Quantitative time-resolved analysis reveals intricate, differential regulation of standard- and immuno-proteasomes

Proteasomal protein degradation is a key determinant of protein half-life and hence of cellular processes ranging from basic metabolism to a host of immunological processes. Despite its importance the mechanisms regulating proteasome activity are only incompletely understood. Here we use an iterative and tightly integrated experimental and modelling approach to develop, explore and validate mechanistic models of proteasomal peptide-hydrolysis dynamics. The 20S proteasome is a dynamic enzyme and its activity varies over time because of interactions between substrates and products and the proteolytic and regulatory sites; the locations of these sites and the interactions between them are predicted by the model, and experimentally supported. The analysis suggests that the rate-limiting step of hydrolysis is the transport of the substrates into the proteasome. The transport efficiency varies between human standard- and immuno-proteasomes thereby impinging upon total degradation rate and substrate cleavage-site usage

Chemical analysis of Greek pollen - Antioxidant, antimicrobial and proteasome activation properties

<p>Abstract</p> <p>Background</p> <p>Pollen is a bee-product known for its medical properties from ancient times. In our days is increasingly used as health food supplement and especially as a tonic primarily with appeal to the elderly to ameliorate the effects of ageing. In order to evaluate the chemical composition and the biological activity of Greek pollen which has never been studied before, one sample with identified botanical origin from sixteen different common plant taxa of Greece has been evaluated.</p> <p>Results</p> <p>Three different extracts of the studied sample of Greek pollen, have been tested, in whether could induce proteasome activities in human fibroblasts. The water extract was found to induce a highly proteasome activity, showing interesting antioxidant properties. Due to this activity the aqueous extract was further subjected to chemical analysis and seven flavonoids have been isolated and identified by modern spectral means. From the methanolic extract, sugars, lipid acids, phenolic acids and their esters have been also identified, which mainly participate to the biosynthetic pathway of pollen phenolics. The total phenolics were estimated with the Folin-Ciocalteau reagent and the total antioxidant activity was determined by the DPPH method while the extracts and the isolated compounds were also tested for their antimicrobial activity by the dilution technique.</p> <p>Conclusions</p> <p>The Greek pollen is rich in flavonoids and phenolic acids which indicate the observed free radical scavenging activity, the effects of pollen on human fibroblasts and the interesting antimicrobial profile.</p

Redox control of protein degradation

Intracellular proteolysis is critical to maintain timely degradation of altered proteins including oxidized proteins. This review attempts to summarize the most relevant findings about oxidant protein modification, as well as the impact of reactive oxygen species on the proteolytic systems that regulate cell response to an oxidant environment: the ubiquitin-proteasome system (UPS), autophagy and the unfolded protein response (UPR). In the presence of an oxidant environment, these systems are critical to ensure proteostasis and cell survival. An example of altered degradation of oxidized proteins in pathology is provided for neurodegenerative diseases. Future work will determine if protein oxidation is a valid target to combat proteinopathies

Jusqu'où le droit réglemente-t-il la naissance des enfants extrêmement prématurés et des bébés-médicaments ? Regards croisés sur ces situations particulières de début de vie en Belgique et en France

Présentation de deux situations particulières de naissance à savoir l'extrême prématurité et le bébé-médicament, d'un point de vue éthique et juridique en Belgique et en France, après une brève explication médicale.Master [120] en droit, Université catholique de Louvain, 201

Criteria to determinate the level and difficulty of written production

The present research aims at highlighting criteria, based on which the language level B and the degree of difficulty (Readability) of a produced text can be measured and determined. For this purpose, 316 texts in Italian of the KPG exams for the periods of May 2015 and November 2016 of levels B1 and B2, produced by Greek users, were digitized in Word format. The READ-IT tool was used for data analysis and the SPSS.24 software for factor analysis. The results showed that the distinction between the two levels of language learning is based on grammar and syntax elements (spelling, use of subordinate clauses, polysyllabic words, etc.), but also on other important factors such as word and sentence coherence, lexical density, exam evaluation, READ-IT Global index etc. More specifically, it was found that the greater the use of the above elements, the higher the language level and the degree of difficulty of the text. Exploring and discovering these new factors will help create tests of written language production in a more reliable and equitable way for foreign candidates of the Italian Language in written production. On the other hand, future researchers are expected to use the present conclusions to discover new factors for levels A and C as well as for other languages.Η παρούσα έρευνα στοχεύει στην ανάδειξη κριτηρίων, βάση των οποίων μπορεί να γίνει μέτρηση και προσδιορισμός του γλωσσικού επιπέδου Β και του βαθμού δυσκολίας (αναγνωσιμότητας) ενός παραγόμενου κειμένου. Γι’ αυτόν τον σκοπό, ψηφιοποιήθηκαν σε μορφή Word 316 κείμενα ιταλικής γλώσσας των εξετάσεων ΚΠΓ περιόδων Μαΐου 2015 και Νοεμβρίου 2016 των επιπέδων Β1 και Β2, παραγόμενα από Έλληνες χρήστες. Για την ανάλυση των δεδομένων έγινε χρήση του εργαλείου READ-IT και για την παραγοντική ανάλυση το λογισμικό SPSS.24. Τα αποτελέσματα έδειξαν ότι η διάκριση μεταξύ των δυο επιπέδων γλωσσομάθειας βασίζεται σε στοιχεία γραμματικής και σύνταξης (ορθογραφία, χρήση δευτερευουσών προτάσεων, πολυσύλλαβες λέξεις κτλ.), αλλά και σε άλλους σημαντικούς παράγοντες όπως η συνοχή των λέξεων και προτάσεων, λεξική πυκνότητα, αξιολόγηση εξεταστών, δείκτης READ-IT Globale κ.α. Πιο συγκεκριμένα, διαπιστώθηκε ότι όσο μεγαλύτερη είναι η χρήση των παραπάνω στοιχείων, τόσο ψηλότερο είναι το γλωσσικό επίπεδο αλλά και ο βαθμός δυσκολίας του κειμένου. Η διερεύνηση και ανακάλυψη αυτών των νέων παραγόντων θα συνδράμουν στην δημιουργία δοκιμασιών παραγωγής γραπτού λόγου με πιο αξιόπιστο και ακριβοδίκαιο τρόπο προς αλλοδαπούς υποψήφιους της ιταλικής Γλώσσας στην παραγωγή του γραπτού λόγου. Από την άλλη, αποτελεί προσδοκία, μελλοντικοί ερευνητές να αξιοποιήσουν τα παρόντα συμπεράσματα, ώστε να ανακαλυφθούν νέοι παράγοντες και για τα επίπεδα Α και Γ όπως και για άλλες γλώσσες.La presente ricerca si propone di evidenziare criteri, in base ai quali viene misurato e determinato il livello linguistico B e il grado di difficoltà (leggibilità) di un testo prodotto. A tal fine sono stati digitalizzati in formato Word 316 testi in italiano degli esami KPG per i periodi di maggio 2015 e novembre 2016 del livello linguistico B1 e B2, prodotti da utenti greci. Lo strumento READ-IT è stato utilizzato per l'analisi dei dati e il software SPSS. 24 per l'analisi fattoriale. I risultati hanno mostrato che la distinzione tra i due livelli di apprendimento delle lingue si basa su elementi grammaticali e di sintassi (ortografia, uso di proposizioni subordinate, parole polisillabiche, ecc.), ma anche su altri fattori importanti come la coerenza di parole e frasi, densità lessicale, valutazione esame, indice READ-IT Globale ecc. Nello specifico, si è riscontrato che maggiore è l'uso degli elementi sopra riportati, maggiore è il livello linguistico e il grado di difficoltà del testo. Esplorare e scoprire questi nuovi fattori aiuterà a creare test di produzione della lingua scritta in modo più affidabile ed equo per i candidati stranieri della lingua italiana nella produzione scritta. D'altra parte, i futuri ricercatori dovrebbero utilizzare le presenti conclusioni per scoprire nuovi fattori per i livelli A e C, nonché per altre lingue

Αntiageing properties of natural compounds in primary human embryonic fibroblasts: study of the function of the proteasome

Replicative senescence in human fibroblasts is accompanied with alterations of various biological processes, including the impaired function of the proteasome. The proteasome, the major cellular non-lysosomal threonine protease, is responsible for the removal of both normal and damaged proteins. Due to its latter function, proteasome is also considered as a representative secondary antioxidant cellular mechanism. The function and the expression of the proteasome are decreased in replicative senescence, while its overexpression delays the senescence in human fibroblasts. Nrf2 is a basic transcription factor responsible for the regulation of the cellular antioxidant response, which regulates several proteasome subunits in mice. We have established in this study the proteasome-related function of Nrf2 in human fibroblasts undergoing replicative senescence. Our study demonstrates that Nrf2 has a declined expression, as well as function in senescence, while its silencing leads to premature senescence. Given the fundamental importance of proper proteasome and Nrf2 function and for the maintenance of cellular homeostasis, the particular research aimed at identifying natural compounds that activate the proteasome and the transcription factor Nrf2. Thus, we studied various extracts and natural compounds, which were isolated by plant of Greek flora, in correlation with their effect in proteasome and the appearance of replicative senescence. In the present study, we have indentified the 18α-glycyrrhetinic acid (18α-GA), a natural compound found in licorice, for its antioxidants and antiaging activities. It was observed tha the 18α-GA enhanced the proteasome activities and increases the proteasome content through the Nrf2 transcription factor. Moreover, treatment by 18α-GA results to enhanced cells’ survival following oxidative stress, while continuous treatment leads to lifespan extension of human fibroblasts. Importantly, the Nrf2-proteasome axis is functional in terminally senescent cultures as these cells retain their responsiveness to the Nrf2 and proteasome stimuli. In summary, the aforementioned data demonstrate the beneficial effect of the 18α-GA on human fibroblasts undergoing replicative senescence and provide new insights towards enhancement and activation of cellular antioxidant mechanisms by natural compounds.Η γήρανση των ανθρωπίνων ινοβλαστών συνοδεύεται από μεταβολές σε διάφορες βιολογικές διαδικασίες, συμπεριλαμβανομένης και της μείωσης της λειτουργίας του πρωτεασώματος. Το πρωτεάσωμα, μια πολυπρωτεϊνική μη πρωτεάση, είναι υπεύθυνο για την αποικοδόμηση φυσιολογικών και μη πρωτεϊνών. H λειτουργία αυτή, το καθιστά έναν από τους κύριους δευτερογενής αντιοξειδωτικούς μηχανισμούς του κυττάρου. Κατά τη φάση της αναδιπλασιαστικής γήρανσης, το πρωτεάσωμα παρουσιάζει μειωμένη έκφραση αλλά και λειτουργία, ενώ η αυξημένη έκφραση του καθυστερεί το φαινόμενο της γήρανση σε ανθρώπινους ινοβλάστες. Ο Nrf2 είναι ένας από τους βασικούς μεταγραφικούς παράγοντες, υπεύθυνος για τη ρύθμιση της αντιοξειδωτικής απόκρισης του κυττάρου, ο οποίος ρυθμίζει την έκφραση διαφόρων πρωτεασωμικών υπομονάδων στο ποντίκι. Στην παρούσα διατριβή, συσχετίστηκε η λειτουργία του Nrf2 με το πρωτεάσωμα κατά την αναδιπλασιαστική γήρανση. Έτσι βρέθηκε ότι ο μεταγραφικός παράγοντας Nrf2 έχει μειωμένη έκφραση καθώς και λειτουργία κατά τη γήρανση, ενώ η αποσιώπησή του οδηγεί σε πρόωρη γήρανση των ανθρωπίνων ινοβλαστών. Η σωστή λειτουργία του πρωτεασώματος και του Nrf2 είναι απαραίτητη για τη διατήρηση της κυτταρικής ομοιοστασίας και για το λόγο αυτό η ταυτοποίηση φυσικών ουσιών που ενεργοποιούν το πρωτεάσωμα και τον μεταγραφικό παράγοντα Nrf2, αποτέλεσε βασικό στόχο της διατριβής. Έτσι, μελετήθηκαν διάφορα εκχυλίσματα και φυσικές ουσίες, που απομονώθηκαν από φυτά της ελληνικής χλωρίδας, σε σχέση με την επίδραση τους στο πρωτεάσωμα και στο φαινόμενο της αναδιπλασιαστικής γήρανσης. Στην παρούσα διδακτορική διατριβή, μελετήθηκε περαιτέρω το 18α-γλυκιρχετινικό οξύ (18α-GA), ένα από τα κύρια συστατικά της γλυκόριζας, ως προς τις αντιοξειδωτικές και αντιγηραντικές του ιδιότητες. Παρατηρήθηκε ότι το 18α-GA ενισχύει τις πρωτεασωμικές ενεργότητες και αυξάνει την ποσότητα του δομημένου πρωτεασώματος, μέσω του μεταγραφικού παράγοντα Nrf2. Επιπλέον, η επίδραση με το 18α-GA αυξάνει την κυτταρική επιβίωση παρουσία οξειδωτικού στρες, ενώ η συνεχής χορήγηση της ουσίας οδηγεί σε αύξηση του πρoσδόκιμου ζωής ανθρωπίνων ινοβλαστών. Ακόμη, ο άξονας πρωτεάσωμα και Nrf2 είναι λειτουργικός σε καλλιέργειες γηρασμένων ινοβλαστών, δεδομένου ότι τα κύτταρα αυτά διατηρούν την απόκρισή τους στην ενεργοποίηση του Nrf2 και του πρωτεασώματος από την ουσία 18α-GA. Συνοπτικά, τα παραπάνω δεδομένα καταδεικνύουν τις ευεργετικές δράσεις του 18α-GA κατά την αναδιπλασιαστική γήρανση και παράλληλα παρέχει νέες δυνατότητες στην ενεργοποίηση κυτταρικών αντιοξειδωτικών μηχανισμών από φυσικές ουσίες

<i>Salmonella</i> contamination, serovars and antimicrobial resistance profiles of cattle slaughtered in South Africa

Antimicrobial resistant Salmonella are among the leading causes of foodborne infections. Our aim was to determine Salmonella contamination during cattle slaughter in South African rural abattoirs (n = 23) and environmental samples. Furthermore, antimicrobial resistance patterns of the Salmonella isolates were determined. Samples of cattle faeces (n = 400), carcass sponges (n = 100), intestinal contents (n = 62), hides (n = 67), and water from the abattoirs (n = 75) were investigated for Salmonella species using microbiological techniques and species-specific polymerase chain reaction targeting the invA gene. In total 92 Salmonella species isolates were recovered. The Salmonella mean frequency of occurrence on hides, carcasses, and intestinal contents was 35.37% (n = 81). Eleven faecal samples (2.75%) tested positive for Salmonella. The predominant serovar was Salmonella Enteritidis. Diverse serovars that were identified on carcasses were not necessarily found on the hides and intestinal contents. The inconsistent occurrence of the diverse Salmonella serovars on hides, carcasses, and intestinal contents implies that in addition to carriage on hides and in intestinal contents, other external factors also play an important role regarding carcass contamination. The 92 Salmonella were serotyped and tested for susceptibility towards the following antimicrobials: ampicillin, cefotaxime, enrofloxacin, kanamycin, and oxytetracycline using the disk diffusion method. Most Salmonella (n = 66; 71.7%) isolates were resistant to at least one antimicrobial with highest resistance observed towards oxytetracycline (51.90%), which highlights the need for strict hygiene during slaughter and prudent antimicrobial use during animal production. In conclusion, cattle slaughtered in South African rural abattoirs harbour diverse Salmonella serovars that are resistant to antimicrobials, which could be a public health risk. The findings should assist policymakers with improving implementation of hygienic slaughter of cattle in rural abattoirs, which is paramount from socioeconomic, public health, and epidemiological standpoints. Keywords: Salmonella; Cattle; Rural abattoirs; slaughter; Multidrug resistance; Environmental sample