DNMT1 regulates expression of MHC class I in post-mitotic neurons

Abstract Major Histocompability Complex I (MHC-I) molecules present cellularly derived peptides to the adaptive immune system. Generally MHC-I is not expressed on healthy post-mitotic neurons in the central nervous system, but it is known to increase upon immune activation such as viral infections and also during neurodegenerative processes. MHC-I expression is known to be regulated by the DNA methyltransferase DNMT1 in non-neuronal cells. Interestingly DNMT1 expression is high in neurons despite these being non-dividing. This suggests a role for DNMT1 in neurons beyond the classical re-methylation of DNA after cell division. We thus investigated whether DNMT1 regulates MHC-I in post-mitotic neurons. For this we used primary cultures of mouse cerebellar granule neurons (CGNs). Our results showed that knockdown of DNMT1 in CGNs caused upregulation of some, but not all subtypes of MHC-I genes. This effect was synergistically enhanced by subsequent IFNγ treatment. Overall MHC-I protein level was not affected by knockdown of DNMT1 in CGNs. Instead our results show that the relative MHC-I expression levels among the different MHC subtypes is regulated by DNMT1 activity. In conclusion, we show that while the mouse H2-D1/L alleles are suppressed in neurons by DNMT1 activity under normal circumstances, the H2-K1 allele is not. This finding is particularly important in two instances. One: in the context of CNS autoimmunity with epitope presentation by specific MHC-I subtypes where this allele specific regulation might become important; and two: in amyotropic lateral sclerosis (ALS) where H2-K but not H2-D protects motor neurons from ALS astrocyte-induced toxicity in a mouse model of ALS

Design of a laboratory bioreactor for engineering articular cartilage based on 3D printed nasal septum-like scaffolds

«Η εκφύλιση των χόνδρων είναι μια σοβαρή πάθηση που επηρεάζει μεγάλο μέρος του πληθυσμού σε όλο το ηλικιακό φάσμα. Επί του παρόντος χρησιμοποιούνται διάφορες τεχνικές αποκατάστασης για μικρής έκτασης βλάβες όπως η αρθροπλαστική απόξεσης και ο υποχονδρικός τρυπανισμός, οι οποίες δεν μπορούν να επιδιορθώσουν βλάβες μεγαλύτερης έκτασης. Η Αναγεννητική Ιατρική προωθεί την Μηχανική Ιστών στο προσκήνιο των σύγχρονων μηχανικών τεχνικών, συνδυάζοντας καινοτόμα βιοσυμβατά υλικά, νέες μεθόδους μηχανικής ιστών όπως η τεχνολογία 3D εκτύπωσης και βιοδιαδικασίες που αποσκοπούν στην δημιουργία ποιοτικών μοσχευμάτων για εκτεταμένες βλάβες των χόνδρων. Κατάλληλο κυτταρικό περιβάλλον για δημιουργία ιστών μπορεί να επιτευχθεί με την ανάπτυξη αυτών των μοσχευμάτων σε βιοαντιδραστήρες. O κάθε βιοαντιδραστήρας χρησιμοποιεί διαφορετικές αρχές καλλιέργειας, και ορισμένοι από αυτούς όπως οι μικτού τύπου και οι βιοαντιδραστήρες διαπότισης επιστρατεύουν την άσκηση μηχανικών δυνάμεων επί του ικριώματος ώστε να επιτευχθεί μεγάλη κυτταρική πυκνότητα και ενισχυμένες μηχανικές ιδιότητες που οδηγούν στην δημιουργία καλύτερης ποιότητας χόνδρου. Αυτές οι ιδιαιτερότητες των βιοαντιδραστήρων μπορούν να αποτελέσουν εφαλτήριο κατασκευής εργαστηριακών βιοαντιδραστήρων, για την καλλιέργεια 3D εκτυπωμένων ρινικών διαφραγμάτων ως ένα λειτουργικό παράδειγμα υαλώδους χόνδρου».Cartilage degeneration is a severe disease affecting a significant part of the population at all ages. Various treatment modalities are currently used for small-sized cartilage defects, such as abrasion arthroplasty and subchondral drilling, but fail to repair larger-scale damages. Regenerative Medicine pushes Tissue Engineering (TE) to the forefront of modern engineering techniques combining novel biocompatible materials, new tissue engineering methods, like 3D printing technology and bioprocesses trying to create quality transplants for large cartilage defects. The appropriate cell environment for engineered tissues can be achieved through growth of the tissue-engineered constructs into bioreactors. Each bioreactor uses different principles for culturing processes, and some of them mostly mixed and perfusion bioreactors, use different kind of mechanical forces on the scaffold to achieve high cell densities, enhanced mechanical properties leading to better quality of engineered cartilage. These advantageous particularities can be used to create a laboratory bioreactor design, for culturing 3D printed nasal septum cartilage as a working example of hyaline cartilage

Translational research for nasal septum cartilage regeneration with chondrocytes derived from differentiated human adipose mesenchymal stem cells

Η εργασία αφορά στη μεταφραστική έρευνα ιστοτεχνολογίας και συγκεκριμένα στη δημιουργία ανθρώπινου ρινικού διαφράγματος με τη χρήση ηλεκτρονικά υποβοηθούμενου σχεδιασμού και τρισδιάστατης εκτύπωσης τρισδιάστατου (3D) πορώδους ικριώματος χιτοζάνης/ζελατίνης (CAD/CAM). Το ικρίωμα θα χρησιμοποιηθεί για να αποικιστεί από χρονδροκύτταρα που προκύπτουν από διαφοροποιημένα μεσεγχυματικά κύτταρα ανθρώπου προερχόμενα από λιπώδη ιστό (Adipose Tissue Mesenchymal Stem Cells-AD- MSCs). Η όλη διαδικασία επιτυγχάνεται με τη χρήση βιοαντιδραστήρα.Τα μεσεγχυματικά κύτταρα είναι πολυδύναμα βλαστοκύτταρα που μπορούν να απομονωθούν από το μυελό των οστώνκαι το λιπώδη ιστό. Τα κύτταρα αυτά έχουν τη δυνατότητα να διαφοροποιούνται, υπό εργαστηριακές συνθήκες, σε οστεοκύτταρα, χονδροκύτταρα, και λιποκύτταρα. Στην παρούσα μελέτη ανθρώπινα μεσεγχυματικά κύτταρα απομονώθηκαν από λιπώδη ιστό και καλλιεργήθηκαν in vitro. Η έκφραση των αντιγόνων επιφανείας CD90, CD73, σε συνδυασμό με την απουσία του μάρτυρα CD45 επιβεβαιώνουν την επιτυχή απομόνωση μεσεγχυματικών βλαστικών κυττάρων, με χρήση κυτταρομετρίας ροής. Έπειτα από 21 ημέρες από την επαγωγή στοχευόμενης διαφοροποίησης τα βλαστοκύτταρα διαφοροποιήθηκαν σε χονδροκύτταρα και χαρακτηρίστηκαν ιστολογικά με χρώση κυανού της τολουιδίνης και μοριακά με RTPCR για δείκτες διαφοροποίησης όπως η αγκρεκάνη. Με τη χρήση του τρισδιάστατου εκτυπωτή δημιουργήθηκε υπό κλίμακα ικρίωμα ρινικού χόνδρου από PLA. Η διαδικασία θα ολοκληρωθεί με την εκτύπωση του υπό διερεύνηση υλικού χιτοζάνης/ζελατίνης σε 3D ικρίωμα και αφού εμποτιστεί με χονδροκύτταρα θα μεταφερθεί στον βιοαντιδραστήρα.Mesenchymal stem cells (MSCs) are multipotent cells isolated from various tissues, mainly from the bone marrow and adipose tissue. Their ability to differentiate into osteoblasts, chondrocytes or adipocytes renders them a promising clinical tool for injury repair and tissue regeneration. In the current study, MSCs were isolated from human adipose tissue (hAD-MSCs) and were triggered to differentiate into chondrocytes in vitro. Expression of mesenchymal stem cell markers, such as CD90 and CD73, in combination with the absence of hematopoietic markers, such as CD45, proves via flow cytometry the successful isolation of MSCs. Histologic staining with Toluidine blue and real time PCR analysis for the expression of the chondrogenic marker aggrecan (ACAN) verified the successful chondrogenic differentiation of AD-MSCs. Using Poly Lactic-Acid as scaffolding material, a three-dimensional scaffold with customized architecture, controlled porosity and interconnected porous structure was fabricated using 3D printing. The produced scaffold represents the morphology of the nasal septum cartilage. We aspire, to see this scaffold with the differentiated chondrocytes and culture the complex under the appropriate micoenvironmental conditions of a bioreactor system in order to regenerate a potential cartilage transplant. This in vitro study expands the potentials of human AD-MSCs to be used in clinic for alleviation of cartilage defects and tissue engineering in Greece and worldwide

A Sandwich‐model experiment with personal response systems on epigenetics: insights into learning gain, student engagement and satisfaction

Current trends in Higher Education Pedagogies include an ongoing discussion about active learning strategies. Technology-based interventions such as personal response systems (PRS) have gained momentum, especially since the advent of cloud-/web-based solutions. One model that supports the transition from traditional lecturing towards active learning by maintaining a balance between instruction and self-learning is the 'Sandwich Model'. In the present study, we investigated the impact of the Sandwich Model combined with PRS in student learning, engagement and satisfaction by a randomised trial in a large undergraduate biomedical/medical sciences class. A teaching session on epigenetics was delivered either as a traditional lecture (C-group) or as a PRS-including Sandwich-based session (S-group). The major finding of our experiment was the significantly enhanced performance of the S-group over the control, suggesting that the Sandwich Model improves learning gain. We also provide strong evidence that the Sandwich Model enhances student engagement and satisfaction. However, the effect of the Sandwich Model in learning gain and student attitudes was not dependent on PRS incorporation per se and students seemed to favour non-PRS activities over PRS, as evidenced by their feedback. Although further experimental research is needed in order to conclusively compare and contrast PRS and non-PRS activities regarding learning gain, we propose the usage of the Sandwich Model with a variety of in-class learning activities, both PRS and non-PRS-based. Altogether, our work shows that the Sandwich Model is a powerful pedagogical approach that exerts a positive impact on student perceptions for learning and satisfaction and that can support the teaching of challenging biomedical concepts, such as epigenetics

Additional file 1: of DNMT1 regulates expression of MHC class I in post-mitotic neurons

Figure S1. Transfection efficiency of Cy3-labelled siRNA in CGNs. CGNs were transfected with Cy3-labelled non-targeting siRNA as described in Materials and Methods. After 72 h incubation the cells were fixated and mounted using mounting media containing DAPI. Epifluorescence images were obtained for DAPI, Cy3-siRNA and overlay. Arrows with thick arrowhead, transfected cell; thin arrowhead, non-transfected cell. Figure S2. Generation of a functional positive control of DNMT1 knockdown. (A) Gene expression of NNAT, CD24A, ICAM1, RUNX1, and S100A10 [46] in CGNs upon knockdown of DNMT1 relative to untreated cells. Bonferroni-corrected one-sample t-test: * p1.5 for NNAT and S100A10 relative to treatment with non-targeting siRNA. (C) Positive control values for three successful and four unsuccessful experiments (mean ± SD). Table S1. siRNAs from DharmaconTM used in the study. Table S2. Taqman probes used in the study. (DOCX 930 kb

A conditional Smg6 mutant mouse model reveals circadian clock regulation through the nonsense-mediated mRNA decay pathway

Nonsense-mediated messenger RNA (mRNA) decay (NMD) has been intensively studied as a surveillance pathway that degrades erroneous transcripts arising from mutations or RNA processing errors. While additional roles in physiological control of mRNA stability have emerged, possible functions in mammalian physiology in vivo remain unclear. Here, we created a conditional mouse allele that allows converting the NMD effector nuclease SMG6 from wild-type to nuclease domain-mutant protein. We find that NMD down-regulation affects the function of the circadian clock, a system known to require rapid mRNA turnover. Specifically, we uncover strong lengthening of free-running circadian periods for liver and fibroblast clocks and direct NMD regulation of Cry2 mRNA, encoding a key transcriptional repressor within the rhythm-generating feedback loop. Transcriptome-wide changes in daily mRNA accumulation patterns in the entrained liver, as well as an altered response to food entrainment, expand the known scope of NMD regulation in mammalian gene expression and physiology

A novel Smg6 mouse model reveals regulation of circadian period and daily CRY2 accumulation through the nonsense-mediated mRNA decay pathway

Nonsense-mediated mRNA decay (NMD) has been intensively studied as a surveillance pathway that degrades erroneous transcripts arising from mutations or RNA processing errors. While additional roles in controlling regular mRNA stability have emerged, possible functions in mammalian physiology in vivo have remained unclear. Here, we report a novel conditional mouse allele that allows converting the NMD effector nuclease SMG6 from wild-type to nuclease domain-mutant protein. We analyzed how NMD downregulation affects the function of the circadian clock, a system known to require rapid mRNA turnover. We uncover strong lengthening of free-running circadian periods for liver and fibroblast clocks, and direct NMD regulation of Cry2 mRNA, encoding a key transcriptional repressor within the rhythm-generating feedback loop. In the entrained livers of Smg6 mutant animals we reveal transcriptome-wide alterations in daily mRNA accumulation patterns, altogether expanding the known scope of NMD regulation in mammalian gene expression and physiology