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Flow Cytometry and Its Applications to Molecular Biology and Diagnosis...
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Flow Cytometry and Its Applications to Molecular Biology and Diagnosis 2.0

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Pathology, Diagnostics, and Therapeutics".

Deadline for manuscript submissions: closed (30 January 2023) | Viewed by 16209

Special Issue Editors

Prof. Dr. Stefano Papa

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Guest Editor
Department of Biomolecular Sciences, University of Urbino Carlo Bo, 61029 Urbino, Italy
Interests: flow cytometry; apoptosis; exosomes; NK cells; melatonin
Special Issues, Collections and Topics in MDPI journals
Prof. Dr. José-Enrique O’Connor

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Guest Editor
Department of Biochemistry, School of Medicine, University of Valencia, 46010 Valencia, Spain
Interests: flow cytometry; cell biology; metabolism; biochemistry; immunology; stem cell biology; macrophage
Special Issues, Collections and Topics in MDPI journals
Prof. Dr. Claudio Ortolani

E-Mail Website
Guest Editor
Department of Biomolecular Sciences, University of Urbino, Urbino, Italy
Interests: flow cytometry; hematology; immunology; clinical diagnostics
Special Issues, Collections and Topics in MDPI journals
Dr. Paula Fernandez

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Guest Editor
Institute for Laboratory Medicine, Kantonsspital Aarau, Aarau, Switzerland
Interests: flow cytometry; hematology; immunology; clinical diagnostics
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Flow Cytometry is the most powerful technology to investigate antigen expression at single cell level, both in living and fixed cells as well as in subcellular bodies like exosomes. The recent evolution of optical benches for the analysis of fluorescence intensity combined with the new spectral cytometry has increased the performance towards an 18-signal analysis in the diagnostic lab. Thanks to multispectral technology, instruments with improved performance allow the number of parameters to increase beyond forty.

This Special Issue will focus upon flow analysis applied to both cellular and subcellular levels, in order to suggest new protocols for these investigations and new findings that could combine research practice with diagnosis in clinical immunology and hematology.

The COVID-19 pandemic has placed flow cytometry at center stage again. The Guest Editors ask researchers and clinicians to suggest and prepare comprehensive reviews regarding the application of flow cytometry in the relevant molecular biology. Moreover, we are inviting colleagues interested in the main aspects of flow analysis applications to contribute to this Special Issue by submitting original research articles, short communications and comprehensive reviews.

Prof. Dr. Stefano Papa
Prof Dr. José-Enrique O’Connor
Prof. Dr. Claudio Ortolani
Dr. Paula Fernandez
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. International Journal of Molecular Sciences is an international peer-reviewed open access semimonthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. There is an Article Processing Charge (APC) for publication in this open access journal. For details about the APC please see here. Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • Flow Cytometry
  • Apoptosis
  • Autophagy
  • Oxidative stress
  • Lysosome pathway
  • Hematological diseases
  • Immunodeficiency
  • COVID-19 immunodepression
  • Immune response

Published Papers (6 papers)

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Editorial

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3 pages, 204 KiB  
Editorial
Flow Cytometry and Its Applications to Molecular Biology and Diagnosis 2.0
by Stefano Papa, Claudio Ortolani, Paula Fernández and José-Enrique O’Connor
Int. J. Mol. Sci. 2023, 24(22), 16215; https://doi.org/10.3390/ijms242216215 - 11 Nov 2023
Viewed by 849
Abstract
Flow cytometry is a single-cell based technology aimed to quantify the scattering of light and the emission of multiple fluorescence signals by individual cells, biological vesicles, or synthetic microscopical particles when examined one by one at high speed using lasers or other suitable [...] Read more.
Flow cytometry is a single-cell based technology aimed to quantify the scattering of light and the emission of multiple fluorescence signals by individual cells, biological vesicles, or synthetic microscopical particles when examined one by one at high speed using lasers or other suitable illumination sources [...] Full article
(This article belongs to the Special Issue Flow Cytometry and Its Applications to Molecular Biology and Diagnosis 2.0)

Research

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24 pages, 4322 KiB  
Article
Extracellular Vesicles from Campylobacter jejuni CDT-Treated Caco-2 Cells Inhibit Proliferation of Tumour Intestinal Caco-2 Cells and Myeloid U937 Cells: Detailing the Global Cell Response for Potential Application in Anti-Tumour Strategies
by Mariele Montanari, Michele Guescini, Ozan Gundogdu, Francesca Luchetti, Paola Lanuti, Caterina Ciacci, Sabrina Burattini, Raffaella Campana, Claudio Ortolani, Stefano Papa and Barbara Canonico
Int. J. Mol. Sci. 2023, 24(1), 487; https://doi.org/10.3390/ijms24010487 - 28 Dec 2022
Cited by 3 | Viewed by 2162
Abstract
Cytolethal distending toxin (CDT) is produced by a range of Gram-negative pathogenic bacteria such as Campylobacter jejuni. CDT represents an important virulence factor that is a heterotrimeric complex composed of CdtA, CdtB, and CdtC. CdtA and CdtC constitute regulatory subunits whilst CdtB [...] Read more.
Cytolethal distending toxin (CDT) is produced by a range of Gram-negative pathogenic bacteria such as Campylobacter jejuni. CDT represents an important virulence factor that is a heterotrimeric complex composed of CdtA, CdtB, and CdtC. CdtA and CdtC constitute regulatory subunits whilst CdtB acts as the catalytic subunit exhibiting phosphatase and DNase activities, resulting in cell cycle arrest and cell death. Extracellular vesicle (EV) secretion is an evolutionarily conserved process that is present throughout all kingdoms. Mammalian EVs play important roles in regular cell-to-cell communications but can also spread pathogen- and host-derived molecules during infections to alter immune responses. Here, we demonstrate that CDT targets the endo-lysosomal compartment, partially evading lysosomal degradation and exploiting unconventional secretion (EV release), which is largely involved in bacterial infections. CDT-like effects are transferred by Caco-2 cells to uninfected heterologous U937 and homologous Caco-2 cells. The journey of EVs derived from CDT-treated Caco-2 cells is associated with both intestinal and myeloid tumour cells. EV release represents the primary route of CDT dissemination, revealing an active toxin as part of the cargo. We demonstrated that bacterial toxins could represent suitable tools in cancer therapy, highlighting both the benefits and limitations. The global cell response involves a moderate induction of apoptosis and autophagic features may play a protective role against toxin-induced cell death. EVs from CDT-treated Caco-2 cells represent reliable CDT carriers, potentially suitable in colorectal cancer treatments. Our data present a potential bacterial-related biotherapeutic supporting a multidrug anticancer protocol. Full article
(This article belongs to the Special Issue Flow Cytometry and Its Applications to Molecular Biology and Diagnosis 2.0)
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19 pages, 3037 KiB  
Article
Flow Cytometric Analysis of Oxidative Stress in Escherichia coli B Strains Deficient in Genes of the Antioxidant Defence
by Beatriz Jávega, Guadalupe Herrera and José-Enrique O’Connor
Int. J. Mol. Sci. 2022, 23(12), 6537; https://doi.org/10.3390/ijms23126537 - 10 Jun 2022
Cited by 3 | Viewed by 3154
Abstract
The detection of reactive oxygen species (ROS) and the analysis of oxidative stress are frequent applications of functional flow cytometry. Identifying and quantifying the ROS species generated during oxidative stress are crucial steps for the investigation of molecular mechanisms underlying stress responses. Currently, [...] Read more.
The detection of reactive oxygen species (ROS) and the analysis of oxidative stress are frequent applications of functional flow cytometry. Identifying and quantifying the ROS species generated during oxidative stress are crucial steps for the investigation of molecular mechanisms underlying stress responses. Currently, there is a wide availability of fluorogenic substrates for such purposes, but limitations in their specificity and sensitivity may affect the accuracy of the analysis. The aim of our work was to validate a new experimental model based in different strains of Escherichia coli B deficient in key genes for antioxidant defense, namely oxyR, sodA and sodB. We applied this model to systematically assess issues of specificity in fluorescent probes and the involvement of different ROS in a bacterial model of oxidative stress, as the probes can react with a variety of oxidants and free radical species. Our results confirm the higher sensitivity and specificity of the fluorescent probe mitochondrial peroxy yellow 1 (MitoPY1) for the detection of H2O2, and its very low capacity for organic hydroperoxides, thus extending MitoPY1′s specificity for H2O2 in mammalian cells to a bacterial model. On the contrary, the fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA) is more sensitive to organic peroxides than to H2O2, confirming the lack of selectivity of H2DCF-DA to H2O2. Treatment with organic peroxides and H2O2 suggests a superoxide-independent oxidation of the fluorescent probe Hydroethidine (HE). We found a positive correlation between the lipophilicity of the peroxides and their toxicity to E. coli, suggesting greater quantitative importance of the peroxidative effects on the bacterial membrane and/or greater efficiency of the protection systems against the intracellular effects of H2O2 than against the membrane oxidative stress induced by organic peroxides. Altogether, our results may aid in preventing or minimizing experimental errors and providing recommendations for the proper design of cytometric studies of oxidative stress, in accordance with current recommendations and guidelines. Full article
(This article belongs to the Special Issue Flow Cytometry and Its Applications to Molecular Biology and Diagnosis 2.0)
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24 pages, 5416 KiB  
Article
Fluorescent Silica Nanoparticles Targeting Mitochondria: Trafficking in Myeloid Cells and Application as Doxorubicin Delivery System in Breast Cancer Cells
by Federica Sola, Mariele Montanari, Mara Fiorani, Chiara Barattini, Caterina Ciacci, Sabrina Burattini, Daniele Lopez, Alfredo Ventola, Loris Zamai, Claudio Ortolani, Stefano Papa and Barbara Canonico
Int. J. Mol. Sci. 2022, 23(6), 3069; https://doi.org/10.3390/ijms23063069 - 12 Mar 2022
Cited by 3 | Viewed by 2391
Abstract
Fluorescent silica nanoparticles (SiNPs) appear to be a promising imaging platform, showing a specific subcellular localization. In the present study, we first investigated their preferential mitochondrial targeting in myeloid cells, by flow cytometry, confocal microscopy and TEM on both cells and isolated mitochondria, [...] Read more.
Fluorescent silica nanoparticles (SiNPs) appear to be a promising imaging platform, showing a specific subcellular localization. In the present study, we first investigated their preferential mitochondrial targeting in myeloid cells, by flow cytometry, confocal microscopy and TEM on both cells and isolated mitochondria, to acquire knowledge in imaging combined with therapeutic applications. Then, we conjugated SiNPs to one of the most used anticancer drugs, doxorubicin (DOX). As an anticancer agent, DOX has high efficacy but also an elevated systemic toxicity, causing multiple side effects. Nanostructures are usually employed to increase the drug circulation time and accumulation in target tissues, reducing undesired cytotoxicity. We tested these functionalized SiNPs (DOX-NPs) on breast cancer cell line MCF-7. We evaluated DOX-NP cytotoxicity, the effect on the cell cycle and on the expression of CD44 antigen, a molecule involved in adhesion and in tumor invasion, comparing DOX-NP to free DOX and stand-alone SiNPs. We found a specific ability to release a minor amount of CD44+ extracellular vesicles (EVs), from both CD81 negative and CD81 positive pools. Modulating the levels of CD44 at the cell surface in cancer cells is thus of great importance for disrupting the signaling pathways that favor tumor progression. Full article
(This article belongs to the Special Issue Flow Cytometry and Its Applications to Molecular Biology and Diagnosis 2.0)
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Review

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10 pages, 423 KiB  
Review
Bioanalytical Assay Strategies and Considerations for Measuring Cellular Kinetics
by Amanda Hays, Jennifer Durham, Bryan Gullick, Nathan Rudemiller and Thomas Schneider
Int. J. Mol. Sci. 2023, 24(1), 695; https://doi.org/10.3390/ijms24010695 - 31 Dec 2022
Cited by 5 | Viewed by 2396
Abstract
A vast evolution of drug modalities has occurred over the last several decades. Novel modalities such as cell and gene therapies have proven to be efficacious for numerous clinical indications–primarily in rare disease and immune oncology. Because of this success, drug developers are [...] Read more.
A vast evolution of drug modalities has occurred over the last several decades. Novel modalities such as cell and gene therapies have proven to be efficacious for numerous clinical indications–primarily in rare disease and immune oncology. Because of this success, drug developers are heavily investing in these novel modalities. Given the complexity of these therapeutics, a variety of bioanalytical techniques are employed to fully characterize the pharmacokinetics of these therapies in clinical studies. Industry trends indicate that quantitative PCR (qPCR) and multiparameter flow cytometry are both valuable in determining the pharmacokinetics, i.e. cellular kinetics, of cell therapies. This manuscript will evaluate the pros and cons of both techniques and highlight regulatory guidance on assays for measuring cellular kinetics. Moreover, common considerations when developing these assays will be addressed. Full article
(This article belongs to the Special Issue Flow Cytometry and Its Applications to Molecular Biology and Diagnosis 2.0)
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19 pages, 1908 KiB  
Review
RNA Flow Cytometry for the Study of T Cell Metabolism
by Alessandra Rossi, Ilenia Pacella and Silvia Piconese
Int. J. Mol. Sci. 2021, 22(8), 3906; https://doi.org/10.3390/ijms22083906 - 09 Apr 2021
Cited by 4 | Viewed by 3930
Abstract
T cells undergo activation and differentiation programs along a continuum of states that can be tracked through flow cytometry using a combination of surface and intracellular markers. Such dynamic behavior is the result of transcriptional and post-transcriptional events, initiated and sustained by the [...] Read more.
T cells undergo activation and differentiation programs along a continuum of states that can be tracked through flow cytometry using a combination of surface and intracellular markers. Such dynamic behavior is the result of transcriptional and post-transcriptional events, initiated and sustained by the activation of specific transcription factors and by epigenetic remodeling. These signaling pathways are tightly integrated with metabolic routes in a bidirectional manner: on the one hand, T cell receptors and costimulatory molecules activate metabolic reprogramming; on the other hand, metabolites modify T cell transcriptional programs and functions. Flow cytometry represents an invaluable tool to analyze the integration of phenotypical, functional, metabolic and transcriptional features, at the single cell level in heterogeneous T cell populations, and from complex microenvironments, with potential clinical application in monitoring the efficacy of cancer immunotherapy. Here, we review the most recent advances in flow cytometry-based analysis of gene expression, in combination with indicators of mitochondrial activity, with the aim of revealing and characterizing major metabolic pathways in T cells. Full article
(This article belongs to the Special Issue Flow Cytometry and Its Applications to Molecular Biology and Diagnosis 2.0)
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