the interdependencies between pathways (pathways cross-talk) has resulted, on one hand, in the growth of informational complexity, on the other, in a strong lack of information coherence. The overall grand challenge remains unchanged: to be able to assemble the knowledge of every "piece of a system in order to figure out the behavior of the whole (integrative approach). In light of these considerations, high performance computing plays a fundamental role in the context of in-silico biology. Stochastic simulation is a renowned analysis tool, which, although widely used, is subject to stringent computational requirements, in particular when dealing with heterogeneous and high dimensional systems. Here, we introduce and discuss a methodology aimed at alleviating the burden of simulating complex biological networks. Such a method, which springs from graph theory, is based on the principle of fragmenting the computational space of a simulation trace and delegating the computation of fragments to a number of parallel processes. © 2012 IEEE.
Xu Z.,New York University |
Chen H.,New York University |
Chen H.,Princeton University |
Ling J.,New York University |
And 5 more authors.
Genes and Development | Year: 2014
In vivo cross-linking studies suggest that the Drosophila transcription factor Bicoid (Bcd) binds to several thousand sites during early embryogenesis, but it is not clear how many of these binding events are functionally important. In contrast, reporter gene studies have identified >60 Bcd-dependent enhancers, all of which contain clusters of the consensus binding sequence TAATCC. These studies also identified clusters of TAATCC motifs (inactive fragments) that failed to drive Bcd-dependent activation. In general, active fragments showed higher levels of Bcd binding in vivo and were enriched in predicted binding sites for the ubiquitous maternal protein Zelda (Zld). Here we tested the role of Zld in Bcd-mediated binding and transcription. Removal of Zld function and mutations in Zld sites caused significant reductions in Bcd binding to known enhancers and variable effects on the activation and spatial positioning of Bcd-dependent expression patterns. Also, insertion of Zld sites converted one of six inactive fragments into a Bcd-responsive enhancer. Genome-wide binding experiments in zld mutants showed variable effects on Bcd-binding peaks, ranging from strong reductions to significantly enhanced levels of binding. Increases in Bcd binding caused the precocious Bcd-dependent activation of genes that are normally not expressed in early embryos, suggesting that Zld controls the genome-wide binding profile of Bcd at the qualitative level and is critical for selecting target genes for activation in the early embryo. These results underscore the importance of combinatorial binding in enhancer function and provide data that will help predict regulatory activities based on DNA sequence. © 2014 Xu et al.
PubMed | University of Trento, CNR Institute of Neuroscience, University of Edinburgh, Fondazione Bruno Kessler and 2 more.
Type: Journal Article | Journal: Nucleic acids research | Year: 2015
Fluctuations in mRNA levels only partially contribute to determine variations in mRNA availability for translation, producing the well-known poor correlation between transcriptome and proteome data. Recent advances in microscopy now enable researchers to obtain high resolution images of ribosomes on transcripts, providing precious snapshots of translation in vivo. Here we propose RiboAbacus, a mathematical model that for the first time incorporates imaging data in a predictive model of transcript-specific ribosome densities and translational efficiencies. RiboAbacus uses a mechanistic model of ribosome dynamics, enabling the quantification of the relative importance of different features (such as codon usage and the 5 ramp effect) in determining the accuracy of predictions. The model has been optimized in the human Hek-293 cell line to fit thousands of images of human polysomes obtained by atomic force microscopy, from which we could get a reference distribution of the number of ribosomes per mRNA with unmatched resolution. After validation, we applied RiboAbacus to three case studies of known transcriptome-proteome datasets for estimating the translational efficiencies, resulting in an increased correlation with corresponding proteomes. RiboAbacus is an intuitive tool that allows an immediate estimation of crucial translation properties for entire transcriptomes, based on easily obtainable transcript expression levels.
PubMed | Section of Invertebrate Zoology and Hydrobiology, CNR Institute of Neuroscience, Center for Integrative Biology and Fondazione Bruno Kessler
Type: | Journal: Journal of insect physiology | Year: 2015
In stressed organisms, strategic proteins are selectively translated even if the global process of protein synthesis is compromised. The determination of protein concentrations in tissues of non-model organisms (thus with limited genomic information) is challenging due to the absence of specific antibodies. Moreover, estimating protein levels quantifying transcriptional responses may be misleading, because translational control mechanisms uncouple protein and mRNAs abundances. Translational control is increasingly recognized as a hub where regulation of gene expression converges to shape proteomes, but it is almost completely overlooked in molecular ecology studies. An interesting approach to study translation and its control mechanisms is the analysis of variations of gene-specific translational efficiencies by quantifying mRNAs associated to ribosomes. In this paper, we propose a robust and streamlined pipeline for purifying ribosome-associated mRNAs and calculating global and gene-specific translation efficiencies from non-model insects species. This method might found applications in molecular ecology to study responses to environmental stressors in non-model organisms.