Glutathione (GSH) is a critical antioxidant in biological systems involved in various cellular processes such as cell proliferation and apoptosis, and is considered as one of the cancer biomarkers. However, the conventional methods for detecting GSH levels often involve complex and time-consuming preparation and sophisticated equipment, posing challenges for rapid and straightforward analysis. Herein, we develop a colorimetric nanosensor using porous single-atom nanozymes (SAzymes), particularly those consisting of atomically dispersed metals on nitrogen-doped carbon supports (M-N-C), to monitor GSH quantitatively. The Mn–N–C SAzymes catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) by hydrogen peroxide (H2O2), resulting in a measurable color change. The high porosity of the Mn–N–C SAzymes offers a large surface area accommodating a high density of accessible active sites for efficient catalysis. The addition of GSH in this system leads to a notable reduction in color intensity, offering an effective method for the quantitative measurement of GSH. The Mn–N–C SAzymes demonstrate high efficacy in the rapid colorimetric detection of GSH, with a low detection limit of 0.70 μM and a broad dynamic range of 0–40 μM. This method is further applied for a simple and rapid colorimetric analysis of the cancer biomarker in various biological samples, including tissues and serum. Demonstrating the potential for diagnostic applications, this approach offers a promising tool for clinical diagnostics, enabling reliable and convenient monitoring of GSH levels, which is crucial for assessing disease progression.

Indium tin oxide (ITO) is a widely used electrode material in electrochemical biosensors owing to its beneficial characteristics such as transparency and chemical inertness. The exceptionally sluggish kinetics of heterogeneous electron transfer in ITO provides an exceptionally low background current, making electron mediators essential for electrochemical sensors. However, the chemical modification of ITO with molecular mediators is still challenging. Few straightforward and credible methods are available to obtain sufficiently thin mediator films firmly anchored on the ITO without losing its inherent benefits, including low background current and transparency. This study demonstrated the anodic electrografting of three osmium complexes, which allowed the formation of molecular mediator monolayers on ITO. The densely packed monolayer of osmium complexes retained their reversible redox processes over hundreds of potential cycles. We confirmed electron mediation by the modified electrodes for 4-aminophenol oxidation, suggesting a simple protocol for developing ITO-based biosensors functionalized with molecular mediators.

L-Glutamate is the most abundant and essential excitatory neurotransmitter in the nervous system. However, its direct electrochemical detection is challenging due to its inherently non-electroactive nature. In this study, we redesigned L-glutamate oxidase (GlutOx) by covalently attaching osmium polypyridyl complexes as electron mediators at selected sites. Most engineered enzymes retained their native catalytic activity, while exhibiting significantly altered catalytic currents during L-glutamate oxidation, depending on the proximity, orientation, and microenvironments of the osmium complexes relative to the FAD cofactors. Notably, two mutants significantly enhanced catalytic currents, revealing selectively and efficiently rerouted electron transfer pathways from the enzyme active site to Os complexes. These findings provide an effective strategy for designing redox-active enzymes for electrochemical biosensors.

Global warming increases methane emissions from Arctic permafrost, which in turn reaccelerates global warming, creating a vicious cycle. Addressing this issue requires innovative solutions, such as electro-assisted methane partial oxidation (EMPO), which can provide an on-site facility for sustainable methane emission reduction in permafrost. In this study, a Co singe-atom catalyst was synthesized as an oxygen reduction reaction (ORR) catalyst that can be practically applied to stand-alone EMPO systems. To address performance degradation and cold weather freezing due to flooding of the electrodes, the hydrophobic polytetrafluoroethylene was mixed into a catalyst layer to regulate the microenvironment near cathodes. Furthermore, hydrophobic cathodes offer a pathway for nonpolar gases to increase the local concentration of methane. The enhanced local methane concentration, combined with an efficient ORR catalyst, yields 8 mmol gcatsingle bond1 formic acid at a low potential bias. Remarkably, the EMPO system exhibits a consistent production even with air and is highly stable. This leads to possibility of on-site facility for methane conversion without external energy at thermokarst lakes.

Direct detection of DNA in complex biological samples bears several challenges regarding the selectivity and sensitivity of analyses. Therefore, DNA pre-extraction from bio-fluids is an emerging tool in biologically related fields. Specifically, a newly developing family of liquid biopsy techniques using PCR detection of circulating tumor DNA from blood serum or blood plasma could be significantly improved by harnessing fast and high-throughput DNA sample preparation. To address these needs, a 3D-printed device and a method based on gel electrophoresis combined with electrodialysis for the time-, cost- and labor-efficient preparative separation of DNA fragments from blood was developed. The proposed system also successfully eliminated large DNA fragments from the samples. Recovery for short DNA fragments was reaching up to 80 %. The method was tested on human genomic DNA and blood and blood serum spiked with DNA standards, and it significantly alleviated the signal of matrix DNA.

Z-DNA is a non-canonical, left-handed helical structure that plays crucial roles in various cellular processes. DNA mismatches, which involve the incorporation of incorrect Watson–Crick base pairs, are present in all living organisms and contribute to the mechanism of Z-DNA formation. However, the impact of mismatches on Z-DNA formation remains poorly understood. Moreover, the combined effect of DNA mismatches and bending, a common biological phenomenon observed in vivo, has not yet been explored due to technological limitations. Here, using single-molecule FRET, we show that a mismatch inside the Z-DNA region, i.e., the CG repeat region, hinders Z-DNA formation. In stark contrast, however, a mismatch in the B–Z junction facilitates Z-DNA formation. When the bending force is applied on double stranded DNA, a mismatch in the B–Z junction releases the bending stress more effectively than one in the CG repeat region. These findings provide mechanical insights into the role of DNA mismatches and bending forces in regulating Z-DNA formation, whether promoting or inhibiting it in biological environments.

Concentrated cations are often employed to promote electrochemical CO2 reduction reaction (CO2RR) selectivity in acidic electrolytes. Here, we investigate the influence of excess cations on the *CO adsorption configuration and the product distribution of the CO2RR. Operando attenuated total reflectance surface-enhanced infrared absorption spectroscopy (ATR-SEIRAS) reveals that increasing the Cs+ concentration shifts the preference of the *CO intermediate on the Cu surface from the atop (*COatop) to the bridge (*CObridge) configuration. This transition leads to a sharp decline in C–C coupling and an increase in the hydrogen evolution reaction at high Cs+ concentrations (0.7 and 1.0 M) under acidic conditions. Time-resolved SEIRAS scans show that *COatop is kinetically dominant and the proportion of *CObridge increases gradually only at high cation concentrations. Density functional theory simulations confirm that Cs+ on the Cu surface can interact electrostatically with *CO and stabilize *CObridge over *COatop on the Cu surface. The evolution of *CObridge is also observed on Ag catalysts, indicating that the effect at high concentrations is not limited to Cu. Furthermore, polymeric binders on the Cu surface mitigate these detrimental effects on the CO2RR and restore C2H4 production by preventing the cation from altering the *CO adsorption sites on the catalyst surface. This study provides new insights into the effects of cations on catalyst performance, with implications for catalyst design and operation.

Our research group developed a novel fluorescence staining strategy based on the DNFC targeting N-Cys in proteins. By treating biological samples with non-fluorogenic citrate and coupling reagents, we achieved strong cyan fluorescence, enabling effective visualization of N-Cys proteins in cells and tissues. The DNFC reaction occurs specifically on N-Cys residues, making it highly ideal for monitoring protein processing events, particularly within the Arg/N-degron pathway. Under hypoxic conditions, DNFC fluorescence is significantly enhanced, likely due to the increased presence of N-Cys-containing proteins. Using immunoassays and mass spectrometry, we identified Class 2 actin as a target protein under hypoxia, emphasizing the utility of 3D histopathology for analyzing actin's spatial distribution. Furthermore, we have identified a novel finding indicating a significant presence of RGS5 in red blood cells (RBCs), a discovery that has not been previously reported. Our fluorescence imaging studies, conducted across various cell types, animal tissues, and human clinical samples suggest that DNFC staining, when combined with tissue-clearing techniques, enables detailed 3D imaging of N-Cys proteins and may offer a means to assess molecular responses to hypoxia within tissues. This study highlights the potential of DNFC as a valuable tool for imaging and quantitative analysis of N-proteomes and providing a foundation for 3D histopathology in hypoxia-related disease research.

This study evaluated the safety, tolerability, and pharmacokinetics of LEM-S401, a novel siRNA therapeutic with DegradaBALL, a mesoporous silica nanoparticle-based delivery system. LEM-S401 is designed to deliver siRNA targeting connective tissue growth factor (CTGF) to fibroblasts for treating hypertrophic scars and keloids, both of which result from abnormal collagen proliferation. LEM-S401, containing unmodified siRNA LEM-17234 encapsulated in DegradaBALL nanoparticles, was administered subcutaneously to healthy adults in a randomized, double-blind, placebo-controlled, single-ascending dose study. Safety and tolerability assessments included vital signs, adverse events (AEs), laboratory tests, and cytokine levels. Pharmacokinetic analysis of LEM-17234 and silicon (Si), the primary component of DegradaBALL, was performed using blood samples collected at specified time points. LEM-S401 demonstrated a favorable safety and tolerability profile with only mild, self-resolving injection site reactions including pain and erythema. No systemic AEs were observed, and cytokine levels showed no significant changes between the treatment and placebo groups. Pharmacokinetic analysis revealed that LEM-17234 was below the plasma detection limit, indicating no notable systemic exposure of siRNA, while Si showed no dose-dependent systemic exposure, suggesting minimal systemic circulation of the mesoporous silica nanoparticles. These findings suggest DegradaBALL effectively encapsulates and delivers siRNA locally without significant systemic exposure. The novel DegradaBALL delivery system enables the stable and targeted delivery of siRNA, which presumably overcomes challenges related to siRNA instability and off-target effects. LEM-S401 has the potential to advance the treatment of fibrotic skin diseases such as keloids and hypertrophic scars by delivering siRNA directly to fibroblasts, thereby inhibiting excessive collagen production.

α-Galactosylceramide (α-GalCer) is a prototypical antigen recognized by natural killer T (NKT) cells, a subset of T cells crucial for immune regulation. Despite its significance, the complex structure-activity relationship of α-GalCer and its analogs remains poorly understood, particularly in defining the structural determinants of NKT cell responses. In this study, we designed and synthesized potential immunomodulatory ligands targeting NKT cells, inspired by glycolipids derived from the gut symbiont Bacteroides fragilis. A series of α-GalCer analogs with terminal iso-branched sphinganine backbones was developed through rational modification of the acyl chain. Our results identified the C3′ hydroxyl group as a structural element that impairs glycolipid presentation by CD1d, as evidenced by reduced IL-2 secretion and weak competition with a potent CD1d ligand. Notably, among C3′-deoxy α-GalCer analogs, those containing an α-chloroacetamide group exhibited robust NKT cell activation with Th2 selectivity. Computational docking and mass spectrometry analyses further confirmed the substantial interaction of α-chloroacetamide analogs to CD1d. These findings underscore the potential of leveraging microbiota-derived glycolipid structures to selectively modulate NKT cell functions for therapeutic purposes.