Institute of Molecular Biology and Biotechnology FORTH

Irákleion, Greece

Institute of Molecular Biology and Biotechnology FORTH

Irákleion, Greece
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Orfanoudaki G.,Institute of Molecular Biology and Biotechnology FoRTH | Orfanoudaki G.,University of Crete | Economou A.,Institute of Molecular Biology and Biotechnology FoRTH | Economou A.,University of Crete | And 2 more authors.
Molecular and Cellular Proteomics | Year: 2014

Cell compartmentalization serves both the isolation and the specialization of cell functions. After synthesis in the cytoplasm, over a third of all proteins are targeted to other subcellular compartments. Knowing how proteins are distributed within the cell and how they interact is a prerequisite for understanding it as a whole. Surface and secreted proteins are important pathogenicity determinants. Here we present the STEP database (STEPdb) that contains a comprehensive characterization of subcellular localization and topology of the complete proteome of Escherichia coli. Two widely used E. coli proteomes (K-12 and BL21) are presented organized into thirteen subcellular classes. STEPdb exploits the wealth of genetic, proteomic, biochemical, and functional information on protein localization, secretion, and targeting in E. coli, one of the best understood model organisms. Subcellular annotations were derived from a combination of bioinformatics prediction, proteomic, biochemical, functional, topological data and extensive literature re-examination that were refined through manual curation. Strong experimental support for the location of 1553 out of 4303 proteins was based on 426 articles and some experimental indications for another 526. Annotations were provided for another 320 proteins based on firm bioinformatic predictions. STEPdb is the first database that contains an extensive set of peripheral IM proteins (PIM proteins) and includes their graphical visualization into complexes, cellular functions, and interactions. It also summarizes all currently known protein export machineries of E. coli K-12 and pairs them, where available, with the secretory proteins that use them. It catalogs the Sec- and TAT-utilizing secretomes and summarizes their topological features such as signal peptides and transmembrane regions, transmembrane topologies and orientations. It also catalogs physicochemical and structural features that influence topology such as abundance, solubility, disorder, heat resistance, and structural domain families. Finally, STEPdb incorporates prediction tools for topology (TMHMM, SignalP, and Phobius) and disorder (IUPred) and implements the BLAST2STEP that performs protein homology searches against the STEPdb. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.


Choulaki C.,University of Crete | Papadaki G.,University of Crete | Repa A.,University of Crete | Kampouraki E.,University of Crete | And 7 more authors.
Arthritis Research and Therapy | Year: 2015

Introduction: Interleukin-1β (IL-1β) is a major inflammatory cytokine, produced predominantly by innate immune cells through NLRP3-inflammasome activation. Both intrinsic and extrinsic danger signals may activate NLRP3. Genetic variations in NLRP3-inflammasome components have been reported to influence rheumatoid arthritis (RA) susceptibility and severity. We sought to assess the activity of NLRP3-inflammasome in patients with active RA compared to healthy individuals. Method: Intracellular protein expression of NLRP3, ASC, pro- and active caspase-1, pro- and active IL-1β was assessed by immunoblotting both at baseline and upon inflammasome activation. NLRP3 function (IL-1β secretion) was assessed upon priming of TLR2 (Pam(3)CysSK(4), TLR3 (poly(I:C)) or TLR4 (LPS) and ATP sequential treatment. We used caspase inhibitors (casp-1, 3/7 and 8) to assess their contribution to IL-1β maturation. All experiments were performed in whole blood cells. Results: Active RA patients (n = 11) expressed higher basal intracellular levels of NLRP3 (p < 0.008), ASC (p < 0.003), active caspase-1 (p < 0.02) and pro-IL-1β (p < 0.001). Upon priming with TLR4 (LPS) and ATP, RA-derived cell extracts (n = 7) displayed increased expression of NLRP3 (p < 0.01) and active caspase-1 (p < 0.001). Secreted IL-1β in culture supernatants from whole blood cells activated with TLR4 (LPS) or TLR3 agonist (poly(I:C)) plus ATP was higher in RA patients (n = 20) versus controls (n = 18) (p < 0.02 for both). Caspase-1 inhibition significantly reduced IL-1β secretion induced by all stimuli, whereas caspase-8 inhibition affected only TLR4 and TLR3 cell priming. Conclusion: Patients with active RA have increased expression of NLRP3 and NLRP3-mediated IL-1β secretion in whole blood cells upon stimulation via TLR3 and TLR4 but not TLR2. In these patients, IL-1β secretion seems to be predominately driven by caspase-1 and caspase-8. Targeting NLRP3 or downstream caspases may be of benefit in suppressing IL-1β production in RA. © 2015 Choulaki et al.


PubMed | University of Crete, Foundation Medicine, Democritus University of Thrace and Institute of Molecular Biology and Biotechnology FORTH
Type: Journal Article | Journal: Human immunology | Year: 2015

Familial Mediterranean fever (FMF) is caused by mutations in pyrin, a protein expressed in innate immune cells that interacts with caspase-1 and other inflammasome components to regulate interleukin (IL)-1 maturation. Since NLRP3 inflammasome represents major source of IL-1, we studied its protein expression and function in FMF. We isolated peripheral white blood cells (WBCs) from 20 symptoms-free FMF patients and 21 healthy individuals. Intracellular protein expression of NLRP3, caspase-1, IL-1 at baseline and after LPS/ATP sequential treatment for NLRP3 activation was assessed by immunoblotting. Secreted IL-1 was quantified by ELISA. THP-1 cells were transfected with wild-type or mutant pyrin and IL-1 secretion was measured. FMF WBCs exhibited lower NLRP3 and active caspase-1 protein expression compared to healthy individuals, and LPS/ATP treatment resulted in significantly lower intracellular IL-1 levels in FMF patients. Likewise, LPS/ATP induced caspase-1-dependent IL-1 release at significantly lower amounts in the FMF group (1182192 versus 2134245pg/mL in controls, p=0.004). Consistently, THP-1 cells transfected with FMF-associated M694V mutant pyrin displayed lower LPS/ATP-induced IL-1 compared with wild-type pyrin-transfected cells. FMF WBCs demonstrate reduced NLRP3-mediated IL-1 production. Additional studies are needed to define whether this finding represents a compensatory mechanism to control inflammation or is directly linked to disease pathogenesis.


Pybus M.,University Pompeu Fabra | Dall'Olio G.M.,University Pompeu Fabra | Luisi P.,University Pompeu Fabra | Uzkudun M.,University Pompeu Fabra | And 6 more authors.
Nucleic Acids Research | Year: 2014

Searching for Darwinian selection in natural populations has been the focus of a multitude of studies over the last decades. Here we present the 1000 Genomes Selection Browser 1.0 (http://hsb.upf.edu) as a resource for signatures of recent natural selection in modern humans. We have implemented and applied a large number of neutrality tests as well as summary statistics informative for the action of selection such as Tajima's D, CLR, Fay and Wu's H, Fu and Li's F* and D*, XPEHH, ΔiHH, iHS, FST, ΔDAF and XPCLR among others to low coverage sequencing data from the 1000 genomes project (Phase 1; release April 2012). We have implemented a publicly available genome-wide browser to communicate the results from three different populations of West African, Northern European and East Asian ancestry (YRI, CEU, CHB). Information is provided in UCSC-style format to facilitate the integration with the rich UCSC browser tracks and an access page is provided with instructions and for convenient visualization. We believe that this expandable resource will facilitate the interpretation of signals of selection on different temporal, geographical and genomic scales. © 2013 The Author(s). Published by Oxford University Press.


Oulas A.,Institute of Molecular Biology and Biotechnology FORTH | Oulas A.,Institute of Marine Biology and Genetics HC MR | Karathanasis N.,Institute of Molecular Biology and Biotechnology FORTH | Karathanasis N.,University of Crete | And 5 more authors.
RNA Biology | Year: 2012

Computational methods for miRNA target prediction vary in the algorithm used; and while one can state opinions about the strengths or weaknesses of each particular algorithm, the fact of the matter is that they fall substantially short of capturing the full detail of physical, temporal and spatial requirements of miRNA:target-mRNA interactions. Here, we introduce a novel miRNA target prediction tool called Targetprofiler that utilizes a probabilistic learning algorithm in the form of a hidden Markov model trained on experimentally verified miRNA targets. Using a large scale protein downregulation data set we validate our method and compare its performance to existing tools. We find that Targetprofiler exhibits greater correlation between computational predictions and protein downregulation and predicts experimentally verified miRNA targets more accurately than three other tools. Concurrently, we use primer extension to identify the mature sequence of a novel miRNA gene recently identified within a cancer associated genomic region and use Targetprofiler to predict its potential targets. Experimental verification of the ability of this small RNA molecule to regulate the expression of CC ND2, a gene with documented oncogenic activity, confirms its functional role as a miRNA. These findings highlight the competitive advantage of our tool and its efficacy in extracting biologically significant results. © 2012 Landes Bioscience.


Papadakis G.,Advanced Materials and Processes | Gizeli E.,Institute Of Molecular Biology And Biotechnology Forth | Gizeli E.,University of Crete
Analytical Methods | Year: 2014

Screening for mutations in the tumor-suppressor genes BRCA1 and BRCA2 is of great importance for breast and ovarian cancer prevention. We describe a methodology for mutation screening and detection based on acoustic wave devices. In particular, we detect four mutations located in BRCA1 and BRCA2 genes using the quartz crystal microbalance technique. The detection is based on measurements of the acoustic ratio of dissipation versus frequency change (ΔD/ΔF) of double-stranded DNA molecules bound to the device surface that are produced after PCR amplification and restriction digestion; the acoustic ratio has been shown to be a measure of the intrinsic viscosity of the attached molecules, which, in turn, depends on the size of the dsDNAs. Novel features of this approach are the lack of a hybridization step, the label free sensing of the length, rather than mass, of the DNA molecules and the direct detection of the digested DNA products without prior purification. The method is generic, simple and capable of detecting single base mutations to long genomic rearrangements; it is also suitable and applicable to a Lab-on-a-chip concept. This journal is © 2014 The Royal Society of Chemistry.


Papanastasiou M.,Institute of Molecular Biology and Biotechnology FORTH | Orfanoudaki G.,Institute of Molecular Biology and Biotechnology FORTH | Orfanoudaki G.,University of Crete | Koukaki M.,Institute of Molecular Biology and Biotechnology FORTH | And 7 more authors.
Molecular and Cellular Proteomics | Year: 2013

Biological membranes are essential for cell viability. Their functional characteristics strongly depend on their protein content, which consists of transmembrane (integral) and peripherally associated membrane proteins. Both integral and peripheral inner membrane proteins mediate a plethora of biological processes. Whereas transmembrane proteins have characteristic hydrophobic stretches and can be predicted using bioinformatics approaches, peripheral inner membrane proteins are hydrophilic, exist in equilibria with soluble pools, and carry no discernible membrane targeting signals. We experimentally determined the cytoplasmic peripheral inner membrane proteome of the model organism Escherichia coli using a multidisciplinary approach. Initially, we extensively re-annotated the theoretical proteome regarding subcellular localization using literature searches, manual curation, and multi-combinatorial bioinformatics searches of the available databases. Next we used sequential biochemical fractionations coupled to direct identification of individual proteins and protein complexes using high resolution mass spectrometry. We determined that the proposed cytoplasmic peripheral inner membrane proteome occupies a previously unsuspected +19% of the basic E. coli BL21(DE3) proteome, and the detected peripheral inner membrane proteome occupies +25% of the estimated expressed proteome of this cell grown in LB medium to mid-log phase. This value might increase when fleeting interactions, not studied here, are taken into account. Several proteins previously regarded as exclusively cytoplasmic bind membranes avidly. Many of these proteins are organized in functional or/and structural oligomeric complexes that bind to the membrane with multiple interactions. Identified proteins cover the full spectrum of biological activities, and more than half of them are essential. Our data suggest that the cytoplasmic proteome displays remarkably dynamic and extensive communication with biological membrane surfaces that we are only beginning to decipher. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.


PubMed | University of Crete, Democritus University of Thrace and Institute of Molecular Biology and Biotechnology FORTH
Type: | Journal: Arthritis research & therapy | Year: 2015

Interleukin-1 (IL-1) is a major inflammatory cytokine, produced predominantly by innate immune cells through NLRP3-inflammasome activation. Both intrinsic and extrinsic danger signals may activate NLRP3. Genetic variations in NLRP3-inflammasome components have been reported to influence rheumatoid arthritis (RA) susceptibility and severity. We sought to assess the activity of NLRP3-inflammasome in patients with active RA compared to healthy individuals.Intracellular protein expression of NLRP3, ASC, pro- and active caspase-1, pro- and active IL-1 was assessed by immunoblotting both at baseline and upon inflammasome activation. NLRP3 function (IL-1 secretion) was assessed upon priming of TLR2 (Pam(3)CysSK(4), TLR3 (poly(I:C)) or TLR4 (LPS) and ATP sequential treatment. We used caspase inhibitors (casp-1, 3/7 and 8) to assess their contribution to IL-1 maturation. All experiments were performed in whole blood cells.Active RA patients (n=11) expressed higher basal intracellular levels of NLRP3 (p<0.008), ASC (p<0.003), active caspase-1 (p<0.02) and pro-IL-1 (p<0.001). Upon priming with TLR4 (LPS) and ATP, RA-derived cell extracts (n=7) displayed increased expression of NLRP3 (p<0.01) and active caspase-1 (p<0.001). Secreted IL-1 in culture supernatants from whole blood cells activated with TLR4 (LPS) or TLR3 agonist (poly(I:C)) plus ATP was higher in RA patients (n=20) versus controls (n=18) (p<0.02 for both). Caspase-1 inhibition significantly reduced IL-1 secretion induced by all stimuli, whereas caspase-8 inhibition affected only TLR4 and TLR3 cell priming.Patients with active RA have increased expression of NLRP3 and NLRP3-mediated IL-1 secretion in whole blood cells upon stimulation via TLR3 and TLR4 but not TLR2. In these patients, IL-1 secretion seems to be predominately driven by caspase-1 and caspase-8. Targeting NLRP3 or downstream caspases may be of benefit in suppressing IL-1 production in RA.


PubMed | Catholic University of Leuven, Technical University of Crete and Institute of Molecular Biology and Biotechnology FORTH
Type: Journal Article | Journal: Journal of proteome research | Year: 2016

Human embryonic stem cells (hESCs) are promising in regenerative medicine (RM) due to their differentiation plasticity and proliferation potential. However, a major challenge in RM is the generation of a vascular system to support nutrient flow to newly synthesized tissues. Here we refined an existing method to generate tight vessels by differentiating hESCs in CD34(+) vascular progenitor cells using chemically defined media and growth conditions. We selectively purified these cells from CD34(-) outgrowth populations also formed. To analyze these differentiation processes, we compared the proteomes of the hESCs with those of the CD34(+) and CD34(-) populations using high resolution mass spectrometry, label-free quantification, and multivariate analysis. Eighteen protein markers validate the differentiated phenotypes in immunological assays; nine of these were also detected by proteomics and show statistically significant differential abundance. Another 225 proteins show differential abundance between the three cell types. Sixty-three of these have known functions in CD34(+) and CD34(-) cells. CD34(+) cells synthesize proteins implicated in endothelial cell differentiation and smooth muscle formation, which support the bipotent phenotype of these progenitor cells. CD34(-) cells are more heterogeneous synthesizing muscular/osteogenic/chondrogenic/adipogenic lineage markers. The remaining >150 differentially abundant proteins in CD34(+) or CD34(-) cells raise testable hypotheses for future studies to probe vascular morphogenesis.


PubMed | University of Crete, Greek National Center For Scientific Research and Institute of Molecular Biology and Biotechnology FORTH
Type: | Journal: Analytica chimica acta | Year: 2016

We present a polymeric microfluidic chip capable of purifying DNA through solid phase extraction. It is designed to be used as a module of an integrated Lab-on-chip platform for pathogen detection, but it can also be used as a stand-alone device. The microfluidic channels are oxygen plasma micro-nanotextured, i.e. randomly roughened in the micro-nano scale, a process creating high surface area as well as high density of carboxyl groups (COOH). The COOH groups together with a buffer that contains polyethylene glycol (PEG), NaCl and ethanol are able to bind DNA on the microchannel surface. The chip design incorporates a mixer so that sample and buffer can be efficiently mixed on chip under continuous flow. DNA is subsequently eluted in water. The chip is able to isolate DNA with high recovery efficiency (9611%) in an extremely large dynamic range of prepurified Salmonella DNA as well as from Salmonella cell lysates that correspond to a range of 5 to 1.910

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