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SummaryHigh-intensity femtosecond pulses from an X-ray free-electron laser enable pump probe experiments for investigating electronic and nuclear changes during light-induced reactions. On time scales ranging from femtoseconds to milliseconds and for a variety of biological systems, time-resolved serial femtosecond crystallography (TR-SFX) has provided detailed structural data for light-induced isomerization, breakage or formation of chemical bonds and electron transfer1. However, all ultra-fast TR-SFX studies to date have employed such high pump laser energies that several photons were nominally absorbed per chromophore2-14. As multiphoton absorption may force the protein response into nonphysiological pathways, it is of great concern15whether this experimental approach16allows valid inferences to be drawn vis-à-vis biologically relevant single-photon-induced reactions17. Here we describe ultrafast pump-probe SFX experiments on photodissociation of carboxymyoglobin, showing that different pump laser fluences yield markedly different results. In particular, the dynamics of structural changes and observed indicators of the mechanistically important coherent oscillations of the Fe-CO bond distance (predicted by recent quantum wavepacket dynamics15) are seen to depend strongly on pump laser energy. Our results confirm both the feasibility and necessity of performing TR-SFX pump probe experiments in the linear photoexcitation regime. We consider this to be a starting point for reassessing design and interpretation of ultrafast TR-SFX pump probe experiments16such that biologically relevant insight emerges.

Regenerative capability in anthozoans is an important adaptive strategy for their survival to environmental disturbance of natural and anthropogenic origin such as predation or anchoring, that can cause injuries or removal of entire parts of the animal body, and it can be also considered indirectly a further tool of innate immune system. In the context of “self”-“non self” recognition, is significant the interaction with the endosymbiont of the genus Symbiodinium and the recognition of pathogens and foreign agents capable of invading the injured tissues. From these premises and the growing stressors that can cause injuries, it is significant to understand how species respond to physical damage and how they manage to recover and regenerate compromised tissues. Our research team, studied in the Mediterranean anthozoan Anemonia viridis (Forsskål, 1775), the natural seasonal variability of its morphology and enzymatic biomarkers involved in inflammatory process, the immune system response following injection of molecules varied in type and dimension. In particular, after the infection of two pathogenic bacteria Escherichia coli and Vibrio alginolyticus a particular and strong reaction was observed. These previous knowledge allowed us to examine the activity of enzymes such as proteases (SDS-PAGE on gelatin and fibrinogen substrate), peroxidase and alkaline phosphatase as biomarkers traditionally involved in wound healing event and in the rearrangement of extracellular matrix. The regenerative process, in this Mediterranean species of anthozoan, was analyzed by subjecting groups of animals to differential tentacle cuts (n = 10, 20, 30) and observing and estimating the regenerative potential after 7, 14 and 21 days from cutting. A morphological and histological observation of the tentacular regrowth event was conducted in addition to the evaluation of the expression of proliferating cell nuclear antigen (PCNA) using the immunoblotting technique. The future goal is to increase the knowledge of the processes that trigger the immune response in inflammation and regeneration, for the considerable interest in basic sciences and for the transferability of results in biotechnological field

Lectins are present in almost all living organisms and are involved in several biological processes, including immune responses. In the present study, a galactose binding lectin (SsGBL) exhibiting an apparent MW of 43 kDa has been characterized and purified from the mucus of the polichaete Sabella spallanzanii

The ""stem cells"" discipline represents one of the most dynamic areas in biomedicine. While adult marine/aquatic invertebrate stem cell (MISC) biology is of prime research and medical interest, studies on stem cells from organisms outside the classical vertebrate (e.g., human, mouse, and zebrafish) and invertebrate (e.g., Drosophila, Caenorhabditis) models have not been pursued vigorously. Marine/aquatic invertebrates constitute the largest biodiversity and the widest phylogenetic radiation on Earth, from morphologically simple organisms (e.g., sponges, cnidarians), to the more complex mollusks, crustaceans, echinoderms, and protochordates. These organisms contain a kaleidoscope of MISC-types that allow the production of a large number of novel bioactive-molecules, many of which are of significant potential interest for human health. MISCs further participate in aging and regeneration phenomena, including whole-body regeneration. For years, the European MISC-community has been highly fragmented and has established scarce ties with biomedical industries in an attempt to harness MISCs for human welfare. Thus, it is important to (i) consolidate the European community of researchers working on MISCs; (ii) promote and coordinate European research on MISC biology; (iii) stimulate young researchers to embark on research in MISC-biology; (iv) develop, validate, and share novel MISC tools and methodologies; (v) establish the MISC discipline as a forefront interest of biomedical disciplines, including nanobiomedicine; and (vi) establish collaborations with industries to exploit MISCs as sources of bioactive molecules. In order to fill the recognized gaps, the EC-COST Action 16203 ""MARISTEM"" has recently been launched. At its initial stage, the consortium unites 26 scientists from EC countries, Cooperating countries, and Near Neighbor Countries.

The diversification of anthozoan toxins played an important role in the ability to colonize various ecological niches. In this study we evaluated the hemolytic activity of HPLC separated fraction of tentacle extracts of sea anemone Actinia equina. Toxic components from acid tissue tentacle extracts were investigated by size exclusion and reverse phase HPLC to characterize cytolytic molecules. A novel low molecular weight active fraction was sequenced by MALDI TOF analysis and a protein correspondent to 5.4 kDa Sodium channel neurotoxin (Ae1) from A. equina was identified. Synthetic Ae1 was assayed and it showed an hemolytic activity against mammalian erythrocytes in a dose dependent manner. Cytolytic activity in addition to neuro-inhibitory function could be a further property of Ae1 toxin repertoire., Invertebrate Survival Journal, Vol 13 No 1 (2016)

Gilthead seabream (Sparus aurata) is the only species of the Sparidae family currently bred on a large scale. As one of the most important species on Mediterranean aquaculture. For this main reason, the knowledge of its immune system and their alterations it is crucial to avoid economic losses worldwide. Fish are in intimate contact with their environment and defending itself by a complex system of innate defence mechanisms, that is divided into physical barriers, cellular and humoral components. The skin as a physical barrier seems to be central to protection from pathogens such as Photobacterium damselae ssp. piscicida. Probiotics are microorganisms (usually bacteria) that promote different benefits on host. The aim of this work was to evaluate the in vitro capacity of Shewanella putrefaciens also known as Pdp11 (a probiotic isolated from skin of gilthead seabream) to improve the skin immune defence against Photobacterium damselae. Thus, we analyzed by qPCR the expression profile of different cytokines in seabream skin explants, in response to an exposure (of 2 and 4 hours) to the probiotic Pdp11 and the pathogen Photobacterium damselae. Our results showed that the different cytokines are modulated by both the pathogen (P. Damselae) and the probiotic (Pdp11), which could, at least in in vitro level, improve the gilthead seabream mucosal health. Further studies are neccesary to unravel if the present findings in vitro can be confirmed through in vivo studies

Immunomodulation and physiological responses of Mytilus galloprovincialis as bioindicators of environmental change Oxygenation level temperature increases and changes in food availability are predicted to occur in the future. In such scenario, a global climate change (GCC), there is growing concern for the health status of wild and farmed organisms. Bivalve molluscs, are important components of coastal marine ecosystems, and as sedentary and filter feeders, are good bioindicators of environmental conditions. The ability of organisms to maintain the immunosurveillance unaltered under adverse environmental conditions may enhance theirs survival capability. Only a few studies have investigated the effects of changing environmental parameters on the mussels immunity. In the present study, the effects of different food concentration, temperature and oxygenation treatments were evaluated on immune parameters of Mytilus galloprovincialis detected on digestive gland and hemocytes. Bivalves were exposed to experimental conditions by increasing of six food treatment, to three different temperatures under conditions of normoxia and anoxia. The multifactorial analysis applied to the responses of the immune variables has showed a direct dependence of various enzymes production by temperature and food concentration. The stability of the lysosomal membrane was altered under conditions of thermal stress and food changing. The protein concentration of the lysate of hemocytes instead was most affected by the lack of adequate oxygenation. In addition, a correlation was carried out between mussels immunological effectors and physiological responses as clearance rate, measured by the removal of suspended particles from water flowing through experimental chambers, food absorption efficiency and rates of oxygen consumption by individual mussels. Overall, information summarized in the present study indicated that climate changes can affect hemocyte and enzymatic functionality and the immune responses of this bivalve could be used as good environmental biomarkers.