Species distribution models of two critically endangered deep-sea octocorals reveal fishing impacts on vulnerable marine ecosystems in central Mediterranean Sea.

Deep-sea coral assemblages are key components of marine ecosystems that generate habitats for fish and invertebrate communities and act as marine biodiversity hot spots. Because of their life history traits, deep-sea corals are highly vulnerable to human impacts such as fishing. They are an indicator of vulnerable marine ecosystems (VMEs), therefore their conservation is essential to preserve marine biodiversity. In the Mediterranean Sea deep-sea coral habitats are associated with commercially important crustaceans, consequently their abundance has dramatically declined due to the effects of trawling. Marine spatial planning is required to ensure that the conservation of these habitats is achieved. Species distribution models were used to investigate the distribution of two critically endangered octocorals (Funiculina quadrangularis and Isidella elongata) in the central Mediterranean as a function of environmental and fisheries variables. Results show that both species exhibit species-specific habitat preferences and spatial patterns in response to environmental variables, but the impact of trawling on their distribution differed. In particular F. quadrangularis can overlap with fishing activities, whereas I. elongata occurs exclusively where fishing is low or absent. This study represents the first attempt to identify key areas for the protection of soft and compact mud VMEs in the central Mediterranean Sea.

Complete mitochondrial genome and evolutionary analysis of Turritopsis dohrnii, the "immortal" jellyfish with a reversible life-cycle.

Turritopsis dohrnii (Cnidaria, Hydrozoa, Hydroidolina, Anthoathecata) is the only known metazoan that is capable of reversing its life cycle via morph rejuvenation from the adult medusa stage to the juvenile polyp stage. Here, we present a complete mitochondrial (mt) genome sequence of T. dohrnii, which harbors genes for 13 proteins, two transfer RNAs, and two ribosomal RNAs. The T. dohrnii mt genome is characterized by typical features of species in the Hydroidolina subclass, such as a high A+T content (71.5%), reversed transcriptional orientation for the large rRNA subunit gene, and paucity of CGN codons. An incomplete complementary duplicate of the cox1 gene was found at the 5' end of the T. dohrnii mt chromosome, as were variable repeat regions flanking the chromosome. We identified species-specific variations (nad5, nad6, cob, and cox1 genes) and putative selective constraints (atp8, nad1, nad2, and nad5 genes) in the mt genes of T. dohrnii, and predicted alterations in tertiary structures of respiratory chain proteins (NADH4, NADH5, and COX1 proteins) of T. dohrnii. Based on comparative analyses of available hydrozoan mt genomes, we also determined the taxonomic relationships of T. dohrnii, recovering Filifera IV as a paraphyletic taxon, and assessed intraspecific diversity of various Hydrozoa species.

Beyond the exome: the role of non-coding somatic mutations in cancer.

The comprehensive identification of mutations contributing to the development of cancer is a priority of large cancer sequencing projects. To date, most studies have scrutinized mutations in coding regions of the genome, but several recent discoveries, including the identification of recurrent somatic mutations in the TERT promoter in multiple cancer types, support the idea that mutations in non-coding regions are also important in tumour development. Furthermore, analysis of whole-genome sequencing data from tumours has elucidated novel mutational patterns and processes etched into cancer genomes. Here, we present an overview of insights gleaned from the analysis of mutations from sequenced cancer genomes. We then review the mechanisms by which non-coding mutations can play a role in cancer. Finally, we discuss recent efforts aimed at identifying non-coding driver mutations, as well as the unique challenges that the analysis of non-coding mutations present in contrast to the identification of driver mutations in coding regions.

Epibiotic Vibrio luminous bacteria isolated from some hydrozoa and bryozoa species.

Luminous bacteria are isolated from both Hydrozoa and Bryozoa with chitinous structures on their surfaces. All the specimens of the examined hydroid species (Aglaophenia kirchenpaueri, Aglaophenia octodonta, Aglaophenia tubiformis, Halopteris diaphana, Plumularia setacea, Ventromma halecioides), observed under blue light excitation, showed a clear fluorescence on the external side of the perisarc (chitinous exoskeleton) around hydrocladia. In the bryozoan Myriapora truncata, luminous bacteria are present on the chitinous opercula. All the isolated luminous bacteria were identified on the basis of both phenotypic and genotypic analysis. The isolates from A. tubiformis and H. diaphana were unambiguously assigned to the species Vibrio fischeri. In contrast, the isolates from the other hydroids, phenotypically assigned to the species Vibrio harveyi, were then split into two distinct species by phylogenetic analysis of 16S rRNA gene sequences and DNA-DNA hybridization experiments. Scanning electron microscopy analysis and results of culture-based and culture-independent approaches enabled us to establish that luminous vibrios represent major constituents of the bacterial community inhabiting the A. octodonta surface suggesting that the interactions between luminous bacteria and the examined hydrozoan and bryozoan species are highly specific. These interactions might have epidemiological as well as ecological implications because of the opportunistic pathogenicity of luminous Vibrio species for marine organisms and the wide-distribution of the hydrozoan and bryozoan functioning as carriers.

Vibrio harveyi associated with Aglaophenia octodonta (Hydrozoa, Cnidaria).

A previously unknown association between a luminous bacterium, Vibrio harveyi, and a benthic hydrozoan, Aglaophenia octodonta, is described. Aglaophenia hydrocladia showed a clear fluorescence in the folds along the hydrocaulus and at the base of the hydrotheca, suggesting the presence of luminous bacteria. This hypothesis was confirmed by isolation of luminous bacteria from Aglaophenia homogenates. Phenotypic characterization of bacterial isolates was performed by several morphological, biochemical, and cultural tests, completed with 16S rDNA sequence analysis. All the isolates were referred to a single species: V. harveyi. The association between V. harveyi and A. octodonta has epidemiological as well as ecological significance. Therefore, A. octodonta may function as habitat "islands" providing a unique set of environmental conditions for luminous bacteria colonization, quite different from those already recorded from the plankton for other Vibrio species.

Morphological and ultrastructural analysis of Turritopsis nutricula during life cycle reversal.

The hydrozoa life cycle is characterized, in normal conditions, by the alternation of a post-larval benthic polyp and an adult pelagic medusa; however, some species of Hydrozoa react to environmental stress by reverting their life cycle: i.e. an adult medusa goes back to the juvenile stage of polyp. This very uncommon life cycle could be considered as some sort of inverted metamorphosis. A morphological study of different stages during the reverted life cycle of Turritopsis nutricula led to the characterization of four different stages: healthy medusa, unhealthy medusa, four-leaf clover and cyst. The ultrastructural study of the cellular modifications (during the life cycle reversion of T. nutricula) showed the presence of both degenerative and apoptotic processes. Degeneration was prevalent during the unhealthy medusa and four-leaf clover stages, while the apoptotic rate was higher during the healthy medusa and cyst stages. The significant presence of degenerative and apoptotic processes could be related to the occurrence of a sort of metamorphosis when an adult medusa transforms itself into a polyp.

Immunogene therapy for murine melanoma using recombinant adenoviral vectors expressing melanoma-associated antigens.

Adenoviral vectors expressing tumor-associated antigens can be used to evoke a specific immune response and inhibit tumor growth. In this study, we tested the efficacy of adenoviral vectors encoding human gp100 (Ad2/hugp100), murine gp100 (Ad2/mugp100), or murine TRP-2 (Ad2/muTRP-2) for their ability to elicit a specific cellular immune response and inhibit the growth of B16 melanoma tumor cells in the mouse. C57BL/6 mice were immunized with Ad2/hugp100, Ad2/mugp100, or Ad2/muTRP-2 either 2 weeks prior to B16-F10 tumor challenge (prophylactic treatment) or 3 days after tumor challenge (active treatment). Ad2/hugp100 and Ad2/muTRP-2 administered to two or more intradermal (i.d.) sites inhibited subsequent subcutaneous tumor growth in > or = 80% of the mice and elicited an antigen-specific cytotoxic T lymphocyte response, whereas other administration routes were not as effective. Ad2/mugp100 administered to two i.d. sites did not inhibit tumor growth or provoke cellular immunity. Immunization was less effective with active treatment where tumor growth was not significantly inhibited by a single dose of either Ad2/muTRP-2 or Ad2/hugp100. However, increasing the number of intradermal immunization sites and the number of doses resulted in progressive improvements in protection from tumor growth in the active treatment model. In conclusion, breaking host tolerance to elicit protective immunity by using adenoviral vectors expressing melanoma-associated antigens is dependent upon the choice of antigen, the site of administration, and the number of doses. These observations provide insights into the clinical applicability of adenoviral vaccines for immunotherapy of malignant diseases.

Gradient elution techniques for capillary electrochromatography.

Capillary electrochromatography (CEC) is a rapidly maturing technique, but still in need of further instrumental development and in need of unique applications that are not possible by traditional pressure-driven LC. We review the development of gradient elution schemes for CEC, beginning with pH gradients initially developed for capillary electrophoresis. Step gradients are the most easily instrumentally implemented, but provide less flexibility in separation than continuous gradients. Pressure-assisted CEC is easily adapted to gradient elution schemes, but does not offer the advantages of very high column efficiency provided by totally electro-driven mobile phases. The development of flow-injection interfaces allows a true solvent gradient to be generated by micro-LC pumps, with the mobile phase drawn into the separation capillary by pure electroosmotic flow. While requiring both a CEC instrument and a traditional pump or pumps capable of generating the gradient, this method offers advantages of greatly reduced column handling, prolonging column lifetimes, and allows simple autosampling. We also discuss voltage gradients, which provide a mobile phase velocity gradient.

Induction of antitumor immunity with dendritic cells transduced with adenovirus vector-encoding endogenous tumor-associated antigens.

Dendritic cells (DCs) are professional Ag-presenting cells that are being considered as potential immunotherapeutic agents to promote host immune responses against tumor Ags. In this study, recombinant adenovirus (Ad) vectors encoding melanoma-associated Ags were used to transduce murine DCs, which were then tested for their ability to activate CTL and induce protective immunity against B16 melanoma tumor cells. Immunization of C57BL/6 mice with DCs transduced with Ad vector encoding the hugp100 melanoma Ag (Ad2/hugp100) elicited the development of gp100-specific CTLs capable of lysing syngeneic fibroblasts transduced with Ad2/hugp100, as well as B16 cells expressing endogenous murine gp100. The induction of gp100-specific CTLs was associated with long term protection against lethal s.c. challenge with B16 cells. It was also possible to induce effective immunity against a murine melanoma self Ag, tyrosinase-related protein-2, using DCs transduced with Ad vector encoding the Ag. The level of antitumor protection achieved was dependent on the dose of DCs and required CD4+ T cell activity. Importantly, immunization with Ad vector-transduced DCs was not impaired in mice that had been preimmunized against Ad to mimic the immune status of the general human population. Finally, DC-based immunization also afforded partial protection against established B16 tumor cells, and the inhibition of tumor growth was improved by simultaneous immunization against two melanoma-associated Ags as opposed to either one alone. Taken together, these results support the concept of cancer immunotherapy using DCs transduced with Ad vectors encoding tumor-associated Ags.

Analysis of recombinant adeno-associated virus packaging and requirements for rep and cap gene products.

Adeno-associated virus (AAV) is a human parvovirus currently being developed as a vector for gene therapy applications. Because the gene transfer vector commonly retains only the AAV terminal repeats, propagation of recombinant AAV (rAAV) requires that the viral replication (Rep) and capsid (Cap) proteins be supplied in trans. In an effort to optimize the production of these vectors, a panel of helper plasmids was constructed to determine if expression of the rep and/or cap genes is a limiting factor for rAAV packaging. Expression of the Rep and Cap proteins was increased by replacing the endogenous AAV promoters, p5 and p40, with the Rous sarcoma virus (RSV) long terminal repeat (LTR) and the cytomegalovirus immediate-early promoter, respectively. Increased synthesis of the Cap proteins resulted in an approximately 10-fold increase in the yield of rAAV, indicating that production of capsid proteins is one limiting factor for rAAV packaging. Expression of the rep gene from the RSV LTR not only failed to increase the yield of rAAV but also prevented activation of p40 transcription with adenovirus infection, resulting in a reduced level of capsid protein synthesis.

The continuity of living matter and the discontinuities of its constituents: do plankton and benthos really exist?

Plankton and benthos are popular concepts identifying two ways of life of aquatic organisms. Their spatial separation led to the development of different sampling techniques and to separate conceptualizations of the principles governing these subsets of the aquatic environment. Reciprocal connections between plankton and benthos, however, are very strong both from a functional (energy fluxes) and a structural (life cycle dynamics) point of view. A full appreciation of such links is forcing marine ecology towards a more integrated approach.

Reversing the Life Cycle: Medusae Transforming into Polyps and Cell Transdifferentiation in Turritopsis nutricula (Cnidaria, Hydrozoa).

Organisms develop through a series of stages leading to sexually mature adults. In a few cases ontogeny reversal is possible, but it does not occur typically after the onset of sexual reproduction. All stages of the medusa Turritopsis nutricula, from newly liberated to fully mature individuals, can transform back into colonial hydroids, either directly or through a resting period, thus escaping death and achieving potential immortality. This is the first metazoan known to revert to a colonial, juvenile morph after having achieved sexual maturity in a solitary stage. Selective excision experiments show that the transformation of medusae into polyps occurs only if differentiated cells of the exumbrellar epidermis and part of the gastrovascular system are present, revealing a transformation potential unparalleled in the animal kingdom.

Functional activation of the cystic fibrosis trafficking mutant delta F508-CFTR by overexpression.

The most common mutation in the gene associated with cystic fibrosis (CF) causes deletion of phenylalanine at residue 508 (delta F508) of the gene product called CFTR. This mutation results in the synthesis of a variant CFTR protein that is defective in its ability to traffic to the plasma membrane. Because earlier studies showed delta F508-CFTR retains significant phosphorylation-regulated chloride (Cl-) channel activity, processes capable of restoring the mislocalized delta F508-CFTR to the correct cellular destination may have therapeutic benefit. Here we report one such process that involves overexpression of the mutant protein and appears to result in the escape of a small amount of delta F508-CFTR to the plasma membrane. In recombinant cells where expression of delta F508-CFTR is controlled by the metallothionein promoter, this effect can be brought about by treatment with sodium butyrate. Although cAMP-activated Cl- channel activity could also be detected in immortalized human airway epithelial cells homozygous for the delta F508 mutation at the single cell level, treatment with butyrate did not generate a measurable cAMP-stimulated Cl- current in polarized monolayers of primary CF airway epithelia. However, the observation that overexpression can effect the presence of recombinant delta F508-CFTR at the plasma membrane suggests that perhaps other butyrate-like compounds that are more potent and more specific for the promoter of the CF gene may be efficacious in alleviating the Cl- channel defect associated with CF.