Fluorescent Proteins

Cell Imaging Platforms (CIPs) are research infrastructures offering support to a number of scientific projects including the choice of adapted fluorescent probes for live cell imaging. What to detect in what type of sample and for how long is a major issue with fluorescent probes and, for this, the "hat-trick" "Probe-Sample-Instrument" (PSI) has to be considered. We propose here to deal with key points usually discussed in CIPs including the properties of fluorescent organic probes, the modality of cell labeling, and the best equipment to obtain appropriate spectral, spatial, and temporal resolution. New strategies in organic synthesis and click chemistry for accessing probes with enhanced photophysical characteristics and targeting abilities will also be addressed. Finally, methods for image processing will be described to optimize exploitation of fluorescence signals.

Cell Imaging Platforms (CIPs) are research infrastructures offering support to a number of scientific projects including the choice of adapted fluorescent probes for live cell imaging. What to detect in what type of sample and for how long is a major issue with fluorescent probes and, for this, the “hat-trick” “Probe–Sample–Instrument” (PSI) has to be considered. We propose here to deal with key points usually discussed in CIPs including the properties of fluorescent organic probes, the modality of cell labeling, and the best equipment to obtain appropriate spectral, spatial, and temporal resolution. New strategies in organic synthesis and click chemistry for accessing probes with enhanced photophysical characteristics and targeting abilities will also be addressed. Finally, methods for image processing will be described to optimize exploitation of fluorescence signals.

Genomes are constantly in flux, undergoing changes due to recombination, repair and mutagenesis. In vivo, many of such changes are studies using reporters for specific types of changes, or through cytological studies that detect changes at the single-cell level. Single molecule assays, which are reviewed here, can detect transient intermediates and dynamics of events. Biochemical assays allow detailed investigation of the DNA and protein activities of each step in a repair, recombination or mutagenesis event. Each type of assay is a powerful tool but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies.

When utilizing a scanning confocal microscope, better resolution and image contrast is achieved when compared to traditional methods. Thin optical sections of living specimens, 100 micrometers thick can be imaged with such devices, which are capable of examining fluorescence emission between 400nm and 750nm. By using laser light of wavelength 470nm as an excitation source, a root cell sample stained with two different fluorescent dyes was examined. Two distinct peaks were notated at 643.98nm (red) and 537.72nm (green). The fluorophores used were deemed to be monolignol (green) and Peridinin Chlorophyll (red) as they matched the ratio of excitation/emission wavelength detected in the experiment.

Genomes are constantly in flux, undergoing changes due to recombination, repair and mutagenesis. In vivo, many of such changes are studies using reporters for specific types of changes, or through cytological studies that detect changes at the single-cell level. Single molecule assays, which are reviewed here, can detect transient intermediates and dynamics of events. Biochemical assays allow detailed investigation of the DNA and protein activities of each step in a repair, recombination or mutagenesis event. Each type of assay is a powerful tool but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies.

To meet the enormous computational needs of live-science research as well as clinical diagnostics and treatment the Hogeschool Rotterdam and the Erasmus Medical Center are currently setting up one of the largest desktop computing grids in the world -The Erasmus Computing Grid. Currently 3 Tera flops are operational and in early production, installation up to the today available maximum capacity of 20 Tera flops in both institutions is planned and partly underway. Thus the Erasmus Computing Grid transforms the existing and sustained huge computer capacity available into usable form via a reliable and secure installing and management system, so that the academic and industrial opportunities depending on such huge computing capacities can be realized for the benefit of society.

In biotechnology, endotoxin (LPS) removal from recombinant proteins is a critical and challenging step in the preparation of injectable therapeutics, as endotoxin is a natural component of bacterial expression systems widely used to manufacture therapeutic proteins. The viability of large-scale industrial production of recombinant biomolecules of pharmaceutical interest significantly depends on the separation and purification techniques used. The aim of this work was to evaluate the use of aqueous two-phase micellar system (ATPMS) for endotoxin removal from preparations containing recombinant proteins of pharmaceutical interest, such as green fluorescent protein (GFPuv). Partition assays were carried out initially using pure LPS, and afterwards in the presence of E. coli cell lysate. The ATPMS technology proved to be effective in GFPuv recovery, preferentially into the micelle-poor phase (K GFPuv \ 1.00), and LPS removal into the micelle-rich phase (%REM LPS [ 98.00%). Therefore, this system can be exploited as the first step for purification in biotechnology processes for removal of higher LPS concentrations. V

Applications at Erasmus MC: E.g. genome sequence analysis, protein structure simulations, chip/array analysis, epidemiology of viral infections, patient diagnostic image analysis (AMI). Applications at Erasmus MC: E.g. genome sequence analysis, protein structure simulations, chip/array analysis, epidemiology of viral infections, patient diagnostic image analysis (AMI). Applications at Erasmus MC: E.g. genome sequence analysis, protein structure simulations, chip/array analysis, epidemiology of viral infections, patient diagnostic image analysis (AMI). To develop an entire novel system-biology oriented genome browser, i.e. integrating the holistic complexity of genome organization in a single easy comprehensible platform has required completely new approaches to represent the genome architecture realistically in combination with the various types experimental data or instant analysis capabilities and annotation.

10 and 13 orders of magnitude concerning length and time scales are bridged. Are and how are all of these organization levels connected to fullfill their obvious functions, e. g. gene regulation or replication, since they are optimized by evolution ? -10 10 2 [s]

and The different organization levels of genomes bridge several orders of magnitude concerning space and time. How all of these organization levels connect to processes like gene regulation, replication, embryogeneses, or cancer development is still unclear?

In biotechnology, endotoxin (LPS) removal from recombinant proteins is a critical and challenging step in the preparation of injectable therapeutics, as endotoxin is a natural component of bacterial expression systems widely used to manufacture therapeutic proteins. The viability of large-scale industrial production of recombinant biomolecules of pharmaceutical interest significantly depends on the separation and purification techniques used. The aim of this work was to evaluate the use of aqueous two-phase micellar system (ATPMS) for endotoxin removal from preparations containing recombinant proteins of pharmaceutical interest, such as green fluorescent protein (GFPuv). Partition assays were carried out initially using pure LPS, and afterwards in the presence of E. coli cell lysate. The ATPMS technology proved to be effective in GFPuv recovery, preferentially into the micelle-poor phase (K GFPuv \ 1.00), and LPS removal into the micelle-rich phase (%REM LPS [ 98.00%). Therefore, this system can be exploited as the first step for purification in biotechnology processes for removal of higher LPS concentrations. V

We describe the facile generation of a stable recombinant antibody with intrinsic red fluorescent properties for qualitative and potentially quantitative immunofluorescence analysis. The REDantibody based on the X-ray crystallographic structures of the anti-sialyl-Tn antibody B72.3 and 3D model of the monomeric red fluorescent protein was designed to retain optimal spatial geometry between the C-and N-termini of the V H and V L chains respectively to mimic the domains interface pairing in antibody Fab fragments and to incorporate the red fluorescent protein as a bridging scaffold. The model was further validated by assembling a REDantibody based on CA19.9 the anti-sialylated Lewis (Le) a blood group antigen and 4D5-8 the anti-p185 HER2 antibodies. The chimeric heavy and light chains containing red fluorescent protein as a bridge were correctly processed and secreted into E. coli periplasm for assembly and disulphide bond formation, further analysis revealed the molecules to be exclusively monomers. Purified antiglycan proteins were used for an immunofluorescent analysis of Trypanosoma cruzi epimastigotes, and the anti-p185 HER2 used to determine the binding properties. The REDantibody platform facilitates rapid generation of scFv chimeras that could be used for screening antibodies against cell surface markers. Furthermore, such modular assembly should permit the interchange of binding sites and of fluorophores to create robust panels of coloured antibodies.

The development of biosensors has seen recent growth owing to their wide range of potential applications from the defending of bioterrorism to the detection of various diseases. Recent investigations in highly sensitive nanomaterials containing nanotechnology suggest the possibility of its advanced applications as biosensors in different biomedical fields. Besides highlighting the characteristic features of different classes of advanced biosensor across different fields of potential application, this article gives a clear overview of some specific investigations related to the surface immobilizations of piezoelectric biosensors employing effective materials and advanced technologies. Further, the current development of piezoelectric biosensors in the detection of potential biomolecules for advanced biomedical applications is critically reviewed. The qualitative and quantitative sensitivity of piezoelectric biosensors, various expressions of resonant frequency shift with changes in mass, thickness, density, and stress for piezoelectric materials are distinctly explored. The importance of several novel nanomaterials for the detection of various diseased biomolecules is clearly illustrated. This study concludes that proper selection of materials is potential to bring improvement in quality as well as reduction in cost of advanced biosensor devices.

Francisella tularensis is a Gram-negative coccobacillus and is the etiological agent of the disease tularemia. Expression of the cytoplasmic membrane protein RipA is required for Francisella replication within macrophages and other cell types; however, the function of this protein remains unknown. RipA is conserved among all sequenced Francisella species, and RipA-like proteins are present in a number of individual strains of a wide variety of species scattered throughout the prokaryotic kingdom. Cross-linking studies revealed that RipA forms homoligomers. Using a panel of RipA-green fluorescent protein and RipA-PhoA fusion constructs, we determined that RipA has a unique topology within the cytoplasmic membrane, with the N and C termini in the cytoplasm and periplasm, respectively. RipA has two significant cytoplasmic domains, one composed roughly of amino acids 1 to 50 and the second flanked by the second and third transmembrane domains and comprising amino acids 104 to 152. RipA ...

Genomes are constantly in flux, undergoing changes due to recombination, repair and mutagenesis. In vivo, many of such changes are studies using reporters for specific types of changes, or through cytological studies that detect changes at the single-cell level. Single molecule assays, which are reviewed here, can detect transient intermediates and dynamics of events. Biochemical assays allow detailed investigation of the DNA and protein activities of each step in a repair, recombination or mutagenesis event. Each type of assay is a powerful tool but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies.

Multicolor bioimaging can be referred to as the imaging method that non-invasively visualizes biological processes using fluorophores. Over the years, this technique has been primarily used in the areas of diagnostic and theranostic applications such as the detection of pathogens, identification of diseasespecific markers, and cancer detection. Fluorophores such as organic dyes are widely used in bioimaging studies. However, organic dyes exhibit a major limitation of excitation and emission spectral overlap, especially when multispectral bioimaging is considered. Quantum dots (QDs), in contrast, hold great potential due to their properties such as size-tunable narrow emission, single excitation in UV, photo and chemical stability, high fluorescence lifetime, simple surface modification process, etc. Thus, QDs can be a good alternative to traditional fluorophores for bioimaging applications. Herein, the previously reported poly dimethyl siloxane (PDMS)-coated CdTe QDs (PQDs) are used to simultaneously image cellular organelles such as lysosomes, mitochondria, nucleus, and actin of HepG2. Briefly, blue, green, yellow, and orange PQDs are conjugated to CD68 ab. (Lysosomes), mitochondria ab., nuclear antigen ab. and smooth muscle actin ab. respectively using EDC-NHS chemistry. The intracellular organelle targeting of the conjugated QDs is assessed by colocalization with the commercially available dyes. Finally, PQD-conjugates are used to simultaneously image four cellular organelles of HepG2 at an excitation wavelength of 405 nm. The present study demonstrates the potential of PQDs as a fluorophore in simultaneous multicolor bioimaging.

The combination of genome sequence and structure, its annotation and experimental data in an accessible and comprehensible way is a major challenge. Increasingly, there are a large number of extremely divergent data sets: the sequence itself, genes, regulatory regions, various forms of reoccurring sequence features and clone sets etc. Currently, one possibility to represent this information in a visual form - and thus to reveal its scientific meaning - is to use genome browsers such as "Ensembl" or "The UCSC Genome Browser". These browsers have been beneficial in the understanding of the complex organization of genomes. However, there are also huge limitations concerning their focus on linear presentation, standardized input and data bank accessibility. Also customisability by a remote user with special requirements is difficult. Therefore, the GLOBE-Consortium has developed the next generation genome viewer - the GLOBE 3D-Viewer - to visualize multi-dimensional ...

Due to its ease of genetic manipulation and transparency, Caenorhabditis elegans (C. elegans) has become a preferred model system to study gene function by microscopy. The use of Aequorea victoria green fluorescent protein (GFP) fused to proteins or targeting sequences of interest, further expanded upon the utility of C. elegans by labeling subcellular structures, which enables following their disposition during development or in the presence of genetic mutations. Fluorescent proteins with excitation and emission spectra different from that of GFP accelerated the use of multifluorophore imaging in real time. We have expanded the repertoire of fluorescent proteins for use in C. elegans by developing a codonoptimized version of Orange2 (CemOrange2). Proteins or targeting motifs fused to CemOr-ange2 were distinguishable from the more common fluorophores used in the nematode; such as GFP, YFP, and mKate2. We generated a panel of CemOrange2 fusion constructs, and confirmed they were targeted to their correct subcellular addresses by colocalization with independent markers. To demonstrate the potential usefulness of this new panel of fluorescent protein markers, we showed that CemOrange2 fusion proteins could be used to: 1) monitor biological pathways, 2) multiplex with other fluorescent proteins to determine colocalization and 3) gain phenotypic knowledge of a human ABCA3 orthologue, ABT-4, trafficking variant in the C. elegans model organism.

(hard cover, 2rd ed.), ISBN 3-00-035858-7, and ISBN 978-3-00-035858-6 (DVD, 2rd ed.), 1998. Knoch, T. A., Münkel, C. & Langowski, J. Three-dimensional organization of chromosome territories and the human cell nucleus-about the structure of a self replicating nano fabrication site.

Condor is used as the standard for batch systems on many real parallel super-computers as well as in many distributed trivial parallel computing grids. It is running in very diverse environments of the highest security levels as e.g. governmental or industry R&D. With a potential capacity of currently ~20 to 25 Tera flops the ECG is one of the biggest grids!

Het Erasmus Medische Centrum (Erasmus MC) en Hogeschool Rotterdam (HR) zijn in 2005 een unieke samenwerking begonnen om 95% van de capaciteit op al haar computers en die van anderen beschikbaar te maken voor onderzoek en onderwijs. Deze samenwerking heeft geleid tot het Erasmus Computing GRID (ECG), een virtuele supercomputer, met op dit moment een capaciteit van 5 Tera FLOPS maar na voltooiing met een capaciteit van 20 Tera (10 12) FLOPS (Floting Point Operations Per Second). Na uitvoerig testen is het ECG in productie gegaan en wordt gebruikt door tal van research groepen bij het Erasmus MC en voor onderwijs bij de HR. Wij geven u géén diepgaande technische uiteenzettingen over grid computing, daarvoor willen we u graag verwijzen naar de uitstekende vakliteratuur en de vele bronnen op het Internet. Wél voorbeelden van toepassingen en een pleidooi om grid computing binnen het onderwijs een betere basis te geven: er is behoefte aan goed opgeleide technici die een grid infrastructuur kunnen bouwen, beheren en gebruiken.

Light-emitting diode (LED) illumination for fluorescence microscopy systems has come a long way since the turn of the century, when the LED sources available had low optical power and a limited range of wavelengths. While the benefits of LED technology were attractive, their limitations prevented large-scale adoption. Twenty years later, advances in the semiconductor industry have eliminated many of those fundamental hurdles. Today, LED technology is rapidly replacing the lamps used in microscopy and fluorescence imaging, even for applications on the extreme ends of the light microscopy spectrum. This article describes the latest advances in illumination for fluorescence imaging, from near-UV to near-IR fluorophores. Case studies demonstrate the new X-Cite ® NOVEM ™ as successful replacements for traditional arc lamps in calcium imaging applications, producing equivalent results with the convenience of LEDs. For its usefulness to the microscopy community, the X-Cite NOVEM system was awarded the prestigious 2021 Microscopy Today Innovation Award.

Multi-wavelength standing wave (SW) microscopy and interference reflection microscopy (IRM) are powerful techniques that use optical interference to study the topographical structure of live and fixed cells. However, the use of more than two wavelengths to image the complex cell surface results in complicated topographical maps and it can be difficult to resolve the three-dimensional contours. We present a simple image processing method to reduce the thickness and spacing of antinodal fringes in multi-wavelength interference microscopy by up to a factor of two, with a view to producing clearer and more precise topographical maps of cellular structures. We first demonstrate this improvement using model non-biological specimens, and we subsequently demonstrate the benefit of our method for reducing the ambiguity of surface topography and revealing obscured features in live and fixed cell specimens imaged with widefield and point-scanning confocal illumination.

High throughput sequencing of eukaryotic, viral and bacterial genomes is providing a huge database of proteins with potential for structure-function analysis. In response to this opportunity, structural proteomics projects, including the Oxford Protein Production Facility have been established focused on high throughput (HTP) structure determination. Crucial to this effort has been the development of technologies for HTP protein production and crystallization. For all projects, there has been an emphasis on parallel processing driven by the need to accommodate relatively large numbers of potential targets at an acceptable cost. The OPPF, in common with most other groups has set up semi-automated liquid handling systems to carry out some of the protocols for protein production [1] and crystallization [2]. However, many of the methods can equally well be carried out manually with appropriate equipment e.g. multi-channel pipette dispensers. The motivation to implement automation is largely to enable processes to be scaleable and sustainable as error-free operations. Target selection in the OPPF is focused on human proteins and those of human pathogens, both viral and bacterial, selected for their direct biomedical relevance. Recent technical developments in OPPF to address some of these challenging targets will be reviewed.

ABSTRACTIn this article, we describe the engineering and X‐ray crystal structure of Thermal Green Protein (TGP), an extremely stable, highly soluble, non‐aggregating green fluorescent protein. TGP is a soluble variant of the fluorescent protein eCGP123, which despite being highly stable, has proven to be aggregation‐prone. The X‐ray crystal structure of eCGP123, also determined within the context of this paper, was used to carry out rational surface engineering to improve its solubility, leading to TGP. The approach involved simultaneously eliminating crystal lattice contacts while increasing the overall negative charge of the protein. Despite intentional disruption of lattice contacts and introduction of high entropy glutamate side chains, TGP crystallized readily in a number of different conditions and the X‐ray crystal structure of TGP was determined to 1.9 Å resolution. The structural reasons for the enhanced stability of TGP and eCGP123 are discussed. We demonstrate the utility...

In fluorescence microscopy, the photophysical properties of the fluorescent markers play a fundamental role. The beauty of phototransformable fluorescent proteins (PTFPs) is that some of these properties can be precisely controlled by light. A wide range of PTFPs have been developed in recent years, including photoactivatable, photoconvertible and photoswitchable fluorescent proteins. These smart labels triggered a plethora of advanced fluorescence methods to scrutinize biological cells or organisms dynamically, quantitatively and with unprecedented resolution. Despite continuous improvements, PTFPs still suffer from limitations, and mechanistic questions remain as to how these proteins precisely work.

Mesophotic coral ecosystems (30-150 m depth) present a high oceanic biodiversity, but remain one of the most understudied reef habitats, especially below 60 m depth. Here, we have assessed the rates of photosynthesis and dissolved inorganic carbon (DIC) and nitrogen (DIN) assimilation by Symbiodiniaceae associated with four soft coral species of the genus Sinularia and two stony coral species of the genus Leptoseris collected respectively at 65 and 80-90 m depth in the Gulf of Eilat. Our study demonstrates that both Leptoseris and Sinularia species have limited autotrophic capacities at mid-lower mesophotic depths. DIC and DIN assimilation rates were overall $ 10 times lower compared to shallow corals from 10 m depth in the same reef. While Leptoseris symbionts transferred at least 50% of the acquired nitrogen to their host after 8-h incubation, most of the nitrogen was retained in the symbionts of Sinularia. In addition, the host tissue of Sinularia species presented a very high structural carbon to nitrogen ratio (C : N) compared to Leptoseris or to the shallow coral species, suggesting nitrogen limitation in these mesophotic soft corals. The limited capacity of soft coral symbionts to acquire DIN and transfer it to the coral animal, as well as the high C : N ratios, might explain the scarcity of symbiotic soft corals at mid-lower mesophotic depths compared to their prevalence in the shallower reef. Overall, this study highlights the significance of DIN for the distribution of the Cnidarian-Symbiodiniaceae association at mesophotic depth.

Mesophotic reef corals remain largely unexplored in terms of the genetic adaptations and physiological mechanisms to acquire, allocate, and use energy for survival and reproduction. In the Hawaiian Archipelago, the Leptoseris species complex form the most spatially extensive mesophotic coral ecosystem known and provide habitat for a unique community. To study how the ecophysiology of Leptoseris species relates to symbiont–host specialization and understand the mechanisms responsible for coral energy acquisition in extreme low light environments, we examined Symbiodinium (endosymbiotic dinoflagellate) photobiological characteristics and the lipids and isotopic signatures from Symbiodinium and coral hosts over a depth‐dependent light gradient (55–7 μmol photons m−2 s−1, 60–132 m). Clear performance differences demonstrate different photoadaptation and photoacclimatization across this genus. Our results also show that flexibility in photoacclimatization depends primarily on Symbiodiniu...

Nowadays a growing number of biosensing systems in microand nanoscale rely in expensive and bulky equipment that must capable acc ur tely to resolve the angle or wavelength of optical interrogation. The costs, size, sensitiv ity, properties and etc which are required to assemble these biosensing tools, may limit the port ability of such systems, and may restrict their usefulness in sensing applications outside of a con trolled environment. This research covers creation and investigations of new piezoelectric ma teri ls based on PVDF and PZT with unique properties, i.e. synthesis, piezoelectric thin film formation, imprint of microperiodic structures, analysis of surface morphology by Atomic Force Micr oscopy, and analysis of diffraction efficiencies using laser diffractometer. These deve lopments of new piezoelectric films have made it possible to understand the strengthening an d deformation mechanisms in microand nanoscales together by bringing new insights in bio sensing applicat...

We demonstrate the applicability of time-correlated single photon counting multiphoton microscopy to the spatiotemporal localisation of protein-protein interactions in situ. An Example of a new fluorescent protein variant with enhanced properties are given and the development of a FRET biosensor for simultaneous measurement of multiple intra-and inter-molecular interactions is illustrated by experimental evidence of an energy transfer cascade via multiple acceptors. The juxtaposition of interacting population and FRET efficiency is elucidated, with a priori knowledge, by multi-exponential analysis.

Fluorescence microscopy in biomedical research is a light microscope technique designed to view fluorescence emission from a biological specimen. It has become an extremely useful tool to localize genes, messenger RNA and proteins within live and fixed cells and tissues and to visualize spatiotemporal variations in intracellular calcium as well as other ions and metabolites.

We study the decay characteristics of Frenkel excitons in solid-state enhanced green fluorescent protein (eGFP) dried from solution. We further monitor the changes of the radiative exciton decay over time by crossing the phase transition from the solved to the solid state. Complex interactions between protonated and deprotonated states in solid-state eGFP can be identified from temperature-dependent and time-resolved fluorescence experiments that further allow the determination of activation energies for each identified process.

The effects of fluorescence saturation on imaging in confocal microscopy have been studied. To include saturation it was necessary to deviate from the widely assumed linear relationship between the fluorescence and the illumination intensity. The lateral response for a point-like object, as well as the optical sectioning power, decreases depending on the degree of saturation. For very high illumination intensities the response for a saturated point object approached that of a conventional fluorescence microscope in which the fluorescence was not saturated. The decrease in the axial confocal response has been confirmed qualitatively by experiment.

 Users may download and print one copy of any publication from the public portal for the purpose of private study or research.  You may not further distribute the material or use it for any profit-making activity or commercial gain  You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.

Anionic phospholipids are essential structural components of cell membranes. Spatiotemporal dynamics of these lipids play central roles in regulating signalling events, membrane trafficking, maintenance of cell-shape, and cargo transport. On the other hand, defects in anionic phospholipid metabolism are linked to multiple diseases. Hence, the ability to visualize these phospholipids and their dynamics in living cells can afford mechanistic insights into vital cell processes, guide the development of therapeutics, and lead to diagnostic agents. In this exciting backdrop, fluorescent sensors that can detect anionic phospholipids become key chemical tools that can be used to image and track these bio-molecules in a confocal microscopy platform. In this review, we highlight existing chemical probes and sensing strategies for anionic phospholipids along with their pros and cons in the context of their applicability toward imaging and tracking these essential lipids in living cells.

Condor is used as the standard for batch systems on many real parallel super-computers as well as in many distributed trivial parallel computing grids. It is running in very diverse environments of the highest security levels as e.g. governmental or industry R&D. With a potential capacity of ~20.000 Gflops the ECG could become one the 20 biggest grids! Based on pool operator reports only! The GLOBE-Consortium: The Erasmus Computing Grid-Building a Super-Computer at Erasmus MC for FREE Knoch, T. A.

Salmonellosis caused by Salmonella sp. has long been reported all over the world. Despite the availability of various diagnostic methods, easy and effective detection systems are still required. This report describes a dialysis membrane electrode interface disc with immobilized specific antibodies to capture antigenic Salmonella cells. The interaction of a specific Salmonella antigen with a mouse anti-Salmonella monoclonal antibody complexed to rabbit anti-mouse secondary antibody conjugated with HRP and the substrate o-aminophenol resulted in a response signal output current measured using two electrode systems (cadmium reference electrode and glassy carbon working electrode) and an agilent HP34401A 6.5 digital multimeter without a potentiostat or applied potential input. A maximum response signal output current was recorded for various concentrations of Salmonella viz., 3, 30, 300, 3000, 30,000 and 300,000 cells. The biosensor has a detection limit of three cells, which is very se...

Background: Genomic and transcriptomic sequence data are essential tools for tackling ecological problems. Using an approach that combines next-generation sequencing, de novo transcriptome assembly, gene annotation and synthetic gene construction, we identify and cluster the protein families from Favia corals from the northern Red Sea. Results: We obtained 80 million 75 bp paired-end cDNA reads from two Favia adult samples collected at 65 m (Fav1, Fav2) on the Illumina GA platform, and generated two de novo assemblies using ABySS and CAP3. After removing redundancy and filtering out low quality reads, our transcriptome datasets contained 58,268 (Fav1) and 62,469 (Fav2) contigs longer than 100 bp, with N50 values of 1,665 bp and 1,439 bp, respectively. Using the proteome of the sea anemone Nematostella vectensis as a reference, we were able to annotate almost 20% of each dataset using reciprocal homology searches. Homologous clustering of these annotated transcripts allowed us to divide them into 7,186 (Fav1) and 6,862 (Fav2) homologous transcript clusters (E-value ≤ 2e-30). Functional annotation categories were assigned to homologous clusters using the functional annotation of Nematostella vectensis. General annotation of the assembled transcripts was improved 1-3% using the Acropora digitifera proteome. In addition, we screened these transcript isoform clusters for fluorescent proteins (FPs) homologs and identified seven potential FP homologs in Fav1, and four in Fav2. These transcripts were validated as bona fide FP transcripts via robust fluorescence heterologous expression. Annotation of the assembled contigs revealed that 1.34% and 1.61% (in Fav1 and Fav2, respectively) of the total assembled contigs likely originated from the corals' algal symbiont, Symbiodinium spp. Conclusions: Here we present a study to identify the homologous transcript isoform clusters from the transcriptome of Favia corals using a far-related reference proteome. Furthermore, the symbiont-derived transcripts were isolated from the datasets and their contribution quantified. This is the first annotated transcriptome of the genus Favia, a major increase in genomics resources available in this important family of corals.