Intracellular Hydrogen Sulfide Research Articles

Specific imaging of intracellular hydrogen sulfide by a positively charged NIR fluorescent probe

The poor water solubility of traditional activatable organic molecular probes usually limits their detection ability in physiological environment. In this work, a positively charged H2S probe was designed, which exhibited a significantly enhanced responsiveness to H2S in the aggregated state due to the increased positive charge density on the aggregate surface. Under physiological conditions, the probe could be activated by H2S with specificity and sensitivity to release near-infrared fluorescence signal. Moreover, endogenous H2S levels in living cells were successfully monitored by using this probe. We expect that this probe can provide a new strategy for the design of activatable probes to break the limitation of poor water solubility of conventional organic molecular probes.

Dual-channel fluorescent probe for simultaneously detecting H2S and viscosity/polarity and its application in non-alcoholic fatty liver, tumor tissue, and food spoilage

Intracellular hydrogen sulfide (H2S) and micro-environments (e.g. viscosity and polarity) are tightly correlated with various physiological/pathological processes, making them potential biomarkers of many diseases. However, the simultaneously detecting H2S, viscosity, and polarity in inflammation, non-alcoholic fatty liver (NAFL) and tumor tissues containing clinical cancer patient samples has not been achieved yet due to the lack of effective tools. Herein, we presented a mitochondria-targeting near-infrared (NIR) fluorescent probe MQA-DNP) for simultaneously monitoring H2S, viscosity/polarity through dual channels. The probe could specifically recognize H2S via far-red emission (λem = 634 nm), and exhibited high sensitivity toward micro-environmental viscosity/polarity in the NIR channel (λem > 714 nm). Facilitated with the probe, we revealed for the first time that inflammation cells and NAFL tissues are accompanied by an up-regulation of H2S, as well as an increase in viscosity level (and/or decrease in polarity degree). Surprisingly, the simultaneous visualization of H2S, viscosity/polarity has also been achieved not only at the cancer cells and tumor models, but also in clinical cancer patients. Compared to detecting a single biomarker, the simultaneous tracking multi-markers through multi-channels seems to be more reliable for visual medical diagnosis of mitochondria-related diseases. Furthermore, inspired by the significant color changes and high sensitivity of MQA-DNP reaction with H2S, MQA-DNP has been successfully used to detect H2S released during food spoilage through test strips, and has great potential for application in complex environments systems.

Potentiometric nanosensor for real-time measurement of hydrogen sulfide in single cell.

One potentiometric nanosensor for monitoring intracellular hydrogen sulfide (H2S) with fast potential response, high selectivity and excellent antifouling properties was developed. This study constructs a powerful tool to real-time track the changes of intracellular H2S in situ, promoting the future studies of physiologically relevant processes.

A New Ratiometric Fluorescent Probe Based on BODIPY for Highly Selective Detection of Hydrogen Sulfide.

Hydrogen sulfide (H2S) as small molecular signal messenger plays key functions in numerous biological processes. The imaging detection of intracellular hydrogen sulfide is of great significance. In this work, a ratiometric fluorescent probe BH based on an asymmetric BODIPY dye for detection of H2S was designed and synthesized. After the interaction with hydrogen sulfide, probe display colorimetric and ratiometric fluorescence response, with its maximum emission fluorescence wavelength red-shifted from 542 nm to 594 nm, which is attributed to the sequential nucleophilic reaction of H2S leading to enhanced molecular conjugation after ring formation of the BODIPY skeleton. A special response mechanism has been fully investigated by NMR titration and MS, so that the probe has excellent detection selectivity. Furthermore, probe BH has low cytotoxicity and fluorescence imaging experiments indicate that it can be used to monitor hydrogen sulfide in living cells.

A biocompatible ratiometric fluorescent nanoprobe for intracellular hydrogen sulfide accurate detection based on rare earth nanoparticle

Hydrogen sulfide (H2S) is an important signal molecule involved in intracellular activities. To understand the role of H2S in cellular physiological and pathological process, the development of sensitive and selective methods, especially biocompatible assays, for efficient monitoring the level of H2S is necessary. Herein, we modified novel rare earth element europium (EU) based fluorescent nanospheres with azide (–N3) based sensor to construct an ingenious ratiometric fluorescent nanoprobe EU-N3. This nanoprobe showed excellent water solubility and high biocompatibility for intracellular H2S accurate detection. Nanoprobe EU-N3 had two obvious emission peaks, the green fluorescence peak at 540 nm increased according to the increasing of H2S concentration and the red fluorescence peak at 616 nm was stable as ratiometric reference. The fluorescence intensity ratio (I540/I616) displayed good linear response (R = 0.99136) in H2S range of 0.5 ∼ 30 μM. The analytes response assay demonstrated that the nanoprobe EU-N3 possessed a better specificity for H2S, compared with other 9 anions and 3 cations. The cell viability assay indicated the nanoprobe EU-N3 had an excellent biocompatibility. The cell imaging showed that the proposed nanoprobe could be applied for detecting the intracellular H2S changes accurately in live cells. Such nanoprobe provided a safe and accurate strategy for intracellular H2S detection, which is helpful for the real-time H2S visualization in the live cell activities.

Sulfhydration of AKT triggers Tau-phosphorylation by activating glycogen synthase kinase 3β in Alzheimer’s disease

In Alzheimer's disease (AD), human Tau is phosphorylated at S199 (hTau-S199-P) by the protein kinase glycogen synthase kinase 3β (GSK3β). HTau-S199-P mislocalizes to dendritic spines, which induces synaptic dysfunction at the early stage of AD. The AKT kinase, once phosphorylated, inhibits GSK3β by phosphorylating it at S9. In AD patients, the abundance of phosphorylated AKT with active GSK3β implies that phosphorylated AKT was unable to inactivate GSK3β. However, the underlying mechanism of the inability of phosphorylated AKT to phosphorylate GSK3β remains unknown. Here, we show that total AKT and phosphorylated AKT was sulfhydrated at C77 due to the induction of intracellular hydrogen sulfide (H2S). The increase in intracellular H2S levels resulted from the induction of the proinflammatory cytokine, IL-1β, which is a pathological hallmark of AD. Sulfhydrated AKT does not interact with GSK3β, and therefore does not phosphorylate GSK3β. Thus, active GSK3β phosphorylates Tau aberrantly. In a transgenic knockin mouse (AKT-KI+/+) that lacked sulfhydrated AKT, the interaction between AKT or phospho-AKT with GSK3β was restored, and GSK3β became phosphorylated. In AKT-KI+/+ mice, expressing the pathogenic human Tau mutant (hTau-P301L), the hTau S199 phosphorylation was ameliorated as GSK3β phosphorylation was regained. This event leads to a decrease in dendritic spine loss by reducing dendritic localization of hTau-S199-P, which improves cognitive dysfunctions. Sulfhydration of AKT was detected in the postmortem brains from AD patients; thus, it represents a posttranslational modification of AKT, which primarily contributes to synaptic dysfunction in AD.

Structural effects of naphthalimide-based fluorescent sensor for hydrogen sulfide and imaging in live zebrafish.

Hydrogen sulfide (H2S) is an important biological messenger, but few biologically-compatible methods are available for its detection in aqueous solution. Herein, we report a highly water-soluble naphthalimide-based fluorescent probe (L1), which is a highly versatile building unit that absorbs and emits at long wavelengths and is selective for hydrogen sulfide over cysteine, glutathione, and other reactive sulfur, nitrogen, and oxygen species in aqueous solution. We describe turn-on fluorescent probes based on azide group reduction on the fluorogenic ‘naphthalene’ moiety to fluorescent amines and intracellular hydrogen sulfide detection without the use of an organic solvent. L1 and L2 were synthetically modified to functional groups with comparable solubility on the N-imide site, showing a marked change in turn-on fluorescent intensity in response to hydrogen sulfide in both PBS buffer and living cells. The probes were readily employed to assess intracellular hydrogen sulfide level changes by imaging endogenous hydrogen sulfide signal in RAW264.7 cells incubated with L1 and L2. Expanding the use of L1 to complex and heterogeneous biological settings, we successfully visualized hydrogen sulfide detection in the yolk, brain and spinal cord of living zebrafish embryos, thereby providing a powerful approach for live imaging for investigating chemical signaling in complex multicellular systems.

A red-emitting fluorescent probe for imaging hydrogen sulphide with a large Stokes shift

A red-emitting (above 600nm) fluorescent probe based on 3-hydroxyflavone has been developed for the detection of hydrogen sulfide with high sensitivity, good selectivity and a large Stokes shift (162nm). When the probe was treated with hydrogen sulfide, a remarkable fluorescence enhancement (112-fold) was obtained. Application of this probe was successfully demonstrated by imaging intracellular hydrogen sulfide in living RAW264.7 cells.

Dual-emissive nanohybrid for ratiometric luminescence and lifetime imaging of intracellular hydrogen sulfide.

We design a nanohybrid for the detection of hydrogen sulfide (H2S) based on mesoporous silica nanoparticles (MSNs). A phosphorescent iridium(III) complex and a specific H2S-sensitive merocyanine derivative are embedded into the nanohybrid. It exhibits a unique dual emission that is ascribed to the iridium(III) complex and the merocyanine derivative, respectively. Upon addition of sodium hydrogen sulfide (NaHS), the emission from the merocyanine derivative is quenched, while the emission from the iridium(III) complex is almost unchanged, which enables the ratiometric detection of H2S. Additionally, the nanohybrid has a long luminescence lifetime and displays a significant change in luminescence lifetime in response to H2S. Intracellular detection of H2S is performed via ratiometric imaging and photoluminescence lifetime imaging microscopy. Compared with the intensity-based method, the lifetime-based detection is independent of the probe concentration and can efficiently distinguish the signals of the probe from the autofluorescence in complex biological samples.

Organization of the Human Mitochondrial Hydrogen Sulfide Oxidation Pathway

Sulfide oxidation is expected to play an important role in cellular switching between low steady-state intracellular hydrogen sulfide levels and the higher concentrations where the physiological effects are elicited. Yet despite its significance, fundamental questions regarding how the sulfide oxidation pathway is wired remain unanswered, and competing proposals exist that diverge at the very first step catalyzed by sulfide quinone oxidoreductase (SQR). We demonstrate that, in addition to sulfite, glutathione functions as a persulfide acceptor for human SQR and that rhodanese preferentially synthesizes rather than utilizes thiosulfate. The kinetic behavior of these enzymes provides compelling evidence for the flow of sulfide via SQR to glutathione persulfide, which is then partitioned to thiosulfate or sulfite. Kinetic simulations at physiologically relevant metabolite concentrations provide additional support for the organizational logic of the sulfide oxidation pathway in which glutathione persulfide is the first intermediate formed.

Hypoxia augments intracellular hydrogen sulfide in LPS‐stimulated murine macrophages (888.3)

Hydrogen sulfide (H2S) is an important signaling molecule, yet it has been technically difficult to measure H2S production, metabolism or distribution at the cellular and sub‐cellular level in tissue. As H2S has been implicated in hypoxic signaling, we hypothesized that H2S produced during an induced inflammatory response would be augmented under hypoxic conditions. Intracellular H2S production was monitored via silicate nanoparticles, PEBBLEs (Photonic Explorers for Bioanalysis with Biologically Localized Embedding). PEBBLES were synthesized with a hydrophilic core that traps fluorescein mercuric acetate (FMA), an analyte‐sensitive dye that becomes irreversibly quenched when bound to sulfur moieties, and a hydrophobic shell permeable to small lipophilic molecules, such as H2S. FMA‐loaded PEBBLES are thus able to provide a historical account of intracellular H2S production. Murine bone marrow‐derived macrophages (BMDM) avidly ingest PEBBLES (BMDM+P) and remain viable as determined by confocal microscopy for an extended period (72 h). Cells incubated in normoxic conditions and treated with Lipopolysaccharide (LPS) showed a slight decrease in fluorescence intensity above the control over 48hrs, which could be blocked by the pre‐treatment with aminooxyacetic acid (AOA) and proparglycine (PPG). However, cells incubated in hypoxic conditions showed much larger decreases in fluorescence intensity, particularly when pre‐treated with LPS, and were less affected by the inhibitors. This supports the hypothesis that LPS stimulates H2S production in BMDM and that this is considerably augmented by hypoxia.Grant Funding Source: Supported by NSF DGE‐1313583 (ERD) and NSF IOS‐1051627 (KRO)