VALUTAZIONE DI ALCUNI BIOMARKERS DI STRESS OSSIDATIVO E DELLA FREQUENZA DEGLI APLOGRUPPI MITOCONDRIALI IN UNA POPOLAZIONE DI PAZIENTI CON MALATTIA DI ANDERSON-FABRY.

La malattia di Fabry comprende una estrema varietà fenotipica in relazione con il grande numero di organi e sistemi coinvolti. La diagnosi di malattia di Fabry è complessa a causa del numero di organi/sistemi coinvolti e dei fenotipi clinici non specifici e della sua rarità. La progressione clinica della malattia si manifesta tra i 30-40 anni quando per i numerosi organi coinvolti compaiono manifestazioni cliniche quali insufficienza cardiaca, renale ed eventi cerebrovascolari. La morte di solito sopraggiunge durante la quarta/quinta decade di vita ed è secondaria all’interessamento cardiaco, renale o cerebrale anche se l’avvento della dialisi e della terapia enzimatica sostitutiva stanno sensibilmente prolungando la vita media dei pazienti. Seppur conosciuto in dettaglio il gene che causa la malattia e la sua funzione, ad oggi appare ancora poco chiaro il perché la malattia presenti un’ampia eterogeneità di presentazione clinica, anche all’interno della stessa famiglia. I fattori che potrebbero giocare un ruolo nella modulazione fenotipica della malattia di Fabry sono molteplici e non ancora studiati (fattori ambientali, fattori epigenetici, altri geni modulatori).mI mitocondri rappresentano “la centralina energetica della cellula” [202). Sono organelli intracellulari che svolgono diverse funzioni, la più importante delle quali è rappresentata dalla produzione di energia sotto forma di ATP mediante la fosforilazione ossidativa. Il mitocondrio è l’unico organellodelle cellule animali dotato di un proprio genoma, il DNA mitocondriale (mtDNA). In ogni mitocondrio si trovano da 2 a 10 copie di mtDNA, ed in ogni cellula più di 1000 copie. Il mtDNA è una molecola circolare di DNA a doppio filamento, costituito da 16569 paia basi, che codifica 37 geni: 13 codificano peptidi della catena respiratoria mitocondriale, 2 RNA ribosomali e 22 RNA transfer (tRNA). Tutte le altre proteine mitocondriali (comprese le 67 subunità che concorrono a formare i cinque complessi della catena respiratoria) sono codificate dal DNA nucleare. L’mtDNA umano è caratterizzato da un elevato tasso evolutivo che è 10 – 20 volte quello dei geni del nucleo; la sua variazione di sequenza si è perciò generata lungo linee di radiazione materna esclusivamente per l’accumulo sequenziale di nuove mutazioni. Nel tempo questo processo di divergenza molecolare ha dato origine ad entità monofiletiche dette aplogruppi. Un valido strumento per determinare se eventuali polimorfismi mitocondriali possano agire come fattori di suscettibilità o di protezione nell’insorgenza di malattie neurodegenerative è stato fornito proprio dall’analisi degli aplogruppi mitocondriali, definiti come clusters di genomi mitocondriali continente-specifici correlati all’evoluzione e definiti in base alla presenza di ancestrali e stabili polimorfismi.. La presenza di stabili siti polimorfici all’interno della regione codificante definisce l’aplogruppo, mentre la maggior parte delle mutazioni che si osservano sia nelle sequenze codificanti che nella regione di controllo e che si realizzano all’interno di preesistenti aplogruppi definiscono il tipo individuale di mtDNA, o aplotipo [172]. È stato ipotizzato che variazioni all’interno del mtDNA possano causare sottili differenze nelle proteine codificate ed indurre una modificazione, seppur minima, nelle attività della fosforilazione ossidativa e nella produzione di specie reattive dell’ossigeno, o viceversa agire come fattori protettivi, con effetto benefico sulla catena di trasporto di elettroni e/o per la produzione di radicali liberi. In questi ultimi anni la ricerca è stata finalizzata alla comprensione di un eventuale ruolo degli aplogruppi nella modulazione dell’espressione dei geni mitocondriali durante l’evoluzione e i processi di adattamento del genere umano; si è iniziato inoltre ad indagare se la variabilità del genoma mitocondriale potesse svolgere un’azione protettiva o, viceversa, agire come fattore di rischio nell’insorgenza di malattie neurodegenerative come il morbo di Parkinson (192), la malattia di Friedreich (203), la Sclerosi Laterale Amiotrofica [196]), la malattia di Alzheimer e Corea di Huntington [172]. Benchè numerose mutazioni del gene GLA siano state associate alle forme classiche e tardive, una definitiva correlazione genotipo-fenotipo none stata dimostrata e la diagnosi e classificazione della malattia on possono essere effettuate solo su base genetica. Il deposito dei substrati è correlato al danno tissutale nella malattia di Fabry; tuttavia, i meccanismi molecolari sottostanti restano non completamente conosciuti. I pazienti affetti da malattia di Fabry presentano un importante danno 44 ossidativo proteico e lipidico, ridotte difese antiossidanti e aumentali livelli di citochine e biomarcatori infiammatori [204–205]. L’eccesso di GB3 intracellulare induce stress ossidativo e un up-regulation dell’espressione delle molecole di adesione cellulare nelle cellule endoteliali vascolari [206]. Inoltre è stato ipotizzato che si verifica uno stato proossidante che è correlato e sembra essere indotto dal GB3 nei pazienti con malattia di Fabry [207]Lo scopo del nostro progetto multicentrico è stato quello di valutare il ruolo dello stress ossidativo nella malattia di Fabry. In particolare abbiamo valutato (I) se vi sono segni di stress ossidativo nel sangue; (II) se esiste un’associazione tra biomarcatori di stress ossidativo e manifestazion i cliniche della malattia e/o una differenza tra forma classica e varianti late onset; (III) se i parametri dello stress ossidativo nel tempo si correlano con il Lyso-Gb3 e l’insorgenza e progressione della malattia, in un sottogruppo di 8 soggetti/paziente naive al trattamento con normali livelli di Lyso-Gb3, al fine di valutare se i biomarkers di stress ossidativo possano presentare marcatori precoci della malattia Questo progetto si proponeva inoltre di valutare la frequenza degli aplogruppi mitocondriali, un loro possibile ruolo di suscettibilità genetica nel determinismo della malattia di Fabry, e se essi potessero influenzare lo stato di stress ossidativo plasmatico in tali pazienti; tuttavia l’analisi dei dati, relativi a questo secondo obiettivo , sul gruppo dei pazienti arruolati non ha permesso di ottenere risultati rilevanti, chiari e definitivi pertanto il nostro gruppo si propone di proseguire un ulteriore approfondimento in questo campo di ricerca scientifica, raccogliere più dati estendendo l’analisi su un maggior numero di pazienti e quindi coinvolgendo più centri.Fabry disease comprises a wide phenotypic variability in relation to the large number of organs and systems involved. The diagnosis of Fabry disease is complex due to the number of organs/systems involved, to the non-specific clinical manifestationa and to its rarity. Clinical progression of the disease occurs between 30-40 years when clinical manifestations because of the involvement of important organs such as heart failure, renal failure and cerebrovascular events. Death usually occurs during the fourth/fifth decade of life and it is secondary to cardiac, renal or cerebral involvement even if the advent of dialysis and enzyme replacement therapy are significantly prolonging the average life of patients. Although the gene that causes the disease and its function is known in detail, it still appears today unclear the wide heterogeneity of clinical presentation that characterizes this disorder, within the same family, too. Factors that might play a role in phenotypic modulation of Fabry disease are multiple and not yet studied (environmental factors, epigenetic factors, other modulatory genes).Mitochondria represent "the energy central unit of the cell" [202].They are intracellular organelles that perform different functions, the most important of which is represented by the production of energy in the form of ATP through oxidative phosphorylation. The mitochondrion is the only organelle with its own genome, mitochondrial DNA (mtDNA). In each mitochondrion there are from 2 to 10 copies of mtDNA, and in each cell more than 1000 copies. The mtDNA is a circular molecule of double-stranded DNA, consisting of 16569 base pairs, encoding 37 genes: 13 encode mitochondrial respiratory chain peptides, 2 ribosomal RNAs and 22 RNAs transfer (tRNA). All other mitochondrial proteins (including the 67 subunits that contribute to form the five complexes of the respiratory chain) are encoded by nuclear DNA. Human mtDNA is characterized by a high evolutionary rate that is 10 – 20 times that of genes of the nucleus; its sequence variation was therefore generated along lines of maternal radiation exclusively for the sequential accumulation of new mutations. Over time this process of molecular divergence has given rise to monophyletic entities called haplogroups. A valid tool to determine if any mitochondrial polymorphisms can act as susceptibility or protective factors in the onset of neurodegenerative diseases was provided precisely from the analysis of mitochondrial haplogroups, defined as clusters of mitochondrial genomes continent-specific related to evolution and defined by the presence of ancestral and stable polymorphisms. The presence of stable polymorphic sites within the coding region defines the haplogroup, on the other hand most of the mutations that are observed are both in the coding sequences and in the control region and that occur within pre-existing haplogroups define the individual mtDNA type, or haplotype [172]. It has been hypothesized that variations within the mtDNA can cause subtle differences in the encoded proteins and induce a modification, albeit minimal, in the activities of oxidative phosphorylation and in the production of reactive species of oxygen, or they act as protective factors, with a beneficial effect on the transport chain of electrons and/or for the production of free radicals. In recent years, research has been aimed at understanding a possible role of haplogroups in the modulation of mitochondrial gene expression during evolution and in the human processes of adaptation ; it has also begun to investigate whether the variability of the genome mitochondrial could perform a protective action or, conversely, it acts as a risk factor in the onset of neurodegenerative diseases such as Parkinson's disease (192), the disease of Friedreich (203), Amyotrophic Lateral Sclerosis [196]), Alzheimer's disease and Huntington [172]. Although numerous mutations of the GLA gene have been associated with the classic and late forms, one definitive genotype-phenotype correlation has not been demonstrated and the diagnosis and classification of disease can only be made on a genetic basis. Substrate deposition is related to tissue damage in Fabry disease; however, the underlying molecular mechanisms remain unclear fully known. Patients with Fabry disease have an important impairment of protein and lipid oxidative processes , reduced antioxidant defenses and increased levels of inflammatory cytokines and biomarkers [204–205]. the high level intracellular GB3 induces oxidative stress and up-regulation of the expression of cell adhesion molecules in vascular endothelial cells [206]. Furthermore it has been hypothesized that a pro-oxidant state occurs which is related and appears to be induced by GB3 in patients with Fabry disease [207]The aim of our multicenter project was to evaluate the role of oxidative stress in Fabry disease. In particular we evaluated (I) whether there are signs of oxidative stress in the blood; (II) whether there is an association between biomarkers of oxidative stress and clinical manifestations of the disease and/or a difference between the classic form and late onset variants; (III) whether the stress parameters over time correlate with Lyso-Gb3 and disease onset and progression, in a subset of 8 treatment-naive subjects/patients with normal Lyso-Gb3 levels, in order to evaluate whether biomarkers of oxidative stress may present early markers of the disease This project also aimed to evaluate the frequency of mitochondrial haplogroups, their possible role of genetic susceptibility in the determinism of Fabry disease, and if they could influence the state of plasma oxidative stress in such patients; however, the analysis of the data, concerning this second aim, on the group of enrolled patients did not allow to obtain relevant , clear and definitive results therefore our group proposes to pursue a further deepening in this field of scientific research, to collect more data by extending the analysis on a greater number of patients and therefore involving more centres

Radiographic and Venographic Appearance of Healthy and Laminitic Feet in Amiata Donkeys

Introduction: Laminitis is a debilitating disorder resulting in irreversible anatomical changes in the feet of equids. Assessing specific anatomical features through radiography and venography provides diagnostic and prognostic information. The reference ranges are well-established in horses, but not in donkeys. It is also uncertain as to whether these ranges can be applied to every donkey breed. The present study characterizes the radiological and venographic hoof anatomy of healthy feet of Amiata donkeys and defines the changes associated with severe and mild laminitis. Materials and Methods: A total of 16 forefeet were evaluated in 8 Amiata jennies. The animals underwent musculoskeletal examination, Obel grading assessment and radiological evaluation. Based on clinical examination and radiographic findings, the forefeet were grouped as healthy, mild or severe laminitic feet, thus the digital venograms were performed according to the group definition. Results: Radiology revealed 7/16 healthy, 4/16 mild laminitic, and 5/16 severe laminitic forefeet. Statistical analysis showed differences between the healthy and laminitic forefeet for the dorsal angle (p <0.0001) and angle of solar aspect (p <0.0001) of the distal phalanx, for deviation between dorsal aspect of distal phalanx and the hoof wall (p <0.0001) and phalangeal rotation angle (p = 0.0032). Venography was abnormal in mild and severe laminitic forefeet. In particular, the vascularization was reduced or absent at the lamellar-circumflex junction dorsally, at the sub-lamellar vascular bed and at the circumflex veins. Coronary plexus vascularization was absent in severe laminitic forefeet. Discussion and Conclusions: This study provides the radiological parameters for the assessment of healthy and laminitic forefeet of Amiata donkeys. The mild laminitic foot venogram showed decreased vascularization mainly on lamellar-circumflex junction and sub-lamellar vascular bed, in latero-medial views. The severe laminitic foot showed very poor or absent vascularization in multiple areas. The technique is easily applicable and provides diagnostic support in laminitis.Peer reviewe

Italian in the Trenches: Linguistic Annotation and Analysis of Texts of the Great War

The paper illustrates the design and development of a textual corpus representative of the historical variants of Italian during the Great War, which was enriched with linguistic (lemmatization and pos-tagging) and meta-linguistic annotation. The corpus, after a manual revision of the linguistic annotation, was used for specializing existing NLP tools to process historical texts with promising results.L’articolo illustra la progettazione e la costruzione di un corpus rappresentativo delle varietà di italiano in uso durante la prima Guerra Mondiale, annotato con dati linguistici (lemmatizzazione, analisi morfo-sintattica) e meta-linguistici. Il corpus, a seguito della revisione manuale dell’annotazione linguistica, è stato utilizzato per l’adattamento degli strumenti NLP esistenti, con risultati promettenti

Brisk walking can be a maximal effort in heart failure patients: a comparison of cardiopulmonary exercise and 6 min walking test cardiorespiratory data

Aims Cardiopulmonary exercise test (CPET) and 6 min walking test (6MWT) are frequently used in heart failure (HF). CPET is a maximal exercise, whereas 6MWT is a self-selected constant load test usually considered a submaximal, and therefore safer, exercise, but this has not been tested previously. The aim of this study was to compare the cardiorespiratory parameters collected during CPET and 6MWT in a large group of healthy subjects and patients with HF of different severity.Methods and results Subjects performed a standard maximal CPET and a 6MWT wearing a portable device allowing breath-by-breath measurement of cardiorespiratory parameters. HF patients were grouped according to their CPET peak oxygen uptake (peak(V) over-dotO(2)). One hundred and fifty-five subjects were enrolled, of whom 40 were healthy (59 +/- 8 years; male 67%) and 115 were HF patients (69 +/- 10 years; male 80%; left ventricular ejection fraction 34.6 +/- 12.0%). CPET peak(V) over-dotO(2) was 13.5 +/- 3.5 ml/kg/min in HF patients and 28.1 +/- 7.4 mL/kg/min in healthy subjects (P &lt; 0.001). 6MWT-(V) over-dotO(2) was 98 +/- 20% of the CPET peak(V) over-dotO(2) values in HF patients, while 72 +/- 20% in healthy subjects (P &lt; 0.001). 6MWT-(V) over-dot was &gt;110% of CPET peak(V) over-dotO(2) in 42% of more severe HF patients (peak(V) over-dotO(2) &lt; 12 mL/kg/min). Similar results have been found for ventilation and heart rate. Of note, the slope of the relationship between (V) over-dotO(2) at 6MWT, reported as a percentage of CPET peak(V) over-dotO(2) vs. 6MWT (V) over-dotO(2) reported as the absolute value, progressively increased as exercise limitation did.Conclusions In conclusion, the last minute of 6MWT must be perceived as a maximal or even supramaximal exercise activity in patients with more severe HF. Our findings should influence the safety procedures needed for the 6MWT in HF

Contribution of MUTYH variants to male breast cancer risk: results from a multicenter study in Italy

Inherited mutations in BRCA1, and, mainly, BRCA2 genes are associated with increased risk of male breast cancer (MBC). Mutations in PALB2 and CHEK2 genes may also increase MBC risk. Overall, these genes are functionally linked to DNA repair pathways, highlighting the central role of genome maintenance in MBC genetic predisposition. MUTYH is a DNA repair gene whose biallelic germline variants cause MUTYH-associated polyposis (MAP) syndrome. Monoallelic MUTYH variants have been reported in families with both colorectal and breast cancer and there is some evidence on increased breast cancer risk in women with monoallelic variants. In this study, we aimed to investigate whether MUTYH germline variants may contribute to MBC susceptibility. To this aim, we screened the entire coding region of MUTYH in 503 BRCA1/2 mutation negative MBC cases by multigene panel analysis. Moreover, we genotyped selected variants, including p.Tyr179Cys, p.Gly396Asp, p.Arg245His, p.Gly264Trpfs*7, and p.Gln338His, in a total of 560 MBC cases and 1,540 male controls. Biallelic MUTYH pathogenic variants (p.Tyr179Cys/p.Arg241Trp) were identified in one MBC patient with phenotypic manifestation of adenomatous polyposis. Monoallelic pathogenic variants were identified in 14 (2.5%) MBC patients, in particular, p.Tyr179Cys was detected in seven cases, p.Gly396Asp in five cases, p.Arg245His and p.Gly264Trpfs*7 in one case each. The majority of MBC cases with MUTYH pathogenic variants had family history of cancer including breast, colorectal, and gastric cancers. In the case-control study, an association between the variant p.Tyr179Cys and increased MBC risk emerged by multivariate analysis [odds ratio (OR) = 4.54; 95% confidence interval (CI): 1.17-17.58; p = 0.028]. Overall, our study suggests that MUTYH pathogenic variants may have a role in MBC and, in particular, the p.Tyr179Cys variant may be a low/moderate penetrance risk allele for MBC. Moreover, our results suggest that MBC may be part of the tumor spectrum associated with MAP syndrome, with implication in the clinical management of patients and their relatives. Large-scale collaborative studies are needed to validate these findings

Voices of the great war: A richly annotated corpus of Italian texts on the first world war

Voci della Grande Guerra (“Voices of the Great War”) is the first large corpus of Italian historical texts dating back to the period ofFirst World War. This corpus differs from other existing resources in several respects. First, from the linguistic point of view it givesaccount of the wide range of varieties in which Italian was articulated in that period, namely from a diastratic (educated vs. uneducatedwriters), diaphasic (low/informal vs. high/formal registers) and diatopic (regional varieties, dialects) points of view. From the historicalperspective, through a collection of texts belonging to different genres it represents different views on the war and the various styles ofnarrating war events and experiences. The final corpus is balanced along various dimensions, corresponding to the textual genre, thelanguage variety used, the author type and the typology of conveyed contents. The corpus is annotated with lemmas, part-of-speech,terminology, and named entities. Significant corpus samples representative of the different “voices” have also been enriched withmeta-linguistic and syntactic information. The layer of syntactic annotation forms the first nucleus of an Italian historical treebankcomplying with the Universal Dependencies standard. The paper illustrates the final resource, the methodology and tools used to buildit, and the Web Interface for navigating it