Green degradation of mycotoxins by biotechnological application of enzymes from Pleurotus spp

Le micotossine sono metaboliti secondari tossici, prodotti da funghi filamentosi che appartengono principalmente ai generi Aspergillus, Penicillium, Fusarium e Alternaria. Sono comuni contaminanti alimentari di cereali, frutta, semi e spezie, prodotti a seguito di contaminazione fungina. La contaminazione da micotossine ha un impatto importante sulla salute mondiale e sulla sua economia. Alcune di esse sono classificate come sostanze cangerogene umane (aflatossine delle serie B e G) o possibili cancerogene umane (aflatossina M1, AFM1, fumonisina B1, FB1 e ocratossina A, OTA) dall’Agenzia Internazionale per la Ricerca sul Cancro (IARC). Inoltre, esplicano una azione tossica, sia acuta che cronica, su umani e animali. A causa della contaminazione da micotossine, milioni di dollari vengono persi ogni anno a causa di commodities invendute, problemi di salute e diminuzione della produttività degli animali e per la messa a punto di sistemi complessi e integrati di lotta e gestione di queste problematiche. Le strategie preventive non sono completamente efficaci e richiedono di essere implementate con nuove strategie di riduzione che agiscano nel post raccolta e che siano in grado di ridurre o di rimuovere le micotossine dai materiali contaminati. Lo scopo di questa tesi è stato quello di valutare e studiare la capacità delle laccasi di ridurre la contaminazione di micotossine, sia in vitro che in materiali contaminati, attraverso un approccio non invasivo sugli alimenti e sull’ambiente. Inoltre, fino a otto diversi mediatori redox sono stati utilizzati insieme alle laccasi nel cosiddetto Sistema Laccasi Mediatore (LMS) per massimizzare la degradazione delle micotossine. Per raggiungere questo scopo, l’attività di due diverse laccasi purificate, la Lac2 da Pleurotus pulmonarius e la ricombinante Ery4 da P. eryngii sono state testate contro le maggiori classi di micotossine. La Lac2 è stata identificata e valutata per la degradazione in vitro delle aflatossine, mentre la Ery4 è stata testata contro AFB1, AFM1, FB1, OTA, deossinivalenolo (DON), zearalenone (ZEN) e tossina T-2. iv Lo screening preliminare ha rivelato che l’inclusione di un mediatore redox nel mix di reazione è necessario per raggiungere un alto livello di degradazione con entrambi gli enzimi. Tuttavia, l’ LMS è risultato non completamente efficace per la degradazione dell’OTA e non efficace per la degradazione del DON. Attraverso l’uso di uno specifico LMS è stata ottenuta anche la degradazione simultanea di due diverse coppie di tossine, AFB1/ZEN e FB1/ tossina T-2. In aggiunta, il trattamento con LMS è stato utilizzato con successo in matrici artificialmente e naturalmente contaminate, latte e farina di mais, per la degradazione di AFM1 e ZEN, rispettivamente. Nonostante le grandi potenzialità mostrate da entrambi gli enzimi nel campo del biorisanamento, le laccasi restano dei biocatalizzatori versatili che possono essere utilizzati in una grande varietà di processi. L’applicazione dell’LMS è stato studiato in dettaglio nel latte per valutarne l’effetto sulle proteine del latte e la possibilità di produrre una cagliata con proprietà tecnologiche e nutrizionali migliorate. I risultati presentati in questa tesi pongono le basi per lo sviluppo di un metodo di biotrasformazione basato su un approccio enzimatico che apre nuove prospettive per l’utilizzo di un biocatalizzatore versatile e green, come la laccasi, nel campo della sicurezza e della qualità delle derrate alimentari contaminate da micotossine.Mycotoxins are toxic secondary metabolites produced by filamentous fungi mainly belonging to Aspergillus, Penicillium, Fusarium and Alternaria genera. They can be found as common contaminants of cereals, fruits, seeds and spices as a result of fungal spoilage. Mycotoxin contamination is an significant health and economic concern worldwide. Some of them were recognized by the International Agency of Research on Cancer (IARC) as carcinogenic (aflatoxin of the B and G series), possible carcinogenic (aflatoxin M1, AFM1; fumonisin B1, FB1; ochratoxin A, OTA) to humans. Moreover, they exert both acute and chronic toxic effects towards humans and animals. Because of mycotoxin contamination, billions of dollars are lost every year due to unsold commodities, decrease of animal health and productivity or to sustain a complex and integrated mycotoxin management system. Prevention strategies are not completely effective and require the implementation of novel post-harvest methods, able to mitigate or remove mycotoxins from contaminated materials. The aim of this thesis was to evaluate and study the capability of laccase enzymes to reduce mycotoxin contamination both in vitro and in contaminated materials through an environmental friendly and mild approach. In addition up to eight different redox mediators were used within the laccase mediator system (LMS) to maximize mycotoxin degradation. Within this purpose, the activity of two different purified LCs, native Lac2 from Pleurotus pulmonarius and the recombinant Ery4 from P. eryngii, was tested towards the main classes of mycotoxins. Lac2 was identified and evaluated for the in vitro degradation of aflatoxins, while Ery4 was tested towards AFB1, AFM1, FB1, OTA, deoxynivalenol (DON), zearalenone (ZEN) and T-2 toxin. The preliminary screening revealed that the inclusion of a toxin - specific redox mediator is required to achieve high levels of degradation with both enzymes. However, the use of the LMS resulted ineffective for DON and not efficient for OTA. i

Understanding and Rethinking Urban's Development Model. Visual Interdisciplinary Methodologies at City-territory Scale (Roma, 18 novembre 2021)

Nell'ambito del progetto di ATENEO 2017 Understanding and rethinking Rome's development model. Proofing interdisciplinary methodologies at city-territory scale, si è tenuto il seminario Audiovisivi e ricerca visuale come metodo di indagine geografica in ambito urbano ed extraurbano. La giornata è stata curata dai/dalle responsabili del progetto: Sandra Leonardi, Riccardo Morri, Riccardo Russo, e da Silvia Aru, Carlo Cellamare, Marco Maggioli, Lidia Piccioni e Tania Rossetto, presso la Facoltà di lettere e filosofia della Sapienza Università di Roma

Fare film geografici: giocare al “ripiglino” con lo spazio urbano

Il gioco del ripiglino si fa intrecciando uno spago tra le dita e passandoselo fra due o più persone per creare configurazioni sempre diverse, in un processo potenzialmente senza fine che, pure se nella sua semplicità, ha un grande valore metaforico. Donna J. Haraway utilizza la metafora del ripiglino (A game of cat’s cradle) per ragionare di modalità di pensiero creative e relazionali che, nell’interazione tra corpi, danno vita a configurazioni inaspettate e imprevedibili della realtà. In questo articolo, portiamo la metafora del ripiglino nelle nostre ricerche geografiche per speculare sui processi di produzione del sapere creativi e relazionali. In particolare, mobilitiamo questa metafora per valorizzare il potenziale del film geografico come metodo di indagine spaziale partecipata in grado di fare emergere molteplici piani di complessità urbana. Supportiamo questo esercizio metaforico comparando diverse fasi del gioco del ripiglino con la realizzazione di tre film collettivi, concepiti e girati durante il workshop di esplorazione delle discriminazioni urbane di genere “Atelier de la Traversée” (Bruxelles 2019-2020)

Cell death induced by mycotoxin fumonisin B1 is accompanied by oxidative stress and transcriptional modulation in Arabidopsis cell culture

Key message: Fumonisin B1 induces rapid programmed cell death in Arabidopsis cells, oxidative and nitrosative bursts, and differentially modulates cell death responsive genes. Glutathione is the main antioxidant involved in the stress response. Abstract: Fumonisin B1 (FB1) is a fungal toxin produced by Fusarium spp. able to exert pleiotropic toxicity in plants. FB1 is known to be a strong inducer of the programmed cell death (PCD); however, the exact mechanism underling the plant–toxin interactions and the molecular events that lead to PCD are still unclear. Therefore, in this work, we provided a comprehensive investigation of the response of the model organism Arabidopsis thaliana at the nuclear, transcriptional, and biochemical level after the treatment with FB1 at two different concentrations, namely 1 and 5 µM during a time-course of 96 h. FB1 induced oxidative and nitrosative bursts and a rapid cell death in Arabidopsis cell cultures, which resembled a HR-like PCD event. Different genes involved in the regulation of PCD, antioxidant metabolism, photosynthesis, pathogenesis, and sugar transport were upregulated, especially during the late treatment time and with higher FB1 concentration. Among the antioxidant enzymes and compounds studied, only glutathione appeared to be highly induced in both treatments, suggesting that it might be an important stress molecule induced during FB1 exposure. Collectively, these findings highlight the complexity of the signaling network of A. thaliana and provide information for the understanding of the physiological, molecular, and biochemical responses to counteract FB1-induced toxicity

SP420LOW PROTEIN DIETS IN CKD: MULTIPLE AND FEASIBLE. A MULTICENTER STUDY (THE TOPI STUDY)

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Glutamine Starvation Affects Cell Cycle, Oxidative Homeostasis and Metabolism in Colorectal Cancer Cells

Cancer cells adjust their metabolism to meet energy demands. In particular, glutamine addiction represents a distinctive feature of several types of tumors, including colorectal cancer. In this study, four colorectal cancer cell lines (Caco-2, HCT116, HT29 and SW480) were cultured with or without glutamine. The growth and proliferation rate, colony-forming capacity, apoptosis, cell cycle, redox homeostasis and metabolomic analysis were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test (MTT), flow cytometry, high-performance liquid chromatography and gas chromatography/mass spectrometry techniques. The results show that glutamine represents an important metabolite for cell growth and that its deprivation reduces the proliferation of colorectal cancer cells. Glutamine depletion induces cell death and cell cycle arrest in the GO/G1 phase by modulating energy metabolism, the amino acid content and antioxidant defenses. Moreover, the combined glutamine starvation with the glycolysis inhibitor 2-deoxy-D-glucose exerted a stronger cytotoxic effect. This study offers a strong rationale for targeting glutamine metabolism alone or in combination with glucose metabolism to achieve a therapeutic benefit in the treatment of colon cancer

Aflatoxin B1 and M1 Degradation by Lac2 from Pleurotus pulmonarius and Redox Mediators

Laccases (LCs) are multicopper oxidases that find application as versatile biocatalysts for the green bioremediation of environmental pollutants and xenobiotics. In this study we elucidate the degrading activity of Lac2 pure enzyme form Pleurotus pulmonarius towards aflatoxin B1 (AFB1) and M1 (AFM1). LC enzyme was purified using three chromatographic steps and identified as Lac2 through zymogram and LC-MS/MS. The degradation assays were performed in vitro at 25 °C for 72 h in buffer solution. AFB1 degradation by Lac2 direct oxidation was 23%. Toxin degradation was also investigated in the presence of three redox mediators, (2,2′-azino-bis-[3-ethylbenzothiazoline-6-sulfonic acid]) (ABTS) and two naturally-occurring phenols, acetosyringone (AS) and syringaldehyde (SA). The direct effect of the enzyme and the mediated action of Lac2 with redox mediators univocally proved the correlation between Lac2 activity and aflatoxins degradation. The degradation of AFB1 was enhanced by the addition of all mediators at 10 mM, with AS being the most effective (90% of degradation). AFM1 was completely degraded by Lac2 with all mediators at 10 mM. The novelty of this study relies on the identification of a pure enzyme as capable of degrading AFB1 and, for the first time, AFM1, and on the evidence that the mechanism of an effective degradation occurs via the mediation of natural phenolic compounds. These results opened new perspective for Lac2 application in the food and feed supply chains as a biotransforming agent of AFB1 and AFM1

Safety and efficacy of direct-acting antivirals in transfusion-dependent thalassemic patients with chronic hepatitis C

Background: Hepatitis C virus (HCV) infection is a major cause of liver-related morbidity and mortality among thalassemic patients. New treatments based on direct-acting antivirals (DAAs) are highly effective and well-tolerated by patients; nonetheless, they have not been studied in thalassemic populations. In this study, we evaluated the safety and efficacy of these treatments in a cohort of Sardinian thalassemic patients with chronic HCV infection. Methods: We consecutively recruited thalassemic patients with HCV infection, who were eligible for DAA therapy at 3 liver units. Different drug combinations, depending on HCV genotype and hepatic disease severity, were used according to the current guidelines. Sustained virological response was assessed at 12 weeks posttreatment. Data regarding the side effects and transfusion requirements were also collected. Results: We recruited 49 patients, including 29 males (59.2%), with the mean age of 43 years (genotype 1, 55.1%). Twenty-one (42.9%) patients had a history of interferon-based treatment. Cirrhosis was detected in 28 (57.1%) patients; only 1 patient had ascites and hypoalbuminemia (Child-Pugh B7). On the other hand, 35 (71.4%) patients received a sofosbuvir-based regimen. Ribavirin treatment was reported in 26 (53.1%) cases. All the patients were followed-up for at least 12 weeks after therapy, and sustained virological response was observed in 98% of the patients. No treatment discontinuation was required due to adverse events. The most common side effects included fatigue (24.5%), headache (10.2%), and anaemia (77%), requiring further blood transfusion in patients receiving ribavirin. Conclusions: This prospective study showed that DAAs are safe and effective agents in thalassemic patients with advanced liver fibrosis, regardless of previous antiviral treatment responses

Discovery of a New Drug-like Series of OGT Inhibitors by Virtual Screening

O-GlcNAcylation is an essential post-translational modification installed by the enzyme O-β- N-acetyl-d-glucosaminyl transferase (OGT). Modulating this enzyme would be extremely valuable to better understand its role in the development of serious human pathologies, such as diabetes and cancer. However, the limited availability of potent and selective inhibitors hinders the validation of this potential therapeutic target. To explore new chemotypes that target the active site of OGT, we performed virtual screening of a large library of commercially available compounds with drug-like properties. We purchased samples of the most promising virtual hits and used enzyme assays to identify authentic leads. Structure-activity relationships of the best identified OGT inhibitor were explored by generating a small library of derivatives. Our best hit displays a novel uridine mimetic scaffold and inhibited the recombinant enzyme with an IC 50 value of 7 µM. The current hit represents an excellent starting point for designing and developing a new set of OGT inhibitors that may prove useful for exploring the biology of OGT