The in vitro cytotoxic effects of natural (fibrous epsomite crystals) and synthetic (Epsom salt) magnesium sulfate.

Exposure to mineral fibers represents an occupational and environmental hazard since particulate inhalation leads to several health disorders. However, few data are available on the effect of fibers with high solubility like natural epsomite, a water-soluble fiber with an inhalable size that allows it to penetrate biological systems, with regard to the respiratory tract. This study evaluated the natural (fibrous epsomite) and synthetic (Epsom salt) magnesium sulfate pathogenicity. Investigations have been performed through morpho-functional and biochemical analyses, in an in vitro cell model that usually grows as monocytes, but that under appropriate conditions differentiates into macrophages. These latter, known as alveolar macrophages, if referred to lungs, represent the first line of defense against harmful inhaled stimuli. Morphological observations reveal that, if Epsom salt induces osmotic stress on cell culture, natural epsomite fibers lead to cellular alterations including thickening of the nuclear envelope and degenerated mitochondria. Moreover, the insoluble fraction (impurities) internalized by cells induces diffuse damage characterized at the highest dosage and exposure time by secondary necrosis or necrotic cell death features. Biochemical analyses confirm this mineral behavior that involves MAPK pathway activation, resulting in many different cellular responses ranging from proliferation control to cell death. Epsom salt leads to MAPK/ERK activation, a marker predictive of overall survival. Unlike, natural epsomite induces upregulation of MAPK/p38 protein involved in the phosphorylation of downstream targets driving necrotic cell death. These findings demonstrate natural epsomite toxicity on U937 cell culture, making the inhalation of these fibers potentially hazardous for human health. RESEARCH HIGHLIGHTS: Natural epsomite and synthetic Epsom salt effects have been evaluated in U937 cell model. Epsom salt induces an osmotic cellular stress. Natural epsomite fibers lead to cellular damage and can be considered potentially dangerous for human health.

Evaluation of the Effects of Electrical Stimulation: A Pilot Experiment on the Marine Benthic Foraminiferal Species Amphistegina lessonii.

Environmental disturbances resulting from anthropogenic energy pollution are intensely growing and represent a concern for the marine environment. Benthic organisms are the significant fauna exposed to this kind of pollution; among them, foraminifera are largely used as pollution bioindicators in marine environments, but studies on the effects induced by electrical stimulation are not documented. In the present research, we evaluated the effects of short-term different electric current densities on the viability of benthic foraminiferal species Amphistegina lessonii by checking the pseudopodial activity and defined the threshold electrical density range. After 3 days of treatment, A. lessonii stimulated with a constant current showed pseudopodial activity at a lower electric current density (0.29, 0.86 µA/cm2) up to 24 h. With increasing stimulation time, the percentages of pseudopodial activity decreased. The pseudopodial activity was absent at high current densities (5.71, 8.57 µA/cm2). The viability of A. lessonii exposed to a pulsed current was higher at a low and middle electric current density (from 0.29 to 5.71 µA/cm2) than at a high electric current density (from 11.43 to 20 µA/cm2). Based on these preliminary results, the selected benthic foraminiferal species seems to better stand pulsed currents than constant ones. These first experiments might provide useful information for the definition of the appropriate electrical density threshold to avoid side effects on a part of the benthic community.

Mercury-Induced Oxidative Stress Response in Benthic Foraminifera: An In Vivo Experiment on Amphistegina lessonii.

The evaluation of the effects of pollution (e.g., Hg pollution) is a difficult task and relies mostly on biomonitoring based on bioindicators. The application of biomarkers may represent a complementary or alternative approach in environmental biomonitoring. Mercury is known to pose a significant health hazard due to its ability to cross cellular membranes, bioaccumulate, and biomagnify. In the present research, the effects of short-term (i.e., 24 h) Hg exposure in the symbiont-bearing benthic foraminiferal species Amphistegina lessonii are evaluated using several biomarkers (i.e., proteins and enzymes). Mercury leads to significant changes in the biochemistry of cells. Its effects are mainly associated with oxidative stress (i.e., production of reactive oxygen species: ROS), depletion of glutathione (GSH), and alteration of protein synthesis. Specifically, our findings reveal that exposure to Hg leads to the consumption of GSH by GPx and GST for the scavenging of ROS and the activation of antioxidant-related enzymes, including SOD and GSH-enzymes (GST, GSR, GPx, and Se-GPx), that are directly related to a defense mechanism against ROS. The Hg exposure also activates the MAPK (e.g., p-p38) and HSP (e.g., HSP 70) pathways. The observed biochemical alterations associated with Hg exposure may represent effective and reliable proxies (i.e., biomarkers) for the evaluation of stress in A. lessonii and lead to a possible application for the detection of early warning signs of environmental stress in biomonitoring.

Could soluble minerals be hazardous to human health? Evidence from fibrous epsomite.

From a toxicological point of view, particulates and fibres with high solubility in water and/or in biological environments have not been considered in detail and the knowledge to date in this area is very scarce. In this study, the water-soluble natural epsomite fibres from Perticara Mine (Italy) were investigated using SEM-EDS, XRPD, ICP-AES and alpha spectrometry measurements which were combined and integrated to characterise the fibres' morphology, crystal chemistry and mineralogy. The morphological and morphometric results showed that most of the fibres are of inhalable size (Dae 5.09 µm) and can be potentially adsorbed from all parts of the respiratory tract. Chemical analysis reveals significant amounts of toxic elements (As, Co, Fe, Mn, Ni, Sr, Ti, Zn) and surprisingly high contents of radioactive isotopes (210Po and 228Th) in epsomite crystals, making the inhalation of these fibres potentially hazardous to human health. Through this study, we want to focus on soluble minerals, such as epsomite, which can be present in both natural and anthropic environments and have never been considered from the point of view of their potential hazard.

Characterisation of potentially toxic natural fibrous zeolites by means of electron paramagnetic resonance spectroscopy and morphological-mineralogical studies.

This study explored the morphological, mineralogical, and physico-chemical features of carcinogenic erionite and other possibly hazardous zeolites, such as mesolite and thomsonite, while also investigating the interacting capability of the mineral surface at the liquid/solid interface. Extremely fibrous erionite is K+ and Ca2+-rich and shows the highest Si/Al ratio (3.38) and specific surface area (8.14 m2/g). Fibrous mesolite is Na+ and Ca2+-rich and displays both a lower Si/Al ratio (1.56) and a smaller specific surface area (1.56 m2/g). The thomsonite composition shows the lowest values of Si/Al ratio (1.23) and specific surface area (0.38 m2/g). Electron paramagnetic resonance data from selected spin probes reveal that erionite has a homogeneous site distribution and interacts well with all spin probes. The surfaces of mesolite and thomsonite are less homogeneous and closer polar sites were found through consequent interaction with the probes. The mesolite surface can also clearly interact but with a lower strength and may represent a potential health hazard for humans, though with a lower degree if compared to erionite. The thomsonite surface is not inert and interacts with the probes with a low-grade capability. We can expect small fragments of thomsonite to interact with the biological environment, though with a low-grade intensity.

Protein Extractions from Amphistegina lobifera: Protocol Development and Optimization.

Proteins are essential to life, and the evaluation of their content, identification, and modification represents a fundamental assay in biochemistry research. Different analytical techniques and protocols have been specifically designed but have rarely been compared. Here, we test and compare a variety of methodologies and treatments for the quantification of proteins in Amphistegina lessonii, a larger symbiont-bearing benthic foraminiferal species. These analyses specifically include (a) lysis buffer (homemade vs. RIPA), (b) protein assays (Lowry, BCA, and Bradford), (c) ultrasonic bath treatment, and (d) protein staining (silver staining vs. Coomassie blue). On the basis of the comparative outcome, we suggest using the homemade lysis buffer, Lowry or BCA assays, ultrasonic bath treatment, and silver stain to maximize the extraction and characterization of protein for A. lessonii. This protocol might be suitable and extended to other benthic foraminiferal species, including the smaller ones.

Alpha-Tocopherol Metabolites (the Vitamin E Metabolome) and Their Interindividual Variability during Supplementation.

The metabolism of α-tocopherol (α-TOH, vitamin E) shows marked interindividual variability, which may influence the response to nutritional and therapeutic interventions with this vitamin. Recently, new metabolomics protocols have fostered the possibility to explore such variability for the different metabolites of α-TOH so far identified in human blood, i.e., the "vitamin E metabolome", some of which have been reported to promote important biological functions. Such advances prompt the definition of reference values and degree of interindividual variability for these metabolites at different levels of α-TOH intake. To this end, a one-week oral administration protocol with 800 U RRR-α-TOH/day was performed in 17 healthy volunteers, and α-TOH metabolites were measured in plasma before and at the end of the intervention utilizing a recently validated LC-MS/MS procedure; the expression of two target genes of α-TOH with possible a role in the metabolism and function of this vitamin, namely pregnane X receptor (PXR) and the isoform 4F2 of cytochrome P450 (CYP4F2) was assessed by immunoblot in peripheral blood leukocytes. The levels of enzymatic metabolites showed marked interindividual variability that characteristically increased upon supplementation. With the exception of α-CEHC (carboxy-ethyl-hydroxychroman) and the long-chain metabolites M1 and α-13'OH, such variability was found to interfere with the possibility to utilize them as sensitive indicators of α-TOH intake. On the contrary, the free radical-derived metabolite α-tocopheryl quinone significantly correlated with the post-supplementation levels of α-TOH. The supplementation stimulated PXR, but not CYP4F2, expression of leucocytes, and significant correlations were observed between the baseline levels of α-TOH and both the baseline and post-supplementation levels of PXR. These findings provide original analytical and molecular information regarding the human metabolism of α-TOH and its intrinsic variability, which is worth considering in future nutrigenomics and interventions studies.

A Bayesian model updating framework for robust seismic fragility analysis of non-isolated historic masonry towers.

Seismic assessment of existing masonry structures requires a numerical model able to both reproduce their nonlinear behaviour and account for the different sources of uncertainties; the latter have to be dealt with since the unavoidable lack of knowledge on the input parameters (material properties, geometry, boundary conditions, etc.) has a relevant effect on the reliability of the seismic response provided by the numerical approaches. The steadily increasing necessity of combining different sources of information/knowledge makes the Bayesian approach an appealing technique, not yet fully investigated for historic masonry constructions. In fact, while the Bayesian paradigm is currently employed to solve inverse problems in several sectors of the structural engineering domain, only a few studies pay attention to its effectiveness for parameter identification on historic masonry structures. This study combines a Bayesian framework with probabilistic structural analyses: starting from the Bayesian finite element model updating by using experimental data it provides the definition of robust seismic fragility curves for non-isolated masonry towers. A comparison between this method and the standard deterministic approach illustrates its benefits. This article is part of the theme issue 'Environmental loading of heritage structures'.

Excitotoxicity, neuroinflammation and oxidant stress as molecular bases of epileptogenesis and epilepsy-derived neurodegeneration: The role of vitamin E.

Glutamate-mediated excitotoxicity, neuroinflammation, and oxidative stress are common underlying events in neurodegeneration. This pathogenic "triad" characterizes the neurobiology of epilepsy, leading to seizure-induced cell death, increased susceptibility to neuronal synchronization and network alterations. Along with other maladaptive changes, these events pave the way to spontaneous recurrent seizures and progressive degeneration of the interested brain areas. In vivo models of epilepsy are available to explore such epileptogenic mechanisms, also assessing the efficacy of chemoprevention and therapy strategies at the pre-clinical level. The kainic acid model of pharmacological excitotoxicity and epileptogenesis is one of the most investigated mimicking the chronicization profile of temporal lobe epilepsy in humans. Its pathogenic cues include inflammatory and neuronal death pathway activation, mitochondrial disturbances and lipid peroxidation of several regions of the brain, the most vulnerable being the hippocampus. The importance of neuroinflammation and lipid peroxidation as underlying molecular events of brain damage was demonstrated in this model by the possibility to counteract the related maladaptive morphological and functional changes of this organ with vitamin E, the main fat-soluble cellular antioxidant and "conditional" co-factor of enzymatic pathways involved in polyunsaturated lipid metabolism and inflammatory signaling. The present review paper provides an overview of the literature supporting the potential for a timely intervention with vitamin E therapy in clinical management of seizures and epileptogenic processes associated with excitotoxicity, neuroinflammation and lipid peroxidation, i.e. the pathogenic "triad".

From Oxidative Stress Damage to Pathways, Networks, and Autophagy via MicroRNAs.

Oxidative stress can alter the expression level of many microRNAs (miRNAs), but how these changes are integrated and related to oxidative stress responses is poorly understood. In this article, we addressed this question by using in silico tools. We reviewed the literature for miRNAs whose expression is altered upon oxidative stress damage and used them in combination with various databases and software to predict common gene targets of oxidative stress-modulated miRNAs and affected pathways. Furthermore, we identified miRNAs that simultaneously target the predicted oxidative stress-modulated miRNA gene targets. This generated a list of novel candidate miRNAs potentially involved in oxidative stress responses. By literature search and grouping of pathways and cellular responses, we could classify these candidate miRNAs and their targets into a larger scheme related to oxidative stress responses. To further exemplify the potential of our approach in free radical research, we used our explorative tools in combination with ingenuity pathway analysis to successfully identify new candidate miRNAs involved in the ubiquitination process, a master regulator of cellular responses to oxidative stress and proteostasis. Lastly, we demonstrate that our approach may also be useful to identify novel candidate connections between oxidative stress-related miRNAs and autophagy. In summary, our results indicate novel and important aspects with regard to the integrated biological roles of oxidative stress-modulated miRNAs and demonstrate how this type of in silico approach can be useful as a starting point to generate hypotheses and guide further research on the interrelation between miRNA-based gene regulation, oxidative stress signaling pathways, and autophagy.

Neurobiological Correlates of Alpha-Tocopherol Antiepileptogenic Effects and MicroRNA Expression Modulation in a Rat Model of Kainate-Induced Seizures.

Seizure-triggered maladaptive neural plasticity and neuroinflammation occur during the latent period as a key underlying event in epilepsy chronicization. Previously, we showed that α-tocopherol (α-T) reduces hippocampal neuroglial activation and neurodegeneration in the rat model of kainic acid (KA)-induced status epilepticus (SE). These findings allowed us to postulate an antiepileptogenic potential for α-T in hippocampal excitotoxicity, in line with clinical evidence showing that α-T improves seizure control in drug-resistant patients. To explore neurobiological correlates of the α-T antiepileptogenic role, rats were injected with such vitamin during the latent period starting right after KA-induced SE, and the effects on circuitry excitability, neuroinflammation, neuronal death, and microRNA (miRNA) expression were investigated in the hippocampus. Results show that in α-T-treated epileptic rats, (1) the number of population spikes elicited by pyramidal neurons, as well as the latency to the onset of epileptiform-like network activity recover to control levels; (2) neuronal death is almost prevented; (3) down-regulation of claudin, a blood-brain barrier protein, is fully reversed; (4) neuroinflammation processes are quenched (as indicated by the decrease of TNF-α, IL-1ß, GFAP, IBA-1, and increase of IL-6); (5) miR-146a, miR-124, and miR-126 expression is coherently modulated in hippocampus and serum by α-T. These findings support the potential of a timely intervention with α-T in clinical management of SE to reduce epileptogenesis, thus preventing chronic epilepsy development. In addition, we suggest that the analysis of miRNA levels in serum could provide clinicians with a tool to evaluate disease evolution and the efficacy of α-T therapy in SE.

Radar detection of pedestrian-induced vibrations on Michelangelo's David.

This paper summarizes the results of a two-day dynamic monitoring of Michelangelo's David subject to environmental loads (city traffic and pedestrian loading induced by tourists visiting the Accademia Gallery). The monitoring was carried out by a no-contact technique using an interferometric radar, whose effectiveness in measuring the resonant frequencies of structures and historic monuments has proved over the last years through numerous monitoring activities. Owing to the dynamic behavior of the measurement system (radar and tripod), an accelerometer has been installed on the radar head to filter out the movement component of the measuring instrument from the measurement of the David's displacement. Measurements were carried out in the presence and absence of visitors, to assess their influence on the dynamic behavior of the statue. A numerical model of the statue was employed to evaluate the experimental results.

α-Tocopherol and Hippocampal Neural Plasticity in Physiological and Pathological Conditions.

Neuroplasticity is an "umbrella term" referring to the complex, multifaceted physiological processes that mediate the ongoing structural and functional modifications occurring, at various time- and size-scales, in the ever-changing immature and adult brain, and that represent the basis for fundamental neurocognitive behavioral functions; in addition, maladaptive neuroplasticity plays a role in the pathophysiology of neuropsychiatric dysfunctions. Experiential cues and several endogenous and exogenous factors can regulate neuroplasticity; among these, vitamin E, and in particular α-tocopherol (α-T), the isoform with highest bioactivity, exerts potent effects on many plasticity-related events in both the physiological and pathological brain. In this review, the role of vitamin E/α-T in regulating diverse aspects of neuroplasticity is analyzed and discussed, focusing on the hippocampus, a brain structure that remains highly plastic throughout the lifespan and is involved in cognitive functions. Vitamin E-mediated influences on hippocampal synaptic plasticity and related cognitive behavior, on post-natal development and adult hippocampal neurogenesis, as well as on cellular and molecular disruptions in kainate-induced temporal seizures are described. Besides underscoring the relevance of its antioxidant properties, non-antioxidant functions of vitamin E/α-T, mainly involving regulation of cell signaling molecules and their target proteins, have been highlighted to help interpret the possible mechanisms underlying the effects on neuroplasticity.

7-Ketocholesterol and 5,6-secosterol induce human endothelial cell dysfunction by differential mechanisms.

7-Ketocholesterol and 5,6-secosterol are cholesterol autoxidation products generated under oxidative stress by two distinct mechanisms. They are present in atherosclerotic plaques and are candidate players in the disease initiation and progression. While 7-ketocholesterol affects at cellular level, in particular apoptosis, are well known and reported on diverse cell lines, 5,6-secosterol is a recently discovered oxysterol with relatively few reports on the potential to affect endothelial cell functions. Endothelial cells have a central role in cardiovascular disease as they provide the barrier between blood and the vessel wall where atherosclerosis starts and progresses. Insults to endothelial cells provoke their dysfunction favoring pro-atherogenic and pro-thrombotic effects. In the present work, we tested 7-ketocholesterol and 5,6-secosterol on endothelial cells - focusing on apoptosis and the associated mitochondrial/lysosome alterations - and on endothelial function using the in vitro model of arterial relaxation of aortic rings. Our data provide evidence that 7-ketocholesterol and 5,6-secosterol are efficient instigators of apoptosis, which for 5,6-secosterol is associated to PKC and p53 up-regulation. In addition 5,6-secosterol is a potent inhibitor of endothelial-dependent arterial relaxation through PKC-dependent mechanisms. This may contribute to pro-atherogenic and pro-thrombotic mechanisms of 5,6-secosterol and highlights the role of cholesterol autoxidation in cardiovascular disease.

A multi-approach study of influence of growth temperature and nutrient deprivation in a strain of Aeromonas hydrophila.

In the present study we investigated the behavior of an Aeromonas hydrophila strain in prolonged nutrient deprivation condition analyzing the possible link among survival, cell morphology and adhesive characteristics and correlating them with the expression of the 43kDa outer membrane protein (OMP). The strain was inoculated in mineral and drinking chlorinated water, and in Nutrient Broth as a control with incubation at 4 and 24°C for 176days. Specimens were analyzed at different times during starvation stress. Viability was assessed by flow cytometry and growth by plate count technique; morphology and adhesivity were detected by optical and electron microscopy. The 43kDa OMP expression at different times was determined after immunoblotting assay using a polyclonal antibody produced in rabbit. The results showed a long-term viability as evidenced by cytofluorimetric analysis; however, the prolonged starvation led to the shift from the normal rod shaped cells to spherical forms in the last phases of incubation especially at 24°C. Concomitantly with the appearance of spherical cells we noted a reduction of the 43kDa OMP content and adhesive ability. Therefore, our results suggest a role of the 43kDa OMP as adhesin in A. hydrophila. In conclusion, we demonstrated that the bacterium can long survive under stress conditions, however adopting strategies which can lead to a loss of some cell surface components involved in the interactions with eukaryotic cells, therefore modifying its virulence properties.

Post-seizure α-tocopherol treatment decreases neuroinflammation and neuronal degeneration induced by status epilepticus in rat hippocampus.

Vitamin E (as α-tocopherol, α-T) was shown to have beneficial effects in epilepsy, mainly ascribed to its antioxidant properties. Besides radical-induced neurotoxicity, neuroinflammation is also involved in the pathophysiology of epilepsy, since neuroglial activation and cytokine production exacerbate seizure-induced neurotoxicity and contribute to epileptogenesis. We previously showed that α-T oral supplementation before inducing status epilepticus, markedly reduces astrocytic and microglial activation, neuronal cell death and oxidative stress in the hippocampus, as observed 4 days after seizure. In order to evaluate the possibility that such a neuroprotective and anti-inflammatory effect may also provide a strategy for an acute intervention in epilepsy, in this study, seizures were induced by single intaperitoneal injection of kainic acid and, starting from 3 h after status epilepticus, rats were treated with an intraperitoneal bolus of α-T (250 mg/kg b.w.; once a day) for 4 days, that was the time after which morphological and biochemical analyses were performed on hippocampus. Post-seizure α-T administration significantly reduced astrocytosis and microglia activation, and decreased neuron degeneration and spine loss; these effects were associated with the presence of a lowered lipid peroxidation in hippocampus. These results confirm and further emphasize the anti-inflammatory and neuroprotective role of α-T in kainic acid-induced epilepsy. Moreover, the findings show that post-seizure treatment with α-T provides an effective secondary prevention against post-seizure inflammation-induced brain damages and possibly against their epileptogenic effects.

Pharmacological doses of melatonin induce alterations in mitochondrial mass and potential, bcl-2 levels and K+ currents in UVB-exposed U937 cells.

Apoptosis is observed in 'actively' dying cells after the exposure to cell stressors such as ultraviolet light irradiation. Since melatonin has been proposed to act under stressful conditions as cell protection factor, in this study we examined the potential of this molecule when used at pharmacological concentrations to control mitochondrial damage and apoptotic signalling of UVB irradiated U937 human leukaemic cells. Moreover, the effect of melatonin treatment on electrophysiological properties and membrane K(+) currents of irradiated U937 cells was investigated as functional aspects relevant to the anti-apoptotic role of melatonin. The general effect is associated with the restoration of mass, number and membrane potential of mitochondria, with a lower caspase activation and bcl-2 upregulation. In the presence of the caspase inhibitor ZVAD-Fmk, melatonin seems to drive UVB stressed cells to follow the mitochondrial intrinsic pathway, interfering just at the mitochondrial level. Moreover, treatment with melatonin, as well as ZVAD-Fmk, prevented the K(+) current reduction observed late following the UVB insult application, by sparing cells from death; this result also indicates that the decrease of K(+) leakage currents could represent a functional feature of apoptotic process in UV-exposed U937 cells.

Motor activity affects adult skeletal muscle re-innervation acting via tyrosine kinase receptors.

Recently, muscle expression of brain-derived neurotrophic factor (BDNF) mRNA and protein under activity control has been reported. BDNF is a neurotrophin known to be involved in axon sprouting in the CNS. Hence, we set out to study the effect of chronic treadmill mid-intensity running on adult rat muscle re-innervation, and to explore the involvement of BDNF and tropomyosin-related kinase (Trk) receptors. After nerve crush, muscle re-innervation was evaluated using intracellular recordings, tension recordings, immunostaining and Western blot analyses. An enhanced muscle multiple innervation was found in running rats that was fully reversed to control values blocking Trk receptors or interrupting the running activity. An increase in muscle multiple innervation was also found in sedentary rats treated with a selective TrkB receptor agonist. The expression of TrkB receptors by intramuscular axons was demonstrated, and increased muscle expression of BDNF was found in running animals. The increase in muscle multiple innervation was consistent with the faster muscle re-innervation that we found in running animals. We conclude that, when regenerating axons contact muscle cells, muscle activity progressively increases modulating BDNF and possibly other growth factors, which in turn, acting via Trk receptors, induce axon sprouting to re-innervate skeletal muscle.

Mitochondrial-dependent anticancer activity of δ-tocotrienol and its synthetic derivatives in HER-2/neu overexpressing breast adenocarcinoma cells.

Anticancer activity and mitochondrial mechanism of the vitamin E form δ-tocotrienol (δ-T3) was investigated in HER-2/neu-overexpressing human SKBR3 and murine TUBO breast cancer cells. δ-T3 was confirmed to possess high cytotoxic and apoptotic activity in SKBR3 cells as compared with all natural forms of vitamin E and several synthetic forms that included novel derivatives with the same backbone of δ-T3 such as δ-tocotrienyl-succinyl amide (δ-T3AS) and the redox-active analogue δ-tocotrienyl amine (δ-T3NH2). As observed in the case of alpha-TOS, a prototypical anticancer drug derived from α-tocopherol, succinylation of δ-T3 enhanced citotoxicity and apoptotic activity of the vitamer. δ-T3 induced apoptosis of SKBR3 cells was associated with mitochondrial destabilization, energy failure, and unbalanced activity of stress/survival MAPKs, namely p38 and ERK1/2 pathways. An increased generation of ROS followed to such a series of early events. Enhanced activity of δ-T3 in this human carcinoma cell line was characterized by the sustained uptake and oxidative transformation to the quinone derivative δ-T3Q, thereby suggesting redox effects in SKBR3 mitochondria by this vitamer. Viability and uptake data show a different pattern of responses in TUBO cells with higher response to synthetic derivatives of δ-T3 than in SKBR3 cells. In conclusion, synthetic derivatives of δ-T3 with enhanced apoptotic activity in breast carcinoma cells are investigated for the first time in this study also describing mechanistic aspects of mitochondrial effects of δ-T3. Further investigation in preclinical models of HER2/neu-high breast adenocarcinoma is underway to identify other and more effective forms of VE in this type of cancer.

Insight on signal transduction pathways involved in phagocytosis in the colonial ascidian Botryllus schlosseri.

Tunicates are chordate invertebrates, closely related to vertebrates, which represent valuable organisms for the study of a variety of biological processes from an evolutionary point of view. As invertebrates, they rely on innate immunity to cope with foreign, potentially pathogenic material. Among tunicates, the compound ascidian Botryllus schlosseri is emerging as a reliable model organism for the study of innate immune responses. However, there is a general lack of knowledge on the signalling pathways activated during immune responses and, in particular, in phagocytosis. In the present work, we carried out a preliminary investigation of the signalling pathways involved in phagocytosis, with particular reference to MAPK activation. We studied in vitro zymosan phagocytosis in the presence of manumycin A, which inhibit the activation of Ras, PD98059, SP600125 and SB202190, inhibitors of Erk, JNK and p38, respectively, parthenolide, N-acetyl-l-cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC), inhibiting NF-kB activation. In addition we carried out immunoblot and immunocytochemistry analysis with the use of anti-pErk1/2, anti-pp38, anti-pJNK, anti-NF-kB (p50) and anti-pan Ras antibodies. Results demonstrate that the recognition of foreign cells triggers a phosphorylation cascade leading to the activation of Ras-like small GTPases, MAPKs and NF-kB and argue in favour of a conservation, also in ascidians, of the main signalling pathways.