The Experts below are selected from a list of 21 Experts worldwide ranked by ideXlab platform
Isaura Font-monclus - One of the best experts on this subject based on the ideXlab platform.
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RESEARCH ARTICLE A Novel High-Content Immunofluorescence Assay as a Tool to Identify at the Single Cell Level γ-Globin Inducing Compounds
2016Co-Authors: Marta Durlak, Cristina Fugazza, Sudharshan Elangovan, Maria Franca Marongiu, Paolo Moi, Ivan Fraietta, Gloria Barbarani, Isaura Font-monclus, Mario Mauri, Sergio OttolenghiAbstract:The identification of drugs capable of reactivating γ-globin to ameliorate β-thalassemia and Sickle Cell anemia is still a challenge, as available γ-globin inducers still have limited clinical indications. High-throughput screenings (HTS) aimed to identify new potentially therapeutic drugs require suitable first-step-screening methods combining the possibility to detect varia-tion in the γ/β globin ratio with the robustness of a cell line. We took advantage of a K562 cell line variant expressing β-globin (β-K562) to set up a new multiplexed high-content immunofluorescence assay for the quantification of γ- and β-globin content at single-cell level. The assay was validated by using the known globin inducers hemin, hydroxyurea and butyric acid and further tested in a pilot screening that confirmed HDACs as targets for γ-globin induction (as proved by siRNA-mediated HDAC3 knockdown and by treatment with HDACs inhibitors entinostat and Dacinostat) and identified Heme-oxygenases as novel can-didate targets for γ-globin induction. Indeed, Heme-oxygenase2 siRNA knockdown as well as its inhibition by Tin protoporphyrin-IX (TinPPIX) greatly increased γ-globin expression. This result is particularly interesting as several metalloporphyrins have already been devel
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High-content γ/β globin analysis as readout of siRNA screening in β-K562 confirms HDAC as targets for γ-globin activation.
2015Co-Authors: Marta Durlak, Cristina Fugazza, Sudharshan Elangovan, Maria Giuseppina Marini, Maria Franca Marongiu, Paolo Moi, Ivan Fraietta, Paolo Cappella, Gloria Barbarani, Isaura Font-monclusAbstract:A) Cells were transfected with a non-targeting oligo (siNTO) as negative control and with a siRNA directed to HDAC3. Two siRNAs were tested, with two technical replicates. C) β-K562 treated with two different HDAC inhibitors: entinostat and Dacinostat (see also S3 Fig). A and C) Immunofluorescence images (Bar = 50μm) and relative scatter plots. Data from three independent experiments are presented and statistically analyzed (B and D) as in Fig 1.
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A Novel High-Content Immunofluorescence Assay as a Tool to Identify at the Single Cell Level γ-Globin Inducing Compounds
2015Co-Authors: Marta Durlak, Cristina Fugazza, Sudharshan Elangovan, Maria Giuseppina Marini, Maria Franca Marongiu, Paolo Moi, Ivan Fraietta, Paolo Cappella, Gloria Barbarani, Isaura Font-monclusAbstract:The identification of drugs capable of reactivating γ-globin to ameliorate β-thalassemia and Sickle Cell anemia is still a challenge, as available γ-globin inducers still have limited clinical indications. High-throughput screenings (HTS) aimed to identify new potentially therapeutic drugs require suitable first-step-screening methods combining the possibility to detect variation in the γ/β globin ratio with the robustness of a cell line. We took advantage of a K562 cell line variant expressing β-globin (β-K562) to set up a new multiplexed high-content immunofluorescence assay for the quantification of γ- and β-globin content at single-cell level. The assay was validated by using the known globin inducers hemin, hydroxyurea and butyric acid and further tested in a pilot screening that confirmed HDACs as targets for γ-globin induction (as proved by siRNA-mediated HDAC3 knockdown and by treatment with HDACs inhibitors entinostat and Dacinostat) and identified Heme-oxygenases as novel candidate targets for γ-globin induction. Indeed, Heme-oxygenase2 siRNA knockdown as well as its inhibition by Tin protoporphyrin-IX (TinPPIX) greatly increased γ-globin expression. This result is particularly interesting as several metalloporphyrins have already been developed for clinical uses and could be tested (alone or in combination with other drugs) to improve pharmacological γ-globin reactivation for the treatment of β-hemoglobinopathies.
Jose Correabasurto - One of the best experts on this subject based on the ideXlab platform.
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insights into structural features of hdac1 and its selectivity inhibition elucidated by molecular dynamic simulation and molecular docking
Journal of Biomolecular Structure & Dynamics, 2019Co-Authors: Yudibeth Sixtolopez, Martiniano Bello, Jose CorreabasurtoAbstract:Histone deacetylases (HDACs) are a family of proteins whose main function is the removal of acetyl groups from lysine residues located on histone and non-histone substrates, which regulates gene transcription and other activities in cells. HDAC1 dysfunction has been implicated in cancer development and progression; thus, its inhibition has emerged as a new therapeutic strategy. Two additional metal binding sites (Site 1 and Site 2) in HDACs have been described that are primarily occupied by potassium ions, suggesting a possible structural role that affects HDAC activity. In this work, we explored the structural role of potassium ions in Site 1 and Site 2 and how they affect the interactions of compounds with high affinities for HDAC1 (AC1OCG0B, Chlamydocin, Dacinostat and Quisinostat) and SAHA (a pan-inhibitor) using molecular docking and molecular dynamics (MD) simulations in concert with a Molecular-Mechanics-Generalized-Born-Surface-Area (MMGBSA) approach. Four models were generated: one with a potassium ion (K+) in both sites (HDAC1k), a second with K+ only at site 1 (HDAC1ks1), a third with K+ only at site 2 (HDAC1ks2) and a fourth with no K+ (HDAC1wk). We found that the presence or absence of K+ not only impacted the structural flexibility of HDAC1, but also its molecular recognition, consistent with experimental findings. These results could therefore be useful for further structure-based drug design studies addressing new HDAC1 inhibitors.
Marta Durlak - One of the best experts on this subject based on the ideXlab platform.
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RESEARCH ARTICLE A Novel High-Content Immunofluorescence Assay as a Tool to Identify at the Single Cell Level γ-Globin Inducing Compounds
2016Co-Authors: Marta Durlak, Cristina Fugazza, Sudharshan Elangovan, Maria Franca Marongiu, Paolo Moi, Ivan Fraietta, Gloria Barbarani, Isaura Font-monclus, Mario Mauri, Sergio OttolenghiAbstract:The identification of drugs capable of reactivating γ-globin to ameliorate β-thalassemia and Sickle Cell anemia is still a challenge, as available γ-globin inducers still have limited clinical indications. High-throughput screenings (HTS) aimed to identify new potentially therapeutic drugs require suitable first-step-screening methods combining the possibility to detect varia-tion in the γ/β globin ratio with the robustness of a cell line. We took advantage of a K562 cell line variant expressing β-globin (β-K562) to set up a new multiplexed high-content immunofluorescence assay for the quantification of γ- and β-globin content at single-cell level. The assay was validated by using the known globin inducers hemin, hydroxyurea and butyric acid and further tested in a pilot screening that confirmed HDACs as targets for γ-globin induction (as proved by siRNA-mediated HDAC3 knockdown and by treatment with HDACs inhibitors entinostat and Dacinostat) and identified Heme-oxygenases as novel can-didate targets for γ-globin induction. Indeed, Heme-oxygenase2 siRNA knockdown as well as its inhibition by Tin protoporphyrin-IX (TinPPIX) greatly increased γ-globin expression. This result is particularly interesting as several metalloporphyrins have already been devel
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High-content γ/β globin analysis as readout of siRNA screening in β-K562 confirms HDAC as targets for γ-globin activation.
2015Co-Authors: Marta Durlak, Cristina Fugazza, Sudharshan Elangovan, Maria Giuseppina Marini, Maria Franca Marongiu, Paolo Moi, Ivan Fraietta, Paolo Cappella, Gloria Barbarani, Isaura Font-monclusAbstract:A) Cells were transfected with a non-targeting oligo (siNTO) as negative control and with a siRNA directed to HDAC3. Two siRNAs were tested, with two technical replicates. C) β-K562 treated with two different HDAC inhibitors: entinostat and Dacinostat (see also S3 Fig). A and C) Immunofluorescence images (Bar = 50μm) and relative scatter plots. Data from three independent experiments are presented and statistically analyzed (B and D) as in Fig 1.
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A Novel High-Content Immunofluorescence Assay as a Tool to Identify at the Single Cell Level γ-Globin Inducing Compounds
2015Co-Authors: Marta Durlak, Cristina Fugazza, Sudharshan Elangovan, Maria Giuseppina Marini, Maria Franca Marongiu, Paolo Moi, Ivan Fraietta, Paolo Cappella, Gloria Barbarani, Isaura Font-monclusAbstract:The identification of drugs capable of reactivating γ-globin to ameliorate β-thalassemia and Sickle Cell anemia is still a challenge, as available γ-globin inducers still have limited clinical indications. High-throughput screenings (HTS) aimed to identify new potentially therapeutic drugs require suitable first-step-screening methods combining the possibility to detect variation in the γ/β globin ratio with the robustness of a cell line. We took advantage of a K562 cell line variant expressing β-globin (β-K562) to set up a new multiplexed high-content immunofluorescence assay for the quantification of γ- and β-globin content at single-cell level. The assay was validated by using the known globin inducers hemin, hydroxyurea and butyric acid and further tested in a pilot screening that confirmed HDACs as targets for γ-globin induction (as proved by siRNA-mediated HDAC3 knockdown and by treatment with HDACs inhibitors entinostat and Dacinostat) and identified Heme-oxygenases as novel candidate targets for γ-globin induction. Indeed, Heme-oxygenase2 siRNA knockdown as well as its inhibition by Tin protoporphyrin-IX (TinPPIX) greatly increased γ-globin expression. This result is particularly interesting as several metalloporphyrins have already been developed for clinical uses and could be tested (alone or in combination with other drugs) to improve pharmacological γ-globin reactivation for the treatment of β-hemoglobinopathies.
Yudibeth Sixtolopez - One of the best experts on this subject based on the ideXlab platform.
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insights into structural features of hdac1 and its selectivity inhibition elucidated by molecular dynamic simulation and molecular docking
Journal of Biomolecular Structure & Dynamics, 2019Co-Authors: Yudibeth Sixtolopez, Martiniano Bello, Jose CorreabasurtoAbstract:Histone deacetylases (HDACs) are a family of proteins whose main function is the removal of acetyl groups from lysine residues located on histone and non-histone substrates, which regulates gene transcription and other activities in cells. HDAC1 dysfunction has been implicated in cancer development and progression; thus, its inhibition has emerged as a new therapeutic strategy. Two additional metal binding sites (Site 1 and Site 2) in HDACs have been described that are primarily occupied by potassium ions, suggesting a possible structural role that affects HDAC activity. In this work, we explored the structural role of potassium ions in Site 1 and Site 2 and how they affect the interactions of compounds with high affinities for HDAC1 (AC1OCG0B, Chlamydocin, Dacinostat and Quisinostat) and SAHA (a pan-inhibitor) using molecular docking and molecular dynamics (MD) simulations in concert with a Molecular-Mechanics-Generalized-Born-Surface-Area (MMGBSA) approach. Four models were generated: one with a potassium ion (K+) in both sites (HDAC1k), a second with K+ only at site 1 (HDAC1ks1), a third with K+ only at site 2 (HDAC1ks2) and a fourth with no K+ (HDAC1wk). We found that the presence or absence of K+ not only impacted the structural flexibility of HDAC1, but also its molecular recognition, consistent with experimental findings. These results could therefore be useful for further structure-based drug design studies addressing new HDAC1 inhibitors.
Paolo Moi - One of the best experts on this subject based on the ideXlab platform.
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RESEARCH ARTICLE A Novel High-Content Immunofluorescence Assay as a Tool to Identify at the Single Cell Level γ-Globin Inducing Compounds
2016Co-Authors: Marta Durlak, Cristina Fugazza, Sudharshan Elangovan, Maria Franca Marongiu, Paolo Moi, Ivan Fraietta, Gloria Barbarani, Isaura Font-monclus, Mario Mauri, Sergio OttolenghiAbstract:The identification of drugs capable of reactivating γ-globin to ameliorate β-thalassemia and Sickle Cell anemia is still a challenge, as available γ-globin inducers still have limited clinical indications. High-throughput screenings (HTS) aimed to identify new potentially therapeutic drugs require suitable first-step-screening methods combining the possibility to detect varia-tion in the γ/β globin ratio with the robustness of a cell line. We took advantage of a K562 cell line variant expressing β-globin (β-K562) to set up a new multiplexed high-content immunofluorescence assay for the quantification of γ- and β-globin content at single-cell level. The assay was validated by using the known globin inducers hemin, hydroxyurea and butyric acid and further tested in a pilot screening that confirmed HDACs as targets for γ-globin induction (as proved by siRNA-mediated HDAC3 knockdown and by treatment with HDACs inhibitors entinostat and Dacinostat) and identified Heme-oxygenases as novel can-didate targets for γ-globin induction. Indeed, Heme-oxygenase2 siRNA knockdown as well as its inhibition by Tin protoporphyrin-IX (TinPPIX) greatly increased γ-globin expression. This result is particularly interesting as several metalloporphyrins have already been devel
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High-content γ/β globin analysis as readout of siRNA screening in β-K562 confirms HDAC as targets for γ-globin activation.
2015Co-Authors: Marta Durlak, Cristina Fugazza, Sudharshan Elangovan, Maria Giuseppina Marini, Maria Franca Marongiu, Paolo Moi, Ivan Fraietta, Paolo Cappella, Gloria Barbarani, Isaura Font-monclusAbstract:A) Cells were transfected with a non-targeting oligo (siNTO) as negative control and with a siRNA directed to HDAC3. Two siRNAs were tested, with two technical replicates. C) β-K562 treated with two different HDAC inhibitors: entinostat and Dacinostat (see also S3 Fig). A and C) Immunofluorescence images (Bar = 50μm) and relative scatter plots. Data from three independent experiments are presented and statistically analyzed (B and D) as in Fig 1.
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A Novel High-Content Immunofluorescence Assay as a Tool to Identify at the Single Cell Level γ-Globin Inducing Compounds
2015Co-Authors: Marta Durlak, Cristina Fugazza, Sudharshan Elangovan, Maria Giuseppina Marini, Maria Franca Marongiu, Paolo Moi, Ivan Fraietta, Paolo Cappella, Gloria Barbarani, Isaura Font-monclusAbstract:The identification of drugs capable of reactivating γ-globin to ameliorate β-thalassemia and Sickle Cell anemia is still a challenge, as available γ-globin inducers still have limited clinical indications. High-throughput screenings (HTS) aimed to identify new potentially therapeutic drugs require suitable first-step-screening methods combining the possibility to detect variation in the γ/β globin ratio with the robustness of a cell line. We took advantage of a K562 cell line variant expressing β-globin (β-K562) to set up a new multiplexed high-content immunofluorescence assay for the quantification of γ- and β-globin content at single-cell level. The assay was validated by using the known globin inducers hemin, hydroxyurea and butyric acid and further tested in a pilot screening that confirmed HDACs as targets for γ-globin induction (as proved by siRNA-mediated HDAC3 knockdown and by treatment with HDACs inhibitors entinostat and Dacinostat) and identified Heme-oxygenases as novel candidate targets for γ-globin induction. Indeed, Heme-oxygenase2 siRNA knockdown as well as its inhibition by Tin protoporphyrin-IX (TinPPIX) greatly increased γ-globin expression. This result is particularly interesting as several metalloporphyrins have already been developed for clinical uses and could be tested (alone or in combination with other drugs) to improve pharmacological γ-globin reactivation for the treatment of β-hemoglobinopathies.
