Copper (II)-catalyzed polydopamine mediated photothermal sensors for visual quantitative point-of-care testing.

BACKGROUND: Temperature sensing is commonly used in point-of-care (POC) detection technologies, yet the portability and convenience of use are frequently compromised by the complexity of thermosensitive processes and signal transduction. Especially, multi-step target recognition reactions and temperature measurement in the reaction vessel present challenges in terms of stability and integration of detection devices. To further combine photothermal reaction and signal readout in one assay, these two processes enable to be integrated into miniaturized microfluidic chips, thereby facilitating photothermal sensing and achieving a simple visual temperature sensing as POC detection. RESULTS: A copper ion (Cu2+)-catalyzed photothermal sensing system integrated onto a microfluidic distance-based analytical device (µDAD), enabling the visual, portable, and sensitive quantitative detection of multiple targets, including ascorbic acid, glutathione, and alkaline phosphatase (ALP). The polydopamine nanoparticles (PDA NPs) were synthesized by the regulation of free Cu2+ through redox or coordination reactions, facilitating the transduction of distinct photothermal response signals and providing the versatile Cu2+-responsive sensing systems. Promoted by integration with a photothermal µDAD, the system combines PDA's photothermal responsiveness and thermosensitive gas production of ammonium bicarbonate for improved sensitivity of ALP detection, reaching the detection limit of 9.1 mU/L. The system has successfully achieved on-chip detection of ALP with superior anti-interference capability and recoveries ranging from 96.8 % to 104.7 %, alongside relative standard deviations below 8.0 %. SIGNIFICANCE AND NOVELTY: The µDAD design accommodated both the photothermal reaction of PDA NPs and thermosensitive gas production reaction, achieving the rapid sensing of visual distance signals. The µDAD-based Cu2+-catalyzed photothermal sensing system holds substantial potential for applications in biochemical analysis and clinical diagnostics, underscored by the versatile Cu2+ regulation mechanism for a broad spectrum of biomarkers.

TME-Responsive Nanoplatform with Glutathione Depletion for Enhanced Tumor-Specific Mild Photothermal/Gene/Ferroptosis Synergistic Therapy.

Background: Triple negative breast cancer (TNBC) is one of the worst prognosis types of breast cancer that urgently needs effective therapy methods. However, cancer is a complicated disease that usually requires multiple treatment modalities. Methods: A tumor microenvironment (TME)-responsive PFC/TRIM37@Fe-TA@HA (abbreviated as PTFTH) nanoplatform was constructed by coating Fe3+ and tannic acid (TA) on the surface of TRIM37-siRNA loaded phase-transition perfluorocarbon (PFC) nanodroplets and further modifying them with hyaluronic acid (HA) to achieve tumor-specific mild photothermal/gene/ferroptosis synergistic therapy (MPTT/GT/ Ferroptosis) in vitro. Once internalized into tumor cells through CD44 receptor-mediated active targeting, the HA shell of PTFTH would be preliminarily disassembled by hyaluronidase (HAase) to expose the Fe-TA metal-phenolic networks (MPNs), which would further degrade in response to an acidic lysosomal environment, leading to HAase/pH dual-responsive release of Fe3+ and PFC/TRIM37. Results: PTFTH showed good biocompatibility in vitro. On the one hand, the released Fe3+ could deplete the overexpressed glutathione (GSH) through redox reactions and produce Fe2+, which in turn converts endogenous H2O2 into highly cytotoxic hydroxyl radicals (•OH) for chemodynamic therapy (CDT). On the other hand, the local hyperthermia generated by PTFTH under 808 nm laser irradiation could not only improve CDT efficacy through accelerating the Fe2+-mediated Fenton reaction, but also enhance TRIM37-siRNA delivery for gene therapy (GT). The consumption of GSH and accumulation of •OH synergistically augmented intracellular oxidative stress, resulting in substantial tumor cell ferroptosis. Moreover, PTFTH possessed outstanding contrast enhanced ultrasound (CEUS), photoacoustic imaging (PAI) and magnetic resonance imaging (MRI) ability. Conclusion: This PTFTH based multiple-mode therapeutic strategy has successfully achieved a synergistic anticancer effect in vitro and has the potential to be translated into clinical application for tumor therapy in future.

A growth type pathway for improving the folding of BPTI.

The in vitro oxidative folding of the protein bovine pancreatic trypsin inhibitor (BPTI) with oxidized dithiothreitol or glutathione has served as a paradigm for protein folding but could take weeks at physiological pH because of the need to escape from kinetic traps via a rearrangement type pathway. The two major kinetic traps are called N' and N* and contain two of the three native disulfide bonds, which occur between residues 5 and 55, 30 and 51, and 14 and 38. N' is missing the disulfide bond between residues 5 and 55 while N* is missing the disulfide bond between residues 30 and 51. By determining rate constant for the reactions of the kinetic traps N* and N' and their mixed disulfides with glutathione and glutathione disulfide, many for the first time, we demonstrate that growth type pathways are feasible and could even be more efficient than rearrangement type pathways. Thus, formally unproductive pathways became productive. Interestingly, under physiological redox conditions both rearrangement and growth type pathways are important highlighting the redundancy of oxidative protein folding. With the new set of rate constants, modeling indicated that in vitro oxidative protein folding of BPTI via a growth type pathway using an oxidation, reduction and oxidation cycle would significantly improve protein folding efficiency, albeit under non-physiological redox conditions. With these changing conditions 91 ± 2% of native BPTI was achieved in 12 h compared to 83% native protein in 24 h using our previous best conditions of 5 mM GSSG and 5 mM GSH. Therefore, changing redox conditions via an oxidation, reduction and oxidation cycle may become an additional methodology for enhancing in vitro protein folding in aqueous solution.

Advancing structural elucidation of conjugation drug metabolites in metabolite profiling with novel electron-activated dissociation.

RATIONALE: This study focuses on the advantage of using the novel electron-activated dissociation (EAD) technology on the QTOF system for structural elucidation of conjugation metabolites. In drug metabolite identification, conceptual "boxes" are generally used to represent potential sites of modifications, which are proposed based on MS/MS data. Electron-activated dissociation (EAD) provides unique fragmentation patterns, potentially allowing for more precise localization of the metabolic modification sites compared to CID, particularly for conjugations. METHOD: Known compounds were incubated with rat liver microsomes in the presence of nicotinamide adenine dinucleotide phosphate (NADPH), uridine dihosphate-glucuronic acid (UDPGA), and glutathione. Conjugation metabolites were analyzed using the QTOF system. High-resolution MS/MS spectra were collected using EAD and CID fragmentations along with TOF MS full scan for tested drugs and metabolites. Fragmentation patterns were compared to evaluate their efficiency in structural elucidation. RESULTS: Metabolite profiling identified conjugation metabolites (glucuronides and GSH adducts), using characteristic mass shifts. A comparison of EAD and CID fragmentation revealed EAD-specific fragments for most conjugates. EAD was able to break the relatively stable bonds on parent drug motifs while keeping relatively weak conjugation bonds intact, despite the generally low intensity of EAD. EAD effectively narrowed the conceptual "box" representing modification sites, providing more definitive information on conjugation sites and facilitating the structural elucidation of conjugated metabolites. CONCLUSION: EAD is a powerful tool for metabolite profiling in drug development, particularly for identifying conjugation sites. EAD-enabled MS/MS spectra offer a greater variety of signature fragments compared to CID, resulting in more comprehensive and unique structural information for metabolic modification analysis. Overall, EAD, complementary to CID, has the potential to narrow down potential modification sites, significantly enhancing the precision of conjugation metabolite structure elucidation.

Simultaneous Mapping of the Nanoscale Organization and Redox State of Extracellular Space in Living Brain Tissue.

The spatial organization characteristics and redox status of the extracellular space (ECS) are crucial in the development of brain diseases. However, it remains a challenge to simultaneously capture dynamic changes in microstructural features and redox states at the submicron level within the ECS. Here, we developed a reversible glutathione (GSH)-responsive nanoprobe (RGN) for mapping the spatial organization features and redox status of the ECS in brain tissues with nanoscale resolution. The RGN is composed of polymer nanoparticles modified with GSH-responsive molecules and amino-functionalized methoxypoly(ethylene glycol), which exhibit exceptional single-particle brightness and excellent free diffusion capability in the ECS of brain tissues. Tracking single RGNs in acute brain slices allowed us to dynamically map spatial organizational features and redox levels within the ECS of brain tissues in disease models. This provides a powerful super-resolution imaging method that offers a potential opportunity to study the dynamic changes in the ECS microenvironment and to understand the physiological and pathological roles of the ECS in vivo.

Gemini Surfactant-Induced Cysteine-Capped Copper Nanoclusters Self-Assembly with Enhanced Peroxidase-Like Activity and Colorimetric Glutathione Sensing.

We have prepared a novel assembly with copper nanoclusters (CuNCs) and imidazolium-based gemini surfactants (different chain lengths). These novel mimic enzymes formed through the assembly of nanocluster-gemini surfactants have been utilized in creating colorimetric sensors to detect biomolecules. Yet, understanding the method for detecting glutathione (GSH) and its sensing mechanism using this specific assembly-based colorimetric sensor poses a significant challenge. Because of the role of surface ligands, the complexes of cysteine-capped CuNCs (Cys-CuNCs) and gemini surfactants exhibit strong amphiphilicity, enabling them to self-assemble like a molecular amphiphile. We have investigated the kinetics and catalytic capabilities of this Cys-CuNCs@gemini surfactant assembly through peroxidase-like activity. Additionally, a sensitive and simple-to-use colorimetric sensing approach for glutathione (GSH) is also disclosed here, demonstrating a low limit of detection, by using this peroxidase-like activity of Cys-CuNCs@gemini surfactant assemblies. Thus, the remarkable advantages of the Cys-CuNCs@gemini surfactant nanozyme make it suitable for the precise colorimetric detection of GSH, demonstrating excellent sensitivity and reliable selectivity. Additionally, it performs well in detecting GSH in various soft drinks.

Improved efficacy of linear glutathione-peptide chaperon complexes on melanogenesis inhibition and transdermal delivery.

Glutathione (GSH) exhibits considerable potential in the cosmetic industry for reducing intracellular tyrosinase activity and inhibiting melanin synthesis. However, its efficacy is hindered by limited permeability, restricting its ability to reach the basal layer of the skin where melanin production occurs. The transdermal enhancer peptide TD1 has emerged as a promising strategy to facilitate the transdermal transfer of proteins or peptides by creating intercellular gaps in keratinocytes, providing access to the basal layer. The primary objective of this study is to enhance the transdermal absorption capacity of GSH while augmenting its inhibitory effect on melanin. Two coupling structures were designed for investigation: linear (TD1-linker-GSH) and branched (TD1-GSH). The study examined the impact of the peptide skeleton on melanin inhibition ability. Our findings revealed that the linear structure not only inhibited synthetic melanin production in B16F10 cells through a direct pathway but also through a paracrine pathway, demonstrating a significant tyrosinase inhibition of nearly 70 %, attributed to the paracrine effect of human keratinocyte HaCaT. In pigmentation models of guinea pigs and zebrafish, the application of TD1-linker-GSH significantly reduced pigmentation. Notably, electric two-photon microscopy demonstrated that TD1-linker-GSH exhibited significant transdermal ability, penetrating 158.67 ± 9.28 µm into the skin of living guinea pigs. Molecular docking analysis of the binding activity with tyrosinase revealed that both TD1-linker-GSH and TD1-GSH occupy the same active pocket, with TD1-linker-GSH binding more tightly to tyrosinase. These results provide a potential foundation for therapeutic approaches aimed at enriched pigmentation and advance our understanding of the mechanisms underlying melanogenesis inhibition.

An endogenous oxygen self-supplied nanoplatform with GSH-depleted and NIR-II triggered electron-hole separation for enhanced photocatalytic anti-tumor therapy.

The use of artificial enzymes and light energy in photocatalytic therapy, a developing drug-free therapeutic approach, can treat malignant tumors in vivo. However, the relatively deficient oxygen concentration in the tumor microenvironment (TME) restrains their further tumor treatment capability. Herein, a novel nanoplatform with Cu7S4@Au nanocatalyst coated by MnO2 was successfully designed. After 1064 nm light irradiation, the designed nanocatalyst can promote the separation of light generated electron-hole pairs, resulting in ROS generation and tumor cell apoptosis. The MnO2 shelled nanoplatform can function as a TME-responsive oxygen self-supplied producer to improve photocatalyst treatment and GSH depletion. In summary, the designed novel nanoplatform shows efficient inhibition of tumor growth via GSH depletion and synergistic photocatalytic therapy, which is of great significance for improving the clinical tumor treatment effect.

Novel fluorescent nanoplatform for all-in-one sensing and removal of acrolein: An ultrasensitive probe to evaluate its removal efficiency.

As a highly toxic aldehyde, acrolein is widely found in diet and environment, and can be produced endogenously, posing a serious threat to human health. Herein, we designed a novel fluorescent nanoplatform integrating carbon dots­manganese dioxide (CDs-MnO2) and glutathione (GSH) for all-in-one sensing and removal of acrolein. By converting Mn4+ to free Mn2+, GSH inhibited the inner filter effect (IFE) of MnO2 nanosheets, and the Michael addition of acrolein with GSH inhibited the GSH-induced Mn4+ conversion, forming an "off-on-off" fluorescence response of CDs. The developed fluorescent nanoplatform exhibited high sensitivity (LOD was 0.067 µM) and selectivity for the simultaneous detection and removal of acrolein. The combination of CDs-MnO2 hydrogels with smartphones realized the point-of-care detection of acrolein, yielding satisfactory results (recovery rates varied between 97.01-104.65%, and RSD ranged from 1.42 to 4.16%). Moreover, the capability of the nanoplatform was investigated for on-site evaluating acrolein scavengers' efficacy, demonstrating excellent potential for practical application.

Glutathione and viscosity double-locked response fluorescent probe for imaging and surgical navigation of hepatocellular carcinoma.

Early diagnosis and precise treatment of hepatocellular carcinoma (HCC) are crucial for human health. Therefore, addressing the potential markers of HCC, glutathione (GSH) and viscosity, we constructed a fluorescent probe (PG-V) activated cascadically by GSH/viscosity. PG-V possessed excellent photophysical properties and biocompatibility, and could specifically illuminate tumor tissue, achieving fluorescence imaging of HCC, and imaging-guided tumor resection.

Dual-Level Reactive Oxygen Species Amplifier for Enhanced Photothermal-Chemodynamic Therapy.

Chemodynamic therapy is an appealing modality in cancer treatment. However, its therapeutic effectiveness is impeded by insufficient catalytic efficiency and overexpression of glutathione (GSH) at the tumor site. In this study, a poly(o-phenylenediamine) (PoPD)@copper sulfide (CuS) nanoplatform was developed as dual-level reactive oxygen species (ROS) amplifier for enhanced photothermal-chemodynamic therapy. The PoPD@CuS nanoplatform exhibited photothermal performance, chemodynamic performance, and GSH-depleting capability. Alongside its improved photothermal conversion efficiency with tumor pH-responsiveness, the photothermal behavior of PoPD@CuS could elevate chemodynamic activity by regulating the temperature spatiotemporally, leading to increased ROS production. Moreover, GSH depletion of PoPD@CuS could suppress ROS scavenging, further enhancing oxidative stress in the tumor region. Consequently, functioning as a dual-level ROS amplifier, PoPD@CuS showcased remarkable effectiveness in photothermal-chemodynamic combination therapy.

Macrophage-Targeted GSH-Depleting Nanocomplexes for Synergistic Chemodynamic Therapy/Gas Therapy/Immunotherapy of Intracellular Bacterial Infection.

Intracellular pathogens can survive inside the macrophages to protect themselves from eradication by the innate immune system and conventional antibiotics, resulting in severe bacterial infections. In this work, an antibiotic-free nanocomplex (HA/GA-Fe@NO-DON), exhibiting macrophage-targeted synergistic gas therapy (nitric oxide, NO)/chemodynamic therapy/immunotherapy, was reported. HA/GA-Fe nanoparticles were synthesized by the strong coordination interactions among carboxyl groups of hyaluronic acid (HA), polyphenol groups of gallic acid (GA), and Fe(II) ions. The hydrophobic glutathione (GSH)-responsive NO donor (NO-DON) was encapsulated in HA/GA-Fe nanoparticles to form the final nanocomplexes (HA/GA-Fe@NO-DON). HA on the nanocomplexes guides the macrophage-specific uptake and intracellular accumulation. After the uptake, HA/GA-Fe@NO-DON nanocomplexes could not only generate highly toxic hydroxyl radicals (•OH) by the Fenton reaction and GSH depletion but also release NO when stimulated by intracellular GSH. Meanwhile, the nanocomplexes could trigger an efficient proinflammation immune response to reinforce the antibacterial activity. This work presents the development of antibiotic-free macrophage-targeted HA/GA-Fe@NO-DON nanocomplexes as an effective adjuvant nanomedicine with synergistic gas therapy/chemodynamic therapy/immunotherapy for eliminating intracellular bacterial infection.

Design of pH/Redox Co-Triggered Degradable Diselenide-Containing Polyprodrug via a Facile One-Pot Two-Step Approach for Tumor-Specific Chemotherapy.

The diselenide bond has attracted intense interest for drug delivery systems (DDSs) for tumor chemotherapy, owing to it possessing higher redox sensitivity than the disulfide one. Various redox-responsive diselenide-containing carriers have been developed for chemotherapeutics delivery. However, the premature drug leakage from these DDSs was significant enough to cause toxic side effects on normal cells. Here, a pH/redox co-triggered degradable polyprodrug was designed as a drug self-delivery system (DSDS) by incorporating drug molecules as structural units in the polymer main chains, using a facile one-pot two-step approach. The proposed PDOX could only degrade and release drugs by breaking both the neighboring acid-labile acylhydrazone and the redox-cleavable diselenide conjugations in the drug's structural units, triggered by the higher acidity and glutathione (GSH) or reactive oxygen species (ROS) levels in the tumor cells. Therefore, a slow solubility-controlled drug release was achieved for tumor-specific chemotherapy, indicating promising potential as a safe and efficient long-acting DSDS for future tumor treatment.

GSH Oligomer-Directed Chiral Au-Helicoid Nanoparticles for Discriminating Penicillamine Enantiomers.

Preparing chiral plasmonic nanoparticles (NPs) with strong chiroptical responses is crucial in numerous fields including constructing optical materials, chiral sensing, and chiral-dependent biological processes. However, precise regulation over the chiral optical activity and chiral configuration of plasmonic NPs is still a challenge. In this work, we report Au helicoid NPs with different chiral structures and reversal chirality directed by the oligomeric structure of inducer glutathione (GSH). By precisely controlling the oligomeric structure of GSH and other synthetic parameters, we successfully prepared chiral Au helicoid NPs with a high anisotropy factor of 0.03. The obtained chiral Au NPs demonstrated an excellent performance in discriminating penicillamine (Pen) enantiomers. Our findings provide a construction strategy for chiral Au NPs and contribute insight into the regulation effect of chiral inducers on the growth of chiral metal NPs.

A Glutathione-Consuming Bimetallic Nano-Bomb with the Combination of Photothermal and Chemodynamic Therapy for Tumors: An in vivo and in vitro Study.

Background: Chemodynamic therapy (CDT) faces challenges of low catalytic ion efficiency and ROS production. We developed a ROS nano-bomb, Cu/ZIF-8@GA-Fe, to address these issues. Methods: The nano-bomb was synthesized by doping copper into ZIF-8 and assembling Fe3+ and gallic acid (GA). It was tested for reactive oxygen species (ROS) generation in acidic conditions and its photothermal properties. Results: In an acidic micro environment, Cu/ZIF-8@GA-Fe effectively released Fe3+ and Cu2+, depleting GSH and generating ROS. The GA-Fe coating provided photothermal heat and was used to enhance Fenton reactions via dual ions for increasing ROS production. In vivo and in vitro experiments, Cu/ZIF-8@GA-Fe inhibited tumor growth with minimal side effects. Conclusion: Cu/ZIF-8@GA-Fe shows promise for safe and effective CDT, offering a synergistic approach to tumor therapy.

Cairo pentagon tessellated covalent organic frameworks with mcm topology for near-infrared phototherapy.

Despite the prevalent of hexagonal, tetragonal, and triangular pore structures in two-dimensional covalent organic frameworks (2D COFs), the pentagonal pores remain conspicuously absent. We herein present the Cairo pentagonal tessellated COFs, achieved through precisely chosen geometry and metrics of the linkers, resulting in unprecedented mcm topology. In each pentagonal structure, porphyrin units create four uniform sides around 15.5 Å with 90° angles, while tetrabiphenyl unit establish a bottom edge about 11.6 Å with 120° angles, aligning precisely with the criteria of Cairo Pentagon. According to the narrow bandgap and strong near-infrared (NIR) absorbance, as-synthesized COFs exhibit the efficient singlet oxygen (1O2) generation and photothermal conversion, resulting in NIR photothermal combined photodynamic therapy to guide cancer cell apoptosis. Mechanistic studies reveal that the good 1O2 production capability upregulates intracellular lipid peroxidation, leading to glutathione depletion, low expression of glutathione peroxidase 4, and induction of ferroptosis. The implementation of pentagonal Cairo tessellations in this work provides a promising strategy for diversifying COFs with new topologies, along with multimodal NIR phototherapy.

In Vivo Self-Sorting of Peptides via In Situ Assembly Evolution.

Despite significant progress achieved in artificial self-sorting in solution, operating self-sorting in the body remains a considerable challenge. Here, we report an in vivo self-sorting peptide system via an in situ assembly evolution for combined cancer therapy. The peptide E3C16-SS-EIY consists of two disulfide-connected segments, E3C16SH and SHEIY, capable of independent assembly into twisted or flat nanoribbons. While E3C16-SS-EIY assembles into nanorods, exposure to glutathione (GSH) leads to the conversion of the peptide into E3C16SH and SHEIY, thus promoting in situ evolution from the nanorods into self-sorted nanoribbons. Furthermore, incorporation of two ligand moieties targeting antiapoptotic protein XIAP and organellar endoplasmic reticulum (ER) into the self-sorted nanoribbons allows for simultaneous inhibition of XIAP and accumulation surrounding ER. This leads to the cytotoxicity toward the cancer cells with elevated GSH levels, through activating caspase-dependent apoptosis and inducing ER dysfunction. In vivo self-sorting of E3C16-SS-EIY decorated with ligand moieties is thoroughly validated by tissue studies. Tumor-bearing mouse experiments confirm the therapeutic efficacy of the self-sorted assemblies for inhibiting tumor growth, with excellent biosafety. Our findings demonstrate an efficient approach to develop in vivo self-sorting systems and thereby facilitating in situ formulation of biomedical agents.

Engineering Magnetic Extracellular Vesicles Mimetics for Enhanced Targeting Chemodynamic Therapy to Overcome Ovary Cancer.

Chemodynamic therapy (CDT), employing metal ions to transform endogenous H2O2 into lethal hydroxyl radicals (•OH), has emerged as an effective approach for tumor treatment. Yet, its efficacy is diminished by glutathione (GSH), commonly overexpressed in tumors. Herein, a breakthrough strategy involving extracellular vesicle (EV) mimetic nanovesicles (NVs) encapsulating iron oxide nanoparticles (IONPs) and ß-Lapachone (Lapa) was developed to amplify intracellular oxidative stress. The combination, NV-IONP-Lapa, created through a serial extrusion from ovarian epithelial cells showed excellent biocompatibility and leveraged magnetic guidance to enhance endocytosis in ovarian cancer cells, resulting in selective H2O2 generation through Lapa catalysis by NADPH quinone oxidoreductase 1 (NQO1). Meanwhile, the iron released from IONPs ionization under acidic conditions triggered the conversion of H2O2 into •OH by the Fenton reaction. Additionally, the catalysis process of Lapa eliminated GSH in tumor, further amplifying oxidative stress. The designed NV-IONP-Lapa demonstrated exceptional tumor targeting, facilitating MR imaging, and enhanced tumor suppression without significant side effects. This study presents a promising NV-based drug delivery system for exploiting CDT against NQO1-overexpressing tumors by augmenting intratumoral oxidative stress.

Structural Analysis of the Drosophila melanogaster GSTome.

Glutathione transferase (GST) is a superfamily of ubiquitous enzymes, multigenic in numerous organisms and which generally present homodimeric structures. GSTs are involved in numerous biological functions such as chemical detoxification as well as chemoperception in mammals and insects. GSTs catalyze the conjugation of their cofactor, reduced glutathione (GSH), to xenobiotic electrophilic centers. To achieve this catalytic function, GSTs are comprised of a ligand binding site and a GSH binding site per subunit, which is very specific and highly conserved; the hydrophobic substrate binding site enables the binding of diverse substrates. In this work, we focus our interest in a model organism, the fruit fly Drosophila melanogaster (D. mel), which comprises 42 GST sequences distributed in six classes and composing its GSTome. The goal of this study is to describe the complete structural GSTome of D. mel to determine how changes in the amino acid sequence modify the structural characteristics of GST, particularly in the GSH binding sites and in the dimerization interface. First, we predicted the 3D atomic structures of each GST using the AlphaFold (AF) program and compared them with X-ray crystallography structures, when they exist. We also characterized and compared their global and local folds. Second, we used multiple sequence alignment coupled with AF-predicted structures to characterize the relationship between the conservation of amino acids in the sequence and their structural features. Finally, we applied normal mode analysis to estimate thermal B-factors of all GST structures of D. mel. Particularly, we extracted flexibility profiles of GST and identify key residues and motifs that are systematically involved in the ligand binding/dimerization processes and thus playing a crucial role in the catalytic function. This methodology will be extended to guide the in silico design of synthetic GST with new/optimal catalytic properties for detoxification applications.

Label-free detection of glutathione and glutathione disulfide in biological fluid by using an alpha-hederin nanopore.

Glutathione (GSH) is indispensable for maintaining redox homeostasis in biological fluids and serves as a key component in cellular defense mechanisms. Accurate assessment of GSH relative to its oxidized counterpart, glutathione disulfide (GSSG), is critical for the early diagnosis and understanding of conditions related to oxidative stress. Despite existing methods for their quantification, the label-free and simultaneous measurement of GSH and GSSG in biological fluid presents significant challenges. Herein, we report the use of an alpha-hederin (Ah) nanopore for the direct measurement of the GSH:GSSG ratio in simulated biological fluid, containing fetal bovine serum (FBS). This system hinges on detecting characteristic relative ion blockades (ΔI/Io) as GSH and GSSG molecules pass through the Ah nanopore under an applied electric field. The distinct current blockage signals derived from the translocation of GSH and GSSG enabled us to determine the molar ratio of GSH and its oxidized form. Notably, the interactions between the hydroxyl groups of the sugar moiety lining the nanopore's inner surface and the sulfhydryl group of GSH significantly influence the translocation dynamics, resulting in a longer translocation time for GSH compared to GSSG. The Ah nanopore technology proposed in this study offers a promising approach for real-time, single molecule-level monitoring of glutathione redox status in biological fluids, eliminating the need for labeling or extensive sample preparation.