Deconvolution of complex G protein-coupled receptor signaling in live cells using dynamic mass redistribution measurements

Label-free biosensor technology based on dynamic mass redistribution (DMR) of cellular constituents promises to translate GPCR signaling into complex optical 'fingerprints' in real time in living cells. Here we present a strategy to map cellular mechanisms that define label-free responses, and we compare DMR technology with traditional second-messenger assays that are currently the state of the art in GPCR drug discovery. The holistic nature of DMR measurements enabled us to (i) probe GPCR functionality along all four G-protein signaling pathways, something presently beyond reach of most other assay platforms; (ii) dissect complex GPCR signaling patterns even in primary human cells with unprecedented accuracy; (iii) define heterotrimeric G proteins as triggers for the complex optical fingerprints; and (iv) disclose previously undetected features of GPCR behavior. Our results suggest that DMR technology will have a substantial impact on systems biology and systems pharmacology as well as for the discovery of drugs with novel mechanisms

Drosophila selenophosphate synthetase 1 regulates vitamin B6 metabolism: prediction and confirmation

<p>Abstract</p> <p>Background</p> <p>There are two selenophosphate synthetases (SPSs) in higher eukaryotes, SPS1 and SPS2. Of these two isotypes, only SPS2 catalyzes selenophosphate synthesis. Although SPS1 does not contain selenophosphate synthesis activity, it was found to be essential for cell growth and embryogenesis in <it>Drosophila</it>. The function of SPS1, however, has not been elucidated.</p> <p>Results</p> <p>Differentially expressed genes in <it>Drosophila </it>SL2 cells were identified using two-way analysis of variance methods and clustered according to their temporal expression pattern. Gene ontology analysis was performed against differentially expressed genes and gene ontology terms related to vitamin B6 biosynthesis were found to be significantly affected at the early stage at which megamitochondria were not formed (day 3) after <it>SPS1 </it>knockdown. Interestingly, genes related to defense and amino acid metabolism were affected at a later stage (day 5) following knockdown. Levels of pyridoxal phosphate, an active form of vitamin B6, were decreased by <it>SPS1 </it>knockdown. Treatment of SL2 cells with an inhibitor of pyridoxal phosphate synthesis resulted in both a similar pattern of expression as that found by <it>SPS1 </it>knockdown and the formation of megamitochondria, the major phenotypic change observed by <it>SPS1 </it>knockdown.</p> <p>Conclusions</p> <p>These results indicate that SPS1 regulates vitamin B6 synthesis, which in turn impacts various cellular systems such as amino acid metabolism, defense and other important metabolic activities.</p

Deconvolution of complex G protein–coupled receptor signaling in live cells using dynamic mass redistribution measurements

Label-free biosensor technology based on dynamic mass redistribution (DMR) of cellular constituents promises to translate GPCR signaling into complex optical 'fingerprints' in real time in living cells. Here we present a strategy to map cellular mechanisms that define label-free responses, and we compare DMR technology with traditional second-messenger assays that are currently the state of the art in GPCR drug discovery. The holistic nature of DMR measurements enabled us to (i) probe GPCR functionality along all four G-protein signaling pathways, something presently beyond reach of most other assay platforms; (ii) dissect complex GPCR signaling patterns even in primary human cells with unprecedented accuracy; (iii) define heterotrimeric G proteins as triggers for the complex optical fingerprints; and (iv) disclose previously undetected features of GPCR behavior. Our results suggest that DMR technology will have a substantial impact on systems biology and systems pharmacology as well as for the discovery of drugs with novel mechanisms

Does Smile Intensity in Photographs Really Predict Longevity? A Replication and Extension of Abel and Kruger (2010)

Abel and Kruger (2010) found that the smile intensity of professional baseball players who were active in 1952, as coded from photographs, predicted these players' longevity. In the current investigation, we sought to replicate this result and to extend the initial analyses. We analyzed (a) a sample that was almost identical to the one from Abel and Kruger's study using the same database and inclusion criteria (N = 224), (b) a considerably larger nonoverlapping sample consisting of other players from the same cohort (N = 527), and (c) all players in the database (N = 13,530 valid cases). Like Abel and Kruger, we relied on categorical smile codings as indicators of positive affectivity, yet we supplemented these codings with subjective ratings of joy intensity and automatic codings of positive affectivity made by computer programs. In both samples and for all three indicators, we found that positive affectivity did not predict mortality once birth year was controlled as a covariate

Biosynthesis and accumulation of ergoline alkaloids in a mutualistic association between Ipomoea asarifolia (Convolvulaceae) and a clavicipitalean fungus

Ergoline alkaloids occur in taxonomically unrelated taxa, such as fungi, belonging to the phylum Ascomycetes and higher plants of the family Convolvulaceae. The disjointed occurrence can be explained by the observation that plant-associated epibiotic clavicipitalean fungi capable of synthesizing ergoline alkaloids colonize the adaxial leaf surface of certain Convolvulaceae plant species. The fungi are seed transmitted. Their capacity to synthesize ergoline alkaloids depends on the presence of an intact differentiated host plant (e.g. Ipomoea asarifolia or Turbina corymbosa [Convolvulaceae]). Here, we present independent proof that these fungi are equipped with genetic material responsible for ergoline alkaloid biosynthesis. The gene (dmaW) for the determinant step in ergoline alkaloid biosynthesis was shown to be part of a cluster involved in ergoline alkaloid formation. The dmaW gene was overexpressed in Saccharomyces cerevisiae, the encoded DmaW protein purified to homogeneity, and characterized. Neither the gene nor the biosynthetic capacity, however, was detectable in the intact I. asarifolia or the taxonomically related T. corymbosa host plants. Both plants, however, contained the ergoline alkaloids almost exclusively, whereas alkaloids are not detectable in the associated epibiotic fungi. This indicates that a transport system may exist translocating the alkaloids from the epibiotic fungus into the plant. The association between the fungus and the plant very likely is a symbiotum in which ergoline alkaloids play an essential role