2019, Articolo in rivista, ENG
Adelfi, Maria Grazia; Vitale, Rosa Maria; d'Ippolito, Giuliana; Nuzzo, Genoveffa; Gallo, Carmela; Amodeo, Pietro; Manzo, Emiliano; Pagano, Dario; Landi, Simone; Picariello, Gianluca; Ferrante, Maria Immacolata; Fontana, Angelo
Diatoms are eukaryotic microalgae that play a pivotal role in biological and geochemical marine cycles. These microorganisms are at the basis of the trophic chain and their lipids are essential components (e.g. eicosapentaenoic acid, EPA) of aquatic food webs. Galactolipids are the primary lipid components of plastid membranes and form the largest lipid family of diatoms. As source of polyunsaturated fatty acids (PUFAs), these compounds are also involved in the synthesis of lipoxygenase (LOX) products such as non-volatile oxylipins and polyunsaturated aldehydes. Here, we report the first identification of two genes, namely PmLAH1 and PaLAH1, coding for lipolytic enzymes in two diatoms of the genus Pseudo-nitzschia. Functional and modeling studies evidence a patatin-like domain endowed with galactolipase and phospholipase activity at the C-terminus of both proteins. Homologues of Pseudo-nitzschia LAH1 genes were retrieved in other diatom species so far sequenced in agreement with conservation of the functional role of these proteins within the lineage.
2019, Articolo in rivista, ENG
Picariello, Gianluca; De Cicco, Maristella; Nocerino, Rita; Paparo, Loretta; Mamone, Gianfranco; Addeo, Francesco; Canani, Roberto Berni
Nanoflow-HPLC-tandem mass spectrometry (MS/MS) was used to analyze the peptide fraction of breast milk samples collected from a single non-atopic donor on different days (10 samples) after receiving an oral load of cow's milk (by drinking 200 mL of bovine milk). In addition, breast milk was sampled from the same lactating mother over a 6-h period at five time points after drinking cow's milk. We aimed to trace the intra-individual variability and to define a time profile of the excretion of dietary peptides into breast milk. Overall, 21 peptides exclusively originating from both bovine caseins and whey proteins with no match within the human milk proteome were identified in the breast milk samples. These peptides were missing in the breast milk obtained from the mother after a prolonged milk- and dairy-free diet (three samples). The time course of cow's milk-derived beta-Lg f(125-135) and beta-casein f(81-92) in breast milk was determined from the MS ion intensity of the peptide signals. No intact cow's milk gene products were detected by HPLC-MS/MS analysis and Western blotting with anti-beta-Lg antibody, but dot-blot analysis confirmed the occurrence of beta-Lg fragments in the enriched peptide fraction of breast milk. These data suggest shifting the analytical perspective for the detection of dietary food allergens in breast milk from intact proteins to digested peptide fragments. The possible sensitization and elicitation potential or the tolerogenic properties of such low amounts of dietary peptides for the breastfed newborns remain to be explored.
2019, Articolo in rivista, ENG
Verhoeckx, Kitty; Bogh, Katrine Lindholm; Dupont, Didier; Egger, Lotti; Gadermaier, Gabriele; Larre, Colette; Mackie, Alan; Menard, Olivia; Adel-Patient, Karine; Picariello, Gianluca; Portmann, Reto; Smit, Joost; Turner, Paul; Untersmayr, Eva; Epstein, Michelle M.
The current allergenicity assessment of novel proteins is based on the EFSA GMO guidance. Recently, EFSA launched a new guidance document on allergenicity assessment of GM plants (2017), This document describes, amongst other topics, the new scientific and regulatory developments on in vitro protein digestibility tests. The EFSA GMO Panel stated that for in vitro protein digestibility tests, additional investigations are needed before any additional recommendation in the form of guidance can be provided. To this end, an interim phase is considered necessary to evaluate the revisions to the in vitro gastrointestinal digestion test, proposed by EFSA. This prompted the establishment of a joint workshop through two COST Action networks: COST Action ImpARAS and COST Acton INFOGEST. In 2017, a workshop was organised to discuss the relevance of digestion in allergenicity risk assessment and how to potentially improve the current methods and readouts. The outcome of the workshop is that there is no rationale for a clear readout that is predictive for allergenicity and we suggest to omit the digestion test from the allergenicity assessment strategy for now, and put an effort into filling the knowledge gaps as summarized in this paper first.
2019, Articolo in rivista, ENG
De Cicco, Maristella; Mamone, Gianfranco; Di Stasio, Luigia; Ferranti, Pasquale; Addeo, Francesco; Picariello, Gianluca
Analyzing an in vitro gastroduodenal digest of whey proteins by high-performance liquid chromatography (HPLC) coupled to high-resolution/high-sensitivity tandem mass spectrometry (MS/MS), we sought to evaluate if state-of-art peptidomics provide comprehensive peptide coverage of food "digestomes". A multitude of small-sized peptides derived from both a-lactalbumin and beta-lactoglobulin as well as disulfide cross-linked hetero-oligomers remained unassigned, even when the digests were compared before and after S-S reduction. The precipitation with 12% trichloroacetic acid demonstrated the occurrence of large-sized polypeptides that escaped the bioinformatic identification. The analysis of a HPLC-MS/MS run with different proteomic search engines generated dissimilar peptide subsets, thus emphasizing the 'demand of refined searching algorithms. Although the MS/MS fragmentation of monocharged ions with exclusion of non-peptide-interfering compounds enlarged the inventory of short peptides, the overall picture of the "digestome" was still incomplete. These findings raise relevant implications for the identification of possible food-derived bioactive peptides or allergenic determinants.
2019, Articolo in rivista, ENG
Rutigliano M.; Picariello G.; Trani A.; Di Luccia A.; la Gatta B.
The molecular weight distribution of protein aggregates from raw meat and cooked pork products was assessed by size exclusion-high performance liquid chromatography (SE-HPLC). Electrophoretic analysis under reducing conditions showed that the high molecular weight SE-HPLC peak (peak 1)of the cooked products contained protein aggregates in addition to high molecular weight muscle proteins, while the second peak (peak 2)still contained aggregates and <50 kDa proteins. The protein aggregates composition was investigated by HPLC-tandem mass spectrometry. Different classes of proteins were identified and the cooked products showed a more complex composition and organization, according to the muscle structure and the technological procedures, respectively. The key role of actin in the building of the protein networks was also confirmed. The different multi-protein systems found in the cooked products suggest protein re-organization in heat-induced supramolecular structures, which might be responsible for the texture and the structural properties of the final products.
2019, Articolo in rivista, ENG
Asledottir T.; Picariello G.; Mamone G.; Ferranti P.; Roseth A.; Devold T.G.; Vegarud G.E.
This work aimed to study the opioid peptide ?-casomorphin-7 (BCM7) degradation or stability during digestion using human gastrointestinal (GI) juices and porcine jejunal brush border membrane (BBM) peptidases. Synthetic BCM7 was subjected to in vitro digestion by GI fluids obtained from human volunteers for 180 min, and to downstream degradation with porcine BBM vesicles. The BCM7 was sampled at 4 time points over 24 h after BBM addition. The digests were profiled by HPLC-electrospray ionization mass spectrometry (ESI/MS) to monitor BCM7 during GI digestion, and intact BCM7 through BBM digestion was quantified by reverse-phase (RP)-HPLC. We found that BCM7 was partly digested with human GI enzymes, as 3 proteolytic fragments in addition to f(60-66) YPFPGPI were detected: f(62-66) FPGPI, f(60-65) YPFPGP and f(61-66) PFPGPI. The RP-HPLC analysis revealed that 42% of the initial peptide was degraded after only 2 h of BBM digestion, and as much as 79% was degraded after 4-h digestion with supplementation of BBM. In conclusion, this study showed that most of BCM7 was degraded during GI and BBM digestion, although a small amount (5%) was still detected after 24-h digestion. It remains to be studied whether the small amount of intact BCM7 detected after in vitro digestion is transported via active transceptors in the human intestinal epithelial cells and enters blood circulation.
2019, Articolo in rivista, ENG
Pieri M.; Silvestre A.; De Cicco M.; Mamone G.; Capasso E.; Addeo F.; Picariello G.
Proteomics was exploited to assess the nature of possible traces of vomit found on the scene of an alleged sexual assault. In the case in point, a woman reported to the police to be raped five days before by a cousin of hers in his car. The woman declared she had vomited in the car before fainting definitely, due to alcohol or possible drugs covertly slipped in her drinks. The suspect confirmed the sexual intercourse, but he claimed consensual sex while the woman was fully conscious. To establish consent and hence subsistence of the crime, the Magistrate requested toxicological analyses on items sampled from the car and from woman's boots. Negative results obtained from toxicological analyses could not exclude the actual assumption of psychoactive substances by the alleged victim, due to sample aging. On the contrary, proteomic analysis disclosed a pattern of 249 gene products including signature endogenous and food-derived proteins along with a multitude of peptide digests, clearly indicative of vomit, thereby supporting the victim's report in the case under examination. Proteomics also provided detailed information about the nature of meal, which might contribute to frame the crime scene in similar cases. Significance: The identification of traces of vomit supported the report of the victim's report according to which she vomited before definitely losing consciousness, so providing key contribution to establish consent for the sexual intercourse. This is the first time that proteomics is used to identify traces of vomit for forensic purposes. In spite of the scantiness of the biological specimen available, proteomics was successful to define a panel of characteristic endogenous proteins as well as to identify partly digested food-proteins arising from a complex meal. Proteomics is increasingly used as a forensic technique, well complementing the existing tools. In general, assessing traces of vomit in biological specimens and characterizing the nature of food ingested at the molecular level could afford probative elements to frame a crime scene.
2019, Articolo in rivista, ENG
Navarro S.A.; Lanza L.; Colombo N.S.R.; de Ullivarri M.F.; Acuna L.; Sosa-Padilla B.; Picariello G.; Bellomio A.; Chalon M.C.
The present paper describes the generation of derivatives from the hybrid peptide called Ent35-MccV, active against Gram-positive and Gram-negative bacteria. This peptide has a triple glycine hinge region between enterocin CRL35 and microcin V. In order to obtain variants of Ent35-MccV with greater biotechnological potential, a saturation mutagenesis was carried out in the hinge region. As a result, we obtained a bank of E. coli strains expressing different mutated hybrid bacteriocins in the central position of the hinge region. From all these variants, we found that the one bearing a tyrosine in the central region of the hinge (Ent35-GYG-MccV) is 2-fold more active against E. coli and 4-fold more active against Listeria than the original peptide Ent35-MccV. This derivative was purified and characterized. The development and evaluation of alternative hinges for Ent35-MccV represents a step forward in the bioengineering of antimicrobial peptides. This approach fosters the rational design of peptides with enhanced antimicrobial activity.
2019, Tesi, ITA
PAGANINI ELISA
Il 21 Agosto 2017, alle 20:57, un terremoto con magnitudo Md= 4.0 ha interessato l'area settentrionale dell'isola di Ischia. L'epicentro è stato localizzato a circa 1 km sud-ovest dal centro abitato di Casamicciola Terme, con una profondità ipocentrale di 1.2 km. Il meccanismo focale dell'evento mostra un piano di faglia diretto, orientato E-W.Lo scopo primario dello studio condotto è stata l'osservazione degli effetti ambientali direttamente correlati all'evento e la loro valutazione attraverso l'uso della scala di intensità ESI 2007, la quale ha promosso un'osservazione obbiettiva e accurata degli stessi. Risulta importante analizzare il terremoto sotto questo aspetto perché gli effetti cosismici ambientali sono caratterizzati da indipendenza rispetto a fattori variabili all'interno del tempo e dello spazio, come la densità di popolazione e le tecniche costruttive. Per una più completa analisi delle conseguenze del terremoto del 21 Agosto 2017, si è voluta redigere una mappa integrata comprendente sia i dati di intensità ESI, sia i dati EMS: nonostante il campo di intensità ESI comprenda una zona geograficamente più ristretta, le due scale convergono sul valore dell'intensità nella zona epicentrale.
2019, Editoriale in rivista, ENG
Siciliano R.A.; Uzzau S.; Mazzeo M.F.
2019, Articolo in rivista, ENG
Etty, Marie-Christine; D'Auria, Sabato; Shankar, Shiv; Salmieri, Stephane; Fraschini, Carole; Lacroix, Monique
This paper presents the application of a new support based on chitosan and reinforced with cellulose nanocrystals (m-CCG) for the detection of Listeria monocytogenes p60 protein in sandwich ELISA. This support consists of biopolymer membrane optimized for the covalent immobilization of the specific monoclonal antibodies against the unique short peptide of eleven amino acids (PepD, QQQTAPKAPTE) of L. monocytogenes p60 protein (anti-PepD mAb). Using the optimized immobilization method of anti-PepD mAb on m-CCG (m-CCG + anti-PepD mAb), the detection of L. monocytogenes culture was improved by 17% in Tryptic soy broth and by 24% in Listeria enrichment broth containing 0.5% of dextrose (modified LEB) on m-CCG compared to standard ELISA support. Moreover, the evaluation of the selectivity of detection of L. monocytogenes by anti-PepD mAb immobilized on m-CCG allowed the absence of any cross-reaction between the support and the nine other bacteria commonly found in food environment.
2019, Articolo in rivista, ENG
Etty, Marie-Christine; D'Auria, Sabato; Shankar, Shiv; Salmieri, Stephane; Coutu, Julie; Baraketi, Amina; Jamshidan, Majid; Fraschini, Carole; Lacroix, Monique
This paper presents the development of a support membrane based on chitosan, cellulose nanocrystals and glycerol (m-CCG) for the antibody immobilization by a covalent crosslinking using glutaraldehyde. The chemical characterization of the support by FTIR showed that m-CCG formation process was stabilized by the formation of hydrogen bonding between each component of m-CCG and the reactive amine groups allowing the antibody immobilization on m-CCG via glutaraldehyde. Moreover, this immobilization on m-CCG was optimized by mathematics modeling approaches, and it exhibited robustness and predictable detection in presence of 0.6% of cellulose nanocrystals (CNCs), 0.5 g of CCG solution per well, after 2 h of antibody immobilization. Results also showed that CNCs (0.6% w/v) was the most important factor of the optimization. At this concentration, CNCs improve the resistance of m-CCG during the crosslinking treatment by a modification of the surface topography and the reinforcement of the tensile strength of m-CCG at > 30%.
2019, Articolo in rivista, ENG
Doulamis, Nikolaos; Voulodimos, Athanasios; Doulamis, Anastasios; Bimpas, Matthaios; Angeli, Aikaterini; Bakalos, Nikolaos; Giusti, Alessandro; Philimis, Panayiotis; Varriale, Antonio; Ausili, Alessio; D'Auria, Sabato; Lampropoulos, George; Baer, Matthias; Schmauss, Bernhard; Freitag, Stephan; Lendl, Bernhard; Mlynarczyk, Krzysztof; Sosna-Glebska, Aleksandra; Trajnerowicz, Artur; Pawluczyk, Jaroslaw; Zbik, Mateusz; Kulakowski, Jacek; Georgiadis, Panagiotis; Blaser, Stephane; Bazzurro, Nicola
In this paper, we present WaterSpy, a project developing an innovative, compact, cost-effective photonic device for pervasive water quality sensing, operating in the mid-IR spectral range. The approach combines the use of advanced Quantum Cascade Lasers (QCLs) employing the Vernier effect, used as light source, with novel, fibre-coupled, fast and sensitive Higher Operation Temperature (HOT) photodetectors, used as sensors. These will be complemented by optimised laser driving and detector electronics, laser modulation and signal conditioning technologies. The paper presents the WaterSpy concept, the requirements elicited, the preliminary architecture design of the device, the use cases in which it will be validated, while highlighting the innovative technologies that contribute to the advancement of the current state of the art.
2019, Articolo in rivista, ENG
Etty, Marie-Christine; D'Auria, Sabato; Fraschini, Carole; Salmieri, Stephane; Lacroix, Monique
This paper presents the effects of the composition of different media (i.e., Tryptic soy broth (TSB), Brain heart infusion (BHI), Listeria enrichment broth (LEB), Fraser broth (FB) and University of Vermont medium (UVM)) on the detection of a short peptide fragment PepD specific to the p60 protein (p60) of L. monocytogenes by a monoclonal antibody (anti-PepD mAb). Expression of the p60 obtained was demonstrated to be proportional to the cellular growth of Listeria monocytogenes regardless of the tested growth medium. However, the early growth of L monocytogenes and the expression of the p60 were negatively affected by the presence of selective agents present in LEB, FB and UVM. Among those three selective enrichment media commonly used for L. monocytogenes, LEB allowed a better expression of L. monocytogenes p60 after an incubation period of 18 h. Optimization of the LEB revealed that the dextrose concentration was the critical factor for improving the expression of p60 and promotes the early expression of p60. Moreover, an optimal dextrose concentration of 0.5% (w/v) in LEB, coupled with anti-PepD mAb immobilized to solid support, reduced the detection of p60 from 18 h to 9 h for an initial concentration of L. monocytogenes of 10(8) CFU/ml. (C) 2019 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
2019, Articolo in rivista, ENG
Montagnese, Concetta; Barattini, Paolo; Giusti, Alessandro; Balka, Gyula; Bruno, Ugo; Bossis, Ioannis; Gelasakis, Athanasios; Bonasso, Matteo; Philmis, Panayiotis; Denes, Lilla; Peransi, Sergio; Rodrigo, Manuel; Simon, Santiago; Griol, Amadeu; Wozniakowski, Grzegorz; Podgorska, Katarzyna; Pugliese, Carolina; Nannucci, Lapo; D'Auria, Sabato; Varriale, Antonio
In this paper, we present the concept of a novel diagnostic device for on-site analyses, based on the use of advanced bio-sensing and photonics technologies to tackle emerging and endemic viruses causing swine epidemics and significant economic damage in farms. The device is currently under development in the framework of the EU Commission co-funded project. The overall concept behind the project is to develop a method for an early and fast on field detection of selected swine viruses by non-specialized personnel. The technology is able to detect pathogens in different types of biological samples, such as oral fluids, faeces, blood or nasal swabs. The device will allow for an immediate on-site threat assessment. In this work, we present the overall concept of the device, its architecture with the technical requirements, and all the used innovative technologies that contribute to the advancements of the current state of the art.
2019, Articolo in rivista, ENG
Di Giovanni, Stefano; Zambrini, Vittorio; Varriale, Antonio; D'Auria, Sabato
Recently, there is an increase in interest to develop user-friendly monitoring devices in healthcare, environmental, and agrofood fields for a fast detection of contaminants. Aflatoxins (AFs) are a group of toxic substances produced by the fungi of species Aspergillus that contaminate cereals and dried fruits. When dairy cows ingest feed contaminated with aflatoxin B1 (AFB1), it is metabolized and transformed in the liver into a carcinogenic form aflatoxin M1 (AFM1), which is eliminated through the milk. In this work, we developed a sensor assay to detect low amounts of AFM1 directly in whole milk. For this purpose, we produced monospecific polyclonal antibody (IgGMS-M1) that was able to bind with high avidity to AFM1. Then, we conjugated the antibody to the invertase enzyme from Saccharomyces cerevisiae. This enzyme is able to convert sucrose into fructose and glucose. After incubation of invertase-conjugated anti-AFM1 antibody with milk containing AFM1, we measured the produced glucose by a glucometer. The produced glucose was then correlated to the amount of AFM1 present in the milk. The obtained results show that the assay is easily customizable as a portable instrument for on-site AFM1 measurements. In addition, the results point out that the assay is very sensitive since it can detect the presence of 27 parts per trillion (ppt) of AFM1 in whole milk, a value lower than the AFM1 quantities in milk and dairy products set by the European Commission (50 ppt).
2019, Articolo in rivista, ENG
Griol, Amadeu; Peransi, Sergio; Rodrigo, Manuel; Hurtado, Juan; Bellieres, Laurent; Ivanova, Teodora; Zurita, David; Sanchez, Carles; Recuero, Sara; Hernandez, Alejandro; Simon, Santiago; Balka, Gyula; Bossis, Ioannis; Capo, Alessandro; Camarca, Alessandra; D'Auria, Sabato; Varriale, Antonio; Giusti, Alessandro
In this paper we introduce a field diagnostic device based on the combination of advanced bio-sensing and photonics technologies, to tackle emerging and endemic viruses causing swine epidemics, and consequently significant economic damage in farms. The device is based on the use of microring resonators fabricated in silicon nitride with CMOS compatible techniques. In the paper, the designed and fabricated photonic integrated circuit (PIC) sensors are presented and characterized, showing an optimized performance in terms of optical losses (30 dB per ring) and extinction ration for ring resonances (15 dB). Furthermore, the results of an experiment for porcine circovirus 2 (PCV2) detection by using the developed biosensors are presented. Positive detection for different virus concentrations has been obtained. The device is currently under development in the framework of the EU Commission co-funded project SWINOSTICS.
2019, Poster, ITA
Giuseppe Sorrentino; Cataldo Pulvento; Giacomo Mele, Anna Tedeschi, Laura Gargiulo, Maria Grazia Volpe, Luca Vitale, Bruno di Matteo, Maria Riccardi, Maria Soprano
Si riporta un nuovomodello di sviluppo nel settore agricolo delle aree interne della Regione campania dopo che dal 2010 è stato abolito il contributo alla coltivazione di tabacco che ha ridotto notevolmente i redditi agricoli. Sono proposti nuovi ordinamenti colturali a basso impatto ambientale con l'introduzione di nuove colture come la canapa per uso alimentare, il sorgo bianco, i legumi ed i grani antichi. Sono state proposte anche nuove colture per la prima volta in Italia come al quinoa. Il modello di sviluppo prevede che oltre al settore alimentare con la canapa si possa avviare anche quello della bioedilizia dall'utilizzazione del canapulo per abbassare notevolmente le emissioni nel settore dell'edilizia civile. Nell'ambito dei borghi in rete si può sostenere il reddito anche con l'introduzione del bioartigianato derivante dall'utilizzo di scarti agricoli. La possibilità di avviare nuove attività economiche è necessaria evitare lo spopolamento di queste aree che hanno perso negli ultimi 10 anni il 30% della popolazione per lo piu giovanile. L'introduzione di queste nuove colture dovrebbe garantire un reddito più elevato rispetto a quelle classiche dell' area basate soprattutto sui cereali.
2019, Altro prodotto, ENG
PORFIDO SABINA
REVIEW OF MANUSCRIPT IN 2019
2019, Articolo in rivista, ENG
Sacchi, Marco; De Natale, Giuseppe; Spiess, Volkhard; Steinmann, Lena; Acocella, Valerio; Corradino, Marta; de Silva, Shanaka; Fedele, Alessandro; Fedele, Lorenzo; Geshi, Nobuo; Kilburn, Christopher; Insinga, Donatella; Jurado, Maria-Jose; Molisso, Flavia; Petrosino, Paola; Passaro, Salvatore; Pepe, Fabrizio; Porfido, Sabina; Scarpati, Claudio; Schmincke, Hans-Ulrich; Somma, Renato; Sumita, Mari; Tamburrino, Stella; Troise, Claudia; Vallefuoco, Mattia; Ventura, Guido
Large calderas are among the Earth's major volcanic features. They are associated with large magma reservoirs and elevated geothermal gradients. Caldera-forming eruptions result from the withdrawal and collapse of the magma chambers and produce large-volume pyroclastic deposits and later-stage deformation related to post-caldera resurgence and volcanism. Unrest episodes are not always followed by an eruption; however, every eruption is preceded by unrest.