Alternative protein sources in the European diets: the contribution of ALTERNATIVA project to One Health perspective

A abordagem como a One Health, baseada em princípios que promovem a coexistência saudável, bem-estar e sustentabilidade entre humanos, animais e ambiente, tem surgido como indispensável para fazer face aos diversos problemas globais associados às alterações climáticas e sus- tentabilidade. A mesma multidisciplinaridade do conceito One Health é, também, uma característica do conceito e aplicação da Avaliação de Risco-Benefício (ARB). Este estudo tem como objetivo demonstrar de que forma é que o projeto ALTERNATIVA – Alternative protein sources in the European diets integrating health risk-benefit and sustainability (Fontes alternativas de proteínas nas dietas europeias – integrando risco-benefício para a saúde e sustentabilidade) contribui para o desenvolvimento e aplicação do conceito One Health, tendo por base a ARB de diferentes dietas alimentares. Realizou-se uma pesquisa bibliográfica sobre o conceito “One Health” através da base de dados Pubmed/U.S. National Library of Medicine e no Google Scholar, nos quais foram efetuadas pesquisas avançadas, que in- cluíram os termos: “One health approach”, “Risk benefit food one health”, “Alternative protein”, “One health food” e “One health food assessment”. Os resultados deste estudo demonstram de que forma é que o projeto ALTERNATIVA, através das suas diferentes atividades desenvolvidas, integra os principais pilares do conceito One Health (Humano, Animal e Ambiental) em todas as suas dimensões. Conclui-se que o projeto ALTERNATIVA aliado ao conceito One Health, constitui uma ferramenta inovadora para apoiar as melhores decisões sobre as dietas do futuro, garantindo a nutrição humana e a saúde planetária, ao contribuir para a mitigação das tendências adversas que estão diretamente associadas às nossas escolhas alimentares.One Health approach, based on principles that promote healthy coexis- tence, well-being and sustainability between humans, animals and the environment, has emerged as crucial to tackle the various global problems associated with climate change and sustainability. The same multidisciplinary of the One Health concept is also a characteristic of the concept and application of the Risk-Benefit Assessment (RBA).This study aims to demonstrate how the ALTERNATIVA Project Alternative protein sources in the European diets – integrating health risk-benefit and sustainability contributes to developing and applying the One Health concept based on the RBA of different diets. Bibliographical research was carried out on the concept One Health through the Pubmed/U.S. National Library of Medicine and Google Scholar, which included advanced searches with the terms: “One health approach”, “Risk-Benefit food one health”, “Alternative protein”, “One health food” and “One health food assessment”. The results of this study demonstrate how the ALTERNATIVA Project, through its different activities, integrates the main pillars of the One Health concept (Human, Animal and Environmental) in all its dimensions. It is concluded that the ALTERNATIVA Project combined with the One Health concept constitutes an innovative tool to support the best decisions about the diets of the future, guaranteeing human nutrition and planetary health, by contributing to the mitigation of the adverse trends directly associated with our food choices.Trabalho desenvolvido no âmbito do projeto projeto ALTERNATIVA − Alternative sources of protein in European diets - integrating risk-benefit for health and sustainability financiado pelas EFSA Partnering Grants (Grant Agreement Number – GP/EFSA/ENCO/2020/03 - GA 2).info:eu-repo/semantics/publishedVersio

Sustainable dietary choices: the contribution of the ALTERNATIVA project as a tool for selecting alternative protein sources

O aumento da população humana e a consequente pressão exercida pelos sistemas alimentares desafiam a saúde e o meio ambiente, cujo impacto se reflete, entre outros, ao nível das alterações climáticas associadas ao aquecimento global, exploração de recursos naturais e da perda de biodiversidade. Entre os fatores que mais contribuem para esse impacto está a produção de proteínas de origem animal, como a carne vermelha e os lacticínios. Neste sentido, urge uma transformação dos sistemas alimentares, sendo que esta transformação deverá ser sustentada numa avaliação dos impactos de fontes alternativas de proteínas, quer na saúde quer na sustentabilidade. Este artigo tem como principal objetivo discutir os desafios colocados aos sistemas alimentares e a necessidade de avaliar o impacto destes, à luz do projeto ALTERNATIVA (Alternative sources of protein in European diets – integrating risk-benefit for health and sustainability) como uma ferra menta para o desenvolvimento sustentável nas diferentes vertentes – ambiental, social e económica, através de uma metodologia que reúne e combina conhecimentos em avaliação do risco-benefício (ARB) de alimentos e a avaliação da sustentabilidade. A utilização de abordagens holísticas, como a que está a ser aplicada no projeto ALTERNATIVA com o objetivo de fornecer ferramentas inovadoras para apoiar decisões sobre as futuras dietas, é fundamental para minimizar os efeitos dos desafios atuais tentando garantir alimentos seguros, economicamente justos, acessíveis, dietas nutricionalmente adequadas e saudáveis com menores impactos ambientais.The growth of human population and the consequent pressure exerted by food systems challenge health and the natural environment, whose impact is reflected, among others, in terms of contribu tion to climate change associated with global warming, exploitation of natural resources and loss of biodiversity. Among the major contributors to this impact is the production of proteins of animal origin, such as red meat and dairy. Therefore, the need of transforming food systems is urgent and should be supported by assessing the overall health impact of alternative protein sources considering also sus tainability aspects. Our main objective is to discuss the challenges posed to food systems and the need to assess their impact under the umbrella of the ALTERNATIVA (Alternative sources of protein in Euro pean diets – integrating risk-benefit for health and sustainability) as a tool for sustainable development in different aspects – health, environmental, social and economic, through a methodology that brings together and combines knowledge in risk-benefit assessment (RBA) and sustainability assessment. The use of holistic approaches, such as the one being applied in the ALTERNATIVA project aiming to provide innovative tools to support decisions about the future diets, is fundamental to minimize the effects of the current challenges trying to guarantee secure, economically fair, affordable, nutritionally adequate and healthy diets having lower environmental impacts.Trabalho desenvolvido no âmbito do projeto ALTERNATIVA (Alternative sources of protein in European diets – integra ting risk-benefit for health and sustainability) financiado pelas EFSA Partnering Grants (Grant Agreement Number – GP/EFSA/ENCO/2020/03 – GA 2.info:eu-repo/semantics/publishedVersio

Proteomic analysis of inducible cell systems Saos2-p21 and U2OS-E2F1

Proteomics is the science concerning the study of proteins. Proteome is the sum of proteins present in an organism, cell, organelle even body fluid, that is determined quantitatively at a certain moment and under precisely defined limiting conditions. Usually, the applied technique in proteomics is two-dimensional polyacrylamide gel electrophoresis (2DE). Using the above technique we can separate as many as 2000 proteins in one run. In this way we can compare the proteomes of control and pathologic samples, to find differences, to better understand diseases, like cancer, and to reveal potential molecular biomarkers. We applied 2DE for the proteomic analysis of the inducible cancer cell systems U2OS ER-E2F1 and Saos2 p21-Tet On. These cancer cell lines derived from patient’s osteosarcoma and are genetically modified so we can control the expression of E2F1 and p21. E2F1 is a transcription factor, whose main function is the promotion of cell cycle from G1 phase to S phase. When is released from his inhibitor, the retinoblastoma protein pRb, during mid G1 phase, E2F1 promotes the transcription of genes necessary for the entrance of the cell to S phase. Beyond this role, E2F1 can promote apoptosis through p53-dependent and p53-independent pathways, when excess damage is present in the cell. To understand the double role of E2F1 we studied the molecular changes of U2OS cells before and after its induction. p21Cip1 is an inhibitor of cycle-dependent kinases and its expression is p53-dependent and p53-independent. The primal role of p21 is inhibition of cell cycle through G1, S and G2 phases. Additionally p21 is implicated in differentiation, cellular senescence and apoptosis. Due to these multiple functions of p21 we studied the molecular changes in Saos2 cells after its induction. For more accurate results we applied proteomic analysis in the parental cell lines U2OS and Saos2. Comparison of proteomes before and after the induction of the genes, E2F1 for U2OS ER-E2F1 and p21 for Saos2 p21-Tet On, as well as those of the parental cell lines, we found differences concerning the hole response of the cell to overexpression of the above genes. Overexpression of p21 in Saos2 cells concequences to induction of 70 proteins and suppression of 25 proteins. Those proteins belong to several functional groups. Some of the most important groups are the group of 14-3-3 proteins, cytoskeletal proteins, proteins implicated in translation/transcription of DNA, metabolism, immunology, adherent proteins and transcription factors. Respectively, activation of E2F1 in U2OS cells led to induction of 52 proteins and suppression of 23 proteins. Consequently, we applied bioinformatic analysis using the Pathway Studio (AriadneGenomics) program and we created molecular networks. Some of the differentially expressed proteins seem to regulate the expression of p53 and ATM. Concluding, proteomic analysis helps in better understanding the cellular function, since it provides a more holistic view of the molecular changes that occur in it. Proteomics is a linkage between the genome and its expression. Application of proteomics in the inducible cell systems Saos2 p21-Tet On and U2OS ER-E2F1 revealed a number of proteins to be affected by p21 and E2F1, respectively in osteosarcoma cells. Finally, those proteins could be potential molecular biomarkers for osteosarcoma and can aid in comprehension of p21 and E2F1 function in cells.Πρωτεωμική είναι η επιστήμη που ασχολείται με τη μελέτη των πρωτεϊνών. Έτσι, πρωτέωμα χαρακτηρίζεται το σύνολο των πρωτεϊνών ενός οργανισμού, κυττάρου, οργανιδίου ακόμα και σωματικού υγρού, που προσδιορίζεται ποσοτικά μια δεδομένη στιγμή και κάτω από αυστηρά καθορισμένες συνθήκες. Για τη μελέτη των πρωτεϊνών αυτών ακολουθείται συνήθως μια τεχνική που ονομάζεται δισδιάστατη ηλεκτροφόρηση σε πήκτωμα πολυακρυλαμίδης (two-dimensional electrophoresis polyacrylamide gel, 2DE). Με την παραπάνω τεχνική μπορούν να διαχωριστούν μέχρι και 2000 πρωτεΐνες σε μια μόνο εφαρμογή. Με αυτό τον τρόπο μπορούν να συγκριθούν τα πρωτεώματα φυσιολογικών και παθολογικών καταστάσεων και να βρεθούν διαφορές μεταξύ τους, οδηγώντας στην καλύτερη κατανόηση των διαφόρων ασθενειών, όπως είναι ο καρκίνος, αλλά και στην εύρεση πιθανών μοριακών δεικτών. Εφαρμόσαμε την παραπάνω μέθοδο για την πρωτεωμική ανάλυση των επαγώγιμων καρκινικών κυτταρικών συστημάτων U2OS ER-E2F1 και Saos2 p21-Tet On. Οι κυτταρικές αυτές σειρές προέρχονται από οστεοσάρκωμα ασθενών και έχουν τροποποιηθεί γενετικά έτσι ώστε να ελέγχεται η έκφραση των γονιδίων E2F1 και p21 αντίστοιχα. Το μόριο E2F1 είναι ένας μεταγραφικός παράγοντας με κύρια δράση του την προαγωγή του κυτταρικού κύκλου από τη G1 φάση στην S. Απελευθερώνεται από τον αναστολέα του, την πρωτεΐνη του ρετινοβλαστώματος pRb, στα μέσα της G1 φάσης, όπου ελεύθερος πια ενεργοποιεί τη μεταγραφή γονιδίων απαραίτητων για την είσοδο των κυττάρων στην S φάση. Πέρα από το ρόλο αυτό, ο E2F1 οδηγεί τα κύτταρα σε απόπτωση μέσω p53-εξαρτώμενων και p53-ανεξάρτητων μονοπατιών, όταν υπάρχουν τεταμένες βλάβες σε αυτό. Λόγω του διττού ρόλου του μελετήθηκε η επίδρασή του στο μοριακό υπόβαθρο των U2OS κυττάρων έπειτα από επαγωγή του. Η πρωτεΐνη p21Cip1 είναι αναστολέας των κυκλινο-εξαρτώμενων κινασών και η έκφρασή του ρυθμίζεται από p53-εξαρτώμενα και p53-ανεξάρτητα μονοπάτια. Βασικός ρόλος της p21 είναι η αναστολή του κυτταρικού κύκλου στις G1, S και G2 φάσεις. Επιπλέον η p21 εμπλέκεται στη διαδικασία διαφοροποίησης των κυττάρων, στην κυτταρική γήρανση καθώς και στην απόπτωση αυτών. Λόγω των πολλαπλών αυτών ρόλων της p21 μελετήθηκαν οι μοριακές αλλαγές που υφίστανται τα Saos2 κύτταρα ύστερα από επαγωγή της. Τέλος για την πιο αξιόπιστη μελέτη των διαφορών που οφείλονται στην επαγωγή και μόνο των μορίων E2F1 και p21 μελετήθηκαν και τα πρωτεώματα των πατρικών καρκινικών κυτταρικών σειρών U2OS και Saos2. Συγκρίνοντας τα πρωτεώματα των κυττάρων πριν και μετά την επαγωγή των γονιδίων, E2F1 για τα U2OS ER-E2F1 και p21 για τα Saos2 p21-Tet On, καθώς και αυτά των πατρικών κυτταρικών σειρών, βρέθηκαν διαφορές οι οποίες οφείλονται στη συνολική απόκριση των κυττάρων στην υπερέκφραση των παραπάνω γονιδίων. Υπερέκφραση της p21 στα Saos2 κύτταρα οδήγησε στην επαγωγή 70 πρωτεϊνών και στη καταστολή 24. Οι πρωτεΐνες αυτές ανήκουν σε διάφορες λειτουργικές ομάδες. Οι πιο σημαντικές από αυτές είναι η ομάδα των 14-3-3 πρωτεϊνών, πρωτεΐνες του κυτταροσκελετού, του μονοπατιού της ουμπικουϊτίνης, πρωτεΐνες σχετιζόμενες με τη μεταγραφή/μετάφραση του DNA, το μεταβολισμό, την ανοσολογία και την προσκόλληση των κυττάρων καθώς και μεταγραφικοί παράγοντες. Αντίστοιχα, ενεργοποίηση του E2F1 στα U2OS κύτταρα οδήγησε στην επαγωγή 52 πρωτεϊνών και στη καταστολή 23 πρωτεϊνών. Στη συνέχεια οι πρωτεΐνες αυτές επεξεργάστηκαν με βιοπληροφορική μελέτη χρησιμοποιώντας το πρόγραμμα Pathway Studio (AriadneGenomics). Έτσι δημιουργήθηκαν μοριακά μονοπάτια που δείχνουν την επίδραση κάποιων από τις πρωτεΐνες που άλλαξαν έκφραση στη λειτουργία του p53 και του ATM. Συμπεραίνοντας, η πρωτεωμική ανάλυση βοηθά στην καλύτερη κατανόηση της λειτουργίας των κυττάρων, αφού παρέχει μια πιο ολική άποψη των μοριακών γεγονότων που συμβαίνουν σε αυτό καθώς συνδέει την πληροφορία που υπάρχει στο γονιδίωμα του κυττάρου με το τελικό αποτέλεσμα της έκφρασης αυτού. Η εφαρμογή της πρωτεωμικής στα επαγώγιμα κυτταρικά συστήματα Saos2 p21-Tet On και U2OS ER-E2F1, είχε σαν αποτέλεσμα την ανάδειξη πρωτεϊνών που επηρεάζονται από την επίδραση των μορίων p21 και E2F1, αντίστοιχα στα κύτταρα του οστεοσαρκώματος. Τέλος οι πρωτεΐνες που είδαμε ότι αλλάζουν έκφραση μπορούν να αποτελέσουν πιθανούς βιολογικούς δείκτες για την καταπολέμηση του καρκίνου των οστών, όπως και να μας βοηθήσουν στην καλύτερη κατανόηση της λειτουργίας των p21 και E2F1 στα κύτταρα

Molecular chaperones and proteostasis regulation during redox imbalance

Free radicals originate from both exogenous environmental sources and as by-products of the respiratory chain and cellular oxygen metabolism. Sustained accumulation of free radicals, beyond a physiological level, induces oxidative stress that is harmful for the cellular homeodynamics as it promotes the oxidative damage and stochastic modification of all cellular biomolecules including proteins. In relation to proteome stability and maintenance, the increased concentration of oxidants disrupts the functionality of cellular protein machines resulting eventually in proteotoxic stress and the deregulation of the proteostasis (homeostasis of the proteome) network (PN). PN curates the proteome in the various cellular compartments and the extracellular milieu by modulating protein synthesis and protein machines assembly, protein recycling and stress responses, as well as refolding or degradation of damaged proteins. Molecular chaperones are key players of the PN since they facilitate folding of nascent polypeptides, as well as holding, folding, and/or degradation of unfolded, misfolded, or non-native proteins. Therefore, the expression and the activity of the molecular chaperones are tightly regulated at both the transcriptional and post-translational level at organismal states of increased oxidative and, consequently, proteotoxic stress, including ageing and various age-related diseases (e.g. degenerative diseases and cancer). In the current review we present a synopsis of the various classes of intra- and extracellular chaperones, the effects of oxidants on cellular homeodynamics and diseases and the redox regulation of chaperones

The effect of precipitation and application rate on dicyandiamide persistence and efficiency in two Irish grassland soils

peer-reviewedThe nitrification inhibitor dicyandiamide (DCD) has had variable success in reducing nitrate (NO3-) leaching and nitrous oxide (N2O) emissions from soils receiving nitrogen (N) fertilisers. Factors such as soil type, temperature and moisture have been linked to the variable efficacy of DCD. Since DCD is water soluble it can be leached from the rooting zone where it is intended to inhibit nitrification. Intact soil columns (15 cm diameter by 35 cm long) were taken from luvic gleysol and haplic cambisol grassland sites and placed in growth chambers. DCD was applied at 15 or 30 kg DCD ha-1, with high or low precipitation. Leaching of DCD, mineral N and the residual soil DCD concentrations were determined over eight weeks High precipitation increased DCD in leachate and decreased recovery in soil. A soil x DCD rate interaction was detected for the DCD unaccounted (proxy for degraded DCD). In the cambisol degradation of DCD was high (circa 81%) and unaffected by DCD rate. In contrast DCD degradation in the gleysol was lower and differentially affected by rate, 67 and 46% for the 15 and 30 kg ha-1 treatments, respectively. Differences DCD degradation rates between soils may be related to differences in organic matter content and associated microbiological activity. Variable degradation rates of DCD in soil, unrelated to temperature or moisture, may contribute to varying DCD efficacy. Soil properties should be considered when tailoring DCD strategies for improving nitrogen use efficiency and crop yields, through the reduction of reactive nitrogen loss.This research was financially supported under the National Development Plan, through the Research Stimulus Fund, administered by the Department of Agriculture, Food and the Marine under grants 07519 and 07545

Phytochemical Study and In Vitro Screening Focusing on the Anti-Aging Features of Various Plants of the Greek Flora

Skin health is heavily affected by ultraviolet irradiation from the sun. In addition, senile skin is characterized by major changes in the collagen, elastin and in the hyaluronan content. Natural products (NPs) have been shown to delay cellular senescence or in vivo aging by regulating age-related signaling pathways. Moreover, NPs are a preferable source of photoprotective agents and have been proven to be useful against the undesirable skin hyperpigmentation. Greek flora harvests great plant diversity with approximately 6000 plant species, as it has a wealth of NPs. Here, we report an extensive screening among hundreds of plant species. More than 440 plant species and subspecies were selected and evaluated. The extracts were screened for their antioxidant and anti-melanogenic properties, while the most promising were further subjected to various in vitro and cell-based assays related to skin aging. In parallel, their chemical profile was analyzed with High-Performance Thin-Layer Chromatography (HPTLC) and/or Ultra-Performance Liquid Chromatography High-Resolution Mass Spectrometry (UPLC-HRMS). A variety of extracts were identified that can be of great value for the cosmetic industry, since they combine antioxidant, photoprotective, anti-melanogenic and anti-aging properties. In particular, the methanolic extracts of Sideritis scardica and Rosa damascena could be worthy of further attention, since they showed interesting chemical profiles and promising properties against specific targets involved in skin aging

Phytochemical Study and In Vitro Screening Focusing on the Anti-Aging Features of Various Plants of the Greek Flora

Skin health is heavily affected by ultraviolet irradiation from the sun. In addition, senile skin is characterized by major changes in the collagen, elastin and in the hyaluronan content. Natural products (NPs) have been shown to delay cellular senescence or in vivo aging by regulating age-related signaling pathways. Moreover, NPs are a preferable source of photoprotective agents and have been proven to be useful against the undesirable skin hyperpigmentation. Greek flora harvests great plant diversity with approximately 6000 plant species, as it has a wealth of NPs. Here, we report an extensive screening among hundreds of plant species. More than 440 plant species and subspecies were selected and evaluated. The extracts were screened for their antioxidant and anti-melanogenic properties, while the most promising were further subjected to various in vitro and cell-based assays related to skin aging. In parallel, their chemical profile was analyzed with High-Performance Thin-Layer Chromatography (HPTLC) and/or Ultra-Performance Liquid Chromatography High-Resolution Mass Spectrometry (UPLC-HRMS). A variety of extracts were identified that can be of great value for the cosmetic industry, since they combine antioxidant, photoprotective, anti-melanogenic and anti-aging properties. In particular, the methanolic extracts of Sideritis scardica and Rosa damascena could be worthy of further attention, since they showed interesting chemical profiles and promising properties against specific targets involved in skin aging

Modulation of the E2F1-Driven Cancer Cell Fate by the DNA Damage Response Machinery and Potential Novel E2F1 Targets in Osteosarcomas

Osteosarcoma is the most common primary bone cancer. Mutations of the RB gene represent the most frequent molecular defect in this malignancy. A major consequence of this alteration is that the activity of the key cell cycle regulator E2F1 is unleashed from the inhibitory effects of pRb. Studies in animal models and in human cancers have shown that deregulated E2F1 overexpression possesses either “oncogenic” or “oncosuppressor” properties, depending on the cellular context. To address this issue in osteosarcomas, we examined the status of E2F1 relative to cell proliferation and apoptosis in a clinical setting of human primary osteosarcomas and in E2F1-inducible osteosarcoma cell line models that are wild-type and deficient for p53. Collectively, our data demonstrated that high E2F1 levels exerted a growth-suppressing effect that relied on the integrity of the DNA damage response network. Surprisingly, induction of p73, an established E2F1 target, was also DNA damage response-dependent. Furthermore, a global proteome analysis associated with bioinformatics revealed novel E2F1-regulated genes and potential E2F1-driven signaling networks that could provide useful targets in challenging this aggressive neoplasm by innovative therapies