A mTurquoise-Based cAMP Sensor for Both FLIM and Ratiometric Read-Out Has Improved Dynamic Range

FRET-based sensors for cyclic Adenosine Mono Phosphate (cAMP) have revolutionized the way in which this important intracellular messenger is studied. The currently prevailing sensors consist of the cAMP-binding protein Epac1, sandwiched between suitable donor- and acceptor fluorescent proteins (FPs). Through a conformational change in Epac1, alterations in cellular cAMP levels lead to a change in FRET that is most commonly detected by either Fluorescence Lifetime Imaging (FLIM) or by Sensitized Emission (SE), e.g., by simple ratio-imaging. We recently reported a range of different Epac-based cAMP sensors with high dynamic range and signal-to-noise ratio. We showed that constructs with cyan FP as donor are optimal for readout by SE, whereas other constructs with green FP donors appeared much more suited for FLIM detection. In this study, we present a new cAMP sensor, termed TEpacVV, which employs mTurquoise as donor. Spectrally very similar to CFP, mTurquoise has about doubled quantum efficiency and unlike CFP, its fluorescence decay is strictly single-exponential. We show that TEpacVV appears optimal for detection both by FLIM and SE, that it has outstanding FRET span and signal-to-noise ratio, and improved photostability. Hence, TEpacVV should become the cAMP sensor of choice for new experiments, both for FLIM and ratiometric detection

Development of infectious cDNA clones of Salmonid alphavirus subtype 3

<p>Abstract</p> <p>Background</p> <p>Salmonid alphavirus (SAV) is a widespread pathogen in European aquaculture of salmonid fish. Distinct viral subtypes have been suggested based on sequence comparisons and some of these have different geographical distributions. In Norway, only SAV subtype 3 have so far been identified. Little is known about viral mechanisms important for pathogenesis and transmission. Tools for detailed exploration of SAV genomes are therefore needed.</p> <p>Results</p> <p>Infectious cDNA clones in which a genome of subtype 3 SAV is under the control of a CMV promoter were constructed. The clones were designed to express proteins that are putatively identical to those previously reported for the SAVH20/03 strain. A polyclonal antiserum was raised against a part of the E2 glycoprotein in order to detect expression of the subgenomic open reading frame (ORF) encoding structural viral proteins. Transfection of the cDNA clone revealed the expression of the E2 protein by IFAT, and in serial passages of the supernatant the presence of infectious recombinant virus was confirmed through RT-PCR, IFAT and the development of a cytopathic effect similar to that seen during infection with wild type SAV. Confirmation that the recovered virus originated from the infectious plasmid was done by sequence identification of an introduced genetic tag. The recombinant virus was infectious also when an additional ORF encoding an EGFP reporter gene under the control of a second subgenomic alphavirus promoter was added. Finally, we used the system to study the effect of selected point mutations on infectivity in Chinook salmon embryo cells. While introduced mutations in nsP2₁₉₇, nsP3₂₆₃and nsP3₃₂₃severely reduced infectivity, a serine to proline mutation in E2₂₀₆appeared to enhance the virus titer production.</p> <p>Conclusion</p> <p>We have constructed infectious clones for SAV based on a subtype 3 genome. The clones may serve as a platform for further functional studies.</p

ATP Changes the Fluorescence Lifetime of Cyan Fluorescent Protein via an Interaction with His148

Recently, we described that ATP induces changes in YFP/CFP fluorescence intensities of Fluorescence Resonance Energy Transfer (FRET) sensors based on CFP-YFP. To get insight into this phenomenon, we employed fluorescence lifetime spectroscopy to analyze the influence of ATP on these fluorescent proteins in more detail. Using different donor and acceptor pairs we found that ATP only affected the CFP-YFP based versions. Subsequent analysis of purified monomers of the used proteins showed that ATP has a direct effect on the fluorescence lifetime properties of CFP. Since the fluorescence lifetime analysis of CFP is rather complicated by the existence of different lifetimes, we tested a variant of CFP, i.e. Cerulean, as a monomer and in our FRET constructs. Surprisingly, this CFP variant shows no ATP concentration dependent changes in the fluorescence lifetime. The most important difference between CFP and Cerulean is a histidine residue at position 148. Indeed, changing this histidine in CFP into an aspartic acid results in identical fluorescence properties as observed for the Cerulean fluorescent based FRET sensor. We therefore conclude that the changes in fluorescence lifetime of CFP are affected specifically by possible electrostatic interactions of the negative charge of ATP with the positively charged histidine at position 148. Clearly, further physicochemical characterization is needed to explain the sensitivity of CFP fluorescence properties to changes in environmental (i.e. ATP concentrations) conditions

No influence of oxygen levels on pathogenesis and virus shedding in Salmonid alphavirus (SAV)-challenged Atlantic salmon (Salmo salar L.)

<p>Abstract</p> <p>Background</p> <p>For more than three decades, diseases caused by salmonid alphaviruses (SAV) have become a major problem of increasing economic importance in the European fish-farming industry. However, experimental infection trials with SAV result in low or no mortality i.e very different from most field outbreaks of pancreas disease (PD). This probably reflects the difficulties in reproducing complex biotic and abiotic field conditions in the laboratory. In this study we looked at the relationship between SAV-infection in salmon and sub-lethal environmental hypoxia as a result of reduced flow-through in tank systems.</p> <p>Results</p> <p>The experiment demonstrated that constant reduced oxygen levels (60-65% oxygen saturation: 6.5-7.0 mg/L) did not significantly increase the severity or the progress of pancreas disease (PD). These conclusions are based upon assessments of a semi-quantitative histopathological lesion score system, morbidities/mortalities, and levels of SAV RNA in tissues and water (measured by 1 MDS electropositive virus filters and downstream real-time RT-PCR). Furthermore, we demonstrate that the fish population shed detectable levels of the virus into the surrounding water during viraemia; 4-13 days after i.p. infection, and prior to appearance of severe lesions in heart (21-35 dpi). After this period, viral RNA from SAV could not be detected in water samples although still present in tissues (gills and hearts) at lasting low levels. Lesions could be seen in exocrine pancreas at 7-21 days post infection, but no muscle lesions were seen.</p> <p>Conclusions</p> <p>In our study, experimentally induced hypoxia failed to explain the discrepancy between the severities reported from field outbreaks of SAV-disease and experimental infections. Reduction of oxygen levels to constant suboptimal levels had no effect on the severity of lesions caused by SAV-infection or the progress of the disease. Furthermore, we present a modified VIRADEL method which can be used to detect virus in water and to supplement experimental infection trials with information related to viral shedding. By using this method, we were able to demonstrate for the first time that shedding of SAV from the fish population into the surrounding water coincides with viraemia.</p

Human Muscle Satellite Cells as Targets of Chikungunya Virus Infection

BACKGROUND: Chikungunya (CHIK) virus is a mosquito-transmitted alphavirus that causes in humans an acute infection characterised by fever, polyarthralgia, head-ache, and myalgia. Since 2005, the emergence of CHIK virus was associated with an unprecedented magnitude outbreak of CHIK disease in the Indian Ocean. Clinically, this outbreak was characterized by invalidating poly-arthralgia, with myalgia being reported in 97.7% of cases. Since the cellular targets of CHIK virus in humans are unknown, we studied the pathogenic events and targets of CHIK infection in skeletal muscle. METHODOLOGY/PRINCIPAL FINDINGS: Immunohistology on muscle biopsies from two CHIK virus-infected patients with myositic syndrome showed that viral antigens were found exclusively inside skeletal muscle progenitor cells (designed as satelllite cells), and not in muscle fibers. To evaluate the ability of CHIK virus to replicate in human satellite cells, we assessed virus infection on primary human muscle cells; viral growth was observed in CHIK virus-infected satellite cells with a cytopathic effect, whereas myotubes were essentially refractory to infection. CONCLUSIONS/SIGNIFICANCE: This report provides new insights into CHIK virus pathogenesis, since it is the first to identify a cellular target of CHIK virus in humans and to report a selective infection of muscle satellite cells by a viral agent in humans

Low Temperature-Dependent Salmonid Alphavirus Glycoprotein Processing and Recombinant Virus-Like Particle Formation

Pancreas disease (PD) and sleeping disease (SD) are important viral scourges in aquaculture of Atlantic salmon and rainbow trout. The etiological agent of PD and SD is salmonid alphavirus (SAV), an unusual member of the Togaviridae (genus Alphavirus). SAV replicates at lower temperatures in fish. Outbreaks of SAV are associated with large economic losses of ∼17 to 50 million $/year. Current control strategies rely on vaccination with inactivated virus formulations that are cumbersome to obtain and have intrinsic safety risks. In this research we were able to obtain non-infectious virus-like particles (VLPs) of SAV via expression of recombinant baculoviruses encoding SAV capsid protein and two major immunodominant viral glycoproteins, E1 and E2 in Spodoptera frugiperda Sf9 insect cells. However, this was only achieved when a temperature shift from 27°C to lower temperatures was applied. At 27°C, precursor E2 (PE2) was misfolded and not processed by host furin into mature E2. Hence, E2 was detected neither on the surface of infected cells nor as VLPs in the culture fluid. However, when temperatures during protein expression were lowered, PE2 was processed into mature E2 in a temperature-dependent manner and VLPs were abundantly produced. So, temperature shift-down during synthesis is a prerequisite for correct SAV glycoprotein processing and recombinant VLP production

«La relation de limitation et d’exception dans le français d’aujourd’hui : excepté, sauf et hormis comme pivots d’une relation algébrique »

L’analyse des emplois prépositionnels et des emplois conjonctifs d’ “excepté”, de “sauf” et d’ “hormis” permet d’envisager les trois prépositions/conjonctions comme le pivot d’un binôme, comme la plaque tournante d’une structure bipolaire. Placées au milieu du binôme, ces prépositions sont forcées par leur sémantisme originaire dûment métaphorisé de jouer le rôle de marqueurs d’inconséquence systématique entre l’élément se trouvant à leur gauche et celui qui se trouve à leur droite. L’opposition qui surgit entre les deux éléments n’est donc pas une incompatibilité naturelle, intrinsèque, mais extrinsèque, induite. Dans la plupart des cas (emplois limitatifs), cette opposition prend la forme d’un rapport entre une « classe » et le « membre (soustrait) de la classe », ou bien entre un « tout » et une « partie » ; dans d’autres (emplois exceptifs), cette opposition se manifeste au contraire comme une attaque de front portée par un « tout » à un autre « tout ». De plus, l’inconséquence induite mise en place par la préposition/conjonction paraît, en principe, tout à fait insurmontable. Dans l’assertion « les écureuils vivent partout, sauf en Australie » (que l’on peut expliciter par « Les écureuils vivent partout, sauf [qu’ils ne vivent pas] en Australie »), la préposition semble en effet capable d’impliquer le prédicat principal avec signe inverti, et de bâtir sur une telle implication une sorte de sous énoncé qui, à la rigueur, est totalement inconséquent avec celui qui le précède (si « les écureuils ne vivent pas en Australie », le fait qu’ils « vivent partout » est faux). Néanmoins, l’analyse montre qu’alors que certaines de ces oppositions peuvent enfin être dépassées, d’autres ne le peuvent pas. C’est, respectivement, le cas des relations limitatives et des relations exceptives. La relation limitative, impliquant le rapport « tout » - « partie », permet de résoudre le conflit dans les termes d’une somme algébrique entre deux sous énoncés pourvus de différent poids informatif et de signe contraire. Les valeurs numériques des termes de la somme étant déséquilibrées, le résultat est toujours autre que zéro. La relation exceptive, au contraire, qui n’implique pas le rapport « tout » - « partie », n’est pas capable de résoudre le conflit entre deux sous énoncés pourvus du même poids informatif et en même temps de signe contraire : les valeurs numériques des termes de la somme étant symétriques et égales, le résultat sera toujours équivalent à zéro

Racine et radical

Présenter les résultats de recherches sur la question de la structure des mots, dans diverses langues naturelles, typologiquement aussi éloignées que le sont le français et l'arabe, le berbère et l'italien. Fournir quelques références pédagogiques aux étudiants intéressés par la linguistique, et plus particulièrement par la morphologie

Complex fluorescence of the cyan fluorescent protein: Comparisons with the H148D variant and consequences for quantitative cell imaging

cited By 37International audienceWe have studied the fluorescence decays of the purified enhanced cyan fluorescent protein (ECFP, with chromophore sequence Thr-Trp-Gly) and of its variant carrying the single H148D mutation characteristic of the brighter form Cerulean. Both proteins exhibit highly complex fluorescence decays showing strong temperature and pH dependences. At neutral pH, the H148D mutation leads (i) to a general increase in all fluorescence lifetimes and (ii) to the disappearance of a subpopulation, estimated to be more than 25% of the total ECFP molecules, characterized by a quenched and red-shifted fluorescence. The fluorescence lifetime distributions of ECFP and its H148D mutant remain otherwise very similar, indicating a high degree of structural and dynamic similarity of the two proteins in their major form. From thermodynamic analysis, we conclude that the multiexponential decay of ECFP cannot be simply ascribed, as is generally admitted, to the slow conformational exchange characterized by NMR and X-ray crystallographic studies [Seifert, M. H., et al. (2002) J. Am. Chem. Soc. 124, 7932-7942; Bae, J. H., et al. (2003) J. Mol. Biol. 328, 1071-1081]. Parallel measurements in living cells show that these fluorescence properties in neutral solution are very similar to those of cytosolic ECFP

Proton beam dosimetry at ultra high dose rates: evaluation of the usability and the dose rate dependency of various radiochromic films

International audienc