Autophagy as a defense strategy against stress: focus on Paracentrotus lividus sea urchin embryos exposed to cadmium

Autophagy is used by organisms as a defense strategy to face environmental stress. This mechanism has been described as one of the most important intracellular pathways responsible for the degradation and recycling of proteins and organelles. It can act as a cell survival mechanism if the cellular damage is not too extensive or as a cell death mechanism if the damage/stress is irreversible; in the latter case, it can operate as an independent pathway or together with the apoptotic one. In this review, we discuss the autophagic process activated in several aquatic organisms exposed to different types of environmental stressors, focusing on the sea urchin embryo, a suitable system recently included into the guidelines for the use and interpretation of assays to monitor autophagy. After cadmium (Cd) exposure, a heavy metal recognized as an environmental toxicant, the sea urchin embryo is able to adopt different defense mechanisms, in a hierarchical way. Among these, autophagy is one of the main responses activated to preserve the developmental program. Finally, we discuss the interplay between autophagy and apoptosis in the sea urchin embryo, a temporal and functional choice that depends on the intensity of stress conditions

Hsp56 protein and mRNA distribution in normal and stressed P.lividus embryos

It was previously demonstrated that Paracentrotus lividus Hsp56 mitochondrial chaperonin is con- stitutively expressed during development, that it increases after heat-shock and cadmium treatment, and that it has a speci\ufb01c territorial distribution, both in normal and heat-shocked embryos, as shown by immunolocalization experiments. In this work, we analyzed by Western blot the territorial distribution of the protein in plutei exposed to heat-shock or sublethal cadmium concentrations, and we found that Hsp56 increases in both ectodermal and en- dodermal cells. Moreover, by \u201cin situ\u201d hybridization, we looked at Hsp56 mRNA during normal development and under stress conditions. We found that the territorial distribution of the messenger changes during development and that its amount is steadily increased in stressed embryos. Finally, by T1 RNase assay, we identi\ufb01ed a cytoplasmic factor that binds to the region of Hsp56 messenger containing the 5\u2019UT

Toxicological Impact of Rare Earth Elements (REEs) on the Reproduction and Development of Aquatic Organisms Using Sea Urchins as Biological Models

The growing presence of lanthanides in the environment has drawn the attention of the scientific community on their safety and toxicity. The sources of lanthanides in the environment include diagnostic medicine, electronic devices, permanent magnets, etc. Their exponential use and the poor management of waste disposal raise serious concerns about the quality and safety of the ecosystems at a global level. This review focused on the impact of lanthanides in marine organisms on reproductive fitness, fertilization and embryonic development, using the sea urchin as a biological model system. Scientific evidence shows that exposure to lanthanides triggers a wide variety of toxic insults, including reproductive performance, fertilization, redox metabolism, embryogenesis, and regulation of embryonic gene expression. This was thoroughly demonstrated for gadolinium, the most widely used lanthanide in diagnostic medicine, whose uptake in sea urchin embryos occurs in a time-and concentration-dependent manner, correlates with decreased calcium absorption and primarily affects skeletal growth, with incorrect regulation of the skeletal gene regulatory network. The results collected on sea urchin embryos demonstrate a variable sensitivity of the early life stages of different species, highlighting the importance of testing the effects of pollution in different species. The accumulation of lanthanides and their emerging negative effects make risk assessment and consequent legislative intervention on their disposal mandatory

Toxicity induced by Gadolinium ions on sea urchin embryos: comparison among phylogenetically distant species and focus on stress response and skeletogenesis.

Pharmaceuticals are a class of emerging environmental contaminants. Gadolinium (Gd) is a lanthanide metal whose chelates are employed as contrast agents for magnetic resonance imaging, and subsequently released into the aquatic environment. We investigated the effects of exposure to sublethal Gd concentrations on the development of four phylogenetically and geographically distant sea urchin species: two Mediterranean, Paracentrotus lividus and Arbacia lixula, and two from Australia, Heliocidaris tuberculata and Centrostephanus rodgersii. Sensitivity to Gd greatly varied, with EC50 ranging from 56 nM to 132 µM across the four species. Measures of the Gd and Ca content inside embryos showed a time- and dose-dependent increase in Gd, in parallel with a reduction in Ca. In all the four species, we observed a general delay of embryo development at 24h post-fertilization, and a strong inhibition of skeleton growth at 48h. Further experiments were carried out on P. lividus embryos: RT-PCR gene expression analysis showed the misregulation of several genes implicated both in the skeletogenic and the left-right axis specification networks. WB analysis showed an increase of the LC3 autophagic marker at 24 and 48h. Confocal microscopy studies confirmed the increased number of autophagosomes and autophagolysosomes and showed no apoptotic induction. The results show the hazard of Gd in the marine environment, indicating that Gd is able to affect three different levels in sea urchin embryos: morphogenesis, stress response such as autophagy, and gene expression. Results highlight that pollution assays based on only one species can be misleading with respect to hazard risk assessment

Measurement of the Bottom-Strange Meson Mixing Phase in the Full CDF Data Set

We report a measurement of the bottom-strange meson mixing phase \beta_s using the time evolution of B0_s -> J/\psi (->\mu+\mu-) \phi (-> K+ K-) decays in which the quark-flavor content of the bottom-strange meson is identified at production. This measurement uses the full data set of proton-antiproton collisions at sqrt(s)= 1.96 TeV collected by the Collider Detector experiment at the Fermilab Tevatron, corresponding to 9.6 fb-1 of integrated luminosity. We report confidence regions in the two-dimensional space of \beta_s and the B0_s decay-width difference \Delta\Gamma_s, and measure \beta_s in [-\pi/2, -1.51] U [-0.06, 0.30] U [1.26, \pi/2] at the 68% confidence level, in agreement with the standard model expectation. Assuming the standard model value of \beta_s, we also determine \Delta\Gamma_s = 0.068 +- 0.026 (stat) +- 0.009 (syst) ps-1 and the mean B0_s lifetime, \tau_s = 1.528 +- 0.019 (stat) +- 0.009 (syst) ps, which are consistent and competitive with determinations by other experiments.Comment: 8 pages, 2 figures, Phys. Rev. Lett 109, 171802 (2012

Defensome against Toxic Diatom Aldehydes in the Sea Urchin Paracentrotus lividus

Many diatom species produce polyunsaturated aldehydes, such as decadienal, which compromise embryonic and larval development in benthic organisms. Here newly fertilized Paracentrotus lividus sea urchins were exposed to low concentration of decadienal and the expression levels of sixteen genes, implicated in a broad range of functional responses, were followed by Real Time qPCR in order to identify potential decadienal targets. We show that at low decadienal concentrations the sea urchin Paracentrotus lividus places in motion different classes of genes to defend itself against this toxic aldehyde, activating hsp60 and two proteases, hat and BP10, at the blastula stage and hsp56 and several other genes (14-3-3ε, p38 MAPK, MTase, and GS) at the prism stage. At this latter stage all genes involved in skeletogenesis (Nec, uni, SM50 and SM30) were also down-expressed, following developmental abnormalities that mainly affected skeleton morphogenesis. Moreover, sea urchin embryos treated with increasing concentrations of decadienal revealed a dose-dependent response of activated target genes. Finally, we suggest that this orchestrated defense system against decadienal represents part of the chemical defensome of P. lividus affording protection from environmental toxicants

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field