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Inorganic Chemistry May 2024Ruthenium(II) polypyridyl complexes have gained significant interest as photochemotherapeutics (PCTs) due to their synthetic viability, strong light absorption, well...
Ruthenium(II) polypyridyl complexes have gained significant interest as photochemotherapeutics (PCTs) due to their synthetic viability, strong light absorption, well understood excited state properties, and high phototoxicity indexes. Herein, we report the synthesis, characterization, electrochemical, spectrochemical, and preliminary cytotoxicity analyses of three series of ruthenium(II) polypyridyl complexes designed to mimic PCTs. The three series have the general structure of [Ru(bpy)(N-N)] (), [Ru(bpy)(dmb)(N-N)] (), and [Ru(dmb)(N-N)] (, where N-N is a bidentate polypyridyl ligand, bpy = 2,2'-bipyridine, and dmb = 6,6'-dimethyl-2,2'-bipyridine). In the three series, the N-N ligand was systematically modified to incorporate increased conjugation and/or electronegative heteroatoms to increase dπ-π* backbonding, red-shifting the lowest energy metal-to-ligand charge transfer (MLCT) absorptions from λ = 454 to λ = 580 nm, nearing the therapeutic window for PCTs (600-1100 nm). In addition, steric bulk was systematically introduced through the series, distorting the Ru(II) octahedra, making the dissociative dd* state thermally accessible at room and body temperatures. This resulted in a 4 orders of magnitude increase in photoinduced ligand ejection kinetics, and demonstrates the ability to modulate both the MLCT* and dd* manifolds in the complexes, which is critical in PCT drug design. Preliminary cell viability assays suggest that the increased steric bulk to lower the dd* states may interfere with the cytotoxicity mechanism, limiting photoinitiated toxicity of the complexes. This work demonstrates the importance of understanding both the MLCT* and dd* manifolds and how they impact the ability of a complex to act as a PCT agent.
PubMed: 38662617
DOI: 10.1021/acs.inorgchem.4c00922 -
ACS Chemical Neuroscience May 2024Disturbances in protein phase transitions promote protein aggregation─a neurodegeneration hallmark. The modular Ran-binding protein 2 (Ranbp2) is a cytosolic molecular...
Proteostatic Remodeling of Small Heat Shock Chaperones─Crystallins by Ran-Binding Protein 2─and the Peptidyl-Prolyl Isomerase and Chaperone Activities of Its Cyclophilin Domain.
Disturbances in protein phase transitions promote protein aggregation─a neurodegeneration hallmark. The modular Ran-binding protein 2 (Ranbp2) is a cytosolic molecular hub for rate-limiting steps of phase transitions of Ran-GTP-bound protein ensembles exiting nuclear pores. Chaperones also regulate phase transitions and proteostasis by suppressing protein aggregation. haploinsufficiency promotes the age-dependent neuroprotection of the chorioretina against phototoxicity by proteostatic regulations of neuroprotective substrates of Ranbp2 and by suppressing the buildup of polyubiquitylated substrates. Losses of peptidyl-prolyl isomerase (PPIase) and chaperone activities of the cyclophilin domain (CY) of Ranbp2 recapitulate molecular effects of haploinsufficiency. These CY impairments also stimulate deubiquitylation activities and phase transitions of 19S cap subunits of the 26S proteasome that associates with Ranbp2. However, links between CY moonlighting activity, substrate ubiquitylation, and proteostasis remain incomplete. Here, we reveal the Ranbp2 regulation of small heat shock chaperones─crystallins in the chorioretina by proteomics of mice with total or selective modular deficits of Ranbp2. Specifically, loss of CY PPIase of Ranbp2 upregulates αA-Crystallin, which is repressed in adult nonlenticular tissues. Conversely, impairment of CY's chaperone activity opposite to the PPIase pocket downregulates a subset of αA-Crystallin's substrates, γ-crystallins. These CY-dependent effects cause age-dependent and chorioretinal-selective declines of ubiquitylated substrates without affecting the chorioretinal morphology. A model emerges whereby inhibition of Ranbp2's CY PPIase remodels crystallins' expressions, subdues molecular aging, and preordains the chorioretina to neuroprotection by augmenting the chaperone capacity and the degradation of polyubiquitylated substrates against proteostatic impairments. Further, the druggable Ranbp2 CY holds -therapeutic potential against proteotoxicity and neurodegeneration.
Topics: Animals; Molecular Chaperones; Mice; Cyclophilins; Proteostasis; Peptidylprolyl Isomerase; Nuclear Pore Complex Proteins; Crystallins
PubMed: 38657106
DOI: 10.1021/acschemneuro.3c00792 -
Journal of Lasers in Medical Sciences 2024This in vitro study was conducted to assess the phototoxic effects of curcumin, nano-curcumin, and erythrosine on the viability of in suspension and biofilm forms....
This in vitro study was conducted to assess the phototoxic effects of curcumin, nano-curcumin, and erythrosine on the viability of in suspension and biofilm forms. Various concentrations of curcumin (1.5 g/L, 3 g/L), nano-curcumin (3 g/L), and erythrosine (100 μM/L, 250 μM/L) were examined for their impact on planktonic and biofilm cultures of , either individually or in conjunction with light irradiation (photodynamic therapy or PDT). A blue light-emitting diode (LED) with a central wavelength of 450 nm served as the light source. The results were compared to 0.12% chlorhexidine digluconate (CHX) as the positive control, and a solution containing neither a photosensitizer (PS) nor a light source as the negative control group. The dependent variable was the number of viable microorganisms per experiment (CFU/mL). Antimicrobial PDT caused a significant reduction in the viability of in both planktonic and biofilm forms, compared to the negative control group (<0.05). The highest cell killing was observed in PDT groups with curcumin 3 g/L or erythrosine 250 μmol/L, although the difference with PDT groups using curcumin 1.5 g/L or erythrosine 100 μmol/L was not significant (>0.05). Antimicrobial treatments were more effective against planktonic S. mutans than the biofilm form. PDT with either curcumin 1.5 g/L or erythrosine 100 μmol/L may be suggested as an alternative to CHX to inactivate the bacteria in dental plaque or deep cavities. Nano-curcumin, at the selected concentration, exhibited lower efficacy in killing compared to Curcumin or erythrosine.
PubMed: 38655044
DOI: 10.34172/jlms.2024.07 -
ACS Applied Materials & Interfaces Apr 2024Phototherapies are promising for noninvasive treatment of aggressive tumors, especially when combining heat induction and oxidative processes. Herein, we show enhanced...
Phototherapies are promising for noninvasive treatment of aggressive tumors, especially when combining heat induction and oxidative processes. Herein, we show enhanced phototoxicity of gold shell-isolated nanorods conjugated with toluidine blue-O (AuSHINRs@TBO) against human colorectal tumor cells (Caco-2) with synergic effects of photothermal (PTT) and photodynamic therapies (PDT). Mitochondrial metabolic activity tests (MTT) performed on Caco-2 cell cultures indicated a photothermal effect from AuSHINRs owing to enhanced light absorption from the localized surface plasmon resonance (LSPR). The phototoxicity against Caco-2 cells was further increased with AuSHINRs@TBO where oxidative processes, such as hydroperoxidation, were also present, leading to a cell viability reduction from 85.5 to 39.0%. The molecular-level mechanisms responsible for these effects were investigated on bioinspired tumor membranes using Langmuir monolayers of Caco-2 lipid extract. Polarization-modulation infrared reflection-absorption spectroscopy (PM-IRRAS) revealed that the AuSHINRs@TBO incorporation is due to attractive electrostatic interactions with negatively charged groups of the Caco-2 lipid extract, resulting in the expansion of surface pressure isotherms. Upon irradiation, Caco-2 lipid extract monolayers containing AuSHINRs@TBO (1:1 v/v) exhibited ca. 1.0% increase in surface area. This is attributed to the generation of reactive oxygen species (ROS) and their interaction with Caco-2 lipid extract monolayers, leading to hydroperoxide formation. The oxidative effects are facilitated by AuSHINRs@TBO penetration into the polar groups of the extract, allowing oxidative reactions with carbon chain unsaturations. These mechanisms are consistent with findings from confocal fluorescence microscopy, where the Caco-2 plasma membrane was the primary site of the cell death induction process.
PubMed: 38652860
DOI: 10.1021/acsami.4c02247 -
Ophthalmic Surgery, Lasers & Imaging... Apr 2024
PubMed: 38648426
DOI: 10.3928/23258160-20240409-02 -
Advanced Science (Weinheim,... Jun 2024The escalating spread of drug-resistant bacteria and viruses is a grave concern for global health. Nucleic acids dominate the drug-resistance and transmission of...
The escalating spread of drug-resistant bacteria and viruses is a grave concern for global health. Nucleic acids dominate the drug-resistance and transmission of pathogenic microbes. Here, imidazolium-type poly(ionic liquid)/porphyrin (PIL-P) based electrospun nanofibrous membrane and its cerium (IV) ion complex (PIL-P-Ce) are developed. The obtained PIL-P-Ce membrane exhibits high and stable efficiency in eradicating various microorganisms (bacteria, fungi, and viruses) and decomposing microbial antibiotic resistance genes and viral nucleic acids under light. The nuclease-mimetic and photocatalytic mechanisms of the PIL-P-Ce are elucidated. Co-infection wound models in mice with methicillin-resistant S. aureus and hepatitis B virus demonstrate that PIL-P-Ce integrate the triple effects of cationic polymer, photocatalysis, and nuclease-mimetic activities. As revealed by proteomic analysis, PIL-P-Ce shows minimal phototoxicity to normal tissues. Hence, PIL-P-Ce has potential as a "green" wound dressing to curb the spread of drug-resistant bacteria and viruses in clinical settings.
Topics: Animals; Mice; Antiviral Agents; Coinfection; Disease Models, Animal; Nanofibers; Anti-Infective Agents; Methicillin-Resistant Staphylococcus aureus; Catalysis; Porphyrins; Ionic Liquids
PubMed: 38647392
DOI: 10.1002/advs.202309590 -
JCI Insight Apr 2024Inherited retinal dystrophies (IRDs) are progressive diseases leading to vision loss. Mutation in the eyes shut homolog (EYS) gene is one of the most frequent causes of...
Inherited retinal dystrophies (IRDs) are progressive diseases leading to vision loss. Mutation in the eyes shut homolog (EYS) gene is one of the most frequent causes of IRD. However, the mechanism of photoreceptor cell degeneration by mutant EYS has not been fully elucidated. Here, we generated retinal organoids from induced pluripotent stem cells (iPSCs) derived from patients with EYS-associated retinal dystrophy (EYS-RD). In photoreceptor cells of RD organoids, both EYS and G protein-coupled receptor kinase 7 (GRK7), one of the proteins handling phototoxicity, were not in the outer segment, where they are physiologically present. Furthermore, photoreceptor cells in RD organoids were vulnerable to light stimuli, and especially to blue light. Mislocalization of GRK7, which was also observed in eys-knockout zebrafish, was reversed by delivering control EYS into photoreceptor cells of RD organoids. These findings suggest that avoiding phototoxicity would be a potential therapeutic approach for EYS-RD.
Topics: Animals; Humans; Eye Proteins; Induced Pluripotent Stem Cells; Light; Mutation; Organoids; Retina; Retinal Dystrophies; Zebrafish
PubMed: 38646933
DOI: 10.1172/jci.insight.174179 -
Scientific Reports Apr 2024Quantitative assessment of cell migration in vitro is often required in fundamental and applied research from different biomedical areas including wound repair, tumor...
Quantitative assessment of cell migration in vitro is often required in fundamental and applied research from different biomedical areas including wound repair, tumor metastasis or developmental biology. A collection of assays has been established throughout the years like the most widely used scratch assay or the so-called barrier assay. It is the principle of these assays to introduce a lesion into an otherwise confluent monolayer in order to study the migration of cells from the periphery into this artificial wound and determine the migration rate from the time necessary for wound closure. A novel assay makes use of photosensitizers doped into a polystyrene matrix. A thin layer of this composite material is coated on the bottom of regular cell culture ware showing perfect biocompatibility. When adherent cells are grown on this coating, resonant excitation of the photosensitizer induces a very local generation of O, which kills the cells residing at the site of illumination. Cells outside the site of illumination are not harmed. When excitation of the photosensitizer is conducted by microscopic illumination, high-precision wounding in any size and geometry is available even in microfluidic channels. Besides proof-of-concept experiments, this study gives further insight into the mechanism of photosensitizer-mediated cell wounding.
Topics: Photosensitizing Agents; Wound Healing; Cell Culture Techniques; Microfluidics; Cell Movement
PubMed: 38643292
DOI: 10.1038/s41598-024-59564-9 -
Journal of Biomedical Optics Jun 2024Three-dimensional quantitative phase imaging (QPI) has rapidly emerged as a complementary tool to fluorescence imaging, as it provides an objective measure of cell...
SIGNIFICANCE
Three-dimensional quantitative phase imaging (QPI) has rapidly emerged as a complementary tool to fluorescence imaging, as it provides an objective measure of cell morphology and dynamics, free of variability due to contrast agents. It has opened up new directions of investigation by providing systematic and correlative analysis of various cellular parameters without limitations of photobleaching and phototoxicity. While current QPI systems allow the rapid acquisition of tomographic images, the pipeline to analyze these raw three-dimensional (3D) tomograms is not well-developed. We focus on a critical, yet often underappreciated, step of the analysis pipeline that of 3D cell segmentation from the acquired tomograms.
AIM
We report the CellSNAP (Cell Segmentation via Novel Algorithm for Phase Imaging) algorithm for the 3D segmentation of QPI images.
APPROACH
The cell segmentation algorithm mimics the gemstone extraction process, initiating with a coarse 3D extrusion from a two-dimensional (2D) segmented mask to outline the cell structure. A 2D image is generated, and a segmentation algorithm identifies the boundary in the plane. Leveraging cell continuity in consecutive -stacks, a refined 3D segmentation, akin to fine chiseling in gemstone carving, completes the process.
RESULTS
The CellSNAP algorithm outstrips the current gold standard in terms of speed, robustness, and implementation, achieving cell segmentation under 2 s per cell on a single-core processor. The implementation of CellSNAP can easily be parallelized on a multi-core system for further speed improvements. For the cases where segmentation is possible with the existing standard method, our algorithm displays an average difference of 5% for dry mass and 8% for volume measurements. We also show that CellSNAP can handle challenging image datasets where cells are clumped and marred by interferogram drifts, which pose major difficulties for all QPI-focused AI-based segmentation tools.
CONCLUSION
Our proposed method is less memory intensive and significantly faster than existing methods. The method can be easily implemented on a student laptop. Since the approach is rule-based, there is no need to collect a lot of imaging data and manually annotate them to perform machine learning based training of the model. We envision our work will lead to broader adoption of QPI imaging for high-throughput analysis, which has, in part, been stymied by a lack of suitable image segmentation tools.
Topics: Humans; Image Processing, Computer-Assisted; Imaging, Three-Dimensional; Quantitative Phase Imaging; Algorithms; Optical Imaging
PubMed: 38638450
DOI: 10.1117/1.JBO.29.S2.S22706 -
Journal of Biomedical Optics Apr 2024Light-sheet fluorescence microscopy (LSFM) has emerged as a powerful and versatile imaging technique renowned for its remarkable features, including high-speed 3D...
SIGNIFICANCE
Light-sheet fluorescence microscopy (LSFM) has emerged as a powerful and versatile imaging technique renowned for its remarkable features, including high-speed 3D tomography, minimal photobleaching, and low phototoxicity. The interference light-sheet fluorescence microscope, with its larger field of view (FOV) and more uniform axial resolution, possesses significant potential for a wide range of applications in biology and medicine.
AIM
The aim of this study is to investigate the interference behavior among multiple light sheets (LSs) in LSFM and optimize the FOV and resolution of the light-sheet fluorescence microscope.
APPROACH
We conducted a detailed investigation of the interference effects among LSs through theoretical derivation and numerical simulations, aiming to find optimal parameters. Subsequently, we constructed a customized system of multi-LSFM that incorporates both interference light sheets (ILS) and noninterference light-sheet configurations. We performed beam imaging and microsphere imaging tests to evaluate the FOV and axial resolution of these systems.
RESULTS
Using our custom-designed light-sheet fluorescence microscope, we captured the intensity distribution profiles of both interference and noninterference light sheets (NILS). Additionally, we conducted imaging tests on microspheres to assess their imaging outcomes. The ILS not only exhibits a larger FOV compared to the NILS but also demonstrates a more uniform axial resolution.
CONCLUSIONS
By effectively modulating the interference among multiple LSs, it is possible to optimize the intensity distribution of the LSs, expand the FOV, and achieve a more uniform axial resolution.
Topics: Microscopy, Fluorescence; Microspheres; Photobleaching
PubMed: 38629030
DOI: 10.1117/1.JBO.29.4.046501