RNA editing of CYFIP2 regulates actin related cellular migration and neuronal development in vitro

Cytoplasmic FMRP Interacting Protein 2 (CYFIP2) è una proteina originariamente identificata come componente del Wave Regulatory Complex e come interattore di Fragile-X Mental Retardation Protein. Il trascritto di CYFIP2 è sottoposto alla reazione di editing dell'RNA, un meccanismo post-trascrizionale catalizzato dagli enzimi ADAR, che porta l'Adenosina alla deaminazione a Inosina. In seguito al processo di traduzione, l'editing di CYFIP2 comporta una sostituzione K/E dell'amminoacido in posizione 320. Questo studio vuole indagare le potenziali implicazioni di questo processo nei fenomeni correlati alla dinamica dell'actina durante la migrazione cellulare, lo sviluppo degli assoni e la sinaptogenesi nelle cellule neurali. I nostri risultati mostrano che l’editing dell'RNA di CYFIP2 aumenta durante lo sviluppo neurale. È un processo attivo solo nel sistema nervoso centrale. Per ottenere maggiori informazioni sul ruolo dell'RNA di CYFIP2, abbiamo inizialmente eliminato il gene CYFIP2 in cellule di neuroblastoma e abbiamo sovraespresso le varianti di CYFIP2 non editate ed editate nelle linee KO. La sovra espressione di entrambe le varianti è in grado di ripristinare il fenotipo morfologico delle cellule analizzate. I nostri risultati mostrano differenze funzionali delle proteine CYFIP2 editate e non editate nella regolazione della migrazione cellulare. La variante editata presenta una maggiore capacità di migrare. Abbiamo inoltre investigato il ruolo delle varianti di CYFIP2 durante lo sviluppo neuronale. A tale scopo, abbiamo utilizzato un consolidato modello di differenziazione neuronale in cellule SH-SY5Y, basato sull'applicazione di acido retinoico (RA) e fattore di crescita neuronale (BDNF) per monitorare la conversione di cellule indifferenziato in cellule con morfologia simil-neuronale altamente polarizzata. I nostri risultati dimostrano chiaramente che le cellule KO per il gene CYFIP2 hanno perso la capacità di produrre una caratteristica morfologia simil-neuronale dopo il differenziamento, mentre la sovra espressione delle varianti di editing ne ripristina la capacità differenziativa. Tuttavia, non è stato possibile rilevare differenze tra le popolazioni di cellule K ed E, indicando come la proteina CYFIP2 sia importante per il differenziamento neuronale, ma il processo di editing potrebbe non avere un ruolo in questo processo. Per approfondire il ruolo delle varianti K e E in un sistema più fisiologico abbiamo deciso di utilizzare colture neuronali primarie di roditore. L'espressione di CYFIP2 endogeno è stata silenziata utilizzando specifici shRNA e le colture cellulari sono state trasdotte con vettori lentivirali esprimenti le varianti K o E di CYFIP2. Durante la maturazione neuronale sono stati analizzati parametri morfologici relativi allo sviluppo assonale delle cellule. Abbiamo riscontrato una differenza statisticamente significativa tra i neuroni che esprimono le varianti non editate ed editate nel numero di ramificazioni, nella lunghezza totale e nell'indice di complessità degli assoni analizzati. Abbiamo inoltre applicato il nostro modello basato sulle colture primarie ippocampali per cercare di capire se l’editing di CYFIP2 possa essere implicato nel processo di sinaptogenesi. I nostri risultati mostrano come la frequenza delle spine venga drasticamente ridotta nelle cellule in cui la proteina CYFIP2 è stata silenziata. La popolazione di cellule che esprimono in maggioranza la variante di CYFIP2 non editata (K) non portano ad un ripristino nella frequenza nelle spine che al contrario si osserva nelle cellule che si sviluppano esprimendo la variante editata di CYFIP2 (E). Nel loro insieme questi risultati mostrano un chiaro ruolo della proteina CYFIP2 nella regolazione dello sviluppo assonale durante le prime fasi dello sviluppo in vitro e durante il processo di sinaptogenesi. La reazione K/E di editing dell'RNA del trascritto risulta inoltre importante in entrambi i processi studiati.Cytoplasmic FMRP Interacting Protein 2 (CYFIP2) is a protein originally identified as a component of Wave Regulatory Complex (WRC) and Fragile-X Mental Retardation Protein (FMRP) interactor. CYFIP2 transcript undergoes RNA editing, a post-transcriptional mechanism catalysed by ADAR enzymes, that leads adenosine to inosine deamination. CYFIP2 editing results in a K/E substitution at amino acid 320. In this study, we aim at investigating the potential implication of this process related to actin dynamics during cell migration and axon development and synaptogenesis in neural cells. Understanding the role of CYFIP2 RNA editing substitution would be fundamental for further analysis in normal and pathological conditions. Our results show that CYFIP2 RNA editing reaction increases during neural development. It is active only in the central nervous system. Taken together these data indicate that a proper CYFIP2 editing regulation is important during neuronal development and function. We have generated neuroblastoma cell lines deleted of CYFIP2 gene. The absence of CYFIP2 clearly modifies SH-SY5Y cellular morphology, leading to acting dynamic dysregulation. We have overexpressed CYFIP2 unedited and edited variants in KO cell lines, using lentivirus transduction. The overexpression of both variants are able to revert the morphological phenotype. Our results show functional differences of the edited and unedited CYFIP2 proteins in regulating cellular migration after wound healing experiments. In particular, the edited variant (CYFIP2-E) presents a stronger capability to migrate and to invade the experimental wound, both in starvation conditions and under growth factors stimulation. These data indicate that the editing reaction might be involved in the acting dynamic underlining cellular migration and proliferation.Since CYFIP2 protein is mainly expressed in neurons and K/E RNA editing reaction is active only in the central nervous system, we investigate the role of CYFIP variants on neuronal development. For this purpose, we use a well-established model of neuronal differentiation based on the application on SH-SY5Y cells of a two-step retinoic acid (RA) and brain-derived neurotrophic factor (BDNF) treatment to monitor the conversion of undifferentiated SH-SY5Y into neuron-like cells with distinctly polarized axon-dendritic morphology. Our results clearly demonstrate that KO cells have lost their ability to produce a characteristic neurite-like morphology after differentiation. However, no differences could be detected between K and E cell populations, indicating that CYFIP2 is important for neuronal differentiation but RNA editing isoform might have a similar function in this process for SH-SY5Y.Since the effect of K/E variants resulted irrelevant in neural maturation process using a previously described model, we took advantage of primary neuronal culture to try to understand the effect of CYFIP2 variants on neuronal maturation and spine dynamics. The expression of endogenous CYFIP2 has been down-regulated by specific shRNA and the cell culture has been transduced with lentiviral vectors expressing CYFIP2 K/E variants. During neuronal maturation a morphological parameters concerning axon development has been analysed. We found a statistically significant difference between neurons carrying K and E variants in the number of axon branches, total axon length and axon complexity index. Taken together these results suggest a clear role of CYFIP2 K/E RNA editing process in regulating the spreading of neuron axon during first stages of in-vitro development.These findings suggest that the K/E editing variations have a functional role that needs further investigation. Additionally, an in-vivo study can be useful to understand the role of CYFIP2 editing variants during neural development and function

House Resolution No. 1074

RESOLVED, BY THE HOUSE OF REPRESENTATIVES OF THE ONE HUNDREDTH GENERAL ASSEMBLY OF THE STATE OF ILLINOIS, we recognize the Illinois Mathematics & Science Academy for hosting the 14th Annual International Student Science Fair and for their commitment to promoting diverse perspectives and lifelong learning; and be it furthe RESOLVED, That a suitable copy of this resolution be presented to The Illinois Mathematics & Science Academy as an expression of our esteem and respect

House Resolution No. 1247

WHEREAS, The members of the Illinois House of Representatives are saddened to learn of the death of Leon Max Lederman, who passed away on October 3, 2018; an

ARCHIVIAZIONE DIGITALE DEL SEGNALE VIDEO DELLE TELECAMERE DI SORVEGLIANZA VISIVA

Dal 1993 l’attuale Sezione di Catania dell’Istituto Nazionale di Geofisica e Vulcanologia (I.N.G.V.) esegue il monitoraggio video dei vulcani attivi della Sicilia orientale (Etna, Stromboli, Vulcano). Fino ad oggi l’archiviazione dei segnali acquisiti era stata effettuata con apparati di videoregistrazione su supporti analogici (cassette VHS), utilizzando la tecnica time lapse. L’utilizzo di nastri magnetici porta con se una serie di problemi intrinseci alla natura del supporto: facile deterioramento, disponibilità di un unico originale, necessità di attrezzature dedicate alla riproduzione, interruzione della registrazione per sostituzione di nastri. Si è quindi pensato di realizzare un’architettura hardware/software per l’acquisizione e l’archiviazione digitale dei segnali video ricevuti dalle telecamere, e la distribuzione delle immagini in diretta su streaming video. Il sistema è in grado di fornire contemporaneamente tre servizi: • streaming video in tempo reale. • archivio immagini fisse acquisite ad intervalli regolari, con un minuto di ritardo sulla diretta. • archivio filmati digitali in time lapse, disponibili con trenta minuti di ritardo sulla diretta. Questa nuova soluzione permette ai vulcanologi in servizio di sorveglianza di esaminare l’attività occorsa utilizzando un qualunque visualizzatore di immagini e filmati video digitali (per le immagini in archivio) e/o client di streaming video (per le immagini in diretta) installati sul proprio personal computer, senza interferire con l’acquisizione

Electron backscattering from stacking faults in SiC by means of \textit{ab initio} quantum transport calculations

We study coherent backscattering phenomena from single and multiple stacking faults (SFs) in 3C- and 4H-SiC within density functional theory quantum transport calculations. We show that SFs give rise to highly dispersive bands within both the valance and conduction bands that can be distinguished for their enhanced density of states at particular wave number subspaces. The consequent localized perturbation potential significantly scatters the propagating electron waves and strongly increases the resistance for $n$-doped systems. We argue that resonant scattering from SFs should be one of the principal degrading mechanisms for device operation in silicon carbide.Comment: 5 pages, 4 figure

FENTANYL VS MORPHINE AS ADJUVANT TO SPINAL ANESTHESIA FOR CAESAREAN SECTION: AN OBSERVATIONAL STUDY

In patients undergoing caesarean section (CS) with spinal anesthesia, the superiority of one intrathecal opioid over another one is not fully established. In order to investigate this, we joined the PAIN OUT Project (NCT02083835) for a two-year period. We surveyed patients undergoing elective CS with intrathecal anesthesia. Patients were asked to complete an anonymous questionnaire. Primary outcomes were: worst pain experienced, time spent in severe pain, relief received by treatment, satisfaction about pain management, wish for more pain treatment. We included 144 patients. The two main pain management combinations used were: bupivacaine-morphine (B-M, n=100) and bupivacaine-fentanyl (B-F, n=32). There were no differences in any of the primary outcomes between the groups. The B-F population received more intravenous/intramuscular opioids during the intraoperative (p<0.01) and the postoperative (p<0.001) period. The choice of morphine or fentanyl as adjunct to local anesthetic in spinal anesthesia for CS does not affect the patient’s experience with regards to pain management

A Possible Circular Approach for Social Perception of Climate Adaptation Action Planning in Metropolitan Cities

AbstractOne of the factors that will affect the livability of cities and the overall citizens' quality of life in the future is certainly climate change. Urban areas will play a fundamental role in the commitment against climate change and will have to develop appropriate adaptation actions, in accordance with the European Strategy against climate change, including the planning and implementation of Green Infrastructures (GIs). They produce various environmental and social benefits in the urban context. Various studies have shown that citizenship involvement at all levels is necessary for the evaluation of the sharing of the proposed projects. The research proposes an innovative methodological model to support administrations in the strategic planning choice of GIs according to a shared and circular approach. To perform a multi-layer assessment, the multi-criteria evaluation will be combined with the circular evaluation model called Green City Circle. The evaluation is set up as a circular process, followed by a first investigative phase, followed by a proactive phase of solutions and an implementation phase up to a final stage of evaluation of the results and strategies for long-term sustainability. The study was carried out in the city of Catania to test a planning and management tool for GIs envisaged by the administration as win–win climate adaptation measures

Structural and electronic characterization of (2,3(3)) bar-shaped stacking fault in 4H-SiC epitaxial layers

Crystallographic, electronic, and energetic analyses of the (2, 3_3) [or (2, 3, 3, 3) in the standard Zhadanov notation] bar-shaped stacking fault, observed in as-grown 4H-SiC epitaxial layers, are presented. The defect has been identified by means of spatially resolved microphotoluminescence (μ-PL) measurements at different emission wavelengths, focusing on the emission peak at 0.3 eV below the conduction band. Low temperature μ-PL measurements have also been performed. The defect has been identified and characterized using high resolution transmission electron microscopy. Experimental results are correlated and validated by the calculations of the Kohn–Sham electronic band structure and the defect formation energy