Literature search – Exploring in silico protein toxicity prediction methods to support the food and feed risk assessment

This report is the outcome of an EFSA procurement (NP/EFSA/GMO/2018/01) reviewing relevant scientific information on in silico prediction methods for protein toxicity, that could support the food and feed risk assessment. Several proteins are associated with adverse (toxic) effects in humans and animals, by a variety of mechanisms. These are produced by plants, animals and bacteria to prevail in hostile environments. In the present report, we present an integrated pipeline to perform a comprehensive literature and database search applied to proteins with toxic effects. \u201cToxin activity\u201d and \u201ctoxin-antitoxin system\u201d strings were used as inputs for this pipeline. UniProtKB was considered as the reference database, and only the UniProtKB curator-reviewed proteins were considered in the pipeline. Experimentally- determined structures and homology-based in silico 3D models were retrieved from protein structures repositories; family-, domain-, motif- and other molecular signature-related information was also obtained from specific databases which are part of the InterPro consortium. Protein aggregation associated with adverse effects was also investigated using different search strategies. This work can serve as the basis for further exploring novel risk assessment strategies for new proteins using in silico predictive methods

Effetti degli oli essenziali di agrumi applicati in post-emergenza sul controllo delle infestanti e sulla comunit\ue0 microbica del suolo

Sebbene gli erbicidi sintetici ad oggi siano il mezzo pi\uf9 utilizzato per il controllo delle erbe infestanti, \ue8 ampiamente dimostrato che essi hanno un impatto negativo sul suolo, sull'ambiente e, inoltre, iniziano a manifestarsi fenomeni di resistenza. Pertanto, \ue8 importante sviluppare mezzi alternativi che siano al contempo efficaci e rispettosi dell\u2019ambiente. Gli olii essenziali estratti dalle piante sono stati da sempre utilizzati per molteplici usi come agenti battericidi, virucidi, fungicidi, insetticidi, medicinali e cosmetici. Diversi studi hanno dimostrato tali attivit\ue0 degli oli essenziali estratti dagli agrumi, ma scarsi sono gli studi che hanno investigato il loro potenziale uso come erbicidi. Inoltre, anche se diversi studi hanno dimostrato un effetto antimicrobico, tali studi sono stati condotti prevalentemente in vitro e non in vivo. Obiettivo del presente lavoro \ue8 stato quello di valutare la potenzialit\ue0 degli oli essenziali estratti dalle bucce di limone e arancia i) per limitare lo sviluppo delle erbe infestanti e ii) il loro effetto sulla comunit\ue0 microbica del suolo. Gli oli essenziali testati sono stati estratti per idrodistillazione e con pressa a freddo: LI e AI, olio essenziale di limone e arancio, rispettivamente, estratti con pressa a freddo; LU e AU, olio essenziale di limone e arancio, rispettivamente, estratti tramite idrodistillazione. Ai fini del presente lavoro, il suolo utilizzato \ue8 stato prelevato presso un campo sperimentale dell'Universit\ue0 degli Studi di Palermo. Dopo il prelievo, il suolo \ue8 stato essiccato all'aria e setacciato con setaccio con fori del diametro di un centimetro. Successivamente, 500 grammi di suolo sono stati posti in 20 vaschette di alluminio (10 7 20 cm) ed incubati in condizioni naturali. Durante l\u2019incubazione il suolo \ue8 stato mantenuto al 50% della sua capacit\ue0 idrica di campo (WHC). Dopo l\u2019emergenza delle piantine, ovvero alla comparsa della terza foglia (ventesimo giorno dall\u2019inizio dell\u2019incubazione), ciascun olio essenziale \ue8 stato applicato nebulizzandolo sulle piantine, a giorni alterni per una settimana (tre applicazioni in totale). Per ogni vaso e per ogni trattamento, sono stati applicati 5 mL di una soluzione contenente 1 L di acqua, 10 mL di olio essenziale e 1 mL di emulsionante. Nel suolo di controllo la soluzione applicata alle piantine non conteneva olio essenziale. Dopo 15 giorni dall\u2019ultima applicazione dei trattamenti, i suoli sono stati campionati in modo totale, setacciati ed analizzati per la determinazione del carbonio della biomassa microbica (metodo fumigazione-estrazione), respirazione basale (emissione di CO2) e struttura della comunit\ue0 microbica (analisi degli acidi grassi dei fosfolipidi). Inoltre, per ogni trattamento sono stati determinati alcuni parametri delle piante come peso secco e fresco totale, numero di specie e numero di piante per specie. In questo lavoro vengono riportati e discussi i principali risultati

New clones and old varieties: Quality of sicilian hillside apple cultivation

Aims: The aim of this study is to evaluate the qualitative characteristics of the new clones according to the Mediterranean hillside growing environment and, at the same time, to highlight the qualitative peculiarities of the old varieties in order to avoid genetic loss. Introduction: Several apple varieties are constantly selected for improved quality traits and introduced for cultivation and marketing in addition to a few traditional and affirmed varieties. On the other side, local genotype and ancient varieties are still valorised due to the request of a niche market. Methods: We have studied the physico-chemical quality and the sensory traits of the fruit obtained in this particular environment. Results: Our study reveals a qualitative response to the environment in a genotype-dependent manner. As expected, the physico-chemical characteristics favour the new clones. Conclusion: Both old varieties and new clones of apple fruit, grown in the Mediterranean area, turned out to be of high quality. Nevertheless, results revealed the better characteristics of new clones for commercialization in large-scale supply chain

What if? Mouse proteomics after gene inactivation

The complex interactions among proteins and of proteins with small molecular weight protein ligands are overturned every time one of the components of the network is missing. For study purposes, animal models lacking one protein are obtained by experimental manipulation of the genome: in the knocking out approach, a gene is altered through the insertion of an artificial DNA sequence, which halts the transcription-translation sequence of events. In this review we have compiled the research papers that analyze the effects of knocking out individual genes on the proteomes of various tissues/organs throughout the body. We have gathered and organized all the available evidence and then compared the proteomic data in order to stress the context-specificity of the outcome every time two or more organs were investigated in the same KO mice. Finally, in a symmetrical approach to the above, we surveyed whether there is any obvious overlap among the effects of different KO on the same organ, marking affection of general pathways or lacking specificity of the gene targeting. Specific attention was put on the possible involvement of cellular stress markers

Modified Atmosphere Packaging and low temperature storage extend marketability of cherimoya (Annona cherimola Mill.)

Cherimoya is a subtropical fruit characterized by a delicious, sweet flavor and beneficial health properties, which found suitable growing conditions in the South of Italy. However, the marketing of this product is halted by its high perishability, which limits the shelf-life of the fresh fruit to few days after harvest and does not allow for commercialization beyond local markets. Studies have shown that storage of this fruit in controlled atmosphere, using Modified Atmosphere Packaging technologies, extended the post-harvest life of Cherimoya, but little is still known about the evolution of its sensory, nutraceutical and microbiological characteristics during such storage period. In this paper, we studied the effect of a 4-days long active-MAP (30% CO2 – 10% O2 – 60% N2) storage period, associated with cold temperatures, on the physico-chemical, sensory, nutraceutical and microbiological quality traits of Italian-grown cherimoya fruits, compared with passive-MAP (Air composition, 21% O2 + 1% CO2 + 78% N2) and simple cold storage. Active-MAP proved effective in delaying the reaching of the optimal consumption point until 10 days from harvest, besides showing absence of microbial growth until after 7 days from harvest. Both active- and passive-MAP treatments maintained better nutraceutical values than control until the end of the trial period, and sensory analysis confirmed that active-MAP treated fruits were at the optimal commercial stage after 10 days from harvest

Novel insights into the transport mechanism of the human amino acid transporter LAT1 (SLC7A5) : probing critical residues for substrate translocation

BACKGROUND: LAT1 (SLC7A5) is the transport competent unit of the heterodimer formed with the glycoprotein CD98 (SLC3A2). It catalyzes antiport of His and some neutral amino acids such as Ile, Leu, Val, Cys, Met, Gln and Phe thus being involved in amino acid metabolism. Interestingly, LAT1 is over-expressed in many human cancers that are characterized by increased demand of amino acids. Therefore LAT1 was recently acknowledged as a novel target for cancer therapy. However, knowledge on molecular mechanism of LAT1 transport is still scarce. METHODS: Combined approaches of bioinformatics, site-directed mutagenesis, chemical modification, and transport assay in proteoliposomes, have been adopted to unravel dark sides of human LAT1 structure/function relationships. RESULTS: It has been demonstrated that residues F252, S342, C335 are crucial for substrate recognition and C407 plays a minor role. C335 and C407 cannot be targeted by SH reagents. The transporter has a preferential dimeric structure and catalyzes an antiport reaction which follows a simultaneous random mechanism. CONCLUSIONS: Critical residues of the substrate binding site of LAT1 have been probed. This site is not freely accessible by molecules other than substrate. Similarly to LeuT, K+ has some regulatory properties on LAT1. GENERAL SIGNIFICANCE: The collected data represent a solid basis for deciphering molecular mechanism underlying LAT1 function

Physicochemical, Nutraceutical and Sensory Traits of Six Papaya (Carica papaya L.) Cultivars Grown in Greenhouse Conditions in the Mediterranean Climate

Six papaya (Carica papaya L.) cultivars, grown in a Mediterranean climate under greenhouse conditions, were screened for physicochemical properties, antioxidant capacity, nutritional and sensory characteristics. The fruits, harvested with more than 50% of yellow surface (between 60% and 77%) were tested for carotenoids content, phenolic content, reducing activity (ABTS) and cellular antioxidant activity (CAA50). The physicochemical traits were measured in terms of the titratable acidity and soluble content whereas proximal composition along with moisture, fats, total sugar, ash, vitamin A, C and E content. Moreover, the sensory profile was analyzed by a semi-trained panel. Although the six analyzed cultivars reached qualitative characteristics to satisfy market needs, significant differences among them were found in a genotype-dependent manner. In particular, Cartagena and Maradol cultivars evidenced the highest values of minerals and vitamins, carotenoids, polyphenols, ABTS and CAA50 and reached the best commercial requisites (size, total soluble solids content/titratable acidity ratio). As for sensory analysis, we observed significate differences only for sweetness, juiciness, odor and flavor of peach and exotic fruits whereas descriptors related to unpleasant defects or sensations always have very low scores in all the observed cultivars. These results highlight the possibility of obtaining quality papaya fruits in a Mediterranean climate using greenhouse growing

Development of an adverse outcome pathway for cranio-facial malformations: A contribution from in silico simulations and in vitro data

Mixtures of substances sharing the same molecular initiating event (MIE) are supposed to induce additive effects. The proposed MIE for azole fungicides is CYP26 inhibition with retinoic acid (RA) local increase, triggering key events leading to craniofacial defects. Valproic acid (VPA) is supposed to imbalance RA-regulated gene expression trough histone deacetylases (HDACs) inhibition. The aim was to evaluate effects of molecules sharing the same MIE (azoles) and of such having (hypothetically) different MIEs but which are eventually involved in the same adverse outcome pathway (AOP). An in silico approach (molecular docking) investigated the suggested MIEs. Teratogenicity was evaluated in vitro (WEC). Abnormalities were modelled by PROAST software. The common target was the branchial apparatus. In silico results confirmed azole-related CYP26 inhibition and a weak general VPA inhibition on the tested HDACs. Unexpectedly, VPA showed also a weak, but not marginal, capability to enter the CYP 26A1 and CYP 26C1 catalytic sites, suggesting a possible role of VPA in decreasing RA catabolism, acting as an additional MIE. Our findings suggest a new more complex picture. Consequently two different AOPs, leading to the same AO, can be described. VPA MIEs (HDAC and CYP26 inhibition) impinge on the two converging AOPs

With or without you - Proteomics with or without major plasma/serum proteins

The first sections of this review compile and discuss strategies and protocols for managing plasma/serum as a source of biomarkers relevant to human disease. In many such cases, depletion of abundant protein(s) is a crucial preliminary step to the procedure; specific conceptual and technical approaches, however, make it possible to effectively use to this purpose whole plasma/serum. The final sections focus instead on the complexity associated with each of the major serum/plasma proteins in terms of both, multiple molecular structures (existence of a number of protein species) and of multiple molecular functions (behavior as multifunctional/multitasking/moonlighting proteins). Reviewing evidence in these and some related fields (regulation of the synthetic pattern by proteins and non-protein compounds and its connection with health and disease) prompts the suggestion/recommendation that information on the abundant components of plasma/serum proteome is routinely obtained and processed/mined as a valuable contribution to the characterization of any non-physiological condition and to the understanding of its mechanisms and of its implications/sequels