Age-associated molecular changes are deleterious and may modulate life span through diet

Transition through life span is accompanied by numerous molecular changes, such as dysregulated gene expression, altered metabolite levels, and accumulated molecular damage. These changes are thought to be causal factors in aging; however, because they are numerous and are also influenced by genotype, environment, and other factors in addition to age, it is difficult to characterize the cumulative effect of these molecular changes on longevity. We reasoned that age-associated changes, such as molecular damage and tissue composition, may influence life span when used in the diet of organisms that are closely related to those that serve as a dietary source. To test this possibility, we used species-specific culture media and diets that incorporated molecular extracts of young and old organisms and compared the influence of these diets on the life span of yeast, fruitflies, and mice. In each case, the “old” diet or medium shortened the life span for one or both sexes. These findings suggest that age-associated molecular changes, such as cumulative damage and altered dietary composition, are deleterious and causally linked with aging and may affect life span through diet

The Role of Glypicans in Wnt Inhibitory Factor-1 Activity and the Structural Basis of Wif1's Effects on Wnt and Hedgehog Signaling

Proper assignment of cellular fates relies on correct interpretation of Wnt and Hedgehog (Hh) signals. Members of the Wnt Inhibitory Factor-1 (WIF1) family are secreted modulators of these extracellular signaling pathways. Vertebrate WIF1 binds Wnts and inhibits their signaling, but its Drosophila melanogaster ortholog Shifted (Shf) binds Hh and extends the range of Hh activity in the developing D. melanogaster wing. Shf activity is thought to depend on reinforcing interactions between Hh and glypican HSPGs. Using zebrafish embryos and the heterologous system provided by D. melanogaster wing, we report on the contribution of glypican HSPGs to the Wnt-inhibiting activity of zebrafish Wif1 and on the protein domains responsible for the differences in Wif1 and Shf specificity. We show that Wif1 strengthens interactions between Wnt and glypicans, modulating the biphasic action of glypicans towards Wnt inhibition; conversely, glypicans and the glypican-binding “EGF-like” domains of Wif1 are required for Wif1's full Wnt-inhibiting activity. Chimeric constructs between Wif1 and Shf were used to investigate their specificities for Wnt and Hh signaling. Full Wnt inhibition required the “WIF” domain of Wif1, and the HSPG-binding EGF-like domains of either Wif1 or Shf. Full promotion of Hh signaling requires both the EGF-like domains of Shf and the WIF domains of either Wif1 or Shf. That the Wif1 WIF domain can increase the Hh promoting activity of Shf's EGF domains suggests it is capable of interacting with Hh. In fact, full-length Wif1 affected distribution and signaling of Hh in D. melanogaster, albeit weakly, suggesting a possible role for Wif1 as a modulator of vertebrate Hh signaling

Biocatalysis as a green route for recycling the recalcitrant plastic polyethylene terephthalate

This paper presents a work in progress focused on facilitation of cross-cultural awareness between citizens of two European cities. We aim to engage visitors of telecom museums in Athens and Luxembourg to learn more about both cities by means of collaborative games played on multitouch tables. We also explore how live video-to-video streaming influences players’ behaviour and collaboration with remote players

Mutations that affect the survival of selected amacrine cell subpopulations define a new class of genetic defects in the vertebrate retina

AbstractAmacrine neurons are among the most diverse cell classes in the vertebrate retina. To gain insight into mechanisms vital to the production and survival of amacrine cell types, we investigated a group of mutations in three zebrafish loci: kleks (kle), chiorny (chy), and bergmann (bgm). Mutants of all three genes display a severe loss of selected amacrine cell subpopulations. The numbers of GABA-expressing amacrine interneurons are sharply reduced in all three mutants, while cell loss in other amacrine cell subpopulations varies and some cells are not affected at all. To investigate how amacrine cell loss affects retinal function, we performed electroretinograms on mutant animals. While the kle mutation mostly influences the function of the inner nuclear layer, unexpectedly the chy mutant phenotype also involves a loss of photoreceptor cell activity. The precise ratios and arrangement of amacrine cell subpopulations suggest that cell–cell interactions are involved in the differentiation of this cell class. To test whether defects of such interactions may be, at least in part, responsible for mutant phenotypes, we performed mosaic analysis and demonstrated that the loss of parvalbumin-positive amacrine cells in chy mutants is due to extrinsic (cell-nonautonomous) causes. The phenotype of another amacrine cell subpopulation, the GABA-positive cells, does not display a clear cell-nonautonomy in chy animals. These results indicate that environmental factors, possibly interactions among different subpopulations of amacrine neurons, are involved in the development of the amacrine cell class

Genome-wide RNAi ionomics screen reveals new genes and regulation of human trace element metabolism

Trace elements are essential for human metabolism and dysregulation of their homoeostasis is associated with numerous disorders. Here we characterize mechanisms that regulate trace elements in human cells by designing and performing a genome-wide high-throughput siRNA/ionomics screen, and examining top hits in cellular and biochemical assays. The screen reveals high stability of the ionomes, especially the zinc ionome, and yields known regulators and novel candidates. We further uncover fundamental differences in the regulation of different trace elements. Specifically, selenium levels are controlled through the selenocysteine machinery and expression of abundant selenoproteins; copper balance is affected by lipid metabolism and requires machinery involved in protein trafficking and post-translational modifications; and the iron levels are influenced by iron import and expression of the iron/haeme-containing enzymes. Our approach can be applied to a variety of disease models and/or nutritional conditions, and the generated data set opens new directions for studies of human trace element metabolism

Dlp and Dally enhance the effects of Wif1 expression in <i>Drosophila</i> wings.

<p>(A–L) <i>nub-Gal4</i> is used to drive transgene expression. (A) Wild-type wing. (B) Overexpression of <i>UAS-dlp</i> results in only slightly fewer bristles along the wing margin, no loss of L1 and no reduction in wing size. For a detailed comparison of bristle numbers, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002503#pgen.1002503.s003" target="_blank">Figure S3</a>. (C) Expression of <i>UAS-wif1</i> eliminates many bristles, interrupts L1 (arrow) and somewhat reduces wing size. (D) Co-expression of <i>UAS-dlp</i> and <i>UAS-wif1</i> almost completely eliminates wing margin bristles and L1, and strongly reduces wing size. (E) Expression of <i>UAS-wif1</i> causes much weaker wing margin defects in <i>dlp^A187/+</i> heterozygotes. (F) Overexpression of <i>UAS-dally</i> results in only very slightly fewer wing margin bristles and no obvious reductions in wing size. For detailed comparison of bristle numbers see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002503#pgen.1002503.s003" target="_blank">Figure S3</a>. (G) Combined expression of <i>UAS-dally</i> and <i>UAS-wif1</i> almost completely eliminates wing margin bristles and L1, and further reduces wing size. (H) Expression of <i>UAS-wif1</i> causes weaker wing margin defects in <i>dally⁸⁰</i>/+ heterozygotes (e.g. more complete anterior L1; compare arrows in C and H). (I–L) EGF-depleted Wif1 is less effective at inhibiting Wg signaling and does not interact with Dlp. Control wings expressing <i>pVal-UAS-wif1</i> show modest defects in margin development (I), which is synergistically enhanced by <i>UAS-dlp</i>; the arrow marks the interruption of L1 and the asterisks mark scalloping of the margin (J). <i>pVal-UAS-wif1</i>Δ<i>EGF</i> is less effective at reducing number of margin bristles than <i>pVal-UAS-wif1</i> and its effects on wing margin development are not enhanced by co-expression of <i>UAS-dlp</i> (J). See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002503#pgen.1002503.s003" target="_blank">Figure S3</a> for comparison of bristle numbers.</p

The EGF-like domains are interchangeable between Wif1 and Shf during Wif1-dependent Wg inhibition.

<p>(A) Domain compositions of Wif1, Shf and the two chimeric constructs. Open boxes show the ‘WIF’ domain, filled boxes the EGF-like domains. (B–F) <i>nub-Gal4</i>-driven misexpression of respective transgenes. (B) <i>UAS-WIF^wif1-EGF^shf</i> strongly reduces the density of anterior wing margin bristles and interrupts L1. Arrow and arrowheads denote L1 or lack of thereof, respectively. (C) Co-expression of <i>UAS-WIF^wif1-EGF^shf</i> and <i>UAS-dlp</i> almost completely eliminates wing margin bristles and L1, and reduces the size of the wing. (D, E) Expression of either <i>UAS-wif1</i> or <i>UAS-WIF^wif1-EGF^shf</i> similarly reduces ex-Wg levels on the surface of prospective margin cells (asterisks) and increases levels proximally. However, compared to <i>UAS-wif1</i>, <i>UAS-WIF^wif1-EGF^shf</i> expression does not increase ex-Wg as far proximally (compare red bars). (F, F′) Expression of two copies of <i>UAS-WIF^shf-EGF^wif1</i> does not alter wing shape or size, and has no measurable effect on margin bristles (anterior margin details in F′).</p

Wif1 stabilizes Wg on Dlp-expressing cells in late third instar wing discs.

<p>Wing pouch regions of wing imaginal discs. (A) <i>wg-lacZ</i> expression along the prospective wing margin (asterisk) where prospective dorsal (D) and ventral (V) wing blade surfaces abut. (B) Extracellular Wg (ex-Wg) from the <i>wg</i>-expressing cells, which is high distally and lower proximally. (C) Pattern of <i>nub-Gal4</i> expression, marked by <i>UAS-GFP</i>. In all subsequent panels, except for I-M, <i>nub-Gal4</i> is used to drive UAS-transgene expression. (D) ex-Wg after expression of <i>UAS-wif1</i>. ex-Wg is higher on proximal cells than on distal ones (red bracket). (E) Anti-Dlp staining in wild-type wing disc. Dlp expression is downregulated in distal cells of the prospective wing margin (red bracket). (F) Anti-Dlp staining after <i>UAS-wif1</i> expression. The width of the prospective wing margin region with reduced staining (red bracket) is narrowed compared to anti-Dlp staining in the wild-type disc in E. (G) ex-Wg staining after co-expression of <i>UAS-wif1</i> and <i>UAS-dlp</i>. ex-Wg is increased at the wing margin (asterisk). (H) ex-Wg staining after co-expression of <i>UAS-wif1</i> and <i>UAS-dlp RNAi</i> is similar to that in the wild-type disc in E. (I) Posterior expression of <i>UAS-dlp</i> (using <i>hh-Gal4</i>). ex-Wg accumulates in the posterior compartment. (J-M) Flpout <i>actin-Gal4</i> (<i>ac</i>) clones marked with <i>UAS-GFP</i> (green). (J) High ex-Wg levels inside and, to a lesser extent, outside clones expressing: <i>UAS-arm^S10</i>, <i>UAS-wif1</i>, and <i>UAS-dlp</i>. (K) Low, largely unchanged ex-Wg levels inside clones expressing <i>UAS-arm^S10</i> and <i>UAS-dlp</i>. (L) Reduced ex-Wg levels in clones expressing <i>UAS-wif1</i> and <i>UAS-arm^S10</i>. zWIF1 increases ex-Wg outside the clone. (M) Reduced ex-Wg levels in clones expressing Arm^S10. (N) After expression of <i>pVal-UAS-wif1</i>, ex-Wg staining is high proximally and low along the wing margin (asterisk). (O) Expression of <i>pVal-UAS-wif1</i>Δ<i>EGF</i> does not lead to a strong increase in proximal ex-Wg, and does not decrease ex-Wg along the wing margin (asterisk).</p

Gpc4 enhances the effects of full-length Wif1 in zebrafish embryos.

<p>Approximately 2 nL of mRNA of a given concentration was injected into one cell stage embryos, and embryos were scored at 28–30 hours post-fertilization. Images show representative examples of the penetrance of the short-tailed phenotype compared to an uninjected control. In top panels dorsal is up and anterior is to the left. Bar graphs show percentage of short-tailed embryos. The data is pooled from two independent experiments; frequencies were scored and their percentages were averaged. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002503#s4" target="_blank">Materials and Methods</a> for information on RNA preparation.</p

Wif1 affects Hh activity in <i>shf</i> discs.

<p>(A–C) Wild-type wing discs, showing the regions with high levels (red bars) of Ci^Act (A) or Ptc (B), or showing the ventral accumulation of Hh-GFP in the posterior compartment after dorsal, <i>ap-Gal4</i>-driven expression of <i>UAS-hh-GFP</i> (C). (D–F) <i>shf</i>/<i>Y</i> wing discs show reductions in the width of domains expressing high levels of Ci^Act (D) or Ptc (E), and also show reduced ventral accumulation of Hh-GFP in the posterior compartment after dorsal <i>ap-Gal4</i>-driven expression of <i>UAS-hh-GFP</i> (F). (G–I) <i>shf</i>/<i>Y</i> wing discs with <i>ap-Gal4</i>-driven expression of <i>UAS-wif1</i> have a broader domain of less intense anti-Ci^Act staining (G) and lower levels of anti-Ptc staining (H), but improve the ventral accumulation of Hh-GFP in the posterior compartment after <i>ap-Gal4</i>-driven expression of <i>UAS-hh-GFP</i> (I). Ventral Hh-GFP was more punctuate than in wild type discs (compare to C). To make it easier to see the differences in ventral Hh-GFP accumulation, levels were increased equally in the boxed regions in C, F, and I. Hh-GFP levels are quantified in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002503#pgen.1002503.s009" target="_blank">Figure S9</a>.</p