In vitro recapitulation of the site-specific editing (to wild-type) of mutant IDS mRNA transcripts, and the characterization of IDS protein translated from the edited mRNAs

The transfer of genomic information into the primary RNA sequence can be altered by RNA editing. We have previously shown that genomic variants can be RNA-edited to wild-type. The presence of distinct “edited” iduronate 2-sulfatase (IDS) mRNA transcripts ex vivo evidenced the correction of a nonsense and frameshift variant, respectively, in three unrelated Hunter syndrome patients. This phenomenon was confirmed in various patient samples by a variety of techniques, and was quantified by single-nucleotide primer extension. Western blotting also confirmed the presence of IDS protein similar in size to the wild-type. Since preliminary experimental evidence suggested that the “corrected” IDS proteins produced by the patients were similar in molecular weight and net charge to their wild-type counterparts, an in vitro system employing different cell types was established to recapitulate the site-specific editing of IDS RNA (uridine to cytidine conversion and uridine deletion), and to confirm the findings previously observed ex vivo in the three patients. In addition, confocal microscopy and flow cytometry analyses demonstrated the expression and lysosomal localization in HEK293 cells of GFP-labeled proteins translated from edited IDS mRNAs. Confocal high-content analysis of the two patients’ cells expressing wild-type or mutated IDS confirmed lysosomal localization and showed no accumulation in the Golgi or early endosomes

Fingerprints of brain disease:connectome identifiability in Alzheimer's disease

Functional connectivity patterns in the human brain, like the friction ridges of a fingerprint, can uniquely identify individuals. Does this "brain fingerprint" remain distinct even during Alzheimer's disease (AD)? Using fMRI data from healthy and pathologically ageing subjects, we find that individual functional connectivity profiles remain unique and highly heterogeneous during mild cognitive impairment and AD. However, the patterns that make individuals identifiable change with disease progression, revealing a reconfiguration of the brain fingerprint. Notably, connectivity shifts towards functional system connections in AD and lower-order cognitive functions in early disease stages. These findings emphasize the importance of focusing on individual variability rather than group differences in AD studies. Individual functional connectomes could be instrumental in creating personalized models of AD progression, predicting disease course, and optimizing treatments, paving the way for personalized medicine in AD management.</p

Prognostic implications of functional mitral regurgitation according to the severity of the underlying chronic heart failure: A long-term outcome study

AimsTo examine the independent prognostic role of functional mitral regurgitation (FMR) and its impact across the severity of chronic heart failure (CHF) in a large population of outpatients with systolic CHF followed at two multidisciplinary clinics.Methods and resultsEchocardiography was performed upon enrolment in 469 CHF patients. Follow-up for death and heart transplant was updated on January 2007. Five-year transplant-free survival was 82.7 in patients with no or Grade I FMR, 64.4 in Grade II, 58.5 in Grade III, and 46.5 in Grade IV (P < 0.0001). There was a strong graded association between FMR and the long-term risk of death and heart transplant, which remained significant after multivariable adjustment (P = 0.0003). The association between FMR and events was strong and independent in patients with less severe symptoms and in those at lower overall risk based on a propensity score analysis, while it was not significant in patients with more advanced CHF or in the high-risk subgroup (P < 0.0001 for interactions).ConclusionThis study clarifies previous apparently discrepant results by demonstrating that FMR is an independent determinant of death and heart transplantation only in less severe CHF and in patients with a lower risk profile. This finding indicates that FMR plays a major role in the early phase of CHF, suggesting that this should be the focus of strategies attempting to reduce it. \uc2\ua9 The Author 2010

Upper limb function in Duchenne muscular dystrophy: 24 month longitudinal data

The aim of the study was to establish 24 month changes in upper limb function using a revised version of the performance of upper limb test (PUL 2.0) in a large cohort of ambulant and non-ambulant boys with Duchenne muscular dystrophy and to identify possible trajectories of progression. Of the 187 patients studied, 87 were ambulant (age range: 7\u201315.8 years), and 90 non-ambulant (age range: 9.08\u201324.78). The total scores changed significantly over time (p&lt;0.001). Non-ambulant patients had lower total scores at baseline (mean 19.7) when compared to the ambulant ones (mean 38.4). They also had also a bigger decrease in total scores over 24 months compared to the ambulant boys (4.36 vs 2.07 points). Multivariate model analysis showed that the Performance of Upper Limb changes reflected the entry level and ambulation status, that were independently associated to the slope of Performance of Upper Limb changes. This information will be of help both in clinical practice and at the time of designing clinical trials

MORFEO enters final design phase

MORFEO (Multi-conjugate adaptive Optics Relay For ELT Observations, formerly MAORY), the MCAO system for the ELT, will provide diffraction-limited optical quality to the large field camera MICADO. MORFEO has officially passed the Preliminary Design Review and it is entering the final design phase. We present the current status of the project, with a focus on the adaptive optics system aspects and expected milestones during the next project phase

Genotype-Phenotype Correlation and Functional Insights for Two Monoallelic TREX1 Missense Variants Affecting the Catalytic Core

The TREX1 exonuclease degrades DNA to prevent aberrant nucleic-acid sensing through the cGAS-STING pathway, and dominant Aicardi&ndash;Gouti&egrave;res Syndrome type 1 (AGS1) represents one of numerous TREX1-related autoimmune diseases. Monoallelic TREX1 mutations were identified in patients showing early-onset cerebrovascular disease, ascribable to small vessel disease, and CADASIL-like neuroimaging. We report the clinical-neuroradiological features of two patients with AGS-like (Patient A) and CADASIL-like (Patient B) phenotypes carrying the heterozygous p.A136V and p.R174G TREX1 variants, respectively. Genetic findings, obtained by a customized panel including 183 genes associated with monogenic stroke, were combined with interferon signature testing and biochemical assays to determine the mutations&rsquo; effects in vitro. Our results for the p.A136V variant are inconsistent with prior biochemistry-pathology correlates for dominant AGS-causing TREX1 mutants. The p.R174G variant modestly altered exonuclease activity in a manner consistent with perturbation of substrate interaction rather than catalysis, which represents the first robust enzymological data for a TREX1 variant identified in a CADASIL-like patient. In conclusion, functional analysis allowed us to interpret the impact of TREX1 variants on patients&rsquo; phenotypes. While the p.A136V variant is unlikely to be causative for AGS in Patient A, Patient B&rsquo;s phenotype is potentially related to the p.R174G variant. Therefore, further functional investigations of TREX1 variants found in CADASIL-like patients are warranted to determine any causal link and interrogate the molecular disease mechanism(s)

Mendelian Genetics of Stroke: an assessment of the broad genetic and phenotypic heterogeneity driven by the identification of underlying pathogenic mechanisms

Being the second cause of death and disability world-wide, approximately 1.1 million inhabitants of Europe suffer a stroke each year, the ischemic stroke accounting for approximately 80% of cases. Stroke presents different prevalence for the diverse range of age: perinatal stroke (1 per 2700), pediatric stroke (1-2 in 100000), juvenile stroke (1 per 10000), adult stroke (95-290 in 100000). Evidence for a genetic aetiology of stroke has largely been described in the literature. Juvenile and pediatric stroke, more seldom neonatal and adult stroke, is thought to be mainly driven by different underlying genetic mechanisms: several mendelian disorders are known to be the cause of monogenic form of stroke. Next Generation Sequencing (NGS) panels represent an excellent option for diagnostic routine. Genotyping of stroke patients is fundamental for focused clinical follow-up and family counselling. Albeit NGS-panels represent an excellent option for diagnostic routine, the mutation detection rate turns out to be 4810%, even for patients affected with a likely monogenic stroke. Whole exome sequencing (WES) embodies straightforward approach in this context, allowing the identification of novel candidate genes. The present project has focused on: i) the identification of the underlying genetic causes of monogenic stroke cases, ii) the improvement of our knowledge about clinically overlapping phenotypes through the genetic characterization of the corresponding patients, iii) the optimization of the diagnostic work-up in order to administer disease specific follow-up and treatments to patients. A custom NGS-panel including 183 genes associated with monogenic forms of stroke was designed according to clinicians and neuroradiologists belonging to the multidisciplinary team of Gaslini Stroke Center. Patients affected by suspected monogenic stroke were selected by different Unit of Gaslini Institute and other Italian children/adult hospitals collaborating with the Gaslini Stroke Center. DNA samples of 102 individuals were extracted and analyzed via the mentioned multigenic panel. Specific functional assays were set up and optimized in order to dissect molecular disease mechanisms in selected subgroup or single patients. Moreover, WES has been applied in specific trios after a negative result of the panel. Up to 16 of 102 harbored pathogenic variants justifying or potentially justifying their clinical and neuroradiological phenotypes. The overall mutational rate is around 16.5%. Specific functional assays opened the way to understand molecular disease mechanism underlying different type of monogenic stroke. As the number of genes involved in the stroke pathogenesis is rapidly growing, a WES approach coupled with an in-silico gene panel has more recently been applied. This not only allow to test a constantly updated gene list but also to look at the rest of the genes of the genome in case the in-silico panel resulted negative. In conclusion, the project of the thesis allowed to identify causative mutation in some of the patients suspected to have a monogenic stroke and to dissect potential molecular mechanisms underlying their stroke subtype. This let the establishment of a more specific follow-up and counselling for families feasible

Risposta vegeto-produttiva di Nerium oleander L. alla concimazione con formulati a lenta cessione e a cessione controllata

L'utilizzo dei concimi a lento effetto è ormai molto diffuso in agricoltura. Il vantaggio di questi formulati, rispetto a quelli tradizionali, è dato dalla loro capacità di alimentare continuamente la pianta, grazie alla particolare struttura dei granuli, e di limitare le perdite di azoto nel substrato di coltura. La solubilizzazione dei concimi a lento rilascio o la liberazione dei principi attivi dei formulati a cessione controllata sono strettamente correlate all'attività microbica ed alle temperature del mezzo di coltura, per cui le piante possono reperire dal substrato quantitativi diversi in funzione delle condizioni ambientali. La ricerca è stata condotta al fine di valutare la risposta di Nerium oleander L., largamente diffuso in ambiente mediterraneo e molto apprezzato come specie da parchi e giardini, alla concimazione con Ornamental (18:6:12; 18 S; 1 Fe) e Osmocote (18:9:10; 2 MgO), concimi rispettivamente a lento effetto e a cessione controllata, somministrati in copertura o incorporati nel substrato in fase di pre-impianto. Lo schema sperimentale adottato è stato la parcella suddivisa. L'impianto è stato realizzato nel febbraio 2006, le piante sono state collocate in vasi del diametro di 18 cm con capacità di 4 l. Il substrato è stato preparato miscelando torba bionda (33%) e bruna (33%) con agriperlite (34%). Ad ogni pianta sono stati somministrati 2,5 kg di concime ogni m3 di substrato (3,75 g pianta-1), indipendentemente dal formulato e dalla modalità di apporto. L’approvvigionamento idrico è stato garantito tramite un impianto di erogazione a microportata con ali gocciolanti dotate di fori posti a 20 cm l’uno dall’altro (uno per pianta). Nel mese di settembre le piante sono state potate in modo da lasciare 3 gemme pianta-1. L'oleandro ha risposto bene all'impiego di entrambi i formulati e le modalità di somministrazione. Le piante hanno fatto osservare un ritmo di accrescimento analogo per tutte le tesi messe a confronto. Il numero di ramificazioni differenziate da ogni pianta non è stato influenzato dai trattamenti nel periodo primaverile-estivo, successivamente, a seguito della potatura a 3 gemme, si sono registrate lievi differenze a favore delle piante in cui il fertilizzante veniva incorporato al substrato. Più significative sono risultate le differenze relative all’interazione dei fattori esaminati, a favore della tesi che prevedeva l’uso del fertilizzante a cessione controllata, incorporato prima dell’impianto. La fioritura è cominciata contemporaneamente in tutte le piante. Le infiorescenze hanno differenziato un maggior numero di fiori nel mese di luglio mentre ad agosto, probabilmente a causa delle alte temperature, la produzione si è ridotta; anche in questo caso non sono state registrate delle differenze statisticamente significative sugli effetti semplici dovuti ai diversi trattamenti. La ricerca ha messo in evidenza la possibilità di adottare, per questa specie, concimi a lento effetto apportandoli in fase di preparazione del substrato o anche successivamente in copertura. La fertilizzazione con questi formulati si è dimostrata capace di provvedere correttamente alla nutrizione minerale delle piante, consentendo al contempo notevoli risparmi di manodopera e per gli impianti di preparazione e distribuzione delle soluzioni nutritive