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Hemorrhois hippocrepis is a colubrid snake with a West Mediterranean distribution. It is widespread in the Iberian Peninsula and Northwest Africa. The only Italian populations are found on the islands of Sardinia and Pantelleria. The phylogenetic relationships of these insular populations have been analysed for the first time on the basis of the mitochondrial DNA cytochrome b gene. The sequences were compared with those available from the geographic range of this species. The analyses showed that the Italian samples are part of a lineage that groups Tunisian and East Algerian samples, with which they share the same haplotype. These results strongly support the hypothesis of a recent origin of the Italian populations of Hemorrhois hippocrepis, probably determined by human-mediated dispersal from North Africa., Acta Herpetologica, Vol. 15 No. 2 (2020): Acta Herpetologica 15(2) 2020

Background and Rationale Some CF patients are compound heterozygous or homozygous for nonsense mutations in the CFTR gene. Mutant CFTR gene coding for transcripts with premature termination codons (PTCs) is responsible for truncated CFTR protein and for a severe form of the disease. In a precision medicine framework the “REPAIRv2” (RNA Editing for Programmable A to I Replacement v2) tool, developed in the laboratory of Dr. Feng Zhang (USA), seems a good alternative to restore the full-length CFTR protein by editing its mRNA containing PTCs. This new approach is based on the possibility of targeting a deaminase enzyme (huADAR2) to a specific Adenosine, to be edited to Inosine (G analogue), on the mutant RNA by a specific guide RNA (gRNA), complementary to the target regions, and a Cas protein. Hypothesis and objectives We applied the new CRISPR/dCas13b based molecular tool of RNA editing (REPAIRv2) to correct the premature stop codon UGA, changing to UGG, in the H2bGFPopal and CFTRW1282X mRNAs with the purpose of recovering the full-length proteins.Essential Methods We designed and cloned the gRNAs needed to target the REPAIRv2 system to the Adenine to be modified. By site-directed mutagenesis we introduced a premature stop codon, W1282X, in the CFTR cDNA. Human HeLa cells expressing the H2BGFPopal mRNA, FRT cells expressing CFTRW1282X and IB3.1 airway epithelial human cells (CFTRΔ508/W12382X) were co-transfected with the plasmids coding for the recombinant protein dCAS13b/ADAR2DD, and for the gRNAs. Fluorescence microscopy was used to analyse the editing results. Results Direct fluorescence microscopy and immunofluorescence analyses detecting the corrected proteins (H2BGFP and CFTR, respectively) suggest that the REPAIRv2 system was able, in different cell lines, to edit the H2BGFPopal and the CFTRW1282X mRNA. However, the rate of editing does not seem high. Indeed, when RNA was purified from transfected cell, retro-transcribed and amplified base correction was not detectable by standard DNA sequencing and western blot. Conclusions Collectively, our results indicate that the REPAIRv2 tool is able to edit the UGA premature stop codon present in the HeLa-H2BGFPopal cells and in engineered FRTW1282X cells harbouring the UGA PTC in the CFTR mRNA. Furthermore, the REPAIRv2 tool worked in the IB3.1 cells suggesting its ability to edit endogenous UGA premature stop codon. Anyway, enhance the delivery of the plasmids as well increase/ stabilize the target mRNA to be edited, seem necessary to improve the efficiency of REPAIRv2. References 1. Cox DBT, Gootenberg JS, Abudayyeh OO, Franklin B, Kellner MJ, Joung J, Zhang F.- RNA editing with CRISPR-Cas13. Science. 2017 Nov 24; 358 (6366):1019-1027) 2. Lentini L, Melfi R, Di Leonardo A, Spinello A, Barone G, Pace A, Palumbo Piccionello A, Pibiri I. Toward a rationale for the PTC124 (Ataluren) promoted readthrough of premature stop codons: a computational approach and GFP-reporter cell-based assay. Mol Pharm. 2014 Mar 3;11(3):653-64. Acknowledgment FFC#5/2018 funded by FFC and supported by Delegazione FFC di Palermo

Optimization of a new lead promoting the readthrough of the nonsense mutations for CFTR rescue in human CF cells Laura Lentini, Raffaella Melfi, Sara Baldassano, Marco Tutone, Aldo Di Leonardo, Andrea Pace, Ivana Pibiri Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF), University of Palermo Background and rationale Cystic Fibrosis patients with nonsense mutations in the CFTR gene have a more severe form of the disease. Nonsense mutations represent about 10% of the mutations that affect the CFTR gene and they are frequently associated to the classical F508 mutation (1). A potential treatment for this genetic alteration is to promote the translational readthrough of premature termination codons (PTCs) by Translational Read-Through-Inducing Drugs (TRIDs) (2-4). Hypothesis and objectives Our objective is to evaluate the functionality of the CFTR channel after treatment with a new molecule that we individuated in a precedent FFC project, and the activity of new lead molecules in cells stably expressing a nonsense-CFTR-mRNA (ns CFTR) in CF cellular model systems. We want also to study the supramolecular interactions among TRIDs, CFTR mRNA and the ribosomal A-site to identify the biological target and the mechanism of action. Essential methods QSAR, carried out on the basis of our preliminary results, will allow to achieve lead optimization and synthesize then a small library of analogs to be tested and compared to the Lead. We will mutagenize the CFTR cDNA by introducing the most diffuse nonsense mutations. Subsequently, FRT cells engineered with the vector expressing mutagenized nsCFTR, and nonsense-CF-human broncoepithelial cells will be grown in the air-liquid culture system to reproduce in vitro the epithelial organization. CFTR expression after treatments with our molecule will be evaluated by biomolecular techniques. CFTR activity will be revealed by specific CFTR-functionality assays. Finally, in vitro-in vivo (Zebrafish model) analyses of the safety profile for the set of synthesized molecules will complete the study. Preliminary results We screened the activity of several molecules synthetized by us in a precedent FFC project, identifying some molecules that showed high readthrough activity associated to the expression of the CFTR protein in ns CF immortalized cells. Expected final results and their significance We are confident that our findings will provide the validation of molecules with readthrough activity for the recovery of the CFTR function. Moreover, our pre-clinical study will assess the presence of toxic effects caused by the molecules in vivo. References 1. Sermet-Gaudelus I, Boeck KD, Casimir GJ, Vermeulen F, Leal T, Mogenet A, Roussel D, Fritsch J, Hanssens L, Hirawat S, Miller NL, Constantine S, Reha A, Ajayi T, Elfring GL, Miller LL. Ataluren (PTC124) induces cystic fibrosis transmembrane conductance regulator protein expression and activity in children with nonsense mutation cystic fibrosis., Am J RespirCrit Care Med. 2010 Nov 15;182(10):1262-72. 2. Lentini L, Melfi R, Di Leonardo A, Spinello A, Barone G, Pace A, Palumbo Piccionello A, Pibiri I. Towards a rationale for the PTC124 (Ataluren) promoted read-through of premature stop codons: a computational approach and GFP-reporter cell-based assay. Mol. Pharm. 2014 11, 653-664. 3. Pibiri I, Lentini L, Melfi R, Gallucci G, Pace A, Spinello A, Barone G, Di Leonardo A. Enhancement of premature stop codon readthrough in the CFTR gene by Ataluren (PTC124) derivatives European Journal of Medicinal Chemistry 06/2015; 101. 4. Nagel-Wolfrum K, Möller F, Penner I, Baasov T4, Wolfrum U. Targeting Nonsense Mutations in Diseases with Translational Read-Through-Inducing Drugs (TRIDs), BioDrugs. 2016 Apr;30(2):49-74.

The Zinc-finger MBF-1 factor is involved in the expression of the early histone genes during devel-opment of the sea urchin embryo (1, 2). In spite of being a transcription activator, the DNA-binding domain of MBF-1 shares high sequence similarity with that of the chromatin organizer CTCF of vertebrates and drosophila (3). On the other hand, extensive in silico analysis failed to identify the sea urchin CTCF ortholog (4). This led us to speculate that MBF-1 somehow could have co-opted the function of CTCF during evolution of the echinoderms. Since in vertebrates CTCF binds Hox chromatin, to support our hypothesis, we first identified high-score putative binding sequences for CTCF/MBF-1 within the single sea urchin Hox gene cluster. Moreover, we observed the full evolu-tionary conservation of these binding sites in S. purpuratus and P. lividus species. Worth of men-tion, by chromatin immunoprecipitation (ChIP) assay, we detected the occupancy of MBF-1 on hox11/13-a, -b, and -c regulatory sequences at distinct stages of development. As expected from the binding of an activator, we found that the association of MBF-1 to the cis-regulatory sequences of both hox11/13-a and -b genes relates to the transcriptional status of these genes. Strikingly, we also mapped the physical binding of MBF-1 to hox11/13-c, which is instead not expressed during em-bryogenesis. Altogether, these observations indeed suggest the possibility that MBF-1, besides be-ing a transcription activator, could also function as a general chromatin organizer. To further support this hypothesis, we are planning ChIP-seq experiments to identify the association of MBF-1 to the sea urchin chromatin at a genome-wide level. 1. Di Caro, V. et al. (2007) J. Mol. Bio.,365, 1285-97. 2. Cavalieri,V et al. (2009) Nucleic Acid Res, 37,7407-7415. 3. Heger , P. et al. (2012) PNAS, 109, 17507–17512. 4. Cavalieri, V. et al. (2013) Plos Genetics, 9, e1003847.

Purpose: Apoptosis is considered a common pathological feature in acute myocardial infarction (MI) and heart failure; however its role in the later phases post MI has not been characterised. The goal of our study was to investigate by pathological examination human hearts at 20 to 30 days post MI and identify signs of ongoing cell apoptosis. Materials and Methods: Two hearts were collected at autopsy from patients who died 20 to 30 days from the onset of MI (Cases 1 and 2). Gross anatomy and light microscopy examination of the hearts was performed to define the infarcted area and the infarct-related artery. The in situ end-labeling of DNA fragmentation (TUNEL) was performed to identify apoptotic cells and the apoptotic rate (AR) was calculated. Results: There were no signs of acute necrosis in any of the specimens examined. A high number of myocardiocyte were positive at TUNEL examination in specimens obtained at sites of infarction, mean AR=44%, but not in specimens derived from the same patients at regions remote from the MI, AR=0. Conclusions: High grade apoptosis is present at sites of infarction and not in regions remote from the infarcted area in the later phases post MI. These data support persistent myocardiocyte loss and identify a possible explanation of progressive left ventricular dysfunction in the subacute phases of MI.

Aims To evaluate safety and effectiveness of clopidogrel reloading in patients on chronic clopidogrel therapy undergoing percutaneous coronary intervention (PCI). Methods and results Five hundred and three patients on >10 days clopidogrel therapy (41% with non-ST-segment elevation acute coronary syndrome, ACS) randomly received 600 mg clopidogrel loading 4–8 h before PCI ( n = 252) or placebo ( n = 251). Primary endpoint was 30-day incidence of major adverse cardiac events (MACE). In the overall population primary endpoint occurred in 6.7% of patients in the reload vs. 8.8% in the placebo arm [odds ratios (OR) 0.75, 95% confidence intervals (CI) 0.37–1.52; P = 0.50]. In stable angina patients, 1-month MACE were not significantly different (7.0 vs. 3.9%; OR 1.84, 0.60–5.88; P = 0.36), whereas ACS patients had significant clinical benefit with reloading (6.4 vs. 16.3%; OR 0.34, 95% CI 0.32–0.90, P = 0.033 at multivariable analysis; interaction test: P = 0.01). There was no excess bleeding in the reload arm (6% in both groups). Conclusion ARMYDA-4 RELOAD reveals no overall benefit from reloading patients on chronic clopidogrel therapy prior to PCI; the benefit observed in ACS patients is a hypothesis-generating finding that needs to be confirmed by larger studies.

We describe the expression of three Paracentrotus lividus homeobox-containing genes of the dispersed class during sea urchin embryogenesis and discuss their possible roles in the mechanisms of cell specification and embryo morphogenesis. PIHbox12 represents the first regulator identified in sea urchin that belongs to the zygotic class of transcription factors. Its early and transient expression and the localization of transcripts suggests that PIHbox12 is involved in cell fate specification of the oral or aboral ectodermal territories at the early cleavage stages. PIHbox9 is expressed just after the completion of gastrulation in a narrow stripe of cells at the ectoderm-endoderm boundary. It probably organizes a novel spatial boundary which definitely separates the archenteron and the aboral ectoderm. Finally, the spatial and temporal expression of the PIOtp gene strongly indicate that this regulator is conditionally activated in few cells of the oral ectoderm and is involved in patterning of this territory at late stages. Furthermore, our data indicate that PIOtp acts upstream of signaling systems that lead to the activation of the primary mesenchyme cell gene expression program and skeletal morphogenesis.

The only Italian population of the Eastern Montpellier snake is found on Lampedusa, a small island in the Strait of Sicily, Mediterranean Sea. This population is currently attributed to Malpolon insignitus based only on morphology. Here, we present the first genetic data on this population, based on mitochondrial cytochrome b and 12S ribosomal RNA data. Our results confirm that the population on Lampedusa belongs to M. insignitus, falling within a clade that also includes Tunisian populations, and presents private haplotypes for both markers. Their slight differentiation from the Tunisian populations suggests an origin linked to natural expansion from Africa, during the latest glaciation events.

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 from northern and central regions of Italy, including two populations, apparently disconnected from the rest of the known range, and analysed their phylogenetic relationships using a portion of the mitochondrial cytochrome b gene. Our results confirm the general phylogenetic arrangement detected in previous studies; specifically for Italian populations, no variability emerged from the Apennine populations, and a slight differentiation could be shown for the Alpine and subalpine ones. This pattern can be explained assuming past spread and recent isolation of C. girondica relict populations in the Alpine region, likely during the Last Glacial Maximum. Later, during the Holocene, the Italian Alps and the Po Plain went through various climatic variations and high anthropization which may have influenced C. girondica distribution through expansion and contraction processes.

The 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). Conversely, individuals collected from the most polluted site (B) showed median PFOA concentrations of 21 ng/g in coelomic fluid, 153 ng/g in coelomocytes, and 195 ng/g in gonads. Calculated bioconcentration factors of log10BCF > 3.7 confirmed the very bioaccumulative nature of PFOA. Significant correlations were found between the PFOA concentration of the coelomic fluid versus the total PFOA concentration of the entire sea urchin. PERMANOVA (p = 0.001) end Welch's t-test (p P. lividus as sentinel species for PFOA biomonitoring.

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 vitellogenesis. PERMANOVA analysis (p = 0.01) confirmed significant differences in the PAEs contamination profiles in the eggs from the four nests. This study confirms the negative impact of plastic related compounds posing questions about the potential adverse effects on organisms and their conservation status.

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 treatment with the selected molecules in cells expressing nonsense&ndash, CFTR&ndash, mRNA. Additionally, the channel functionality was measured by the halide sensitive yellow fluorescent protein (YFP) quenching assay. All three of the new TRIDs displayed high readthrough activity and low toxicity and can be considered for further evaluation as a therapeutic approach toward the second major cause of CF.

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 reptile species. To define the identity of this distinctive lineage, as well as the Macroprotodon taxonomic structure, further sampling efforts within the undersampled distribution area of this genus and more extensive analyses will be necessary.

[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 synthesized and tested using the Fluc reporter harboring the UGA PTC. Moreover, the cytotoxic effect and the expression of the CFTR protein were evaluated. These compounds, a benzimidazole derivative (NV2899), a benzoxazole derivative (NV2913), a thiazole derivative (NV2909), and a benzene-1,3-disulfonate derivative (NV2907), were shown to be potential new lead compounds as TRIDs, boosting further efforts to address the optimization of the chemical scaffolds.

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 to aminoglycosides. Additionally, recent results on the efficiency of new candidate TRIDs in restoring the production of the cystic fibrosis transmembrane regulator (CFTR) protein will be presented. Finally, a prospectus on complementary strategies to enhance the effect of TRIDs will be illustrated together with a conclusive paragraph about perspectives, opportunities, and caveats in developing small molecules as TRIDs.

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 nonsense mutation in the bronchial epithelial IB3.1 cell line carrying the W1282X mutation in the CFTR gene.

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 working group” (SPwg), which aims at monitoring the distribution and impact of the species in Sicily. To date, the SPwg found the red swamp crayfish in five new sites on the island, thus doubling the number of local sites of occurrence. The new Procambarus clarkii sites lie in different river basins, some of them located several hundred kilometres from the invaded areas known to date, suggesting the existence of multiple independent releases of the species in the wild. The need of better informing the local population on the risks exerted by invasive species on biological diversity, and of carefully monitoring the impact of P. clarkii on the Sicilian inland water biota is briefly stressed.

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 raised a specific antibody against the sea urchin GAF and used it in whole-mount immunofluorescence assays, showing that the protein was distributed predominantly to the nuclei of the whole embryo. By Chromatin Immunoprecipitation experiments, we showed that the sea urchin GAF not only binds the sns5 insulator, which is essential for the regulation of the sea urchin early-histone genes (2,3), but also to other GAGA containing sequences in the early-histone gene cluster. 1 Matharu NK et al. 2010 JMB 400 (3): 434–447 2 Melfi R et Al. 2001 JMB (1); 304:753-63 3 Cavalieri V et al. 2013 Nucleic Acids Res 37: 7407-7415

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. Here we used chromatin immunoprecipitation to demonstrate that MBF-1 occupies its site regardless of the transcriptional state of the H2A gene. In addition, we have mapped two nucleosomes specifically positioned on the enhancer and promoter regions of the repressed H2A gene. Interestingly, insertion of a 26 bp oligonucleotide between the enhancer and the TATA box, led to up-regulation of the H2A gene at gastrula stage, possibly by changing the position of the TATA nucleosome. Finally, we found association of histone de-acetylase and de-acetylation and methylation of K9 of histone H3 on the promoter and insulator of the repressed H2A chromatin. These data argue for a role of a defined positioned nucleosome in the promoter and histone tail post-translational modifications, in the 3' insulator and 5' regulatory regions, in the repression of the alpha-H2A gene despite the presence of the MBF-1 activator bound to the enhancer.

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 importance of H-bonding and stacking π-π interactions. To improve the readthrough activity we changed the fluorine number and position in the PTC124 fluoroaryl moiety. The readthrough ability of these PTC124 derivatives was tested in human cells harboring reporter plasmids with premature stop codons in H2BGFP and FLuc genes as well as in cystic fibrosis (CF) IB3.1 cells with a nonsense mutation. Maintaining low toxicity, three of these molecules showed higher efficacy than PTC124 in the readthrough of the UGA premature stop codon and in recovering the expression of the CFTR protein in IB3.1 cells from cystic fibrosis patient. Molecular dynamics simulations performed with mutated CFTR mRNA fragments and active or inactive derivatives are in agreement with the suggested interaction of PTC124 with mRNA.

Enhancer 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 binds specifically to Polycomb protein.

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.

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 codons by using a new reporter. The reporter vector was based on a plasmid harboring the H2B histone coding sequence fused in frame with the green fluorescent protein (GFP) cDNA, and a TGA stop codon was introduced in the H2B-GFP gene by site-directed mutagenesis. Additionally, an unprecedented computational study on the putative supramolecular interaction between PTC124 and an 11-codon (33-nucleotides) sequence corresponding to a CFTR mRNA fragment containing a central UGA nonsense mutation showed a specific interaction between PTC124 and the UGA codon. Altogether, the H2B-GFP-opal based assay and the molecular dynamics (MD) simulation support the hypothesis that PTC124 is able to promote the specific readthrough of internal TGA premature stop codons.

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.

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, previously characterized in sea urchin, are also specific binding sites for human proteins. We conclude that sns interferes with enhancer promoter interaction also in a human chromatin context. The relatively small size, evolutionary conservation and apparent lack of enhancer specificity might result useful in gene transfer experiments in human cells. © 2001 Academic Press.

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 azione di questa molecola (stabilizzazione proteica, superamento del codone stop o altri meccanismi) (Welch, 2007; Auld, 2009). Al fine di chiarirlo abbiamo introdotto nel gene codificante la proteina GFP del plasmide pBOS-H2BGFP un codone di stop TGA mediante mutagenesi sito diretta. Cellule HeLa trasfettate con questo vettore e cellule IB3.1 (FC) sono state utilizzate per valutare la capacità di indurre readthrough del PTC124.

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 muscular dystrophy), amber (TAG; -thalassemia, emphysema, cystic fibrosis) and ochre mutations (TAA; APC gastric cancer, Haemophilia B, Hypothyroidism) (3). Messenger RNA containing a nonsense mutation is often degraded rapidly through the process of nonsense-mutation-mediated decay (NMD) resulting in the lack of the protein (4). 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 (5). Several aminoglycosides (gentamicin, amikacin, hygromycin, etc.) can suppress the accurate identification of translation termination codons in cultured eukaryotic cells. Unfortunately, aminoglycoside action lacks specificity resulting in readthrough of many correctly positioned stop codons. Consequently, long- term use of aminoglycosides may originate toxic aggregates or dominant negative readthrough products (6). By an high throughput screening it was identified the PTC124 (Ataluren), a small molecule that has been suggested to allow PTCs readthrough (7). However, despite the results obtained on opal mutation it was shown that it has a lower activity against ochre and amber nonsense mutations (7). In the attempt to identify molecules with an activity against ochre and amber nonsense mutations, we designed and synthesized new PTC124 derivatives to be tested in human cultured cells to see if they have higher and wider activity towards PTCs than PTC124. To this aim we generated a reporter vector with non-sense mutation by introducing in the pBOS-H2BGFP plasmid a TAG codon (amber) and TAA codon (ochre) by site-directed mutagenesis. PCR and sequencing analyses confirmed the presence of the stop codons in the plasmids that were transfected in HeLa cells to explore the ability of the derivatives to promote the translational read-through. Immunofluorescence analyses showed that one of the analyzed derivatives was able to restore GFP fluorescence in HeLa H2BGFP-amber cells, as indicated by GFP-localization in the nuclei of treated cells (figure 1). This positive response also confirmed the correct functioning of the model system which will allow us to perform the screening of a greater number of molecules with read-through action.