Superoxide radical production by sponges Sycon sp.

AbstractUsing the catechol Tiron as an O−⋅2 scavenger, we showed that sea sponges (Sycon sp.) produce superoxide radicals in sea water at a high rate without any stimuli added. The rate of O−⋅2 outflow from sponges to their water surroundings reaches a value of 0.5 nmol/min per sponge at pH 6.5. The generation of O−⋅2 was inhibited by Cu,Zn-superoxide dismutase, and restored by the addition of KCN. We also confirmed the abiotic production of O−⋅2 in sea water, detected earlier with a different method by Petasne and Zika [Nature 325 (1987) 516–518]

Single fluorescent protein-based Ca2+ sensors with increased dynamic range

<p>Abstract</p> <p>Background</p> <p>Genetically encoded sensors developed on the basis of green fluorescent protein (GFP)-like proteins are becoming more and more popular instruments for monitoring cellular analytes and enzyme activities in living cells and transgenic organisms. In particular, a number of Ca²⁺sensors have been developed, either based on FRET (Fluorescence Resonance Energy Transfer) changes between two GFP-mutants or on the change in fluorescence intensity of a single circularly permuted fluorescent protein (cpFP).</p> <p>Results</p> <p>Here we report significant progress on the development of the latter type of Ca²⁺sensors. Derived from the knowledge of previously reported cpFP-based sensors, we generated a set of cpFP-based indicators with different spectral properties and fluorescent responses to changes in Ca²⁺concentration. Two variants, named Case12 and Case16, were characterized by particular high brightness and superior dynamic range, up to 12-fold and 16.5-fold increase in green fluorescence between Ca²⁺-free and Ca²⁺-saturated forms. We demonstrated the high potential of these sensors on various examples, including monitoring of Ca²⁺response to a prolonged glutamate treatment in cortical neurons.</p> <p>Conclusion</p> <p>We believe that expanded dynamic range, high brightness and relatively high pH-stability should make Case12 and Case16 popular research tools both in scientific studies and high throughput screening assays.</p

Promotion of manual drilling in Guinea

In the last decade UNICEF has supported manual drilling in several countries as a possible low cost and sustainable strategy to increase adequate water supply for the population. In partnership with local authorities and other stakeholders, UNICEF has implemented different activities to ensure high professional level in manual drilling: mapping of suitable zones, capacity building in construction of drilling tools and application of different drilling techniques, good practice in manual drilling, organization management. In Guinea manual drilling was unknown before 2011; at that time the joint program of SNAPE (National Water Authority) and UNICEF aiming to create an efficient manual drilling sector started, and after 3 years Guinea can be considered one of the most positive example of implementation of this program

Identification and In Vivo Characterization of NvFP-7R, a Developmentally Regulated Red Fluorescent Protein of Nematostella vectensis

In recent years, the sea anemone Nematostella vectensis has emerged as a critical model organism for comparative genomics and developmental biology. Although Nematostella is a member of the anthozoan cnidarians (known for producing an abundance of diverse fluorescent proteins (FPs)), endogenous patterns of Nematostella fluorescence have not been described and putative FPs encoded by the genome have not been characterized.We described the spatiotemporal expression of endogenous red fluorescence during Nematostella development. Spatially, there are two patterns of red fluorescence, both restricted to the oral endoderm in developing polyps. One pattern is found in long fluorescent domains associated with the eight mesenteries and the other is found in short fluorescent domains situated between tentacles. Temporally, the long domains appear simultaneously at the 12-tentacle stage. In contrast, the short domains arise progressively between the 12- and 16-tentacle stage. To determine the source of the red fluorescence, we used bioinformatic approaches to identify all possible putative Nematostella FPs and a Drosophila S2 cell culture assay to validate NvFP-7R, a novel red fluorescent protein. We report that both the mRNA expression pattern and spectral signature of purified NvFP-7R closely match that of the endogenous red fluorescence. Strikingly, the red fluorescent pattern of NvFP-7R exhibits asymmetric expression along the directive axis, indicating that the nvfp-7r locus senses the positional information of the body plan. At the tissue level, NvFP-7R exhibits an unexpected subcellular localization and a complex complementary expression pattern in apposed epithelia sheets comprising each endodermal mesentery.These experiments not only identify NvFP-7R as a novel red fluorescent protein that could be employed as a research tool; they also uncover an unexpected spatio-temporal complexity of gene expression in an adult cnidarian. Perhaps most importantly, our results define Nematostella as a new model organism for understanding the biological function of fluorescent proteins in vivo

Monitoring of Gene Expression in Bacteria during Infections Using an Adaptable Set of Bioluminescent, Fluorescent and Colorigenic Fusion Vectors

A family of versatile promoter-probe plasmids for gene expression analysis was developed based on a modular expression plasmid system (pZ). The vectors contain different replicons with exchangeable antibiotic cassettes to allow compatibility and expression analysis on a low-, midi- and high-copy number basis. Suicide vector variants also permit chromosomal integration of the reporter fusion and stable vector derivatives can be used for in vivo or in situ expression studies under non-selective conditions. Transcriptional and translational fusions to the reporter genes gfpmut3.1, amCyan, dsRed2, luxCDABE, phoA or lacZ can be constructed, and presence of identical multiple cloning sites in the vector system facilitates the interchange of promoters or reporter genes between the plasmids of the series. The promoter of the constitutively expressed gapA gene of Escherichia coli was included to obtain fluorescent and bioluminescent expression constructs. A combination of the plasmids allows simultaneous detection and gene expression analysis in individual bacteria, e.g. in bacterial communities or during mouse infections. To test our vector system, we analyzed and quantified expression of Yersinia pseudotuberculosis virulence genes under laboratory conditions, in association with cells and during the infection process

The evolution of multiple active site configurations in a designed enzyme

Developments in computational chemistry, bioinformatics, and laboratory evolution have facilitated the de novo design and catalytic optimization of enzymes. Besides creating useful catalysts, the generation and iterative improvement of designed enzymes can provide valuable insight into the interplay between the many phenomena that have been suggested to contribute to catalysis. In this work, we follow changes in conformational sampling, electrostatic preorganization, and quantum tunneling along the evolutionary trajectory of a designed Kemp eliminase. We observe that in the Kemp Eliminase KE07, instability of the designed active site leads to the emergence of two additional active site configurations. Evolutionary conformational selection then gradually stabilizes the most efficient configuration, leading to an improved enzyme. This work exemplifies the link between conformational plasticity and evolvability and demonstrates that residues remote from the active sites of enzymes play crucial roles in controlling and shaping the active site for efficient catalysis

forums

E-learning standards as a basis for contextua

Qualitative proteomic analysis of Tipula oleracea nudivirus occlusion bodies

Nudiviruses are arthropod-specific large double-stranded circular DNA viruses, related to baculoviruses, which replicate in the nucleus of the cells they infect. Up to date six fully sequenced nudiviral genomes are available in databases and protein profile from nudivirus particles were mainly characterized by polyacrylamide gel electrophoresis. However, only few direct matches were completed between genomic and proteomic data to the exception of the major occlusion body protein from Penaeus monodon nudivirus (PmNV) and four nucleocapsid proteins from Helicoverpa zea nudivirus 2 (HzNV-2). Function of predicted nudiviral proteins is still inferred from what is known from baculoviruses or endogenous nudiviruses (i.e. bracoviruses). Tipula oleracea nudivirus (ToNV) is the causative agent of crane fly nucleopolyhedrosis. With PmNV, ToNV is the second fully sequenced nudivirus to be described as forming occlusion bodies. Protein profile revealed by Coomassie-stained SDS-PAGE is quite similar to those observed for other nudiviruses with five major protein bands of about 75, 48, 35, 25 and 12 kDa. Proteomic analysis using on-line nanoflow liquid chromatography tandem high resolution mass spectrometry revealed ToNV occlusion bodies are composed of 52 viral proteins, the most abundant of which are the functional homolog of baculovirus polyhedrin/granulin and the homologs of three HzNV-2 predicted proteins: the two virion structural proteins 34K (Hz2V052, the baculovirus capsid protein VP39 homolog) and 11K (Hz2V025); and the hypothetical protein Hz2V079, a newly identified nudivirus core gene product