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This study investigates the occurrence of anthropogenic fibres inside wild Paracentrotus lividus at a Mediterranean coastal area in 2020. From each sea urchin, the coelomic fluid was directly analysed while digestive tracts and gonads were removed, pre-treated with trypsin (0.3%) and digested with H 2 O 2 (10%) before analysis. A total of 260 fibres and 1 fragment were found in 100 specimens, with an average of 2.6 items/individual. Fibres were more abundant in the digestive system, less in gonads and in the coelomic fluid, respectively. Fourier transform infrared (FTIR) analysis of representative fibres identified 67% natural (cotton-based) and 33% synthetic polymers (polyester) suggesting their origin from textiles, possibly released from laundry sewages. Overall, these results encourage further in-depth investigations on fibres accumulation and potential transfer through the trophic chain up to humans., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Ovothiols are π-methyl-5-thiohistidines produced in great amounts in sea urchin eggs, where they can act as protective agents against the oxidative burst at fertilization and environmental stressors during development. Here we examined the biological relevance of ovothiol during the embryogenesis of the sea urchin Paracentrotus lividus by assessing the localization of the key biosynthetic enzyme OvoA, both at transcript and protein level, and perturbing its protein translation by morpholino antisense oligonucleotide-mediated knockdown experiments. In addition, we explored the possible involvement of ovothiol in the inflammatory response by assessing ovoA gene expression and protein localization following exposure to bacterial lipopolysaccharide. The results of the present study suggest that ovothiol may be a key regulator of cell proliferation in early developing embryos. Moreover, the localization of OvoA in key larval cells and tissues, in control and inflammatory conditions, suggests that ovothiol may ensure larval skeleton formation and mediate inflammatory processes triggered by bacterial infection. This work significantly contributes to the understanding of the biological function of ovothiols in marine organisms, and may provide new inspiration for the identification of the biological activities of ovothiols in humans, considering the pharmacological potential of these molecules.

Introduction: Short-term and long-term steroid contraceptive systems are widely employed in adolescents and premenopausal women; they could induce variation in bone metabolism, but whether these changes increase the overall fracture risk is not yet clear., Evidence Acquisition: A systematic search of scientific publications about "hormonal contraceptives" and "bone metabolism" in reproductive age women was conducted., Evidence Synthesis: In adolescent girl, combined oral contraceptives could have a deleterious effect on bone health when their onset is within three years after menarche and when they contain ethinyl estradiol at the dose of 20 mcg. In perimenopausal women, steroid contraceptives seem not influence bone health nor increase osteoporotic fractures risk in menopause. The oral progestogens intake is not related to negative effects on skeletal health. Depot medroxyprogesterone acetate (DMPA) induce a prolonged hypoestrogenism with secondary detrimental effect on healthy bone; the higher bone loss was observed at the DMPA dose of 150 mg intramuscular such as after long-term DMPA-users. Progestin-based implants and intrauterine devices have not negative effect on bone health., Conclusions: Since sex-steroid drugs induce variations in hormonal circulating concentrations, they may negatively affect bone metabolism. Contraceptive choice should be tailored evaluating any possible effect on bone health. Clinicians should always perform a precontraceptive counselling to identify any coexisting condition that may affect bone health. Further randomized studies are needed to confirm these results.

Objective: Many unmet rehabilitation needs of patients with stroke can be addressed effectively using technology. However, technological solutions have not yet been seamlessly incorporated into clinical care. The purpose of this pilot study was to examine how to bridge the gaps between the recovery process, technology, and clinical practice to impact stroke rehabilitation meaningfully., Design: Semistructured interviews were performed using a grounded theory approach with purposive sampling of 17 diverse expert providers in acute care, inpatient, and outpatient stroke rehabilitation settings. Common themes were identified from qualitative analyses of the transcribed conversations to develop a guiding framework from the emerging concepts., Results: Four core themes emerged that addressed major barriers in stroke rehabilitation and technology-assisted solutions to overcome these barriers: (1) accessibility to quality rehabilitation, (2) adaptability to patient differences, (3) accountability or compliance with rehabilitation, and (4) engagement with rehabilitation., Conclusions: The results suggest a four-pronged framework, the A3E framework that stands for Accessibility, Adaptability, Accountability, and Engagement, to comprehensively address existing barriers in providing rehabilitation services. This framework can guide technology developers and clinicians in designing and deploying technology-assisted rehabilitation solutions for poststroke rehabilitation, particularly using telerehabilitation., Competing Interests: Financial disclosure statements have been obtained, and no conflicts of interest have been reported by the authors or by any individuals in control of the content of this article., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

The sea urchin Paracentrotus lividus (P. lividus) was exposed to either virgin or biofilm-covered polystyrene microbeads (micro-PS, 45 μm) in order to test the effect of microbial colonization on the uptake, biodistribution, and immune response. The biofilm was dominated by bacteria, as detected by scanning electron microscopy and 16S rRNA sequencing. A higher internalization rate of colonized micro-PS inside sea urchins compared to virgin ones was detected, suggesting a role of the plastisphere in the interaction. Colonized and virgin micro-PS showed the same biodistribution pattern by accumulating mainly in the digestive system with higher levels and faster egestion rates for the colonized. However, a significant increase of catalase and total antioxidant activity was observed only in the digestive system of colonized micro-PS-exposed individuals. Colonized micro-PS also induced a significant decrease in the number of coelomocytes with a significant increase in vibratile cells, compared to control and virgin micro-PS-exposed animals. Moreover, a general time-dependent increase in the red/white amoebocytes ratio and reactive oxygen species and a decrease in nitrogen ones were observed upon exposure to both colonized and virgin micro-PS. Overall, micro-PS colonization clearly affected the uptake and toxicological responses of the Mediterranean sea urchin P. lividus in comparison to virgin micro-PS.

Progestin-only contraceptive implants provide long-acting, highly effective reversible contraception. We searched the medical publications in PubMed, CENTRAL, and EMBASE for relevant articles on hormonal implants published in English between 1990 and 2021. Levonorgestrel (LNG) 6-capsule subdermal implants represented the first effective system approved for reversible contraception. The etonogestrel (ENG) single rod dispositive has been widely employed in clinical practice, since it is a highly effective and safe contraceptive method. Abnormal menstrual bleeding is a common ENG side effect, representing the main reason for its premature discontinuation. Emerging evidence demonstrated that it is possible to extend the use of the ENG implant beyond the three-year period for which it is approved. The ENG implant could be an effective and discrete alternative to the IUD in young girls, such as post-partum/post-abortion. Implants should be inserted by trained skilled clinicians who previously provide adequate counselling about their contraceptive effect, benefits, and any possible adverse events. More studies are needed to validate the extended use of the ENG implant for up to 5 years.

Background: Residual motor deficits of the upper limb in patients with chronic stroke are common and have a negative impact on autonomy, participation and quality of life. Music-Supported Therapy (MST) is an effective intervention to enhance motor and cognitive function, emotional well-being and quality of life in chronic stroke patients. We have adapted the original MST training protocol to a home-based intervention, which incorporates increased training intensity and variability, group sessions, and optimisation of learning to promote autonomy and motivation., Methods: A randomised controlled trial will be conducted to test the effectiveness of this enriched MST (eMST) protocol in improving motor functions, cognition, emotional well-being and quality of life of chronic stroke patients when compared to a program of home-based exercises utilizing the Graded Repetitive Arm Supplementary Program (GRASP). Sixty stroke patients will be recruited and randomly allocated to an eMST group (n = 30) or a control GRASP intervention group (n = 30). Patients will be evaluated before and after a 10-week intervention, as well as at 3-month follow-up. The primary outcome of the study is the functionality of the paretic upper limb measured with the Action Research Arm Test. Secondary outcomes include other motor and cognitive functions, emotional well-being and quality of life measures as well as self-regulation and self-efficacy outcomes., Discussion: We hypothesize that patients treated with eMST will show larger improvements in their motor and cognitive functions, emotional well-being and quality of life than patients treated with a home-based GRASP intervention., Trial Registration: The trial has been registered at ClinicalTrials.gov and identified as NCT04507542 on 8 August 2020.

Nitric oxide synthase is ubiquitously present in metazoans and is involved in a wide range of biological processes. Three distinct Nos genes have been so far identified in vertebrates exhibiting a complex expression pattern and transcriptional regulation. Nevertheless, although independent events of Nos duplication have been observed in several taxa, only few studies described the regulatory mechanisms responsible for their activation in non-vertebrate animals. To shed light on the mechanisms underlying neuronal-type Nos expression, we focused on two non-vertebrate chordates: the cephalochordate Branchiostoma lanceolatum and the tunicate Ciona robusta. Here, throughout transphyletic and transgenic approaches, we identified genomic regions in both species acting as Nos functional enhancers during development. In vivo analyses of Nos genomic fragments revealed their ability to recapitulate the endogenous expression territories. Therefore, our results suggest the existence of evolutionary conserved mechanisms responsible for neuronal-type Nos regulation in non-vertebrate chordates. In conclusion, this study paves the way for future characterization of conserved transcriptional logic underlying the expression of neuronal-type Nos genes in chordates.

Objective: to evaluate the impact of the COVID-19 pandemic on the obstetrics and gynecology residency training program in Italy., Study Design: This was a cross-sectional survey study aimed to assess the impact of the COVID-19 pandemic on the obstetrics and gynecology residency training program in Italy. An online survey with 45 questions was sent and completed anonymously by residents after accepting an informed consent. The invitation to the online survey was sent to all the Italian residents in obstetrics and gynecology. Those on maternity leave at the time of the study were excluded. Residents were asked about their routinely activity before the COVID-19 pandemic, and to report the reduction in their clinical practice. They were also asked about psychological impact of COVID-19 on their clinical practice., Results: 933 Italian residents in obstetrics and gynecology, were invited for this survey study. Four-hundred and seventy-six (51 %) completed the survey and were included in the study. Three-hundred and eighty-seven (81.3 %) were female, and 89 (18.7 %) were male. Residents age ranged from 25 to 42. In 71,8 % (342/476) of the cases residents work in a COVID-19 reference Hospitals. One-hundred and eighty-four out of 76 residents (38.6 %) were tested on RT-PCR assay of nasal and pharyngeal swab specimens, and of them 12/184 (6.5 %) were positive to SARS-COV-2. Regarding the use of personal protective equipment (PPE), 267 (56.1 %) reported to receive adequate device, and 379 (79.6 %) felt to be well informed about prevention and management protocols. Three-hundred and thirty-one residents (69.5 %) reported to have managed COVID-19 positive patients. For 54,7 % of respondent residents, training activity in general decreased significantly during the COVID-19 epidemic. A one-third reduction was reported in 31,4 % of the cases, whereas a total suspension of the training in 9,9 % of the cases. In 89,3 % of cases the reduction was caused by the reorganization of work. Anxiety about the professional future was reported in 84 % of the residents, and 59 % of them had the perception that their training was irreversibly compromised., Conclusions: Among Italian residents in obstetrics and gynecology, COVID-19 pandemic was associated with a significant training impairment., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Diatoms are one of the most widespread groups of microalgae on Earth. They possess extraordinary metabolic capabilities, including a great ability to adapt to different light conditions. Recently, we have discovered that the diatom Skeletonema marinoi produces the natural antioxidant ovothiol B, until then identified only in clams. In this study, we investigated the light-dependent modulation of ovothiol biosynthesis in S. marinoi . Diatoms were exposed to different light conditions, ranging from prolonged darkness to low or high light, also differing in the velocity of intensity increase (sinusoidal versus square-wave distribution). The expression of the gene encoding the key ovothiol biosynthetic enzyme, ovoA , was upregulated by high sinusoidal light mimicking natural conditions. Under this situation higher levels of reactive oxygen species and nitric oxide as well as ovothiol and glutathione increase were detected. No ovoA modulation was observed under prolonged darkness nor low sinusoidal light. Unnatural conditions such as continuous square-wave light induced a very high oxidative stress leading to a drop in cell growth, without enhancing ovoA gene expression. Only one of the inducible forms of nitric oxide synthase, nos2 , was upregulated by light with consequent production of NO under sinusoidal light and darkness conditions. Our data suggest that ovothiol biosynthesis is triggered by a combined light stress caused by natural distribution and increased photon flux density, with no influence from the daily light dose. These results open new perspectives for the biotechnological production of ovothiols, which are receiving a great interest for their biological activities in human model systems.

Plastic pollution represents one of the major threats to the marine environment. A wide range of marine organisms has been shown to ingest microplastics due to their small dimensions (less than 1 mm). This negatively affects some biological processes, such as feeding, energy reserves and reproduction. Very few studies have been performed on the effect of microplastics on sea urchin development and virtually none on adults. The aim of this work was to evaluate the uptake and distribution of fluorescent labelled polystyrene microbeads (micro-PS) in the Mediterranean sea urchin Paracentrotus lividus and the potential impact on circulating immune cells. Differential uptake was observed in the digestive and water vascular systems as well as in the gonads based on microbeads size (10 and 45 μm in diameter). Treatment of sea urchins with particles of both sizes induced an increase of the total number of immune cells already after 24 h. No significant differences were observed among immune cell types. However, the ratio between red and white amoebocytes, indicative of sea urchin healthy status, increased with both particles. This effect was detectable already at 24 h upon exposure to smaller micro-PS (10 μm). An increase of intracellular levels of reactive oxygen and nitrogen species was observed at 24 h upon both micro-PS exposure, whereas at later time these levels became comparable to those of controls. A significant increase of total antioxidant capacity was observed after treatment with 10 μm micro-PS. Overall data provide the first evidence on polystyrene microbeads uptake and tissue distribution in sea urchins, indicating a stress-related impact on circulating immune cells., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Ovothiols are histidine-derived thiols that are receiving a great interest for their biological activities in human model systems. Thanks to the position of the thiol group on the imidazole ring of histidine, these compounds exhibit unusual antioxidant properties. They have been revealing a very promising pharmacological potential due to their anti-proliferative and anti-inflammatory properties, as well as anti-fibrotic activities not always related to their antioxidant power. Ovothiols occur in three differentially methylated forms (A, B and C), isolated from ovary, eggs and biological fluids of many marine invertebrates, mollusks, microalgae, and pathogenic protozoa. These molecules are synthesized by two enzymes: the sulfoxide synthase OvoA and the sulfoxide lyase OvoB. OvoA catalyzes the insertion of the sulfur atom of cysteine on the imidazole ring of histidine, leading to the formation of a sulfoxide intermediate. This is then cleaved by OvoB, giving 5-thiohistidine, finally methylated on the imidazole ring thanks to the methyltransferase domain of OvoA. Recent studies have shown that OvoA homologs are encoded in a wide variety of genomes suggesting that ovothiol biosynthesis is much more widespread in nature than initially thought. Here we have investigated the OvoA occurrence in diatoms, one of the most abundant group of microalgae, dominating marine and freshwater environments. They are considered a very good model system for both biology/photophysiology studies and for biotechnological applications. We have performed comparative sequence and phylogenetic analyses of OvoA from diatoms, highlighting a high degree of conservation of the canonical domain architecture in the analyzed species, as well as a clear clustering of OvoA in the two different morphological groups, i.e. centric and pennate diatoms. The in silico analyses have also revealed that OvoA gene expression is modulated by growth conditions. More importantly, we have characterized the thiol fraction from cultures of the coastal centric diatom Skeletonema marinoi, providing the first evidence of ovothiol B biosynthesis in diatoms., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

We utilized volcanic CO 2 vents at Castello Aragonese off Ischia Island as a natural laboratory to investigate the effect of lowered pH/elevated CO 2 on the bioactivities of extracts from fleshy brown algae Sargassum vulgare C. Agardh. We analysed the carbohydrate levels, antioxidant capacity, antibacterial, antifungal, antiprotozoal, anticancer properties and antimutagenic potential of the algae growing at the acidified site (pH ∼ 6.7) and those of algae growing at the nearby control site Lacco Ameno (pH∼8.1). The results of the present study show that the levels of polysaccharides fucoidan and alginate were higher in the algal population at acidified site. In general, extracts for the algal population from the acidified site showed a higher antioxidant capacity, antilipidperoxidation, antibacterial, antifungal, antiprotozoal, anticancer activities and antimutagenic potential compared to the control population. The increased bioactivity in acidified population could be due to elevated levels of bioactive compounds of algae and/or associated microbial communities. In this snapshot study, we performed bioactivity assays but did not characterize the chemistry and source of presumptive bioactive compounds. Nevertheless, the observed improvement in the medicinal properties of S. vulgare in the acidified oceans provides a promising basis for future marine drug discovery., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Caesarean section (CS) is usually perceived as a simple and safe alternative to natural birth, but in some instances can be technically difficult with consequent health hazards for both the mother and the fetus. We have proposed an evidence-based literature review of the most common difficult CS scenarios, with the aim to provide useful information about their management, possible prevention and resolution of complications., Methods: We identified articles through a reserch in PubMed, Scopus, Web of Science and Ovid MEDLINE for studies published between 1979 and 2019. We included the best available evidence, such as RCTs, non-randomised controlled clinical trials, case-control studies, cohort studies, and case series. About sixty articles were included in this review, four hundred and thirty-six were excluded after reviewing the title or abstract or because they weren't in English., Findings: The possible causes of "difficult" caesarean sections were divided into four categories: difficult access to the lower uterine segment; complicated fetal extraction, laceration or organ damage and abnormal placentation., Conclusions: Knowing in advance the potential technical difficulties and resulting risks allows the surgeon to plan appropriate strategies., Competing Interests: Declaration of Competing Interest The authors report no conflict of interest., (Published by Elsevier B.V.)

Gamma-glutamyl transpeptidase (GGT) is a cell surface enzyme involved in glutathione metabolism and maintenance of redox homeostasis. High expression of GGT on tumor cells is associated with an increase of cell proliferation and resistance against chemotherapy. GGT inhibitors that have been evaluated in clinical trials are too toxic for human use. We have previously identified ovothiols, 5(Nπ)-methyl-thiohistidines of marine origin, as non-competitive-like inhibitors of GGT that are more potent than the known GGT inhibitor, 6-diazo-5-oxo-l-norleucine (DON), and are not toxic for human embryonic cells. We extended these studies to the desmethylated form of ovothiol, 5-thiohistidine, and confirmed that this ovothiol derivative also acts as a non-competitive-like GGT inhibitor, with a potency comparable to ovothiol. We also found that both 5-thiohistidine derivatives act as reversible GGT inhibitors compared to the irreversible DON. Finally, we probed the interactions of 5-thiohistidines with GGT by docking analysis and compared them with the 2-thiohistidine ergothioneine, the physiological substrate glutathione, and the DON inhibitor. Overall, our results provide new insight for further development of 5-thiohistidine derivatives as therapeutics for GGT-positive tumors., Competing Interests: The authors declare no conflict of interest.

γ-Glutamyl transpeptidase (GGT) is an enzyme located on the surface of cellular membranes and involved in GSH metabolism and maintenance of redox homeostasis. High GGT expression on tumor cells is associated with increased cell proliferation and resistance against chemotherapy. GGT inhibitors evaluated so far in clinical trials are too toxic for human use. In this study, using enzyme kinetics analyses, we demonstrate that ovothiols, 5( N π)-methyl thiohistidines of marine origin, act as noncompetitive inhibitors of GGT, with an apparent K i of 21 μm, when we fixed the concentrations of the donor substrate. We found that these compounds are more potent than the known GGT inhibitor 6-diazo-5-oxo-l-norleucine and are not toxic toward human embryonic cells. In particular, cellular process-specific fluorescence-based assays revealed that ovothiols induce a mixed cell-death phenotype of apoptosis and autophagy in GGT-overexpressing cell lines, including human liver cancer and chronic B leukemic cells. The findings of our study provide the basis for further development of 5-thiohistidines as therapeutics for GGT-positive tumors and highlight that GGT inhibition is involved in autophagy., (© 2019 Brancaccio et al.)

The effects of ocean acidification, a major anthropogenic impact on marine life, have been mainly investigated in laboratory/mesocosm experiments. We used the CO 2 vents at Ischia as a natural laboratory to study the long-term effects of ocean acidification on the sea urchin Paracentrotus lividus population resident in low-pH (7.8 ± 0.2) compared to that at two control sites (pH 8.02 ± 0.00; 8.02 ± 0.01). The novelty of the present study is the analysis of the sea urchin immune cells, the sentinels of environmental stress responses, by a wide-ranging approach, including cell morphology, biochemistry and proteomics. Immune cell proteomics showed that 311 proteins were differentially expressed in urchins across sites with a general shift towards antioxidant processes in the vent urchins. The vent urchin immune cells showed higher levels of total antioxidant capacity, up-regulation of phagosome and microsomal proteins, enzymes of ammonium metabolism, amino-acid degradation, and modulation of carbon metabolism proteins. Lipid-hydroperoxides and nitric oxide levels were not different in urchins from the different sites. No differences in the coelomic fluid pH, immune cell composition, animal respiration, nitrogen excretion and skeletal mineralogy were observed. Our results reveal the phenotypic plasticity of the immune system of sea urchins adapted to life at vent site, under conditions commensurate with near-future ocean acidification projections., (Copyright © 2019. Published by Elsevier B.V.)

Soil pollution and toxicity have been investigated in the Gardanne area (southern France) at a range of sites around a recognized pollution source, a bauxite processing plant (BPP), and a power plant (PP). Soil samples were submitted to inorganic and organic analyses and tested for toxicity in two invertebrate models. Inorganic analysis was based on determining elemental concentrations by ICP-MS, encompassing a total of 26 elements including 13 rare earth elements (REEs), of the soil samples and their leachates after 24 or 48 h in seawater. Organic analyses were performed by measuring the sums of 16 polycyclic aromatic hydrocarbons (PAHs) and of total hydrocarbons (C-10 to C-40). Bioassays were carried out on the early life stages of three sea urchin species (Arbacia lixula, Paracentrotus lividus and Sphaerechinus granularis), and on a nematode (Caenorhabditis elegans). Sea urchin bioassays were evaluated by the effects of soil samples (0.1-0.5% dry wt/vol) on developing embryos and on sperm, and scored as: a) % developmental defects, b) inhibition of sperm fertilization success and offspring damage, and c) frequencies of mitotic aberrations. C. elegans 24 h-mortality assay showed significant toxicity associated with soil samples. The effects of soil samples showed heightened toxicity at two groups of sites, close to the BPP main entrance and around the PP, which was consistent with the highest concentrations found for metals and PAHs, respectively. Total hydrocarbon concentrations displayed high concentrations both close to BPP main entrance and to the PP. Further studies of the health effects of such materials in Gardanne are warranted., (Copyright © 2018. Published by Elsevier Inc.)

Adverse environmental conditions in the Taranto area (southern Italy) were investigated in studies of air, marine sediment, and human health. The present study aimed at providing unprecedented information on soil pollution and toxicity in a set of sites around recognized pollution sources in the Taranto area, since previous studies were focused on marine or air pollution, or on human health effects. The investigated area included a steel foundry and a power plant, as well as some sites located in an adjacent neighborhood. Surface soil samples and urban dust were collected and submitted to inorganic and organic analyses and tested for toxicity in two invertebrate bioassay models; a sea urchin (Sphaerechinus granularis) and an annelid (Caenorhabditis elegans). Inorganic analysis was carried out using ICP-MS for elemental composition for a total of 34 elements, whose levels were evaluated as a function of bioassay data analyzed through principal component analysis (PCA). Other analyses included asbestos search by powder X-ray diffraction (PXRD) and organic analysis for polycyclic aromatic hydrocarbons (PAHs) and aliphatic compounds (C10-C40). Toxicity bioassays were carried out on a sea urchin (Sphaerechinus granularis), and an annelid (Caenorhabditis elegans). Sea urchin bioassays evaluated effects of topsoil or street dust sample exposures (0.1 to 0.5% dry wt/vol) on developing embryos and on sperm, and scored as (a) % developmental defects, (b) inhibition of fertilization success and offspring damage, and (c) frequencies of mitotic aberrations. C. elegans mortality assay displayed significant toxicity associated with soil samples. The overall effects of samples showed very high toxicity at four out of nine sites. These effects were consistent with the highest levels measured for metals and PAHs. Further studies of health effects related to dust exposures in residential areas are warranted. Graphical abstract ᅟ.

Liver fibrosis is a complex process caused by chronic hepatic injury, which leads to an excessive increase in extracellular matrix protein accumulation and fibrogenesis. Several natural products, including sulfur-containing compounds, have been investigated for their antifibrotic effects; however, the molecular mechanisms underpinning their action are partially still obscure. In this study, we have investigated for the first time the effect of ovothiol A, π -methyl-5-thiohistidine, isolated from sea urchin eggs on an in vivo murine model of liver fibrosis. Mice were intraperitoneally injected with carbon tetrachloride (CCl 4 ) to induce liver fibrosis and treated with ovothiol A at the dose of 50 mg/kg 3 times a week for 2 months. Treatment with ovothiol A caused a significant reduction of collagen fibers as observed by histopathological changes and serum parameters compared to mice treated with control solution. This antifibrotic effect was associated to the decrease of fibrogenic markers involved in liver fibrosis progression, such as the transforming growth factor (TGF- β ), the α -smooth muscle actin (α-SMA), and the tissue metalloproteinases inhibitor (TIMP-1). Finally, we provided evidence that the attenuation of liver fibrosis by ovothiol A treatment can be regulated by the expression and activity of the membrane-bound γ -glutamyl-transpeptidase (GGT), which is a key player in maintaining intracellular redox homoeostasis. Overall, these findings indicate that ovothiol A has significant antifibrotic properties and can be considered as a new marine drug or dietary supplement in potential therapeutic strategies for the treatment of liver fibrosis.

Most of the studies regarding the impact of ocean acidification on macroalgae have been carried out for short-term periods, in controlled laboratory conditions, thus hampering the possibility to scale up the effects on long-term. In the present study, the volcanic CO 2 vents off Ischia Island were used as a natural laboratory to investigate the metabolic response of the brown alga Sargassum vulgare to acidification at different time scales. For long-term effects, algal populations naturally growing at acidified and control sites were compared. For short-term responses, in situ reciprocal transplants from control to acidified site and vice-versa were performed. Changes in the levels of sugars, fatty acids (FAs), amino acids (AAs), antioxidants, and phenolic compounds were examined. Our main finding includes variable metabolic response of this alga at different time scales to natural acidification. The levels of sugars, FAs, and some secondary metabolites were lower in the natural population at the acidified site, whereas the majority of AAs were higher than those detected in thalli growing at control site. Moreover, in algae transplanted from control to acidified site, soluble sugars (glucose and mannose), majority of AAs, and FAs increased in comparison to control plants transplanted within the same site. The differences in the response of the macroalga suggest that the metabolic changes observed in transplants may be due to acclimation that supports algae to cope with acidification, thus leading to adaptation to lowered pH in long time scale., (Copyright © 2018. Published by Elsevier B.V.)

The major yolk protein toposome plays crucial roles during gametogenesis and development of sea urchins. We previously found that nitration of toposome increases in the gonads of a Paracentrotus lividus population living in a marine protected area affected by toxic blooms of Ostreospsis cf. ovata, compared to control populations. This modification is associated with ovatoxin accumulation, high levels of nitric oxide in the gonads, and a remarkable impairment of progeny development. However, nothing is known about the environmental-mediated-regulation of the structure and biological function of toposome. Here, we characterize through wide-ranging biochemical and structural analyses the nitrated toposome of sea urchins exposed to the bloom, and subsequently detoxified. The increased number of nitrated tyrosines in toposome of sea urchins collected during algal bloom induced structural changes and improvement of the Ca 2+ -binding affinity of the protein. After 3 months' detoxification, ovatoxin was undetectable, and the number of nitric oxide-modified tyrosines was reduced. However, the nitration of specific residues was irreversible and occurred also in embryos treated with metals, used as a proxy of environmental pollutants. The structural and functional changes of toposome caused by nitration under adverse environmental conditions may be related to the defective development of sea urchins' progeny.

The widespread use of rare earth elements (REEs) in a number of technological applications raises unanswered questions related to REE-associated adverse effects. We have previously reported on the multiple impact of some REEs on the early life stages of the sea urchin Paracentrotus lividus. The present investigation was to evaluate REE toxicity to early life stages in two unrelated sea urchin species, Sphaerechinus granularis and Arbacia lixula. The comparative toxicities were tested of seven REEs, namely yttrium, lanthanum, cerium, neodymium, samarium, europium and gadolinium as chloride salts at concentrations ranging from 10 -7 to 10 -4  M. The evaluated endpoints included developmental defects and cytogenetic anomalies in REE-exposed embryos/larvae, and decreased fertilization success and offspring damage following sperm exposure. The results showed different toxicity patterns for individual REEs that varied according to test species and to treatment protocol, thus showing toxicity scaling for the different REEs. Further, the observed effects were compared with those reported for P. lividus either following embryo or sperm exposures. S. granularis showed a significantly higher sensitivity both compared to A. lixula and to P. lividus. This study provides clear-cut evidence for distinct toxicity patterns among a series of REEs. The differences in species sensitivity at micromolar REE levels may warrant investigations on species susceptibility to impacts along polluted coasts.

The development of the mouth in animals has fascinated researchers for decades, and a recent study proposed the modern view of recurrent evolution of protostomy and deuterostomy. Here we expanded our knowledge about conserved traits of mouth formation in chordates, testing the hypothesis that nitric oxide (NO) is a potential regulator of this process. In the present work we show for the first time that NO is an essential cell signaling molecule for cephalochordate mouth formation, as previously shown for vertebrates, indicating its conserved ancestral role in chordates. The experimental decrease of NO during early amphioxus Branchiostoma lanceolatum development impaired the formation of the mouth and gill slits, demonstrating that it is a prerequisite in pharyngeal morphogenesis. Our results represent the first step in the understanding of NO physiology in non-vertebrate chordates, opening new evolutionary perspectives into the ancestral importance of NO homeostasis and acquisition of novel biological roles during evolution.

Studies regarding macroalgal responses to ocean acidification (OA) are mostly limited to short-term experiments in controlled conditions, which hamper the possibility to scale up the observations to long-term effects in the natural environment. To gain a broader perspective, we utilized volcanic CO 2 vents as a "natural laboratory" to study OA effects on Sargassum vulgare at different time scales. We measured photosynthetic rates, oxidative stress levels, antioxidant contents, antioxidant enzyme activities, and activities of oxidative metabolic enzymes in S. vulgare growing at a natural acidified site (pH 6.7) compared to samples from a site with current pH (pH 8.2), used as a control one. These variables were also tested in plants transplanted from the control to the acidified site and vice-versa . After short-term exposure, photosynthetic rates and energy metabolism were increased in S. vulgare together with oxidative damage. However, in natural populations under long-term conditions photosynthetic rates were similar, the activity of oxidative metabolic enzymes was maintained, and no sign of oxidative damages was observed. The differences in the response of the macroalga indicate that the natural population at the acidified site is adapted to live at the lowered pH. The results suggest that this macroalga can adopt biochemical and physiological strategies to grow in future acidified oceans.

Background: Heavy rare earth elements (HREEs) have been scarcely studied for their toxicity, in spite of their applications in several technologies. Thus HREEs require timely investigations for their adverse health effects., Methods: Paracentrotus lividus and Arbacia lixula embryos and sperm were exposed to trichloride salts of five HREEs (Dy, Ho, Er, Yb and Lu) and to Ce(III) as a light REE (LREE) reference to evaluate: 1) developmental defects (% DD) in HREE-exposed larvae or in the offspring of HREE-exposed sperm; 2) mitotic anomalies; 3) fertilization success; and 4) reactive oxygen species (ROS) formation, and nitric oxide (NO) and malondialdehyde (MDA) levels. Nominal HREE concentrations were confirmed by inductively coupled plasma mass spectrometry (ICP-MS)., Results: HREEs induced concentration-related DD increases in P. lividus and A. lixula larvae, ranging from no significant DD increase at 10 -7 M HREEs up to ≅100% DD at 10 -5 M HREE. Larvae exposed to 10 -5 M Ce(III) resulted in less severe DD rates compared to HREEs. Decreased mitotic activity and increased aberration rates were found in HREE-exposed P. lividus embryos. Significant increases in ROS formation and NO levels were found both in HREE-exposed and in Ce(III) embryos, whereas only Ce(III), but not HREEs resulted in significant increase in MDA levels. Sperm exposure to HREEs (10 -5 -10 -4 M) resulted in a concentration-related decrease in fertilization success along with increase in offspring damage. These effects were significantly enhanced for Dy(III), Ho(III), Er(III) and Yb(III), compared to Lu(III) and to Ce(III)., Conclusion: HREE-associated toxicity affected embryogenesis, fertilization, cytogenetic and redox endpoints showing different toxicities of tested HREEs., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Ocean acidification is an emerging problem that is expected to impact ocean species to varying degrees. Currently, little is known about its effect on molecular mechanisms induced in fleshy macroalgae. To elucidate genome wide responses to acidification, a transcriptome analysis was carried out on Sargassum vulgare populations growing under acidified conditions at volcanic CO 2 vents and compared with populations in a control site. Several transcripts involved in a wide range of cellular and metabolic processes were differentially expressed. No drastic changes were observed in the carbon acquisition processes and RuBisCO level. Moreover, relatively few stress genes, including those for antioxidant enzymes and heat-shock proteins, were affected. Instead, increased expression of transcripts involved in energy metabolism, photosynthetic processes and ion homeostasis suggested that algae increased energy production to maintain ion homeostasis and other cellular processes. Also, an increased allocation of carbon to cell wall and carbon storage was observed. A number of genes encoding proteins involved in cellular signalling, information storage and processing and transposition were differentially expressed between the two conditions. The transcriptional changes of key enzymes were largely confirmed by enzymatic activity measurements. Altogether, the changes induced by acidification indicate an adaptation of growth and development of S. vulgare at the volcanic CO 2 vents, suggesting that this fleshy alga exhibits a high plasticity to low pH and can adopt molecular strategies to grow also in future more acidified waters., (© 2017 John Wiley & Sons Ltd.)

Polyunsaturated aldehydes (PUAs) are fatty-acid-derived metabolites produced by some microalgae, including different diatom species. PUAs are mainly produced as a wound-activated defence mechanism against microalgal predators or released from senescent cells at the end of a bloom. PUAs, including 2,4-trans-decadienal (DD), induce deleterious effects on embryonic and larval development of several planktonic and benthic organisms. Here, we report on the effects of DD on larval development and metamorphosis of the ascidian Ciona intestinalis. Ciona larval development is regulated by the cross-talking of different molecular events, including nitric oxide (NO) production, ERK activation and caspase 3-dependent apoptosis. We report that treatment with DD at the competence larval stage results in a delay in metamorphosis. DD affects redox balance by reducing total glutathione and NO levels. By biochemical and quantitative gene expression analysis, we identify the NO-signalling network affected by DD, including the upregulation of ERK phosphatase mkp1 and consequent reduction of ERK phosphorylation, with final changes in the expression of downstream ERK target genes. Overall, these results give new insights into the molecular pathways induced in marine organisms after exposure to PUAs during larval development, demonstrating that this aldehyde affects key checkpoints of larval transition from the vegetative to the reproductive life stage.

Cephalopods have been utilised in neuroscience research for more than 100 years particularly because of their phenotypic plasticity, complex and centralised nervous system, tractability for studies of learning and cellular mechanisms of memory (e.g. long-term potentiation) and anatomical features facilitating physiological studies (e.g. squid giant axon and synapse). On 1 January 2013, research using any of the about 700 extant species of "live cephalopods" became regulated within the European Union by Directive 2010/63/EU on the "Protection of Animals used for Scientific Purposes", giving cephalopods the same EU legal protection as previously afforded only to vertebrates. The Directive has a number of implications, particularly for neuroscience research. These include: (1) projects will need justification, authorisation from local competent authorities, and be subject to review including a harm-benefit assessment and adherence to the 3Rs principles (Replacement, Refinement and Reduction). (2) To support project evaluation and compliance with the new EU law, guidelines specific to cephalopods will need to be developed, covering capture, transport, handling, housing, care, maintenance, health monitoring, humane anaesthesia, analgesia and euthanasia. (3) Objective criteria need to be developed to identify signs of pain, suffering, distress and lasting harm particularly in the context of their induction by an experimental procedure. Despite diversity of views existing on some of these topics, this paper reviews the above topics and describes the approaches being taken by the cephalopod research community (represented by the authorship) to produce "guidelines" and the potential contribution of neuroscience research to cephalopod welfare.

Nitric oxide (NO) is essential to many physiological functions and operates in several signaling pathways. It is not understood how and when the different isoforms of nitric oxide synthase (NOS), the enzyme responsible for NO production, evolved in metazoans. This study investigates the number and structure of metazoan NOS enzymes by genome data mining and direct cloning of Nos genes from the lamprey. In total, 181 NOS proteins are analyzed from 33 invertebrate and 63 vertebrate species. Comparisons among protein and gene structures, combined with phylogenetic and syntenic studies, provide novel insights into how NOS isoforms arose and diverged. Protein domains and gene organization--that is, intron positions and phases--of animal NOS are remarkably conserved across all lineages, even in fast-evolving species. Phylogenetic and syntenic analyses support the view that a proto-NOS isoform was recurrently duplicated in different lineages, acquiring new structural configurations through gains and losses of protein motifs. We propose that in vertebrates a first duplication took place after the agnathan-gnathostome split followed by a paralog loss. A second duplication occurred during early tetrapod evolution, giving rise to the three isoforms--I, II, and III--in current mammals. Overall, NOS family evolution was the result of multiple gene and genome duplication events together with changes in protein architecture.

The presence of vertebrate-like steroids, steroidogenic enzymes and steroid receptors has been reported exclusively in cephalopods gonads. The role played by these steroids has been also recently investigated. We here give evidence of steroidogenic activity in the brain of cephalopods. The activity of two key steroidogenic enzymes: 3beta-hydroxysteroid dehydrogenase (HSD) and 17beta-HSD is present in the lobes of the nervous system of both Sepia and Octopus. Such enzymes convert pregnenolone to progesterone and androstenedione to testosterone respectively. Binding experiments seem to assign a functional role to the androgens in the brain of cephalopods. According to the present results, the absence of any progesterone binding moiety supports the hypothesis that progesterone may be a metabolite product along the steroidogenic chain leading to androgens. The presence of steroidogenic enzymes in specific lobes of the central nervous system is discussed in terms of the possible role that steroids can play in the sexual differentiation of the brain and in influencing some coded behaviours of cephalopods, such as learning processes.

Among the various melanin-producing systems, the ink gland of the cuttlefish (Sepia officinalis) has traditionally been regarded as a most convenient model system for the studies of melanogenesis. The ink gland is a highly specialized organ with immature cells in the inner portion, from where the cells gradually mature, migrate towards the outer portion of the gland and become competent to produce melanin giving rise to particulate melanosomes. When cell maturation is complete, melanin is secreted into the lumen of the gland, accumulated into the ink sac and ejected on demand. Biochemical studies carried out over the past two decades have shown that the ink gland contains a variety of melanogenic enzymes, including tyrosinase, a peculiar dopachrome rearranging enzyme (which catalyses the rearrangement of dopachrome to 5,6-dihydroxyindole) and a peroxidase (presumably involved in the later stages of melanin biosynthesis). These enzymes are functionally interactive in close subcellular compartments of ink gland cells and appear to act in a concerted fashion during the process of melanogenesis in the mature portion of the gland. More recent studies have revealed that ink production and ejection are affected and modulated by the N-methyl-D-aspartate (NMDA)-nitric oxide (NO)-cyclic GMP (cGMP) signalling pathway. Glutamate NMDA receptor and NO synthase, the enzyme responsible for the synthesis of NO, have been detected by biochemical and immunohistochemical techniques in immature ink gland cells. Stimulation of NMDA receptors caused a marked elevation of cGMP levels, activation of tyrosinase and increased melanin synthesis in the mature portion of the gland, via the NO-guanylyl cyclase interaction. This signalling is also present in different regions of the nervous system in Sepia and in certain neural pathways controlling contraction of the ink sac sphincters and wall muscle in the ejection mechanism. Overall, these and other findings allowed elaboration of an improved model of melanin formation in Sepia, which underscores the complex interplay of melanogenic enzymes and regulatory factors, highlighting both the similarities and the differences with melanogenesis in mammals.