2023
|
Mazzini G; Danova M Histochemistry in Advanced Cytometry: From Fluorochromes to Mass Probes Book Chapter In: Marco Biggiogera Carlo Pellicciari, Manuela Malatesta (Ed.): vol. 2556, pp. 1-25, Humana New York, NY, Histochemistry of Single Molecules Methods and Protocols, 2023. @inbook{%a1.%Yb_73,
title = {Histochemistry in Advanced Cytometry: From Fluorochromes to Mass Probes},
author = {Mazzini G and Danova M},
editor = {Carlo Pellicciari, Marco Biggiogera, Manuela Malatesta},
url = {https://link.springer.com/protocol/10.1007/978-1-0716-2675-7_1},
doi = {10.1007/978-1-0716-2675-7_1},
year = {2023},
date = {2023-03-09},
journal = {Methods in molecular biology - Histochemistry of Single Molecules },
volume = {2556},
pages = {1-25},
publisher = {Humana New York, NY},
edition = {Histochemistry of Single Molecules Methods and Protocols},
abstract = {For over half a century, fluorescence has been the milestone of most of the quantitative approaches in various fields from chemistry and biochemistry to microscopy. This latter also evolved into cytometry, thanks to the development of fluorescence techniques. The dyes of classical cytochemistry were replaced by fluorochromes, and the pioneer microphotometry was replaced by microfluorometry. The latter has great advantages in terms of simplicity, sensitivity, and accuracy. The extensive research and availability of new fluorochromes as well as the technological evolution contributed to the success of microfluorometry. The development of flow cytometry in the 1960s gave a giant boost to cell analysis and in particular to the clinical diagnostics. The synergy between flow cytometry and the subsequent development of monoclonal antibodies allowed the setup of multiparametric analytical panels that are today popular and irreplaceable in many clinical and research laboratories. Multiparametric analysis has required the application of an increasing number of fluorochromes, but their simultaneous use creates problems of mutual contamination, hence the need to develop new fluorescent probes. Semiconductor and nanotechnology research enabled the development of new probes called nanocrystals or quantum dots, which offered great advantages to the multiparametric analysis: in fact, thanks to their spectrofluorometric peculiarities, dozens of quantum dots may be simultaneously used without appreciable crosstalk between them. New analytical horizons in cytometry seem to be associated with a new concept of analysis that replaces fluorescence toward new markers with (non-radiative) isotopes of heavy metals. Thus, the mass flow cytometry was born, which seems to guarantee the simultaneous compensation-free analysis of up to 100 markers on a single sample aliquot.},
keywords = {},
pubstate = {published},
tppubtype = {inbook}
}
For over half a century, fluorescence has been the milestone of most of the quantitative approaches in various fields from chemistry and biochemistry to microscopy. This latter also evolved into cytometry, thanks to the development of fluorescence techniques. The dyes of classical cytochemistry were replaced by fluorochromes, and the pioneer microphotometry was replaced by microfluorometry. The latter has great advantages in terms of simplicity, sensitivity, and accuracy. The extensive research and availability of new fluorochromes as well as the technological evolution contributed to the success of microfluorometry. The development of flow cytometry in the 1960s gave a giant boost to cell analysis and in particular to the clinical diagnostics. The synergy between flow cytometry and the subsequent development of monoclonal antibodies allowed the setup of multiparametric analytical panels that are today popular and irreplaceable in many clinical and research laboratories. Multiparametric analysis has required the application of an increasing number of fluorochromes, but their simultaneous use creates problems of mutual contamination, hence the need to develop new fluorescent probes. Semiconductor and nanotechnology research enabled the development of new probes called nanocrystals or quantum dots, which offered great advantages to the multiparametric analysis: in fact, thanks to their spectrofluorometric peculiarities, dozens of quantum dots may be simultaneously used without appreciable crosstalk between them. New analytical horizons in cytometry seem to be associated with a new concept of analysis that replaces fluorescence toward new markers with (non-radiative) isotopes of heavy metals. Thus, the mass flow cytometry was born, which seems to guarantee the simultaneous compensation-free analysis of up to 100 markers on a single sample aliquot. |
Casali C; Galgano L; Zannino L; Siciliani S; Cavallo M; Mazzini G; Biggiogera M Impact of heat and cold shock on epigenetics and chromatin structure Journal Article In: European journal of cell biology, vol. 103, iss. 1, pp. 151373, 2023. @article{%a1.%Y_124,
title = {Impact of heat and cold shock on epigenetics and chromatin structure},
author = {Casali C and Galgano L and Zannino L and Siciliani S and Cavallo M and Mazzini G and Biggiogera M},
url = {https://www.sciencedirect.com/science/article/pii/S0171933523000882?via%3Dihub},
doi = {10.1016/j.ejcb.2023.151373},
year = {2023},
date = {2023-12-02},
urldate = {2024-01-28},
journal = {European journal of cell biology},
volume = {103},
issue = {1},
pages = {151373},
abstract = {Cells are continuously exposed to various sources of insults, among which temperature variations are extremely common. Epigenetic mechanisms, critical players in gene expression regulation, undergo alterations due to these stressors, potentially leading to health issues. Despite the significance of DNA methylation and histone modifications in gene expression regulation, their changes following heat and cold shock in human cells remain poorly understood. In this study, we investigated the epigenetic profiles of human cells subjected to hyperthermia and hypothermia, revealing significant variations. Heat shock primarily led to DNA methylation increments and epigenetic modifications associated with gene expression silencing. In contrast, cold shock presented a complex scenario, with both methylation and demethylation levels increasing, indicating different epigenetic responses to the opposite thermal stresses. These temperature-induced alterations in the epigenome, particularly their impact on chromatin structural organization, represent an understudied area that could offer important insights into genome function and potential prospects for therapeutic targets.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Cells are continuously exposed to various sources of insults, among which temperature variations are extremely common. Epigenetic mechanisms, critical players in gene expression regulation, undergo alterations due to these stressors, potentially leading to health issues. Despite the significance of DNA methylation and histone modifications in gene expression regulation, their changes following heat and cold shock in human cells remain poorly understood. In this study, we investigated the epigenetic profiles of human cells subjected to hyperthermia and hypothermia, revealing significant variations. Heat shock primarily led to DNA methylation increments and epigenetic modifications associated with gene expression silencing. In contrast, cold shock presented a complex scenario, with both methylation and demethylation levels increasing, indicating different epigenetic responses to the opposite thermal stresses. These temperature-induced alterations in the epigenome, particularly their impact on chromatin structural organization, represent an understudied area that could offer important insights into genome function and potential prospects for therapeutic targets. |
2021
|
Pelizzo G; Riva F; Croce S; Avanzini MA; Acquafredda G; de Silvestri A; Mazzon E; Bramanti P; Zuccotti G; Mazzini G; Calcaterra V Proliferation Pattern of Pediatric Tumor-Derived Mesenchymal Stromal Cells and Role in Cancer Dormancy: A Perspective of Study for Surgical Strategy Journal Article In: Frontiers in pediatrics, vol. 9, pp. 766610, 2021. @article{%a1:%Yb_69,
title = {Proliferation Pattern of Pediatric Tumor-Derived Mesenchymal Stromal Cells and Role in Cancer Dormancy: A Perspective of Study for Surgical Strategy},
author = {Pelizzo G and Riva F and Croce S and Avanzini MA and Acquafredda G and de Silvestri A and Mazzon E and Bramanti P and Zuccotti G and Mazzini G and Calcaterra V},
editor = {Pelizzo G and Riva F and Croce S and Avanzini MA and Acquafredda G and de Silvestri A and Mazzon E and Bramanti P and Zuccotti G and Mazzini G and Calcaterra V},
url = {https://www.frontiersin.org/articles/10.3389/fped.2021.766610/full},
doi = {10.3389/fped.2021.766610},
year = {2021},
date = {2021-12-14},
urldate = {2021-12-14},
journal = {Frontiers in pediatrics},
volume = {9},
pages = {766610},
abstract = {The explanation for cancer recurrence still remains to be fully elucidated. Moreover, tumor dormancy, which is a process whereby cells enter reversible G0 cell cycle arrest, appears to be a critical step in this phenomenon. We evaluated the cell cycle proliferation pattern in pediatric tumor-derived mesenchymal stromal cells (MSCs), in order to provide a better understanding of the complex mechanisms underlying cancer dormancy. Specimens were obtained from 14 pediatric patients diagnosed with solid tumors and submitted to surgery. Morphology, phenotype, differentiation, immunological capacity, and proliferative growth of tumor MSCs were studied. Flow cytometric analysis was performed to evaluate the cell percentage of each cell cycle phase. Healthy donor bone marrow-derived mesenchymal stromal cells (BM-MSCs) were employed as controls. It was noted that the DNA profile of proliferating BM-MSC was different from that of tumor MSCs. All BM-MSCs expressed the typical DNA profile of proliferating cells, while in all tumor MSC samples, ≥70% of the cells were detected in the G0/G1 phase. In particular, seven tumor MSC samples displayed intermediate cell cycle behavior, and the other seven tumor MSC samples exhibited a slow cell cycle. The increased number of tumor MSCs in the G0-G1 phase compared with BM-MSCs supports a role for quiescent MSCs in tumor dormancy regulation. Understanding the mechanisms that promote dormant cell cycle arrest is essential in identifying predictive markers of recurrence and to promote a dedicated surgical planning.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The explanation for cancer recurrence still remains to be fully elucidated. Moreover, tumor dormancy, which is a process whereby cells enter reversible G0 cell cycle arrest, appears to be a critical step in this phenomenon. We evaluated the cell cycle proliferation pattern in pediatric tumor-derived mesenchymal stromal cells (MSCs), in order to provide a better understanding of the complex mechanisms underlying cancer dormancy. Specimens were obtained from 14 pediatric patients diagnosed with solid tumors and submitted to surgery. Morphology, phenotype, differentiation, immunological capacity, and proliferative growth of tumor MSCs were studied. Flow cytometric analysis was performed to evaluate the cell percentage of each cell cycle phase. Healthy donor bone marrow-derived mesenchymal stromal cells (BM-MSCs) were employed as controls. It was noted that the DNA profile of proliferating BM-MSC was different from that of tumor MSCs. All BM-MSCs expressed the typical DNA profile of proliferating cells, while in all tumor MSC samples, ≥70% of the cells were detected in the G0/G1 phase. In particular, seven tumor MSC samples displayed intermediate cell cycle behavior, and the other seven tumor MSC samples exhibited a slow cell cycle. The increased number of tumor MSCs in the G0-G1 phase compared with BM-MSCs supports a role for quiescent MSCs in tumor dormancy regulation. Understanding the mechanisms that promote dormant cell cycle arrest is essential in identifying predictive markers of recurrence and to promote a dedicated surgical planning. |
2020
|
Gaipa G; Erba E; Danova M; Mazzini G; Venditti A; Buldini B; Specchia G; Maglia O; Kunkl A; Ciriello MM; Arpinati M; Mannelli F; Lanza F; Riccioni R; Pistotti V; Apolone G; Società Italiana di Citometria (GIC) Health technology assessment-based approach to flow cytometric immunophenotyping of acute leukemias: a literature classification Journal Article In: Tumori, vol. 106, no. 3, pp. 249-256, 2020. @article{%a1:%Y__510,
title = {Health technology assessment-based approach to flow cytometric immunophenotyping of acute leukemias: a literature classification},
author = {Gaipa G and Erba E and Danova M and Mazzini G and Venditti A and Buldini B and Specchia G and Maglia O and Kunkl A and Ciriello MM and Arpinati M and Mannelli F and Lanza F and Riccioni R and Pistotti V and Apolone G and {Società Italiana di Citometria (GIC)}},
url = {https://journals.sagepub.com/doi/10.1177/0300891620904412?url_ver=Z39.88 2003&rfr_id=ori%3Arid%3Acrossref.org&rfr_dat=cr_pub++0pubmed&},
doi = {10.1177/0300891620904412.},
year = {2020},
date = {2020-03-12},
journal = {Tumori},
volume = {106},
number = {3},
pages = {249-256},
abstract = {Objective: Acute leukemia (AL) is a broad, heterogeneous group of malignant diseases. The diagnostic workup of AL is based on several clinical and laboratory findings, including flow cytometric immunophenotyping. However, the role of this assay in the diagnosis of AL has not been systematically investigated. The aim of this study was to determine the accuracy and utility of flow cytometric immunophenotyping in the identification, characterization, and staging of AL. Methods: We performed a systematic selection and classification of the literature since 1980, focused on flow cytometric immunophenotyping of AL. We applied a 6-variables model to cover both the technical capabilities and the clinical value of flow cytometric immunophenotyping in the diagnosis of AL. Results: Using 3 key words (acute leukemia, immunophenotyping, flow cytometry), we screened the literature from January 1985 to April 2015 in PubMed and Embase databases and found 1010 articles. A total of 363 were selected and submitted to the expert panel, which selected a final data set of 248 articles to be analyzed. Of these, 160 were focused on clinical and biological issues, 55 were technical articles, and 31 were reviews. These 248 articles were then analyzed according to the 6-variables model and definitively classified. Conclusions: We assessed the literature on flow cytometric immunophenotyping of AL over 3 decades as the first step toward an evidence-based analysis of the impact of this technology on the clinical management of patients with AL.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Objective: Acute leukemia (AL) is a broad, heterogeneous group of malignant diseases. The diagnostic workup of AL is based on several clinical and laboratory findings, including flow cytometric immunophenotyping. However, the role of this assay in the diagnosis of AL has not been systematically investigated. The aim of this study was to determine the accuracy and utility of flow cytometric immunophenotyping in the identification, characterization, and staging of AL. Methods: We performed a systematic selection and classification of the literature since 1980, focused on flow cytometric immunophenotyping of AL. We applied a 6-variables model to cover both the technical capabilities and the clinical value of flow cytometric immunophenotyping in the diagnosis of AL. Results: Using 3 key words (acute leukemia, immunophenotyping, flow cytometry), we screened the literature from January 1985 to April 2015 in PubMed and Embase databases and found 1010 articles. A total of 363 were selected and submitted to the expert panel, which selected a final data set of 248 articles to be analyzed. Of these, 160 were focused on clinical and biological issues, 55 were technical articles, and 31 were reviews. These 248 articles were then analyzed according to the 6-variables model and definitively classified. Conclusions: We assessed the literature on flow cytometric immunophenotyping of AL over 3 decades as the first step toward an evidence-based analysis of the impact of this technology on the clinical management of patients with AL. |
2019
|
Buzzella A; Mazzini G; Vicini R; Angelinetta C; Pastoris O A preliminary study of an alternative method for evaluating skin sensitizing potential of chemicals. Journal Article In: International journal of cosmetic science, vol. 41, no. 3, pp. 257-264, 2019. @article{%a1:%Y_81,
title = {A preliminary study of an alternative method for evaluating skin sensitizing potential of chemicals.},
author = {Buzzella A and Mazzini G and Vicini R and Angelinetta C and Pastoris O},
url = {https://onlinelibrary.wiley.com/doi/full/10.1111/ics.12530},
doi = {10.1111/ics.12530},
year = {2019},
date = {2019-02-20},
journal = {International journal of cosmetic science},
volume = {41},
number = {3},
pages = {257-264},
abstract = {BACKGROUND: In order to comply with the European legislation concerning the risk assessment of skin sensitizers, considerable progress has been made in developing alternative methods, such as human cell line activation test (h-CLAT). H-CLAT is based on cytometric measurement of fluorescence emitted by anti-CD54 and anti-CD86 antibodies in THP-1 cells. Following this method, a range of substances have been analyzed; the emitted fluorescence, generally at low intensity, has caused problems concerning the interpretation of results. AIM: Find an alternative parameter to h-CLAT for evaluating the sensitizing potential of chemicals. MATERIALS AND METHODS: Cells have been analyzed with flow cytometry after treatment with sensitizing compounds administered at non-cytotoxic concentrations. RESULTS: Sensitizers were able to inducealterations in cell morphology to a more 'condensed' one allowing the identification of cells under microscope as a 'sensitized' subpopulation. These variations cause similar modifications in 'scattering' parameters, making cells easily monitorable by flow cytometry. No changes have been observed in cells treated with non-sensitizers or in untreated cells. CONCLUSION: This method based on the analysis of forward scatter and side scatter parameters, can be used as an alternative method for identifying sensitization potential of chemical compounds. 2019 The Authors. International Journal of Cosmetic Science published by John Wiley & Sons Ltd on behalf of Society of Cosmetic Scientists and the Societe Française de Cosmetologie.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
BACKGROUND: In order to comply with the European legislation concerning the risk assessment of skin sensitizers, considerable progress has been made in developing alternative methods, such as human cell line activation test (h-CLAT). H-CLAT is based on cytometric measurement of fluorescence emitted by anti-CD54 and anti-CD86 antibodies in THP-1 cells. Following this method, a range of substances have been analyzed; the emitted fluorescence, generally at low intensity, has caused problems concerning the interpretation of results. AIM: Find an alternative parameter to h-CLAT for evaluating the sensitizing potential of chemicals. MATERIALS AND METHODS: Cells have been analyzed with flow cytometry after treatment with sensitizing compounds administered at non-cytotoxic concentrations. RESULTS: Sensitizers were able to inducealterations in cell morphology to a more 'condensed' one allowing the identification of cells under microscope as a 'sensitized' subpopulation. These variations cause similar modifications in 'scattering' parameters, making cells easily monitorable by flow cytometry. No changes have been observed in cells treated with non-sensitizers or in untreated cells. CONCLUSION: This method based on the analysis of forward scatter and side scatter parameters, can be used as an alternative method for identifying sensitization potential of chemical compounds. 2019 The Authors. International Journal of Cosmetic Science published by John Wiley & Sons Ltd on behalf of Society of Cosmetic Scientists and the Societe Française de Cosmetologie. |
2018
|
Aredia F; Carpignano F; Surdo S; Barillaro G; Mazzini G; Scovassi AI; Merlo S An Innovative Cell Microincubator for Drug Discovery Based on 3D Silicon Structures Journal Article In: Journa of Nanomaterials, vol. 2016, pp. 8236539, 2018. @article{%a1:%Y_244,
title = {An Innovative Cell Microincubator for Drug Discovery Based on 3D Silicon Structures},
author = {Aredia F and Carpignano F and Surdo S and Barillaro G and Mazzini G and Scovassi AI and Merlo S},
url = {https://www.hindawi.com/journals/jnm/2016/8236539/},
doi = {10.1155/2016/8236539},
year = {2018},
date = {2018-02-16},
journal = {Journa of Nanomaterials},
volume = {2016},
pages = {8236539},
abstract = {Recently, we applied three-dimensional (3D) silicon microstructures (SMSs), consisting of arrays of ?3 "m-thick silicon walls separated by 50 "m-deep, ?5 "m-wide gaps, as microincubators suitable for monitoring the biomechanical properties of tumor cells in culture.The same structures were here employed to investigate the in vitro behavior of tumor cells driven to apoptosis by a chemotherapeutic compound. HT1080 human fibrosarcoma cells were grown on silicon dice incorporating the 3D-SMSs, treated with the proapoptotic drug Bleomycin (200 "g/mL for 24 h, 48 h, and 72 h), and fixed and stained for fluorescence microscopy analyses. Results of our investigation demonstrated that HT1080 cells exhibited a great ability to colonize the grooves, entering the narrow, deep gaps of the 3D-SMS. We also observed that cells grown on 3D-SMS, when treated with the DNA damaging agent Bleomycin under conditions leading to apoptosis, tended to shrink, reducing their volume and mimicking the normal behavior of apoptotic cells, and to leave the gaps. Visual inspection performed bymeans of fluorescencemicroscopy allowed us to demonstrate that cells grown on 3D-SMS exhibited the morphological alterations typical of apoptosis. In view of future applications of the 3DSMS as the core element of a lab-on-a-chip suitable for screening the effect of new molecules potentially able to kill tumor cells, we also performed label-free detection of a cell adherent to the vertical siliconwall, inside the gap of 3D-SMS, by exploitingOptical Low Coherence Reflectometry using infrared, low power radiation.This kind of lab-on-a-chip combined with label-free cell detection may become a new tool for increasing automation in the drug discovery area.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Recently, we applied three-dimensional (3D) silicon microstructures (SMSs), consisting of arrays of ?3 "m-thick silicon walls separated by 50 "m-deep, ?5 "m-wide gaps, as microincubators suitable for monitoring the biomechanical properties of tumor cells in culture.The same structures were here employed to investigate the in vitro behavior of tumor cells driven to apoptosis by a chemotherapeutic compound. HT1080 human fibrosarcoma cells were grown on silicon dice incorporating the 3D-SMSs, treated with the proapoptotic drug Bleomycin (200 "g/mL for 24 h, 48 h, and 72 h), and fixed and stained for fluorescence microscopy analyses. Results of our investigation demonstrated that HT1080 cells exhibited a great ability to colonize the grooves, entering the narrow, deep gaps of the 3D-SMS. We also observed that cells grown on 3D-SMS, when treated with the DNA damaging agent Bleomycin under conditions leading to apoptosis, tended to shrink, reducing their volume and mimicking the normal behavior of apoptotic cells, and to leave the gaps. Visual inspection performed bymeans of fluorescencemicroscopy allowed us to demonstrate that cells grown on 3D-SMS exhibited the morphological alterations typical of apoptosis. In view of future applications of the 3DSMS as the core element of a lab-on-a-chip suitable for screening the effect of new molecules potentially able to kill tumor cells, we also performed label-free detection of a cell adherent to the vertical siliconwall, inside the gap of 3D-SMS, by exploitingOptical Low Coherence Reflectometry using infrared, low power radiation.This kind of lab-on-a-chip combined with label-free cell detection may become a new tool for increasing automation in the drug discovery area. |
Danova M; Torchio M; Comolli G; Sbrana A; Antonuzzo A; Mazzini G The role of automated cytometry in the new era of cancer immunotherapy. Journal Article In: Molecular and clinical oncology, vol. 9, no. 4, pp. 355-361, 2018. @article{%a1:%Y_130,
title = {The role of automated cytometry in the new era of cancer immunotherapy.},
author = {Danova M and Torchio M and Comolli G and Sbrana A and Antonuzzo A and Mazzini G},
url = {10.3892/mco.2018.1701},
doi = {10.3892/mco.2018.1701},
year = {2018},
date = {2018-10-19},
journal = {Molecular and clinical oncology},
volume = {9},
number = {4},
pages = {355-361},
abstract = {The introduction in the clinical practice of several new approaches to cancer immunotherapy has greatly increased the interest in analytical methodologies that can define the immunological profile of patients in the clinical setting. This requires huge effort to obtain reliable monitoring tools that could be used to improve the patient's clinical outcome. The clinical applications of flow cytometry (FCM) in oncology started with the measurement of DNA content for the evaluation of both ploidy and cell cycle profile as potential prognostic parameters in the majority of human solid cancer types. The availability of monoclonal antibodies widely broadened the spectrum of clinical applications of this technique, which rapidly became a fundamental tool for the diagnosis and prognosis of malignant hematological diseases. Among the emerging clinical applications of FCM, the study of minimal residual disease in hematological malignancies, the quantification of blood dendritic cells in various types of tumors, the study of metastatic spread in solid tumors throughout both the analysis of circulating endothelial progenitor cells and the identification and characterization of circulating tumor cells, all appear very promising. More recently, an advanced single cell analysis technique has been developed that combines the precision of mass spectrometry with the unique advantages of FCM. This approach, termed mass cytometry, utilizes antibodies conjugated to heavy metal ions for the analysis of cellular proteins by a mass spectrometer. It provides measurement of over 100 simultaneous cellular parameters in a single sample and has evolved from a promising technology to a high recognized platform for multi-dimensional single-cell analysis. Should a careful standardization of the analytical procedures be reached, both FCM and mass cytometry could effectively become ideal tools for the optimization of new immunotherapeutic approaches in cancer patients.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The introduction in the clinical practice of several new approaches to cancer immunotherapy has greatly increased the interest in analytical methodologies that can define the immunological profile of patients in the clinical setting. This requires huge effort to obtain reliable monitoring tools that could be used to improve the patient's clinical outcome. The clinical applications of flow cytometry (FCM) in oncology started with the measurement of DNA content for the evaluation of both ploidy and cell cycle profile as potential prognostic parameters in the majority of human solid cancer types. The availability of monoclonal antibodies widely broadened the spectrum of clinical applications of this technique, which rapidly became a fundamental tool for the diagnosis and prognosis of malignant hematological diseases. Among the emerging clinical applications of FCM, the study of minimal residual disease in hematological malignancies, the quantification of blood dendritic cells in various types of tumors, the study of metastatic spread in solid tumors throughout both the analysis of circulating endothelial progenitor cells and the identification and characterization of circulating tumor cells, all appear very promising. More recently, an advanced single cell analysis technique has been developed that combines the precision of mass spectrometry with the unique advantages of FCM. This approach, termed mass cytometry, utilizes antibodies conjugated to heavy metal ions for the analysis of cellular proteins by a mass spectrometer. It provides measurement of over 100 simultaneous cellular parameters in a single sample and has evolved from a promising technology to a high recognized platform for multi-dimensional single-cell analysis. Should a careful standardization of the analytical procedures be reached, both FCM and mass cytometry could effectively become ideal tools for the optimization of new immunotherapeutic approaches in cancer patients. |
2017
|
Mazzini G; Danova M Fluorochromes for DNA Staining and Quantitation. Journal Article In: Methods in molecular biology - Histochemistry of Single Molecules Methods and Protocols, vol. 1560, pp. 239-259, 2017. @article{%a1:%Y_188,
title = {Fluorochromes for DNA Staining and Quantitation.},
author = {Mazzini G and Danova M},
url = {https://link.springer.com/protocol/10.1007%2F978-1-4939-6788-9_18},
doi = {10.1007/978-1-4939-6788-9_18},
year = {2017},
date = {2017-02-28},
journal = {Methods in molecular biology - Histochemistry of Single Molecules Methods and Protocols},
volume = {1560},
pages = {239-259},
abstract = {In these last few decades the great explosion of the molecular approaches has casted a little shadow on the DNA quantitative analysis. Nevertheless DNA cytochemistry represented a long piece of history in cell biology since the advent of the Feulgen reaction. This discovery was really the milestone of the emerging quantitative cytochemistry, and scientists from all over the world produced a very large literature on this subject. This first era of quantitation (histochemistry followed by cytochemistry) started by means of absorption measurements (histophotometry and cytophotometry). The successive introduction of fluorescence microscopy gave a great boost to quantitation, making easier and faster the determination of cell components by means of cytofluorometry. The development of flow cytometry further contributed to the importance of quantitative cytochemistry. At its beginning, the mission of flow cytometry was still DNA quantitation. For a decade the Feulgen reaction had been the reference methodology for both conventional and flow cytofluorometry; the advent of Shiff-type reagents contributed to expand the variety of possible fluorochromes excitable in the entire visible spectrum as well as in the ultraviolet region. The fluorescence scenario was progressively enriched by new probes among which are the intercalating dyes which made DNA quantitation simple and fast, thus spreading it worldwide. The final explosion of cytofluorometry was made possible by the availability of a large variety of probes directly binding DNA structure. In addition, immunofluorescence allowed to correlate the cell cycle-related DNA content to other cell markers. In the clinical application of flow cytometry, this promoted the introduction of multiparametric analyses aimed at describing the cytokinetic characteristics of a given cell subpopulation defined by a specific immunophenotype setting.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
In these last few decades the great explosion of the molecular approaches has casted a little shadow on the DNA quantitative analysis. Nevertheless DNA cytochemistry represented a long piece of history in cell biology since the advent of the Feulgen reaction. This discovery was really the milestone of the emerging quantitative cytochemistry, and scientists from all over the world produced a very large literature on this subject. This first era of quantitation (histochemistry followed by cytochemistry) started by means of absorption measurements (histophotometry and cytophotometry). The successive introduction of fluorescence microscopy gave a great boost to quantitation, making easier and faster the determination of cell components by means of cytofluorometry. The development of flow cytometry further contributed to the importance of quantitative cytochemistry. At its beginning, the mission of flow cytometry was still DNA quantitation. For a decade the Feulgen reaction had been the reference methodology for both conventional and flow cytofluorometry; the advent of Shiff-type reagents contributed to expand the variety of possible fluorochromes excitable in the entire visible spectrum as well as in the ultraviolet region. The fluorescence scenario was progressively enriched by new probes among which are the intercalating dyes which made DNA quantitation simple and fast, thus spreading it worldwide. The final explosion of cytofluorometry was made possible by the availability of a large variety of probes directly binding DNA structure. In addition, immunofluorescence allowed to correlate the cell cycle-related DNA content to other cell markers. In the clinical application of flow cytometry, this promoted the introduction of multiparametric analyses aimed at describing the cytokinetic characteristics of a given cell subpopulation defined by a specific immunophenotype setting. |
Vetro A; Savasta S; Russo Raucci A; Cerqua C; Sartori G; Limongelli I; Forlino A; Maruelli S; Perucca P; Vergani D; Mazzini G; Mattevi A; Stivala LA; Salviati L; Zuffardi O MCM5: a new actor in the link between DNA replication and Meier-Gorlin syndrome. Journal Article In: European journal of human genetics, vol. 25, no. 5, pp. 646-650, 2017. @article{%a1:%Y_221,
title = {MCM5: a new actor in the link between DNA replication and Meier-Gorlin syndrome.},
author = {Vetro A and Savasta S and Russo Raucci A and Cerqua C and Sartori G and Limongelli I and Forlino A and Maruelli S and Perucca P and Vergani D and Mazzini G and Mattevi A and Stivala LA and Salviati L and Zuffardi O},
url = {http://www.nature.com/ejhg/journal/vaop/ncurrent/full/ejhg20175a.html},
doi = {10.1038/ejhg.2017.5},
year = {2017},
date = {2017-05-18},
journal = {European journal of human genetics},
volume = {25},
number = {5},
pages = {646-650},
abstract = {Meier-Gorlin syndrome (MGORS) is a rare disorder characterized by primordial dwarfism, microtia, and patellar aplasia/hypoplasia. Recessive mutations in ORC1, ORC4, ORC6, CDT1, CDC6, and CDC45, encoding members of the pre-replication (pre-RC) and pre-initiation (pre-IC) complexes, and heterozygous mutations in GMNN, a regulator of cell-cycle progression and DNA replication, have already been associated with this condition. We performed whole-exome sequencing (WES) in a patient with a clinical diagnosis of MGORS and identified biallelic variants in MCM5. This gene encodes a subunit of the replicative helicase complex, which represents a component of the pre-RC. Both variants, a missense substitution within a conserved domain critical for the helicase activity, and a single base deletion causing a frameshift and a premature stop codon, were predicted to be detrimental for the MCM5 function. Although variants of MCM5 have never been reported in specific human diseases, defect of this gene in zebrafish causes a phenotype of growth restriction overlapping the one associated with orc1 depletion. Complementation experiments in yeast showed that the plasmid carrying the missense variant was unable to rescue the lethal phenotype caused by mcm5 deletion. Moreover cell-cycle progression was delayed in patient's cells, as already shown for mutations in the ORC1 gene. Altogether our findings support the role of MCM5 as a novel gene involved in MGORS, further emphasizing that this condition is caused by impaired DNA replication.European Journal of Human Genetics advance online publication, 15 February 2017; doi:10.1038/ejhg.2017.5.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Meier-Gorlin syndrome (MGORS) is a rare disorder characterized by primordial dwarfism, microtia, and patellar aplasia/hypoplasia. Recessive mutations in ORC1, ORC4, ORC6, CDT1, CDC6, and CDC45, encoding members of the pre-replication (pre-RC) and pre-initiation (pre-IC) complexes, and heterozygous mutations in GMNN, a regulator of cell-cycle progression and DNA replication, have already been associated with this condition. We performed whole-exome sequencing (WES) in a patient with a clinical diagnosis of MGORS and identified biallelic variants in MCM5. This gene encodes a subunit of the replicative helicase complex, which represents a component of the pre-RC. Both variants, a missense substitution within a conserved domain critical for the helicase activity, and a single base deletion causing a frameshift and a premature stop codon, were predicted to be detrimental for the MCM5 function. Although variants of MCM5 have never been reported in specific human diseases, defect of this gene in zebrafish causes a phenotype of growth restriction overlapping the one associated with orc1 depletion. Complementation experiments in yeast showed that the plasmid carrying the missense variant was unable to rescue the lethal phenotype caused by mcm5 deletion. Moreover cell-cycle progression was delayed in patient's cells, as already shown for mutations in the ORC1 gene. Altogether our findings support the role of MCM5 as a novel gene involved in MGORS, further emphasizing that this condition is caused by impaired DNA replication.European Journal of Human Genetics advance online publication, 15 February 2017; doi:10.1038/ejhg.2017.5. |
2016
|
Bandiera L; Pasini A; Pasotti L; Zucca S; Mazzini G; Magni P; Giordano E; Furini S Experimental measurements and mathematical modeling of biological noise arising from transcriptional and translational regulation of basic synthetic gene circuits. Journal Article In: Journal of Theoretical Biology, vol. 395, pp. 153-160, 2016. @article{%a1:%Y_249,
title = {Experimental measurements and mathematical modeling of biological noise arising from transcriptional and translational regulation of basic synthetic gene circuits.},
author = {Bandiera L and Pasini A and Pasotti L and Zucca S and Mazzini G and Magni P and Giordano E and Furini S},
url = {http://www.sciencedirect.com/science/article/pii/S0022519316000941},
doi = {10.1016/j.jtbi.2016.02.004},
year = {2016},
date = {2016-04-21},
journal = {Journal of Theoretical Biology},
volume = {395},
pages = {153-160},
abstract = {The small number of molecules, unevenly distributed within an isogenic cell population, makes gene expression a noisy process, and strategies have evolved to deal with this variability in protein concentration and to limit its impact on cellular behaviors. As translational efficiency has a major impact on biological noise, a possible strategy to control noise is to regulate gene expression processes at the post-transcriptional level. In this study, fluctuations in the concentration of a green fluorescent protein were compared, at the single cell level, upon transformation of an isogenic bacterial cell population with synthetic gene circuits implementing either a transcriptional or a post-transcriptional control of gene expression. Experimental measurements showed that protein variability is lower under post-transcriptional control, when the same average protein concentrations are compared. This effect is well reproduced by stochastic simulations, supporting the hypothesis that noise reduction is due to the control mechanism acting on the efficiency of translation. Similar strategies are likely to play a role in noise reduction in natural systems and to be useful for controlling noise in synthetic biology applications. Copyright 2016 Elsevier Ltd. All rights reserved.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The small number of molecules, unevenly distributed within an isogenic cell population, makes gene expression a noisy process, and strategies have evolved to deal with this variability in protein concentration and to limit its impact on cellular behaviors. As translational efficiency has a major impact on biological noise, a possible strategy to control noise is to regulate gene expression processes at the post-transcriptional level. In this study, fluctuations in the concentration of a green fluorescent protein were compared, at the single cell level, upon transformation of an isogenic bacterial cell population with synthetic gene circuits implementing either a transcriptional or a post-transcriptional control of gene expression. Experimental measurements showed that protein variability is lower under post-transcriptional control, when the same average protein concentrations are compared. This effect is well reproduced by stochastic simulations, supporting the hypothesis that noise reduction is due to the control mechanism acting on the efficiency of translation. Similar strategies are likely to play a role in noise reduction in natural systems and to be useful for controlling noise in synthetic biology applications. Copyright 2016 Elsevier Ltd. All rights reserved. |
Danova M; Comolli G; Manzoni M; Torchio M; Mazzini G Flow cytometric analysis of circulating endothelial cells and endothelial progenitors for clinical purposes in oncology: A critical evaluation. Journal Article In: Molecular and Clinical Oncology, vol. 4, no. 6, pp. 909-917, 2016. @article{%a1:%Y_265,
title = {Flow cytometric analysis of circulating endothelial cells and endothelial progenitors for clinical purposes in oncology: A critical evaluation.},
author = {Danova M and Comolli G and Manzoni M and Torchio M and Mazzini G},
url = {http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4888001/},
doi = {10.3892/mco.2016.823},
year = {2016},
date = {2016-02-04},
journal = {Molecular and Clinical Oncology},
volume = {4},
number = {6},
pages = {909-917},
abstract = {Malignant tumors are characterized by uncontrolled cell growth and metastatic spread, with a pivotal importance of the phenomenon of angiogenesis. For this reason, research has focused on the development of agents targeting the vascular component of the tumor microenvironment and regulating the angiogenic switch. As a result, the therapeutic inhibition of angiogenesis has become an important component of anticancer treatment, however, its utility is partly limited by the lack of an established methodology to assess its efficacy in vivo. Circulating endothelial cells (CECs), which are rare in healthy subjects and significantly increased in different tumor types, represent a promising tool for monitoring the tumor clinical outcome and the treatment response. A cell population circulating into the blood also able to form endothelial colonies in vitro and to promote vasculogenesis is represented by endothelial progenitor cells (EPCs). The number of both of these cell types is extremely low and they cannot be identified using a single marker, therefore, in absence of a definite consensus on their phenotype, require discrimination using combinations of antigens. Multiparameter flow cytometry (FCM) is ideal for rapid processing of high numbers of cells per second and is commonly utilized to quantify CECs and EPCs, however, remains technically challenging since there is as yet no standardized protocol for the identification and enumeration of these rare events. Methodology in studies on CECs and/or EPCs as clinical biomarkers in oncology is heterogeneous and data have been obtained from different studies leading to conflicting conclusions. The present review presented a critical review of the issues that limit the comparability of results of the most significant studies employing FCM for CEC and/or EPC detection in patients with cancer.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Malignant tumors are characterized by uncontrolled cell growth and metastatic spread, with a pivotal importance of the phenomenon of angiogenesis. For this reason, research has focused on the development of agents targeting the vascular component of the tumor microenvironment and regulating the angiogenic switch. As a result, the therapeutic inhibition of angiogenesis has become an important component of anticancer treatment, however, its utility is partly limited by the lack of an established methodology to assess its efficacy in vivo. Circulating endothelial cells (CECs), which are rare in healthy subjects and significantly increased in different tumor types, represent a promising tool for monitoring the tumor clinical outcome and the treatment response. A cell population circulating into the blood also able to form endothelial colonies in vitro and to promote vasculogenesis is represented by endothelial progenitor cells (EPCs). The number of both of these cell types is extremely low and they cannot be identified using a single marker, therefore, in absence of a definite consensus on their phenotype, require discrimination using combinations of antigens. Multiparameter flow cytometry (FCM) is ideal for rapid processing of high numbers of cells per second and is commonly utilized to quantify CECs and EPCs, however, remains technically challenging since there is as yet no standardized protocol for the identification and enumeration of these rare events. Methodology in studies on CECs and/or EPCs as clinical biomarkers in oncology is heterogeneous and data have been obtained from different studies leading to conflicting conclusions. The present review presented a critical review of the issues that limit the comparability of results of the most significant studies employing FCM for CEC and/or EPC detection in patients with cancer. |
Carpignano F; Rigamonti G; Mazzini G; Merlo S Low-Coherence Reflectometry for Refractive Index Measurements of Cells in Micro-Capillaries. Journal Article In: Sensors, vol. 16, no. 10, pp. E1670, 2016. @article{%a1:%Y_257,
title = {Low-Coherence Reflectometry for Refractive Index Measurements of Cells in Micro-Capillaries.},
author = {Carpignano F and Rigamonti G and Mazzini G and Merlo S},
url = {http://www.mdpi.com/1424-8220/16/10/1670},
doi = {10.3390/s16101670},
year = {2016},
date = {2016-10-11},
journal = {Sensors},
volume = {16},
number = {10},
pages = {E1670},
abstract = {The refractive index of cells provides insights into their composition, organization and function. Moreover, a good knowledge of the cell refractive index would allow an improvement of optical cytometric and diagnostic systems. Although interferometric techniques undoubtedly represent a good solution for quantifying optical path variation, obtaining the refractive index of a population of cells non-invasively remains challenging because of the variability in the geometrical thickness of the sample. In this paper, we demonstrate the use of infrared low-coherence reflectometry for non-invasively quantifying the average refractive index of cell populations gently confined in rectangular glass micro-capillaries. A suspension of human red blood cells in plasma is tested as a reference. As a use example, we apply this technique to estimate the average refractive index of cell populations belonging to epithelial and hematological families.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The refractive index of cells provides insights into their composition, organization and function. Moreover, a good knowledge of the cell refractive index would allow an improvement of optical cytometric and diagnostic systems. Although interferometric techniques undoubtedly represent a good solution for quantifying optical path variation, obtaining the refractive index of a population of cells non-invasively remains challenging because of the variability in the geometrical thickness of the sample. In this paper, we demonstrate the use of infrared low-coherence reflectometry for non-invasively quantifying the average refractive index of cell populations gently confined in rectangular glass micro-capillaries. A suspension of human red blood cells in plasma is tested as a reference. As a use example, we apply this technique to estimate the average refractive index of cell populations belonging to epithelial and hematological families. |
Terzaghi L; Tessaro I; Raucci F; Merico V; Mazzini G; Garagna S; Zuccotti M; Franciosi F; Lodde V PGRMC1 participates in late events of bovine granulosa cells mitosis and oocyte meiosis. Journal Article In: Cell Cycle, vol. 15, no. 15, pp. 2019-2032, 2016. @article{%a1:%Y_313,
title = {PGRMC1 participates in late events of bovine granulosa cells mitosis and oocyte meiosis.},
author = {Terzaghi L and Tessaro I and Raucci F and Merico V and Mazzini G and Garagna S and Zuccotti M and Franciosi F and Lodde V},
url = {http://www.tandfonline.com/doi/abs/10.1080/15384101.2016.1192731?journalCode=kccy20},
doi = {10.1080/15384101.2016.1192731},
year = {2016},
date = {2016-08-02},
journal = {Cell Cycle},
volume = {15},
number = {15},
pages = {2019-2032},
abstract = {Progesterone Receptor Membrane Component 1 (PGRMC1) is expressed in both oocyte and ovarian somatic cells, where it is found in multiple cellular sub-compartments including the mitotic spindle apparatus. Strikingly, PGRMC1 localization in the maturing bovine oocytes mirrors its localization in mitotic cells, suggesting a possible common action in mitosis and meiosis. To test the hypothesis that altering PGRMC1 activity leads to similar defects in mitosis and meiosis, PGRMC1 function was perturbed in cultured bovine granulosa cells (bGC) and maturing oocytes and the effect on mitotic and meiotic progression assessed. RNA interference-mediated PGRMC1 silencing in bGC significantly reduced cell proliferation, with a concomitant increase in the percentage of cells arrested at G2/M phase, which is consistent with an arrested or prolonged M-phase. This observation was confirmed by time-lapse imaging that revealed defects in late karyokinesis. In agreement with a role during late mitotic events, a direct interaction between PGRMC1 and Aurora Kinase B (AURKB) was observed in the central spindle at of dividing cells. Similarly, treatment with the PGRMC1 inhibitor AG205 or PGRMC1 silencing in the oocyte impaired completion of meiosis I. Specifically the ability of the oocyte to extrude the first polar body was significantly impaired while meiotic figures aberration and chromatin scattering within the ooplasm increased. Finally, analysis of PGRMC1 and AURKB localization in AG205-treated oocytes confirmed an altered localization of both proteins when meiotic errors occur. The present findings demonstrate that PGRMC1 participates in late events of both mammalian mitosis and oocyte meiosis, consistent with PGRMC's localization at the mid-zone and mid-body of the mitotic and meiotic spindle.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Progesterone Receptor Membrane Component 1 (PGRMC1) is expressed in both oocyte and ovarian somatic cells, where it is found in multiple cellular sub-compartments including the mitotic spindle apparatus. Strikingly, PGRMC1 localization in the maturing bovine oocytes mirrors its localization in mitotic cells, suggesting a possible common action in mitosis and meiosis. To test the hypothesis that altering PGRMC1 activity leads to similar defects in mitosis and meiosis, PGRMC1 function was perturbed in cultured bovine granulosa cells (bGC) and maturing oocytes and the effect on mitotic and meiotic progression assessed. RNA interference-mediated PGRMC1 silencing in bGC significantly reduced cell proliferation, with a concomitant increase in the percentage of cells arrested at G2/M phase, which is consistent with an arrested or prolonged M-phase. This observation was confirmed by time-lapse imaging that revealed defects in late karyokinesis. In agreement with a role during late mitotic events, a direct interaction between PGRMC1 and Aurora Kinase B (AURKB) was observed in the central spindle at of dividing cells. Similarly, treatment with the PGRMC1 inhibitor AG205 or PGRMC1 silencing in the oocyte impaired completion of meiosis I. Specifically the ability of the oocyte to extrude the first polar body was significantly impaired while meiotic figures aberration and chromatin scattering within the ooplasm increased. Finally, analysis of PGRMC1 and AURKB localization in AG205-treated oocytes confirmed an altered localization of both proteins when meiotic errors occur. The present findings demonstrate that PGRMC1 participates in late events of both mammalian mitosis and oocyte meiosis, consistent with PGRMC's localization at the mid-zone and mid-body of the mitotic and meiotic spindle. |
2015
|
Mazzini G; Carpignano F; Surdo S; Aredia F; Torchio M; Erba E; Danova M; Scovassi AI; Barillaro G; Merlo S 3D silicon microstructures: a new tool for evaluating biological aggressiveness of tumour cells. Journal Article In: IEEE Trans Nanobioscience , vol. 14, no. 7, pp. 797-805, 2015. @article{%a1:%Y_324,
title = {3D silicon microstructures: a new tool for evaluating biological aggressiveness of tumour cells.},
author = {Mazzini G and Carpignano F and Surdo S and Aredia F and Torchio M and Erba E and Danova M and Scovassi AI and Barillaro G and Merlo S},
url = {https://ieeexplore.ieee.org/document/7239627},
doi = {10.1109/TNB.2015.2476351},
year = {2015},
date = {2015-02-12},
urldate = {2015-02-12},
journal = {IEEE Trans Nanobioscience },
volume = {14},
number = {7},
pages = {797-805},
abstract = {In this work, silicon micromachined structures (SMS), consisting of arrays of 3-μm-thick silicon walls separated by 50-μm-deep, 5-μm-wide gaps, were applied to investigate the behavior of eight tumor cell lines, with different origins and biological aggressiveness, in a three-dimensional (3D) microenvironment. Several cell culture experiments were performed on 3D-SMS and cells grown on silicon were stained for fluorescence microscopy analyses. Most of the tumor cells lines recognized in the literature as highly aggressive (OVCAR-5, A375, MDA-MB-231, and RPMI-7951) exhibited a great ability to enter and colonize the narrow deep gaps of the SMS, whereas less aggressive cell lines (OVCAR-3, Capan-1, MCF7, and NCI-H2126) demonstrated less penetration capability and tended to remain on top of the SMS. Quantitative image analyses of several fluorescence microscopy fields of silicon samples were performed for automatic cell recognition and count, in order to quantify the fraction of cells inside the gaps, with respect to the total number of cells in the examined field. Our results show that higher fractions of cells in the gaps are obtained with more aggressive cell lines, thus supporting in a quantitative way the observation that the behavior of tumor cells on the 3D-SMS depends on their aggressiveness level.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
In this work, silicon micromachined structures (SMS), consisting of arrays of 3-μm-thick silicon walls separated by 50-μm-deep, 5-μm-wide gaps, were applied to investigate the behavior of eight tumor cell lines, with different origins and biological aggressiveness, in a three-dimensional (3D) microenvironment. Several cell culture experiments were performed on 3D-SMS and cells grown on silicon were stained for fluorescence microscopy analyses. Most of the tumor cells lines recognized in the literature as highly aggressive (OVCAR-5, A375, MDA-MB-231, and RPMI-7951) exhibited a great ability to enter and colonize the narrow deep gaps of the SMS, whereas less aggressive cell lines (OVCAR-3, Capan-1, MCF7, and NCI-H2126) demonstrated less penetration capability and tended to remain on top of the SMS. Quantitative image analyses of several fluorescence microscopy fields of silicon samples were performed for automatic cell recognition and count, in order to quantify the fraction of cells inside the gaps, with respect to the total number of cells in the examined field. Our results show that higher fractions of cells in the gaps are obtained with more aggressive cell lines, thus supporting in a quantitative way the observation that the behavior of tumor cells on the 3D-SMS depends on their aggressiveness level. |
Manzoni M; Comolli G; Torchio M; Mazzini G; Danova M Circulating endothelial cells and their subpopulations: role as predictive biomarkers in antiangiogenic therapy for colorectal cancer. Journal Article In: Clinical Colorectal Cancer, vol. 14, no. 1, pp. 11-17, 2015. @article{%a1:%Y_347,
title = {Circulating endothelial cells and their subpopulations: role as predictive biomarkers in antiangiogenic therapy for colorectal cancer.},
author = {Manzoni M and Comolli G and Torchio M and Mazzini G and Danova M},
url = {https://www.sciencedirect.com/science/article/pii/S1533002814001339?via%3Dihub},
doi = {10.1016/j.clcc.2014.12.002},
year = {2015},
date = {2015-02-12},
journal = {Clinical Colorectal Cancer},
volume = {14},
number = {1},
pages = {11-17},
abstract = {Several anticancer therapies have been developed to block angiogenesis, a key mechanism in tumor growth and metastasis. The predominantly cytostatic action of these compounds makes an assessment of their clinical activities inadequate if based only on the reduction of the tumor dimensions, as this may not reflect their true biologic efficacy. Thus, it is crucial to identify biomarkers that permit the recognition of potentially responsive subjects and to spare toxicity in those who are unlikely to benefit from treatment. Circulating endothelial cells (CECs) have been recently indicated as potential surrogate biomarkers of angiogenesis in several types of cancer. The possibility of rapidly quantifying these cells represents a promising tool for monitoring the clinical outcome of tumors with the potential to assess response to various treatments. However, the identification and quantification of CECs is technically difficult and not well standardized. A variety of methods to detect CECs in patients with solid tumors have been used; these are based on different technical approaches, combinations of surface markers, sample handling, and staining protocols. With an expanding interest in the field of potential clinical applications for CECs in oncology, the development of standardized protocols for analysis is mandatory. The aim of this review was to critically summarize the available data concerning the clinical value of CECs and their subpopulations as biomarkers of antiangiogenic therapy in patients with metastatic colorectal cancer. Copyright 2015 Elsevier Inc. All rights reserved.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Several anticancer therapies have been developed to block angiogenesis, a key mechanism in tumor growth and metastasis. The predominantly cytostatic action of these compounds makes an assessment of their clinical activities inadequate if based only on the reduction of the tumor dimensions, as this may not reflect their true biologic efficacy. Thus, it is crucial to identify biomarkers that permit the recognition of potentially responsive subjects and to spare toxicity in those who are unlikely to benefit from treatment. Circulating endothelial cells (CECs) have been recently indicated as potential surrogate biomarkers of angiogenesis in several types of cancer. The possibility of rapidly quantifying these cells represents a promising tool for monitoring the clinical outcome of tumors with the potential to assess response to various treatments. However, the identification and quantification of CECs is technically difficult and not well standardized. A variety of methods to detect CECs in patients with solid tumors have been used; these are based on different technical approaches, combinations of surface markers, sample handling, and staining protocols. With an expanding interest in the field of potential clinical applications for CECs in oncology, the development of standardized protocols for analysis is mandatory. The aim of this review was to critically summarize the available data concerning the clinical value of CECs and their subpopulations as biomarkers of antiangiogenic therapy in patients with metastatic colorectal cancer. Copyright 2015 Elsevier Inc. All rights reserved. |
Cansolino L; Clerici AM; Zonta C; Dionigi P; Mazzini G; Di Liberto R; Altieri S; Ballarini F; Bortolussi S; Carante MP; Ferrari M; Gonzalez SJ; Postuma I; Protti N; Santa Cruz GA; Ferrari C Comparative study of the radiobiological effects induced on adherent vs suspended cells by BNCT, neutrons and gamma rays treatments. Journal Article In: Applied Radiation and Isotopes, vol. 106, pp. 226-232, 2015. @article{%a1:%Y_349,
title = {Comparative study of the radiobiological effects induced on adherent vs suspended cells by BNCT, neutrons and gamma rays treatments.},
author = {Cansolino L and Clerici AM and Zonta C and Dionigi P and Mazzini G and {Di Liberto R} and Altieri S and Ballarini F and Bortolussi S and Carante MP and Ferrari M and Gonzalez SJ and Postuma I and Protti N and Santa Cruz GA and Ferrari C},
url = {https://www.sciencedirect.com/science/article/pii/S0969804315301366?via%3Dihub},
doi = {10.1016/j.apradiso.2015.07.054},
year = {2015},
date = {2015-01-08},
journal = {Applied Radiation and Isotopes},
volume = {106},
pages = {226-232},
abstract = {The present work is part of a preclinical in vitro study to assess the efficacy of BNCT applied to liver or lung coloncarcinoma metastases and to limb osteosarcoma. Adherent growing cell lines can be irradiated as adherent to the culture flasks or as cell suspensions, differences in radio-sensitivity of the two modalities of radiation exposure have been investigated. Dose related cell survival and cell cycle perturbation results evidenced that the radiosensitivity of adherent cells is higher than that of the suspended ones.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The present work is part of a preclinical in vitro study to assess the efficacy of BNCT applied to liver or lung coloncarcinoma metastases and to limb osteosarcoma. Adherent growing cell lines can be irradiated as adherent to the culture flasks or as cell suspensions, differences in radio-sensitivity of the two modalities of radiation exposure have been investigated. Dose related cell survival and cell cycle perturbation results evidenced that the radiosensitivity of adherent cells is higher than that of the suspended ones. |
Vigone G; Merico V; Redi CA; Mazzini G; Garagna S; Zuccotti M FSH and LH receptors are differentially expressed in cumulus cells surrounding developmentally competent and incompetent mouse fully grown antral oocytes. Journal Article In: Reproduction, fertility and development, vol. 27, no. 3, pp. 497-503, 2015. @article{%a1:%Y_362,
title = {FSH and LH receptors are differentially expressed in cumulus cells surrounding developmentally competent and incompetent mouse fully grown antral oocytes.},
author = {Vigone G and Merico V and Redi CA and Mazzini G and Garagna S and Zuccotti M},
doi = {10.1071/RD13251},
year = {2015},
date = {2015-03-19},
journal = {Reproduction, fertility and development},
volume = {27},
number = {3},
pages = {497-503},
abstract = {Cumulus cells (CCs) maintain strict functional relationships with the enclosed antral oocyte and are thought to reflect its developmental competence. Several studies have described a correlation between CC gene expression and oocyte quality. Herein, we tested whether CC-specific FSH and LH receptors (FSHR and LHR, respectively) are differentially expressed in CCs enclosing developmentally competent or incompetent oocytes. To this end, mouse fully grown cumulus-oocyte complexes were isolated and their CCs and oocytes analysed separately. Based on their chromatin organisation, oocytes were classified as those with a surrounded nucleolus (SN) or a non-surrounded nucleolus (NSN), the former being developmentally competent, whereas the latter arrest at the 2-cell stage. The CCs were then analysed to compare the pattern of expression of the Fshr and Lhr genes and their proteins. Quantitative reverse transcription-polymerase chain reaction analysis revealed that only Lhr is significantly differentially expressed. Immunofluorescence analysis revealed that both FSHR and LHR proteins are significantly upregulated in CCs surrounding oocytes arrested at the 2-cell stage, reflecting their developmental incompetence.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Cumulus cells (CCs) maintain strict functional relationships with the enclosed antral oocyte and are thought to reflect its developmental competence. Several studies have described a correlation between CC gene expression and oocyte quality. Herein, we tested whether CC-specific FSH and LH receptors (FSHR and LHR, respectively) are differentially expressed in CCs enclosing developmentally competent or incompetent oocytes. To this end, mouse fully grown cumulus-oocyte complexes were isolated and their CCs and oocytes analysed separately. Based on their chromatin organisation, oocytes were classified as those with a surrounded nucleolus (SN) or a non-surrounded nucleolus (NSN), the former being developmentally competent, whereas the latter arrest at the 2-cell stage. The CCs were then analysed to compare the pattern of expression of the Fshr and Lhr genes and their proteins. Quantitative reverse transcription-polymerase chain reaction analysis revealed that only Lhr is significantly differentially expressed. Immunofluorescence analysis revealed that both FSHR and LHR proteins are significantly upregulated in CCs surrounding oocytes arrested at the 2-cell stage, reflecting their developmental incompetence. |
Paolini A; Curti V; Pasi F; Mazzini G; Nano R; Capelli E Gallic acid exerts a protective or an anti-proliferative effect on glioma T98G cells via dose-dependent epigenetic regulation mediated by miRNAs. Journal Article In: International Journal of Oncology, vol. 46, no. 4, pp. 1491-1497, 2015. @article{%a1:%Y_363,
title = {Gallic acid exerts a protective or an anti-proliferative effect on glioma T98G cells via dose-dependent epigenetic regulation mediated by miRNAs.},
author = {Paolini A and Curti V and Pasi F and Mazzini G and Nano R and Capelli E},
url = {https://www.spandidos-publications.com/ijo/46/4/1491},
doi = {10.3892/ijo.2015.2864},
year = {2015},
date = {2015-02-20},
journal = {International Journal of Oncology},
volume = {46},
number = {4},
pages = {1491-1497},
abstract = {Glioblastoma multiforme (GBM) is the most malignant primary brain tumor in adulthood, characterized by very high recurrence. Following the limited results for conventional therapies, novel therapeutic agents are under investigation. Among the putative new molecules, gallic acid (GA) represents a promising new anticancer drug. The anticancer effect of this drug has been based on its antioxidant effects. The aim of the present study was to investigate the toxic effects of GA on the T98G human glioblastoma cell line and its capacity to modulate the expression of microRNAs targeting the genes involved in tumor growth and invasion. Cytotoxicity, clonogenic ability and cell migration after GA treatment were tested. Moreover, the expression of miRNAs that target genes for antioxidant mitochondrial enzymes (mir-17-3p), p-21 protein (mir-21-5p) and ATM (mir-421-5p) was determined by qRT-PCR. The results confirmed in the T98G cells the anti-proliferative effect of GA reported for other glioma cell lines and showed that the miRNA expression changes depending on GA concentrations. Different GA concentrations can determine a protective or a toxic effect on tumor cells. Thus, the key for GA to induce a specific anticancer action is to use an optimal concentration that avoids these twin effects.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Glioblastoma multiforme (GBM) is the most malignant primary brain tumor in adulthood, characterized by very high recurrence. Following the limited results for conventional therapies, novel therapeutic agents are under investigation. Among the putative new molecules, gallic acid (GA) represents a promising new anticancer drug. The anticancer effect of this drug has been based on its antioxidant effects. The aim of the present study was to investigate the toxic effects of GA on the T98G human glioblastoma cell line and its capacity to modulate the expression of microRNAs targeting the genes involved in tumor growth and invasion. Cytotoxicity, clonogenic ability and cell migration after GA treatment were tested. Moreover, the expression of miRNAs that target genes for antioxidant mitochondrial enzymes (mir-17-3p), p-21 protein (mir-21-5p) and ATM (mir-421-5p) was determined by qRT-PCR. The results confirmed in the T98G cells the anti-proliferative effect of GA reported for other glioma cell lines and showed that the miRNA expression changes depending on GA concentrations. Different GA concentrations can determine a protective or a toxic effect on tumor cells. Thus, the key for GA to induce a specific anticancer action is to use an optimal concentration that avoids these twin effects. |
Pasotti L; Zucca S; Casanova M; Politi N; Massaiu I; Mazzini G; Micoli G; Calvio C; Cusella De Angelis MG; Magni P Methods for genetic optimization of biocatalysts for biofuel production from dairy waste through synthetic biology. Journal Article In: Conference proceedings : Annual International Conference of the IEEE Engineering in Medicine and Biology Society., vol. 2015, pp. 953-956, 2015. @article{%a1:%Y_380,
title = {Methods for genetic optimization of biocatalysts for biofuel production from dairy waste through synthetic biology.},
author = {Pasotti L and Zucca S and Casanova M and Politi N and Massaiu I and Mazzini G and Micoli G and Calvio C and Cusella De Angelis MG and Magni P},
url = {https://ieeexplore.ieee.org/document/7318521},
doi = {10.1109/EMBC.2015.7318521},
year = {2015},
date = {2015-08-26},
journal = {Conference proceedings : Annual International Conference of the IEEE Engineering in Medicine and Biology Society.},
volume = {2015},
pages = {953-956},
abstract = {Whey is an abundant by-product of cheese production process and it is considered a special waste due to its high nutritional load and hypertrophic potential. Technologies for whey valorization are available. They can convert such waste into high-value products, like whey proteins. However, the remaining liquid (called permeate) is still considered as a polluting waste due to its high lactose concentration. The alcoholic fermentation of lactose into ethanol will simultaneously achieve two important goals: safe disposal of a pollutant waste and green energy production. This methodology paper illustrates the workflow carried out to design and realize an optimized microorganism that can efficiently perform the lactose-to-ethanol conversion, engineered via synthetic biology experimental and computational approaches.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Whey is an abundant by-product of cheese production process and it is considered a special waste due to its high nutritional load and hypertrophic potential. Technologies for whey valorization are available. They can convert such waste into high-value products, like whey proteins. However, the remaining liquid (called permeate) is still considered as a polluting waste due to its high lactose concentration. The alcoholic fermentation of lactose into ethanol will simultaneously achieve two important goals: safe disposal of a pollutant waste and green energy production. This methodology paper illustrates the workflow carried out to design and realize an optimized microorganism that can efficiently perform the lactose-to-ethanol conversion, engineered via synthetic biology experimental and computational approaches. |
Zucca S; Pasotti L; Politi N; Casanova M; Mazzini G; Cusella De Angelis MG; Magni P Multi-Faceted Characterization of a Novel LuxR-Repressible Promoter Library for Escherichia coli. Journal Article In: Plos One, vol. 10, no. 5, pp. e0126264, 2015. @article{%a1:%Y_384,
title = {Multi-Faceted Characterization of a Novel LuxR-Repressible Promoter Library for Escherichia coli.},
author = {Zucca S and Pasotti L and Politi N and Casanova M and Mazzini G and Cusella De Angelis MG and Magni P},
url = {https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0126264},
doi = {10.1371/journal.pone.0126264},
year = {2015},
date = {2015-05-26},
journal = {Plos One},
volume = {10},
number = {5},
pages = {e0126264},
abstract = {The genetic elements regulating the natural quorum sensing (QS) networks of several microorganisms are widely used in synthetic biology to control the behaviour of single cells and engineered bacterial populations via ad-hoc constructed synthetic circuits. A number of novel engineering-inspired biological functions have been implemented and model systems have also been constructed to improve the knowledge on natural QS systems. Synthetic QS-based parts, such as promoters, have been reported in literature, to provide biological components with functions that are not present in nature, like modified induction logic or activation/repression by additional molecules. In this work, a library of promoters that can be repressed by the LuxR protein in presence of the QS autoinducer N-3-oxohexanoyl-L-homoserine lactone (AHL) was reported for Escherichia coli, to expand the toolkit of genetic parts that can be used to engineer novel synthetic QS-based systems. The library was constructed via polymerase chain reaction with highly constrained degenerate oligonucleotides, designed according to the consensus -35 and -10 sequences of a previously reported constitutive promoter library of graded strength, to maximize the probability of obtaining functional clones. All the promoters have a lux box between the -35 and -10 regions, to implement a LuxR-repressible behaviour. Twelve unique library members of graded strength (about 100-fold activity range) were selected to form the final library and they were characterized in several genetic contexts, such as in different plasmids, via different reporter genes, in presence of a LuxR expression cassette in different positions and in response to different AHL concentrations. The new obtained regulatory parts and corresponding data can be exploited by synthetic biologists to implement an artificial AHL-dependent repression of transcription in genetic circuits. The target transcriptional activity can be selected among the available library members to meet the design specifications of the biological system.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The genetic elements regulating the natural quorum sensing (QS) networks of several microorganisms are widely used in synthetic biology to control the behaviour of single cells and engineered bacterial populations via ad-hoc constructed synthetic circuits. A number of novel engineering-inspired biological functions have been implemented and model systems have also been constructed to improve the knowledge on natural QS systems. Synthetic QS-based parts, such as promoters, have been reported in literature, to provide biological components with functions that are not present in nature, like modified induction logic or activation/repression by additional molecules. In this work, a library of promoters that can be repressed by the LuxR protein in presence of the QS autoinducer N-3-oxohexanoyl-L-homoserine lactone (AHL) was reported for Escherichia coli, to expand the toolkit of genetic parts that can be used to engineer novel synthetic QS-based systems. The library was constructed via polymerase chain reaction with highly constrained degenerate oligonucleotides, designed according to the consensus -35 and -10 sequences of a previously reported constitutive promoter library of graded strength, to maximize the probability of obtaining functional clones. All the promoters have a lux box between the -35 and -10 regions, to implement a LuxR-repressible behaviour. Twelve unique library members of graded strength (about 100-fold activity range) were selected to form the final library and they were characterized in several genetic contexts, such as in different plasmids, via different reporter genes, in presence of a LuxR expression cassette in different positions and in response to different AHL concentrations. The new obtained regulatory parts and corresponding data can be exploited by synthetic biologists to implement an artificial AHL-dependent repression of transcription in genetic circuits. The target transcriptional activity can be selected among the available library members to meet the design specifications of the biological system. |
Main research line is the development of fluorescent staining for quantitative cytochemistry and flow cytometry. Fluorescence histochemistry had been the “mile-stone” in the “story” of the CNR-Centre even if nowadays histo has been largely replaced by cells and molecules. Quantitative aspect of cytochemistry had been also a foundamental research theme of the Centre. Among the various cell components considered and studied, the DNA had been and still is in focus, in both basic and applied research programs. Since many decades DNA quantitation had been the main data, useful to understand many biological phenomena, including cell proliferation, cell death (apoptosis), malignant transformation and related radio-chemo treatments. A great methodological/instrumental development know-how, very well established at international level, had always characterized the research group.
