Rapporto tecnico sulla ottimizzazione del processo di biosintesi di una Green Fluorescent Protein ricombinante estratta da A. sulcata

L’espressione di geni eterologhi in Escherichia coli rappresenta uno dei metodi più veloci, semplici ed economici per la produzione di ampie quantità di proteine target. Tuttavia, meccanismi di folding e le modifiche post traduzionali inducono a volte un non corretto ripiegamento delle proteine nella conformazione nativa, con successiva aggregazione in quelli che vengono definiti corpi di inclusione. Nel nostro caso, l’attenzione è stata focalizzata su una Green Fluorescent Protein (GFP) di 228 aa estratta da Anemonia sulcata, contenente un fluoroforo composto da tre amminoacidi Gln63, Tyr64, Gly65 all'interno di una struttura a barile. Il corretto folding della proteina era correlato strettamente alla funzionalità del fluoroforo. Il nostro obiettivo è stato quello, quindi, di ottimizzare il processo di biosintesi della GFP espressa in E. coli, ovviando alla formazione di corpi di inclusione contenenti la proteina (non funzionale), definendo e standardizzando inoltre, le condizioni che consentivano di produrre la più alta percentuale di GFP correttamente ripiegata (in condizioni non denaturanti) e quindi funzionale

Environmental Conditions along Tuna Larval Dispersion: Insights on the Spawning Habitat and Impact on Their Development Stages

Estimated larval backward trajectories of three Tuna species, namely, Atlantic Bluefin Tuna (Thunnus thynnus, Linnaeus, 1758), Bullet Tuna (Auxis Rochei, Risso, 1801) and Albacore Tuna (Thunnus alalunga, Bonnaterre, 1788) in the central Mediterranean Sea, were used to characterize their spawning habitats, and to assess the impact of changes due to the major environmental parameters (i.e., sea surface temperature and chlorophyll-a concentration) on larval development during their advection by surface currents. We assumed that the environmental variability experienced by larvae along their paths may have influenced their development, also affecting their survival. Our results showed that the Tuna larvae underwent an accelerated growth in favorable environmental conditions, impacting on the notochord development. In addition, further updated information on spawning and larval retention habitats of Atlantic Bluefin Tuna, Bullet and Albacore Tunas in the central Mediterranean Sea were delivered

Characterization of Cystatin B Interactome in Saliva from Healthy Elderly and Alzheimer's Disease Patients

Cystatin B is a small, multifunctional protein involved in the regulation of inflammation, innate immune response, and neuronal protection and found highly abundant in the brains of patients with Alzheimer's disease (AD). Recently, our study demonstrated a significant association between the level of salivary cystatin B and AD. Since the protein is able to establish protein-protein interaction (PPI) in different contexts and aggregation-prone proteins and the PPI networks are relevant for AD pathogenesis, and due to the relevance of finding new AD markers in peripheral biofluids, we thought it was interesting to study the possible involvement of cystatin B in PPIs in saliva and to evaluate differences and similarities between AD and age-matched elderly healthy controls (HC). For this purpose, we applied a co-immunoprecipitation procedure and a bottom-up proteomics analysis to purify, identify, and quantify cystatin B interactors. Results demonstrated for the first time the existence of a salivary cystatin B-linked multi-protein complex composed by 82 interactors and largely expressed in the body. Interactors are involved in neutrophil activation, antimicrobial activity, modulation of the cytoskeleton and extra-cellular matrix (ECM), and glucose metabolism. Preliminary quantitative data showed significantly lower levels of triosophosphate isomerase 1 and higher levels of mucin 7, BPI, and matrix Gla protein in AD with respect to HC, suggesting implications associated with AD of altered glucose metabolism, antibacterial activities, and calcification-associated processes. Data are available via ProteomeXchange with identifiers PXD039286 and PXD030679

A top-down proteomic approach reveals a salivary protein profile able to classify Parkinson's disease with respect to Alzheimer's disease patients and to healthy controls

Parkinson's disease (PD) is a complex neurodegenerative disease with motor and non-motor symptoms. Diagnosis is complicated by lack of reliable biomarkers. To individuate peptides and/or proteins with diagnostic potential for early diagnosis, severity and discrimination from similar pathologies, the salivary proteome in 36 PD patients was investigated in comparison with 36 healthy controls (HC) and 35 Alzheimer's disease (AD) patients. A top-down platform based on HPLC-ESI-IT-MS allowed characterizing and quantifying intact peptides, small proteins and their PTMs (overall 51). The three groups showed significantly different protein profiles, PD showed the highest levels of cystatin SA and antileukoproteinase and the lowest of cystatin SN and some statherin proteoforms. HC exhibited the lowest abundance of thymosin & beta;4, short S100A9, cystatin A, and dimeric cystatin B. AD patients showed the highest abundance of & alpha;-defensins and short oxidized S100A9. Moreover, different proteoforms of the same protein, as S-cysteinylated and S-glutathionylated cystatin B, showed opposite trends in the two pathological groups. Statherin, cystatins SA and SN classified accurately PD from HC and AD subjects. & alpha;-defensins, histatin 1, oxidized S100A9, and P-B fragments were the best classifying factors between PD and AD patients. Interestingly statherin and thymosin & beta;4 correlated with defective olfactory functions in PD patients. All these outcomes highlighted implications of specific proteoforms involved in the innate-immune response and inflammation regulation at oral and systemic level, suggesting a possible panel of molecular and clinical markers suitable to recognize subjects affected by PD

Tecniche di campionamento di sostanze bioattive da aculei di Scorpaena porcus

Nel presente report vengono descritte le tecniche utilizzate per l'estrazione del veleno dagli aculei della specie ittica Scorpaena porcus. Gli estratti grezzi ottenuti sono successivamente processati al fine di ottenere frazioni da utilizzare in saggi biochimici, per lo studio di possibili attività biologiche

DNA Barcoding as a tool for Zoological Taxonomy: Identification of bony fish in the Mediterranean Sea

The description of all the species present in nature is a vast task to be fulfilled by using the classical approach of morphological description of the organisms. In recent years, the traditional taxonomy, based primarily on identification keys of species, has shown a number of limitations in the use of the distinctive features in many animal taxa and inconsistencies with the genetic data. Furthermore, the increasing need to get a true estimate of biodiversity has led Zoological Taxonomy to seek new approaches and methodologies to support the traditional methods. The classification procedure has added modern criteriasuch as the evolutionary relationships and the genetic, biochemical and morphological characteristics of the organisms.Until now the Linnean binomial was the only abbreviated code associated with the description of the morphology of a species. The new technologies aim to achieve a short nucleotide sequence of the DNA to be used as an unique and solely label for a particular species, a specific genetic barcode. For both morphological and genetic approaches, skills and experience are required. Taxonomy is one of zoological disciplines that has been benefited from the achievements reached by modern molecular biotechnology. Using a molecular approach it is possible to identify cryptic species, to establish a family relationship between species and their membership of taxonomic categories or to reconstruct the evolutionary history of a taxon

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

Exploiting videos for science communication: the edulab experience

Nowadays, science communication and popularisation are part of a scientist’s everiday life because because divulgation disseminates scientific information to the general pubblic, helping himto under stand specific scientific subject and the advancements of research, as well as inspiring critical reflection, awareness and environmentally responsible behaviours. A team of scientists, researches and technicians of the IAS-CNR of Capo Granitola welcomed this challenge, founding the EDULab group. Its core activities are addressed in science divulgation and experimentation of innovative approaches using emerging digital technologies. Among a variety of science comunication products, videos have proved to be one of the best ways to litterally «immerse» the audience in scientific research and make the experience fun, in particular for non scientic audience and young people

Valutazione dello stato della biodiversità in aree antropizzate: applicazione della metodologia BRUV (Baited Remote Underwater Video) per lo studio della componente nectonica dell'ecosistema

Uno studio di valutazione dello stato della biodiversità focalizzato sulla componente nectonica e finalizzato alla determinazione dell’abbondanza e della composizione in specie del popolamento marino costiero si può avvalere di diversi strumenti e metodologie, le quali possono essere più o meno invasive sull’ecosistema oggetto dello studio. È possibile ad esempio applicare delle metodologie di pesca sperimentale, i.e. di prelievo degli organismi dal loro ambiente al fine di riconoscere e quantificare la presenza delle specie nectoniche nell’area di studio. Esistono tuttavia metodologie meno distruttive che permettono la raccolta di dati attraverso una stima visiva dell’abbondanza e della composizione delle specie direttamente nel loro ambiente naturale. Di queste metodologie fanno parte i censimenti visivi in situ (Samoilys M.A. 1997), i quali hanno il vantaggio di permettere la raccolta dati nel rispetto della biodiversità, evitando eventi mortali e/o fortemente stressanti agli individui oggetto dello studio. Questo fa si che tali metodologie risultino particolarmente utili in ambienti vulnerabili e/o soggetti a particolari vincoli protezionistici volti alla conservazione dell’ambiente marino, come le Aree Marine Protette (AMP), le quali in questo studio rappresentano delle zone di confronto caratterizzate da un limitato impatto antropico. Per tali ragioni, nell’ambito del progetto CISAS è stato scelto di portare avanti la valutazione dello stato del popolamento ittico attraverso una particolare tecnica di censimento visivo, la quale viene effettuata attraverso il sistema BRUV (Baited Remote Underwater Video)