Raffaella Melfi - Academia.edu (original) (raw)
Papers by Raffaella Melfi
International Journal of Molecular Sciences, Oct 10, 2023
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
PubMed, 2000
We describe the expression of three Paracentrotus lividus homeobox-containing genes of the disper... more 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. PlHbox12 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 PlHbox12 is involved in cell fate specification of the oral or aboral ectodermal territories at the early cleavage stages. PlHbox9 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 PlOtp 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 PlOtp acts upstream of signaling systems that lead to the activation of the primary mesenchyme cell gene expression program and skeletal morphogenesis.
Enhancers are DNA elements which increase the transcription of associated gene in a position and ... more 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\u2019-H4-H2B-H3-H2A-H1-3\u2019. 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 binds to a 30 bp sequence of the H2A modulator, the only cis-acting element of the histone unit for which an enhancer activity has unambiguously demonstrated. Here we assessed the specificity of the MBF-1 factor and the molecular mechanisms that eventually limit its interaction with the adjacent promoters. We find by Chromatin ImmunePrecipitation and in vivo functional studies that the MBF-1 activator binds both the H3 and H2A regulatory sequences, suggesting that this activator is needed for the maximal expression of both genes. In addition, we find that the enhancer blocking activity of the sns5 insulator buffers the downstream H1 promoter from the H2A enhancer bound MBF-1 factor. These results suggest that the block of the H2A enhancer by the sns5 insulator might be the mechanism that regulate the ratio of 2 to 1 of core to linker histones during early development of the sea urchin embryo
Nonsense mutations cause several genetic diseases such as Cystic fibrosis, Duchenne muscular dyst... more Nonsense mutations cause several genetic diseases such as Cystic fibrosis, Duchenne muscular dystrophy, β-thalassemia, and Shwachman-Diamond syndrome. These mutations induce the formation of a premature termination codon (PTC) inside the mRNA sequence that leads to the aberrant translation, resulting in the synthesis of truncated polypeptides. Nonsense suppression therapy mediated by translational readthrough-inducing drugs (TRIDs) is a promising approach to correct these genetic defects. TRIDs generate a ribosome miscoding of the PTC named “translational readthrough” and restore the synthesis of full-length and potentially functional protein. The new oxadiazole-core TRIDs NV848, NV914, and NV930 showed translational readthrough activity in nonsense-related in vitro systems. In this work, the possible off-target effect of NV molecules on natural termination codons (NTCs) was investigated. Two different in vitro approaches were used to assess if the NV molecules treatment induce NTCs...
Journal of Cystic Fibrosis
Circa il 10% dei pazienti affetti da fibrosi cistica (FC) presenta nel gene CFTR mutazioni non se... more 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 gi\ue0 note come il G418, abbiano effetti collaterali ridotti ed una maggiore specificit\ue0 per uno specifico codone. Una piccola molecola che sembra possedere una tale attivit\ue0 \ue8 il PTC124 (Welch, 2007). Ad oggi per\uf2 non c\u2019\ue8 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 capacit\ue0 di indurre readthrough del PTC124
Biotecnologie Ricerca di Base Interdisciplinare Traslazionale in ambito Biomedico 2018, 2018
In Cystic fibrosis (CF) disease nonsense mutations in the CFTR gene cause absence of the CFTR pro... more 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) showing high readthrough activity. Moreover, we evaluated quantitatively the CFTR functionality after 2a/NV2445 treatment in CF model systems and in cells expressing a nonsense-CFTR-mRNA. Finally, we studied the supramolecular interactions among readthrough inducing drugs and CFTR mRNA to assess the biological target/mechanism and further we used the QikProp program to compare the ADME properties of 2a/NV2445 and Ataluren
PROCEEDINGS OF THE 17th ITALIAN CONVENTION OF FFC INVESTIGATORS IN CYSTIC FIBROSIS, 2020
Every year, the Italian Cystic Fibrosis Research Foundation (FFC) brings together all its funded ... more Every year, the Italian Cystic Fibrosis Research Foundation (FFC) brings together all its funded researchers from across Italy and beyond, in a Convention where results from FFC projects are shared and debated. These projects are either newly funded, ongoing or recently concluded research. The Proceedings of the 17th Italian Convention of FFC Investigators in Cystic Fibrosis report the results of the concluded research projects.
International Journal of Molecular Sciences
It is reported that about 10% of cystic fibrosis (CF) patients worldwide have nonsense (stop) mut... more It is reported that about 10% of cystic fibrosis (CF) patients worldwide have nonsense (stop) mutations in the CFTR gene, which cause the premature termination of CFTR protein synthesis, leading to a truncated and non-functional protein. To address this issue, we investigated the possibility of rescuing the CFTR nonsense mutation (UGA) by sequence-specific RNA editing in CFTR mutant CFF-16HBEge, W1282X, and G542X human bronchial cells. We used two different base editor tools that take advantage of ADAR enzymes (adenosine deaminase acting on RNA) to edit adenosine to inosine (A-to-I) within the mRNA: the REPAIRv2 (RNA Editing for Programmable A to I Replacement, version 2) and the minixABE (A to I Base Editor). Immunofluorescence experiments show that both approaches were able to recover the CFTR protein in the CFTR mutant cells. In addition, RT-qPCR confirmed the rescue of the CFTR full transcript. These findings suggest that site-specific RNA editing may efficiently correct the UGA...
Cystic Fibrosis (CF) is an autosomal recessive genetic disease caused by mutations in the CFTR ge... more 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 ribosome’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 investigate a possible mechanism of action (MOA) by which our recently synthesized TRIDs, namely NV848, NV914, and NV930, could exert their readthrough activity by in silico analysis and in vitro studies. Our results suggest a likely inhibition of FTSJ1, a tryptophan tRNA-specific 2’-O-methyltransferase.
Acta Herpetologica
The Southern smooth snake, Coronella girondica, is a small-sized colubrid found in Northwest Afri... more 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 ...
Environmental Science and Pollution Research
Perfluorooctanoic acid (PFOA) has been largely used in the manufacturing industry but a few years... more Perfluorooctanoic 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’ physiological pathways, we analysed the expression profile of some marker genes both in the gonads and in the embryos obtained from parents exposed to PFOA. Our results suggest that PFOA is a persistent, bioaccumulative compound that adversely affects the health of the exposed organisms and their offspring ...
Biomedicine & Pharmacotherapy
To dissect the role of ISWI-mediated chromatin remodeler in controlling stem celi self-renewal, I... more To dissect the role of ISWI-mediated chromatin remodeler in controlling stem celi self-renewal, I developed a strategy to purify a large numbers of pure GSCs from the Drosophila ovary. Using this approach I generated a genome-wide transcriptome and chromatin-binding profile of ISWl on GSCs chromatin. To identify the potential regions of the genome that are bound by ISWI in GSCs, I conducted a ChIP-Seq analysis and found nearly 7000 ISWI bound coding genes. Moreover, RNA-Seq experiments conducted in ISWI mutant GSCs revealed ISWI as major regulator of about 70 % of its target genes in GSCs. Furthermore, by gene ontology analysis I identifìed specific gene networks under the control of ISWI. Particularly, I found that the ISWI regualtes genes playing an essential role in the maintenance of GSCs.. Our data suggest that the ATP-dependent chromatin remodeler ISWI works as a master regulator of GSCs self-renewal in the Drosophila ovary.
VII Congresso Nazionale del …, 2009
... PNAS USA, 91, 12322-6 5. Di Caro, D, Melfi, R, Alessandro, C, Serio, G, Di Caro, V, Cavalieri... more ... PNAS USA, 91, 12322-6 5. Di Caro, D, Melfi, R, Alessandro, C, Serio, G, Di Caro, V, Cavalieri, V, Palla, F, Spinelli, G (2004). J Mol Biol, 342, 1367-77 6. D'Apolito, D, Baiamonte, E, Bagliesi, M, Di Marzo, R, Calzolari, R, Ferro, L, Franco, V, Spinelli, G, Maggio, A, Acuto, S (2009). ...
Environmental Science and Pollution Research
The bioaccumulation of phthalates was studied in fragments of Ulva lactuca exposed for a maximum ... more 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 accumulation (biota-sediment accumulation factor (log10BSAF) ranging from 3.75 to 4.02) and photodegradation (higher than 70% removal for all phthalates in 8 h) are more efficient at lower concentration levels. These results suggest the potential use of the algal matrices for environmental bioremediation, in o...
ACS Medicinal Chemistry Letters, 2020
Translational readthrough-inducing drugs (TRIDs) rescue the functional full-length protein expres... more 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.
The presence of Premature Stop Codons (PTCs) in mRNA results in protein truncation that is respon... more The presence of Premature Stop Codons (PTCs) in mRNA results in protein truncation that is responsible for inherited (genetic) diseases. Approximately 10% (worldwide) of patients affected by cystic fibrosis (CF) have nonsense mutations (UAA, UAG or UGA) in the CF trans-membrane regulator (CFTR) gene. CFTR mutations in the two genes (alleles) of a patient can be different, with one mutation being delta-F508 and the other a nonsense mutation. Pharmacological approaches aimed to rescue protein function have been proposed to directly overcome nonsense mutations. PTC124 (Ataluren) a small molecule that mimic the activity of aminoglycosides has been suggested to allow PTCs readthrough (Welch EM et al. Nature. 2007 May 3;447(7140):87-91.). However, despite the results obtained from "in vitro" and "in vivo" experiments as well the advanced clinical trials done with Ataluren, some caveats exist. In fact Ataluren has a lower activity against UAA and UAG than UGA nonsense mutations and also there is no general consensus about its mechanism of action. We think that is very important to develop drugs capable of promoting better PTCs read-through found in CF patients. Our project is aimed to design and synthesize new small molecules possessing wider activity towards PTCs than Ataluren. To this end Ataluren derivatives with the geometrical requirements to match the hydrogen bonding of the PTCs present in the mRNA will be synthesized. To evaluate these small molecules we will use human cultured cells engineerized with plasmids harboring PTCs in the H2BGFP, FLuc and RLuc reporter genes (Auld DS et al. Proc Natl Acad Sci U S A. 2009 Mar 3;106(9):3585-90.). Ability of the new molecules to promote PTCs read-through will be assessed by detecting fluorescence of the H2BGFP reporter gene by microscopy and by luminometer, and at molecular level by immunoblotting and Real time RT-PCR. Validation will consist in assessing CFTR status showed by IB3-1epithelial cells after treatment with the newly synthesized small molecules. We expect to obtain newly synthesized compounds displaying, in preclinical settings, better bioavailability and better activity than Ataluren to restore expression of the full-length proteins. The project deals with innovative research and its results could open up new avenues for the understanding of the mechanism of action of these small molecules as well for the development of new drugs for PTCs caused pathology
The only Italian population of false smooth snakes is found on Lampedusa, a small island located ... more 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.
International Journal of Molecular Sciences, Oct 10, 2023
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
PubMed, 2000
We describe the expression of three Paracentrotus lividus homeobox-containing genes of the disper... more 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. PlHbox12 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 PlHbox12 is involved in cell fate specification of the oral or aboral ectodermal territories at the early cleavage stages. PlHbox9 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 PlOtp 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 PlOtp acts upstream of signaling systems that lead to the activation of the primary mesenchyme cell gene expression program and skeletal morphogenesis.
Enhancers are DNA elements which increase the transcription of associated gene in a position and ... more 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\u2019-H4-H2B-H3-H2A-H1-3\u2019. 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 binds to a 30 bp sequence of the H2A modulator, the only cis-acting element of the histone unit for which an enhancer activity has unambiguously demonstrated. Here we assessed the specificity of the MBF-1 factor and the molecular mechanisms that eventually limit its interaction with the adjacent promoters. We find by Chromatin ImmunePrecipitation and in vivo functional studies that the MBF-1 activator binds both the H3 and H2A regulatory sequences, suggesting that this activator is needed for the maximal expression of both genes. In addition, we find that the enhancer blocking activity of the sns5 insulator buffers the downstream H1 promoter from the H2A enhancer bound MBF-1 factor. These results suggest that the block of the H2A enhancer by the sns5 insulator might be the mechanism that regulate the ratio of 2 to 1 of core to linker histones during early development of the sea urchin embryo
Nonsense mutations cause several genetic diseases such as Cystic fibrosis, Duchenne muscular dyst... more Nonsense mutations cause several genetic diseases such as Cystic fibrosis, Duchenne muscular dystrophy, β-thalassemia, and Shwachman-Diamond syndrome. These mutations induce the formation of a premature termination codon (PTC) inside the mRNA sequence that leads to the aberrant translation, resulting in the synthesis of truncated polypeptides. Nonsense suppression therapy mediated by translational readthrough-inducing drugs (TRIDs) is a promising approach to correct these genetic defects. TRIDs generate a ribosome miscoding of the PTC named “translational readthrough” and restore the synthesis of full-length and potentially functional protein. The new oxadiazole-core TRIDs NV848, NV914, and NV930 showed translational readthrough activity in nonsense-related in vitro systems. In this work, the possible off-target effect of NV molecules on natural termination codons (NTCs) was investigated. Two different in vitro approaches were used to assess if the NV molecules treatment induce NTCs...
Journal of Cystic Fibrosis
Circa il 10% dei pazienti affetti da fibrosi cistica (FC) presenta nel gene CFTR mutazioni non se... more 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 gi\ue0 note come il G418, abbiano effetti collaterali ridotti ed una maggiore specificit\ue0 per uno specifico codone. Una piccola molecola che sembra possedere una tale attivit\ue0 \ue8 il PTC124 (Welch, 2007). Ad oggi per\uf2 non c\u2019\ue8 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 capacit\ue0 di indurre readthrough del PTC124
Biotecnologie Ricerca di Base Interdisciplinare Traslazionale in ambito Biomedico 2018, 2018
In Cystic fibrosis (CF) disease nonsense mutations in the CFTR gene cause absence of the CFTR pro... more 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) showing high readthrough activity. Moreover, we evaluated quantitatively the CFTR functionality after 2a/NV2445 treatment in CF model systems and in cells expressing a nonsense-CFTR-mRNA. Finally, we studied the supramolecular interactions among readthrough inducing drugs and CFTR mRNA to assess the biological target/mechanism and further we used the QikProp program to compare the ADME properties of 2a/NV2445 and Ataluren
PROCEEDINGS OF THE 17th ITALIAN CONVENTION OF FFC INVESTIGATORS IN CYSTIC FIBROSIS, 2020
Every year, the Italian Cystic Fibrosis Research Foundation (FFC) brings together all its funded ... more Every year, the Italian Cystic Fibrosis Research Foundation (FFC) brings together all its funded researchers from across Italy and beyond, in a Convention where results from FFC projects are shared and debated. These projects are either newly funded, ongoing or recently concluded research. The Proceedings of the 17th Italian Convention of FFC Investigators in Cystic Fibrosis report the results of the concluded research projects.
International Journal of Molecular Sciences
It is reported that about 10% of cystic fibrosis (CF) patients worldwide have nonsense (stop) mut... more It is reported that about 10% of cystic fibrosis (CF) patients worldwide have nonsense (stop) mutations in the CFTR gene, which cause the premature termination of CFTR protein synthesis, leading to a truncated and non-functional protein. To address this issue, we investigated the possibility of rescuing the CFTR nonsense mutation (UGA) by sequence-specific RNA editing in CFTR mutant CFF-16HBEge, W1282X, and G542X human bronchial cells. We used two different base editor tools that take advantage of ADAR enzymes (adenosine deaminase acting on RNA) to edit adenosine to inosine (A-to-I) within the mRNA: the REPAIRv2 (RNA Editing for Programmable A to I Replacement, version 2) and the minixABE (A to I Base Editor). Immunofluorescence experiments show that both approaches were able to recover the CFTR protein in the CFTR mutant cells. In addition, RT-qPCR confirmed the rescue of the CFTR full transcript. These findings suggest that site-specific RNA editing may efficiently correct the UGA...
Cystic Fibrosis (CF) is an autosomal recessive genetic disease caused by mutations in the CFTR ge... more 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 ribosome’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 investigate a possible mechanism of action (MOA) by which our recently synthesized TRIDs, namely NV848, NV914, and NV930, could exert their readthrough activity by in silico analysis and in vitro studies. Our results suggest a likely inhibition of FTSJ1, a tryptophan tRNA-specific 2’-O-methyltransferase.
Acta Herpetologica
The Southern smooth snake, Coronella girondica, is a small-sized colubrid found in Northwest Afri... more 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 ...
Environmental Science and Pollution Research
Perfluorooctanoic acid (PFOA) has been largely used in the manufacturing industry but a few years... more Perfluorooctanoic 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’ physiological pathways, we analysed the expression profile of some marker genes both in the gonads and in the embryos obtained from parents exposed to PFOA. Our results suggest that PFOA is a persistent, bioaccumulative compound that adversely affects the health of the exposed organisms and their offspring ...
Biomedicine & Pharmacotherapy
To dissect the role of ISWI-mediated chromatin remodeler in controlling stem celi self-renewal, I... more To dissect the role of ISWI-mediated chromatin remodeler in controlling stem celi self-renewal, I developed a strategy to purify a large numbers of pure GSCs from the Drosophila ovary. Using this approach I generated a genome-wide transcriptome and chromatin-binding profile of ISWl on GSCs chromatin. To identify the potential regions of the genome that are bound by ISWI in GSCs, I conducted a ChIP-Seq analysis and found nearly 7000 ISWI bound coding genes. Moreover, RNA-Seq experiments conducted in ISWI mutant GSCs revealed ISWI as major regulator of about 70 % of its target genes in GSCs. Furthermore, by gene ontology analysis I identifìed specific gene networks under the control of ISWI. Particularly, I found that the ISWI regualtes genes playing an essential role in the maintenance of GSCs.. Our data suggest that the ATP-dependent chromatin remodeler ISWI works as a master regulator of GSCs self-renewal in the Drosophila ovary.
VII Congresso Nazionale del …, 2009
... PNAS USA, 91, 12322-6 5. Di Caro, D, Melfi, R, Alessandro, C, Serio, G, Di Caro, V, Cavalieri... more ... PNAS USA, 91, 12322-6 5. Di Caro, D, Melfi, R, Alessandro, C, Serio, G, Di Caro, V, Cavalieri, V, Palla, F, Spinelli, G (2004). J Mol Biol, 342, 1367-77 6. D'Apolito, D, Baiamonte, E, Bagliesi, M, Di Marzo, R, Calzolari, R, Ferro, L, Franco, V, Spinelli, G, Maggio, A, Acuto, S (2009). ...
Environmental Science and Pollution Research
The bioaccumulation of phthalates was studied in fragments of Ulva lactuca exposed for a maximum ... more 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 accumulation (biota-sediment accumulation factor (log10BSAF) ranging from 3.75 to 4.02) and photodegradation (higher than 70% removal for all phthalates in 8 h) are more efficient at lower concentration levels. These results suggest the potential use of the algal matrices for environmental bioremediation, in o...
ACS Medicinal Chemistry Letters, 2020
Translational readthrough-inducing drugs (TRIDs) rescue the functional full-length protein expres... more 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.
The presence of Premature Stop Codons (PTCs) in mRNA results in protein truncation that is respon... more The presence of Premature Stop Codons (PTCs) in mRNA results in protein truncation that is responsible for inherited (genetic) diseases. Approximately 10% (worldwide) of patients affected by cystic fibrosis (CF) have nonsense mutations (UAA, UAG or UGA) in the CF trans-membrane regulator (CFTR) gene. CFTR mutations in the two genes (alleles) of a patient can be different, with one mutation being delta-F508 and the other a nonsense mutation. Pharmacological approaches aimed to rescue protein function have been proposed to directly overcome nonsense mutations. PTC124 (Ataluren) a small molecule that mimic the activity of aminoglycosides has been suggested to allow PTCs readthrough (Welch EM et al. Nature. 2007 May 3;447(7140):87-91.). However, despite the results obtained from "in vitro" and "in vivo" experiments as well the advanced clinical trials done with Ataluren, some caveats exist. In fact Ataluren has a lower activity against UAA and UAG than UGA nonsense mutations and also there is no general consensus about its mechanism of action. We think that is very important to develop drugs capable of promoting better PTCs read-through found in CF patients. Our project is aimed to design and synthesize new small molecules possessing wider activity towards PTCs than Ataluren. To this end Ataluren derivatives with the geometrical requirements to match the hydrogen bonding of the PTCs present in the mRNA will be synthesized. To evaluate these small molecules we will use human cultured cells engineerized with plasmids harboring PTCs in the H2BGFP, FLuc and RLuc reporter genes (Auld DS et al. Proc Natl Acad Sci U S A. 2009 Mar 3;106(9):3585-90.). Ability of the new molecules to promote PTCs read-through will be assessed by detecting fluorescence of the H2BGFP reporter gene by microscopy and by luminometer, and at molecular level by immunoblotting and Real time RT-PCR. Validation will consist in assessing CFTR status showed by IB3-1epithelial cells after treatment with the newly synthesized small molecules. We expect to obtain newly synthesized compounds displaying, in preclinical settings, better bioavailability and better activity than Ataluren to restore expression of the full-length proteins. The project deals with innovative research and its results could open up new avenues for the understanding of the mechanism of action of these small molecules as well for the development of new drugs for PTCs caused pathology
The only Italian population of false smooth snakes is found on Lampedusa, a small island located ... more 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.