Characterization and functional implications of the RNA binding properties of nuclear factor TDP-43, a novel splicing regulator of CFTR exon 9.

Variations in a polymorphic (TG)m sequence near exon 9 of the human CFTR gene have been associated with variable proportions of exon skipping and occurrence of disease. We have recently identified nuclear factor TDP-43 as a novel splicing regulator capable of binding to this element in the CFTR pre-mRNA and inhibiting recognition of the neighboring exon. In this study we report the dissection of the RNA binding properties of TDP-43 and their functional implications in relationship with the splicing process. Our results show that this protein contains two fully functional RNA recognition motif (RRM) domains with distinct RNA/DNA binding characteristics. Interestingly, TDP-43 can bind a minimum number of six UG (or TG) single-stranded dinucleotide stretches, and binding affinity increases with the number of repeats. In particular, the highly conserved Phe residues in the first RRM region play a key role in nucleic acid recognition

The multiple roles of TDP-43 in pre-mRNA processing and gene expression regulation.

Heterogeneous ribonucleoproteins (hnRNPs) are multifunctional RNA-binding proteins (RBPs) involved in many cellular processes. They participate in most gene expression pathways, from DNA replication and repair to mRNA translation. Among this class of proteins, TDP-43 (and more recently FUS/TLS) have received considerable attention due to their involvement in several neurodegenerative diseases. This finding has prompted many research groups to focus on the gene expression pathways that are regulated by these proteins. The results have uncovered a considerable complexity of TDP-43 and FUS/TLS functions due to the many independent mechanisms by which they may act to influence various cellular processes (such as DNA transcription, pre-mRNA splicing, mRNA export/import). The aim of this chapter will be to review especially some of the novel functions that have been uncovered, such as role in miRNA synthesis, regulation of transcript levels, and potential autoregulatory mechanisms in order to provide the basis for further investigations

CCD photometry of 2060 Chiron, 1991 January

Observations of 2060 Chiron was performed on 7 to 8 Jan. 1991 with the Mt. Palomar 1.52 m telescope in the Gunn-R passband. On-chip field stars were used to perform differential reductions. The repeatability of the 5.9 hour light curve was excellent, both within a night and from night to night. No evidence for short-term secular variations similar to those seen last year by both Luu and Jewitt (1990) and Buratti and Dunbar (1991) is seen in the new light curve. Chiron's rotational light curve appears strikingly similar to that obtained a year earlier by Luu and Jewitt (1990), both in amplitude and shape. Both light curves show strongly correlated changes over a timescale of perhaps 15 minutes. These same features were marginally visible in the 1986 light curve. Such behavior is believed to be evidence that Chiron may be more aspherical than the 4 percent intensity variation might otherwise indicate, and favors a viewing geometry where the subearth latitude is rather low. Chiron was much fainter in 1985, when a partial light curve was obtained by Marcialis. Due to the lower sampling rate of these early data, no conclusions can be made regarding the high-frequency light curve structure back then. All three of these light curves differ significantly from that obtained by Buratti and Dunbar (1991), one week before the observations of Luu and Jewitt. The Chiron field was calibrated using Landolt standards on Ut 15 Mar. 1991. A mean R-magnitude of 15.6 + or - 0.1 was found. Variability of 2060 Chiron was demonstrated over timescales of minutes, hours, and years. An intense campaign was urged to monitor the photometric behavior of Chiron throughout the 1990s

Defective splicing, disease and therapy: searching for master checkpoints in exon definition

The number of aberrant splicing processes causing human disease is growing exponentially and many recent studies have uncovered some aspects of the unexpectedly complex network of interactions involved in these dysfunctions. As a consequence, our knowledge of the various cis- and trans-acting factors playing a role on both normal and aberrant splicing pathways has been enhanced greatly. However, the resulting information explosion has also uncovered the fact that many splicing systems are not easy to model. In fact we are still unable, with certainty, to predict the outcome of a given genomic variation. Nonetheless, in the midst of all this complexity some hard won lessons have been learned and in this survey we will focus on the importance of the wide sequence context when trying to understand why apparently similar mutations can give rise to different effects. The examples discussed in this summary will highlight the fine ‘balance of power’ that is often present between all the various regulatory elements that define exon boundaries. In the final part, we shall then discuss possible therapeutic targets and strategies to rescue genetic defects of complex splicing systems

An Amyloid-Like Pathological Conformation of TDP-43 Is Stabilized by Hypercooperative Hydrogen Bonds

TDP-43 is an essential RNA-binding protein forming aggregates in almost all cases of sporadic amyotrophic lateral sclerosis (ALS) and many cases of frontotemporal lobar dementia (FTLD) and other neurodegenerative diseases. TDP-43 consists of a folded N-terminal domain with a singular structure, two RRM RNA-binding domains, and a long disordered C-terminal region which plays roles in functional RNA regulatory assemblies as well as pernicious aggregation. Evidence from pathological mutations and seeding experiments strongly suggest that TDP-43 aggregates are pathologically relevant through toxic gain-of-harmful-function and/or harmful loss-of-native-function mechanisms. Recent, but not early, microscopy studies and the ability of TDP-43 aggregates to resist harsh treatment and to seed new pathological aggregates in vitro and in cells strongly suggest that TDP-43 aggregates have a self-templating, amyloid-like structure. Based on the importance of the Gln/Asn-rich 341–367 residue segment for efficient aggregation of endogenous TDP-43 when presented as a 12X-repeat and extensive spectroscopic and computational experiments, we recently proposed that this segment adopts a beta-hairpin structure that assembles in a parallel with a beta-turn configuration to form an amyloid-like structure. Here, we propose that this conformer is stabilized by an especially strong class of hypercooperative hydrogen bonding unique to Gln and Asn sidechains. The clinical existence of this conformer is supported by very recent LC-MS/MS characterization of TDP-43 from ex vivo aggregates, which show that residues 341–367 were protected in vivo from Ser phosphorylation, Gln/Asn deamidation and Met oxidation. Its distinct pattern of SDS-PAGE bands allows us to link this conformer to the exceptionally stable seed of the Type A TDP-43 proteinopathy.This work was supported by Grants SAF2013-49179-C2-2-R (DVL) and EU JPND AC14/00037 (DVL) and EU JPND RiModFTD, Italy, Ministero della Sanita’ (EB), and the Thierry Latran Foundation REHNPALS (EB).Peer reviewedPeer Reviewe

Studio di matrici alternative nell’analisi tossicologica forense

La tossicologia forense è una disciplina nata nell’ambito della medicina legale per la necessità di indagine su materiale cadaverico per la ricerca dei veleni. Attualmente ha ampliato molto il suo campo di interesse e si è affermata sempre di più. Si può quindi ritenere che il compito specifico della tossicologia forense sia quello di studiare il rapporto tra uomo e agente tossico attraverso l’identificazione delle sostanze tossiche in qualsiasi substrato, biologico o meno, in riferimento a fattispecie giuridiche. La ricerca può essere svolta su matrici biologiche differenti a seconda della finalità ultima dell’analisi. In caso di estrema putrefazione o carbonizzazione dei resti, quando tessuti molli e fluidi corporei non sono disponibili, possono essere utili capelli, ossa, denti e unghie definiti anche come matrici alternative o non convenzionali. Queste matrici sono le più resistenti alla decomposizione. Esse possono rappresentare una fonte di dati di confronto addizionale, possono fornire informazioni temporali retroattive, e la loro conservazione non necessita di particolari accorgimenti. Le unghie in particolar modo hanno un’origine cellulare comune a quella dei capelli, ma presentano specifiche differenze se analizzate allo stato maturo. Per poter sfruttare le nuove matrici alla ricerca di sostanze esogene è necessario sviluppare tecnologie analitiche in grado di ottenere alti livelli di sensibilità. In questo lavoro di tesi sono 5 state ricercate alcune sostanze stupefacenti (basiche) tramite l’analisi in HPLC, una tecnica cromatografica altamente sensibile che sta prendendo piede negli ultimi anni, che richiede un minor pretrattamento del campione grazie alle sue caratteristiche. Basandomi anche su altri precedenti lavori su matrici pilifere ho voluto aggiungere al metodo di estrazione per sostanze basiche in HPLC una seconda estrazione specifica per le sostanze acide, come i cannabinoidi che sono stati poi analizzati in gas cromatografia. Il metodo estrattivo utilizzato sui campioni prelevati da materiale cadaverico e da vivente si è rivelato efficace ed efficiente, in quanto tutte le sostanze ricercate sono state individuate e quantificate. Il metodo di accumulo delle sostanze all’interno della matrice ungueale non è ancora ben compreso, e ulteriori approfondimenti nel prossimo futuro vanno intrapresi per far luce sulle zone d’ombra ancora presenti in questo ambito

Parameter estimation and uncertainty quantification of a fiber-reinforced concrete model by means of a multi-level Bayesian approach

The paper presents a procedure for the stochastic calibration of a cracked hinge model on the basis of an extensive experimental campaign performed on a large group of nominally identical fiber-reinforced specimens. The calibration is carried out in a multi-level Bayesian framework that allows to quantify and separate several uncertainty contributions affecting model parameters. Indeed, the variability in the experimental response for nominally identical specimens due to the material heterogeneity represents a significant uncertainty contribution as well as model error. The former can be quantified at the hyper-parameter level of the multi-level framework. The presented results highlight the good agreement of the numerical predictions with the experimental data and the superior performance of the multi-level framework compared to that of the classical single-level framework. We also perform analyses to explore the impact of the prior parameter model conditioned on hyper-parameters and assess the minimum number of specimen datasets needed to quantify the inherent variability of model parameters

Mutational Analysis of the Different Bulge Regions of Hepatitis C Virus Domain II and Their Influence on Internal Ribosome Entry Site Translational Ability

The hepatitis C virus (HCV) 5′-untranslated region and, in particular, domains II to IV are involved in the internal ribosome entry site (IRES) structure. Recent structural evidence has shown that the function of domain II may be to hold the coding RNA in position until the translational machinery is correctly assembled on the decoding site. However, a comprehensive mutational and functional study concerning the importance of the different RNA regions that compose domain II is not yet available. Therefore, we have taken advantage of the recently proposed secondary structure of domain II to design a series of specific mutants. The bulge regions present in the latest secondary structure prediction of domain II were selectively deleted, and the effects of these mutations on IRES translation efficiency were analyzed. Our results show that the introduction of these mutations can variably affect the degree of HCV translation, causing a moderate to total loss of translation ability that correlates with the severity of changes induced in the RNA secondary structure and degree of p25 ribosomal protein UV cross-linking, but not with the ability of the 40S ribosomal subunit to bind the IRES. These findings support the proposed structural role of domain II in HCV translation

Evolution of physicochemical, morphological and aromatic characteristics of Italian PDO dry-cured hams during processing

The aim of this work was to follow the evolution of physicochemical (dry matter, NaCl concentration, pH, water activity), morphological (image analysis) and aromatic (e-nose) characteristics of the three main Italian PDOs during processing, from slaughtering to end of ripening. Main phenomena distinguishing the PDOs are NaCl concentration increase, which is higher in Toscano than in Parma and San Daniele hams, starting from the salting phase. Water activity values decrease during processing, and the lowest values are detected in Toscano ham. Changes in morphological parameters (area, shape) and in color progressively occur during processing and are more pronounced in Toscano ham. A clear evolution of aroma of the three PDOs has been observed by e-nose, and the complexity of the aromatic profile of the ripened hams is clearly highlighted