Analisi funzionale del C-terminale di Xotx2 e Xotx5 nello sviluppo precoce di Xenopus laevis

I geni <i style='mso-bidi-font-style: normal'>Otx dei Vertebrati, omologhi di <i style='mso-bidi-font-style:normal'>orthodenticle (<i style='mso-bidi-font-style: normal'>otd) di Drosophila, svolgono un ruolo di primaria importanza nella specificazione della regione rostrale del sistema nervoso centrale (SNC) dei Vertebrati. Studi su questi geni sono stati condotti dapprima in topo dove sono stati inizialmente caratterizzati Otx1 e <i style='mso-bidi-font-style:normal'>Otx2. Successivamente sono stati isolati Otx3 in Zebrafish e Xotx1, Xotx2, <i style='mso-bidi-font-style: normal'>Xotx4 ed Xotx5 in <i style='mso-bidi-font-style:normal'>Xenopus laevis. Nei mammiferi <i style='mso-bidi-font-style:normal'>otx5 non è presente, ma si trova un altro gene della famiglia otx detto <i style='mso-bidi-font-style:normal'>crx (cone rod homeobox), la cui relazione con otx5 non è definitivamente chiarita. Crx è implicato nello sviluppo dell’occhio e mutazioni di <i style='mso-bidi-font-style: normal'>Crx provocano nell’uomo diverse malattie della retina, che comprendono l’amaurosi congenita di Leber (una grave degenerazione dei coni e dei bastoncelli nell’infanzia), la distrofia dei coni e dei bastoncelli nell’adulto e la retinite pigmentosa. In questo lavoro di tesi <span class=GramE>abbiamo concentrato la nostra attenzione sui geni <i style='mso-bidi-font-style:normal'>Xotx2 e <i style='mso-bidi-font-style: normal'>Xotx5 di Xenopus, nell’ambito di un progetto di ricerca rivolto a chiarire il loro ruolo nello sviluppo dell’occhio. Come tutti i geni della classe <i style='mso-bidi-font-style:normal'>otd/otx, <i style='mso-bidi-font-style: normal'>Xotx2 e Xotx5 contengono un omeodominio della classe bicoid, caratterizzato da uno specifico residuo di lisina in posizione 50 (corrispondente alla posizione 9 dell’elica di riconoscimento) che conferisce loro la specificità di legame al DNA. Esperimenti condotti in topo hanno mostrato che <i style='mso-bidi-font-style: normal'>Otx2 è espresso nell’intero epiblasto di embrioni precoci. Successivamente la sua espressione diviene sempre più ristretta alle regioni più anteriori che includono il neuroectoderma corrispondente alle regioni telencefaliche, diencefaliche e mesencefaliche. A <span class=GramE>sottolineare l’importanza di tale gene vi è il fatto che topi Otx2-/- muoiono precocemente nell’embriogenesi per la totale assenza di tali strutture e per malformazioni generalizzate del piano corporeo (dovuti a movimenti anomali durante la gastrulazione). L’espressione di Xotx2 nell’embriogenesi di Xenopus è simile a quella osservata nel topo, ed interessa inizialmente la regione dell’organizzatore di Spemann, e successivamente le regioni rostrali del SNC, incluso l’occhio. Dal canto suo <i style='mso-bidi-font-style:normal'>Xotx5 ha un’espressione nell’embrione precoce simile a quella di Xotx2. Si trova infatti nella regione dell’organizzatore di Spemann allo stadio di gastrula precoce e, poco dopo, anche nel neurectoderma anteriore. Successivamente alla chiusura del tubo neurale però, si ritrova esclusivamente espresso nell’occhio e nella ghiandola pineale, entrambe strutture deputate alla fotorecezione. Esperimenti di sovraespressione in embrioni precoci hanno mostrato effetti simili alla sovraespressione di Xotx2, producendo riduzioni delle strutture posteriori e l’induzione di strutture anteriori e tessuto neurale nell’intero embrione. L’espressione di questi geni durante lo sviluppo dell’occhio ha spinto a ulteriori approfondimenti che hanno mostrato il ruolo chiave dei due geni nel differenziamento di tale struttura. <span class=GramE>Infatti l’espressione di Xotx5, durante lo sviluppo dell’occhio si restringe progressivamente sia alle cellule bipolari che ai fotorecettori. Invece l’espressione di <i style='mso-bidi-font-style: normal'>Xotx2 viene progressivamente ristretta alle sole cellule bipolari. Esperimenti di lipotrasfezione di precursori retinici con costrutti per i due geni mostrano che <i style='mso-bidi-font-style: normal'>Xotx2 spinge i precursori verso un destino bipolare, mentre <i style='mso-bidi-font-style:normal'>Xotx5 spinge le cellule verso un destino di fotorecettori. Scopo del presente lavoro di tesi è stato quello di analizzare l’importanza delle varie porzioni del C-terminale nell’azione <span class=GramE>di transattivazione di questi fattori di trascrizione. A questo scopo, si è provveduto alla creazione di vari costrutti <i style='mso-bidi-font-style:normal'>Xotx2 e <i style='mso-bidi-font-style: normal'>Xotx5, deleti al C-terminale, tramite PCR. Successivamente, il loro mRNA è stato trascritto “in vitro” e microiniettato in embrioni di <i style='mso-bidi-font-style:normal'>Xenopus allo stadio di 4 blastomeri. Gli embrioni sono stati poi caratterizzati in base al fenotipo osservato e sottoposti ad analisi con opportuni markers molecolari allo scopo di evidenziare strutture ectopiche indotte in seguito all’iniezione. L’analisi degli effetti di tali costrutti è preliminare a successivi esperimenti di lipotrasfezione di precursori cellulari retinici; l’intento è di identificare quali domini funzionali di Xotx2 e Xotx5 siano essenziali per la loro azione di specificazione di tipi cellulari in retina.</p

Relationship of Oligomerization to DNA Binding of Wheat Dwarf Virus RepA and Rep Proteins

AbstractMembers of the genus Mastrevirus (family Geminiviridae) produce a complementary-sense (c-sense) transcription unit with the potential to encode two proteins, RepA and Rep. In the present work, we have studied the DNA–protein complexes formed by the Wheat dwarf virus (WDV) RepA protein within the WDV large intergenic region. WDV RepA forms large nucleoprotein complexes near the TATA boxes of the viral complementary-sense and virion-sense (v-sense) promoters (the RepA C- and V-complexes, respectively), a location similar to those of WDV Rep–DNA complexes but with distinct DNase I footprints. We have also studied the relationship of oligomerization of WDV RepA and Rep proteins to DNA–protein complex formation. Using chemical cross-linking, we have determined that both WDV proteins can form oligomers in solution. Interestingly, the pH is critical for the monomer–oligomer equilibrium and small changes produce a displacement in such a way that at pH ≤ 7.0, the predominant species is an octamer while at pH ≥ 7.4 it is a monomer. Complex formation is also strongly affected by pH and occurs more efficiently at pH 7.0–7.4. We found that preformed oligomers interact very poorly with DNA. Thus, our data are consistent with a stepwise model for protein–DNA complex assembly in which monomers interact with DNA and then with other monomers to assemble an oligomeric structure on the DNA. These results may be relevant for studies on the DNA binding, replication, and transcription properties of geminivirus proteins

Neuroinflammation: A Signature or a Cause of Epilepsy?

: Epilepsy can be both a primary pathology and a secondary effect of many neurological conditions. Many papers show that neuroinflammation is a product of epilepsy, and that in pathological conditions characterized by neuroinflammation, there is a higher probability to develop epilepsy. However, the bidirectional mechanism of the reciprocal interaction between epilepsy and neuroinflammation remains to be fully understood. Here, we attempt to explore and discuss the relationship between epilepsy and inflammation in some paradigmatic neurological and systemic disorders associated with epilepsy. In particular, we have chosen one representative form of epilepsy for each one of its actual known etiologies. A better understanding of the mechanistic link between neuroinflammation and epilepsy would be important to improve subject-based therapies, both for prophylaxis and for the treatment of epilepsy

Analysis of immunization time, amplitude, and adverse events of seven different vaccines against SARS-CoV-2 across four different countries

BackgroundScarce information exists in relation to the comparison of seroconversion and adverse events following immunization (AEFI) with different SARS-CoV-2 vaccines. Our aim was to correlate the magnitude of the antibody response to vaccination with previous clinical conditions and AEFI.MethodsA multicentric comparative study where SARS-CoV-2 spike 1-2 IgG antibodies IgG titers were measured at baseline, 21-28 days after the first and second dose (when applicable) of the following vaccines: BNT162b2 mRNA, mRNA-1273, Gam-COVID-Vac, Coronavac, ChAdOx1-S, Ad5-nCoV and Ad26.COV2. Mixed model and Poisson generalized linear models were performed.ResultsWe recruited 1867 individuals [52 (SD 16.8) years old, 52% men]. All vaccines enhanced anti-S1 and anti-S2 IgG antibodies over time (p&lt;0.01). The highest increase after the first and second dose was observed in mRNA-1273 (p&lt;0.001). There was an effect of previous SARS-CoV-2 infection; and an interaction of age with previous SARS-CoV-2 infection, Gam-COVID-Vac and ChAdOx1-S (p&lt;0.01). There was a negative correlation of Severe or Systemic AEFI (AEs) of naïve SARS-CoV-2 subjects with age and sex (p&lt;0.001); a positive interaction between the delta of antibodies with Gam-COVID-Vac (p=0.002). Coronavac, Gam-COVID-Vac and ChAdOx1-S had less AEs compared to BNT162b (p&lt;0.01). mRNA-1273 had the highest number of AEFIs. The delta of the antibodies showed an association with AEFIs in previously infected individuals (p&lt;0.001).ConclusionsThe magnitude of seroconversion is predicted by age, vaccine type and SARS-CoV-2 exposure. AEs are correlated with age, sex, and vaccine type. The delta of the antibody response only correlates with AEs in patients previously exposed to SARS-CoV-2.Registration numberClinicalTrials.gov, identifier NCT05228912

Simultaneous two-photon imaging of intracellular chloride concentration and pH in mouse pyramidal neurons in vivo

Intracellular chloride ([Cl-](i)) andpH(pH(i)) are fundamental regulators of neuronal excitability. They exert wide-ranging effects on synaptic signaling and plasticity and on development and disorders of the brain. The ideal technique to elucidate the underlying ionic mechanisms is quantitative and combined two-photon imaging of [Cl-](i) and pH(i), but this has never been performed at the cellular level in vivo. Here, by using a genetically encoded fluorescent sensor that includes a spectroscopic reference (an element insensitive to Cl-and pH), we show that ratiometric imaging is strongly affected by the optical properties of the brain. We have designed a method that fully corrects for this source of error. Parallel measurements of [Cl-](i) and pH(i) at the single-cell level in the mouse cortex showed the in vivo presence of the widely discussed developmental fall in [Cl-](i) and the role of the K-Cl cotransporter KCC2 in this process. Then, we introduce a dynamic two-photon excitation protocol to simultaneously determine the changes of pHi and [Cl-](i) in response to hypercapnia and seizure activity.Peer reviewe

Guidance on aneugenicity assessment

The EFSA Scientific Committee was asked to provide guidance on the most appropriate in vivo tests to follow up on positive in vitro results for aneugenicity, and on the approach to risk assessment for substances that are aneugenic but not clastogenic nor causing gene mutations. The Scientific Committee confirmed that the preferred approach is to perform an in vivo mammalian erythrocyte micronucleus test with a relevant route of administration. If this is positive, it demonstrates that the substance is aneugenic in vivo. A negative result with evidence that the bone marrow is exposed to the test substance supports a conclusion that aneugenic activity is not expressed in vivo. If there is no evidence of exposure to the bone marrow, a negative result is viewed as inconclusive and further studies are required. The liver micronucleus assay, even though not yet fully validated, can provide supporting information for substances that are aneugenic following metabolic activation. The gastrointestinal micronucleus test, conversely, to be further developed, may help to assess aneugenic potential at the initial site of contact for substances that are aneugenic in vitro without metabolic activation. Based on the evidence in relation to mechanisms of aneugenicity, the Scientific Committee concluded that, in principle, health-based guidance values can be established for substances that are aneugenic but not clastogenic nor causing gene mutations, provided that a comprehensive toxicological database is available. For situations in which the toxicological database is not sufficient to establish health-based guidance values, some approaches to risk assessment are proposed. The Scientific Committee recommends further development of the gastrointestinal micronucleus test, and research to improve the understanding of aneugenicity to support risk assessment

The N-Terminal Domain of ERK1 Accounts for the Functional Differences with ERK2

The Extracellular Regulated Kinase 1 and 2 transduce a variety of extracellular stimuli regulating processes as diverse as proliferation, differentiation and synaptic plasticity. Once activated in the cytoplasm, ERK1 and ERK2 translocate into the nucleus and interact with nuclear substrates to induce specific programs of gene expression. ERK1/2 share 85% of aminoacid identity and all known functional domains and thence they have been considered functionally equivalent until recent studies found that the ablation of either ERK1 or ERK2 causes dramatically different phenotypes. To search a molecular justification of this dichotomy we investigated whether the different functions of ERK1 and 2 might depend on the properties of their cytoplasmic-nuclear trafficking. Since in the nucleus ERK1/2 is predominantly inactivated, the maintenance of a constant level of nuclear activity requires continuous shuttling of activated protein from the cytoplasm. For this reason, different nuclear-cytoplasmic trafficking of ERK1 and 2 would cause a differential signalling capability. We have characterised the trafficking of fluorescently tagged ERK1 and ERK2 by means of time-lapse imaging in living cells. Surprisingly, we found that ERK1 shuttles between the nucleus and cytoplasm at a much slower rate than ERK2. This difference is caused by a domain of ERK1 located at its N-terminus since the progressive deletion of these residues converted the shuttling features of ERK1 into those of ERK2. Conversely, the fusion of this ERK1 sequence at the N-terminus of ERK2 slowed down its shuttling to a similar value found for ERK1. Finally, computational, biochemical and cellular studies indicated that the reduced nuclear shuttling of ERK1 causes a strong reduction of its nuclear phosphorylation compared to ERK2, leading to a reduced capability of ERK1 to carry proliferative signals to the nucleus. This mechanism significantly contributes to the differential ability of ERK1 and 2 to generate an overall signalling output