48 results on '"Gioria S"'
Search Results
2. Solar desiccant cooling AHU coupled with chilled ceiling: description of a new installation at DREAM in Palermo
- Author
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BECCALI, Marco, FINOCCHIARO P, NOCKE B, GIORIA S., BECCALI M, FINOCCHIARO P, NOCKE B, and GIORIA S
- Published
- 2007
3. Personalizing services on board a car. An application for tourist information
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Console, Luca, Torre, Ilaria, Lombardi, I., Gioria, S., and Surano, V.
- Published
- 2003
4. Squaraine as a bright, stable and environment-sensitive far-red label for receptor-specific cellular imaging.
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Karpenko, J., Klymchenko, A. S., Gioria, S., Kreder, R., Shulov, I., Villa, P., Mély, Y., Hibert, M., and Bonnet, D.
- Subjects
SQUARAINES ,LECTINS ,CELL imaging - Abstract
Herein, we show that a far-red arylidene–squaraine dye is stable against nucleophiles, in contrast to arene–squaraines. Owing to the fluorescence enhancement in apolar media together with high brightness and photostability, this dye was successfully applied to detect the oxytocin G protein-coupled receptor and monitor its internalization in living cells. [ABSTRACT FROM AUTHOR]
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- 2015
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5. Adaptation and Personalization on Board Cars: A Framework and Its Application to Tourist Services.
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Console, L., Gioria, S., Lombardi, I., Surano, V., and Torre, I.
- Abstract
In this paper we analyse the goals and problems that should be taken into account when designing adaptive/personalized services that must run onboard vehicles. This is, in fact, a very interesting and promising area of application where adaptation and personalization can provide unique advantages. We then introduce a framework and a multi-agent architecture for on-board services supporting different forms of user and context modelling and different forms of adaptation and personalization. Finally, to support our claims and framework, we discuss a specific prototype system for on-board tourist services. [ABSTRACT FROM AUTHOR]
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- 2006
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6. HEMA-Lysine-Based Cryogels for Highly Selective Heparin Neutralization.
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Mecca T, Spitaleri F, La Spina R, Gioria S, Giglio V, and Cunsolo F
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- Humans, Heparin, Low-Molecular-Weight chemistry, Heparin Antagonists chemistry, Heparin chemistry, Heparin adverse effects, Cryogels chemistry, Anticoagulants chemistry, Lysine chemistry
- Abstract
Unfractionated heparin (UFH) and its low-molecular-weight fragments (LMWH) are widely used as anticoagulants for surgical procedures and extracorporeal blood purification therapies such as cardiovascular surgery and dialysis. The anticoagulant effect of heparin is essential for the optimal execution of extracorporeal blood circulation. However, at the end of these procedures, to avoid the risk of bleeding, it is necessary to neutralize it. Currently, the only antidote for heparin neutralization is protamine sulphate, a highly basic protein which constitutes a further source of serious side events and is ineffective in neutralizing LMWH. Furthermore, dialysis patients, due to the routine administration of heparin, often experience serious adverse effects, among which HIT (heparin-induced thrombocytopenia) is one of the most severe. For this reason, the finding of new heparin antagonists or alternative methods for heparin removal from blood is of great interest. Here, we describe the synthesis and characterization of a set of biocompatible macroporous cryogels based on poly(2-hydroxyethyl methacrylate) (pHEMA) and L -lysine with strong filtering capability and remarkable neutralization performance with regard to UFH and LMWH. These properties could enable the design and creation of a filtering device to rapidly reverse heparin, protecting patients from the harmful consequences of the anticoagulant.
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- 2024
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7. Analytical Ultracentrifugation to Assess the Quality of LNP-mRNA Therapeutics.
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Guerrini G, Mehn D, Scaccabarozzi D, Gioria S, and Calzolai L
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- Humans, Particle Size, COVID-19 Vaccines, Nanoparticles chemistry, Ultracentrifugation methods, RNA, Messenger genetics, SARS-CoV-2 genetics, COVID-19 virology
- Abstract
The approval of safe and effective LNP-mRNA vaccines during the SARS-CoV-2 pandemic is catalyzing the development of the next generation of mRNA therapeutics. Proper characterization methods are crucial for assessing the quality and efficacy of these complex formulations. Here, we show that analytical ultracentrifugation (AUC) can measure, simultaneously and without any sample preparation step, the sedimentation coefficients of both the LNP-mRNA formulation and the mRNA molecules. This allows measuring several quality attributes, such as particle size distribution, encapsulation efficiency and density of the formulation. The technique can also be applied to study the stability of the formulation under stress conditions and different buffers.
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- 2024
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8. Nano(bio)Materials Do Not Affect Macrophage Phenotype-A Study Conducted by the REFINE Project.
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David CAW, Vermeulen JP, Gioria S, Vandebriel RJ, and Liptrott NJ
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- Humans, Cells, Cultured, Macrophages metabolism, Macrophages drug effects, Cell Differentiation drug effects, Monocytes metabolism, Monocytes cytology, Biocompatible Materials, Phenotype
- Abstract
Macrophages are well known for their involvement in the biocompatibility, as well as biodistribution, of nano(bio)materials. Although there are a number of rodent cell lines, they may not fully recapitulate primary cell responses, particularly those of human cells. Isolation of tissue-resident macrophages from humans is difficult and may result in insufficient cells with which to determine the possible interaction with nano(bio)materials. Isolation of primary human monocytes and differentiation to monocyte-derived macrophages may provide a useful tool with which to further study these interactions. To that end, we developed a standard operating procedure for this differentiation, as part of the Regulatory Science Framework for Nano(bio)material-based Medical Products and Devices (REFINE) project, and used it to measure the secretion of bioactive molecules from M1 and M2 differentiated monocytes in response to model nano(bio)materials, following an initial assessment of pyrogenic contamination, which may confound potential observations. The SOP was deployed in two partner institutions with broadly similar results. The work presented here shows the utility of this assay but highlights the relevance of donor variability in responses to nano(bio)materials. Whilst donor variability can provide some logistical challenges to the application of such assays, this variability is much closer to the heterogeneous cells that are present in vivo, compared to homogeneous non-human cell lines.
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- 2024
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9. Quality assessment of LNP-RNA therapeutics with orthogonal analytical techniques.
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Parot J, Mehn D, Jankevics H, Markova N, Carboni M, Olaisen C, Hoel AD, Sigfúsdóttir MS, Meier F, Drexel R, Vella G, McDonagh B, Hansen T, Bui H, Klinkenberg G, Visnes T, Gioria S, Urban-Lopez P, Prina-Mello A, Borgos SE, Caputo F, and Calzolai L
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- Reproducibility of Results, Lipids chemistry, RNA, Small Interfering genetics, Liposomes, Particle Size, Nanoparticles chemistry, Nucleic Acids
- Abstract
The availability of analytical methods for the characterization of lipid nanoparticles (LNPs) for in-vivo intracellular delivery of nucleic acids is critical for the fast development of innovative RNA therapies. In this study, analytical protocols to measure (i) chemical composition, (ii) drug loading, (iii) particle size, concentration, and stability as well as (iv) structure and morphology were evaluated and compared based on a comprehensive characterization strategy linking key physical and chemical properties to in-vitro efficacy and toxicity. Furthermore, the measurement protocols were assessed either by testing the reproducibility and robustness of the same technique in different laboratories, or by a correlative approach, comparing measurement results of the same attribute with orthogonal techniques. The characterization strategy and the analytical measurements described here will have an important role during formulation development and in determining robust quality attributes ultimately supporting the quality assessment of these innovative RNA therapeutics., Competing Interests: Declaration of competing interest Roland Drexel and Florian Meier are employees of Postnova Analytics GmbH; Hanna Jankevics, Natalia Markova, Michele Carboni are employees of Malvern Panalytical Ltd. The other authors do not have any conflict of interest to declare., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)
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- 2024
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10. Analytical Ultracentrifugation Detects Quaternary Rearrangements and Antibody-Induced Conformational Selection of the SARS-CoV-2 Spike Trimer.
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Guerrini G, Mehn D, Fumagalli F, Gioria S, Pedotti M, Simonelli L, Bianchini F, Robbiani DF, Varani L, and Calzolai L
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- Humans, Antibodies, Neutralizing immunology, Antibodies, Viral immunology, Epitopes chemistry, Epitopes immunology, Ultracentrifugation, Protein Domains, SARS-CoV-2 chemistry, SARS-CoV-2 immunology, Spike Glycoprotein, Coronavirus chemistry, Spike Glycoprotein, Coronavirus immunology
- Abstract
Analytical ultracentrifugation (AUC) analysis shows that the SARS-CoV-2 trimeric Spike (S) protein adopts different quaternary conformations in solution. The relative abundance of the "open" and "close" conformations is temperature-dependent, and samples with different storage temperature history have different open/close distributions. Neutralizing antibodies (NAbs) targeting the S receptor binding domain (RBD) do not alter the conformer populations; by contrast, a NAb targeting a cryptic conformational epitope skews the Spike trimer toward an open conformation. The results highlight AUC, which is typically applied for molecular mass determination of biomolecules as a powerful tool for detecting functionally relevant quaternary protein conformations.
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- 2023
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11. Differences in Physico-Chemical Properties and Immunological Response in Nanosimilar Complex Drugs: The Case of Liposomal Doxorubicin.
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Lipsa D, Magrì D, Della Camera G, La Spina R, Cella C, Garmendia-Aguirre I, Mehn D, Ruiz-Moreno A, Fumagalli F, Calzolai L, and Gioria S
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- Excipients, Liposomes, Doxorubicin pharmacology, Polyethylene Glycols
- Abstract
This study aims to highlight the impact of physicochemical properties on the behaviour of nanopharmaceuticals and how much carrier structure and physiochemical characteristics weigh on the effects of a formulation. For this purpose, two commercially available nanosimilar formulations of Doxil and their respective carriers were compared as a case study. Although the two formulations were "similar", we detected different toxicological effects (profiles) in terms of in vitro toxicity and immunological responses at the level of cytokines release and complement activation (iC3b fragment), that could be correlated with the differences in the physicochemical properties of the formulations. Shedding light on nanosimilar key quality attributes of liposome-based materials and the need for an accurate characterization, including investigation of the immunological effects, is of fundamental importance considering their great potential as delivery system for drugs, genes, or vaccines and the growing market demand.
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- 2023
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12. Induction of Innate Memory in Human Monocytes Exposed to Mixtures of Bacterial Agents and Nanoparticles.
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Della Camera G, Liu T, Yang W, Li Y, Puntes VF, Gioria S, Italiani P, and Boraschi D
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- Humans, Monocytes, Cytokines, Immune Tolerance, Immunity, Innate, Lipopolysaccharides pharmacology, Nanoparticles
- Abstract
We assessed whether concomitant exposure of human monocytes to bacterial agents and different engineered nanoparticles can affect the induction of protective innate memory, an immune mechanism that affords better resistance to diverse threatening challenges. Monocytes were exposed in vitro to nanoparticles of different chemical nature, shape and size either alone or admixed with LPS, and cell activation was assessed in terms of production of inflammatory (TNFα, IL-6) and anti-inflammatory cytokines (IL-10, IL-1Ra). After return to baseline conditions, cells were re-challenged with LPS and their secondary "memory" response measured. Results show that nanoparticles alone are essentially unable to generate memory, while LPS induced a tolerance memory response (less inflammatory cytokines, equal or increased anti-inflammatory cytokines). LPS-induced tolerance was not significantly affected by the presence of nanoparticles during the memory generation phase, although with substantial donor-to-donor variability. This suggests that, despite the overall lack of significant effects on LPS-induced innate memory, nanoparticles may have donor-specific effects. Thus, future nanosafety assessment and nanotherapeutic strategies will need a personalized approach in order to ensure both the safety and efficacy of nano medical compounds for individual patients.
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- 2022
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13. Monitoring Anti-PEG Antibodies Level upon Repeated Lipid Nanoparticle-Based COVID-19 Vaccine Administration.
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Guerrini G, Gioria S, Sauer AV, Lucchesi S, Montagnani F, Pastore G, Ciabattini A, Medaglini D, and Calzolai L
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- Antibodies, Viral, Humans, Immunoglobulin G, Liposomes, Polyethylene Glycols, COVID-19 prevention & control, COVID-19 Vaccines adverse effects, Nanoparticles
- Abstract
PEGylated lipids are one of the four constituents of lipid nanoparticle mRNA COVID-19 vaccines. Therefore, various concerns have been raised on the generation of anti-PEG antibodies and their potential role in inducing hypersensitivity reactions following vaccination or in reducing vaccine efficacy due to anti-carrier immunity. Here, we assess the prevalence of anti-PEG antibodies, in a cohort of vaccinated individuals, and give an overview of their time evolution after repeated vaccine administrations. Results indicate that, in our cohort, the presence of PEG in the formulation did not influence the level of anti-Spike antibodies generated upon vaccination and was not related to any reported, serious adverse effects. The time-course analysis of anti-PEG IgG showed no significant booster effect after each dose, whereas for IgM a significant increase in antibody levels was detected after the first and third dose. Data suggest that the presence of PEG in the formulation does not affect safety or efficacy of lipid-nanoparticle-based COVID-19 vaccines.
- Published
- 2022
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14. The SARS-CoV-2 Nucleoprotein Induces Innate Memory in Human Monocytes.
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Urbán P, Italiani P, Boraschi D, and Gioria S
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- Humans, Monocytes, Nucleocapsid Proteins, Nucleoproteins, COVID-19, SARS-CoV-2
- Abstract
The interaction of SARS-CoV-2 with the human immune system is at the basis of the positive or negative outcome of the infection. Monocytes and macrophages, which are major innate immune/inflammatory effector cells, are not directly infected by SARS-CoV-2, however they can react to the virus and mount a strong reaction. Whether this first interaction and reaction may bias innate reactivity to re-challenge, a phenomenon known as innate memory, is currently unexplored and may be part of the long-term sequelae of COVID-19. Here, we have tested the capacity of SARS-CoV-2 and some of its proteins to induce innate memory in human monocytes in vitro . Our preliminary results show that the Spike protein subunits S1 and S2 and the entire heat-inactivated virus have no substantial effect. Conversely, monocytes pre-exposed to the nucleocapsid N protein react to subsequent viral or bacterial challenges with an increased production of anti-inflammatory IL-1Ra, a response profile suggesting a milder response to new infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Urbán, Italiani, Boraschi and Gioria.)
- Published
- 2022
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15. In Vitro High-Throughput Toxicological Assessment of Nanoplastics.
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Tolardo V, Magrì D, Fumagalli F, Cassano D, Athanassiou A, Fragouli D, and Gioria S
- Abstract
Sub-micrometer particles derived from the fragmentation of plastics in the environment can enter the food chain and reach humans, posing significant health risks. To date, there is a lack of adequate toxicological assessment of the effects of nanoplastics (NPs) in mammalian systems, particularly in humans. In this work, we evaluated the potential toxic effects of three different NPs in vitro: two NPs obtained by laser ablation (polycarbonate (PC) and polyethylene terephthalate (PET1)) and one (PET2) produced by nanoprecipitation. The physicochemical characterization of the NPs showed a smaller size, a larger size distribution, and a higher degree of surface oxidation for the particles produced by laser ablation. Toxicological evaluation performed on human cell line models (HePG2 and Caco-2) showed a higher toxic effect for the particles synthesized by laser ablation, with PC more toxic than PET. Interestingly, on differentiated Caco-2 cells, a conventional intestinal barrier model, none of the NPs produced toxic effects. This work wants to contribute to increase knowledge on the potential risks posed by NPs.
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- 2022
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16. Characterization of nanoparticles-based vaccines for COVID-19.
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Guerrini G, Magrì D, Gioria S, Medaglini D, and Calzolai L
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- COVID-19 Vaccines therapeutic use, Humans, Nanomedicine methods, COVID-19 prevention & control, Nanoparticles chemistry, Nanoparticles therapeutic use, Vaccines chemistry
- Abstract
Several vaccines against COVID-19 use nanoparticles to protect the antigen cargo (either proteins or nucleic acids), increase the immunogenicity and ultimately the efficacy. The characterization of these nanomedicines is challenging due to their intrinsic complexity and requires the use of multidisciplinary techniques and competencies. The accurate characterization of nanovaccines can be conceptualized as a combination of physicochemical, immunological and toxicological assays. This will help to address key challenges in the preclinical characterization, will guide the rapid development of safe and effective vaccines for current and future health crises, and will streamline the regulatory process., (© 2022. Springer Nature Limited.)
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- 2022
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17. Impact of Viral Decontamination Method on Cytokine Profile of COVID-19 Patients.
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Magrì D, Navarro A, Bergami F, Percivalle E, Ferrari A, Lettieri T, Calzolai L, Piralla A, Baldanti F, and Gioria S
- Abstract
COVID-19 related morbidity and mortality have been often attributed to an exaggerated immune response. The role of cytokines and chemokines in COVID-19 and their contributions to illness severity are known, and thus their profiling from patient bronchoalveolar lavage (BAL) samples would help in understanding the disease progression. To date, limited studies have been performed on COVID-19 BAL samples, as the manipulation of such specimens (potentially containing live viruses) requires several laboratorial precautions, such as personnel training and special equipment, a requirement that not all laboratories can fulfil. Here, we assessed two fast and easily applicable methods (ultrafiltration and ultraviolet-C irradiation) for their impact on viral load removal or inactivation, respectively and on cytokine profiles preservation. Eight samples of BAL fluids from SARS-CoV2 patients with high viral load were tested. For both methods, complete removal was confirmed by lack of viral replication in Vero E6 cells and by RT-qPCR. Although both methods showed to remove completely the active SARS-CoV2 viral load, only UVC treatment has little or no quantitative effect on total cytokines/chemokines measurements, however cytokines profile and relative ratios are preserved or minimally altered when compared data obtained by the two different decontamination methods. Sample preparation and manipulation can greatly affect the analytical results; therefore, understanding if changes occurred after sample processing is of outmost importance for reliable data and can be useful to improve clinical practice.
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- 2021
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18. Publisher Correction: Bispecific IgG neutralizes SARS-CoV-2 variants and prevents escape in mice.
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De Gasparo R, Pedotti M, Simonelli L, Nickl P, Muecksch F, Cassaniti I, Percivalle E, Lorenzi JCC, Mazzola F, Magrì D, Michalcikova T, Haviernik J, Honig V, Mrazkova B, Polakova N, Fortova A, Tureckova J, Iatsiuk V, Di Girolamo S, Palus M, Zudova D, Bednar P, Bukova I, Bianchini F, Mehn D, Nencka R, Strakova P, Pavlis O, Rozman J, Gioria S, Sammartino JC, Giardina F, Gaiarsa S, Pan-Hammarström Q, Barnes CO, Bjorkman PJ, Calzolai L, Piralla A, Baldanti F, Nussenzweig MC, Bieniasz PD, Hatziioannou T, Prochazka J, Sedlacek R, Robbiani DF, Ruzek D, and Varani L
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- 2021
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19. Personalised Profiling of Innate Immune Memory Induced by Nano-Imaging Particles in Human Monocytes.
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Della Camera G, Madej M, Ferretti AM, La Spina R, Li Y, Corteggio A, Heinzl T, Swartzwelter BJ, Sipos G, Gioria S, Ponti A, Boraschi D, and Italiani P
- Subjects
- Cell Differentiation drug effects, Cells, Cultured, Cytokines immunology, Diagnostic Imaging, Humans, Lipopolysaccharides pharmacology, Macrophages drug effects, Macrophages immunology, Monocytes metabolism, Particle Size, Ferric Compounds administration & dosage, Gold administration & dosage, Immunity, Innate drug effects, Immunologic Memory drug effects, Monocytes drug effects, Nanoparticles administration & dosage
- Abstract
Engineered nanoparticles used for medical purposes must meet stringent safety criteria, which include immunosafety, i.e. , the inability to activate possibly detrimental immune/inflammatory effects. Even medical nanomaterials devoid of direct immunotoxic or inflammatory effects may have an impact on human health if able to modify innate memory, which is the ability to "prime" future immune responses towards a different, possibly more detrimental reactivity. Although innate memory is usually protective, anomalous innate memory responses may be at the basis of immune pathologies. In this study, we have examined the ability of two nanomaterials commonly used for diagnostic imaging purposes, gold and iron oxide nanoparticles, to induce or modulate innate memory, using an in vitro model based on human primary monocytes. Monocytes were exposed in culture to nanoparticles alone or together with the bacterial agent LPS (priming phase/primary response), then rested for six days (extinction phase), and eventually challenged with LPS (memory/secondary response). The memory response to the LPS challenge was measured as changes in the production of inflammatory (TNFα, IL-6) and anti-inflammatory cytokines (IL-10, IL-1Ra), as compared to unprimed monocytes. The results show that both types of nanoparticles can have an effect in the induction of memory, with changes observed in the cytokine production. By comparing nanomaterials of different shapes (spherical vs . rod-shaped gold particles) and different size (17 vs . 22 nm diameter spherical iron oxide particles), it was evident that innate memory could be differentially induced and modulated depending on size, shape and chemical composition. However, the main finding was that the innate memory effect of the particles was strongly donor-dependent, with monocytes from each donor showing a distinct memory profile upon priming with the same particles, thereby making impossible to draw general conclusions on the particle effects. Thus, in order to predict the effect of imaging nanoparticles on the innate memory of patients, a personalised profiling would be required, able to take in consideration the peculiarities of the individual innate immune reactivity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Della Camera, Madej, Ferretti, La Spina, Li, Corteggio, Heinzl, Swartzwelter, Sipos, Gioria, Ponti, Boraschi and Italiani.)
- Published
- 2021
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20. Physicochemical Characterization Cascade of Nanoadjuvant-Antigen Systems for Improving Vaccines.
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Guerrini G, Vivi A, Gioria S, Ponti J, Magrì D, Hoeveler A, Medaglini D, and Calzolai L
- Abstract
Adjuvants have been used for decades to enhance the immune response to vaccines, in particular for the subunit-based adjuvants. Physicochemical properties of the adjuvant-protein antigen complexes, such as size, morphology, protein structure and binding, influence the overall efficacy and safety of the vaccine. Here we show how to perform an accurate physicochemical characterization of the nanoaluminum-ovalbumin complex. Using a combination of existing techniques, we developed a multi-staged characterization strategy based on measurements of increased complexity. This characterization cascade has the advantage of being very flexible and easily adaptable to any adjuvant-protein antigen combinations. It will contribute to control the quality of antigen-adjuvant complexes and immunological outcomes, ultimately leading to improved vaccines.
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- 2021
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21. Bispecific IgG neutralizes SARS-CoV-2 variants and prevents escape in mice.
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De Gasparo R, Pedotti M, Simonelli L, Nickl P, Muecksch F, Cassaniti I, Percivalle E, Lorenzi JCC, Mazzola F, Magrì D, Michalcikova T, Haviernik J, Honig V, Mrazkova B, Polakova N, Fortova A, Tureckova J, Iatsiuk V, Di Girolamo S, Palus M, Zudova D, Bednar P, Bukova I, Bianchini F, Mehn D, Nencka R, Strakova P, Pavlis O, Rozman J, Gioria S, Sammartino JC, Giardina F, Gaiarsa S, Pan-Hammarström Q, Barnes CO, Bjorkman PJ, Calzolai L, Piralla A, Baldanti F, Nussenzweig MC, Bieniasz PD, Hatziioannou T, Prochazka J, Sedlacek R, Robbiani DF, Ruzek D, and Varani L
- Subjects
- Angiotensin-Converting Enzyme 2 antagonists & inhibitors, Angiotensin-Converting Enzyme 2 genetics, Angiotensin-Converting Enzyme 2 metabolism, Animals, Antibodies, Bispecific therapeutic use, Antibodies, Monoclonal immunology, Antibodies, Neutralizing therapeutic use, Body Weight, COVID-19 prevention & control, Dependovirus genetics, Disease Models, Animal, Epitopes, B-Lymphocyte chemistry, Epitopes, B-Lymphocyte immunology, Female, Humans, Immune Evasion genetics, Mice, Mice, Inbred C57BL, SARS-CoV-2 genetics, Spike Glycoprotein, Coronavirus antagonists & inhibitors, Spike Glycoprotein, Coronavirus chemistry, Spike Glycoprotein, Coronavirus immunology, COVID-19 Drug Treatment, Antibodies, Bispecific immunology, Antibodies, Neutralizing immunology, COVID-19 immunology, COVID-19 virology, Immunoglobulin G immunology, SARS-CoV-2 immunology
- Abstract
Neutralizing antibodies that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein are among the most promising approaches against COVID-19
1,2 . A bispecific IgG1-like molecule (CoV-X2) has been developed on the basis of C121 and C135, two antibodies derived from donors who had recovered from COVID-193 . Here we show that CoV-X2 simultaneously binds two independent sites on the RBD and, unlike its parental antibodies, prevents detectable spike binding to the cellular receptor of the virus, angiotensin-converting enzyme 2 (ACE2). Furthermore, CoV-X2 neutralizes wild-type SARS-CoV-2 and its variants of concern, as well as escape mutants generated by the parental monoclonal antibodies. We also found that in a mouse model of SARS-CoV-2 infection with lung inflammation, CoV-X2 protects mice from disease and suppresses viral escape. Thus, the simultaneous targeting of non-overlapping RBD epitopes by IgG-like bispecific antibodies is feasible and effective, and combines the advantages of antibody cocktails with those of single-molecule approaches.- Published
- 2021
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22. Bispecific antibody neutralizes circulating SARS-CoV-2 variants, prevents escape and protects mice from disease.
- Author
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De Gasparo R, Pedotti M, Simonelli L, Nickl P, Muecksch F, Cassaniti I, Percivalle E, Lorenzi JCC, Mazzola F, Magrì D, Michalcikova T, Haviernik J, Honig V, Mrazkova B, Polakova N, Fortova A, Tureckova J, Iatsiuk V, Girolamo SD, Palus M, Zudova D, Bednar P, Bukova I, Bianchini F, Mehn D, Nencka R, Strakova P, Pavlis O, Rozman J, Gioria S, Camilla Sammartino J, Giardina F, Gaiarsa S, Hammarström QP, Barnes CO, Bjorkman PJ, Calzolai L, Piralla A, Baldanti F, Nussenzweig MC, Bieniasz PD, Hatziioannou T, Prochazka J, Sedlacek R, Robbiani DF, Ruzek D, and Varani L
- Abstract
Neutralizing antibodies targeting the receptor binding domain (RBD) of the SARS-CoV-2 Spike (S) are among the most promising approaches against coronavirus disease 2019 (COVID-19)
1,2 . We developed a bispecific, IgG1-like molecule (CoV-X2) based on two antibodies derived from COVID-19 convalescent donors, C121 and C1353 . CoV-X2 simultaneously binds two independent sites on the RBD and, unlike its parental antibodies, prevents detectable S binding to Angiotensin-Converting Enzyme 2 (ACE2), the virus cellular receptor. Furthermore, CoV-X2 neutralizes SARS-CoV-2 and its variants of concern, as well as the escape mutants generated by the parental monoclonals. In a novel animal model of SARS-CoV-2 infection with lung inflammation, CoV-X2 protects mice from disease and suppresses viral escape. Thus, simultaneous targeting of non-overlapping RBD epitopes by IgG-like bispecific antibodies is feasible and effective, combining into a single molecule the advantages of antibody cocktails.- Published
- 2021
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23. A Step-by-Step Approach to Improve Clinical Translation of Liposome-Based Nanomaterials, a Focus on Innate Immune and Inflammatory Responses.
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Della Camera G, Lipsa D, Mehn D, Italiani P, Boraschi D, and Gioria S
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- 1,2-Dipalmitoylphosphatidylcholine chemistry, Cell Survival drug effects, Cells, Cultured, Cholesterol chemistry, Cytokines metabolism, Hep G2 Cells, Humans, Inflammation Mediators metabolism, Leukocytes, Mononuclear cytology, Leukocytes, Mononuclear drug effects, Leukocytes, Mononuclear metabolism, Liposomes chemistry, Liposomes pharmacology, Particle Size, Phosphatidylcholines chemistry, Cytokines immunology, Immunity, Innate immunology, Inflammation Mediators immunology, Liposomes immunology, Nanostructures chemistry, Translational Research, Biomedical methods
- Abstract
This study aims to provide guidelines to design and perform a robust and reliable physical-chemical characterization of liposome-based nanomaterials, and to support method development with a specific focus on their inflammation-inducing potential. Out of eight differently functionalized liposomes selected as "case-studies", three passed the physical-chemical characterization ( in terms of size-distribution, homogeneity and stability) and the screening for bacterial contamination (sterility and apyrogenicity). Although all three were non-cytotoxic when tested in vitro, they showed a different capacity to activate human blood cells. HSPC/CHOL-coated liposomes elicited the production of several inflammation-related cytokines, while DPPC/CHOL- or DSPC/CHOL-functionalized liposomes did not. This work underlines the need for accurate characterization at multiple levels and the use of reliable in vitro methods, in order to obtain a realistic assessment of liposome-induced human inflammatory response, as a fundamental requirement of nanosafety regulations.
- Published
- 2021
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24. Nano-enabled medicinal products: time for an international advanced community?
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Gioria S, Caputo F, and Mehn D
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- Animals, Drug Development, Drug and Narcotic Control, Humans, Nanotechnology, Nanomedicine, Nanoparticles therapeutic use
- Published
- 2019
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25. SynAggreg: A Multifunctional High-Throughput Technology for Precision Study of Amyloid Aggregation and Systematic Discovery of Synergistic Inhibitor Compounds.
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Aviolat H, Nominé Y, Gioria S, Bonhoure A, Hoffmann D, Ruhlmann C, Nierengarten H, Ruffenach F, Villa P, Trottier Y, and Klein FAC
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- Amyloidogenic Proteins antagonists & inhibitors, Animals, Drug Evaluation, Preclinical, Drug Synergism, Humans, Kinetics, Amyloidogenic Proteins chemistry, High-Throughput Screening Assays methods, Small Molecule Libraries pharmacology
- Abstract
Numerous proteins can coalesce into amyloid self-assemblies, which are responsible for a class of diseases called amyloidoses, but which can also fulfill important biological functions and are of great interest for biotechnology. Amyloid aggregation is a complex multi-step process, poorly prone to detailed structural studies. Therefore, small molecules interacting with amyloids are often used as tools to probe the amyloid aggregation pathway and in some cases to treat amyloidoses as they prevent pathogenic protein aggregation. Here, we report on SynAggreg, an in vitro high-throughput (HT) platform dedicated to the precision study of amyloid aggregation and the effect of modulator compounds. SynAggreg relies on an accurate bi-fluorescent amyloid-tracer readout that overcomes some limitations of existing HT methods. It allows addressing diverse aspects of aggregation modulation that are critical for pathomechanistic studies, such as the specificity of compounds toward various amyloids and their effects on aggregation kinetics, as well as the co-assembly propensity of distinct amyloids and the influence of prion-like seeding on self-assembly. Furthermore, SynAggreg is the first HT technology that integrates tailored methodology to systematically identify synergistic compound combinations-an emerging strategy to improve fatal amyloidoses by targeting multiple steps of the aggregation pathway. To this end, we apply analytical combinatorial scores to rank the inhibition efficiency of couples of compounds and to readily detect synergism. Finally, the SynAggreg platform should be suited for the characterization of a broad class of amyloids, whether of interest for drug development purposes, for fundamental research on amyloid functions, or for biotechnological applications., (Copyright © 2018 Elsevier Ltd. All rights reserved.)
- Published
- 2018
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26. Proteomics study of silver nanoparticles on Caco-2 cells.
- Author
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Gioria S, Urbán P, Hajduch M, Barboro P, Cabaleiro N, La Spina R, and Chassaigne H
- Subjects
- Caco-2 Cells, Cell Survival drug effects, Humans, Intestine, Small metabolism, Proteomics, Silver Nitrate toxicity, Metal Nanoparticles toxicity, Proteome drug effects, Silver toxicity
- Abstract
Silver nanoparticles (AgNPs) have been incorporated into several consumer products. While these advances in technology are promising and exciting, the effects of these nanoparticles have not equally been studied. Due to the size, AgNPs can penetrate the body through oral exposure and reach the gastrointestinal tract. The present study was designed as a comparative proteomic analysis of Caco-2 cells, used as an in vitro model of the small intestine, exposed to 30 nm citrate stabilized-silver nanoparticles (AgNPs) for 24 or 72 h. Using two complementary proteomic approaches, 2D gel-based and label-free mass spectrometry, we present insight into the effects of AgNPs at proteins level. Exposure of 1 or 10 μg/mL AgNPs to Caco-2 cells resulted in 56 and 88 altered proteins at 24 h and 72 h respectively, by 2D gel-based technique. Ten of these proteins were found to be common between the two time-points. Using label-free mass spectrometry technique, 291 and 179 altered proteins were found at 24 h and 72 h, of which 24 were in common. Analysis of the proteomes showed several major biological processes altered, from which, cell cycle, cell morphology, cellular function and maintenance were the most affected., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2018
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27. Are existing standard methods suitable for the evaluation of nanomedicines: some case studies.
- Author
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Gioria S, Caputo F, Urbán P, Maguire CM, Bremer-Hoffmann S, Prina-Mello A, Calzolai L, and Mehn D
- Subjects
- Chemistry, Pharmaceutical, Drug Carriers adverse effects, Drug Carriers therapeutic use, Drug Liberation, Humans, Nanomedicine, Particle Size, Pharmaceutical Preparations administration & dosage, Drug Carriers chemistry, Nanoparticles chemistry, Pharmaceutical Preparations chemistry
- Abstract
The use of nanotechnology in medical products has been demonstrated at laboratory scale, and many resulting nanomedicines are in the translational phase toward clinical applications, with global market trends indicating strong growth of the sector in the coming years. The translation of nanomedicines toward the clinic and subsequent commercialization may require the development of new or adaptation of existing standards to ensure the quality, safety and efficacy of such products. This work addresses some identified needs, and illustrates the shortcomings of currently used standardized methods when applied to medical-nanoparticles to assess particle size, drug loading, drug release and in vitro safety. Alternative physicochemical, and in vitro toxicology methods, with the potential to qualify as future standards supporting the evaluation of nanomedicine are provided.
- Published
- 2018
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28. Development of an in vitro co-culture model to mimic the human intestine in healthy and diseased state.
- Author
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Kämpfer AAM, Urbán P, Gioria S, Kanase N, Stone V, and Kinsner-Ovaskainen A
- Subjects
- Caco-2 Cells, Coculture Techniques, Humans, In Vitro Techniques, Inflammatory Bowel Diseases, Intestinal Mucosa, THP-1 Cells, Intestinal Diseases pathology, Intestines physiology
- Abstract
The intestine forms the largest interface between the environment and the human organism. Its integrity and functioning are crucial for the uptake of nutrients while preventing access of harmful antigens. Inflammatory conditions can significantly change the normal functioning of the intestine. In vitro models that adequately reproduce both healthy and inflamed intestinal tissue could provide a useful tool for studying the mechanisms of intestinal inflammation and investigating new therapeutic drugs. We established a co-culture of Caco-2 and PMA-differentiated THP-1 cells that mimics the intestine in healthy and controlled inflamed states. In homoeostatic conditions without stimulation, Caco-2 and THP-1 cells were co-cultured for 48h without affecting the barrier integrity and with no increase in the release of cytokines, nitric oxide or lactate dehydrogenase. To simulate the inflamed intestine, the Caco-2 barrier was primed with IFN-γ and THP-1 cells were pre-stimulated with LPS and IFN-γ. In these conditions a significant but temporary reduction in barrier integrity was measured, and large concentrations of pro-inflammatory cytokines and cytotoxicity markers detected. With its ability to feature numerous hallmarks of intestinal inflammation the presented co-culture model of epithelial cells and macrophages offers a unique possibility to study exposure effects in relation to the health status of the intestine., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2017
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29. The agglomeration state of nanoparticles can influence the mechanism of their cellular internalisation.
- Author
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Halamoda-Kenzaoui B, Ceridono M, Urbán P, Bogni A, Ponti J, Gioria S, and Kinsner-Ovaskainen A
- Subjects
- Caco-2 Cells, Dimerization, Humans, Microscopy, Electron, Transmission, Nanoparticles analysis, Nanoparticles ultrastructure, Particle Size, Silicon Dioxide analysis, Endocytosis, Nanoparticles metabolism, Silicon Dioxide metabolism
- Abstract
Background: Significant progress of nanotechnology, including in particular biomedical and pharmaceutical applications, has resulted in a high number of studies describing the biological effects of nanomaterials. Moreover, a determination of so-called "critical quality attributes", that is specific physicochemical properties of nanomaterials triggering the observed biological response, has been recognised as crucial for the evaluation and design of novel safe and efficacious therapeutics. In the context of in vitro studies, a thorough physicochemical characterisation of nanoparticles (NPs), also in the biological medium, is necessary to allow a correlation with a cellular response. Following this concept, we examined whether the main and frequently reported characteristics of NPs such as size and the agglomeration state can influence the level and the mechanism of NP cellular internalization., Results: We employed fluorescently-labelled 30 and 80 nm silicon dioxide NPs, both in agglomerated and non-agglomerated form. Using flow cytometry, transmission electron microscopy, the inhibitors of endocytosis and gene silencing we determined the most probable routes of cellular uptake for each form of tested silica NPs. We observed differences in cellular uptake depending on the size and the agglomeration state of NPs. Caveolae-mediated endocytosis was implicated particularly in the internalisation of well dispersed silica NPs but with an increase of the agglomeration state of NPs a combination of endocytic pathways with a predominant role of macropinocytosis was noted., Conclusions: We demonstrated that the agglomeration state of NPs is an important factor influencing the level of cell uptake and the mechanism of endocytosis of silica NPs.
- Published
- 2017
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30. The adaptors Grb10 and Grb14 are calmodulin-binding proteins.
- Author
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García-Palmero I, Pompas-Veganzones N, Villalobo E, Gioria S, Haiech J, and Villalobo A
- Subjects
- Adaptor Proteins, Signal Transducing chemistry, Adaptor Proteins, Signal Transducing genetics, Amino Acid Sequence, Binding Sites, Calmodulin chemistry, Chromatography, Affinity, Conserved Sequence, GRB10 Adaptor Protein chemistry, GRB10 Adaptor Protein genetics, GRB7 Adaptor Protein chemistry, GRB7 Adaptor Protein genetics, GRB7 Adaptor Protein metabolism, Gene Deletion, HEK293 Cells, Humans, Kinetics, Mutagenesis, Site-Directed, Peptide Fragments chemistry, Peptide Fragments genetics, Peptide Fragments metabolism, Protein Conformation, Protein Interaction Domains and Motifs, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins metabolism, Sequence Homology, Amino Acid, Structural Homology, Protein, Adaptor Proteins, Signal Transducing metabolism, Calcium Signaling, Calmodulin metabolism, GRB10 Adaptor Protein metabolism
- Abstract
We identified the Grb7 family members, Grb10 and Grb14, as Ca
2+ -dependent CaM-binding proteins using Ca2+ -dependent CaM-affinity chromatography as we previously did with Grb7. The potential CaM-binding sites were identified and experimentally tested using fluorescent-labeled peptides corresponding to these sites. The apparent affinity constant of these peptides for CaM, and the minimum number of calcium ions bound to CaM that are required for effective binding to these peptides were also determined. We prepared deletion mutants of the three adaptor proteins lacking the identified sites and determined that they lost or strongly diminished their CaM-binding capacity following the sequence Grb7 > > Grb14 > Grb10. More than one CaM-binding site and/or accessory CaM-binding sites appear to exist in Grb10 and Grb14, as compared to a single one present in Grb7., (© 2017 Federation of European Biochemical Societies.)- Published
- 2017
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31. Erratum to: Quantification of the cellular dose and characterization of nanoparticle transport during in vitro testing.
- Author
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Rischitor G, Parracino M, La Spina R, Urbán P, Ojea-Jiménez I, Bellido E, Valsesia A, Gioria S, Capomaccio R, Kinsner-Ovaskainen A, Gilliland D, Rossi F, and Colpo P
- Published
- 2016
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32. Quantification of the cellular dose and characterization of nanoparticle transport during in vitro testing.
- Author
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Rischitor G, Parracino M, La Spina R, Urbán P, Ojea-Jiménez I, Bellido E, Valsesia A, Gioria S, Capomaccio R, Kinsner-Ovaskainen A, Gilliland D, Rossi F, and Colpo P
- Subjects
- Dose-Response Relationship, Drug, Humans, In Vitro Techniques, Spectrophotometry, Ultraviolet, Nanoparticles toxicity
- Abstract
Background: The constant increase of the use of nanomaterials in consumer products is making increasingly urgent that standardized and reliable in vitro test methods for toxicity screening be made available to the scientific community. For this purpose, the determination of the cellular dose, i.e. the amount of nanomaterials effectively in contact with the cells is fundamental for a trustworthy determination of nanomaterial dose responses. This has often been overlooked in the literature making it difficult to undertake a comparison of datasets from different studies. Characterization of the mechanisms involved in nanomaterial transport and the determination of the cellular dose is essential for the development of predictive numerical models and reliable in vitro screening methods., Results: This work aims to relate key physico-chemical properties of gold nanoparticles (NPs) to the kinetics of their deposition on the cellular monolayer. Firstly, an extensive characterization of NPs in complete culture cell medium was performed to determine the diameter and the apparent mass density of the formed NP-serum protein complexes. Subsequently, the kinetics of deposition were studied by UV-vis absorbance measurements in the presence or absence of cells. The fraction of NPs deposited on the cellular layer was found to be highly dependent on NP size and apparent density because these two parameters influence the NP transport. The NP deposition occurred in two phases: phase 1, which consists of cellular uptake driven by the NP-cell affinity, and phase 2 consisting mainly of NP deposition onto the cellular membrane., Conclusion: The fraction of deposited NPs is very different from the initial concentration applied in the in vitro assay, and is highly dependent of the size and density of the NPs, on the associated transport rate and on the exposure duration. This study shows that an accurate characterization is needed and suitable experimental conditions such as initial concentration of NPs and liquid height in the wells has to be considered since they strongly influence the cellular dose and the nature of interactions of NPs with the cells.
- Published
- 2016
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33. A combined proteomics and metabolomics approach to assess the effects of gold nanoparticles in vitro.
- Author
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Gioria S, Lobo Vicente J, Barboro P, La Spina R, Tomasi G, Urbán P, Kinsner-Ovaskainen A, François R, and Chassaigne H
- Subjects
- Biomarkers metabolism, Caco-2 Cells, Cell Culture Techniques, Cytoplasm drug effects, Cytoplasm metabolism, Humans, Gold toxicity, Metabolomics methods, Metal Nanoparticles toxicity, Proteomics methods
- Abstract
Omics technologies, such as proteomics or metabolomics, have to date been applied in the field of nanomaterial safety assessment to a limited extent. To address this dearth, we developed an integrated approach combining the two techniques to study the effects of two sizes, 5 and 30 nm, of gold nanoparticles (AuNPs) in Caco-2 cells. We observed differences in cells exposed for 72 h to each size of AuNPs: 61 responsive (up/down-regulated) proteins were identified and 35 metabolites in the cell extract were tentatively annotated. Several altered biological pathways were highlighted by integrating the obtained multi-omics data with bioinformatic tools. This provided a unique set of molecular information on the effects of nanomaterials at cellular level. This information was supported by complementary data obtained by immunochemistry, microscopic analysis, and multiplexed assays. A part from increasing our knowledge on how the cellular processes and pathways are affected by nanomaterials (NMs), these findings could be used to identify specific biomarkers of toxicity or to support the safe-by-design concept in the development of new nanomedicines.
- Published
- 2016
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34. Dispersion Behaviour of Silica Nanoparticles in Biological Media and Its Influence on Cellular Uptake.
- Author
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Halamoda-Kenzaoui B, Ceridono M, Colpo P, Valsesia A, Urbán P, Ojea-Jiménez I, Gioria S, Gilliland D, Rossi F, and Kinsner-Ovaskainen A
- Subjects
- Caco-2 Cells, Cell Line, Cell Proliferation drug effects, Cell Survival drug effects, Flow Cytometry, Humans, Particle Size, Silicon Dioxide chemistry, Surface Properties, Culture Media chemistry, Nanoparticles chemistry, Silicon Dioxide pharmacokinetics
- Abstract
Given the increasing variety of manufactured nanomaterials, suitable, robust, standardized in vitro screening methods are needed to study the mechanisms by which they can interact with biological systems. The in vitro evaluation of interactions of nanoparticles (NPs) with living cells is challenging due to the complex behaviour of NPs, which may involve dissolution, aggregation, sedimentation and formation of a protein corona. These variable parameters have an influence on the surface properties and the stability of NPs in the biological environment and therefore also on the interaction of NPs with cells. We present here a study using 30 nm and 80 nm fluorescently-labelled silicon dioxide NPs (Rubipy-SiO2 NPs) to evaluate the NPs dispersion behaviour up to 48 hours in two different cellular media either supplemented with 10% of serum or in serum-free conditions. Size-dependent differences in dispersion behaviour were observed and the influence of the living cells on NPs stability and deposition was determined. Using flow cytometry and fluorescence microscopy techniques we studied the kinetics of the cellular uptake of Rubipy-SiO2 NPs by A549 and CaCo-2 cells and we found a correlation between the NPs characteristics in cell media and the amount of cellular uptake. Our results emphasize how relevant and important it is to evaluate and to monitor the size and agglomeration state of nanoparticles in the biological medium, in order to interpret correctly the results of the in vitro toxicological assays.
- Published
- 2015
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35. Changes in Caco-2 cells transcriptome profiles upon exposure to gold nanoparticles.
- Author
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Bajak E, Fabbri M, Ponti J, Gioria S, Ojea-Jiménez I, Collotta A, Mariani V, Gilliland D, Rossi F, and Gribaldo L
- Subjects
- Cell Survival drug effects, Computational Biology, Gene Expression Regulation, Neoplastic drug effects, Gold metabolism, Humans, Microarray Analysis, Nanoparticles metabolism, Particle Size, Caco-2 Cells drug effects, Gold toxicity, Nanoparticles toxicity, Transcriptome drug effects
- Abstract
Higher efficacy and safety of nano gold therapeutics require examination of cellular responses to gold nanoparticles (AuNPs). In this work we compared cellular uptake, cytotoxicity and RNA expression patterns induced in Caco-2 cells exposed to AuNP (5 and 30nm). Cellular internalization was dose and time-dependent for both AuNPs. The toxicity was observed by colony forming efficiency (CFE) and not by Trypan blue assay, and exclusively for 5nm AuNPs, starting at the concentration of 200μM (24 and 72h of exposure). The most pronounced changes in gene expression (Agilent microarrays) were detected at 72h (300μM) of exposure to AuNPs (5nm). The biological processes affected by smaller AuNPs were: RNA/zinc ion/transition metal ion binding (decreased), cadmium/copper ion binding and glutathione metabolism (increased). Some Nrf2 responsive genes (several metallothioneins, HMOX, G6PD, OSGIN1 and GPX2) were highly up regulated. Members of the selenoproteins were also differentially expressed. Our findings indicate that exposure to high concentration of AuNPs (5nm) induces metal exposure, oxidative stress signaling pathways, and might influence selenium homeostasis. Some of detected cellular responses might be explored as potential enhancers of anti-cancer properties of AuNPs based nanomedicines., (Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.)
- Published
- 2015
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36. A proteomic approach to investigate AuNPs effects in Balb/3T3 cells.
- Author
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Gioria S, Chassaigne H, Carpi D, Parracino A, Meschini S, Barboro P, and Rossi F
- Subjects
- Animals, BALB 3T3 Cells, Cell Fractionation, Chromatography, High Pressure Liquid, Collagen Type I biosynthesis, Collagen Type I genetics, Databases, Protein, Electrophoresis, Gel, Two-Dimensional, Fibronectins biosynthesis, Fibronectins genetics, Gene Expression Regulation drug effects, Hydrolysis, Image Processing, Computer-Assisted, Mass Spectrometry, Mice, Microscopy, Electron, Transmission, Peptides chemistry, Phosphorylation, Signal Transduction drug effects, Surface Plasmon Resonance, Gold toxicity, Metal Nanoparticles toxicity, Proteomics methods
- Abstract
Although gold nanoparticles (AuNPs) are currently used in several industrial products and biomedical applications, information about their biological effects is very limited. Thus, it is becoming crucial to assess their safety and adequately investigate the complexity of cell-nanoparticles interactions. In this work, the Balb/3T3 mouse fibroblast cell line was selected as an in vitro model to study the effects of AuNPs. Alteration of cellular processes and biochemical pathways caused by AuNPs exposure was investigated by analysing the differentially expressed proteome. Of interest was the difference observed in the protein pattern expression of cells exposed to AuNPs. It was found that 88 and 83 proteins were de-regulated after exposure to 5 and 15nm AuNPs, respectively. Analysis of the proteome revealed that AuNPs triggers several pathways related to cellular growth and proliferation, cell morphology, cell cycle regulation, cellular function and maintenance, oxidative stress, and inflammatory response. Moreover, SPR analysis showed an increase of ECM proteins biosynthesis in cells exposed to AuNPs. We observed by TEM analysis that NPs are internalized and confined mainly in autophagosomes. Endoplasmic reticulum stressed and modification at mitochondrial level occurred. This study aims to improve existing knowledge necessary for a correct assessment of the balance between AuNPs potential adverse and beneficial effects and might have important implications for biomedical applications (e.g. nanomedicine). To conclude proteomics link to system biology analysis is a valuable tool to understand and predict nanoparticles' toxicity, furthermore it has the potential to reveal pathways that may not be immediately evident with classical toxicological assays., (Copyright © 2014 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.)
- Published
- 2014
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37. Silica nanoparticle uptake induces survival mechanism in A549 cells by the activation of autophagy but not apoptosis.
- Author
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Nowak JS, Mehn D, Nativo P, García CP, Gioria S, Ojea-Jiménez I, Gilliland D, and Rossi F
- Subjects
- Cell Line, Tumor, Cell Survival drug effects, Humans, Silicon Dioxide pharmacokinetics, Apoptosis drug effects, Autophagy drug effects, Nanoparticles toxicity, Silicon Dioxide toxicity
- Abstract
We report here an in vitro evaluation of silica nanoparticle uptake by lung epithelial cells (A549), the cytotoxic effect of the particles and we propose autophagy as possible survival strategy. The effect of surface charge, serum proteins and the influence of inhibitors on the uptake of 20 nm monodispersed nanoparticles with various functional groups are discussed. Uptake rate of the particles with various functional groups is demonstrated to be similar in the presence of serum proteins, while the uptake rate ranking is COOH>NH2>OH under serum free conditions. Our results suggest an actin-dependent, macropinocytotic uptake process that was also confirmed by scanning and transmission electron microscopy. In spite of the intensive active uptake, significant cytotoxic effect is detected only at relatively high concentrations (above 250 μg/mL). Blebbing of the cell surface is observed already at 5h of exposure and is shown to be related to autophagy rather than apoptotic cell death. The A549 cells display elevated levels of autophagosomes, however they do not express typical apoptosis markers such as increased amount of active caspase-3 and release of mitochondrial cytochrome C. Based on these results, we propose here an autophagic activity and cross-talk between autophagic and apoptotic pathways as a mechanism allowing the survival of A549 cells under exposure to silica nanoparticles., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)
- Published
- 2014
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38. A general framework to characterize inhibitors of calmodulin: use of calmodulin inhibitors to study the interaction between calmodulin and its calmodulin binding domains.
- Author
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Audran E, Dagher R, Gioria S, Tsvetkov PO, Kulikova AA, Didier B, Villa P, Makarov AA, Kilhoffer MC, and Haiech J
- Subjects
- Allosteric Site, Binding Sites, Binding, Competitive, Calcium Channels, L-Type metabolism, Fluorescence Polarization, Humans, Molecular Structure, Peptide Library, Protein Binding, Thermodynamics, Calcium metabolism, Calmodulin antagonists & inhibitors, Calmodulin metabolism, Cell Membrane metabolism, Peptide Fragments pharmacology
- Abstract
The prominent role of Ca(2+) in cell physiology is mediated by a whole set of proteins involved in Ca(2+)-signal generation, deciphering and arrest. Among these intracellular proteins, calmodulin (CaM) known as a prototypical calcium sensor, serves as a ubiquitous carrier of the intracellular calcium signal in all eukaryotic cell types. CaM is assumed to be involved in many diseases including Parkinson, Alzheimer, and rheumatoid arthritis. Defects in some of many reaction partners of CaM might be responsible for disease symptoms. Several classes of drugs bind to CaM with unwanted side effects rather than specific therapeutic use. Thus, it may be more promising to concentrate at searching for pharmacological interferences with the CaM target proteins, in order to find tools for dissecting and investigating CaM-regulatory and modulatory functions in cells. In the present study, we have established a screening assay based on fluorescence polarization (FP) to identify a diverse set of small molecules that disrupt the regulatory function of CaM. The FP-based CaM assay consists in the competition of two fluorescent probes and a library of chemical compounds for binding to CaM. Screening of about 5300 compounds (Strasbourg Academic Library) by displacement of the probe yielded 39 compounds in a first step, from which 6 were selected. Those 6 compounds were characterized by means of calorimetry studies and by competitive displacement of two fluorescent probes interacting with CaM. Moreover, those small molecules were tested for their capability to displace 8 different CaM binding domains from CaM. Our results show that these CaM/small molecules interactions are not functionally equivalent. The strategy that has been set up for CaM is a general model for the development and validation of other CaM interactors, to decipher their mode of action, or rationally design more specific CaM antagonists. Moreover, this strategy may be used for other protein binding assays intended to screen for molecules with preferred binding activity. This article is part of a Special Issue entitled: 12th European Symposium on Calcium., (Copyright © 2013 Elsevier B.V. All rights reserved.)
- Published
- 2013
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39. Size-dependent toxicity and cell interaction mechanisms of gold nanoparticles on mouse fibroblasts.
- Author
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Coradeghini R, Gioria S, García CP, Nativo P, Franchini F, Gilliland D, Ponti J, and Rossi F
- Subjects
- Animals, BALB 3T3 Cells, Blotting, Western, Caveolin 1 metabolism, Cell Survival drug effects, Clathrin Heavy Chains metabolism, Endocytosis drug effects, Fibroblasts metabolism, Fibroblasts ultrastructure, Immunohistochemistry, Mice, Microscopy, Electron, Transmission, Particle Size, Fibroblasts drug effects, Gold toxicity, Metal Nanoparticles toxicity
- Abstract
Gold nanoparticles (AuNPs) are currently used in several fields including biomedical applications, although no conclusive information on their cytotoxicity is available. For this reason this work has investigated the effects of AuNPs in vitro on Balb/3T3 mouse fibroblasts. Results obtained exposing cells for 72 h to AuNPs 5 and 15 nm citrate stabilized, revealed cytotoxic effects only for AuNPs 5 nm at concentration ≥ 50 μM if measured by colony forming efficiency (CFE). To understand the differences in cytotoxicity observed for the two AuNPs sizes, we investigated the uptake and the intracellular distribution of the nanoparticles. By TEM it was observed that 5 and 15 nm AuNPs are internalized by Balb/3T3 cells and located within intracellular endosomal compartments. Quantification of the uptake by ICP-MS showed that AuNPs internalization enhanced even up to 72 h. Disruption of the actin cytoskeleton was evident, with cell footprints narrow and contracted; effects more remarkable in cells exposed to 5 nm AuNP. The mechanism of NPs cell internalization was investigated using immunocytochemistry and western blot. No significant effect was observed in the expression level of caveolin, while reduction of the expression and degradation of the clathrin heavy chain was observed in cells exposed for 72 h to AuNPs., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)
- Published
- 2013
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40. Morphological transformation induced by multiwall carbon nanotubes on Balb/3T3 cell model as an in vitro end point of carcinogenic potential.
- Author
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Ponti J, Broggi F, Mariani V, De Marzi L, Colognato R, Marmorato P, Gioria S, Gilliland D, Pascual Garcìa C, Meschini S, Stringaro A, Molinari A, Rauscher H, and Rossi F
- Subjects
- Animal Testing Alternatives, Animals, BALB 3T3 Cells, Carcinogenicity Tests, Cell Transformation, Neoplastic pathology, Dose-Response Relationship, Drug, Mice, Micronuclei, Chromosome-Defective chemically induced, Micronucleus Tests, Risk Assessment, Time Factors, Carcinogens toxicity, Cell Shape drug effects, Cell Transformation, Neoplastic chemically induced, Nanotubes, Carbon toxicity
- Abstract
In this work we investigated the toxicological effects of nude and chemically functionalised (-NH(2), -OH and -COOH groups) multiwall carbon nanotubes (mwCNTs) using immortalised mouse fibroblasts cell line (Balb/3T3) as in vitro model, alternative to the use of animals, to assess basal cytotoxicity, carcinogenic potential, genotoxicity and cell interaction of nanomaterials (NM). Combining in vitro tests such as cell transformation assay and micronucleus with physicochemical and topological analysis, we obtained results showing no cytotoxicity and genotoxicity. Carcinogenic potential and mwCNTs interaction with cells were instead evident. We stressed the importance that different toxicological end points have to be considered when studying NM, therefore, assays able to detect long-term effects, such as carcinogenicity, must be taken into account together with a panel of tests able to detect more immediate effects like basal cytotoxicity or genotoxicity.
- Published
- 2013
- Full Text
- View/download PDF
41. New aspects of calmodulin-calmodulin binding domains recognition.
- Author
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Audran E, Dagher R, Gioria S, Kilhoffer MC, and Haiech J
- Subjects
- Amino Acid Sequence, Buffers, Fluorescence Polarization, Humans, Models, Molecular, Molecular Sequence Data, Peptides chemical synthesis, Peptides chemistry, Peptides metabolism, Protein Binding, Protein Structure, Tertiary, Calmodulin chemistry, Calmodulin metabolism
- Abstract
Understanding the role of calmodulin (CaM) in calcium signal transduction implies to describe the -calcium-dependent molecular mechanism of interaction of CaM with the various CaM-binding domains (CBD). In order to fulfill this aim, we have developed a new strategy and the afferent techniques to quantify the interaction of CaM with any CBD as a function of calcium concentration. Excel software has been used to deconvolute the experimental data and to obtain the macroscopic constants characterizing the system. We are illustrating our approach on six different CaM/CBD. This strategy may be used to analyze the interaction between any calcium-binding protein and its targets.
- Published
- 2013
- Full Text
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42. Online monitoring of cell metabolism to assess the toxicity of nanoparticles: the case of cobalt ferrite.
- Author
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Mariani V, Ponti J, Giudetti G, Broggi F, Marmorato P, Gioria S, Franchini F, Rauscher H, and Rossi F
- Subjects
- Animals, BALB 3T3 Cells, Cell Culture Techniques, Cell Hypoxia drug effects, Cell Proliferation drug effects, Cell Survival drug effects, Cobalt chemistry, Culture Media, Ferric Compounds chemistry, Magnetite Nanoparticles chemistry, Mice, Microscopy, Electron, Transmission, Oxygen Consumption drug effects, Particle Size, Surface Properties, Toxicity Tests instrumentation, Cobalt toxicity, Ferric Compounds toxicity, Fibroblasts drug effects, Fibroblasts metabolism, Magnetite Nanoparticles toxicity, Toxicity Tests methods
- Abstract
Different in vitro assays are successfully used to determine the relative cytotoxicity of a broad range of compounds. Nevertheless, different research groups have pointed out the difficulty in using the same tests to assess the toxicity of nanoparticles (NPs). In this study, we evaluated the possible use of a microphysiometer, Bionas 2500 analyzing system Bionas GmbH®, to detect in real time changes in cell metabolisms linked to NPs exposure. We focused our work on response changes of fibroblast cultures linked to exposure by cobalt ferrite NPs and compared the results to conventional in vitro assays. The measurements with the microphysiometer showed a cobalt ferrite cytotoxic effect, confirmed by the Colony Forming Efficiency assay. In conclusion, this work demonstrated that the measurement of metabolic parameters with a microphysiometer is a promising method to assess the toxicity of NPs and offers the advantage to follow on-line the cell metabolic changes.
- Published
- 2012
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43. Identification of Nonpeptide Oxytocin Receptor Ligands by Receptor-Ligand Fingerprint Similarity Search.
- Author
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Weill N, Valencia C, Gioria S, Villa P, Hibert M, and Rognan D
- Published
- 2011
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44. A general strategy to characterize calmodulin-calcium complexes involved in CaM-target recognition: DAPK and EGFR calmodulin binding domains interact with different calmodulin-calcium complexes.
- Author
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Dagher R, Peng S, Gioria S, Fève M, Zeniou M, Zimmermann M, Pigault C, Haiech J, and Kilhoffer MC
- Subjects
- Amino Acid Sequence, Death-Associated Protein Kinases, Fluorescent Dyes metabolism, Kinetics, Molecular Sequence Data, Mutant Proteins chemistry, Mutant Proteins metabolism, Peptides chemistry, Peptides metabolism, Protein Binding, Protein Structure, Tertiary, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Static Electricity, Thermodynamics, Apoptosis Regulatory Proteins chemistry, Apoptosis Regulatory Proteins metabolism, Biochemistry methods, Calcium metabolism, Calcium-Calmodulin-Dependent Protein Kinases chemistry, Calcium-Calmodulin-Dependent Protein Kinases metabolism, Calmodulin metabolism, ErbB Receptors chemistry, ErbB Receptors metabolism
- Abstract
Calmodulin (CaM) is a ubiquitous Ca(2+) sensor regulating many biochemical processes in eukaryotic cells. Its interaction with a great variety of different target proteins has led to the fundamental question of its mechanism of action. CaM exhibits four "EF hand" type Ca(2+) binding sites. One way to explain CaM functioning is to consider that the protein interacts differently with its target proteins depending on the number of Ca(2+) ions bound to it. To test this hypothesis, the binding properties of three entities known to interact with CaM (a fluorescent probe and two peptide analogs to the CaM binding sites of death associated protein kinase (DAPK) and of EGFR) were investigated using a quantitative approach based on fluorescence polarization (FP). Probe and peptide interactions with CaM were studied using a titration matrix in which both CaM and calcium concentrations were varied. Experiments were performed with SynCaM, a hybrid CaM able to activate CaM dependent enzymes from mammalian and plant cells. Results show that the interaction between CaM and its targets is regulated by the number of calcium ions bound to the protein, namely one for the DAPK peptide, two for the probe and four for the EGFR peptide. The approach used provides a new tool to elaborate a typology of CaM-targets, based on their recognition by the various CaM-Ca(n) (n=0-4) complexes. This article is part of a Special Issue entitled: 11th European Symposium on Calcium., (2010 Elsevier B.V. All rights reserved.)
- Published
- 2011
- Full Text
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45. Colony Forming Efficiency and microscopy analysis of multi-wall carbon nanotubes cell interaction.
- Author
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Ponti J, Colognato R, Rauscher H, Gioria S, Broggi F, Franchini F, Pascual C, Giudetti G, and Rossi F
- Subjects
- Animals, Cell Line, Humans, Kinetics, Mice, Microscopy, Atomic Force, Microscopy, Electron, Scanning, Nanotubes, Carbon ultrastructure, Colony-Forming Units Assay, Nanotubes, Carbon toxicity, Toxicity Tests methods
- Abstract
In this work, we present a complete physicochemical characterization of multi-wall carbon nanotubes (mwCNTs) in order to assess their potential toxicological effects in in vitro cell models using Colony Forming Efficiency (CFE) assay. We verified that Dimethyl Sulfoxide (DMSO) was a more suitable solvent to disperse mwCNTs compared to culture medium guaranteeing reproducibility in the preparation of testing dilutions. The CFE assay was carried out on five mammalian cell lines representing the potentially exposed and/or target organs for nanomaterials (lung, liver, kidney, intestine, skin), as well as on mouse fibroblasts cell line, which usually is considered a sensitive model to verify in vitro cytotoxicity of test compounds. A statistically significant toxic effect was found only in human alveolar basal epithelial cells and immortalized mouse fibroblasts, for which the interaction between mwCNTs and cells was additionally studied by Atomic Force and Scanning Electron Microscopy. In this study, we considered and suggested the CFE assay as a promising test for screening studies of cytotoxicity. In addition, combining in vitro tests with physicochemical analysis, this work underlines basic points to be considered when research on nanomaterials has to be carried out, to set up, in our opinion, well-defined and suitable experimental planning and procedures., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)
- Published
- 2010
- Full Text
- View/download PDF
46. Toward metrological traceability for DNA fragment ratios in GM quantification. 1. Effect of DNA extraction methods on the quantitative determination of Bt176 corn by real-time PCR.
- Author
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Corbisier P, Broothaerts W, Gioria S, Schimmel H, Burns M, Baoutina A, Emslie KR, Furui S, Kurosawa Y, Holden MJ, Kim HH, Lee YM, Kawaharasaki M, Sin D, and Wang J
- Subjects
- Bacillus thuringiensis Toxins, DNA, Plant analysis, Seeds genetics, Bacterial Proteins genetics, Bacterial Toxins genetics, DNA, Plant isolation & purification, Endotoxins genetics, Food, Genetically Modified, Hemolysin Proteins genetics, Plants, Genetically Modified genetics, Polymerase Chain Reaction methods, Zea mays genetics
- Abstract
An international CCQM-P60 pilot study involving eight national metrological institutes was organized to investigate if the quantification of genetically modified (GM) corn powder by real-time PCR was affected by the DNA extraction method applied. Four commonly used extraction methods were compared for the extraction of DNA from a GM Bt176 corn powder. The CTAB-based method yielded the highest DNA template quantity and quality. A difference in the 260 nm/230 nm absorbance ratio was observed among the different extraction methods. Real-time amplification of sequences specific for endogenous genes zein and hmg as well as transgenic sequences within the cryIA(b) gene and a fragment covering the junction between the transformed DNA and the plant genome were used to determine the GM percentage. The detection of the transgenic gene was affected by the quantity and quality of template used for the PCR reaction. The Bt176 percentages measured on diluted or purified templates were statistically different depending on the extraction method applied.
- Published
- 2007
- Full Text
- View/download PDF
47. FRET and colocalization analyzer--a method to validate measurements of sensitized emission FRET acquired by confocal microscopy and available as an ImageJ Plug-in.
- Author
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Hachet-Haas M, Converset N, Marchal O, Matthes H, Gioria S, Galzi JL, and Lecat S
- Subjects
- Arrestins metabolism, Fluorescent Dyes, Humans, Receptors, G-Protein-Coupled metabolism, Software, beta-Arrestin 2, beta-Arrestins, Fluorescence Resonance Energy Transfer, Image Processing, Computer-Assisted, Microscopy, Confocal methods
- Abstract
Fluorescence resonance energy transfer (FRET) between an adequate pair of fluorophores is an indication of closer proximity than colocalization and is used by biologists to study fluorescently modified protein interactions inside cells. We present a method for visualization of FRET images acquired by confocal sensitized emission, involving excitation of the donor fluorophore and detection of the energy transfer as an emission from the acceptor fluorophore into the FRET channel. Authentic FRET signal measurements require the correction from the FRET channel of the undesired bleed-through signals (BT) resulting from both the leak-through of the donor emission and the direct acceptor emission. Our method reduces the interference of the user to a minimum by analyzing the entire image, pixel by pixel. It proposes imaging treatments and the display of control images to validate the BT calculation and the image corrections. It displays FRET images as a function of the colocalization of the two fluorescent partners. Finally, it proposes an alternative to normalization of the FRET intensities to compare FRET signal variations between samples. This method called "FRET and Colocalization Analyzer" has been implemented in a Plug-in of the freely available ImageJ software. It is particularly adapted when transient expression of the fluorescent proteins is used thereby giving very variable expression levels or when the colocalization of the two partners is varying in proportion, in amount, and in size, as a function of time. The method and program are validated using the analysis of the spatio-temporal interactions between a G-protein coupled receptor, the tachykinin NK2 receptor, and the beta-arrestin 2 as an example., (Copyright (c) 2006 Wiley-Liss, Inc.)
- Published
- 2006
- Full Text
- View/download PDF
48. Pseudo-prolines: induction of cis/trans-conformational interconversion by decreased transition state barriers.
- Author
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Mutter M, Wöhr T, Gioria S, and Keller M
- Subjects
- Chymotrypsin chemistry, Circular Dichroism, Crystallography, X-Ray, Isomerism, Kinetics, Molecular Conformation, Molecular Structure, Peptides chemistry, Proline chemistry, Protein Structure, Secondary, Proteins chemistry, Thermodynamics, Proline analogs & derivatives
- Abstract
Molecular events such as cis/trans isomerization of Xaa-Pro tertiary amide bonds in peptides and proteins are slow on the overall time scale of the formation of a final biostructure and are, therefore, rate limiting. In order to pursue a better understanding of the molecular events underlying such slow interconversions, we applied the recently introduced pseudo-proline (PsiPro) concept as a tool to study the dynamics of Xaa-Pro bonds by determining the kinetics and thermodynamics of cis/trans isomerism. We show that enhanced isomerization rates of tertiary amide bonds prior to a PsiPro unit in short model peptides is due to lowered transition state barriers. In addition, pronounced effects upon the dynamics of the reversible transition between helix I and II of oligoprolines containing one or several PsiPro units were observed., (Copyright 1999 John Wiley & Sons, Inc.)
- Published
- 1999
- Full Text
- View/download PDF
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