Carmelo Bennici

Comprehensive Analysis of a Vibrio parahaemolyticus Strain Extracellular Serine Protease VpSP37.

Proteases play an important role in the field of tissue dissociation combined with regenerative medicine. During the years new sources of proteolytic enzymes have been studied including proteases from different marine organisms both eukaryotic and prokaryotic. Herein we have purified a secreted component of an isolate of Vibrio parahaemolyticus, with electrophoretic mobilities corresponding to 36 kDa, belonging to the serine proteases family. Sequencing of the N-terminus enabled the in silico identification of the whole primary structure consisting of 345 amino acid residues with a calculated molecular mass of 37.4 KDa. The purified enzyme, named VpSP37, contains a Serine protease domain between residues 35 and 276 and a canonical Trypsin/Chimotrypsin 3D structure. Functional assays were performed to evaluate protease activity of purified enzyme. Additionally the performance of VpSP37 was evaluated in tissue dissociations experiments and the use of such enzyme as a component of enzyme blend for tissue dissociation procedures is strongly recommended.

Development of a fast DNA extraction method for sea food and marine species identification

The authentication of food components is one of the key issues in food safety. Similarly taxonomy, population and conservation genetics as well as food web structure analysis, also rely on genetic analyses including the DNA barcoding technology. In this scenario we developed a fast DNA extraction method without any purification step from fresh and processed seafood, suitable for any PCR analysis. The protocol allows the fast DNA amplification from any sample, including fresh, stored and processed seafood and from any waste of industrial fish processing, independently of the sample storage method. Therefore, this procedure is particularly suitable for the fast processing of samples and to carry out investigations for the authentication of seafood by means of DNA analysis.

Evolutionary conserved mechanisms pervade structure and transcriptional modulation of allograft inflammatory factor-1 from sea anemone Anemonia viridis

Abstract Gene family encoding allograft inflammatory factor‐1 (AIF‐1) is well conserved among organisms; however, there is limited knowledge in lower organisms. In this study, the first AIF‐1 homologue from cnidarians was identified and characterised in the sea anemone Anemonia viridis. The full‐length cDNA of AvAIF‐1 was of 913 bp with a 5′ ‐untranslated region (UTR) of 148 bp, a 3′‐UTR of 315 and an open reading frame (ORF) of 450 bp encoding a polypeptide with149 amino acid residues and predicted molecular weight of about 17 kDa. The predicted protein possesses evolutionary conserved EF hand Ca2+ binding motifs, post‐transcriptional modification sites and a 3D structure which can be superimposed with human members of AIF‐1 family. The AvAIF‐1 transcript was constitutively expressed in all tested tissues of unchallenged sea anemone, suggesting that AvAIF‐1 could serve as a general protective factor under normal physiological conditions. Moreover, we profiled the transcriptional activation of AvAIF‐1 after challenges with different abiotic/biotic stresses showing induction by warming conditions, heavy metals exposure and immune stimulation. Thus, mechanisms associated to inflammation and immune challenges up‐regulated AvAIF‐1 mRNA levels. Our results suggest its involvement in the inflammatory processes and immune response of A. viridis. HighlightsAvAIF‐1 is the first cnidarians homologue of this family.AvAIF‐1 possess EF hand Ca2+ binding motifs and modification sites.Evidence for the evolutionary maintenance of the 3D structure are provided.AvAIF‐1 is transcriptionally regulated during immune response.

Partially Purified Extracts of Sea Anemone Anemonia viridis Affect the Growth and Viability of Selected Tumour Cell Lines

In the last few years, marine species have been investigated for the presence of natural products with anticancer activity. Using reversed phase chromatography, low molecular weight proteins were fractionated from the sea anemone Anemonia viridis. Four different fractions were evaluated for their cytotoxic activity by means of erythrocyte haemolysis test, MTS, and LDH assays. Finally, the antiproliferative activities of three of these fractions were studied on PC3, PLC/PRF/5, and A375 human cancer cell lines. Our analysis revealed that the four fractions showed different protein contents and diverse patterns of activity towards human PBMC and cancer cell lines. Interestingly, fractions III and IV exerted cytotoxic effects on human cells. Conversely, fractions I and II displayed very low toxic effects associated with antiproliferative activities on cancer cell lines.

Characterization of Translationally Controlled Tumour Protein from the Sea Anemone Anemonia viridis and Transcriptome Wide Identification of Cnidarian Homologues

Gene family encoding translationally controlled tumour protein (TCTP) is defined as highly conserved among organisms; however, there is limited knowledge of non-bilateria. In this study, the first TCTP homologue from anthozoan was characterised in the Mediterranean Sea anemone, Anemonia viridis. The release of the genome sequence of Acropora digitifera, Exaiptasia pallida, Nematostella vectensis and Hydra vulgaris enabled a comprehensive study of the molecular evolution of TCTP family among cnidarians. A comparison among TCTP members from Cnidaria and Bilateria showed conserved intron exon organization, evolutionary conserved TCTP signatures and 3D protein structure. The pattern of mRNA expression profile was also defined in A. viridis. These analyses revealed a constitutive mRNA expression especially in tissues with active proliferation. Additionally, the transcriptional profile of A. viridis TCTP (AvTCTP) after challenges with different abiotic/biotic stresses showed induction by extreme temperatures, heavy metals exposure and immune stimulation. These results suggest the involvement of AvTCTP in the sea anemone defensome taking part in environmental stress and immune responses.