Medical ozone alleviates acute lung injury by enhancing phagocytosis targeting NETs via AMPK/SR-A1 axis.

Acute lung injury (ALI) linked to sepsis has a high mortality rate, with limited treatment options available. In recent studies, medical ozone has shown promising results in alleviating inflammation and infection. Here, we aimed to evaluate the therapeutic potential of medical ozone in sepsis-induced ALI using a mouse model, measuring behavioral assessments, lung function, and blood flow. Western blot was used to quantify the levels of protein. In vitro, experiments on BMDM cells examine the impact of AMPK inhibitors and agonists on phagocytic activity. Results indicate that medical ozone can enhance the survival rate, ameliorate lung injury, and improve lung function and limb microcirculation in mice with ALI. Notably, it inhibits NETs formation, a crucial player in ALI development. Medical ozone also counteracts elevated TF, MMP-9, and IL-1ß levels. In ALI mice, the effects of ozone are nullified and BMDMs exhibit impaired engulfment of NETs following Sr-a1 knockout. Under normal physiological conditions, the use of an AMPK antagonist produces similar effects to Sr-a1 knockout, significantly inhibiting the phagocytosis of NETs by BMDMs. On the contrary, AMPK agonists enhance this phagocytic process. In conclusion, medical ozone can alleviate sepsis-induced lung injury via the AMPK/SR-A1 pathway, thereby enhancing phagocytosis of NETs by macrophages.

Application of heart-cutting two-dimensional liquid chromatography-mass spectrometry to the characterization of highly polar impurities in calcium gluconate injection.

The separation and characterization of small polar impurities in polar drugs such as calcium gluconate products are always challenging, due to their poor retention on traditional reversed phase (RP) columns. Although ion-pair reversed-phase liquid chromatography (IPRP-LC) and hydrophilic interaction liquid chromatography (HILIC) are commonly used methods for polar compound analysis, both methods have some drawbacks. For example, IPRP-LC is incompatible with mass spectrometry (MS) due to the presence of non-volatile salts in its mobile phase and HILIC has limited sensitivity due to the poor solubility of polar drugs in the organic-rich sample diluents used in HILIC separations. In order to characterize the highly polar impurities in calcium gluconate injections, a heart-cutting two-dimensional liquid chromatography (2D-LC) method coupled with quadrupole time-of-flight mass spectrometry (Q-TOF/MS) was developed in this study. An IPRP-LC method in the first dimension (1D) provided the selectivity for the separation of polar analytes, using a 100% aqueous mobile phase containing phosphate buffer and ion-pair reagent. Heart cuts of target peaks were collected with sample loops and transferred to the second dimension (2D) HILIC column using an organic-rich mobile phase. In order to solve the mobile phase mismatch problem between the two dimensions, a make-up flow module was introduced in the 2D-LC system to dilute the 1D-water-rich fractions with acetonitrile before entering the sample loops. By optimizing the loop size and dilution factor, good retention and peak shape of the highly polar impurities were obtained on the 2D-HILIC column, and the ion suppression effect for MS detection from the ion-pair reagent and non-volatile salt in the 1D-effluent was minimized. A total of five impurities were identified through fragmentation studies by Q-TOF/MS analysis and their fragmentation pathways were proposed. Four of them were further confirmed by reference substances. This study not only provided useful information for quality control of calcium gluconate injections, but also provided an alternative method for polar impurity characterization in pharmaceuticals.

Stimuli-Responsive and Reversible Nanoassemblies of G-Triplexes.

G-triplex (G3) structures formed with three consecutive G-tracts have recently been identified as a new emerging guanine-rich DNA fold. There could likely be a wide range of biological functions for G3s as occurring for G-quadruplex (G4) structures formed with four consecutive G-tracts. However, in comparison to the many reports on G4 nanoassemblies that organize monomers together in a controllable manner, G3-favored nanoassemblies have yet to be explored. In this work, we found that a natural alkaloid of sanguinarine can serve as a dynamic ligand glue to reversibly switch the dimeric nanoassemblies of the thrombin binding aptamer G3 (TBA-G3). The glue planarity was considered to be a crucial factor for realizing this switching. More importantly, external stimuli including pH, sulfite, O2 and H2 O2 can be employed as common regulators to easily modulate the glue's adhesivity for constructing and destructing the G3 nanoassemblies as a result of the ligand converting between isoforms. However, this assembly behavior does not occur with the counterpart TBA-G4. Our work demonstrates that higher-order G3 nanoassemblies can be reversibly operated by manipulating ligand adhesivity. This provides an alternative understanding of the unique behavior of guanine-rich sequences and focuses attention on the G3 fold since the nanoassembly event investigated herein might occur in living cells.

G-quadruplex apurinic site-programmed chiral cyanine assemblies for specifically recognizing guanosine and guanine.

DNA-tuned dye assemblies have received considerable attention toward developing various devices. Owing to easy conformation implementation, G-quadruplexes (G4s) have been extensively used as initiators to grow dye assemblies with controllable chiralities. However, programmed chirality regulation of dye assemblies for a given G4 sequence has not been realized in a straightforward manner. In this work, we replaced a middle guanine in the G-tracts of a human telomeric G4 with an apurinic site (AP site) to meet the programmed dye assemblies. Although all of the AP site replacements altered the G4 conformation from the hybrid to the antiparallel folding, the handedness of pinacyanol (PIN) assemblies grown on the AP site-containing G4 was programmably regulated. The G4 with the AP site at the 5'-most G-tract grew right-handed assemblies, while that with the AP site at the 3'-most G-tract grew left-handed assemblies. The handedness of assemblies almost totally mirrored each other within 450-700 nm. Interestingly, we found that the AP site provided a specific binding site for guanosine and guanine, and this binding event sensitively broke the chiral assemblies. Thus, dye assembly-based sensors can be easily established based on the chiral responses with a high selectivity and sensitivity. Our work first demonstrates the AP site programmed chirality regulation of G4-grown dye assemblies and will find wide application in chiral devices.

Concurrent formation of H- and J-aggregates of dyes with chiralities individually determined by G-quadruplex handedness.

DNA templated dye assemblies pave an easy way to regulate the optical properties of molecular aggregates. G-quadruplexes (G4s) provide versatile DNA platforms for the dye assemblies since their foldings can be easily tuned by cation ions and sequences. In this work, we found that the G4 handedness can be used to control the aggregate chirality of a dye of 3,3'-diethylthiacarbocyanine (DiSC2(3)). The left-handed and right-handed G4s can template the concurrent formation of the J- and H-aggregates of DiSC2(3) with emergence of the featured absorption spectra. However, the chiral J-aggregate of DiSC2(3) can be formed only on the left-handed G4s, while the chiral H-aggregate is otherwise grown only on the right-handed G4s, as confirmed by the induced circular dichroism (ICD) spectra with the characteristic splitting bands. Additionally, these G4s even at tens of nM level are efficient to produce these chiral aggregates, demonstrating the high sensitivity of G4s in creating these optically active dye assemblies. The possible growth sites of the aggregates are proposed by the sequence length-dependent assemblies. Our work will provide a new way to control the chiral assemblies of dye aggregates via the G4 handedness.

Metalloenzyme-mimic innate G-quadruplex DNAzymes using directly coordinated metal ions as active centers.

G-quadruplex DNAs (G4s) have been reported to exhibit the DNAzyme activities by binding with some metal complexes and functional organic ligands. However, there is a challenge to develop metalloenzyme-mimic G4-based innate DNAzymes using the complexed metal ions directly serving as the active centers. This will diversify DNAzymes for developing novel devices since G4 structures are more polymorphic than the other DNA foldings. In this work, we found that the lanthanide trivalent cerium ion of Ce3+ can bind to the human telomere G4 (htG4) according to a 1 : 2 binding mode favorable for creating metalloenzymes-mimic G4 DNAzymes. This Ce3+-G4 entity exhibits a peroxidase activity towards the oxidation of the substrate of 3,3,5,5-tetramethylbenzidine (TMB) by hydrogen peroxide. The 5' G4 tetrads with the orderly arranged carbonyl oxygen atoms are believed to be the coordination sites for Ce3+ and favor the conversion between Ce3+ and Ce4+. Our work provides an alternative feasibility in developing the G4-based innate DNAzymes for variant applications.

A pH-triggered G-triplex switch with K+ tolerance.

A pH-triggered G-triplex (G3) switch is demonstrated to operate in K+ using a planar ligand enabling reversible iminium-alkanolamine conversion as the G3 structuring-destructuring initiator.

Characterization of intermolecular G-quadruplex formation over intramolecular G-triplex for DNA containing three G-tracts.

G-triplex (G3) has been recognized as a popular intermediate during the folding of G-quadruplex (G4). This has raised interest to anticipate the ultimate formation of G3 by shortening the G4-forming oligonucleotides with the remaining three G-tracts. Some G3 structures have been validated and their stability has been found to be affected by the loop sequences similar to G4s. In this work, however, we first found that an intermolecular parallel G4 structure was preferred in K+ for the oligonucleotide 5'-TGGGTAGGGCGGG-3' (DZ3) containing only three G-tracts. We screened auramine O (AO) as the appropriate fluorophore with a molecular rotor feature to target this G4 structure. AO bound with DZ3 in a 1 : 4 ratio, as confirmed by isothermal titration calorimetry experiments, suggesting the formation of a tetramolecular G4 structure (4erG4). The excimer emission from the labelled pyrene and the DNA melting behavior at various pHs in the presence of Ag+ proved the formation of the 4erG4 structure rather than the prevalent intramolecular G3 folding. This work demonstrates that one should be cautious while putatively predicting a G3 structure from an oligonucleotide containing three G-tracts.

Multicolorfully probing intramolecular G-Quadruplex tandem interface.

A long guanine-rich oliogonucleotide sequence can form multiple G-quadruplex (G4) tandem individuals in a single molecule with internal G4-G4 (inG4-G4) interfaces. The interface can exist at the stacked (s-inG4-G4) or unstacked (us-inG4-G4) state, dependent of the G4 conformation and environment. Because of the vital bioactivity of the G4 interface state, there is a great demand for developing a reliable multicolor fluorescence method to identify the interface state using a fluorophore that can emit at the individual wavelength for a specific interface. Herein, we found that a porphyrin with four dihydroxyphenyl substituents (OH2PP) can multicolorfully recognize the s-inG4-G4 dimer interface against the us-inG4-G4 dimer one. The s-inG4-G4 dimer cause significant red shifts in the excitation and emission bands of OH2PP in contrast to the us-inG4-G4 dimer and G4 monomers. OH2PP adopts a 1:1 binding mode with the s-inG4-G4 dimer, whereas a 2:1 binding mode occurs to the us-inG4-G4 dimer. The limit of detection (LOD) for the s-inG4-G4 structure is about tens of nM level. The observed binding dependence of OH2PP on the linker length between the G4 individuals suggests the interface binding with the s-inG4-G4 dimer. Deformation of the porphyrin macrocycle within the s-inG4-G4 interface confinement most likely contributes to the multicolorful response with the hyperporphyrin effect. Our work demonstrates that OH2PP is a promising fluorophore to fluorescently recognize the G4 multimer with an ideal interface-sensitive multicolor response.

A Fluorescently Ratiometric Natural Probe for Selective Detection of Sulfur Dioxide Derivative and Host-Guest Supramolecular Regulation.

Natural sanguinarine (SG) was first used as a fluorescent probe to develop a novel ratiometric sensor for selective HSO3- detection. The nucleophilic addition reaction of HSO3- occurs at the C=N+ group of SG, and subsequent breakage of the conjugated π cycle leads to a decrease in the SG iminium fluorescence that is accompanied by an increase in the alkanolamine fluorescence. Therefore, a ratiometric fluorescence method with a large wavelength shift can be established for HSO3- detection. Furthermore, cucurbit[8]uril was used as an efficient host to encapsulate SG for an improved selectivity for HSO3- detection over H2S. Our method benefits include little interference from other common anions and cations for HSO3- detection, suggesting a promising application in real sample analysis. Besides sensor development, the interaction of the natural SG with HSO3- was first demonstrated in this work to further get an insight into SG's pharmacology.

Polarity inversion sensitized G-quadruplex metal sensors with K+ tolerance.

Due to the high abundance of K+ in environments and K+-induced high stability of G-quadruplex (G4), developing a selective G4-based fluorescent sensor for other metal ions with K+ tolerance is a great challenge. Herein, we found that even in the presence of 15000-fold excess of K+, Ba2+ exhibits a highly specific binding with a human telomeric G4 (htG4) in comparison with other G4-binding metal ions such as Pb2+ and Sr2+. This specific binding event can be recognized by a natural fluorophore of hypericin with a lighting-up fluorescence response. Interestingly, inverting the polarity of the most 3' G in htG4 can sensitize the Ba2+ response with the retaining Ba2+ specificity and K+ tolerance. This polarity inversion of htG4 causes a G4 conformation change in K+ and the polarity-inverted htG4 tends to favorably dimerize in response to the Ba2+ specific binding. To our knowledge, this is the first report that polarity inversion of G4 can be applied to construct a selective metal sensor with K+ tolerance. Our findings will open a new way to conveniently regulate the G4 conformation and stability by polarity inversion towards developing high-performance sensors.

Fluorescently probing site-specific and self-catalyzed DNA depurination.

Depurination occurs via hydrolysis of the purine-deoxyribose glycosyl bond and causes nucleic acid damage. In particular, the DNA sequences that can undergo a self-catalyzed depurination (SCD) will cause a great uncertainty in duplicating, separating, purifying, and storing the DNA samples. Therefore, there is a great demand to develop a rapid detection method for SCD events. Herein, the use of a convenient fluorescence method to follow the site-specific SCD was demonstrated. We found that the resultant apurine site (AP site) from depurination can be selectively recognized by a fluorescent probe of palmatine (PAL) with a turn-on fluorescence response. The dependence of SCD on the bases of the depurination site, pH, metal ions, and time shows that our method can be used to rapidly evaluate the depurination process. Furthermore, the depurination process can be photo-switched using a photoacid as an external initiator. Our work will find wide applications in preliminarily identifying the DNA depurination.

Mitochondrial Dysfunction in Neural Injury.

Mitochondria are the double membrane organelles providing most of the energy for cells. In addition, mitochondria also play essential roles in various cellular biological processes such as calcium signaling, apoptosis, ROS generation, cell growth, and cell cycle. Mitochondrial dysfunction is observed in various neurological disorders which harbor acute and chronic neural injury such as neurodegenerative diseases and ischemia, hypoxia-induced brain injury. In this review, we describe how mitochondrial dysfunction contributes to the pathogenesis of neurological disorders which manifest chronic or acute neural injury.