Comparative Analysis Of Residual Factor VIII Expression from Recurrent F8 Nonsense Mutations Indicates that Localization in the B- domain Favours Readthrough- mediated Protein Output

Background: Nonsense mutations, inserting premature termination codons (PTCs), might undergo, with low frequency (<0.01%), spontaneous suppression (readthrough) with production of full-length proteins upon amino acid insertion at the PTC. This process, dictated by nucleotide/protein sequence features, might have implications for hemophilia A (HA) patients. Aims: To investigate residual factor VIII (FVIII) expression through complementary studies in HA patients’ plasma and exploiting a sensitive in-vitro expression platform. Methods: Detection of plasma FVIII levels (ELISA, aPTT), and expression studies (HEK293 cells) with a highly-sensitive naturally-secreted luciferase (Gaussia, GL) fused to FVIII (FVIII-GL). Results: Plasma samples from HA patients affected by six nonsense mutations (p.R446X, p.R814X, p.K1289X, p.W1726X, p.R1985X, p.R2135X) revealed traces of FVIII. Strikingly, the two B-domain variants (p.R814X, p.K1289X) showed the highest FVIII levels, suggesting a position-dependent effect. Expression studies with the FVIII-GL variants showed that those of the B-domain produced the highest luciferase activity levels, thus supporting in vivo findings. Accordingly, the predicted readthrough-deriving amino acid changes (R446W, R814W, K1289Q/Y, W1726Y, R1985W, R2135W) showed a minor impact for those affecting the B-domain. To verify further our hypothesis, the panel of F8 mutations was rationally expanded to be representative of the majority of patients with nonsense mutations (60%), including the most frequent (50% of patients) in the B-domain. Through our sensitive platform we observed that all F8 nonsense variants led to detectable luciferase activity (0.4-6%). Strikingly, when categorized in two groups (B-domain, n=21; other domains, n=26), secreted luciferase activity of B-domain variants was significantly higher (p<0.0001) as compared with variants located in the other FVIII domains. Conclusions: Our findings for the first time indicate that nonsense mutations in the B-domain, known to tolerate missense changes as those potentially arising from readthrough, are favoured in terms of readthrough-mediated protein output, which might have pathophysiological implications for HA patients

Agronomic performance of 21 new disease resistant winegrape varieties grown in northeast Italy

The goal of the field trial was to evaluate the agronomic performance of 21 (10 red and 11 white) winegrape varieties obtained from recent breeding programmes for disease resistance developed in Hungary, Germany, and Italy. The tested red varieties were as follows: ‘Cabernet Carbon’, ‘Cabernet Eidos’, ‘Cabernet Volos’, ‘Julius’, ‘Merlot Khorus’, ‘Merlot Kanthus’, ‘Monarch’, ‘Prior’, UD. 31.103, ‘Vinera’. The tested white varieties were as follows: ‘Aromera’, ‘Bronner’, ‘Fleurtai’, ‘Johanniter’, ‘Muscaris’, ‘Souvignier Gris’, ‘Sauvignon Kretos’, ‘Sauvignon Nepis’, ‘Sauvignon Rytos’, ‘Solaris’, ‘Soreli’. ‘Merlot’ (red) and ‘Glera’ (white) were included as control. The experimental vineyard was established in Castelfranco Veneto on the plain, in 2014. Spray treatments were applied against downy and powdery mildew, by using only copper and sulphur. Grape production, grape quality, and phenology were recorded over a six-year-period, while disease resistance (downy mildew, powdery mildew, black rot and anthracnose) was detected only during a few years. The most significant findings were: a) all varieties showed a good level of downy mildew resistance, especially ‘Cabernet Carbon’, ‘Monarch’, ‘Prior’, UD 31.103, ‘Muscaris’, ‘Solaris’, ‘Souvignier Gris’, ‘Bronner’, ‘Fleurtai’, ‘Aromera’; b) no powdery mildew attacks were detected in any variety; c) ‘Monarch’, ‘Muscaris’, ‘Solaris’ and ‘Souvignier Gris’ also showed a high level of resistance towards black rot and anthracnose; d) red grape varieties had an earlier bud burst as compared to ‘Merlot’, and, concerning ripening, some varieties were earlier than ‘Merlot’, other ones were later; e) white varieties had a later bud burst but an earlier ripening time as compared to ‘Glera’; f) grape production and quality changed significantly depending on the varieties, being titratable acidity higher than 6.4 g L-1 tartaric acid and pH lower than 3.5; also the year affected in a significant way those parameters as well as the interaction between the genotype and the year. In conclusion, the tested varieties behaved positively in terms of environmental sustainability

F9 Missense mutations impairing factor ix activation are associated with pleiotropic plasma phenotypes

Background: Circulating dysfunctional factor IX (FIX) might modulate distribution of infused FIX in haemophilia B (HB) patients. Recurrent substitutions at FIX activation sites (R191-R226, >300 patients) are associated with variable FIX activity and antigen (FIXag) levels. Objectives: To investigate i) expression of a complete panel of missense mutations at FIX activation sites and ii) contribution of F9 genotypes on the FIX pharmacokinetics (PK). Methods: FIXag and activity assays in plasma and after recombinant expression of FIX variants. Analysis of infused FIX PK parameters in patients (n=30), mostly enrolled in the F9 Genotype and PK HB Italian Study (GePKHIS; EudraCT ID2017-003902-42). Results: The variable FIXag amounts and good relation between biosynthesis and activity of multiple R191 variants result in graded moderate-to-mild severity of the R191C>L>P>H substitutions. Recombinant expression may predict the absence in the HB mutation database of the benign R191Q/W/K and R226K substitutions. Equivalent changes at R191/R226 produced higher FIXag levels for R226Q/W/P substitutions, as also observed in p.R226W female carrier plasma. PK analysis in patients suggested that infused FIX Alpha distribution and Beta elimination phases positively correlated with endogenous FIXag levels. Mean residence time was particularly prolonged (79.4 hrs, 95% CI 44.3-114.5) in patients (n=7) with the R191/R226 substitutions, which in regression analysis were independent predictors (β coefficient 0.699, p=0.004) of Beta half-life, potentially prolonged by the increasing over time ratio between endogenous and infused FIX. Conclusions: FIXag levels and specific features of the dysfunctional R191/R226 variants may exert pleiotropic effects both on HB patients’ phenotypes and substitutive treatment

Videoriprese non investigative e tutela della riservatezza

L’utilizzo delle videoriprese nel processo non è oggetto di specifica disciplina legislativa. Tale lacuna è stata affrontata dalla giurisprudenza la quale, però, si è in prevalenza occupata di delineare gli argini entro cui l’autorità investigativa può realizzare riprese audiovisive, trascurando quelle effettuate al di fuori dal procedimento. L’assenza di regole circa l’utilizzo delle videoriprese non investigative non può, tuttavia, legittimare indiscriminate compressioni della riservatezza la cui tutela va necessariamente bilanciata con le esigenze dell’accertamento penale

Impossibilità di confiscare il profitto illecito conseguito dalla società e sequestro per equivalente sui beni degli amministratori

La Corte di cassazione ha statuito che, nei confronti dell’amministratore di una società, il sequestro preventivo finalizzato alla confisca per equivalente può essere eseguito solo se è impossibile aggredire in via diretta il profitto del reato; ma occorre prima realizzare la ricerca del profitto tra i beni della persona giuridica destinataria dell’illecito arricchimento. Il principio espresso dalla Suprema corte ha il pregio di tracciare i presupposti del sequestro finalizzato alla confisca per equivalente, in particolare valorizzando il vincolo di sussidiarietà previsto dall’art. 322-ter c.p

Note sull’uso di contenitori informatici in carcere da parte del difensore,

La Corte di legittimità impone al difensore che intende utilizzare il proprio personal computer durante un colloquio in carcere con l’assistito in vinculis, di indicare le ragioni che ne rendono indispensabile l’impiego per soddisfare le esigenze difensive. Si tratta però di un obbligo non previsto da alcuna disposizione normativa, determinando così una ingiustificata compressione delle prerogative difensive

Il dies a quo per la costituzione della parte civile: aspetti critici e prassi devianti

L’assenza di regole chiare circa l’individuazione del dies a quo per la costituzione di parte civile nel processo penale, genera orientamenti contrastanti; solo una interpretazione sistematica della norma consente di poter dirimere la questione e di censurare prassi applicative che, in modo ingiustificato, ostacolano la costituzione della parte civile

I riflessi della prescrizione del reato sulla decisione in appello per gli interessi civili

Le sezioni unite chiariscono la sorte della decisione sugli effetti civili qualora il giudice di appello, nel riformare la sentenza di condanna, accerti che il reato era già prescritto durante il giudizio di primo grado. L’intervento nomofilattico, nel valorizzare l’accessorietà della domanda risarcitoria all’azione penale, offre l’occasione per svolgere alcune riflessioni sistematiche sul rapporto tra le due actiones, nonché, anche alla luce dei valori europei, sull’effettività degli strumenti offerti alla “vittima” per ottenere il ristoro dei danni causati dal reat

Mutamento del giudice e nuova istruttoria: note sull’involuzione interpretativa

L’immutabilità del giudice è un aspetto caratterizzante il giusto processo ed è, implicitamente, prevista dall’art. 111, comma 3, Cost.; pertanto, la rinnovazione del dibattimento in caso di mutamento del giudice è attività doverosa. Nondimeno, l’assenza di una norma esplicita circa le modalità di rinnovazione istruttoria consente, ancora, tentativi volti ad eludere l’applicazione della regola. Il presente contributo, alla luce degli orientamenti della Corte di cassazione, evidenzia la necessità che la prova orale sia assunta dinanzi al giudice chiamato a decidere