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A central aspect of nervous system development and function is the post-transcriptional regulation of mRNA fate, which implies time- and site-dependent translation, in response to cues originating from cell-to-cell crosstalk. Such events are fundamental for the establishment of brain cell asymmetry, as well as of long-lasting modifications of synapses (long-term potentiation: LTP), responsible for learning, memory, and higher cognitive functions. Post-transcriptional regulation is in turn dependent on RNA-binding proteins that, by recognizing and binding brief RNA sequences, base modifications, or secondary/tertiary structures, are able to control maturation, localization, stability, and translation of the transcripts. Notably, most RBPs contain intrinsically disordered regions (IDRs) that are thought to be involved in the formation of membrane-less structures, probably due to liquid–liquid phase separation (LLPS). Such structures are evidenced as a variety of granules that contain proteins and different classes of RNAs. The other side of the peculiar properties of IDRs is, however, that, under altered cellular conditions, they are also prone to form aggregates, as observed in neurodegeneration. Interestingly, RBPs, as part of both normal and aggregated complexes, are also able to enter extracellular vesicles (EVs), and in doing so, they can also reach cells other than those that produced them.

Using the timely re-activation of WNT signalling in neuralizing human induced pluripotent stem cells (hiPSCs), we have produced neural progenitor cells with a gene expression profile typical of human embryonic dentate gyrus (DG) cells. Notably, in addition to continuous WNT signalling, a specific laminin isoform is crucial to prolonging the neural stem state and to extending progenitor cell proliferation for over 200 days in vitro. Laminin 511 is indeed specifically required to support proliferation and to inhibit differentiation of hippocampal progenitor cells for extended time periods when compared with a number of different laminin isoforms assayed. Global gene expression profiles of these cells suggest that a niche of laminin 511 and WNT signalling is sufficient to maintain their capability to undergo typical hippocampal neurogenesis. Moreover, laminin 511 signalling sustains the expression of a set of genes responsible for the maintenance of a hippocampal neurogenic niche. Finally, xenograft of human DG progenitors into the DG of adult immunosuppressed host mice produces efficient integration of neurons that innervate CA3 layer cells spanning the same area of endogenous hippocampal neuron synapses.

It is known that in breast cancer biology, autophagy mainly plays a cytoprotective and anti-apoptotic role in vitro, being conceivably responsible for cell resistance to drug exposure and a higher metastatic attitude in vivo. Thus, the development of novel autophagy-targeting agents represents a valuable strategy to improve the efficacy of anticancer interventions. It is widely acknowledged that the enormous biodiversity of marine organisms represents a highly promising reserve for the isolation of bioactive primary and secondary metabolites targeting one or several specific molecular pathways and displaying active pharmacological properties against a variety of diseases. The aim of this review is to pick up selected studies that report the extraction and identification of marine animal-derived extracts or isolated compounds which exert a modulatory effect on the autophagic process in breast cancer cells and list them with respect to the taxonomical hierarchy of the producing species. Where available, the molecular and biochemical aspects associated with the molecules or extracts under discussion will be also summarized.

Pyrrolomycins (PMs) are a family of naturally occurring antibiotic agents, isolated from the fermentation broth of Actinosporangium and Streptomyces species. Pursuing our studies on pyrrolomycins, we performed the total synthesis of the F-series pyrrolomycins (1–4) by microwave-assisted synthesis (MAOS), thus obtaining the title compounds in excellent yields (63–69%). Considering that there is no evidence so far of the anticancer effect of this class of compounds, we investigated PMs for their antiproliferative activity against HCT116 and MCF-7 cancer cell lines. PMs showed anticancer activity at submicromolar level with a minimal effect on normal epithelial cell line (hTERT RPE-1), and they were able to induce several morphological changes including elongated cells, cytoplasm vacuolization, long and thin filopodia as well as the appearance of tunneling nanotubes (TNTs). These data suggest that PMs could act by impairing the cell membranes and the cytoskeleton organization, with subsequent increase of ROS generation and the activation of different forms of non-apoptotic cell death.

Purpose To evaluate the correlation between the DNA Fragmentation Index (DFI) and sperm morphology in patients undergoing ICSI, as a predictive parameter in reproductive outcomes. Methods A retrospective study was conducted on 125 infertile patients enrolled in a fertility clinic. Seminal characteristics were measured following the WHO guidelines (2010) for the examination of the seminal fluid. After collecting motile sperm population by pellet swim up, DFI was calculated and simultaneously associated with sperm morphology using in situ TUNEL assay and an image analyzer software in at least 250 spermatozoa for each patient. Results All subjects were divided into two groups according to a cutoff established, by choice, of the sperm DFI (15%): group A (< 15%) consisting of 65 patients and group B (≥ 15%) of 60 patients. Data were analyzed using non-parametric statistical methods. The results demonstrate that there is no statistical difference between the two groups in seminal characteristics. The collective data show a high significant correlation, suggesting that spermatozoa with abnormal morphology are the best candidates to contain DNA damage (p < 0.001). Also, when group A is compared with group B, an increased percentage of morphologically normal spermatozoa with fragmented DNA was observed in patients, with DFI values ≥ 15% (p < 0.001). Conclusion These results are aimed at providing an exact value of DFI in morphologically normal spermatozoa, which will be helpful to the embryologist in evaluating the risk of transferring, during the ICSI procedure, a spermatozoon whit normal morphology but fragmented DNA.

Abstract The prenatal exposure to ethanol (Eth), fluconazole (FLUCO) and sodium valproate (VPA) is related to effects on development, producing characteristic syndromic pictures. Among embryotoxic effects described for the three molecules, the alteration on craniofacial morphogenesis is a common feature in humans and animal models, including rodent embryos developed in vitro. The aim of the present work is to evaluate the developmental effects of low Eth serum concentration (17 mM, corresponding to the legal limit to drive in UK, USA, Canada, and many other countries) in mixture with increasing realistic concentrations of the antifungal drug FLUCO (62.5–500 µM) or with increasing realistic concentrations of the antiepileptic drug VPA (31.25–250 µM). Groups exposed to Eth alone (17–127.5 mM), FLUCO alone (62.5–500 µM) or VPA alone (31.25–750 µM) were also included. The chosen alternative animal model was the post-implantation rat whole embryo culture (WEC). E9.5 embryos were exposed in vitro to the test molecules during the whole test period (48 h, corresponding to the developmental stages characteristics of any vertebrate, for human embryos post-fertilization days 23–31). Data were statistically analyzed and processed for modelling applying the benchmark dose (BMD) and relative potency factor (RPF) approaches. Concentration-related effects on facial outcomes were observed in all experimental groups, with a significant enhancement in the groups co-exposed with Eth in comparison to the single exposures. Data obtained by the present work suggest an additional alert for the assumption of even low levels of alcohol in pregnant women during FLUCO or VPA therapy.

Indoxyl sulphate (IS) is a uremic toxin accumulating in the plasma of chronic kidney disease (CKD) patients. IS accumulation induces side effects in the kidneys, bones and cardiovascular system. Most studies assessed IS effects on cell lines by testing higher concentrations than those measured in CKD patients. Differently, we exposed a human microvascular endothelial cell line (HMEC-1) to the IS concentrations measured in the plasma of healthy subjects (physiological) or CKD patients (pathological). Pathological concentrations reduced cell proliferation rate but did not increase long-term oxidative stress level. Indeed, total protein thiols decreased only after 24 h of exposure in parallel with an increased Nrf-2 protein expression. IS induced actin cytoskeleton rearrangement with formation of stress fibres. Proteomic analysis supported this hypothesis as many deregulated proteins are related to actin filaments organization or involved in the endothelial to mesenchymal transition. Interestingly, two proteins directly linked to cardiovascular diseases (CVD) in in vitro and in vivo studies underwent deregulation: COP9 signalosome complex subunit 9 and thrombomodulin. Future experiments will be needed to investigate the role of these proteins and the signalling pathways in which they are involved to clarify the possible link between CKD and CVD.

It is known that the intake of alcoholic beverages may impair genetic and epigenetic regulatory events with consequent crucial effects on cell phenotypes and that its association with selected genotypes can lead to a different risk of cancer in the population. The aim of this review is to pick up selected studies on this topic and recapitulate some of the biochemical and nutrigenetic/nutrigenomic aspects involved in the impact of dietary low-dose alcohol consumption on the switching-on or -off of tumorigenic pathways. These include i) the existence of predisposing or protective human genotypes and the relationship between dietary compounds and alcohol in the promotion or inhibition of carcinogenesis; ii) the effects of other components of alcoholic drinks in the modulation of the expression of oncogenes and oncosuppressors, the autophagic flux and the onset of apoptosis, with examples of their cytospecificity; and iii) the role of alcoholic beverage consumption within particular dietary regimens, including the Mediterranean diet. Taking all the data into account, several alcohol-associated bioactive dietary compounds appear capable to modulate peculiar intracellular pathways predisposing to or protecting from cancer. Advances in the nutrigenetic, nutrigenomic and nutriepigenetic knowledge complementing the biochemical and molecular approaches will help in unveiling their impact on health outcome.

Clinical embryologists are highly trained laboratory professionals with multiple roles, including laboratory, clinical, biobanking and quality system management. In most European countries, clinical embryologists are trained to work in Medically Assisted Reproduction (MAR) centres without a specifically dedicated educational path. The criteria required for employment vary according to the educational structure and the public or private nature of the centre. We have herein described the educational profile required by Italian clinical embryologists to work in MAR centres of the National Health System (NHS). Public centres currently represent 36% of all the Italian MAR clinics. According to the Italian law, a future clinical embryologist must achieve a 3-4 year unpaid post-graduate specialization in a different field, choosing from Genetics, Microbiology, Clinical Pathology or Nutrition. Accesses to the above-mentioned post-graduate courses are themselves very limited. Clinical embryologists are basically trained by senior colleagues. This situation makes inevitably difficult to recruit laboratory staff in NHS centres. Moreover, it represents an emblematic example of the need for an equal training curriculum, possibly ensuring a comparable education quality, mobility of trainees and dissemination of skills for clinical embryologists all over Europe.

Aqueous extracts from Posidonia oceanica’s green and brown (beached) leaves and rhizomes were prepared, submitted to phenolic compound and proteomic analysis, and examined for their potential cytotoxic effect on HepG2 liver cancer cells in culture. The chosen endpoints related to survival and death were cell viability and locomotory behavior, cell-cycle analysis, apoptosis and autophagy, mitochondrial membrane polarization, and cell redox state. Here, we show that 24 h exposure to both green-leaf- and rhizome-derived extracts decreased tumor cell number in a dose–response manner, with a mean half maximal inhibitory concentration (IC50) estimated at 83 and 11.5 μg of dry extract/mL, respectively. Exposure to the IC50 of the extracts appeared to inhibit cell motility and long-term cell replicating capacity, with a more pronounced effect exerted by the rhizome-derived preparation. The underlying death-promoting mechanisms identified involved the down-regulation of autophagy, the onset of apoptosis, the decrease in the generation of reactive oxygen species, and the dissipation of mitochondrial transmembrane potential, although, at the molecular level, the two extracts appeared to elicit partially differentiating effects, conceivably due to their diverse composition. In conclusion, P. oceanica extracts merit further investigation to develop novel promising prevention and/or treatment agents, as well as beneficial supplements for the formulation of functional foods and food-packaging material with antioxidant and anticancer properties.

Specimens of Sparus aurata were exposed to sub-lethal concentrations of Hg and Cd for 25 days and the levels of both metals were investigated in organs and tissues. Bioaccumulation of Hg decreased as follow: gills > kidney > liver > skin > muscle, while the order of Cd bioaccumulation was: liver > kidney > gills > skin > muscle. Immediately after exposure, both metals showed the highest bioaccumulation in gills and skin indicating that these organs are reliable targets for biomonitoring studies after short term exposure. Metals introduction caused a significant time-dependent concentrations increase in kidney and liver, while in the muscle a significant in-crease of Hg was recorded only at the end of the experimentation.The effects of exposure were also investigated, at biochemical level, in the liver, which represents the main target of xenobiotics biotransformation and metabolism in fish. Exposed fishes exhibited a reduction of total lipid level, a decrease of polyunsaturated fatty acids (PUFA), together with a MDA increase. This suggests a direct effect of contaminants on oxidative stress induction that, through the MDA increase, altered the membrane fatty acids composition decreasing the PUFA content. As it regards molecular markers related to oxidative stress and lipid metanolism, a significant increase of Nrf2, Hif- 1 alpha and Ampk and a decrease of Fas were observed after exposure to both metals, while an Nf-kB increase was recorded in specimens exposed to Hg, docuemnting a correlation with oxidative stress and consequent metabolism adaptation. Finally, these results suggest the pos-sibility to adopt these biomarkers to explore fish metabolic responses to environmental pollution.

To dissect the TBX5 regulatory circuit, we focused on microRNAs (miRNAs) that collectively contribute to make TBX5 a pivotal cardiac regulator. We profiled miRNAs in hearts isolated from wild-type, CRE, Tbx5lox/+and Tbx5del/+ mice using a Next Generation Sequencing (NGS) approach. TBX5 deficiency in cardiomyocytes increased the expression of the miR-183 cluster family that is controlled by Kruppel-like factor 4, a transcription factor repressed by TBX5. MiR-182-5p, the most highly expressed miRNA of this family, was functionally analyzed in zebrafish. Transient overexpression of miR-182-5p affected heart morphology, calcium handling and the onset of arrhythmias as detected by ECG tracings. Accordingly, several calcium channel proteins identified as putative miR-182-5p targets were downregulated in miR-182-5p overexpressing hearts. In stable zebrafish transgenic lines, we demonstrated that selective miRNA-182-5p upregulation contributes to arrhythmias. Moreover, cardiac-specific down-regulation of miR-182-5p rescued cardiac defects in a zebrafish model of Holt–Oram syndrome. In conclusion, miR-182-5p exerts an evolutionarily conserved role as a TBX5 effector in the onset of cardiac propensity for arrhythmia, and constitutes a relevant target for mediating the relationship between TBX5, arrhythmia and heart development. To dissect the TBX5 regulatory circuit, we focused on microRNAs (miRNAs) that collectively contribute to make TBX5 a pivotal cardiac regulator. We profiled miRNAs in hearts isolated from wild-type, CRE, Tbx5lox/+and Tbx5del/+ mice using a Next Generation Sequencing (NGS) approach. TBX5 deficiency in cardiomyocytes increased the expression of the miR-183 cluster family that is controlled by Kruppel-like factor 4, a transcription factor repressed by TBX5. MiR-182-5p, the most highly expressed miRNA of this family, was functionally analyzed in zebrafish. Transient overexpression of miR-182-5p affected heart morphology, calcium handling and the onset of arrhythmias as detected by ECG tracings. Accordingly, several calcium channel proteins identified as putative miR-182-5p targets were downregulated in miR-182-5p overexpressing hearts. In stable zebrafish transgenic lines, we demonstrated that selective miRNA-182-5p upregulation contributes to arrhythmias. Moreover, cardiac-specific down-regulation of miR-182-5p rescued cardiac defects in a zebrafish model of Holt–Oram syndrome. In conclusion, miR-182-5p exerts an evolutionarily conserved role as a TBX5 effector in the onset of cardiac propensity for arrhythmia, and constitutes a relevant target for mediating the relationship between TBX5, arrhythmia and heart development.

SummaryActivated pERK1/2 and pAKT are key players in supporting cell survival and proliferation pathways. Translocation of pERK1/2 into the nucleus, where it interacts with transcription factors and DNA itself, is instrumental in exerting an anti-apoptotic effect. In this study, pAKT levels, pERK1/2 nuclear localization and DNA fragmentation index (DFI) in cumulus cells of single cumulus–oocyte complexes of patients undergoing in vitro fertilization programmes were evaluated and correlated with the clinical outcome of the related embryos. For a positive clinical outcome of blastocyst development, pERK1/2 nuclear localization and DFI value had a significant inverse relationship, whereas the former and the intracellular accumulation of pAKT had a significant direct relationship. This relationship was not observed for the negative clinical outcome of the arrested embryos. Moreover, intracellular accumulation of pAKT and DFI value had a significant inverse relationship in all samples examined. The obtained data suggest that the intranuclear relocation of pERK1/2, along with an enhanced intracellular accumulation of pAKT, may exert a survival effect and increase cell viability, therefore providing a novel marker tool to choose the best oocyte to be fertilized and submitted to an intracytoplasmic sperm injection cycle.

Dehydrated lemon peel (DLP) at two levels (1.5% and 3%) was included for 30days in the diet of gilthead seabream (Sparus aurata L.) and after 15 and 30days the serum and skin mucus of fish were analysed. In serum, both experimental diets led to an initial decrease in glucose and lactate after which the levels became comparable to the control. Dietary DLP modulated the activity of transaminases, alanine aminotransferase and aspartate aminotransferase. The total antioxidant status showed a progressive increase in relation to levels of inclusion of DLP and time of administration (p'.05). In skin mucus, some biomolecular markers related to general stress, oxidative stress and apoptosis exhibited variations that suggest an adaptive response of fish to the new metabolic situation, resulting from the experimental diet. The inclusion of DLP in the diet seems to positively modulate the central metabolism and welfare of gilthead seabream.

In this study, the carotenoid astaxanthin was obtained by supercritical fluid extraction (SFE) from shrimp by-products (SBP). Its bioactive properties were evaluated in vitro in human normal and cancerous cells lines. The antioxidant activity of the extracted astaxanthin of the SFE fraction (ASTA) was tested in fibroblast cells (HS-68), by inducing oxidative stress and by evaluating the protective effect of the pre-treatment with different levels of ASTA against toxicity. The anti-proliferative activity was evaluated in hepatoma cells (HEP-G2), treated with increased concentrations of ASTA and measuring the effects on vitality and on some biomolecular markers related to oxidative stress, cell cycle, and apoptosis. It was found that pre-treating normal fibroblast cells with ASTA resulted in a marked increase in cell viability in a dose-dependent manner (P < 0.05) attesting its antioxidant power; in cancer cell line, increased concentrations of ASTA caused a time-dose-dependent decrease in the vitality, attesting its anti-proliferative activity (P < 0.05). The increased levels of the protein p-53 and the reduced levels of the proteins c-Jun and c-Fos at higher concentrations of ASTA, as well as, suggest the pro-apoptotic and anti-cancerous effects that this extract has on hepatocellular carcinomas, confirmed also by caspase-3 activation. These findings suggest biotechnological utilisation of marine by-products for nutraceutical and pharmaceutical applications avoiding the employment of organic solvents for extraction.

Echinoderms have an extensive endoskeleton composed of magnesian calcite and occluded matrix proteins. As biomineralization in sea urchin larvae is sensitive to the Magnesium:Calcium ratio of seawater, we investigated the effects of magnesium deprivation on development and skeletogenesis in the sea urchin Arbacia lixula. We focused on the localization of the skeletogenic cells (primary mesenchyme cells) and the spatial expression of associated genes. Embryos reared in Mg-free seawater exhibited developmental delay from 6-h post-fertilization and at 24 h embryos showed complete lack of biomineral formation. Larvae (48–72 h) exhibited severe skeleton malformations. Fluorescent labelling revealed that the primary mesenchyme cells and the developing skeleton of treated embryos were in an abnormal ectopic location. Expression of the skeleton matrix protein gene (msp130) in the primary mesenchyme cells as seen using in situ hybridization was normal at 24 h. At 48 h this gene was down-regulated in control larvae, but not in treated larvae. Development of the pigment cells, immune cells that, like the skeleton, are mesodermal derivatives, was also impaired. Our results highlight the essential role of Mg in skeleton formation in sea urchin embryos with an indication that this element is also generally important for the development of mesoderm. Abbreviations: hpf: hours post fertilization; PMCs: primary mesenchyme cells; ACC: amorphous calcium carbonate; MgFSW: magnesium-free seawater; FSW: filtered seawater Impact statement Echinoderms have an extensive endoskeleton composed of magnesian calcite We investigated the effects of magnesium deprivation on development and skeletogenesis in the sea urchin Arbacia lixula Magnesium deprivation caused developmental delay and skeleton malformations Primary mesenchyme cells of treated embryos were in an abnormal ectopic location The spatial and temporal expression profile of the skeleton matrix protein gene (msp130) was found to be different from controls Our results highlight the essential role of Mg across developmental processes in sea urchin embryos.

Echinoderms are an acknowledged source of bioactive compounds exerting various beneficial effects on human health. Here, we examined the potential in vitro anti-hepatocarcinoma effects of aqueous extracts of the cell-free coelomic fluid obtained from the sea urchin Arbacia lixula using the HepG2 cell line as a model system. This was accomplished by employing a combination of colorimetric, microscopic and flow cytometric assays to determine cell viability, cell cycle distribution, the possible onset of apoptosis, the accumulation rate of acidic vesicular organelles, mitochondrial polarization, cell redox state and cell locomotory ability. The obtained data show that exposed HepG2 cells underwent inhibition of cell viability with impairment of cell cycle progress coupled to the onset of apoptotic death, the induction of mitochondrial depolarization, the inhibition of reactive oxygen species production and acidic vesicular organelle accumulation, and the block of cell motile attitude. We also performed a proteomic analysis of the coelomic fluid extract identifying a number of proteins that are plausibly responsible for anti-cancer effects. Therefore, the anti-hepatocarcinoma potentiality of A. lixula’s preparation can be taken into consideration for further studies aimed at the characterization of the molecular mechanism of cytotoxicity and the development of novel prevention and/or treatment agents.

Nonalcoholic fatty liver disease (NAFLD) is characterized by the accumulation of lipids in the liver. Given the high prevalence of NAFLD, its evolution to nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC) is of global concern. Therapies for managing NASH-driven HCC can benefit from targeting factors that play a continuous role in NAFLD evolution to HCC. Recent work has shown that postprandial liver translation exacerbates lipid accumulation through the activity of a translation factor, eukaryotic initiation factor 6 (eIF6). Here, we test the effect of eIF6 inhibition on the progression of HCC. Mice heterozygous for eIF6 express half the level of eIF6 compared to wt mice and are resistant to the formation of HCC nodules upon exposure to a high fat/high sugar diet combined with liver damage. Histology showed that nodules in eIF6 het mice were smaller with reduced proliferation compared to wt nodules. By using an in vitro model of human HCC, we confirm that eIF6 depletion reduces the growth of HCC spheroids. We also tested three pharmacological inhibitors of eIF6 activity—eIFsixty-1, eIFsixty-4, and eIFsixty-6—and all three reduced eIF6 binding to 60S ribosomes and limited the growth of HCC spheroids. Thus, inhibition of eIF6 activity is feasible and limits HCC formation.

Uncontrolled cell proliferation is a hallmark of cancer as a result of rapid and deregulated progression through the cell cycle. The inhibition of cyclin-dependent kinases (CDKs) activities is a promising therapeutic strategy to block cell cycle of tumor cells. In this work we reported a new example of nanocomposites based on halloysite nanotubes (HNTs)/pyrazolo[3,4-d]pyrimidine derivatives (Si306 and Si113) as anticancer agents and CDK inhibitors. HNTs/Si306 and HNTs/Si113 nanocomposites were synthesized and characterized. The release kinetics were also investigated. Antitumoral activity was evaluated on three cancer cell lines (HeLa, MDA-MB-231 and HCT116) and the effects on cell cycle arrest in HCT116 cells were evaluated. Finally, molecular dynamics simulations were performed of the complexes between Si113 or Si306 and the active site of both CDK 1 and 2., The work was financially supported by the University of Palermo. The work was carried out in the frame of the PON “AIM: Attrazione e Mobilità Internazionale” No. 1808223 project. SS and GG wish to acknowledge the AIRC project 2019 code 23725. The authors would thank Dr. Susanna Guernelli (University of Bologna) for TEM and SEM measurements.

Gliomas are complex and heterogeneous tumors that originate from the glial cells of the brain. The malignant cells undergo deep modifications of their metabolism, and acquire the capacity to invade the brain parenchyma and to induce epigenetic modifications in the other brain cell types. In spite of the efforts made to define the pathology at the molecular level, and to set novel approaches to reach the infiltrating cells, gliomas are still fatal. In order to gain a better knowledge of the cellular events that accompany astrocyte transformation, we developed three increasingly transformed astrocyte cell lines, starting from primary rat cortical astrocytes, and analyzed them at the cytogenetic and epigenetic level. In parallel, we also studied the expression of the differentiation-related H1.0 linker histone variant to evaluate its possible modification in relation with transformation. We found that the most modified astrocytes (A-FC6) have epigenetic and chromosomal alterations typical of cancer, and that the other two clones (A-GS1 and A-VV5) have intermediate properties. Surprisingly, the differentiation-specific somatic histone H1.0 steadily increases from the normal astrocytes to the most transformed ones. As a whole, our results suggest that these three cell lines, together with the starting primary cells, constitute a potential model for studying glioma development.

Polybrominated diphenyl ethers (PBDEs) are flame retardants, characterized by elevated stability in the marine environment, where are accumulated by organisms, inducing a wide panel of negative effects. In this study, some biochemical patterns related to toxicity, biotransformation and oxidative stress, were studied in the marine model system, Mytilus galloprovincialis, exposed to BDE-47. Mussels were fed with microalgae, previously treated with increasing concentrations of PBDEs (maximum dose 100 ng L-1 of BDE-47 per day). After 15 days of treatment, mussels were fed with the same diet without BDE-47, for additional 15 days. Gills and digestive glands were analyzed at T 0, at 15 and 30 days. Histopathological lesions were assessed in digestive glands of contaminated mussels, while expression of genes, related to cell cycle, multidrug resistance, oxidative stress and detoxification was evaluated on both gills and digestive glands. After 15 days, BDE-47 exposure significantly affected the cell activity in digestive gland and, at 30 days, only mussels exposed to the lower doses showed a certain recovery. Regarding the gene expression, both gills and digestive glands showed a significant down-regulation of the target genes at 15 days, although most of them were up-regulated at 30 days in digestive gland. The results on BDE-47 accumulation in mussels revealed a dose-dependent concentration in tissues, which remained elevated after further 15 days of depuration. This trend supports the responses of the biomarkers, indicating that exposure, at environmentally realistic concentrations of BDE-47, strongly modulates oxidative stress and related patterns of gene expression, suggesting concerns for long-term effect in the biota.

In our previous work, we reported alterations in protamines/histones ratio, in DNA binding of these proteins and their involvement in DNA oxidative damage in 84% of the young men living in the Land of Fires. In the present work, we extended our findings, evaluating any alterations in spermatozoa of a family case, a father and son, living in this area, to also give a first look at the possibility of transgenerational inherited effects of environmental contaminants on the molecular alterations of sperm nuclear basic proteins (SNBP), DNA and semen parameters. In the father and son, we found a diverse excess of copper and chromium in the semen, different alterations in SNBP content and low DNA binding affinity of these proteins. In addition, DNA damage, in the presence of CuCl2 and H2O2, increased by adding both the father and son SNBP. Interestingly, son SNBP, unlike his father, showed an unstable DNA binding and were able to produce DNA damage even without external addition of CuCl2, in line with a lower seminal antioxidant activity than the father. The peculiarity of some characteristics of son semen could be a basis for possible future studies on transgenerational effects of pollutants on fertility.

Neuronal hyperexcitability linked to an increase in glutamate signalling is a peculiar trait of the early stages of Alzheimer's disease (AD) and tauopathies, however, a progressive reduction in glutamate release follows in advanced stages. We recently reported that in the early phases of the neurodegenerative process, soluble, non-aggregated Tau accumulates in the nucleus and modulates the expression of disease-relevant genes directly involved in glutamatergic transmission, thus establishing a link between Tau instability and altered neurotransmission. Here we report that while the nuclear translocation of Tau in cultured cells is not impaired by its own aggregation, the nuclear amyloid inclusions of aggregated Tau abolish Tau-dependent increased expression of the glutamate transporter. Remarkably, we observed that in the prefrontal cortex (PFC) of AD patient brain, the glutamate transporter is upregulated at early stages and is downregulated at late stages. The Gene Set Enrichment Analysis indicates that the modulation of Tau-dependent gene expression along the disease progression can be extended to all protein pathways of the glutamatergic synapse. Together, this evidence links the altered glutamatergic function in the PFC during AD progression to the newly discovered function of nuclear Tau.

Although the fishing and wine industries undoubtedly contribute significantly to the economy, they also generate large waste streams with considerable repercussions on both economic and environmental levels. Scientific literature has shown products can be extracted from these streams which have properties of interest to the cosmetics, pharmaceutical and food industries. Antimicrobial activity is undoubtedly among the most interesting of these properties, and particularly useful in the production of food packaging to increase the shelf life of food products. In this study, film for food packaging was produced for the first time using chitosan extracted from the exoskeletons of red shrimp (Aristomorpha foliacea) and oil obtained from red grape seeds (Vitis vinifera). The antimicrobial activity of two films was analyzed: chitosan-only film and chitosan film with the addition of red grape seed oil at two different concentrations (0.5 mL and 1 mL). Our results showed noteworthy antimicrobial activity resulting from functionalized chitosan films; no activity was observed against pathogen and spoilage Gram-positive and Gram-negative bacteria, although the antimicrobial effects observed were species-dependent. The preliminary results of this study could contribute to developing the circular economy, helping to promote the reuse of waste to produce innovative films for food packaging.

The valorization of side streams from fishery and aquaculture value-chains is a valuable solution to address one of the challenges of the circular economy: turning wastes into profit. Side streams produced after filleting of sea cage fattened bluefin tuna (Thunnus thynnus) were analyzed for proximate composition and fatty acid profile to evaluate the possibility of producing tuna oil (TO) as a valuable source of ω-3 polyunsaturated fatty acids (PUFA) and testing its bioactivity in vitro. Ethyl esters of total fatty acids (TFA), obtained from TO, were pre-enriched by urea complexation (PUFA-Ue) and then enriched by short path distillation (SPD) up to almost 85% of the PUFA fraction (PUFA-SPe). The bioactivity of TFA, PUFA-SPe, and ethyl esters of depleted PUFA (PUFA-SPd) were tested in vitro, through analysis of lipid metabolism genes, in gilthead sea bream (Sparus aurata) fibroblast cell line (SAF-1) exposed to oils. TFA and PUFA-SPd upregulated transcription factors (pparβ and pparγ) and lipid metabolism-related genes (D6D, fas, fabp, fatp1, and cd36), indicating the promotion of adipogenesis. PUFA-SPe treated cells were similar to control. PUFA-SPe extracted from farmed bluefin tuna side streams could be utilized in fish feed formulations to prevent excessive fat deposition, contributing to improving both the sustainability of aquaculture and the quality of its products.

Metal pharmaceutical residues often represent emerging toxic pollutants of the aquatic environment, as wastewater treatment plants do not sufficiently remove these compounds. Recently, vanadium (V) derivatives have been considered as potential therapeutic factors in several diseases, however, only limited information is available about their impact on aquatic environments. This study used sea urchin embryos (Paracentrotus lividus) to test V toxicity, as it is known they are sensitive to V doses from environmentally relevant to very cytotoxic levels (50 nM; 100 nM; 500 nM; 1 µM; 50 µM; 100 µM; 500 µM; and 1 mM). We used two approaches: The fertilization test (FT) and a protease detection assay after 36 h of exposure. V affected the fertilization percentage and increased morphological abnormalities of both egg and fertilization envelope, in a dose-dependent manner. Moreover, a total of nine gelatinases (with apparent molecular masses ranging from 309 to 22 kDa) were detected, and their proteolytic activity depended on the V concentration. Biochemical characterization shows that some of them could be aspartate proteases, whereas substrate specificity and the Ca2+/Zn2+ requirement suggest that others are similar to mammalian matrix metalloproteinases (MMPs).

The effect of six salts of different geographical areas on the quality of salted anchovies was evaluated. The crude salts were chemically characterized by determination of inorganic and volatile organic compounds (VOCs). Salted anchovies, corresponding to six experimental trials, were subjected to microbiological, chemical (including histamine content) and sensory analysis during the entire period of ripening (150 days). The salts were characterized by marked differences in terms of major cations and trace element amounts. Among the 27 VOCs detected, octadecane was the most abundant compound and the main differences of the salts were registered for alkanes and alcohols. During maturation, significant microbiological differences between the salts were found for the levels of total aerobic mesophilic microorganisms, lactic acid bacteria, Staphylococcaceae and Enterobacteriaceae counts. All salted anchovies contained histamine below the thresholds allowed by current regulations, but statistical differences were registered for the concentrations of the different trials. Consistent differences were also revealed for their sensory profiles, in particular concerning odour and taste and overall acceptability. Several differences were also detected for dryness, brown colour, putrid odour, rancid and raw blood taste sensory attributes. Especially the differences in the composition (chemical and VOC's) of the raw salts used for the production of salted anchovies has a significant effect on the sensory characteristics of the final product.

Physical activity (PA) has been central in the life of our species for most of its history, and thus shaped our physiology during evolution. However, only recently the health consequences of a sedentary lifestyle, and of highly energetic diets, are becoming clear. It has been also acknowledged that lifestyle and diet can induce epigenetic modifications which modify chromatin structure and gene expression, thus causing even heritable metabolic outcomes. Many studies have shown that PA can reverse at least some of the unwanted effects of sedentary lifestyle, and can also contribute in delaying brain aging and degenerative pathologies such as Alzheimer’s Disease, diabetes, and multiple sclerosis. Most importantly, PA improves cognitive processes and memory, has analgesic and antidepressant effects, and even induces a sense of wellbeing, giving strength to the ancient principle of “mens sana in corpore sano” (i.e., a sound mind in a sound body). In this review we will discuss the potential mechanisms underlying the effects of PA on brain health, focusing on hormones, neurotrophins, and neurotransmitters, the release of which is modulated by PA, as well as on the intra- and extra-cellular pathways that regulate the expression of some of the genes involved.

The grape pomace (GP) is an important by-product of winemaking, accounting for about 13-25% of the grapes processed. The aim of this work was to investigate the nutritional and antioxidant composition of GP obtained from Nero d’Avola (NA) grape, one of the most important indigenous varieties in Italy, in order to verify application in nutraceutics. Seeds and skin of the GP were studied for their nutritional and antioxidants composition, fatty acid and polyphenols profile, bioactives properties in vitro, by gravimetric, spectrophotometric and chromatographic techniques. The results showed that NAGP seeds are rich in the beneficial polyunsaturated fatty acids and that the polyphenols extracted from the GP skin present a strong antiradical and antiproliferative activity, attested also in vitro, in human skin fibroblast (HS-68) and in hepatoma cell line (Hep-G2). Obtained results underline the possibility to employ this residual biomass for nutraceuticals application, contributing also to increase the sustainability.

The present study analyzed the ability of primary rat astrocytes to colonize a porous scaffold, mimicking the reticular structure of the brain parenchyma extracellular matrix, as well as their ability to grow, survive and differentiate on the scaffold. Scaffolds were prepared using poly-L-lactic acid (PLLA) via thermally-induced phase separation. Firstly, the present study studied the effects of scaffold morphology on the growth of astrocytes, evaluating their capability to colonize. Specifically, two different morphologies were tested, which were obtained by changing the polymer concentration in the starting solution. The structures were characterized by scanning electron microscopy, and a pore size of 20 µm (defined as the average distance between the pore walls) was detected. For comparison, astrocytes were also cultured in the traditional 2D culture system that we have been using since 2003. Then the effects of different substrates, such as collagen I and IV, and fibronectin were analyzed. The results revealed that the PLLA scaffolds, coated with collagen IV, served as very good matrices for astrocytes, which were observed to adhere, grow and colonize the matrix, acquiring their typical morphology. In addition, under these conditions, they secreted extracellular vesicles (EVs) that were compatible in size with exosomes. Their ability to produce exosomes was also suggested by transmission electron microscopy pictures which revealed both EVs and intracellular structures that could be interpreted as multivesicular bodies. The fact that these cells were able to adapt to the PLLA scaffold, together with our previous results, which demonstrated that brain capillary endothelial cells can grow and differentiate on the same scaffold, could support the future use of 3D brain cell co-culture systems.

Vanadium, a naturally occurring element widely distributed in soil, water and air, has received considerable interest because its compounds are often used in different applications, from industry to medicine. While the possible medical use of vanadium compounds is promising, its potential harmful effects on living organisms are still unclear. Here, for the first time, we provide a toxicological profile induced by vanadium on Paracentrotus lividus sea urchin embryos, reporting an integrated and comparative analysis of the detected effects reflecting vanadium-toxicity. At the morphological level we found a dose-dependent induction of altered phenotypes and of skeletal malformations. At the molecular levels, vanadium-exposed embryos showed the activation of the cellular stress response, in particular, autophagy and a high degree of cell-selective apoptosis in a dose-dependent manner. The stress response mediated by heat shock proteins seems to counteract the damage induced by low and intermediate concentrations of vanadium while the high cytotoxic concentrations induce more marked cell death mechanisms. Our findings, reporting different mechanisms of toxicity induced by vanadium, contribute to increase the knowledge on the possible threat of vanadium for marine organisms and for both environmental and human health.

Wounds in living organisms trigger tissue-repair mechanisms. The sea cucumber (Holoturia tubulosa) is an excellent model species for achieving a better understanding of the humoral and cellular aspects involved in such healing processes. Consequently, this study assesses data on its morphometric, physiological and humoral responses 1, 2, 6, 24 and 48h after wound induction. In particular, morphometric data on the weight, width, length and coelomic-fluid volume of the species were estimated at different times during our experiments. In addition, the humoral aspects related to the enzymatic activity of esterase, alkaline phosphatase and peroxidase, as well as the cytotoxic activity of cell lysates (CL) and cell-free coelomic fluids (CfCf) are evaluated for the first time. Our results reveal a significant decrease in body length and weight, along with time-dependent, significant changes in the esterase, alkaline phosphatase, peroxidase and cytotoxic activity in both the CL and CfCf. The data obtained lead to the pioneering finding that there is an important time-dependent involvement of morphometric (changes in weight and length) and humoral (enzymatic and cytotoxic) responses in wound healing.

The carbazole compounds PK9320 (1-(9-ethyl-7-(furan-2-yl)-9H-carbazol-3-yl)-N-methylmethanamine) and PK9323 (1-(9-ethyl-7-(thiazol-4-yl)-9H-carbazol-3-yl)-N-methylmethanamine), second-generation analogues of PK083 (1-(9-ethyl-9H-carbazol-3-yl)-N-methylmethanamine), restore p53 signaling in Y220C p53-mutated cancer cells by binding to a mutation-induced surface crevice and acting as molecular chaperones. In the present paper, these three molecules have been tested for mutant p53-independent genotoxic and epigenomic effects on wild-type p53 MCF-7 breast adenocarcinoma cells, employing a combination of Western blot for phospho-γH2AX histone, Comet assay and methylation-sensitive arbitrarily primed PCR to analyze their intrinsic DNA damage-inducing and DNA methylation-changing abilities. We demonstrate that small modifications in the substitution patterns of carbazoles can have profound effects on their intrinsic genotoxic and epigenetic properties, with PK9320 and PK9323 being eligible candidates as “anticancer compounds” and “anticancer epi-compounds” and PK083 a “damage-corrective” compound on human breast adenocarcinoma cells. Such different properties may be exploited for their use as anticancer agents and chemical probes.

Opuntia ficus-indica fruit and cladodes are a source of mucilage, a carbohydrate complex, as well as of phytochemicals and other nutrients, useful in the food industry. Despite the environmental-based composition variability has been studied, there is no information on its seasonal variability, which is particularly important for its industrial use. In this study, some technological, nutritional, and bioactive properties of O. ficus-indica cladodes mucilage were analyzed during the seasonal growth period. Total proteins did not change during the whole period and the lipid content significantly decreased from winter to the onset of summer. The fatty acid profile showed high levels of palmitic (16:0), oleic (18:1n9), and linoleic (18:2n6) acids and that the polyunsaturated fatty acid class (PUFA) is the most abundant fatty acid class, which showed a significant increase during the growth period. In this class, the presence of 20:5n-3 (EPA) and 22:6n-3 (DHA), two of the most important human nutrition essential fatty acids, is worth noting as well as the positive effect of the omega-3 series founder, α-linolenic acid 18:3 n-3. Total polyphenol values indicated that extract quality changes with the sampling period and is not closely related to the total extract yield. All the descriptors of the antioxidant power seem to indicate that the best properties are exhibited during the summer. The principal component analysis can significantly help identify the technical, nutritional, and bioactive factors that make it possible to distinguish the quality of O. ficus-indica mucilage, in order to harvest it in the most appropriate season.

Quality and shelf-life of whole and filleted Coryphaena hippurus, stored with modified atmosphere packaging (MAP) and natural antioxidants from halophytes (HAL), were investigated. Fillets were divided into control, simply sealed in trays; MAP, preserved by modified atmosphere (45% CO2, 50% N-2, 5% O-2); and MAP-HAL, pretreated with antioxidants and preserved by MAP. Whole and filleted fish were stored at -1 +/- 0.5 degrees C for 18days. The quality of the samples was analyzed at the time of packaging and after 3, 6, 9, 12, 15, and 18days. The MAP and MAP-HAL groups maintained the best sensorial profile, pH, and drip loss with respect to the untreated fillets. Higher levels of total volatile basic nitrogen (TVB-N) and oxidized proteins were observed in untreated samples with respect to the MAP and MAP-HAL groups. The principle component analysis revealed a different quality profile for untreated and MAP-treated fish.

This study aimed to determine whether isotopic ratio mass spectrometry (IRMS) can discriminate farmed European sea bass according to different farming systems and geographic origins. Dicentrarchus labrax of commercial size from three different rearing systems (concrete tank inland, sea cages, and extensive methods in valleys or salt works) were collected at the trading period (autumn&ndash, winter). For each farming type, different locations spread over Italy were monitored. Once the fish were harvested, the muscle and feed were sampled. For both muscle and feed, &delta, 13C and &delta, 15N were measured by continuous flow elemental analyzer isotope ratio mass spectrometry (CF-EA-IRMS) with the goal of discriminating samples based on the rearing system. Additional &delta, 2H and &delta, 18O measurements of fish samples were performed by continuous flow total combustion elemental analyzer isotope ratio mass spectrometry (CF-TC/EA-IRMS) to track the geographical origin. The measurements of &delta, 15N made it possible to discriminate cultured sea bass from different farming systems (extensive vs. intensive) reared at different geographical sites in Italy. Additional information was obtained from &delta, 18O and &delta, 2H, which enabled the geographical areas of origin of the sea bass farmed extensively and intensively (in cages) to be distinguished.

During neuronal development, neuronal cells read extracellular stimuli from the micro/nano-environment within which they exist, retrieving essential directionality and wiring information. Here, focal adhesions (FAs-protein clusters anchoring integrins to cytoskeleton) act as sensors, by integrating signals from both the extracellular matrix environment and chemotactic factors, contributing to the final neuronal pathfinding and migration. In the processes that orchestrate neuronal development, the important function of ubiquitin E3A ligase (UBE3A) is emerging. UBE3A has crucial functions in the brain and changes in its expression levels lead to neurodevelopmental disorders: the lack of UBE3A leads to Angelman syndrome (AS, OMIN 105830), while its increase causes autisms (Dup15q-autism). By using nano/micro-structured anisotropic substrates we previously showed that UBE3A-deficient neurons have deficits in contact guidance (Tonazzini et al, Mol Autism 2019). Here, we investigate the adhesion and migration dynamics of UBE3A-silenced SH-SY5Y neuroblastoma cells in vitro by exploiting nano/micro-grooved substrates. We analyze the molecular processes regulating the development of FAs by transfection with EGFP-vector encoding for paxillin, a protein of FA clusters, and by live-cell total-internal-reflection-fluorescence microscopy. We show that UBE3A-silenced SH-SY5Y cells have impaired FA morphological development and pathway activation, which lead to a delayed adhesion and also explain the defective contact guidance in response to directional topographical stimuli. However, UBE3A-silenced SH-SY5Y cells show an overall normal migration behavior, in terms of speed and ability to follow the GRs directional stimulus. Only the collective cell migration upon cell gaps was slightly delayed for UBE3Ash SHs. Overall, the deficits of UBE3Ash SHS-SY5Y cells in FA maturation/sensing and in collective migration may have patho-physiological implications, in AS condition, considering the much more complex stimuli that neurons find in vivo during the neurodevelopment.

Two mononuclear Pd(II) complexes [PdCl2(pfptp)] (1) and [PdCl2(pfhtp)] (2), with ligands 2-(3-perfluoropropyl-1-methyl-1,2,4-triazole-5yl)-pyridine (pfptp) and 2-(3-perfluoroheptyl-1-methyl-1,2,4-triazole-5yl)-pyridine (pfhtp), were synthesized and structurally characterized. The two complexes showed a bidentate coordination of the ligand occurring through N atom of pyridine ring and N4 atom of 1,2,4-triazole. Both complexes showed antimicrobial activity when tested against both Gram-negative and Gram-positive bacterial strains.

Hormones, i.e., the products of specialized endocrine cells which spread throughout the body via the bloodstream, control the normal development and growth of organisms at the embryo-fetal stage and, in adult life, regulate, integrate, and coordinate a range of different physiological processes which concern virtually all body tissues. They exert their biological effects by interacting with either surface or intracellular receptors, thereby activating signalization pathways [1]. For example, steroid hormones, such as those released by the adrenal glands, testes and ovaries, once freely crossed through the plasmalemma, bind to receptors that act as ligand-dependent transcriptional regulators and influence the expression of a plethora of target genes responsible for diversified biological responses, including sexual differentiation, osmoregulation, metabolism and developmental roles in various fetal systems among others [2,3].

The Mediterranean Diet (MD), UNESCO Intangible Cultural Heritage of Humanity, has become a scientific topic of high interest due to its health benefits. The aim of this review is to pick up selected studies that report nutrigenomic or nutrigenetic data and recapitulate some of the biochemical/genomic/genetic aspects involved in the positive health effects of the MD. These include (i) the antioxidative potential of its constituents with protective effects against several diseases; (ii) the epigenetic and epigenomic effects exerted by food components, such as Indacaxanthin, Sulforaphane, and 3-Hydroxytyrosol among others, and their involvement in the modulation of miRNA expression; (iii) the existence of predisposing or protective human genotypes due to allelic diversities and the impact of the MD on disease risk. A part of the review is dedicated to the nutrigenomic effects of the main cooking methods used in the MD and also to a comparative analysis of the nutrigenomic properties of the MD and other diet regimens and non-MD-related aliments. Taking all the data into account, the traditional MD emerges as a diet with a high antioxidant and nutrigenomic modulation power, which is an example of the “Environment-Livings-Environment” relationship and an excellent patchwork of interconnected biological actions working toward human health.

Polybrominated diphenyl ethers (PBDEs) are a class of persistent contaminants which are found all over the world in the marine environment. Sparus aurata fibroblast cell line (SAF-1) was exposed to increasing concentrations of PBDEs 47 and 99, until 72 h to evaluate the cytotoxicity, reactive oxygen species (ROS) production and the expression of some selected molecular markers related to cell cycle, cell signaling, energetic balance and oxidative stress (p53, erk-1, hif-1α and nrf-2), by real-time PCR. Furthermore, SAF-1 cells were exposed for 7 and 15 days to sub-lethal concentrations, in order to evaluate the response of some biomarkers by immunoblotting (p53, ERK-1, AMPK, HIF-1α and NRF-2). After 48 and 72 h, the cells showed a significant decrease of cell vitality as well as an increase of intracellular ROS production. Gene expression analysis showed that sub-lethal concentrations of BDE-99 and 47, after 72 h, up-regulated cell cycle and oxidative stress biomarkers, although exposure to 100 μmol L−1 down-regulated the selected markers related to cell cycle, cell signaling, energetic balance. After 7 and 15 days of sub-lethal doses exposure, all the analyzed markers resulted affected by the contaminants. Our results suggest that PBDEs influence the cells homeostasis first of all via oxidative stress, reducing the cell response and defense capacity and affecting its energetic levels. This situation of stress and energy imbalance could represents a condition that, modifying some of the analyzed biochemical pathways, would predispose to cellular transformation.

A wide range of contaminants, industrial by-products, plastics, and pharmaceutics belonging to various categories, have been found in sea water. Although these compounds are detected at concentrations that might be considered as sub-lethal, under certain conditions they could act synergistically producing unexpected effects in term of toxicity or perturbation of biochemical markers leading to standard pathway. In this study, the Sparus aurata fibroblast cell line SAF-1, was exposed to increasing concentrations of carbamazepine (CBZ), polybrominated diphenyl ether 47 (BDE-47) and cadmium chloride (CdCl2) until 72 h, to evaluate the cytotoxicity and the expression of genes related to antioxidant defense, cell cycle and energetic balance. In general, both vitality and gene expression were affected by the exposure to the different toxicants, in terms of antioxidant defense and cell cycle control, showing the most significant effects in cells exposed to the mixture of the three compounds, respect to the single compounds separately. The synergic effect of the compounds on the analyzed biomarkers, underlie the potential negative impact of the contaminants on health of marine organisms.

The persistent pollutants polybrominated diphenyl ethers (PBDEs) have been demonstrated to produce several negative effects on marine organisms. Although Mytilus galloprovincialis was extensively studied as model system, the effects of PBDEs on the innate immune system of mussels remains unclear. In this study, except for the control treatment, specimens of M. galloprovincialis were fed with microalgae treated with increasing concentrations of PBDEs (maximum level 100 ng L−1 of BDE-47 per day). BDE-47 treatment was maintained for 15 days and then the animals were fed with the same control diet, without contaminants, for 15 days. Samples of haemolymph (HL) were obtained at T0, T15 and T30 days of the experiment to evaluate different parameters related to immunity, such as neutral red retention time, and peroxidase, protease, antiprotease, lysozyme and bactericidal activities. BDE-47 exposure for 15 days affected both the stability of haemocytes and humoral parameters. In addition, the obtained results indicated that, at 30 days, after 15 days of culture without contaminant, the immune parameters were still affected, as some of them did not return to the basal levels, and others remained stimulated. Overall the results indicate that BDE-47 exposures at environmentally realistic levels may affect various aspects of immune function in M. galloprovincialis, acting as stressor that can compromise the general welfare.

In this work, we studied the nervous and sensory system of the colonial ascidian Botryllus schlosseri during the development of embryos, buds, and in adult individuals belonging to young and old colonies. B. schlosseri is a colonial ascidian in which individuals (blastozooids) are organized in star-shaped systems. In a colony, sexual and asexual reproductions are coordinated and cyclical. Moreover, three generations of zooids coexist: adults (filter-feeding individuals), their buds (primary buds), and the budlets (secondary buds) produced by buds. Cyclically, during a phase called takeover, adult zooids are reabsorbed by the colony and are replaced in physiological activities by their buds that become adults. Cuncurrently, budlets mature into buds and produce a new generation of budlets. We first reviewed historical studies concerning B. schlosseri with the intent of characterizing the colony life cycle and bud development. We then compared nervous system formation in two different developmental pathways, embryogenesis and blastogenesis. Since B. schlosseri has internal fertilization and development, we developed a method for culturing embryos in vitro. Using a combination of in vivo, confocal, histological observations, and 3D reconstructions based on serial sections, we described the embryonic development and drafted a timetable. Next, we sequenced transcriptomes of embryos and buds at several stages and illuminated the strict temporal relationship between morphogenetic events and the expression pattern of genes associated with the nervous system during the formation of the larval brain, its degeneration at metamorphosis, the adult brain formation in embryo and bud, and its degeneration at takeover. We also studied the nervous system in adult individuals belonging to colonies of different ages. We observed that the number of brain cells changes throughout the adult zooid life following a specific trend. Transmission electron microscopy and TUNEL assays on adult brains showed that apoptosis is involved in neurodegeneration and the number of immunocytes contacting or infiltrating the brain increase in number during the adult life. Changes in brain cell number parallel changes in sensory cell number. We developed two novel behavioural experiments for B. schlosseri, which showed that zooid ability to respond to mechanical stimuli parallels changes in the number of brain and sensory cells. Then we compared adult individuals belonging to young and old colonies and found that aging influences both nervous system morphology and behaviour. We analysed differentially expressed genes in brains of individuals belonging to young and old colonies and found that the old colonies exhibit a gene pattern associated with several human neurodegenerative diseases, such as the Alzheimer’s disease. Finally, we studied, the coronal organ, a mechanoreceptor located on the tentacles, in the oral siphon. We analysed its cytodifferentiation during asexual reproduction and we documented the mechanosensorial impairment caused by gentamicin at morphological and behavioural level. In mammals gentamicin can destroy both hair cells and their innervating neurons. The pre-treatment with fenofibrate, a gentamicin protector, followed by a treatment with gentamicin, resulted in no significant effect on animal behaviour. These results support the hypothesis of homology between vertebrate hair cells and tunicate coronal sensory cells. In conclusion, the research presented here shows that B. schlosseri can be considered a useful model species for analysing the development of the central nervous system and sensory system, as well as its degeneration as caused by drugs, metamorphosis, takeover and aging. Additionally, the species’ different developmental pathways allow for interesting evolutionary comparisons, at both the morphological and molecular level, that can help improve scientific understanding of the origin of the animal phenotype.

Mitochondrial DNA depletion syndromes (MDS) are a group of rare autosomal recessive disorders with early onset and no cure available. MDS are caused by mutations in several nuclear genes, involved in mitochondrial DNA (mtDNA) maintenance, characterized by a strong reduction of mtDNA copy number in affected tissues and severe defects in mitochondrial functionality. Mutations in MPV17, a nuclear gene encoding a mitochondrial inner membrane protein, have been specifically associated with hepatocerebral forms of MDS. However, MPV17 protein function is still unclear, although it has been suggested a primary role in mtDNA maintenance. Zebrafish represents a model to clarify this biological question: a mpv17 null mutant (roy orbison) shows a 19 bp deletion resulting in aberrant splicing between the exons 2 and 3 of mpv17 gene and lacks the guanine-based reflective skin cells named iridophores. In our work, we have characterized in details the mitochondrial phenotype of roy larvae and found early severe ultrastructural alterations in liver mitochondria; we could also observe a significant impairment of respiratory chain complexes leading to mitochondrial quality control activation. Our results provide evidences for Mpv17 being really essential in mitochondrial cristae maintenance and OXPHOS functionality, while its effect on mtDNA maintenance seems to be consequential, considering that mtDNA depletion only appeared at later stages of development. Moreover, taking into account that in roy orbison it has been previously postulated a role for Mpv17 in purines availability, and that embryos blocked in their pyrimidine synthesis resemble roy phenotype, we investigated the two alternatives by administrating purine and pyrimidine precursors to homozygous mutant embryos. Interestingly, orotic acid (OA) administration ameliorated roy phenotype, hence linking the loss of Mpv17 to pyrimidine de novo synthesis. In particular, the treatment with OA, currently used as food supplement, significantly increased not only iridophores number but also mtDNA content of mpv17 null mutants, thus opening up a new simple therapeutic approach for MPV17-related MDS.

Matrix metalloproteases (MMPS) are a family of well-known enzymes which operate prevalently in the extracellular domain, where they fulfil the function of remodeling the extracellular matrix. Within the about 26 family members, encoded by 24 genes in humans, MMP-2 and MMP-9, have been regarded as the primary responsibility for the basement membrane and pericellular ECM rearrangement. In cases of infiltrating carcinomas, which arise from the epithelial tissues of a gland or of an internal organ, a marked alteration of the expression and the activity levels of both MMPs is known to occur. Present investigation represents the continuation and upgrading of our previous studies, now focusing on the occurrence and intensity levels of MMP-2 and -9, and their proteomic correlations, in a cohort of 80 breast cancer surgical tissues

We investigated the effects of natural antioxidants and modified atmosphere packaging (MAP) on the quality and shelf-life of Coryphaena hippurus (common dolphinfish) fillets. Fillets of the control group (CO) were simply placed on trays and not sealed. Fillets of the second group (MAP) were preserved in modified atmosphere (45% CO2, 50% N2, 5% O2), and fillets of the third group (MAP-AOX) were preserved by combining natural antioxidants from halophytes with modified atmosphere. All samples were stored in a refrigerator at 1 ± 0.5 C for 18 days. Analyses were conducted at T0 and after 3, 6, 12, 15 and 18 days of storage. According to sensory tests and colour analyses, the fillets in the MAP and MAP-AOX groups maintained the best appearance. In the CO group, we observed a significant decrease of omega-3 polyunsaturated fatty acids (FAs) (P < 0.05), which indicates the oxidation of these FAs. FAs in the MAP and MAP-AOX groups were protected against oxidation. Further, lower peroxide value (PV) and malondialdehyde (MDA) content were observed in the MAP and MAP-AOX groups compared to the CO group. The sensory properties of the fillets were correlated with changes in FA content, PV and MDA content.

The present investigation has been conducted on one hundred tissue fragments of breast cancer, collected and immediately cryopreserved following the surgical resection. The specimens were selected from patients with invasive ductal carcinoma of the breast, the most frequent and potentially aggressive type of mammary cancer, with the objective to increase the knowledge of breast cancer molecular markers potentially useful for clinical applications. The proteomic screening; by 2D-IPG and mass spectrometry; allowed us to identify two main classes of protein clusters: proteins expressed ubiquitously at high levels in all patients; and proteins expressed sporadically among the same patients. Within the group of ubiquitous proteins, glycolytic enzymes and proteins with anti-apoptotic activity were predominant. Among the sporadic ones, proteins involved in cell motility, molecular chaperones and proteins involved in the detoxification appeared prevalent. The data of the present study indicates that the primary tumor growth is reasonably supported by concurrent events: the inhibition of apoptosis and stimulation of cellular proliferation, and the increased expression of glycolytic enzymes with multiple functions. The second phase of the evolution of the tumor can be prematurely scheduled by the occasional presence of proteins involved in cell motility and in the defenses of the oxidative stress. We suggest that this approach on large-scale 2D-IPG proteomics of breast cancer is currently a valid tool that offers the opportunity to evaluate on the same assay the presence and recurrence of individual proteins, their isoforms and short forms, to be proposed as prognostic indicators and susceptibility to metastasis in patients operated on for invasive ductal carcinoma of the breast.