Presence and biodistribution of perfluorooctanoic acid (PFOA) in Paracentrotus lividus highlight its potential application for environmental biomonitoring

AbstractThe first determination of presence and biodistribution of PFOA in ninety specimens of sea urchin Paracentrotus lividus from two differently contaminated sites along Palermo’s coastline (Sicily) is reported. Analyses were performed on the sea urchins’ coelomic fluids, coelomocytes, gonads or mixed organs, as well as on seawater and Posidonia oceanica leaves samples from the collection sites. PFOA concentration ranged between 1 and 13 ng/L in seawater and between 0 and 794 ng/g in P. oceanica. The analyses carried out on individuals of P. lividus from the least polluted site (A) showed PFOA median values equal to 0 in all the matrices (coelomic fluid, coelomocytes and gonads). Conver…

Premature termination codon 124 derivatives as a novel approach to improve the read-through of premature amber and ochre stop codons

Nucleotide changes within an exon may alter the trinucleotide normally encoding a particular amino acid, such that a new stop signal is transcribed into the mRNA open reading frame. A recent approach to directly overcome the deleterious effects caused by nonsense mutations is represented by readthrough strategies which take advantage of the known properties of aminoglycosides that can suppress stop codons.

Nonsense codons suppression. An acute toxicity study of three optimized TRIDs in murine model, safety and tolerability evaluation.

Stop mutations cause 11% of the genetic diseases, due to the introduction of a premature termination codon (PTC) in the mRNA, followed by the production of a truncated protein. A promising therapeutic approach is the suppression therapy by Translational Readthrough Inducing Drugs (TRIDs), restoring the expression of the protein. Recently, three new TRIDs (NV848, NV914, NV930) have been proposed, and validated by several in vitro assays, for the rescue of the CFTR protein, involved in Cystic Fibrosis disease. In this work, an acute toxicological study for the three TRIDs was conducted in vivo on mice, according to the OECD No.420 guidelines. Animals were divided into groups and treated with …

Identification and validation of novel molecules obtained by integrated computational and experimental approaches for the read-through of PTCs in CF cells

Dinamica della cromatina nella regolazione del gene con homeobox Hbox12 in P. lividus.

Homeobox-containing gene transiently expressed in a spatially restricted pattern in the early sea urchin embryo

In the sea urchin embryo, the lineage founder cells whose polyclonal progenies will give rise to five different territories are segregated at the sixth division. To investigate the mechanisms by which the fates of embryonic cells are first established, we looked for temporal and spatial expression of homeobox genes in the very early cleavage embryos. We report evidence that PlHbox12, a paired homeobox-containing gene, is expressed in the embryo from the 4-cell stage. The abundance of the transcripts reaches its maximum when the embryo has been divided into the five polyclonal territories--namely at the 64-cell stage--and it abruptly declines at later stages of development. Blastomere dissoc…

Caffeine boosts Ataluren's readthrough activity

Abstract The readthrough of nonsense mutations by small molecules like Ataluren is considered a novel therapeutic approach to overcome the gene defect in several genetic diseases as cystic fibrosis (CF). This pharmacological approach suppresses translation termination at premature termination codons (PTCs readthrough) thus restoring the expression of a functional protein. However, readthrough might be limited by the nonsense-mediated mRNA decay (NMD), a cell process that reduces the amount/level of PTCs containing mRNAs. Here we investigate the combined action of Ataluren and caffeine to enhance the readthrough of PTCs. IB3.1 CF cells with a nonsense mutation were treated with caffeine to a…

Ruolo delle HDAC sul silenziamento genico nell’embrione di riccio di mare

Establishment of Polycomb silencing requires a transient interaction between PC and ESC

Two distinct types of Polycomb complexes have been identified in flies and in vertebrates, one containing ESC and one containing PC. Using LexA fusions, we show that PC and ESC can establish silencing of a reporter gene but that each requires the presence of the other. In early embryonic extracts, we find PC transiently associated with ESC in a complex that includes EZ, PHO, PH, GAGA, and RPD3 but not PSC. In older embryos, PC is found in a complex including PH, PSC, GAGA, and RPD3, whereas ESC is in a separate complex including EZ, PHO, and RPD3.

Identification of a new molecule with readthrough activity to rescue CFTR protein function

In Cystic fibrosis (CF) disease nonsense mutations in the CFTR gene cause absence of the CFTR protein expression and a more severe form of the disease. About 10% of patient affected by CF show a nonsense mutation. A potential treatment of this alteration is to promote translational readthrough of premature termination codons (PTCs) by translational readthrough inducing drugs such as Ataluren. In this context we aimed to compare the 1,2,4-oxadiazole core of Ataluren with a slightly different scaffold, the 1,3,4oxadiazole core. By a validated protocol consisting of computational screening, synthesis and biological tests we identified, a new small molecule with 1,3,4-oxadiazole core (2a/NV2445…

Constitutive Promoter Occupancy by the MBF-1 Activator and Chromatin Modification of the Developmental Regulated Sea Urchin α-H2A Histone Gene

The tandemly repeated sea urchin alpha-histone genes are developmentally regulated. These genes are transcribed up to the early blastula stage and permanently silenced as the embryos approach gastrulation. As previously described, expression of the alpha-H2A gene depends on the binding of the MBF-1 activator to the 5' enhancer, while down-regulation relies on the functional interaction between the 3' sns 5 insulator and the GA repeats located upstream of the enhancer. As persistent MBF-1 binding and enhancer activity are detected in gastrula embryos, we have studied the molecular mechanisms that prevent the bound MBF-1 from trans-activating the H2A promoter at this stage of development. Her…

Enhancement of premature stop codon readthrough in the CFTR gene by Ataluren (PTC124) derivatives.

Abstract Premature stop codons are the result of nonsense mutations occurring within the coding sequence of a gene. These mutations lead to the synthesis of a truncated protein and are responsible for several genetic diseases. A potential pharmacological approach to treat these diseases is to promote the translational readthrough of premature stop codons by small molecules aiming to restore the full-length protein. The compound PTC124 (Ataluren) was reported to promote the readthrough of the premature UGA stop codon, although its activity was questioned. The potential interaction of PTC124 with mutated mRNA was recently suggested by molecular dynamics (MD) studies highlighting the importanc…

PTC124 derivatives as a novel approach to improve the readthrough of premature stop codons in the CFTR gene.

Background Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). Approximately 10% (worldwide) of patients have in-frame nonsense mutations (UAA, UAG or UGA class I mutations) in the CF trans-membrane regulator (CFTR) gene that result in premature stop codons (PTCs) in the messenger RNA (mRNA) generating truncated CFTR protein responsible for a severe CF phenotype. Pharmacological approaches have been proposed to directly overcome PTCs. Ataluren (PTC124) a small molecule that mimics the activity of aminoglycosides has been suggested to allow PTCs readthrough and to partially restore the protein function. However, des…

“Epigenetics of silencing of Paracentrotus lividus α-H2A gene”

Down-regulation of early sea urchin histone H2A gene relies on cis regulative sequences located in the 5' and 3' regions and including the enhancer blocker sns.

The tandem repeated sea urchin alpha-histone genes are developmentally regulated by gene-specific promoter elements. Coordinate transcription of the five genes begins after meiotic maturation of the oocyte, continues through cleavage, and reaches its maximum at morula stage, after which these genes are shut off and maintained in a silenced state for the life cycle of the animal. Although cis regulative sequences affecting the timing and the level of expression of these genes have been characterized, much less is known about the mechanism of their repression. Here we report the results of a functional analysis that allowed the identification of the sequence elements needed for the silencing …

Enhancer blocking activity located near the 3′ end of the sea urchin early H2A histone gene

The sea urchin early histone repeating unit contains one copy of each of the five histone genes whose coordinate expression during development is regulated by gene-specific elements. To learn how within the histone repeating unit a gene-specific activator can be prevented to communicate with the heterologous promoters, we searched for domain boundaries by using the enhancer blocking assay. We focused on the region near the 3′ end of the H2A gene where stage-specific nuclease cleavage sites appear upon silencing of the early histone genes. We demonstrated that a DNA fragment of 265 bp in length, defined as sns (for silencing nucleoprotein structure), blocked the enhancer activity of the H2A…

Uptake and photoinduced degradation of phthalic acid esters (PAEs) in Ulva lactuca highlight its potential application in environmental bioremediation

Abstract The bioaccumulation of phthalates was studied in fragments of Ulva lactuca exposed for a maximum of 31 days at different concentrations of a solution of six phthalic acid esters (PAEs). The algal matrix showed rapid uptake since the first sampling, which increased over the time of the experimental period, at the end of which seaweed’s bioaccumulation potential was also evaluated. After the uptake, the algal matrix was subjected to UV irradiation in order to verify the removal of the phthalates. PAEs with higher octanol–water partition coefficients (logKow) and molecular weights were preferentially uptaken by U. lactuca in all the exposure experiments. It was observed that both accu…

Impact of Heavy Metals in Eggs and Tissues of C. caretta along the Sicilian Coast (Mediterranean Sea)

In this study we compared the heavy metal concentration found in different tissues and eggs of the loggerhead sea turtle and evaluated the potential ecotoxicological risk for this important species. Eighteen heavy metal elements were determined in different tissues (liver, gonads, fat, kidney, heart, brain, and spleen) of nine individuals of Caretta caretta found stranded along the coasts of Messina (Sicily, Italy) and in the shell and yolk of six eggs from the island of Linosa (Sicily, Italy). For the analysis of the heavy metals, we used the analytical procedures in accordance with the EPA 200.8 method supplemented by EPA 6020b with three replicates for each measurement. The elements anal…

Investigating the inhibition of FTSJ1 a tryptophan tRNA-specific 2’-O-methyltransferase by NV TRIDs, as a mechanism of readthrough in nonsense mutated CFTR

Abstract: Cystic Fibrosis (CF) is an autosomal recessive genetic disease caused by mutations in the CFTR gene, coding for the CFTR chloride channel. About 10% of the CFTR gene mutations are "stop" mutations, which generate a Premature Termination Codon (PTC), thus synthesizing a truncated CFTR protein. A way to bypass PTC relies on ribosome readthrough, which is the ri-bosome’s capacity to skip a PTC, thus generating a full-length protein. “TRIDs” are molecules exerting ribosome readthrough; for some, the mechanism of action is still under debate. We in-vestigate a possible mechanism of action (MOA) by which our recently synthesized TRIDs, namely NV848, NV914, and NV930, could exert their r…

LA DINAMICA DELLA CROMATINA NELLA REGOLAZIONE DELL’ESPRESSIONEGENICA DELL’EMBRIONE DI RICCIO DI MARE

Sea urchin HSF activity in vitro and in transgenic embryos.

Evidence is provided for the presence at the physiological temperature of 20 degrees C of a heat shock transcriptor factor, HSF, in the nuclei of P.lividus embryos. This HSF is able to specifically bind in vitro the heat shock element, HSE, of the promoter of the hsp70 gene i.v., as suggested by DNA-protein binding reactions and DNAse I protection assays. Upon heat-shock, at the temperature of 31 degrees C, its ability to bind the HSE units becomes much higher. The HSF activated by heat-shock drives in vivo the transcription of the beta-galactosidase reporter gene in transgenic sea urchin gastrulae. An ATF-like transcription factor, widely described in other organisms but not at all in sea …

Promoter activity of the sea urchin (Paracentrotus lividus) nucleosomal H3 and H2A and linker H1 a-histone genes is modulated by enhancer and chromatin insulator

Core promoters and chromatin insulators are key regulatory elements that may direct a transcriptional enhancer to prefer a specific promoter in complex genetic loci. Enhancer and insulator flank the sea urchin (Paracentrotus lividus) alpha-histone H2A transcription unit in a tandem repeated cluster containing the five histone genes. This article deals with the specificity of interaction between the H2A enhancer-bound MBF-1 activator and histone gene promoters, and with the mechanism that leads the H1 transcripts to peak at about one-third of the value for nucleosomal H3 and H2A mRNAs. To this end, in vivo competition assays of enhancer and insulator functions were performed. Our evidence su…

The Sea Urchin sns Insulator Blocks CMV Enhancer following Integration in Human Cells

Insulators are a new class of genetic elements that attenuate enhancer function directionally. Previously, we characterized in sea urchin a 265-bp-long insulator, termed sns. To test insulator activity following stable integration in human cells, we placed sns between the CMV enhancer and a tk promoter up-stream of a GFP transgene of plasmid or retroviral vectors. In contrast to controls, cells transfected or transduced with insulated constructs displayed a barely detectable fluorescence. Southern blot and PCR ruled out vector rearrangement following integration into host DNA; RNase protection confirmed the enhancer blocking activity. Finally, we demonstrate that two cis-acting sequences, p…

Valutazione dell'azione readthorough della molecola PTC124 su sistemi modello cellulari contenenti mutazioni non senso e in cellule epiteliali bronchiali IB3.1 (delF508/W1282X) derivate da pazienti affetti da fibrosi cistica.

Circa il 10% dei pazienti affetti da fibrosi cistica (FC) presenta nel gene CFTR mutazioni non senso (o 'stop': UGA, UAG or UAA, mutazioni di classe I) che bloccano prematuramente la sintesi della proteina. Attualmente non esiste una cura per questo tipo di mutazioni ma si sta cercando di individuare delle molecole che siano in grado di indurre la traduzione di codoni di stop prematuri (readthrough) che, rispetto a molecole già note come il G418, abbiano effetti collaterali ridotti ed una maggiore specificità per uno specifico codone. Una piccola molecola che sembra possedere una tale attività è il PTC124 (Welch, 2007). Ad oggi però non c’è ancora un consenso generale sul meccanismo di azio…

Rescuing CFTR Protein Function: 1,3,4-oxadiazoles versus 1,2,4-oxadiazoles as readthrough inducing drugs

In Cystic fibrosis (CF) disease nonsense mutations in the CFTR gene cause the absence of the CFTR protein expression and a more severe form of the disease. About 10% of patient affected by CF show a nonsense mutation. A potential treatment of this alteration is to promote translational readthrough of premature termination codons (PTCs) by translational readthrough inducing drugs such as Ataluren (1). We reported a rationale for Ataluren promoted readthrough of PTCs by computational approach and GFP-reporter cell-based assay (2) and the observed enhancement of readthrough activity by some Ataluren derivatives (3, 4). In this context we aimed to compare the 1,2,4-oxadiazole core of Ataluren w…

Characterization of a nuclear factor associated to the chromatin of sea urchin histone genes

Translational readthrough inducing drugs: a study of toxicity in mice models and in vitro safety validation of the specific readthrough process.

Objective Nonsense mutations are responsible for 15% of Cystic Fibrosis (CF) patients due to the introduction of a premature stop codon (PTC) in the mRNA and the production of a truncated CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) protein1. A promising therapeutic approach for stop mutations is the suppression therapy by Translational Readthrough Inducing Drugs (TRIDs) to restore the expression of the protein2,3. Recently three new TRIDS (NV848, NV914, NV930) have been proposed and validated by several assays. Our work was focused on TRIDs NV848, NV914, NV930. Important aspects of TRIDs to be evaluated are their specificity towards PTC, to demonstrate that TRIDs do not inter…

The genetic identity of the only Italian population of the genus Macroprotodon Guichenot, 1850 on the island of Lampedusa, Sicily

The only Italian population of false smooth snakes is found on Lampedusa, a small island located in the Sicilian Channel and part of the African continental shelf. The taxonomic identity of this population is currently uncertain, although it is most often attributed to Macroprotodon cucullatus textilis on a morphological basis. We present here the first genetic data on this population. The analysis carried out on the mitochondrial cytochrome b gene shows that the Lampedusan false smooth snake belongs to a clade shared with a single sample from central Tunisia. The genetic distance between this lineage and its sister group (M. abubakeri) is comparable to or higher than that found among many …

Drosophila Enhancer of Zeste/ESC Complexes Have a Histone H3 Methyltransferase Activity that Marks Chromosomal Polycomb Sites

AbstractEnhancer of Zeste is a Polycomb Group protein essential for the establishment and maintenance of repression of homeotic and other genes. In the early embryo it is found in a complex that includes ESC and is recruited to Polycomb Response Elements. We show that this complex contains a methyltransferase activity that methylates lysine 9 and lysine 27 of histone H3, but the activity is lost when the E(Z) SET domain is mutated. The lysine 9 position is trimethylated and this mark is closely associated with Polycomb binding sites on polytene chromosomes but is also found in centric heterochromatin, chromosome 4, and telomeric sites. Histone H3 methylated in vitro by the E(Z)/ESC complex …

Derivati Ossadiazolici per il trattamento della fibrosi cistica: Readthrough di mutazioni nonsense

Investigating REPAIRv2 as a Tool to Edit CFTR mRNA with Premature Stop Codons

Cystic fibrosis (CF) is caused by mutations in the gene encoding the transmembrane conductance regulator (CFTR) protein. Some CF patients are compound heterozygous or homozygous for nonsense mutations in the CFTR gene. This implies the presence in the transcript of premature termination codons (PTCs) responsible for a truncated CFTR protein and a more severe form of the disease. Aminoglycoside and PTC124 derivatives have been used for the read-through of PTCs to restore the full-length CFTR protein. However, in a precision medicine framework, the CRISPR/dCas13b-based molecular tool &ldquo

Spatially restricted expression of PlOtp, a Paracentrotus lividus Orthopedia-related homeobox gene, is correlated with oral ectodermal patterning and skeletal morphogenesis in late-cleavage sea urchin embryos

ABSTRACT Several homeobox genes are expressed in the sea urchin embryo but their roles in development have yet to be elucidated. Of particular interest are homologues of homeobox genes that in mouse and Drosophila are involved in patterning the developing central nervous system (CNS). Here, we report the cloning of an orthopedia (Otp)-related gene from Paracentrotus lividus, PlOtp. Otp is a single copy zygotic gene that presents a unique and highly restricted expression pattern. Transcripts were first detected at the mid-gastrula stage in two pairs of oral ectoderm cells located in a ventrolateral position, overlying primary mesenchyme cell (PMC) clusters. Increases in both transcript abund…

Identification and validation of novel molecules obtained by integrated computational and experimental approaches for the readthrough of PTCs in CF cells

Cystic Fibrosis patients with nonsense-mutation in h-CFTR gene generally make virtually no CFTR protein and thus often have a more severe form of CF. Ataluren (PTC124) was suggested to induce read-through of premature but not normal termination codons. Despite the promising results there is not a general consensus on the mechanism of its action (protein stabilization or codon read-through) and its efficacy, the identification of new PTC124 analogues and the study of the mechanism of action may led to a new strategy for the development of a pharmacologic approach to the cure of CF.

Tracking the invasion of the red swamp crayfish Procambarus clarkii (Girard, 1852) (Decapoda Cambaridae) in Sicily: a “citizen science” approach

Author(s): Faraone, Francesco Paolo; Giacalone, Gabriele; Canale, Domenica Emanuela; D'Angelo, Stefania; Favaccio, Giorgio; Garozzo, Vincenzo; Giancontieri, Giacoma Lidia; Isgro, Carmelo; Melfi, Raffaella; Morello, Bruno; Navarria, Federica; Russo, Giuseppe; Tinnirello, Viviana; Torre, Antonio; Torre, Daniele; Torre, Giancarlo; Urso, Giuseppe; Vinci, Pierluigi; Zizzo, Maria Grazia; Marrone, Federico | Abstract: The first record of the red swamp crayfish in Sicily dates back to 2003 and, since then, the species seemed to be confined to a few localities in western Sicily. A small “citizen science” project carried out from November 2016 onwards led to the creation of the “Sicilian Procambarus …

Strategies against nonsense: oxadiazoles as translational readthrough-inducing drugs (TRIDs)

This review focuses on the use of oxadiazoles as translational readthrough-inducing drugs (TRIDs) to rescue the functional full-length protein expression in mendelian genetic diseases caused by nonsense mutations. These mutations in specific genes generate premature termination codons (PTCs) responsible for the translation of truncated proteins. After a brief introduction on nonsense mutations and their pathological effects, the features of various classes of TRIDs will be described discussing differences or similarities in their mechanisms of action. Strategies to correct the PTCs will be presented, particularly focusing on a new class of Ataluren-like oxadiazole derivatives in comparison …

Preliminary genetic characterisation of Southern Smooth Snake Coronella girondica (Serpentes, Colubridae) populations in Italy, with some considerations on their alpine distribution

The Southern smooth snake, Coronella girondica, is a small-sized colubrid found in Northwest Africa and Southwest Europe. Mitochondrial DNA-based studies showed that the species can be split into five clades: two from Northwest Africa (one Moroccan and one Tunisian-Algerian) and three from Europe (one in the south-west of the Iberian Peninsula, one in the south-east of Spain and one in the rest of the European range). With regards to Italy, to date, only two samples have been analysed both from the Province of Pisa, Tuscany, pointing at that fact that genetic characterisation of Italian populations is still lacking. Accordingly, we have increased the sampling coverage with 19 new samples fr…

Identification of a putative regulator of early-histone gene expression of Paracentrotus lividus

Drosophila GAGA factor (GAF) is a nuclear protein, conserved along evolution, with multiple roles in gene regulation, chromatin remodelling, Polycomb-dependent silencing, and insulator functions (1). GAF recognizes and specifically binds GAGAG consensus DNA motif by its C2H2-type zinc-finger domain, and interacts with other regulatory factors by its BTB/POZ domain. We have identified plGaga, a cDNA coding for a putative GAF of the sea urchin P. lividus, which showed significant sequence similarity with Drosophila and vertebrate GAFs. Real time RT-PCR revealed that the plGaga RNA is always present during embryo development decreasing rapidly in abundance at the larval pluteus stage. We raise…

Rescuing the CFTR protein function: Introducing 1,3,4-oxadiazoles as translational readthrough inducing drugs.

Nonsense mutations in the CFTR gene prematurely terminate translation of the CFTR mRNA leading to the production of a truncated protein that lacks normal function causing a more severe form of the cystic fibrosis (CF) disease. About 10% of patients affected by CF show a nonsense mutation. A potential treatment of this alteration is to promote translational readthrough of premature termination codons (PTCs) by Translational Readthrough Inducing Drugs (TRIDs) such as PTC124. In this context we aimed to compare the activity of PTC124 with analogues differing in the heteroatoms position in the central heterocyclic core. By a validated protocol consisting of computational screening, synthesis an…

Can phthalates move into the eggs of the loggerhead sea turtle Caretta caretta? The case of the nests on the Linosa Island in the Mediterranean Sea

During the monitoring of Caretta caretta nests on the island of Linosa, 30 unhatched eggs from four nests were collected to study the presence of phthalates in their three components (shell, yolk, and albumen). Four phthalates, namely diethyl (DEP), dibutyl (DBP), di-(2-ethylhexyl) (DEHP), and dioctyl (DOTP) phthalic acid esters (PAE), which are widely used as additives in plastics, were detected in all egg components. The most frequently found phthalate was DBP, followed by DEHP in eggshell and yolk. Dimethyl- (DMP) and butylbenzyl-phthalate (BBP) were below the limits of detection for all samples. The high total phthalate recorded in the yolk suggests that contamination could arise by vit…

Pharmacophore-Based Design of New Chemical Scaffolds as Translational Readthrough-Inducing Drugs (TRIDs)

[Image: see text] Translational readthrough-inducing drugs (TRIDs) rescue the functional full-length protein expression in genetic diseases, such as cystic fibrosis, caused by premature termination codons (PTCs). Small molecules have been developed as TRIDs to trick the ribosomal machinery during recognition of the PTC. Herein we report a computational study to identify new TRID scaffolds. A pharmacophore approach was carried out on compounds that showed readthrough activity. The pharmacophore model applied to screen different libraries containing more than 87000 compounds identified four hit-compounds presenting scaffolds with diversity from the oxadiazole lead. These compounds have been s…

The sea urchin histone H2A enhancer-binding protein MBF-1 is needed for maximal expression also for the H3 gene, while is buffered by the sns5 insulator on its interaction with the downstream H1 promoter.

Enhancers are DNA elements which increase the transcription of associated gene in a position and distance independent manner relative to the transcription initiation site. Molecular mechanisms must operate to direct enhancers to specific promoters in complex genetic loci. The sea urchin a-histone genes are organized in several hundred tandem repeated units, each containing one copy of the five histone genes in the order 5’-H4-H2B-H3-H2A-H1-3’. Transcription is limited to the early cleavage and reaches its maximum at morula stage. After hatching these genes are repressed and maintained in the silenced state for whole life cycle of the animal. In Paracentrotus lividus, the MBF-1 activator bin…

AZIONE READTHROUGH DI DERIVATI DEL PTC124 SU SISTEMI MODELLO CELLULARI E IN CELLULE DI EPITELIO BRONCHIALE-FC IB3.1 (CFTR F508/W1282X )

Obiettivi specifici: Le mutazioni nonsenso (mutazioni STOP), un difetto genetico frequente negli individui affetti da Fibrosi Cistica (CF), causano la sintesi di proteine CFTR tronche e non funzionanti che sono associate ad un fenotipo più severo della CF (McKone EF. et al., Chest 2006). L’obiettivo del nostro studio è stato quello di disegnare derivati dell’Ataluren (PTC124), una ‘small molecule’ a cui è stata attribuita attività readthrough, e valutarne l’attività su tre differenti sistemi modello sperimentali contenenti codoni di STOP prematuri (UGA, UAG, UAA). Materiali e metodi: Sono state sintetizzate 24 molecole derivate dal PTC124 e analizzate mediante tecniche spettroscopiche per v…

Gene expression during early embryogenesis of sea urchin: The histone and homeobox genes

Transcriptional regulators are thought to play a key role in cell fate determination and territorial specification in sea urchin. Our goals are to clone transcription factors for studying embryonic development. One approach has been to use promoter binding and gene transfer technology to investigate the mechanisms of transcriptional activation and repression of the early H2A histone gene. By this analysis we identified a transcriptional activator, the MBF-1, that binds to the modulator element of the H2A gene and enhances the activity of the H2A promoter. However, the enhancer activity of the modulator and its interaction with MBF-1 also occurs at the gastrula stage when the early histone g…

Enhancer, chromatin insulator, non-coding RNA and α-histone gene expression during embryogenesis of the sea urchin Paracentrotus lividus.

Core promoters and chromatin insulators (ins) may direct a transcriptional enhancer (enh) to prefer a specific promoter in complex genetic loci. Enh and ins flank the sea urchin Paracentrotus lividus α-histone H2A transcription unit in a tandem repeated cluster containing the five histone genes. In vivo competition assays of enh and ins functions reveal that the H2A enh-bound MBF-1 activator participates also in the expression of the H3 gene and that the sns5 ins buffers the downstream H1 promoter from the H2A enh. These results suggest that both the H2A enh and the sns5 ins may account for the diverse accumulation of the linker vs core nucleosomal histones during early development of the s…

PTC124 DERIVATIVES AS A NOVEL APPROACH TO IMPROVE THE READTHROUGH OF PREMATURE AMBER AND OCHRE STOP CODONS

Nucleotide changes within an exon may alter the trinucleotide normally encoding a particular amino acid, such that a new “stop” signal is transcribed into the mRNA open reading frame. This causes the ribosome to prematurely terminate its reading of the mRNA, leading to the lack of production of a normal full-length protein. Such premature termination codon (PTC) mutations occur in an estimated 10% to 15% of many genetically based disorders (1). Pathological nonsense mutations resulting in TAG (40.4%), TGA (38.5%), and TAA (21.1%) occur in different proportions to naturally occurring stop codons (2). Several genetic disorders are characterized by opal (TGA; Cystic fibrosis, Duchenne/Becker m…

X CONVENTION OF INVESTIGATORS IN CYSTIC FIBROSIS.

Background Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). Approximately 10% (worldwide) of patients have in-frame nonsense mutations (UAA, UAG or UGA class I mutations) in the CF trans-membrane regulator (CFTR) gene that result in premature stop codons (PTCs) in the messenger RNA (mRNA) generating truncated CFTR protein responsible for a severe CF phenotype. Pharmacological approaches have been proposed to directly overcome PTCs. Ataluren (PTC124) a small molecule that mimics the activity of aminoglycosides has been suggested to allow PTCs readthrough and to partially restore the protein function. However, des…

Exploring the readthrough of nonsense mutations by non-acidic Ataluren analogues selected by ligand-based virtual screening

Abstract Ataluren, also known as PTC124, is a 5-(fluorophenyl)-1,2,4-oxadiazolyl-benzoic acid suggested to suppress nonsense mutations by readthrough of premature stop codons in the mRNA. Potential interaction of PTC124 with mRNA has been recently studied by molecular dynamics simulations highlighting the importance of H-bonding and stacking π−π interactions. A series of non-acidic analogues of PTC124 were selected from a large database via a ligand-based virtual screening approach. Eight of them were synthesized and tested for their readthrough activity using the Fluc reporter harboring the UGA premature stop codon. The most active compound was further tested for suppression of the UGA non…

The Compass-like Locus, Exclusive to the Ambulacrarians, Encodes a Chromatin Insulator Binding Protein in the Sea Urchin Embryo

Chromatin insulators are eukaryotic genome elements that upon binding of specific proteins display barrier and/or enhancer-blocking activity. Although several insulators have been described throughout various metazoans, much less is known about proteins that mediate their functions. This article deals with the identification and functional characterization in Paracentrotus lividus of COMPASS-like (CMPl), a novel echinoderm insulator binding protein. Phylogenetic analysis shows that the CMPl factor, encoded by the alternative spliced Cmp/Cmpl transcript, is the founder of a novel ambulacrarian-specific family of Homeodomain proteins containing the Compass domain. Specific association of CMPl…

Regulation of the sea urchin early H2A histone gene expression depends on the modulator element and on sequences located near the 3' end

Abstract Transcription of the sea urchin early histone genes occurs transiently during early cleavage, reaching the maximum at the morula stage and declining to an undetectable level at the gastrula stage. To identify the regulatory elements responsible for the timing and the levels of transcription of the H2A gene, we used promoter binding studies in nuclear extracts and microinjection of a CAT transgene driven by the early H2A promoter. We found that morula and gastrula nuclear proteins produced indistinguishable DNase I footprint patterns on the H2A promoter. Two sites of interactions, centred on the modulator/enhancer and on the CCAAT box respectively, were detected. Deletion of the mod…

CHOMATIN DYNAMIC AND REGULATION OF GENE EXSPRESSION IN THE SEA URCHIN EMBRYO

OXADIAZOLE DERIVATIVES FOR THE TREATMENT OF GENETIC DISEASES DUE TO NONSENSE MUTATIONS

Are disclosed oxadiazole derivatives, their use as medicaments and in particular for the treatment of diseases associated with the presence of a nonsense mutation in the gene or a premature stop codon in the mRNA, pharmaceutical formulation comprising said oxadiazole derivatives and prodrug or mixture thereof and the methods for the preparation of said Oxadiazole derivatives.

Expression of homeobox-containing genes in the sea urchin (Parancentrotus lividus) embryo

Two homeobox-containing genes that belong to different homeodomain classes have been isolated from a sea urchin geonomic library. One, PlHbox11, is the sea urchin homologue of the human and mouse Hox B3 gene, the other, PlHbox12, shows about 55% identity with paired class genes. Expression profile analysis of the two sea urchin Hbox genes suggests that they play different roles during embryogenesis. In fact, PlHbox11 transcripts are rare and are detected only in the pluteus larva and in the Aristotle's lantern and intestine of the adult. The PlHbox12 gene is, on the contrary, transiently expressed in the very early embryo already at the four cell stage; it accumulates at the 64 cell stage a…

Readthrough Inducing Drugs (TRIDs) in human fibroblasts harboring the c.5047 C>T (R1683*) nonsense mutation

Toward a Rationale for the PTC124 (Ataluren) Promoted Readthrough of Premature Stop Codons: A Computational Approach and GFP-Reporter Cell-Based Assay

The presence in the mRNA of premature stop codons (PTCs) results in protein truncation responsible for several inherited (genetic) diseases. A well-known example of these diseases is cystic fibrosis (CF), where approximately 10% (worldwide) of patients have nonsense mutations in the CF transmembrane regulator (CFTR) gene. PTC124 (3-(5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl)-benzoic acid), also known as Ataluren, is a small molecule that has been suggested to allow PTC readthrough even though its target has yet to be identified. In the lack of a general consensus about its mechanism of action, we experimentally tested the ability of PTC124 to promote the readthrough of premature termination c…

Functional characterization of the enhancer blocking element of the sea urchin early histone gene cluster reveals insulator properties and three essential cis-acting sequences

Insulator elements can be functionally identified by their ability to shield promoters from regulators in a position-dependent manner or their ability to protect adjacent transgenes from position effects. We have previously reported the identification of a 265 bp sns DNA fragment at the 3' end of the sea urchin H2A early histone gene that blocked expression of a reporter gene in transgenic embryos when placed between the enhancer and the promoter. Here we show that sns interferes with enhancer-promoter interaction in a directional manner. When sns is placed between the H2A modulator and the inducible tet operator, the modulator is barred from interaction with the basal promoter. However, th…

Identification of a putative GAGA factor in P. lividus embryos

Targeting Nonsense: Optimization of 1,2,4-Oxadiazole TRIDs to Rescue CFTR Expression and Functionality in Cystic Fibrosis Cell Model Systems

Cystic fibrosis (CF) patients develop a severe form of the disease when the cystic fibrosis transmembrane conductance regulator (CFTR) gene is affected by nonsense mutations. Nonsense mutations are responsible for the presence of a premature termination codon (PTC) in the mRNA, creating a lack of functional protein. In this context, translational readthrough-inducing drugs (TRIDs) represent a promising approach to correct the basic defect caused by PTCs. By using computational optimization and biological screening, we identified three new small molecules showing high readthrough activity. The activity of these compounds has been verified by evaluating CFTR expression and functionality after…

Controlled uptake of PFOA in adult specimens of Paracentrotus lividus and evaluation of gene expression in their gonads and embryos

AbstractPerfluorooctanoic acid (PFOA) has been largely used in the manufacturing industry but a few years ago it turned out to be a dangerous pollutant which is now of concern for terrestrial and aquatic environments. Here, we investigated the bioaccumulation of PFOA in the sea urchin Paracentrotus lividus after exposure to different concentrations of the pollutant for 28 days. We observed rapid uptake of PFOA in the coelomic fluid collected weekly during the exposure period and high bioaccumulation in gonads at the end of the experiment. Interestingly, animals were also able to fast depurate when relocated to a clean environment. In addition, to assess the effect of PFOA on sea urchins’ ph…