EMT/MET at the crossroad of stemness, regeneration and oncogenesis. The Ying-Yang equilibrium recapitulated in cell spheroids

The epithelial-to-mesenchymal transition (EMT) is an essential trans-differentiation process, which plays a critical role in embryonic development, wound healing, tissue regeneration, organ fibrosis, and cancer progression. It is the fundamental mechanism by which epithelial cells lose many of their characteristics while acquiring features typical of mesenchymal cells, such as migratory capacity and invasiveness. Depending on the contest, EMT is complemented and balanced by the reverse process, the mesenchymal-to-epithelial transition (MET). In the saving economy of the living organisms, the same (Ying-Yang) tool is integrated as a physiological strategy in embryonic development, as well as in the course of reparative or disease processes, prominently fibrosis, tumor invasion and metastasis. These mechanisms and their related signaling (e.g., TGF-β and BMPs) have been effectively studied in vitro by tissue-derived cell spheroids models. These three-dimensional (3D) cell culture systems, whose phenotype has been shown to be strongly dependent on TGF-β-regulated EMT/MET processes, present the advantage of recapitulating in vitro the hypoxic in vivo micro-environment of tissue stem cell niches and their formation. These spheroids, therefore, nicely reproduce the finely regulated Ying-Yang equilibrium, which, together with other mechanisms, can be determinant in cell fate decisions in many pathophysiological scenarios, such as differentiation, fibrosis, regeneration, and oncogenesis. In this review, current progress in the knowledge of signaling pathways affecting EMT/MET and stemness regulation will be outlined by comparing data obtained from cellular spheroids systems, as ex vivo niches of stem cells derived from normal and tumoral tissues. The mechanistic correspondence in vivo and the possible pharmacological perspective will be also explored, focusing especially on the TGF-β-related networks, as well as others, such as SNAI1, PTEN, and EGR1. This latter, in particular, for its ability to convey multiple types of stimuli into relevant changes of the cell transcriptional program, can be regarded as a heterogeneous "stress-sensor" for EMT-related inducers (growth factor, hypoxia, mechano-stress), and thus as a therapeutic target

The prion protein regulates glutamate-mediated Ca2+ entry and mitochondrial Ca2+ accumulation in neurons

The cellular prion protein (PrPC) whose conformational misfolding leads to the production of deadly prions, has a still-unclarified cellular function despite decades of intensive research. Following our recent finding that PrPC limits Ca2+ entry via store-operated Ca2+ channels in neurons, we investigated whether the protein could also control the activity of ionotropic glutamate receptors (iGluRs). To this end, we compared local Ca2+ movements in primary cerebellar granule neurons and cortical neurons transduced with genetically encoded Ca2+ probes and expressing, or not expressing, PrPC. Our investigation demonstrated that PrPC downregulates Ca2+ entry through each specific agonist-stimulated iGluR and after stimulation by glutamate. We found that, although PrP-knockout (KO) mitochondria were displaced from the plasma membrane, glutamate addition resulted in a higher mitochondrial Ca2+ uptake in PrP-KO neurons than in their PrPC-expressing counterpart. This was because the increased Ca2+ entry through iGluRs in PrP-KO neurons led to a parallel increase in Ca2+-induced Ca2+ release via ryanodine receptor channels. These data thus suggest that PrPC takes part in the cell apparatus controlling Ca2+ homeostasis, and that PrPC is involved in protecting neurons from toxic Ca2+ overloads

Contribution and Effectiveness of Laboratory Testing in the Diagnostic Assessment of Juvenile Ischemic Stroke and Transient Ischemic Attack

Introduction Strokes in young people require an extensive diagnostic workup to detect their possible several etiopathogenetic mechanisms. There is no consensus indicating what and when it should be tested. The clinical benefit and cost-effectiveness ratio of laboratory tests is unclear as well.Methods In one series of 104 consecutive juvenile ischemic stroke patients, under 45 years old, admitted between January 1, 2012, and December 31, 2017, we considered a wide panel of laboratory biomarkers exploring both the patient's basal status and specific risk factors for thrombotic disorders. To combine conventional and unconventional risk factors, structural defects, and other stroke-related diseases, we defined four categories of etiologic probability. We then studied the contribution of laboratory testing in changing the rate of "definite or probable stroke etiology" and the "proportion of patients with at least one additional risk factor" for stroke.Results The mere clinical assessment clarified stroke etiopathogenesis in 31% of cases. Abnormal values of the panel of biomarkers we considered were found in 30.1% of young ischemic strokes, while 11.5% of patients had unclear or borderline values. The benefit of laboratory assessment consisted of a relevant 14% gain in patients with a "definite or probable stroke etiology." Conclusion Several areas of uncertainty are still pending and herein discussed, such as the low re-testing rate during follow-up and the neglect of some relevant biomarkers. However, our results support the importance of laboratory testing in this setting. An improvement of diagnostic protocols in juvenile ischemic stroke would even increase their effectiveness, and this is still an unsolved issue in the field of cerebrovascular diseases. The same age limit, conventionally considered for juvenile stroke, could be better defined according to the effectiveness of both laboratory and clinical assessment in identifying unconventional stroke risk factors

SPLICS: a split green fluorescent protein-based contact site sensor for narrow and wide heterotypic organelle juxtaposition

Contact sites are discrete areas of organelle proximity that coordinate essential physiological processes across membranes, including Ca2+ signaling, lipid biosynthesis, apoptosis, and autophagy. However, tools to easily image inter-organelle proximity over a range of distances in living cells and in vivo are lacking. Here we report a split-GFP-based contact site sensor (SPLICS) engineered to fluoresce when organelles are in proximity. Two SPLICS versions efficiently measured narrow (8\u201310 nm) and wide (40\u201350 nm) juxtapositions between endoplasmic reticulum and mitochondria, documenting the existence of at least two types of contact sites in human cells. Narrow and wide ER\u2013mitochondria contact sites responded differently to starvation, ER stress, mitochondrial shape modifications, and changes in the levels of modulators of ER\u2013mitochondria juxtaposition. SPLICS detected contact sites in soma and axons of D. rerio Rohon Beard (RB) sensory neurons in vivo, extending its use to analyses of organelle juxtaposition in the whole anim

L'apprendimento della competenza procedurale in simulazione negli studenti del corso di laurea in infermieristica. Confronto tra due diverse modalità

Problema Nell’ambito della formazione degli infermieri, l’acquisizione di competenze clinico/procedurali è sempre complessa, sia nel procedimento di acquisizione, sia nell’effettuazione in contesto reale. Questa complessità è data da due componenti fondamentali: 1. I diversi fabbisogni di professionalità necessari a sviluppare e mantenere competenze procedurali; 2. La necessaria gradualità finalizzata ad una acquisizione che tuteli correttezza e sicurezza di tutti gli attori. 1. Le competenze procedurali da erogare al paziente comprendono delle conoscenze preliminari (fornite in aula), delle abilità manuali (da acquisire in laboratorio e da affinare nella pratica e nel tempo) e un atteggiamento adeguato verso il paziente (di cui acquisire le basi teoriche e da contestualizzare nella pratica clinica). 2. Le fasi di acquisizione dell’attività procedurale comprendono: · Lo studio delle fasi e delle relative motivazioni; · L’osservazione iniziale di una dimostrazione; · Una prima ripetizione dei singoli passaggi procedurali con prova in ambiente protetto su manichino con correzioni da parte degli esercitatori; · Più ripetizioni sempre in ambiente protetto; · La valutazione in ambiente protetto; · L’applicazione graduale e guidata della procedura in contesto reale. Obiettivo La ricerca si propone di valutare il metodo di insegnamento della peer education all’interno del corso di Laurea in Infermieristica. A questo fine si è fatto un confronto generale tra due anni accademici, il 2013/2014 e l’anno successivo, durante i quali la stessa procedura infermieristica è stata trasmessa agli studenti discenti da due diverse categorie di insegnanti. Ci si è proposti quindi di valutare la riuscita o meno dell’insegnamento della procedura nell’anno accademico 2014/2015, in cui si è voluta dimostrare la validità del metodo della peer education, generalmente utilizzato per studenti adolescenti frequentanti le scuole superiori, all’interno della struttura dell’università

A Fusarium graminearum PG is required for virulence during wheat infection.

Fusarium graminearum is a relevant pathogen of monocot species and is the main causal agent of Fusarium Head Blight (FHB), a devastating disease which commonly affects cereals. Besides, this fungus produces in the infected grains significant levels of mycotoxins, such as the trichotecenes deoxinivalenol (DON). F. graminearum penetrates in the host tissue during anthesis and once inside the tissue is able to spread systemically through the vascular vessels. During penetration and colonization of wheat spike tissues, the fungus secrete several cell wall degrading enzymes (CWDE). In particular, endo-polygalacturonases (PGs) have been shown to be early secreted in wheat plants, but, their role during the infection process has not been ascertained yet. Two pg encoding genes have been previously identified in the F. graminearum genome database and the isoforms encoded by these two genes (named PG1 and PG2) have been characterized both in vitro and in vivo. Aim of the present work was to clarify the importance of these F. graminearum PGs during infection of host wheat plants through the knock-out of their corresponding encoding genes, obtained by targeted homologous recombination. Two ∆PG1 mutants and two ∆PG2 mutants strains (producing only PG2 and PG1, respectively) were obtained and characterised both in vitro and during plant infection. The PG activity produced in liquid culture by the ΔPG1 mutants was negligible compared to that produced by WT and ΔPG2 mutants. However, no difference in dry weight was obtained when growing WT and mutant strains in the presence of pectin as the sole carbon source. Infection experiments of wheat host plants were also performed. We observed that all the knock-out mutants tested maintained the capacity to infect the plant, but the ΔPG1 strains showed a significant reduction of virulence (about 75% less infected spikelets) compared to WT strain, while no reduction of virulence was observed with the ΔPG2 mutants. We also quantified the amount of DON mycotoxin produced by the fungus during wheat infection: the ∆PG1 strains produced from 8 to 15 times less DON mycotoxin compared to WT and ΔPG2 strains, probably due to their reduced colonization of the plant tissue. F.graminearum WT and a ∆PG1 mutant strain were also transformed to constitutively express the green fluorescent protein (GFP). The obtained mutants were used to perform a comparative study of the early events of wheat colonization. Compared to WT, the ΔPG1 mutant colonized more slowly the ovaries, tissues rich of pectin,, and also the colonization of the vascular vessels of the rachis by this mutant was strongly delayed compared to WT. In particular, the retarded colonization of the ovary by the ΔPG1 mutant might be due to the reduced production of PG activity. The fungal growth in the infected wheat tissue could result therefore slowed down allowing the plant to initiate its defence reactions. Taken together, these results seems to indicate that PG1 play an important role during pathogenesis and therefore can be considered a virulence factor.F. graminearum è l’agente causale della fusariosi della spiga, una malattia descritta per la prima volta in Inghilterra nel 1884 ed ora una delle più studiate in quanto colpisce le tre principale risorse di cibo mondiale frumento, mais e riso. Questo fungo può causare la distruzione del raccolto in poche settimane determinando ingenti perdite economiche. In aggiunta, il prodotto ricavato dalle piante infette presenta elevati livelli di micotossine, in particolare tricoteceni quali il deossinivalenolo (DON). Questa tossina è un potente inibitore delle sintesi proteica negli eucarioti e risulta dannoso per la salute umana e animale. F. graminearum penetra nell’ospite durante la fase di antesi attraverso l’ovario e colonizza la superficie interna di lemma e palea. Una volta penetrato esso è in grado di diffondere nella spiga in modo sistemico. Durante la penetrazione e la colonizzazione dei tessuti della spiga, il fungo produce e rilascia numerosi enzimi degradativi della parete cellulare; tra questi, le endo-poligalacturonasi (PGs) sono tra i primi enzimi secreti ma il loro ruolo durante l’infezione non è stato ancora chiarito. Nel database genomico di F. graminearum sono stati identificati due putativi geni codificanti pg. Le isoforme codificate da questi geni (chiamate PG1 and PG2) sono state caratterizzate in vitro e in vivo (Tomassini et al., 2009). L’obiettivo del presente lavoro di tesi è chiarire l’importanza di queste PG prodotte durante l’infezione della pianta ospite attraverso l’ottenimento di mutanti knock-out ottenuti attraverso ricombinazione omologa target del gene target. Due mutanti ∆PG1 e due mutanti ∆PG2 (producenti rispettivamente solo PG2 e PG1) sono stati ottenuti e caratterizzati in vitro e durante l’infezione della pianta ospite. In coltura liquida il mutante ΔPG1 è risultato produrre scarsa attività PG rispetto al fungo WT e al mutante ΔPG2. Tuttavia i mutanti analizzati non hanno mostrato differenze in peso secco dopo crescita in terreno liquido contenente pectina come unica fonte di carbonio. I mutanti sono stati stati in seguito saggiati in esperimenti di infezione di piante di frumento. Si è osservato che tutti i mutanti testati mantenevano la loro capacità di infettare la spiga. Tuttvia, il mutante ΔPG1 presentava una ridotta virulenza (circa 75% in meno di spighe infettate) rispetto al WT mentre il mutante ΔPG2 non mostrava alcuna riduzione di virulenza. E’ stata inoltre quantificata la micotossina DON prodotta dai mutanti durante l’infezione della pianta ospite: il mutante ∆PG1 produceva da 8 a 15 volte meno DON rispetto a WT e mutante ΔPG2, probabilmente a causa della ridotta colonizzazione della spighetta da parte di questo mutante. Il ceppo WT e un mutante ∆PG1 sono stati inoltre trasformati in modo da esprimere costitutivamente la green fluorescent protein (GFP). I mutanti così ottenuti sono stati utilizzati per studi istologici delle prime fasi di infezione. Rispetto al WT il mutante ΔPG1 era in grado di colonizzare l’ovario, un tessuto ricco in pectina, molto più lentamente; anche la colonizzazione da parte del mutante del tessuto conduttore nel rachide risultava più lenta. Questo rallentamento nella crescita del fungo nella spiga potrebbe essere dovuto alla ridotta attività PG da parte del mutante ΔPG1 e potrebbe permettere alla pianta di attivare più efficacemente le risposte di difesa. Nel complesso i risultati ottenuti nel presente lavoro sembrano indicare che la PG1 di F. graminearum svolge un importante ruolo durante la patogenesi e può quindi essere considerato un fattore di virulenza

Dal singolo edificio all'analisi della trama urbana: ricerche sull'edilizia medievale di Padova e del suo territorio

Obbiettivi, metodologia e comunicazione del progetto Armep sulle architetture residenciali di Padov

A Fusarium graminearum polygalacturonase gene is required for full virulence during wheat infection

Fusarium graminearum is the main causal agent of Fusarium head blight (FHB) in cereals. In wheat, FHB causes remarkable yield and quality losses because of mycotoxins accumulation in the infected kernels. F. graminearum is known to produce two endo-polygalacturonases (PGs) in liquid culture and during infection of wheat plants. The role played by these PGs during the infection process has not been ascertained yet and it has been often neglected because graminaceous plant tissues have a cell wall consisting mainly of cellulose and xylan. In order to establish the role of these PGs in pathogenesis, we have disrupted by targeted homologous recombination the pg encoding genes of this fungus. When grown in liquid culture containing pectin as the sole carbon source, the PG activity produced by the \u394PG1 mutant resulted negligible compared to that produced by wild-type and \u394PG2 mutant strains; however, the dry weight of wild-type and of both mutant strains was comparable. The virulence of each mutant has been evaluated by infecting wheat plants: results indicate that both pg knock-out mutants maintain the capability to infect wheat, although the \u394PG1 mutant shows a significant reduction of virulence compared to the wild-type strain (about 50% less infected spikelets), while no reduction of virulence is observed with the \u394PG2 mutant. Therefore, PG1 can be considered a virulence factor of F. graminearum during spike infection