Induction of fetal hemoglobin (HbF) is considered a promising strategy to treat β-thalassemia, wh... more Induction of fetal hemoglobin (HbF) is considered a promising strategy to treat β-thalassemia, where the production of adult hemoglobin (HbA) is impaired by mutations affecting the β-globin gene. Recent results indicate that B-cell lymphoma/leukemia 11A (BCL11A) is a major repressor of γ-globin gene expression. Therefore, disrupting the binding of the BCL11A transcriptional repressor complex to the γ-globin gene promoter provides a novel approach for inducing expression of the γ-globin genes. In order to develop a cellular screening system for the identification of BCL11A inhibitors, we have produced K562 cell clones with integrated copies of a BCL11A-XL expressing vector. We have characterized twelve K562 clones expressing different levels of BCL11A-XL and found that a clear association does exist between the levels of BCL11A-XL and the extent of hemoglobinization induced by a panel of HbF inducers. Using mithramycin as inducer, we found that this molecule was the only HbF inducer efficient in rescuing the ability to differentiate along the erythroid program, even the K562 cell clones expressing high levels of BCL11A-XL, suggesting that BCL11A-XL activity is counteracted by mithramycin.
The analysis of polymerase-chain reaction (PCR) products by surface plasmon resonance (SPR) -base... more The analysis of polymerase-chain reaction (PCR) products by surface plasmon resonance (SPR) -based biospecific interaction analysis (BIA) using biosensors; could be an excellent tool for specific and sensitive detection of genetically modified organisms (GMO). While this technology has several important advantages, a major drawback is that, in some instances, secondary structures of the PCR products to be analysed lead to low efficiency in binding to the specific probes. Cross-hybridization between PCR products and probes could also occur, especially when standard BIA conditions are employed. Here we demonstrate that biospecific interaction analysis, employing surface plasmon resonance and biosensor technologies, is suitable to detect Bt-176 "maximizer" sequences in maize. Not all the probes and PCR products were found to be suitable for SPR-based BIA. However, we designed and characterized probes able to selectively identify and discriminate Bt-176 and zein gene sequences...
Peptide nucleic acids (PNAs) are DNA mimics constituted by a pseudopeptide backbone composed of N... more Peptide nucleic acids (PNAs) are DNA mimics constituted by a pseudopeptide backbone composed of N-(2-aminoethyl)glycine units. PNAs hybridize with high affinity to complementary sequences of single-stranded RNA and DNA, forming Watson-Crick double helices and are resistant to both nucleases and proteases. While applications of PNAs as antisense and antigene molecules have been described, PNA/DNA and PNA/PNA hybrids are not useful for transcription factor decoy (TFD) pharmacotherapy. By contrast, PNA-DNA-PNA (PDP) chimeras, constituted of sequential PNA, DNA and PNA stretches, are potent decoy molecules in vitro. Interestingly, PDP-based decoys a) are more soluble than PNAs, b) are more resistant than synthetic oligonucleotides to enzymatic activity present in cellular extracts and serum and c) can be delivered with liposomes. In the present study we demonstrated that double-stranded PNA-DNA-PNA chimeras targeting NF-kappaB transcription factors induce apoptosis of human primary oste...
Nucleic-acid amplification is a crucial step in nucleic-acid-sequence-detection assays. The use o... more Nucleic-acid amplification is a crucial step in nucleic-acid-sequence-detection assays. The use of digital microfluidic devices to miniaturize amplification techniques reduces the required sample volume and the analysis time and offers new possibilities for process automation and integration in a single device. The recently introduced droplet polymerase-chain-reaction (PCR) amplification methods require repeated cycles of two or three temperature-dependent steps during the amplification of the nucleic-acid target sequence. In contrast, low-temperature isothermal-amplification methods have no need for thermal cycling, thus requiring simplified microfluidic-device features. Here, the combined use of digital microfluidics and molecular-beacon (MB)-assisted isothermal circular-strand-displacement polymerization (ICSDP) to detect microRNA-210 sequences is described. MicroRNA-210 has been described as the most consistently and predominantly upregulated hypoxia-inducible factor. The nmol L(-1)-pmol L(-1) detection capabilities of the method were first tested by targeting single-stranded DNA sequences from the genetically modified Roundup Ready soybean. The ability of the droplet-ICSDP method to discriminate between full-matched, single-mismatched, and unrelated sequences was also investigated. The detection of a range of nmol L(-1)-pmol L(-1) microRNA-210 solutions compartmentalized in nanoliter-sized droplets was performed, establishing the ability of the method to detect as little as 10(-18) mol of microRNA target sequences compartmentalized in 20 nL droplets. The suitability of the method for biological samples was tested by detecting microRNA-210 from transfected K562 cells.
We studied the effects of rapamycin on cultures of erythroid progenitors derived from the periphe... more We studied the effects of rapamycin on cultures of erythroid progenitors derived from the peripheral blood of 10 beta-thalassaemia patients differing widely with respect to their potential to produce foetal haemoglobin (HbF). For this, we employed the two-phase liquid culture procedure for growing erythroid progenitors, high performance liquid chromatography for analysis of HbF production and reverse transcription polymerase chain reaction for quantification of the accumulation of globin mRNAs. The results demonstrated that rapamycin induced an increase of HbF in cultures from all the beta-thalassaemia patients studied and an increase of their overall Hb content/cell. The inducing effect of rapamycin was restricted to gamma-globin mRNA accumulation, being only minor for beta-globin and none for alpha-globin mRNAs. The ability of rapamycin to preferentially increase gamma-globin mRNA content and production of HbF in erythroid precursor cells from beta-thalassaemia patients is of grea...
We studied the effects of rapamycin on cultures of erythroid progenitors derived from the periphe... more We studied the effects of rapamycin on cultures of erythroid progenitors derived from the peripheral blood of 10 beta-thalassaemia patients differing widely with respect to their potential to produce foetal haemoglobin (HbF). For this, we employed the two-phase liquid culture procedure for growing erythroid progenitors, high performance liquid chromatography for analysis of HbF production and reverse transcription polymerase chain reaction for quantification of the accumulation of globin mRNAs. The results demonstrated that rapamycin induced an increase of HbF in cultures from all the beta-thalassaemia patients studied and an increase of their overall Hb content/cell. The inducing effect of rapamycin was restricted to gamma-globin mRNA accumulation, being only minor for beta-globin and none for alpha-globin mRNAs. The ability of rapamycin to preferentially increase gamma-globin mRNA content and production of HbF in erythroid precursor cells from beta-thalassaemia patients is of great importance as this agent (also known as sirolimus or rapamune) is already in clinical use as an anti-rejection agent following kidney transplantation. These data suggest that rapamycin warrants further evaluation as a potential therapeutic drug in beta-thalassaemia and sickle cell anaemia.
Congenital myopathies are genetically and clinically heterogeneous conditions causing severe musc... more Congenital myopathies are genetically and clinically heterogeneous conditions causing severe muscle weakness and mutations in the ryanodine receptor gene (RYR1) represent the most frequent cause of these conditions. A common feature of diseases caused by recessive RYR1 mutations is a decrease of ryanodine receptor 1 protein content in muscle. The aim of the present investigation was to gain mechanistic insight into the causes of this reduced ryanodine receptor 1. We found that muscle biopsies of patients with recessive RYR1 mutations exhibit decreased expression of muscle specific microRNAs, increased DNA methylation and increased expression of class II histone deacetylases. Transgenic mouse muscle fibres over-expressing HDAC-4/HDAC-5 exhibited decreased expression of RYR1 and of muscle specific miRNAs, while acute knock-down of RYR1 in mouse muscle fibers by siRNA caused up-regulation of HDAC-4/HDAC-5. Intriguingly, increased class II HDAC expression and decreased ryanodine recepto...
Seeds of C. libani have been extracted through ultrasound-assisted maceration and evaluated in or... more Seeds of C. libani have been extracted through ultrasound-assisted maceration and evaluated in order to check their antiproliferative and erythroid differentiation properties. Preliminary chemical compositions of the extracts have been also performed with the object to identify the chemical classes mainly responsible of the bioactivity. Chloroform and ethanol extracts were tested for growth inhibition and erythroid differentiation induction in K562 cells. Both the extracts were then analysed by GC and GCMS. Chloroform extracts evidenced the predominant presence of terpenes, the monoterpenes α- and β- pinene checked as the most abundant (34.42±1.22 % and 33.28±1.08 % respectively), and most probably directly involved in the antiproliferative activity against K562 cells (IC50=69.20±1.69 μg/ml). Ethanol extracts evidenced a slightly higher antiproliferative activity (IC50=40.57±1.16 μg/ml) which could be mainly related to abietane diterpenoids and to the less abundant palmitic acid (0....
Expression and regulation of microRNAs is an emerging issue in erythroid differentiation and glob... more Expression and regulation of microRNAs is an emerging issue in erythroid differentiation and globin gene expression in hemoglobin disorders. In the first part of this study microarray analysis was performed both in mithramycin-induced K562 cells and erythroid precursors from healthy subjects or β-thalassemia patients producing low or high levels of fetal hemoglobin. We demonstrated that: (a) microRNA-210 expression is higher in erythroid precursors from β-thalassemia patients with high production of fetal hemoglobin; (b) microRNA-210 increases as a consequence of mithramycin treatment of K562 cells and human erythroid progenitors both from healthy and β-thalassemia subjects; (c) this increase is associated with erythroid induction and elevated expression of γ-globin genes; (d) an anti-microRNA against microRNA-210 interferes with the mithramycin-induced changes of gene expression. In the second part of the study we have obtained convergent evidences suggesting raptor mRNA as a putat...
In vitro evaluation of the biological activity of Lebanese medicinal plants extracts against herp... more In vitro evaluation of the biological activity of Lebanese medicinal plants extracts against herpes simplex virus type 1 Medicinal plants extracts are interesting novel drugs for use as antimicrobial and antiviral agents. In this study we investigate the in vitro antiviral activity of eight ethanol medicinal plant extracts against Herpes simplex virus (HSV-1) infection on monkey kidney cel-ls. Acyclovir, an antiviral agent currently applied for treatment of herpes virus type 1 infection, was used to compare the plant extracts therapeutic activity. The inhibitory concentrations (IC 50) were determi-ned for eight medicinal plants extracts obtained from the following plants: Calamintha origanifolia, Satu-reja thymbra, Prangos aspurela, Sidiritis Perfoliata, Aspurela glomerata, Erythreae Centaurium, Hysso-pus officinalis and Salvia accetabulosa. Cytotoxicity was evaluated by MTT assay in Vero cells. The selec-tive index (SI) of these medicinal plant extracts was used to prove the therap...
β-thalassemias are caused by nearly 300 mutations of the β-globin gene, leading to low or absent ... more β-thalassemias are caused by nearly 300 mutations of the β-globin gene, leading to low or absent production of adult hemoglobin. Achievements have been recently obtained on innovative therapeutic strategies for β-thalassemias, based on studies focusing on the transcriptional regulation of the γ-globin genes, epigenetic mechanisms governing erythroid differentiation, gene therapy and genetic correction of the mutations. The objective of this review is to describe recently published approaches (the review covers the years 2011 - 2014) useful for the development of novel therapeutic strategies for the treatment of β-thalassemia. Modification of β-globin gene expression in β-thalassemia cells was achieved by gene therapy (eventually in combination with induction of fetal hemoglobin [HbF]) and correction of the mutated β-globin gene. Based on recent areas of progress in understanding the control of γ-globin gene expression, novel strategies for inducing HbF have been proposed. Furthermor...
Induction of fetal hemoglobin (HbF) is considered a promising strategy to treat β-thalassemia, wh... more Induction of fetal hemoglobin (HbF) is considered a promising strategy to treat β-thalassemia, where the production of adult hemoglobin (HbA) is impaired by mutations affecting the β-globin gene. Recent results indicate that B-cell lymphoma/leukemia 11A (BCL11A) is a major repressor of γ-globin gene expression. Therefore, disrupting the binding of the BCL11A transcriptional repressor complex to the γ-globin gene promoter provides a novel approach for inducing expression of the γ-globin genes. In order to develop a cellular screening system for the identification of BCL11A inhibitors, we have produced K562 cell clones with integrated copies of a BCL11A-XL expressing vector. We have characterized twelve K562 clones expressing different levels of BCL11A-XL and found that a clear association does exist between the levels of BCL11A-XL and the extent of hemoglobinization induced by a panel of HbF inducers. Using mithramycin as inducer, we found that this molecule was the only HbF inducer efficient in rescuing the ability to differentiate along the erythroid program, even the K562 cell clones expressing high levels of BCL11A-XL, suggesting that BCL11A-XL activity is counteracted by mithramycin.
The analysis of polymerase-chain reaction (PCR) products by surface plasmon resonance (SPR) -base... more The analysis of polymerase-chain reaction (PCR) products by surface plasmon resonance (SPR) -based biospecific interaction analysis (BIA) using biosensors; could be an excellent tool for specific and sensitive detection of genetically modified organisms (GMO). While this technology has several important advantages, a major drawback is that, in some instances, secondary structures of the PCR products to be analysed lead to low efficiency in binding to the specific probes. Cross-hybridization between PCR products and probes could also occur, especially when standard BIA conditions are employed. Here we demonstrate that biospecific interaction analysis, employing surface plasmon resonance and biosensor technologies, is suitable to detect Bt-176 "maximizer" sequences in maize. Not all the probes and PCR products were found to be suitable for SPR-based BIA. However, we designed and characterized probes able to selectively identify and discriminate Bt-176 and zein gene sequences...
Peptide nucleic acids (PNAs) are DNA mimics constituted by a pseudopeptide backbone composed of N... more Peptide nucleic acids (PNAs) are DNA mimics constituted by a pseudopeptide backbone composed of N-(2-aminoethyl)glycine units. PNAs hybridize with high affinity to complementary sequences of single-stranded RNA and DNA, forming Watson-Crick double helices and are resistant to both nucleases and proteases. While applications of PNAs as antisense and antigene molecules have been described, PNA/DNA and PNA/PNA hybrids are not useful for transcription factor decoy (TFD) pharmacotherapy. By contrast, PNA-DNA-PNA (PDP) chimeras, constituted of sequential PNA, DNA and PNA stretches, are potent decoy molecules in vitro. Interestingly, PDP-based decoys a) are more soluble than PNAs, b) are more resistant than synthetic oligonucleotides to enzymatic activity present in cellular extracts and serum and c) can be delivered with liposomes. In the present study we demonstrated that double-stranded PNA-DNA-PNA chimeras targeting NF-kappaB transcription factors induce apoptosis of human primary oste...
Nucleic-acid amplification is a crucial step in nucleic-acid-sequence-detection assays. The use o... more Nucleic-acid amplification is a crucial step in nucleic-acid-sequence-detection assays. The use of digital microfluidic devices to miniaturize amplification techniques reduces the required sample volume and the analysis time and offers new possibilities for process automation and integration in a single device. The recently introduced droplet polymerase-chain-reaction (PCR) amplification methods require repeated cycles of two or three temperature-dependent steps during the amplification of the nucleic-acid target sequence. In contrast, low-temperature isothermal-amplification methods have no need for thermal cycling, thus requiring simplified microfluidic-device features. Here, the combined use of digital microfluidics and molecular-beacon (MB)-assisted isothermal circular-strand-displacement polymerization (ICSDP) to detect microRNA-210 sequences is described. MicroRNA-210 has been described as the most consistently and predominantly upregulated hypoxia-inducible factor. The nmol L(-1)-pmol L(-1) detection capabilities of the method were first tested by targeting single-stranded DNA sequences from the genetically modified Roundup Ready soybean. The ability of the droplet-ICSDP method to discriminate between full-matched, single-mismatched, and unrelated sequences was also investigated. The detection of a range of nmol L(-1)-pmol L(-1) microRNA-210 solutions compartmentalized in nanoliter-sized droplets was performed, establishing the ability of the method to detect as little as 10(-18) mol of microRNA target sequences compartmentalized in 20 nL droplets. The suitability of the method for biological samples was tested by detecting microRNA-210 from transfected K562 cells.
We studied the effects of rapamycin on cultures of erythroid progenitors derived from the periphe... more We studied the effects of rapamycin on cultures of erythroid progenitors derived from the peripheral blood of 10 beta-thalassaemia patients differing widely with respect to their potential to produce foetal haemoglobin (HbF). For this, we employed the two-phase liquid culture procedure for growing erythroid progenitors, high performance liquid chromatography for analysis of HbF production and reverse transcription polymerase chain reaction for quantification of the accumulation of globin mRNAs. The results demonstrated that rapamycin induced an increase of HbF in cultures from all the beta-thalassaemia patients studied and an increase of their overall Hb content/cell. The inducing effect of rapamycin was restricted to gamma-globin mRNA accumulation, being only minor for beta-globin and none for alpha-globin mRNAs. The ability of rapamycin to preferentially increase gamma-globin mRNA content and production of HbF in erythroid precursor cells from beta-thalassaemia patients is of grea...
We studied the effects of rapamycin on cultures of erythroid progenitors derived from the periphe... more We studied the effects of rapamycin on cultures of erythroid progenitors derived from the peripheral blood of 10 beta-thalassaemia patients differing widely with respect to their potential to produce foetal haemoglobin (HbF). For this, we employed the two-phase liquid culture procedure for growing erythroid progenitors, high performance liquid chromatography for analysis of HbF production and reverse transcription polymerase chain reaction for quantification of the accumulation of globin mRNAs. The results demonstrated that rapamycin induced an increase of HbF in cultures from all the beta-thalassaemia patients studied and an increase of their overall Hb content/cell. The inducing effect of rapamycin was restricted to gamma-globin mRNA accumulation, being only minor for beta-globin and none for alpha-globin mRNAs. The ability of rapamycin to preferentially increase gamma-globin mRNA content and production of HbF in erythroid precursor cells from beta-thalassaemia patients is of great importance as this agent (also known as sirolimus or rapamune) is already in clinical use as an anti-rejection agent following kidney transplantation. These data suggest that rapamycin warrants further evaluation as a potential therapeutic drug in beta-thalassaemia and sickle cell anaemia.
Congenital myopathies are genetically and clinically heterogeneous conditions causing severe musc... more Congenital myopathies are genetically and clinically heterogeneous conditions causing severe muscle weakness and mutations in the ryanodine receptor gene (RYR1) represent the most frequent cause of these conditions. A common feature of diseases caused by recessive RYR1 mutations is a decrease of ryanodine receptor 1 protein content in muscle. The aim of the present investigation was to gain mechanistic insight into the causes of this reduced ryanodine receptor 1. We found that muscle biopsies of patients with recessive RYR1 mutations exhibit decreased expression of muscle specific microRNAs, increased DNA methylation and increased expression of class II histone deacetylases. Transgenic mouse muscle fibres over-expressing HDAC-4/HDAC-5 exhibited decreased expression of RYR1 and of muscle specific miRNAs, while acute knock-down of RYR1 in mouse muscle fibers by siRNA caused up-regulation of HDAC-4/HDAC-5. Intriguingly, increased class II HDAC expression and decreased ryanodine recepto...
Seeds of C. libani have been extracted through ultrasound-assisted maceration and evaluated in or... more Seeds of C. libani have been extracted through ultrasound-assisted maceration and evaluated in order to check their antiproliferative and erythroid differentiation properties. Preliminary chemical compositions of the extracts have been also performed with the object to identify the chemical classes mainly responsible of the bioactivity. Chloroform and ethanol extracts were tested for growth inhibition and erythroid differentiation induction in K562 cells. Both the extracts were then analysed by GC and GCMS. Chloroform extracts evidenced the predominant presence of terpenes, the monoterpenes α- and β- pinene checked as the most abundant (34.42±1.22 % and 33.28±1.08 % respectively), and most probably directly involved in the antiproliferative activity against K562 cells (IC50=69.20±1.69 μg/ml). Ethanol extracts evidenced a slightly higher antiproliferative activity (IC50=40.57±1.16 μg/ml) which could be mainly related to abietane diterpenoids and to the less abundant palmitic acid (0....
Expression and regulation of microRNAs is an emerging issue in erythroid differentiation and glob... more Expression and regulation of microRNAs is an emerging issue in erythroid differentiation and globin gene expression in hemoglobin disorders. In the first part of this study microarray analysis was performed both in mithramycin-induced K562 cells and erythroid precursors from healthy subjects or β-thalassemia patients producing low or high levels of fetal hemoglobin. We demonstrated that: (a) microRNA-210 expression is higher in erythroid precursors from β-thalassemia patients with high production of fetal hemoglobin; (b) microRNA-210 increases as a consequence of mithramycin treatment of K562 cells and human erythroid progenitors both from healthy and β-thalassemia subjects; (c) this increase is associated with erythroid induction and elevated expression of γ-globin genes; (d) an anti-microRNA against microRNA-210 interferes with the mithramycin-induced changes of gene expression. In the second part of the study we have obtained convergent evidences suggesting raptor mRNA as a putat...
In vitro evaluation of the biological activity of Lebanese medicinal plants extracts against herp... more In vitro evaluation of the biological activity of Lebanese medicinal plants extracts against herpes simplex virus type 1 Medicinal plants extracts are interesting novel drugs for use as antimicrobial and antiviral agents. In this study we investigate the in vitro antiviral activity of eight ethanol medicinal plant extracts against Herpes simplex virus (HSV-1) infection on monkey kidney cel-ls. Acyclovir, an antiviral agent currently applied for treatment of herpes virus type 1 infection, was used to compare the plant extracts therapeutic activity. The inhibitory concentrations (IC 50) were determi-ned for eight medicinal plants extracts obtained from the following plants: Calamintha origanifolia, Satu-reja thymbra, Prangos aspurela, Sidiritis Perfoliata, Aspurela glomerata, Erythreae Centaurium, Hysso-pus officinalis and Salvia accetabulosa. Cytotoxicity was evaluated by MTT assay in Vero cells. The selec-tive index (SI) of these medicinal plant extracts was used to prove the therap...
β-thalassemias are caused by nearly 300 mutations of the β-globin gene, leading to low or absent ... more β-thalassemias are caused by nearly 300 mutations of the β-globin gene, leading to low or absent production of adult hemoglobin. Achievements have been recently obtained on innovative therapeutic strategies for β-thalassemias, based on studies focusing on the transcriptional regulation of the γ-globin genes, epigenetic mechanisms governing erythroid differentiation, gene therapy and genetic correction of the mutations. The objective of this review is to describe recently published approaches (the review covers the years 2011 - 2014) useful for the development of novel therapeutic strategies for the treatment of β-thalassemia. Modification of β-globin gene expression in β-thalassemia cells was achieved by gene therapy (eventually in combination with induction of fetal hemoglobin [HbF]) and correction of the mutated β-globin gene. Based on recent areas of progress in understanding the control of γ-globin gene expression, novel strategies for inducing HbF have been proposed. Furthermor...
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