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

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

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Apoptosis in human sperm as a quality marker for a new diagnostic approach of infertile patients

The quality of the sperm DNA is one of the most important molecular markers of male reproductive potential (Bosco, L et al., 2018, Environ Toxicol Pharmacol 58:243-249). The aim of this study was to evaluate sperm DNA fragmentation according to morphology, to predict the probability of selecting a sperm with normal morphology and intact DNA. An observational study was performed on 70 patients with oligoasthenoteratozoospermia. Sperm DNA Fragmentation Index (DFI) and semen parameters such as sperm density, motility and morphology were evaluated in all patients. DFI was calculated using the in situ TUNEL assay. DFI can be determined and thus used as a marker of sperm quality for a diagnostic approach of infertile patients. For each patient, out of the total of sperm DFI, we calculated the proportion between sperm with normal morphology and with abnormal morphology. Among 35 patients (A Group), the DFI was inferior than 15% (average value was 8.1%), while 35 patients (B Group) had a DFI higher than 15% (average value was 24.6%). When the analysis was restricted only to spermatozoa with normal morphology, it was observed that among patients of B Group the DFI value was 13.6%, while in A Group the average was 2.2%. DFI calculated on sperm of normal morphology can provide an important information on the risk of injecting, during ICSI procedure, a sperm with normal morphology but with DNA fragmentation. This risk is higher if the semen sample has a baseline DFI higher than 15%

Magnesium deprivation affects development and biomineralization in the sea urchin arba-cia lixula

Skeletogenesis is a key morphogenetic event in the life of marine invertebrates. Marine calcifiers secrete their calcareous skeletons taking up ions from seawater. Marine biominerals include aragonite and calcite, the latter of which in some taxa (e.g. echinoderms, coralline algae) can have a substantial magnesium (Mg) component. Echinoderms have an extensive endoskeleton composed of high magnesian calcite and occluded matrix proteins1. As biomineralization in sea urchin larvae is sensitive to the Magnesium:Calcium ratio of sea water, we investigated the effects of magnesium deprivation on development and skeletogenesis in the Mediterranean sea urchin Arbacia lixula. Microscopic inspection revealed that embryos reared in Mg-free seawater exhibited developmental delay from 6 hours post-fertilization, complete lack of skeleton formation at 24 hours, and severe skeleton malformations in larvae (48-72 hours). We subsequently focused on the localization of the skeletogenic cells (primary mesenchyme cells) and the spatial expression of associated genes. Immunocytochemistry revealed abnormal ectopic location of the primary mesenchyme cells (PMCs) and of the developing skeleton of treated embryos. Expression of msp130, an important skeleton matrix protein gene expressed only in PMCs, detected by in situ hybridization, was normal at 24 hours, but this gene was not down-regulated at 48 hours, as in controls2. Strikingly, development of the pigment cells, immune cells that, like the skeleton, are mesodermal derivatives, was also impaired. These results suggest the essential role of Mg in skeleton formation in sea urchin embryos with an indication that this element is also generally important for development of mesoderm. 1. Smith AM et al. Mar Ecol Prog Ser 2016, 561:1–16 2. Martino C et al. Aq tox 2018, 194:57-66

EVALUATION OF OOCYTE QUALITY IN GRANULOSA AND CUMULUS CELLS OF PATIENTS UNDERGOING PMA

Background. We investigate the apoptosis rate of individual granulosa cell–oocyte and cumulus cell–oocyte (COC), associated with the levels of molecules playing a critical role in the regulation of cell death or survival. These molecular analyses have been done to verify the difference of competence between oocytes producing embryos able to reach the blastocyst stage compared with embryos arrested during the in vitro culture. Methods. From each single follicle: granulosa cells were processed for Western blotting analyses, using the following antibodies: pAKT, ERK 1/2, pERK 1/2; cumulus cells were used for in situ immunofluorescence with the same antibodies. DNA fragmentation rate was measured by TUNEL assay. Results. We have involved 58 patients and recovered 255 MII oocytes, of which 197 were fertilized and the derived embryos had the following evolution: 117 transferred, 57 vitrified and 23 arrested; 58 oocytes failed the fertilization or were in GV or MI stages. In the cumulus cells: we found a significant inverse correlation between oocytes resulting in transferred and arrested embryos in the ratio pAKT/TUNEL; nuclear localization of pERK1/2 showed a significant inverse correlation pERK1/2/TUNEL and a significant direct correlation with the intracellular accumulation of pERK1/2/pAKT. In granulosa cells: oocytes able to produce blastocysts, ERK1/2 /TUNEL ratio was higher than in cells of arrested embryos. Conclusions. Cumulus and granulosa cells showed different levels of expression of the investigated molecules. We found that in the cumulus cells of the oocytes able to produce blastocysts, the pAKT/TUNEL ratio is higher than in cumulus cells of arrested embryos, indicating that pAKT is involved in survival pathways. Moreover, pERK1/2 has an anti-apoptotic effect, when translocated into the nucleus. In granulosa cells: ERK1/2 indicates that it is involved in survival pathways. Briefly, we demonstrated that DNA fragmentation rate related to specific molecular levels could be considered a molecular marker of oocyte competence, for the evaluation of a prognostic pattern of blastocyst formation

Correlation among pAKT, pERK1/2 and DNA fragmentation index in human cumulus cells to determine oocyte competence

The aim was to correlate specific biological aspects of cumulus cells isolated from individual cumulus-oocyte complexes (COCs) with the clinical outcome of the related embryos

Sperm DNA integrity and human papillomavirus (HPV) infections: a controversy that could be resolved by a new molecular approach

Study question: The aim was to determine if HPVs affect spermatozoa DNA integrity. To resolve the discrepancy regarding the association between HPV infections and sperm DNA damage. Summary answer: To elucidate if HPV impairs DNA integrity, we suggest to investigate HPV DNA positivity in spermatozoa by a differential lysis procedure before TUNEL assay