A comparison between two brine shrimp assays to detect in vitro cytotoxicity in marine natural products

Background  

The brine shrimp lethality assay is considered a useful tool for preliminary assessment of toxicity. It has also been suggested for screening pharmacological activities in plant extracts. However, we think that it is necessary to evaluate the suitability of the brine shrimp methods before they are used as a general bio-assay to test natural marine products for pharmacological activity.     

Inhibition of cholesterol transport impairs Cav-1 trafficking and small extracellular vesicles secretion,promoting amphisome formation in melanoma cells

Caveolin-1 (Cav-1) is a fundamental constituent of caveolae, whose functionality and structure are strictly dependent on cholesterol. In this work the U18666A inhibitor was used to study the role of cholesterol transport in the endosomal degradative-secretory system in a metastatic human melanoma cell line (WM266-4). We found that U18666A induces a shift of Cav-1 from the plasma membrane to the endolysosomal compartment, which is involved, through Multi Vesicular Bodies (MVBs), in the formation and release of small extracellular vesicles (sEVs). Moreover, this inhibitor induces an increase in the production of sEVs with chemical–physical characteristics similar to control sEVs but with a different protein composition (lower expression of Cav-1 and increase of LC3II) and reduced transfer capacity on target cells. Furthermore, we determined that U18666A affects mitochondrial function and also cancer cell aggressive features, such as migration and invasion. Taken together, these results indicate that the blockage of cholesterol transport, determining the internalization of Cav-1, may modify sEVs secretory pathways through an increased fusion between autophagosomes and MVBs to form amphisome, which in turn fuses with the plasma membrane releasing a heterogeneous population of sEVs to maintain homeostasis and ensure correct cellular functionality.     

Rapid shift in thermal resistance between generations through maternal heat exposure

Given the current rapid climate change, understanding the mechanisms underlying heat tolerance and its plasticity is an important goal of global change biology. Soil fauna communities are especially vulnerable because of their limited dispersal ability. It is generally recognized that transgenerational effects can contribute to the expression of phenotypic plasticity. Nevertheless, transgenerational plasticity in belowground organisms has received relatively little attention in the context of climate change, despite their major role in soil functioning. Here we test for transgenerational effects of heat shock exposure in the soil arthropod Orchesella cincta, a springtail species that regularly experiences heat stress conditions in its natural environment. We exposed females to heat stress, and subsequently investigated the effects of the same stress on the survival of their offspring. Thermal resistance of the progeny from treated and untreated mothers was compared at three life stages: egg, juvenile and adult. We provide evidence that exposure to heat shock induces a life stage‐dependent increase in thermal resistance in the subsequent generation. The induced adaptive maternal effect persisted into the adult stage of the progeny. However, there is also a tradeoff resulting in reduced clutch size of treated females. These results are of broad significance to understanding the potential of organisms to cope with a changing climate.     

CaMKII activity is reduced in skeletal muscle during sepsis

Exercise‐induced muscle hypertrophy is associated with increased calcium/calmodulin‐dependent protein kinase II (CaMKII) expression and activity. In contrast, the influence of muscle atrophy‐related conditions on CaMKII is poorly understood. Here, we tested the hypothesis that sepsis‐induced muscle wasting is associated with reduced CaMKII expression and activity. Sepsis, induced by cecal ligation and puncture in rats, and treatment of rats with TNFα, resulted in reduced total CaMKII activity in skeletal muscle whereas autonomous CaMKII activity was unaffected. The expression of CaMKIIδ, but not β and γ, was reduced in septic muscle. In additional experiments, treatment of cultured myotubes with TNFα resulted in reduced total CaMKII activity and decreased levels of phosphorylated glycogen synthase kinase (GSK)‐3β, a downstream target of CaMKII. The present results suggest that sepsis‐induced muscle wasting is associated with reduced CaMKII activity and that TNFα may be involved in the regulation of CaMKII activity in skeletal muscle. Decreased phosphorylation (consistent with activation) of GSK‐3β may be a consequence of reduced CaMKII activity, indicating that inhibited CaMKII activity may be involved in the catabolic response to sepsis. J. Cell. Biochem. 114: 1294–1305, 2013. © 2012 Wiley Periodicals, Inc.