130 results on '"Cosa G"'
Search Results
2. meso-Acetoxymethyl BODIPY dyes for photodynamic therapy: improved photostability of singlet oxygen photosensitizers
- Author
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Lincoln, R., Durantini, A. M., Greene, L. E., Martínez, S. R., Knox, R., Becerra, M. C., and Cosa, G.
- Published
- 2017
- Full Text
- View/download PDF
Catalog
3. Primary syphilis of the rectum—Endoscopic and clinical features: Report of a case
- Author
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Bassi, O., Cosa, G., Colavolpe, A., and Argentieri, R.
- Published
- 1991
- Full Text
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4. A169 FLUORESCENT LABELING OF THE HCV HELICASE TO MONITOR NUCLEIC ACID UNWINDING BY FRET
- Author
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Ablenas, C, primary, Powdrill, M, additional, Shaw, T, additional, Cosa, G, additional, and Gotte, M, additional
- Published
- 2018
- Full Text
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5. Photophysical Properties of Fluorescent DNA-dyes Bound to Single- and Double-stranded DNA in Aqueous Buffered Solution¶
- Author
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Cosa, G., primary, Focsaneanu, K.-S., additional, McLean, J. R. N., additional, McNamee, J. P., additional, and Scaiano, J. C., additional
- Published
- 2007
- Full Text
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6. Intrazeolite Photochemistry. 26. Photophysical Properties of Nanosized TiO2 Clusters Included in Zeolites Y, β, and Mordenite
- Author
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Corrent, S., primary, Cosa, G., additional, Scaiano, J. C., additional, Galletero, Maria S., additional, Alvaro, Mercedes, additional, and Garcia, H., additional
- Published
- 2001
- Full Text
- View/download PDF
7. Photophysical Properties of Fluorescent DNA-dyes Bound to Single- and Double-stranded DNA in Aqueous Buffered Solution¶
- Author
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Cosa, G., primary, Focsaneanu, K.-S., additional, McLean, J. R. N., additional, McNamee, J. P., additional, and Scaiano, J. C., additional
- Published
- 2001
- Full Text
- View/download PDF
8. Direct determination of single-to-double stranded DNA ratio in solution applying time-resolved fluorescence measurements of dye–DNA complexes
- Author
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Cosa, G., primary, Focsaneanu, K.-S., additional, Scaiano, J. C., additional, and McLean, J. R. N., additional
- Published
- 2000
- Full Text
- View/download PDF
9. Photocatalytic Activity of a Multicomponent System Assembled within Zeolites: Case of 2,4,6-Triphenylpyrylium or Ruthenium Tris(bipyridyl) Photosensitizers and Titanium Dioxide Relays within Zeolite Y
- Author
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Cosa, G., Chretien, M. N., Galletero, M. S., Fornes, V., Garcia, H., and Scaiano, J. C.
- Abstract
Samples of zeolite Y containing 2,4,6-triphenylpyrylium or tris(bipyridyl)ruthenium(II) have been prepared and their photophysical and photochemical properties compared to those of the same samples containing encapsulated nanoscopic TiO
2 clusters as relays. The emission of encapsulated Ru(bpy)3 2+ and TP+ photolumophores undergoes static quenching in the presence of codoped TiO2 clusters. This is the expected behavior for systems, where photosensitizer and relay lack diffusional mobility. Time-resolved diffuse reflectance laser flash photolysis shows the transient generation of TP• pyranyl radical and Ru(bpy)3 3+, upon irradiation of TP+ and Ru(bpy)3 2+ in the presence of TiO2 , respectively. This provides firm support for the occurrence of photoinduced electron transfer between excited TP+ as acceptor and TiO2 clusters as donors and from Ru(bpy)3 2+ to the conduction band of TiO2 . Using the photoinactivation of horseradish peroxidase as a test reaction in aqueous medium, a synergism has been found with respect to the activity of Ru(bpy)3 2+ or TP+ photocatalysts when incorporated inside the supercages of zeolite Y in the presence of TiO2 nanoclusters. more...- Published
- 2002
- Full Text
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10. Estimation of the Bimolecular Rate Constant for Exciplex Formation from the Analysis of Its Emission Spectrum
- Author
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Cosa, G. and Chesta, C. A.
- Abstract
The bimolecular rate constant for the quenching of 1-methylnaphthalene by triethylamine was measured in series of solvents covering a large range of solvent polarity (ε = 2−37). The formation of a fluorescent exciplex was observed in all the solvents studied. Analyzing the emission of the exciplex, it is possible to estimate the relevant parameters associated with the radiative back electron transfer process, namely, the solvent dependent Gibbs energy change, the solvent reorganization energy, and the internal reorganization energy. These parameters are used to calculate, according to Marcus theory, the rate constant for the photoinduced forward electron transfer reaction. A good correlation between calculated and experimental quenching rate constants is observed. more...
- Published
- 1997
11. Absolute Rate Constants for Water Protonation of 1-(3-Benzoylphenyl)alkyl Carbanions
- Author
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Cosa, G., Llauger, L., Scaiano, J. C., and Miranda, M. A.
- Abstract
Efficient photodecarboxylation of (3-benzoylphenyl)alkanoic acids with formation of carbanions has enabled the determination of their protonation rate constants in water; the values obtained show that the reactivity toward protonation is determined by the size of the alkyl groups attached to the carbanion center. more...- Published
- 2002
12. Direct determination of single-to-double stranded DNA ratio in solution applying time-resolved fluorescence measurements of dyeDNA complexes
- Author
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Cosa, G., Focsaneanu, K.-S., Scaiano, J. C., and McLean, J. R. N.
- Abstract
We report the fluorescence lifetimes of the DNA-stain dye PicoGreen and discuss the difference exhibited in the upon binding to single-stranded vs. double-stranded DNA; we here developed a direct method for determining single-to-double stranded DNA ratios in solution by measurement of the pre-exponential factors in the fluorescence decay traces of dyeDNA complexes. more...
- Published
- 2000
13. Azatriangulene-Based Conductive C═C Linked Covalent Organic Frameworks with Near-Infrared Emission.
- Author
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Hamzehpoor E, Ghamari P, Tao Y, Rafique MG, Zhang Z, Salehi M, Stein RS, Ramos-Sanchez J, Laramée AW, Cosa G, Pellerin C, Seifitokaldani A, Khaliullin RZ, and Perepichka DF
- Abstract
Two near-infrared (NIR) emissive π-conjugated covalent organic frameworks (COFs) pTANG1 and pTANG2 are synthesized using Knoevenagel condensation of trioxaazatriangulenetricarbaldehyde (TATANG) with benzene- and biphenyldiacetonitriles, respectively. The morphology of the COFs is affected by the size of TATANG precursor crystals. Donor-acceptor interactions in these COFs result in small bandgaps (≈1.6 eV) and NIR emission (λ
max = 789 nm for pTANG1). pTANG1 can absorb up to 9 molecules of water per unit cell, which is accompanied by a marked quenching of the NIR emission, suggesting applications as humidity sensors. p-Doping with magic blue significantly increases the electrical conductivities of the COFs by up to 8 orders of magnitude, with the room temperature conductivity of pTANG1 reaching 0.65 S cm-1 , the highest among reported C═C linked COFs.1 H NMR relaxometry, temperature-dependent fluorescence spectroscopy, and DFT calculations reveal that the higher rigidity of the shorter phenylene linker is responsible for the more extended conjugation (red-shifted emission, higher electrical conductivity) of pTANG1 compared to pTANG2., (© 2024 The Author(s). Advanced Materials published by Wiley‐VCH GmbH.) more...- Published
- 2024
- Full Text
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14. Automated Synthesis of DNA Nanostructures.
- Author
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Islas P, Platnich CM, Gidi Y, Karimi R, Ginot L, Saliba D, Luo X, Cosa G, and Sleiman HF
- Subjects
- Nanotubes chemistry, Nanotechnology methods, Nanostructures chemistry, DNA chemistry, Automation
- Abstract
DNA nanotechnology has revolutionized the ability to position matter at the nanoscale, but the preparation of DNA-based architectures remains laborious. To facilitate the formation of custom structures, a fully automated method is reported to produce sequence- and size-defined DNA nanotubes. By programming the sequential addition of desired building blocks, rigid DX-tile-based DNA nanotubes and flexible wireframe DNA structures are attained, where the total number of possible constructs increases as a power function of the number of different units available. Using single-molecule fluorescence imaging, the kinetics and yield of each synthetic step can be quantitatively determined, revealing differences in self-assembly dynamics as the nanotube is built up from the solid support and providing new insights into DNA self-assembly. The exploitation of automation for both assembly and analysis (through an ad-hoc developed K-means clustering algorithm) facilitates a workflow wherein the synthesis parameters may be iteratively improved upon, demonstrating how a single-molecule "assembly-analysis-optimization" sequence can be used to generate complex, noncovalent materials in good yield. The presented synthetic strategy is generalizable, making use of equipment already available in most standard laboratories and represents the first fully automated supramolecular assembly on a solid support., (© 2024 The Author(s). Advanced Materials published by Wiley‐VCH GmbH.) more...
- Published
- 2024
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15. Heat-activated growth of metastable and length-defined DNA fibers expands traditional polymer assembly.
- Author
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Dore MD, Rafique MG, Yang TP, Zorman M, Platnich CM, Xu P, Trinh T, Rizzuto FJ, Cosa G, Li J, Guarné A, and Sleiman HF
- Subjects
- Hydrophobic and Hydrophilic Interactions, DNA chemistry, Hot Temperature, Polymers chemistry
- Abstract
Biopolymers such as nucleic acids and proteins exhibit dynamic backbone folding, wherein site-specific intramolecular interactions determine overall structure. Proteins then hierarchically assemble into supramolecular polymers such as microtubules, that are robust yet dynamic, constantly growing or shortening to adjust to cellular needs. The combination of dynamic, energy-driven folding and growth with structural stiffness and length control is difficult to achieve in synthetic polymer self-assembly. Here we show that highly charged, monodisperse DNA-oligomers assemble via seeded growth into length-controlled supramolecular fibers during heating; when the temperature is lowered, these metastable fibers slowly disassemble. Furthermore, the specific molecular structures of oligomers that promote fiber formation contradict the typical theory of block copolymer self-assembly. Efficient curling and packing of the oligomers - or 'curlamers' - determine morphology, rather than hydrophobic to hydrophilic ratio. Addition of a small molecule stabilises the DNA fibers, enabling temporal control of polymer lifetime and underscoring their potential use in nucleic-acid delivery, stimuli-responsive biomaterials, and soft robotics., (© 2024. The Author(s).) more...
- Published
- 2024
- Full Text
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16. Lipid-derived electrophiles inhibit the function of membrane channels during ferroptosis.
- Author
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Van Kessel ATM and Cosa G
- Subjects
- Humans, Multidrug Resistance-Associated Proteins metabolism, Glutathione metabolism, Ferroptosis drug effects, Lipid Peroxidation drug effects, Aldehydes pharmacology, Aldehydes metabolism
- Abstract
The therapeutic targeting of ferroptosis requires full understanding of the molecular mechanism of this regulated cell death pathway. While lipid-derived electrophiles (LDEs), including 4-hydroxy-2-nonenal (4-HNE), are important biomarkers of ferroptosis, a functional role for these highly reactive species in ferroptotic cell death execution has not been established. Here, through mechanistic characterization of LDE-detoxification impairment, we demonstrate that LDEs mediate altered protein function during ferroptosis. Applying live cell fluorescence imaging, we first identified that export of glutathione-LDE-adducts through multidrug resistance-associated protein (MRP) channels is inhibited following exposure to a panel of ferroptosis inducers (FINs) with different modes of action (type I-IV FINs erastin, RSL3, FIN56, and FINO
2 ). This channel inhibition was recreated by both initiation of lipid peroxidation and treatment with 4-HNE. Importantly, treatment with radical-trapping antioxidants prevented impaired LDE-adduct export when working with both FINs and lipid peroxidation initiators but not 4-HNE, pinpointing LDEs as the cause of this inhibited MRP activity observed during ferroptosis. Our findings, when combined with reports of widespread LDE alkylation of key proteins following ferroptosis induction, including MRP1, set a precedent for LDEs as critical mediators of ferroptotic cell damage. Lipid hydroperoxide breakdown to form truncated phospholipids and LDEs may fully explain membrane permeabilization and modified protein function downstream of lipid peroxidation, offering a unified explanation of the molecular cell death mechanism of ferroptosis., Competing Interests: Competing interests statement:The authors declare no competing interest. more...- Published
- 2024
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17. Two-Pronged Dormant Photosensitizer-Antibiotic Bacterial Inactivation: Mechanism, Dosage, and Cellular Evolution Visualized at the Single-Cell Level.
- Author
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McCain J, Martínez SR, Fungo F, Sakaya A, and Cosa G
- Subjects
- Escherichia coli metabolism, Reactive Oxygen Species metabolism, Bacteria metabolism, Photosensitizing Agents pharmacology, Anti-Bacterial Agents pharmacology
- Abstract
Innovative therapeutic approaches are required to battle the rise of antibiotic-resistant bacterial strains. Tapping on reactive oxygen species (ROS) generation in bacteria induced by bactericidal antibiotics, here we report a two-pronged strategy for bacterial inactivation relying on the synergistic combination of a bactericidal antibiotic and newly designed dormant photosensitizers (DoPSs) that activate in the presence of ROS. Intramolecular quenching renders DoPS inert in the presence of light. ROS trapping by DoPS aborts the quenching mechanism unmasking, in equal proportions, singlet oxygen (
1 O2 ) sensitization and fluorescence emission. Juxtaposed antioxidant-prooxidant activity built within our DoPS enables (i) initial activation of a few molecules by ROS and (ii) subsequent rapid activation of all DoPS in a bacterium via a domino effect mediated by photogenerated1 O2 . Bulk colony forming unit studies employing the minimum inhibitory concentration of the antibiotic illustrate rapid and selective inactivation of Escherichia coli and Pseudomonas aeruginosa only in the presence of light, antibiotic, and DoPS. Single-cell, real-time imaging studies on E. coli reveal an autocatalytic progression of DoPS activation from focal points, providing a unique amplification system for sensing. Single-cell analysis further illustrates the impact of DoPS cellular loading on the rate of DoPS activation and cell death times and on the1 O2 dosing necessary for cell death to occur. Our two-pronged therapy discriminates based on cell metabolites and has the potential to result in lower toxicity, pave the way to reduced drug resistance, and provide insightful mechanistic information about bacterial membrane response to1 O2 . more...- Published
- 2023
- Full Text
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18. Minimalist Design of Wireframe DNA Nanotubes: Tunable Geometry, Size, Chirality, and Dynamics.
- Author
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Luo X, Saliba D, Yang T, Gentile S, Mori K, Islas P, Das T, Bagheri N, Porchetta A, Guarne A, Cosa G, and Sleiman HF
- Subjects
- Nanotechnology, DNA chemistry, DNA Replication, Nanotubes chemistry, Biosensing Techniques
- Abstract
DNA nanotubes (NTs) have attracted extensive interest as artificial cytoskeletons for biomedical, synthetic biology, and materials applications. Here, we report the modular design and assembly of a minimalist yet robust DNA wireframe nanotube with tunable cross-sectional geometry, cavity size, chirality, and length, while using only four DNA strands. We introduce an h-motif structure incorporating double-crossover (DX) tile-like DNA edges to achieve structural rigidity and provide efficient self-assembly of h-motif-based DNA nanotube (H-NT) units, thus producing programmable, micrometer-long nanotubes. We demonstrate control of the H-NT nanotube length via short DNA modulators. Finally, we use an enzyme, RNase H, to take these structures out of equilibrium and trigger nanotube assembly at a physiologically relevant temperature, underlining future cellular applications. The minimalist H-NTs can assemble at near-physiological salt conditions and will serve as an easily synthesized, DNA-economical modular template for biosensors, plasmonics, or other functional materials and as cost-efficient drug-delivery vehicles for biomedical applications., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.) more...
- Published
- 2023
- Full Text
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19. Superior Photoprotection of Cyanine Dyes with Thio-imidazole Amino Acids.
- Author
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Gidi Y, Ramos-Sanchez J, Lovell TC, Glembockyte V, Cheah IK, Schnermann MJ, Halliwell B, and Cosa G
- Subjects
- Amino Acids, Fluorescent Dyes, Imidazoles, Ionophores, Ergothioneine, Quinolines
- Abstract
Preventing fluorophore photobleaching and unwanted blinking is crucial for single-molecule fluorescence (SMF) studies. Reductants achieve photoprotection via quenching excited triplet states, yet either require counteragents or, for popular alkyl-thiols, are limited to cyanine dye Cy3 protection. Here, we provide mechanistic and imaging results showing that the naturally occurring amino acid ergothioneine and its analogue dramatically enhance photostability for Cy3, Cy5, and their conformationally restrained congeners, providing a biocompatible universal solution for demanding fluorescence imaging. more...
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- 2023
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20. Binding and Sliding Dynamics of the Hepatitis C Virus Polymerase: Hunting the 3' Terminus.
- Author
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Gidi Y, Robert A, Tordo A, Lovell TC, Ramos-Sanchez J, Sakaya A, Götte M, and Cosa G
- Subjects
- Humans, Nucleotidyltransferases, RNA, Viral genetics, RNA-Dependent RNA Polymerase metabolism, Viral Nonstructural Proteins metabolism, Hepacivirus genetics, Hepacivirus metabolism, Hepatitis C
- Abstract
The hepatitis C virus (HCV) nonstructural protein 5B (NS5B) polymerase catalyzes the replication of the (+) single-stranded RNA genome of HCV. In vitro studies have shown that replication can be performed in the absence of a primer. However, the dynamics and mechanism by which NS5B locates the 3'-terminus of the RNA template to initiate de novo synthesis remain elusive. Here, we performed single-molecule fluorescence studies based on protein-induced fluorescence enhancement reporting on NS5B dynamics on a short model RNA substrate. Our results suggest that NS5B exists in a fully open conformation in solution wherefrom it accesses its binding site along RNA and then closes. Our results revealed two NS5B binding modes: an unstable one resulting in rapid dissociation, and a stable one characterized by a larger residence time on the substrate. We associate these bindings to an unproductive and productive orientation, respectively. Addition of extra mono (Na
+ )- and divalent (Mg2+ ) ions increases the mobility of NS5B along its RNA substrate. However, only Mg2+ ions induce a decrease in NS5B residence time. Dwell times of residence increase with the length of the single-stranded template, suggesting that NS5B unbinds its substrate by unthreading the template rather than by spontaneous opening. more...- Published
- 2023
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21. Alkylation converts riboflavin into an efficient photosensitizer of phospholipid membranes.
- Author
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Sosa MJ, Fonseca JL, Sakaya A, Urrutia MN, Petroselli G, Erra-Balsells R, Quindt MI, Bonesi SM, Cosa G, Vignoni M, and Thomas AH
- Subjects
- Riboflavin, Unilamellar Liposomes, Alkylation, Phospholipids, Photosensitizing Agents
- Abstract
A new decyl chain [-(CH
2 )9 CH3 ] riboflavin conjugate has been synthesized and investigated. A nucleophilic substitution (S <subscript>N 2) reaction was used for coupling the alkyl chain to riboflavin (Rf), a model natural photosensitizer. As expected, the alkylated compound (decyl-Rf) is significantly more lipophilic than its precursor and efficiently intercalates within phospholipid bilayers, increasing its fluorescence quantum yield. The oxidative damage to lipid membranes photoinduced by decyl-Rf was investigated in large and giant unilamellar vesicles (LUVs and GUVs, respectively) composed of different phospholipids. Using a fluorogenic probe, fast radical formation and singlet oxygen generation was demonstrated upon UVA irradiation in vesicles containing decyl-Rf. Photosensitized formation of conjugated dienes and hydroperoxides, and membrane leakage in LUVs rich in poly-unsaturated fatty acids were also investigated. The overall assessment of the results shows that decyl-Rf is a significantly more efficient photosensitizer of lipids than its unsubstituted precursor and that the association to lipid membranes is key to trigger phospholipid oxidation. Alkylation of hydrophilic photosensitizers as a simple and general synthetic tool to obtain efficient photosensitizers of biomembranes, with potential applications, is discussed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.) more...- Published
- 2023
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22. Triarylamines as catalytic donors in light-mediated electron donor-acceptor complexes.
- Author
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Castillo-Pazos DJ, Lasso JD, Hamzehpoor E, Ramos-Sánchez J, Salgado JM, Cosa G, Perepichka DF, and Li CJ
- Abstract
Recently, photochemistry of Electron Donor-Acceptor (EDA) complexes employing catalytic amounts of electron donors have become of interest as a new methodology in the catalysis field, allowing for decoupling of the electron transfer (ET) from the bond-forming event. However, examples of practical EDA systems in the catalytic regime remain scarce, and their mechanism is not yet well-understood. Herein, we report the discovery of an EDA complex between triarylamines and α-perfluorosulfonylpropiophenone reagents, catalyzing C-H perfluoroalkylation of arenes and heteroarenes under visible light irradiation in pH- and redox-neutral conditions. We elucidate the mechanism of this reaction using a detailed photophysical characterization of the EDA complex, the resulting triarylamine radical cation, and its turnover event., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.) more...
- Published
- 2023
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23. Singlet Oxygen Flux, Associated Lipid Photooxidation, and Membrane Expansion Dynamics Visualized on Giant Unilamellar Vesicles.
- Author
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Sakaya A, Bacellar IOL, Fonseca JL, Durantini AM, McCain J, Xu L, Vignoni M, Thomas AH, Baptista MS, and Cosa G
- Subjects
- Hydrogen Peroxide, Antioxidants chemistry, Lipids chemistry, Unilamellar Liposomes chemistry, Singlet Oxygen
- Abstract
The physical properties of lipid membranes depend on their lipid composition. Photosensitized singlet oxygen (
1 O2 ) provides a handle to spatiotemporally control the generation of lipid hydroperoxides via the ene reaction, enabling fundamental studies on membrane dynamics in response to chemical composition changes. Critical to relating the physical properties of the lipid membrane to hydroperoxide formation is the availability of a sensitive reporter to quantify the arrival of1 O2 . Here, we show that a fluorogenic α-tocopherol analogue, H4 BPMHC, undergoes a >360-fold emission intensity enhancement in liposomes following a reaction with1 O2 . Rapid quenching of1 O2 by the probe ( kq = 4.9 × 108 M-1 s-1 ) ensures zero-order kinetics of probe consumption. The remarkable intensity enhancement of H4 BPMHC upon1 O2 trapping, its linear temporal behavior, and its protective role in outcompeting membrane damage provide a sensitive and reliable method to quantify the1 O2 flux on lipid membranes. Armed with this probe, fluorescence microscopy studies were devised to enable (i) monitoring the flux of photosensitized1 O2 into giant unilamellar vesicles (GUVs), (ii) establishing the onset of the ene reaction with the double bonds of monounsaturated lipids, and (iii) visualizing the ensuing collective membrane expansion dynamics associated with molecular changes in the lipid structure upon hydroperoxide formation. A correlation was observed between the time for antioxidant H4 BPMHC consumption by1 O2 and the onset of membrane fluctuations and surface expansion. Together, our imaging studies with H4 BPMHC in GUVs provide a methodology to explore the intimate relationship between photosensitizer activity, chemical insult, membrane morphology, and its collective dynamics. more...- Published
- 2023
- Full Text
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24. Live-cell imaging reveals impaired detoxification of lipid-derived electrophiles is a hallmark of ferroptosis.
- Author
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Van Kessel ATM, Karimi R, and Cosa G
- Abstract
The central mechanism in ferroptosis linking lipid hydroperoxide accumulation with cell death remains poorly understood. Although lipid hydroperoxides are known to break down to reactive lipid-derived electrophiles (LDEs), the ability of cells to detoxify increasing LDE levels during ferroptosis has not been studied. Here, we developed an assay (ElectrophileQ) correlating the cellular retention vs. excretion of a fluorogenic lipophilic electrophile (AcroB) that enables live-cell assessment of the glutathione-mediated LDE conjugation and adduct export steps of the LDE detoxification pathway. This method revealed that during ferroptosis, LDE detoxification failure occurs through decreased conjugation or export impairment, amplifying cellular electrophile accumulation. Notably, ferroptosis susceptibility was increased following exacerbation of LDE-adduct export impairment through export channel inhibition. Our results expand understanding of the ferroptosis molecular cell death mechanism to position the LDE detoxification pathway as a ferroptosis-relevant therapeutic target. We envision the ElectrophileQ assay becoming an invaluable tool for studying ferroptosis and cellular health., Competing Interests: The authors declare no conflicts of interest., (This journal is © The Royal Society of Chemistry.) more...
- Published
- 2022
- Full Text
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25. An evolutionary conserved detoxification system for membrane lipid-derived peroxyl radicals in Gram-negative bacteria.
- Author
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Naguib M, Feldman N, Zarodkiewicz P, Shropshire H, Biamis C, El-Halfawy OM, McCain J, Dezanet C, Décout JL, Chen Y, Cosa G, and Valvano MA
- Subjects
- Anti-Bacterial Agents pharmacology, Gram-Negative Bacteria, Lipocalins, Aldehyde Reductase, Membrane Lipids
- Abstract
How double-membraned Gram-negative bacteria overcome lipid peroxidation is virtually unknown. Bactericidal antibiotics and superoxide ion stress stimulate the transcription of the Burkholderia cenocepacia bcnA gene that encodes a secreted lipocalin. bcnA gene orthologs are conserved in bacteria and generally linked to a conserved upstream gene encoding a cytochrome b561 membrane protein (herein named lcoA, lipocalin-associated cytochrome oxidase gene). Mutants in bcnA, lcoA, and in a gene encoding a conserved cytoplasmic aldehyde reductase (peroxidative stress-associated aldehyde reductase gene, psrA) display enhanced membrane lipid peroxidation. Compared to wild type, the levels of the peroxidation biomarker malondialdehyde (MDA) increase in the mutants upon exposure to sublethal concentrations of the bactericidal antibiotics polymyxin B and norfloxacin. Microscopy with lipid peroxidation-sensitive fluorescent probes shows that lipid peroxyl radicals accumulate at the bacterial cell poles and septum and peroxidation is associated with a redistribution of anionic phospholipids and reduced antimicrobial resistance in the mutants. We conclude that BcnA, LcoA, and PsrA are components of an evolutionary conserved, hitherto unrecognized peroxidation detoxification system that protects the bacterial cell envelope from lipid peroxyl radicals., Competing Interests: The authors have declared that no competing interests exist. more...
- Published
- 2022
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26. Fluorescence-Amplified Detection of Redox Turnovers in Supported Lipid Bilayers Illuminates Redox Processes of α-Tocopherol.
- Author
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Sakaya A, Durantini AM, Gidi Y, Šverko T, Wieczny V, McCain J, and Cosa G
- Subjects
- Fluorescence, Kinetics, Oxidation-Reduction, Lipid Bilayers, alpha-Tocopherol
- Abstract
Electron-transfer processes in lipid membranes are key to biological functions, yet challenging to study because of the intrinsic heterogeneity of the systems. Here, we report spectro-electrochemical measurements on indium tin oxide-supported lipid bilayers toward the selective induction and sensing of redox processes in membranes. Working at neutral pH with a fluorogenic α-tocopherol analogue, the dynamics of the two-electron oxidation of the chromanol to a chromanone and the rapid thermal decay of the latter to a chromoquinone are recorded as a rapid surge and drop in intensity, respectively. Continuous voltage cycling reveals rapid chromoquinone two-electron, two-proton reduction to dihydrochromoquinone at negative bias, followed by slow regeneration of the former at positive bias. The kinetic parameters of these different transitions are readily obtained as a function of applied potentials. The sensitivity and selectivity afforded by the reported method enables monitoring signals equivalent to femtoampere currents with a high signal-to-background ratio. The study provides a new method to monitor membrane redox processes with high sensitivity and minimal concentrations and unravels key dynamic aspects of α-tocopherol redox chemistry. more...
- Published
- 2022
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27. Chemically Tuned, Reversible Fluorogenic Electrophile for Live Cell Nanoscopy.
- Author
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Lincoln R, Zhang W, Lovell TC, Jodko-Piórecka K, Devlaminck PA, Sakaya A, Van Kessel A, and Cosa G
- Subjects
- Cyanoacrylates, Endoplasmic Reticulum, Microscopy, Fluorescence methods, Fluorescent Dyes chemistry, Mitochondria
- Abstract
We report a chemically tuned fluorogenic electrophile designed to conduct live-cell super-resolution imaging by exploiting its stochastic reversible alkylation reaction with cellular nucleophiles. Consisting of a lipophilic BODIPY fluorophore tethered to an electrophilic cyanoacrylate warhead, the new probe cyanoAcroB remains nonemissive due to internal conversion along the cyanoacrylate moiety. Intermittent fluorescence occurs following thiolate Michael addition to the probe, followed by retro-Michael reaction, tuned by the cyano moiety in the acrylate warhead and BODIPY decoration. This design enables long-term super-resolved imaging of live cells by preventing fluorescent product accumulation and background increase, while preserving the pool of the probe. We demonstrate the imaging capabilities of cyanoAcroB via two methods: (i) single-molecule localization microscopy imaging with nanometer accuracy by stochastic chemical activation and (ii) super-resolution radial fluctuation. The latter tolerates higher probe concentrations and low imaging powers, as it exploits the stochastic adduct dissociation. Super-resolved imaging with cyanoAcroB reveals that electrophile alkylation is prevalent in mitochondria and endoplasmic reticulum. The 2D dynamics of these organelles within a single cell are unraveled with tens of nanometers spatial and sub-second temporal resolution through continuous imaging of cyanoAcroB extending for tens of minutes. Our work underscores the opportunities that reversible fluorogenic probes with bioinspired warheads bring toward illuminating chemical reactions with super-resolved features in live cells. more...
- Published
- 2022
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28. A dissipative pathway for the structural evolution of DNA fibres.
- Author
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Rizzuto FJ, Platnich CM, Luo X, Shen Y, Dore MD, Lachance-Brais C, Guarné A, Cosa G, and Sleiman HF
- Subjects
- Hydrogen-Ion Concentration, Indoles chemistry, Indoles radiation effects, Light, Polymerization radiation effects, Triazines chemistry, DNA chemistry
- Abstract
Biochemical networks interconnect, grow and evolve to express new properties as different chemical pathways are selected during a continuous cycle of energy consumption and transformation. In contrast, synthetic systems that push away from equilibrium usually return to the same self-assembled state, often generating waste that limits system recyclability and prevents the formation of adaptable networks. Here we show that annealing by slow proton dissipation selects for otherwise inaccessible morphologies of fibres built from DNA and cyanuric acid. Using single-molecule fluorescence microscopy, we observe that proton dissipation influences the growth mechanism of supramolecular polymerization, healing gaps within fibres and converting highly branched, interwoven networks into nanocable superstructures. Just as the growth kinetics of natural fibres determine their structural attributes to modulate function, our system of photoacid-enabled depolymerization and repolymerization selects for healed materials to yield organized, robust fibres. Our method provides a chemical route for error-checking, distinct from thermal annealing, that improves the morphologies and properties of supramolecular materials using out-of-equilibrium systems., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.) more...
- Published
- 2021
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29. Room Temperature Phosphorescence vs Triplet-Triplet Annihilation in N-Substituted Acridone Solids.
- Author
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Hamzehpoor E, Ruchlin C, Tao Y, Ramos-Sanchez JE, Titi HM, Cosa G, and Perepichka DF
- Subjects
- Electrons, Models, Molecular, Molecular Conformation, Acridones chemistry, Luminescence, Temperature
- Abstract
Organic room temperature phosphorescent (ORTP) compounds have recently emerged as a promising class of emissive materials with a multitude of potential applications. However, the number of building blocks that give rise to efficient ORTP materials is still limited, and the rules for engineering phosphorescent properties in organic solids are not well understood. Here, we report ORTP in a series of N -substituted acridone derivatives with electron-donating, electron-withdrawing, and sterically bulky substituents. X-ray crystallography shows that the solid-state packing varies progressively between coparallel and antiparallel π-stacking and separated π-dimers, depending on the size of the substituent. The detailed photophysical studies supported by DFT calculations reveal complex dynamics of singlet and triplet excited states, depending on the electronic effects of substituents and solid-state packing. The programmable molecular packing provides a lever to control the triplet-triplet annihilation that is manifested as delayed fluorescence in acridone derivatives with continuous (both parallel and antiparallel) π-stacking. more...
- Published
- 2021
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30. Connecting the "Dots": From Free Radical Lipid Autoxidation to Cell Pathology and Disease.
- Author
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Foret MK, Lincoln R, Do Carmo S, Cuello AC, and Cosa G
- Subjects
- Alzheimer Disease pathology, Animals, Antioxidants chemistry, Free Radicals chemistry, Free Radicals metabolism, Humans, Lipid Peroxidation, Alzheimer Disease metabolism, Antioxidants metabolism
- Abstract
Our understanding of lipid peroxidation in biology and medicine is rapidly evolving, as it is increasingly implicated in various diseases but also recognized as a key part of normal cell function, signaling, and death (ferroptosis). Not surprisingly, the root and consequences of lipid peroxidation have garnered increasing attention from multiple disciplines in recent years. Here we "connect the dots" between the fundamental chemistry underpinning the cascade reactions of lipid peroxidation (enzymatic or free radical), the reactive nature of the products formed (lipid-derived electrophiles), and the biological targets and mechanisms associated with these products that culminate in cellular responses. We additionally bring light to the use of highly sensitive, fluorescence-based methodologies. Stemming from the foundational concepts in chemistry and biology, these methodologies enable visualizing and quantifying each reaction in the cascade in a cellular and ultimately tissue context, toward deciphering the connections between the chemistry and physiology of lipid peroxidation. The review offers a platform in which the chemistry and biomedical research communities can access a comprehensive summary of fundamental concepts regarding lipid peroxidation, experimental tools for the study of such processes, as well as the recent discoveries by leading investigators with an emphasis on significant open questions. more...
- Published
- 2020
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31. Real-Time Single-Cell Imaging Reveals Accelerating Lipid Peroxyl Radical Formation in Escherichia coli Triggered by a Fluoroquinolone Antibiotic.
- Author
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Martínez SR, Durantini AM, Becerra MC, and Cosa G
- Subjects
- Anti-Bacterial Agents pharmacology, Fluorescent Dyes, Fluoroquinolones, Lipids, Peroxides, Antioxidants, Escherichia coli
- Abstract
The formation of reactive oxygen species (ROS) induced by bactericidal antibiotics has been associated with a common, nonspecific mechanism of cellular death. Herein, we report real-time single-cell fluorescence studies on Escherichia coli stained with a fluorogenic probe for lipid peroxyl radicals showing the generation of this form of ROS when exposed to the minimum inhibitory concentration (MIC) and 10× MIC of the fluoroquinolone antibiotic ciprofloxacin (3 and 30 μM, respectively). Single-cell intensity-time trajectories show an induction period followed by an accelerating phase for cells treated with antibiotic, where initial and maximum intensity achieved following 3.5 h of incubation with antibiotic showed dose-dependent average values. A large fraction of bacteria remains viable after the studies, indicating ROS formation is occurring a priori of cell death. Punctate structures are observed, consistent with membrane blebbing. The addition of a membrane embedding lipid peroxyl radical scavenger, an α-tocopherol analogue, to the media increased the MIC of ciprofloxacin. Lipid peroxyl radical formation precedes E. coli cell death and may be invoked in a cascade event including membrane disruption and consequent cell wall permeabilization. Altogether, our work illustrates that lipid peroxidation is caused by ciprofloxacin in E. coli and suppressed by α-tocopherol analogues. Lipid peroxidation may be invoked in a cascade event including membrane disruption and consequent cell wall permeabilization. Our work provides a methodology to assess antibiotic-induced membrane peroxidation at the single-cell level; this methodology provides opportunities to explore the scope and nature of lipid peroxidation in antibiotic-induced cell lethality. more...
- Published
- 2020
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32. Unifying Mechanism for Thiol-Induced Photoswitching and Photostability of Cyanine Dyes.
- Author
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Gidi Y, Payne L, Glembockyte V, Michie MS, Schnermann MJ, and Cosa G
- Subjects
- Density Functional Theory, Molecular Structure, Photochemical Processes, Carbocyanines chemistry, Fluorescent Dyes chemistry, Sulfhydryl Compounds chemistry
- Abstract
Cyanines (Cy3, Cy5, Cy3B) are the most utilized dyes for single-molecule fluorescence and localization-based super-resolution imaging. These modalities exploit cyanines' versatile photochemical behavior with thiols. A mechanism reconciling seemingly divergent results and enabling control over cyanine photoreactivity is however missing. Utilizing single-molecule fluorescence on Cy5 and Cy5B, transient-absorption spectroscopy, and DFT modeling on a range of cyanine dyes, herein we show that photoinduced electron transfer (P e T) from a thiolate to Cy in their triplet excited state and then triplet-to-singlet intersystem crossing in the nascent geminate radical pair are crucial steps. Next, a bifurcation occurs, yielding either back electron transfer and regeneration of ground state Cy, required for photostabilization, or Cy-thiol adduct formation, necessary for super-resolution microscopy. Cy regeneration via photoinduced thiol elimination is favored by adduct absorption spectra broadening. Elimination is also shown to occur through an acid-catalyzed reaction. Overall, our work provides a roadmap for designing fluorophores, photoswitching agents, and triplet excited state quenchers for single-molecule and super-resolution imaging. more...
- Published
- 2020
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33. Single-molecule methods in structural DNA nanotechnology.
- Author
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Platnich CM, Rizzuto FJ, Cosa G, and Sleiman HF
- Subjects
- Biosensing Techniques, Microscopy, Atomic Force, Microscopy, Electron, Microscopy, Fluorescence, Nucleic Acid Conformation, DNA chemistry, Nanostructures chemistry, Single Molecule Imaging methods
- Abstract
Single molecules can now be visualised with unprecedented precision. As the resolution of single-molecule experiments improves, so too does the breadth, quantity and quality of information that can be extracted using these methodologies. In the field of DNA nanotechnology, we use programmable interactions between nucleic acids to generate complex, multidimensional structures. We can use single-molecule techniques - ranging from electron and fluorescence microscopies to electrical and force spectroscopies - to report on the structure, morphology, robustness, sample heterogeneity and other properties of these DNA nanoconstructs. In this Tutorial Review, we will detail how complementarity between static and dynamic single-molecule techniques can provide a unified image of DNA nanoarchitectures. The single-molecule methods that we discuss provide unprecedented insight into chemical and structural behaviour, yielding not just an average outcome but reporting on the distribution of values, ultimately showing how bulk properties arise from the collective behaviour of individual structures. As the fields of both DNA nanotechnology and single-molecule characterisation intertwine, a feedback loop is generated between disciplines, providing new opportunities for the development and operation of DNA-based materials as sensors, delivery vehicles, machinery and structural scaffolds. more...
- Published
- 2020
- Full Text
- View/download PDF
34. Self-Healing Dyes-Keeping the Promise?
- Author
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Isselstein M, Zhang L, Glembockyte V, Brix O, Cosa G, Tinnefeld P, and Cordes T
- Subjects
- DNA, Luminescent Proteins, Microscopy, Fluorescence, Photobleaching, Fluorescent Dyes
- Abstract
Self-healing dyes have emerged as a new promising class of fluorescent labels. They consist of two units, a fluorescent dye and a photostabilizer. The latter heals whenever the fluorescent dye is in danger of taking a reaction pathway toward photobleaching. We describe the underlying concepts and summarize the developmental history and state-of-the-art, including latest applications in high-resolution microscopy, live-cell, and single-molecule imaging. We further discuss remaining limitations, which are (i) lower photostabilization of most self-healing dyes when compared to solution additives, (ii) limited mechanistic understanding on the influence of the biochemical environment and molecular oxygen on self-healing, and (iii) the lack of cheap and facile bioconjugation strategies. Finally, we provide ideas on how to further advance self-healing dyes, show new data on redox blinking caused by double-stranded DNA, and highlight forthcoming work on intramolecular photostabilization of fluorescent proteins. more...
- Published
- 2020
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35. Quantifying Heme-Protein Maturation from Ratiometric Fluorescence Lifetime Measurements on the Single Fluorophore in Its GFP Fusion.
- Author
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Dastpeyman S, Godin R, Cosa G, and English AM
- Abstract
Protein maturation by heme insertion is a common post-translation modification of key biological importance. Nonetheless, where and when this maturation occurs in eukaryotic cells remain unknown for most heme proteins. Here, we demonstrate for the first time that the maturation of a chromosomally expressed, endogenous heme protein fused to a green fluorescent protein (GFP) can be tracked in live cells. Selecting yeast cytochrome c peroxidase (Ccp1) as our model heme-binding protein, we first characterized the emission in vitro of recombinant Ccp1-GFP with GFP fused C-terminally to Ccp1 by the linker GRRIPGLIN. Time-correlated single-photon counting reveals a single fluorescence lifetime for heme-free apoCcp1-GFP, τ
0 = 2.84 ± 0.01 ns. Heme bound to Ccp1 only partially quenches GFP fluorescence since holoCcp1-GFP exhibits two lifetimes, τ1 = 0.95 ± 0.02 and τ2 = 2.46 ± 0.03 ns with fractional amplitudes a1 = 38 ± 1.5% and a2 = 62 ± 1.5%. Also, τ and a are independent of Ccp1-GFP concentration and solution pH between 5.5 and 8.0, and a standard plot of a1 vs % holoCcp1-GFP in mixtures with apoCcp1-GFP is linear, establishing that the fraction of Ccp1-GFP with heme bound can be determined from a1 . Fluorescence lifetime imaging microscopy (FLIM) of live yeast cells chromosomally expressing the same Ccp1-GFP fusion revealed 30% holoCcp1-GFP (i.e., mature Ccp1) and 70% apoCcp1-GFP in agreement with biochemical measurements on cell lysates. Thus, ratiometric fluorescence lifetime measurements offer promise for probing heme-protein maturation in live cells, and we can dispense with the reference fluorophore required for ratiometric intensity-based measurements. more...- Published
- 2020
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36. Hepatitis C Virus Helicase Binding Activity Monitored through Site-Specific Labeling Using an Expanded Genetic Code.
- Author
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Ablenas CJ, Gidi Y, Powdrill MH, Ahmed N, Shaw TA, Mesko M, Götte M, Cosa G, and Pezacki JP
- Subjects
- Azides chemistry, Binding Sites, DNA metabolism, DNA Helicases chemistry, DNA Helicases metabolism, Escherichia coli genetics, Fluorescence Resonance Energy Transfer, Genetic Code, Hepacivirus genetics, Models, Molecular, Mutagenesis, Site-Directed, Phenylalanine analogs & derivatives, Phenylalanine chemistry, Protein Binding, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Viral Nonstructural Proteins chemistry, Hepacivirus enzymology, Single Molecule Imaging methods, Viral Nonstructural Proteins genetics, Viral Nonstructural Proteins metabolism
- Abstract
The mechanism of unwinding catalyzed by the hepatitis C virus nonstructural protein 3 helicase (NS3h) has been a subject of considerable interest, with NS3h serving as a prototypical enzyme in the study of helicase function. Recent studies support an ATP-fueled, inchworm-like stepping of NS3h on the nucleic acid that would result in the displacement of the complementary strand of the duplex during unwinding. Here, we describe the screening of a site of incorporation of an unnatural amino acid in NS3h for fluorescent labeling of the enzyme to be used in single-molecule Förster resonance energy transfer (FRET) experiments. From the nine potential sites identified in NS3h for incorporation of the unnatural amino acid, only one allowed for expression and fluorescent labeling of the recombinant protein. Incorporation of the unnatural amino acid was confirmed via bulk assays to not interfere with unwinding activity of the helicase. Binding to four different dsDNA sequences bearing a ssDNA overhang segment of varying length (either minimal 6 or 7 base length overhang to ensure binding or a long 24 base overhang) and sequence was recorded with the new NS3h construct at the single-molecule level. Single-molecule fluorescence displayed time intervals with anticorrelated donor and acceptor emission fluctuations associated with protein binding to the substrates. An apparent FRET value was estimated from the binding events showing a single FRET value of ∼0.8 for the 6-7 base overhangs. A smaller mean value and a broad distribution was in turn recorded for the long ssDNA overhang, consistent with NS3h exploring a larger physical space while bound to the DNA construct. Notably, intervals where NS3h binding was recorded were exhibited at time periods where the acceptor dye reversibly bleached. Protein induced fluorescence intensity enhancement in the donor channel became apparent at these intervals. Overall, the site-specific fluorescent labeling of NS3h reported here provides a powerful tool for future studies to monitor the dynamics of enzyme translocation during unwinding by single-molecule FRET. more...
- Published
- 2019
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37. Advancing Wireframe DNA Nanostructures Using Single-Molecule Fluorescence Microscopy Techniques.
- Author
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Platnich CM, Hariri AA, Sleiman HF, and Cosa G
- Subjects
- Microscopy, Fluorescence, Particle Size, DNA chemistry, Nanostructures chemistry, Nanotechnology
- Abstract
DNA nanotechnology relies on the molecular recognition properties of DNA to produce complex architectures through self-assembly. The resulting DNA nanostructures allow scientists to organize functional materials at the nanoscale and have therefore found applications in many domains of materials science over the past several years. These scaffolds have been used to position proteins, nanoparticles, carbon nanotubes, and other nanomaterials with high spatial resolution. In addition to their remarkable performance as frameworks for other species, DNA constructs also possess interesting dynamic properties, which have led to their use in logic circuits, drug delivery vehicles, and molecular walkers. Although DNA nanostructures have become increasingly complex, the development of tools to study them has lagged. Currently, gel electrophoresis, dynamic light scattering, and ensemble fluorescence measurements are widely used to characterize DNA-based assemblies. Unfortunately, ensemble averaging in these methods obscures malformed structures and may mask properties associated with structure, length, and shape in polydisperse samples. While atomic force microscopy allows for the determination of morphology at the single-molecule level, this technique cannot typically be used to assess the dynamic properties of these constructs. To analyze the function of DNA-based devices such as molecular motors and reconfigurable nanostructures in real time, new single-molecule techniques are required. This Account details the work from our laboratories toward developing single-molecule fluorescence (SMF) methodologies for the structural and dynamic characterization of wireframe DNA nanostructures, one at a time. The methods described herein provide us with two separate yet related sets of information: First, we can statically examine the nanostructures one by one to assess their robustness, structural fidelity, and morphology. This is primarily done using two-color stepwise photobleaching, wherein we can examine the subunit stoichiometry of our assemblies before and after various perturbations to the structures. For example, we can introduce length mismatches to cause the nanotube to bend or perform strand displacement reactions to generate single-stranded, flexible analogues of our materials. Second, due to the unmatched spatiotemporal resolution of SMF techniques, we can study the dynamic character of these assemblies by implementing structural changes to the nanotube and monitoring them in real time. With this structural and dynamic information in hand, our groups have additionally developed new tools for the improved construction of DNA nanotubes, inspired by solid-phase DNA synthesis. By assembling the nanotubes in a stepwise manner, highly monodisperse nanostructures of any desired length can be made without a template strand. In this way, unique building blocks can also be added sequence-specifically, allowing for the production of user-defined scaffolds to organize nanoscale materials in three dimensions. This method, in combination with our imaging and analysis protocols, may be extended to assemble and inspect other supramolecular constructs in a controlled manner. Overall, by combining synthesis, characterization, and analysis, these single-molecule techniques hold the potential to advance the study of DNA nanostructures and dynamic DNA-based devices. more...
- Published
- 2019
- Full Text
- View/download PDF
38. A High-Throughput Image Correlation Method for Rapid Analysis of Fluorophore Photoblinking and Photobleaching Rates.
- Author
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Sehayek S, Gidi Y, Glembockyte V, Brandão HB, François P, Cosa G, and Wiseman PW
- Abstract
Super-resolution fluorescence imaging based on localization microscopy requires tuning the photoblinking properties of fluorescent dyes employed. Missing is a rapid way to analyze the blinking rates of the fluorophore probes. Herein we present an ensemble autocorrelation technique for rapidly and simultaneously measuring photoblinking and bleaching rate constants from a microscopy image time series of fluorescent probes that is significantly faster than individual single-molecule trajectory analysis approaches. Our method is accurate for probe densities typically encountered in single-molecule studies as well as for higher density systems which cannot be analyzed by standard single-molecule techniques. We also show that we can resolve characteristic blinking times that are faster than camera detector exposure times, which cannot be accessed by threshold-based single-molecule approaches due to aliasing. We confirm this through computer simulation and single-molecule imaging data of DNA-Cy5 complexes. Finally, we demonstrate that with sufficient sampling our technique can accurately recover rates from stochastic optical reconstruction microscopy super-resolution data. more...
- Published
- 2019
- Full Text
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39. A dormant BODIPY-acrolein singlet oxygen photosensitizer intracellularly activated upon adduct formation with cysteine residues.
- Author
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Lincoln R, Van Kessel ATM, Zhang W, and Cosa G
- Subjects
- Acrolein chemistry, Boron Compounds chemistry, Cell Death drug effects, Coloring Agents chemical synthesis, Coloring Agents chemistry, Cysteine chemistry, HeLa Cells, Humans, Light, Microscopy, Fluorescence, Molecular Structure, Photosensitizing Agents chemical synthesis, Photosensitizing Agents chemistry, Quantum Theory, Singlet Oxygen chemistry, Acrolein pharmacology, Boron Compounds pharmacology, Coloring Agents pharmacology, Cysteine pharmacology, Photosensitizing Agents pharmacology, Singlet Oxygen pharmacology
- Abstract
Here we report the activatable photosensitizer BromoAcroB, a brominated BODIPY dye incorporating a reactive acrolein warhead. The acrolein moiety serves as an intramolecular switch, deactivating the BODIPY dye in its singlet and triplet excited states via internal conversion. Thiolate addition to this moiety disables the intramolecular quenching mechanism restoring the photosensitizing properties of the parent dye, characterized by a quantum yield of singlet oxygen photosensitization of 0.69 ± 0.02. In cell cultures, and upon thiol adduct formation, BromoAcroB induced light-dependent cell death in MRC-5 and HeLa cell lines. Using fluorescence microscopy and upon measuring the low yet non-negligible emission of the activated compound, we show that the phototoxicity of the dormant photosensitizer correlated with the quantity of BromoAcroB adducts generated. BromoAcroB thus serves as a dormant photosensitizer sensitive to intracellular electrophile response. Our results highlight the effective control of a triplet state process by modulation of an unsaturated moiety on the BODIPY scaffold and underscore the mechanistic opportunities arising for controlled singlet oxygen production in cells specifically sensitive to electrophile stress. more...
- Published
- 2019
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40. Tuning Photoinduced Electron Transfer Efficiency of Fluorogenic BODIPY-α-Tocopherol Analogues.
- Author
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Greene LE, Lincoln R, and Cosa G
- Abstract
Fluorogenic analogues of α-tocopherol developed by our group have been instrumental in monitoring reactive oxygen species (ROS) within lipid membranes. Prepared as two-segment trap-reporter (chromanol-BODIPY) probes, photoinduced electron transfer (PeT) was utilized to provide these probes with an off/on switch mechanism warranting the necessary sensitivity. Herein, we rationalize within the context of Marcus theory of electron transfer how substituents on the BODIPY core and linker length joining the trap and reporter segments, tune PeT efficiency. DFT and electrochemical studies were used to estimate the thermodynamic driving force of PeT in our constructs. By tuning the redox potential over a 400 mV range, we observed over an order of magnitude increase in PeT efficiency. Increasing the linker length between the chromanol and BODIPY by 2.8 angstroms, in turn, decreased PeT efficiency 2.7-fold. Our results illustrate how substituent and linker choice enable "darkening" the off state of fluorogenic probes based on BODIPY fluorophores, by favoring PeT over radiative emission from the singlet excited state manifold. Ultimately, our work brings light to the sensitivity ceiling one may achieve in developing fluorogenic antioxidant analogues of α-tocopherol. The work provides general guidelines applicable to those developing fluorogenic probes based on PeT., (© 2018 The American Society of Photobiology.) more...
- Published
- 2019
- Full Text
- View/download PDF
41. Effect of antioxidant supplements on lipid peroxidation levels in primary cortical neuron cultures.
- Author
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Foret MK, Do Carmo S, Lincoln R, Greene LE, Zhang W, Cuello AC, and Cosa G
- Subjects
- Animals, Antioxidants chemical synthesis, Antioxidants chemistry, Borates chemical synthesis, Borates chemistry, Fluorescent Dyes chemical synthesis, Fluorescent Dyes chemistry, Fluorescent Dyes pharmacology, Humans, Liposomes chemistry, Liposomes pharmacology, Molecular Imaging methods, Neocortex diagnostic imaging, Neocortex metabolism, Neocortex pathology, Oxidative Stress drug effects, Peroxides chemistry, Peroxides metabolism, Primary Cell Culture, Rats, Reactive Oxygen Species metabolism, Antioxidants pharmacology, Borates pharmacology, Lipid Peroxidation drug effects, Neurons drug effects
- Abstract
Oxidative stress, specifically lipid peroxidation, is a major driving force in neurodegenerative processes. However, the exact role of lipid peroxidation remains elusive as reliable real-time detection and quantification of lipid peroxyl radicals proves to be challenging in vitro and in vivo. Motivated by this methodological limitation, we have optimized conditions for real-time imaging and quantification of lipid peroxyl radical generation in primary neuron cultures using the lipophilic fluorogenic antioxidant H
4 BPMHC (8-((6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)-methyl)-1,5-di(3-chloropropyl)-pyrromethene fluoroborate), an α-tocopherol analog probe. By subjecting neurons to different antioxidant conditions in the presence and absence of lipid peroxidation inducing stressors (Haber-Weiss reagents), we maximized H4 BPMHC sensitivity and confirmed its potential to temporally resolve subtle and marked differences in lipid peroxidation levels in real-time. Herein we report imaging and quantification of homeostatic and induced lipid peroxidation in primary neuron cultures, supporting the use of this probe for investigating healthy and diseased states. Overall these results provide the necessary foundation and impetus towards using H4 BPMHC for elucidating and mapping lipid peroxyl radical contributions to ROS-associated pathological processes in neurons., (Copyright © 2018 Elsevier Inc. All rights reserved.) more...- Published
- 2019
- Full Text
- View/download PDF
42. Kinetics of Strand Displacement and Hybridization on Wireframe DNA Nanostructures: Dissecting the Roles of Size, Morphology, and Rigidity.
- Author
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Platnich CM, Hariri AA, Rahbani JF, Gordon JB, Sleiman HF, and Cosa G
- Subjects
- Kinetics, Particle Size, Polymers chemistry, Surface Properties, DNA chemistry, Nanostructures chemistry
- Abstract
Dynamic wireframe DNA structures have gained significant attention in recent years, with research aimed toward using these architectures for sensing and encapsulation applications. For these assemblies to reach their full potential, however, knowledge of the rates of strand displacement and hybridization on these constructs is required. Herein, we report the use of single-molecule fluorescence methodologies to observe the reversible switching between double- and single-stranded forms of triangular wireframe DNA nanotubes. Specifically, by using fluorescently labeled DNA strands, we were able to monitor changes in intensity over time as we introduced different sequences. This allowed us to extract detailed kinetic information on the strand displacement and hybridization processes. Due to the polymeric nanotube structure, the ability to individually address each of the three sides, and the inherent polydispersity of our samples as a result of the step polymerization by which they are formed, a library of compounds could be studied independently yet simultaneously. Kinetic models relying on mono-exponential decays, multi-exponential decays, or sigmoidal behavior were adjusted to the different constructs to retrieve erasing and refilling kinetics. Correlations were made between the kinetic behavior observed, the site accessibility, the nanotube length, and the structural robustness of wireframe DNA nanostructures, including fully single-stranded analogs. Overall, our results reveal how the length, morphology, and rigidity of the DNA framework modulate the kinetics of strand displacement and hybridization as well as the overall addressability and structural stability of the structures under study. more...
- Published
- 2018
- Full Text
- View/download PDF
43. Highly Photostable and Fluorescent Microporous Solids Prepared via Solid-State Entrapment of Boron Dipyrromethene Dyes in a Nascent Metal-Organic Framework.
- Author
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Glembockyte V, Frenette M, Mottillo C, Durantini AM, Gostick J, Štrukil V, Friščić T, and Cosa G
- Abstract
We report a strategy to synthesize highly emissive, photostable, microporous materials by solid-state entrapment of boron dipyrromethene (BODIPY) fluorophores in a metal-organic framework. Solvent-free mechanochemistry or accelerated aging enabled quantitative capture and dispersal of the PM605 dye within the ZIF-8 framework starting from inexpensive, commercial materials. While the design of emissive BODIPY solids is normally challenged by quenching in a densely packed environment, herein reported PM605@ZIF-8 materials show excellent emissive properties and to the best of our knowledge an unprecedented ∼10-fold enhancement of BODIPY photostability. Time-resolved and steady-state fluorescence studies of PM605@ZIF-8 show that interchromophore interactions are minimal at low dye loadings, but at higher ones lead to through-pore energy transfer between chromophores and to aggregate species. more...
- Published
- 2018
- Full Text
- View/download PDF
44. Efficient One-Step PEG-Silane Passivation of Glass Surfaces for Single-Molecule Fluorescence Studies.
- Author
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Gidi Y, Bayram S, Ablenas CJ, Blum AS, and Cosa G
- Subjects
- DNA chemistry, Fluorescence, Hydrolysis, Microscopy, Atomic Force, Protein Binding, Proteins chemistry, Surface Properties, Surface-Active Agents chemistry, Temperature, Glass chemistry, Microscopy, Fluorescence methods, Polyethylene Glycols chemistry, Silanes chemistry
- Abstract
Surface passivation to inhibit nonspecific interactions is a key requirement for in vitro single-molecule fluorescent studies. Although the standard passivation methods involve the covalent attachment of poly(ethylene glycol) (PEG) in two steps preferably over quartz surfaces, this protocol and improvements thereon require extensive labor and chemicals. Herein, we report an efficient one-step surface grafting of PEG-silane that yields enhanced passivation, as evidenced by reduced nonspecific interactions, over the conventional method at a minimal time and reagent cost and on glass surfaces. Our method is rooted in a mechanistic understanding of the silane reaction with the silanol groups on the glass surface. Single-molecule fluorescence studies with fluorescently tagged proteins and DNA on PEG-silane-functionalized glass surfaces validate the enhanced performance of the method. Combined with atomic force microscopy surface characterization, our study further illustrates that few remaining pinhole defects, plausibly from defects on the glass, on PEG-silane glass-coated surfaces account for the minimal background, where typically no more than one molecule is nonspecifically attached in a given diffraction-limited spot on the surface. more...
- Published
- 2018
- Full Text
- View/download PDF
45. Spatio-temporal monitoring of lipid peroxyl radicals in live cell studies combining fluorogenic antioxidants and fluorescence microscopy methods.
- Author
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Greene LE, Lincoln R, and Cosa G
- Subjects
- Animals, Humans, Cell Membrane metabolism, Fluorescent Dyes chemistry, Lipid Peroxidation, Lipids analysis, Microscopy, Fluorescence methods, Peroxides analysis, Spatio-Temporal Analysis
- Abstract
Lipid peroxidation of polyunsaturated fatty acids in cells may occur via their catalytic autoxidation through peroxyl radicals under oxidative stress conditions. Lipid peroxidation is related to a number of pathologies, and may be invoked in new forms of regulated cell death, yet it may also have beneficial roles in cell signaling cascades. Antioxidants are a natural line of defense against lipid peroxidation, and may accordingly impact the biological outcome associated with the redox chemistry of lipid peroxidation. Critical to unraveling the physiological and pathological role of lipid peroxidation is the development of novel probes with the partition, chemical sensitivity and more importantly, molecular specificity, enabling the spatial and temporal imaging of peroxyl radicals in the lipid membranes of live cells, reporting on the redox status of the cell membrane. This review describes our recent progress to visualize lipid peroxidation in model membrane systems and in live cell studies. Our work portrays the mechanistic insight leading to the development of a highly sensitive probe to monitor lipid peroxyl radicals (LOO
• ). It also describes technical aspects including reagents and fluorescence microscopy methodologies to consider in order to achieve the much sought after monitoring of rates of lipid peroxyl radical production in live cell studies, be it under oxidative stress but also under cell homeostasis. This review seeks to bring attention to the study of lipid redox reactions and to lay the groundwork for the adoption of fluorogenic antioxidant probeshancement and maximum intensity recorded in turn provide a benchmark to estimate, when compared to the control BODIPY dye lacking the intramolecular PeT based switch, the overall exte and related fluorescence microscopy methods toward gaining rich spatiotemporal information on lipid peroxidation in live cells., (Copyright © 2018 Elsevier Inc. All rights reserved.) more...- Published
- 2018
- Full Text
- View/download PDF
46. Tris -N-Nitrilotriacetic Acid Fluorophore as a Self-Healing Dye for Single-Molecule Fluorescence Imaging.
- Author
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Glembockyte V, Wieneke R, Gatterdam K, Gidi Y, Tampé R, and Cosa G
- Subjects
- Microscopy, Fluorescence, Molecular Structure, Fluorescent Dyes chemistry, Nitrilotriacetic Acid chemistry, Optical Imaging
- Abstract
The photostability of fluorescent labels comprises one of the main limitations in single-molecule fluorescence (SMF) and super-resolution imaging. An attractive strategy to increase the photostability of organic fluorophores relies on their coupling to photostabilizers, e.g., triplet excited state quenchers, rendering self-healing dyes. Herein we report the self-healing properties of trisNTA-Alexa647 fluorophores (NTA, N-nitrilotriacetic acid). Primarily designed to specifically label biomolecules containing an oligohistidine tag, we hypothesized that the increased effective concentration of Ni(II) triplet state quenchers would lead to their improved photostability. We evaluated photon output, survival time, and photon count rate of different Alexa647-labeled trisNTA constructs differing in the length and rigidity of the fluorophore- trisNTA linker. Maximum photon output enhancements of 25-fold versus Alexa647-DNA were recorded for a short tetraproline linker, superseding the solution based photostabilization by Ni(II). Steady-state and time-resolved studies illustrate that trisNTA self-healing role is associated with a dynamic excited triplet state quenching by Ni(II). Here improved photophysical/photochemical properties require for a judicious choice of linker length and rigidity, and in turn a balance between rapid dynamic triplet excited state quenching versus dynamic/static singlet excited state quenching. TrisNTA fluorophores offer superior properties for SMF allowing specific labeling and increased photostability, making them ideal candidates for extended single-molecule imaging techniques. more...
- Published
- 2018
- Full Text
- View/download PDF
47. Photosensitized Membrane Permeabilization Requires Contact-Dependent Reactions between Photosensitizer and Lipids.
- Author
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Bacellar IOL, Oliveira MC, Dantas LS, Costa EB, Junqueira HC, Martins WK, Durantini AM, Cosa G, Di Mascio P, Wainwright M, Miotto R, Cordeiro RM, Miyamoto S, and Baptista MS
- Abstract
Although the general mechanisms of lipid oxidation are known, the chemical steps through which photosensitizers and light permeabilize lipid membranes are still poorly understood. Herein we characterized the products of lipid photooxidation and their effects on lipid bilayers, also giving insight into their formation pathways. Our experimental system was designed to allow two phenothiazinium-based photosensitizers (methylene blue, MB, and DO15) to deliver the same amount of singlet oxygen molecules per second to 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine liposome membranes, but with a substantial difference in terms of the extent of direct physical contact with lipid double bonds; that is, DO15 has a 27-times higher colocalization with ω-9 lipid double bonds than MB. Under this condition, DO15 permeabilizes membranes at least 1 order of magnitude more efficiently than MB, a result that was also valid for liposomes made of polyunsaturated lipids. Quantification of reaction products uncovered a mixture of phospholipid hydroperoxides, alcohols, ketones, and aldehydes. Although both photosensitizers allowed the formation of hydroperoxides, the oxidized products that require direct reactions between photosensitizer and lipids were more prevalent in liposomes oxidized by DO15. Membrane permeabilization was always connected with the presence of lipid aldehydes, which cause a substantial decrease in the Gibbs free energy barrier for water permeation. Processes depending on direct contact between photosensitizers and lipids were revealed to be essential for the progress of lipid oxidation and consequently for aldehyde formation, providing a molecular-level explanation of why membrane binding correlates so well with the cell-killing efficiency of photosensitizers. more...
- Published
- 2018
- Full Text
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48. DNA Nanotubes with Hydrophobic Environments: Toward New Platforms for Guest Encapsulation and Cellular Delivery.
- Author
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Rahbani JF, Vengut-Climent E, Chidchob P, Gidi Y, Trinh T, Cosa G, and Sleiman HF
- Subjects
- HeLa Cells, Humans, Hydrophobic and Hydrophilic Interactions, Nanotechnology methods, Coated Materials, Biocompatible chemistry, Coated Materials, Biocompatible pharmacology, DNA chemistry, DNA pharmacology, Drug Delivery Systems methods, Nanotubes chemistry
- Abstract
Natural systems combine different supramolecular interactions in a hierarchical manner to build structures. In contrast, DNA nanotechnology relies almost exclusively on DNA base pairing for structure generation. Introducing other supramolecular interactions can expand the structural and functional range of DNA assemblies, but this requires an understanding of the interplay between these interactions. Here, an economic strategy to build DNA nanotubes functionalized with lipid-like polymers is reported. When these polymers are linked to the nanotube using a spacer, they fold inside to create a hydrophobic environment within the nanotube; the nanotube can encapsulate small molecules and conditionally release them when specific DNA strands are added, as monitored by single-molecule fluorescence microscopy. When the polymers are directly linked to the nanostructure without spacers, they interact intermolecularly to form a network of DNA bundles. This morphological switch can be directly observed using a strand displacement strategy. The two association modes result in different cellular uptake behavior. Nanotubes with internal hydrophobic association show dye-mediated mitochondrial colocalization inside cells; while the bundles disassemble into smaller polymer-coated structures that reduce the extent of nonspecific cellular uptake. This approach uncovers parameters to direct the hierarchical assembly of DNA nanostructures, and produces promising materials for targeted drug delivery., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.) more...
- Published
- 2018
- Full Text
- View/download PDF
49. Cigarette smoke activates CFTR through ROS-stimulated cAMP signaling in human bronchial epithelial cells.
- Author
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Wong FH, AbuArish A, Matthes E, Turner MJ, Greene LE, Cloutier A, Robert R, Thomas DY, Cosa G, Cantin AM, and Hanrahan JW
- Subjects
- Aminophenols pharmacology, Aminopyridines pharmacology, Autocrine Communication drug effects, Benzodioxoles pharmacology, Bronchi metabolism, Bronchi pathology, Calcium Signaling drug effects, Cell Line, Cystic Fibrosis drug therapy, Cystic Fibrosis genetics, Cystic Fibrosis metabolism, Cystic Fibrosis pathology, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Epithelial Cells metabolism, Epithelial Cells pathology, Humans, Mutation, Quinolones pharmacology, Receptors, Prostaglandin E, EP4 Subtype agonists, Receptors, Prostaglandin E, EP4 Subtype metabolism, Second Messenger Systems drug effects, Secretory Pathway drug effects, Bronchi drug effects, Cyclic AMP metabolism, Cystic Fibrosis Transmembrane Conductance Regulator agonists, Epithelial Cells drug effects, Oxidative Stress drug effects, Reactive Oxygen Species metabolism, Tobacco Smoke Pollution adverse effects
- Abstract
Air pollution stimulates airway epithelial secretion through a cholinergic reflex that is unaffected in cystic fibrosis (CF), yet a strong correlation is observed between passive smoke exposure in the home and impaired lung function in CF children. Our aim was to study the effects of low smoke concentrations on cystic fibrosis transmembrane conductance regulator (CFTR) function in vitro. Cigarette smoke extract stimulated robust anion secretion that was transient, mediated by CFTR, and dependent on cAMP-dependent protein kinase activation. Secretion was initiated by reactive oxygen species (ROS) and mediated by at least two distinct pathways: autocrine activation of EP4 prostanoid receptors and stimulation of Ca
2+ store-operated cAMP signaling. The response was absent in cells expressing the most common disease-causing mutant F508del-CFTR. In addition to the initial secretion, prolonged exposure of non-CF bronchial epithelial cells to low levels of smoke also caused a gradual decline in CFTR functional expression. F508del-CFTR channels that had been rescued by the CF drug combination VX-809 (lumacaftor) + VX-770 (ivacaftor) were more sensitive to this downregulation than wild-type CFTR. The results suggest that CFTR-mediated secretion during acute cigarette smoke exposure initially protects the airway epithelium while prolonged exposure reduces CFTR functional expression and reduces the efficacy of CF drugs. more...- Published
- 2018
- Full Text
- View/download PDF
50. Development of a Fluorogenic Reactivity Palette for the Study of Nucleophilic Addition Reactions Based on meso -Formyl BODIPY Dyes.
- Author
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Greene LE, Lincoln R, Krumova K, and Cosa G
- Abstract
We describe herein a fluorescence-based assay to characterize and report on nucleophilic addition to carbonyl moieties and highlight the advantages a fluorescence-based assay and multiplex analysis can offer. The assay relies on the fluorogenic properties of meso -formyl boron-dipyrromethene (BODIPY) dyes that become emissive following nucleophilic addition. A reactivity palette is assembled based on the increasing electrophilic character of five meso -formyl BODIPY compounds tested. We show that increasing rates of emission enhancement correlate with the decreasing electrophilic character of BODIPY dyes in the presence of an acid catalyst and a nucleophile. These results are consistent with the rate-limiting step involving activation of the electrophile. Increasing product formation is shown to correlate with the increasing electrophilic character of the BODIPY dyes, as expected based on thermodynamics. In addition to providing rates of reaction, analysis of the fluorescence parameters for the reaction mixtures, including emission quantum yields and fluorescence lifetimes, enables us to determine the extent of reactant conversion at equilibrium (in our case the estimated yield of a transient species) and the presence of different products, without the need for isolation. We anticipate that our reactivity palette approach, combined with the in-depth fluorescence analysis discussed herein, will provide guidelines toward developing fluorogenic assays of reactivity offering multiplex information, beyond fluorescence intensity., Competing Interests: The authors declare no competing financial interest. more...
- Published
- 2017
- Full Text
- View/download PDF
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