Biothiols-activated near-infrared frequency up-conversion luminescence probe for early evaluation of drug-induced hepatotoxicity.

A novel biothiols-sensitive near-infrared (NIR) fluorescent probe RhDN based on a rhodamine skeleton was developed for early detection of drug-induced hepatotoxicity in living mice. RhDN can be used not only as a conventional large stokes shift fluorescent (FL) probe, but also as a kind of anti-Stokes frequency upconversion luminescence (FUCL) molecular probe, which represents a long wavelength excitation (808 nm) to short wavelength emission (760 nm), and response to Cys/Hcy/GSH with high sensitivity. Compared with traditional FL methods, the FUCL method exhibited a lower detection limit of Cys, Hcy, and GSH in 75.1 nM, 101.8 nM, and 84.9 nM, respectively. We exemplify RhDN for tracking endogenously biothiols distribution in living cells and further realize real-time in vivo bioimaging of biothiols activity in mice with dual-mode luminescence system. Moreover, RhDN has been successfully applied to visualize the detection of drug-induced hepatotoxicity in living mice. Overall, this report presents a unique approach to the development of large stokes shift NIR FUCL molecular probes for in vitro and in vivo biothiols biosensing.

S-scheme heterojunction phthalocyanine/TiO2 photoelectrochemical sensor for innovative glutathione detection.

A novel photoelectrochemical sensor, employing an S-scheme heterojunction of phthalocyanine and TiO2 nanoparticles, has been developed to enable highly sensitive determination of glutathione. By integrating the favorable stability, environmental benignity, and electronic properties of the TiO2 matrix with the unique photoactivity of phthalocyanine species, the designed sensor presents a substantial linear dynamic range and a low detection limit for the quantification of glutathione. The sensitivity is attributed to efficient charge transfer and separation across the staggered heterojunction energy levels, which generates measurable photocurrent signals. Systematic variation of phthalocyanine content reveals an optimal composition that balances light harvesting capacity and electron-hole recombination rates. The incorporation of phosphotungstic acid (PTA) in sample preparation effectively minimizes interference from compounds like L-cysteine and others. Consequently, this leads to an improvement in accuracy through the reduction of impurity levels. Appreciable photocurrent enhancements are observed upon introduction of both oxidized and reduced glutathione at the optimized composite photoanode. Coupled with advantageous features of photoelectrochemical transduction such as simplicity, cost-effectiveness, and resistance to fouling, this sensor holds great promise for practical applications in complex biological media.

Biocompatibility FeOOH QD@ATP-BODIPY nanocomposite for glutathione detection and intracellular imaging.

Monitoring of glutathione has attracted considerable attention owing to its biological and clinical significance. An eco-friendly, economic, simple, biocompatible probe with excellent sensitivity and selectivity is very important. Herein, FeOOH QD@ATP-BODIPY nanocomposite was fabricated from one-step synthesized FeOOH quantum dots (FeOOH QD) and commercial boron-dipyrromethene-conjugated adenosine 5'-triphosphate (ATP-BODIPY) for glutathione (GSH) sensing in solutions and living cells. Three fascinate merits of FeOOH QD were confirmed: (a) as fluorescence quencher for ATP-BODIPY, (b) as selective recognizer of GSH and (c) with carrier effects and membrane permeability. The construction and response mechanism of the nanocomposite was based on the competitive coordination chemistry and redox reaction of FeOOH QD between GSH and phosphate group of ATP-BODIPY. Under the optimal conditions, the detection limit for GSH was as low as 68.8 nM. Excellent linear range of 0.2-400 µM was obtained. Furthermore, the chemical response of the nanocomposite exhibits high selectivity toward GSH over other electrolytes and biomolecules. It was successfully applied for GSH determination in human serum samples. The MTT assay exhibited FeOOH QD@ATP-BODIPY nanocomposite own good biocompatibility. FeOOH QD@ATP-BODIPY respond to GSH in living cells in situ was also proved via fluorescence imaging. These suggested that the FeOOH QD@ATP-BODIPY nanocomposite had potential application in biological and clinical applications.

A metal-phenolic coordination framework nanozyme exhibits dual enzyme mimicking activity and its application is effective for colorimetric detection of biomolecules.

Biomolecules play vital roles in many biological processes and diseases, making their identification crucial. Herein, we present a colorimetric sensing method for detecting biomolecules like cysteine (Cys), homocysteine (Hcy), and glutathione (GSH). This approach is based on a reaction system whereby colorless 3,3',5,5'-tetramethylbenzidine (TMB) undergoes catalytic oxidation to form blue-colored oxidized TMB (ox-TMB) in the presence of hydrogen peroxide (H2O2), utilizing the peroxidase and catalase-mimicking activities of metal-phenolic coordination frameworks (MPNs) of Cu-TA, Co-TA, and Fe-TA nanospheres. The Fe-TA nanospheres demonstrated superior activity, more active sites and enhanced electron transport. Under optimal conditions, the Fe-TA nanospheres were used for the detection of biomolecules. When present, biomolecules inhibit the reaction between TMB and H2O2, causing various colorimetric responses at low detection limits of 0.382, 0.776 and 0.750 µM for Cys, Hcy and GSH. Furthermore, it was successfully applied to real water samples with good recovery results. The developed sensor not only offers a rapid, portable, and user-friendly technique for multi-target analysis of biomolecules at low concentrations but also expands the potential uses of MPNs for other targets in the environmental field.

A detection system using sensing motif-tethered oligodeoxynucleotides for multiplex biomolecular analysis.

We developed a system to detect multiple target biomolecules through sensing motif-tethered oligodeoxynucleotides. DNA-based molecular probes gave the primary amine motif upon reaction with the target biomolecules, glutathione (GSH) and H2O2. After labelling with biotin, the product DNAs were selectively collected to be quantified by qPCR.

Lanthanide coordination polymers@CuO nanoparticles: Enhanced self-cascade nanoenzyme activity and ratiometric fluorescence assay of glutathione.

Tandem enzyme can catalyze some cascade reactions with high efficiency, and some few tandem enzyme-like mimics have been discovered recently. Further improving the catalytic efficiency of tandem nanoenzymes with facile method may undoubtedly promote and broaden their applications in various fields. In this work, cupric oxide nanoparticles (CuO NPs) with dual-functional enzyme mimics were synthesized using the rapid deposition method in advance, which simultaneously combined with lanthanide infinite coordination polymers (Ln ICPs) during the self-assemble of Tb3+, guanine-5'-triphosphate (GTP) and auxiliary ligand terephthalic acid (TA). Excitingly, the obtained Tb-GTP/TA@CuO ICPs, not only displayed obviously enhanced tandem catalytic activity compared with pure CuO NPs, but also provided a versatile ratiometric platform for ultrahigh selective and sensitive detection of glutathione (GSH) under single-wavelength excitation. A good linear relationship between the ratio signal and the GSH concentration was spanning from 0.001 to 20 µM with an impressive detection limit of 0.50 nM. This study opens a new and universal avenue for preparing integrated multifunctional probes by coupling of nanoenzyme catalytic activity with superior luminescent Ln ICPs through facile method.

Glutathione­iron hybrid nanozyme-based colorimetric sensor for specific and stable detection of thiram pesticide on fruit juices.

Given the potential dangers of thiram to food safety, constructing a facile sensor is significantly critical. Herein, we presented a colorimetric sensor based on glutathione­iron hybrid (GSH-Fe) nanozyme for specific and stable detection of thiram. The GSH-Fe nanozyme exhibits good peroxidase-mimicking activity with comparable Michaelis constant (Km = 0.551 mM) to the natural enzyme. Thiram pesticides can specifically limit the catalytic activity of GSH-Fe nanozyme via surface passivation, causing the change of colorimetric signal. It is worth mentioning that the platform was used to prepare a portable hydrogel kit for rapid qualitative monitoring of thiram. Coupling with an image-processing algorithm, the colorimetric image of the hydrogel reactor is converted into the data information for accurate quantification of thiram with a detection limit of 0.3 µg mL-1. The sensing system has good selectivity and high stability, with recovery rates in fruit juice samples ranging from 92.4% to 106.9%.

A two-photon fluorescent probe for highly selective detection of Cys over GSH and Hcy based on the Michael addition and transcyclization mechanism and its application in bioimaging and protein straining in SDS-PAGE.

BACKGROUND: Cysteine (Cys), glutathione (GSH), and homocysteine (Hcy), as three major biothiols are involved in a variety of physiological processes and play a crucial role in plant growth. Abnormal levels of Cys can cause plants to fail to grow properly. To date, although a very large number of fluorescent probes have been reported for the detection of biothiols, very few of them can be used for the selective discrimination of Cys from GSH and Hcy due to their structural similarity, and only a few of them can be used for plant imaging. RESULTS: Here, three fluorescent probes (o-/m-/p-TMA) based on TMN fluorophore and the ortho-/meta-/para-substituted maleimide recognition groups were constructed to investigate the selective response effect of Cys. Compared to the o-/m-TMA, p-TMA can selectively detect Cys over GSH and Hcy with a rapid response time (10 min) and a low detection limit (0.26 µM). The theoretical calculation confirmed that the intermediate p-TMA-Cys-int has shorter interatomic reaction distances (3.827 Å) compared to o-/m-TMA-Cys (5.533/5.287 Å), making it more suitable for further transcyclization reactions. Additionally, p-TMA has been employed for selective tracking of exogenous and endogenous Cys in Arabidopsis thaliana using both single-/two-photon fluorescence imaging. Furthermore, single cell walls produced obvious two-photon fluorescence signals, indicating that p-TMA can be used for high-concentration Cys analysis in single cells. Surprisingly, p-TMA can be used as a fluorescent dye for protein staining in SDS-PAGE with higher sensitivity (7.49 µg/mL) than classical Coomassie brilliant blue (14.11 µg/mL). SIGNIFICANCE: The outstanding properties of p-TMA make it a promising multifunctional molecular tool for the highly selective detection of Cys over GSH and Hcy in various complex environments, including water solutions, zebrafish, and plants. Additionally, it has the potential to be developed as a fluorescent dye for a simple and fast SDS-PAGE fluorescence staining method.

Cu-BTC Derived Mesoporous CuS Nanomaterial as Nanozyme for Colorimetric Detection of Glutathione.

In this paper, Cu-BTC derived mesoporous CuS nanomaterial (m-CuS) was synthesized via a two-step process involving carbonization and sulfidation of Cu-BTC for colorimetric glutathione detection. The Cu-BTC was constructed by 1,3,5-benzenetri-carboxylic acid (H3BTC) and Cu2+ ions. The obtained m-CuS showed a large specific surface area (55.751 m2/g), pore volume (0.153 cm3/g), and pore diameter (15.380 nm). In addition, the synthesized m-CuS exhibited high peroxidase-like activity and could catalyze oxidation of the colorless substrate 3,3',5,5'-tetramethylbenzidine to a blue product. Peroxidase-like activity mechanism studies using terephthalic acid as a fluorescent probe proved that m-CuS assists H2O2 decomposition to reactive oxygen species, which are responsible for TMB oxidation. However, the catalytic activity of m-CuS for the oxidation of TMB by H2O2 could be potently inhibited in the presence of glutathione. Based on this phenomenon, the colorimetric detection of glutathione was demonstrated with good selectivity and high sensitivity. The linear range was 1-20 µM and 20-300 µM with a detection limit of 0.1 µM. The m-CuS showing good stability and robust peroxidase catalytic activity was applied for the detection of glutathione in human urine samples.

Anthelmintic efficacy of an organic fraction from Guazuma ulmifolia leaves and evaluation of reactive oxidative stress on Haemonchus contortus.

This study describes the anthelmintic efficacy of an organic fraction (EtOAc-F) from Guazuma ulmifolia leaves and the evaluation of its reactive oxidative stress on Haemonchus contortus. The first step was to assess the anthelmintic effect of EtOAc-F at 0.0, 3.5, 7.0 and 14 mg kg of body weight (BW) in gerbil's (Meriones unguiculatus) artificially infected with H. contortus infective larvae (L3). The second step was to evaluate the preliminary toxicity after oral administration of the EtOAc-F in gerbils. Finally, the third step was to determine the relative expression of biomarkers such as glutathione (GPx), catalase (CAT), and superoxide dismutase (SOD) against H. contortus L3 post-exposition to EtOAc-F. Additionally, the less-polar compounds of EtOAc-F were identified by gas mass spectrophotometry (GC-MS). The highest anthelmintic efficacy (97.34%) of the organic fraction was found in the gerbils treated with the 14 mg/kg of BW. Histopathological analysis did not reveal changes in tissues. The relative expression reflects overexpression of GPx (p<0.05, fold change: 14.35) and over expression of SOD (p≤0.05, fold change: 0.18) in H. contortus L3 exposed to 97.44 mg/mL of EtOAc-F compared with negative control. The GC-MS analysis revealed the presence of 4-hydroxybenzaldehyde (1), leucoanthocyanidin derivative (2), coniferyl alcohol (3), ferulic acid methyl ester acetate (4), 2,3,4-trimethoxycinnamic acid (5) and epiyangambin (6) as major compounds. According to these results, the EtOAc-F from G. ulmifolia leaves exhibit anthelmintic effect and increased the stress biomarkers on H. contortus.

Nitrodopamine modified MnO2 NS-MoS2QDs hybrid nanocomposite for the extracellular and intracellular detection of glutathione.

We have developed a highly sensitive and reliable fluorescence resonance energy transfer (FRET) probe using nitro-dopamine (ND) and dopamine (DA) coated MnO2 nanosheet (ND@MnO2 NS and DA@MnO2 NS) as an energy acceptor and MoS2 quantum dots (QDs) as an energy donor. By employing surface-modified MnO2 NS, we can effectively reduce the fluorescence intensity of MoS2 QDs through FRET. It can reduce MnO2 NS to Mn2+ and facilitate the fluorescence recovery of the MoS2 QDs. This ND@MnO2 NS@MoS2 QD-based nanoprobe demonstrates excellent sensitivity to GSH, achieving an LOD of 22.7 nM in an aqueous medium while exhibiting minimal cytotoxicity and good biocompatibility. Moreover, our sensing platform shows high selectivity to GSH towards various common biomolecules and electrolytes. Confocal fluorescence imaging revealed that the nanoprobe can image GSH in A549 cells. Interestingly, the ND@MnO2 NS nanoprobe demonstrates no cytotoxicity in living cancer cells, even at concentrations up to 100 µg mL-1. Moreover, the easy fabrication and eco-friendliness of ND@MnO2 NS make it a rapid and simple method for detecting GSH. We envision the developed nanoprobe as an incredible platform for real-time monitoring of GSH levels in both extracellular and intracellular mediums, proving valuable for biomedical research and clinical diagnostics.

Foldable paper-based photoelectrochemical biosensor based on etching reaction of CoOOH nanosheets-coated laser-induced PbS/CdS/graphene for sensitive detection of ampicillin.

Timely and rapid detection of antibiotic residues in the environment is conducive to safeguarding human health and promoting an ecological virtuous cycle. A foldable paper-based photoelectrochemical (PEC) sensor was successfully developed for the detection of ampicillin (AMP) based on glutathione/zirconium dioxide hollow nanorods/aptamer (GSH@ZrO2 HS@apt) modified cellulose paper as a reactive zone with laser direct-writing lead sulfide/cadmium sulfide/graphene (PbS/CdS/LIG) as photoelectrode and cobalt hydroxide (CoOOH) as a photoresist material. Initially, AMP was introduced into the paper-based reaction zone as a biogate aptamer, which specifically recognized the target and then left the ZrO2 HS surface, releasing glutathione (GSH) encapsulated inside. Subsequently, the introduction of GSH into the reaction region and etching of CoOOH nanosheets to expose the PbS/CdS/LIG photosensitive material increased photocurrent. Under optimal conditions, the paper-based PEC biosensor showed a linear response to AMP in the range of 5.0 - 2 × 104 pM with a detection limit of 1.36 pM (S/N = 3). In addition, the constructed PEC sensing platform has excellent selectivity, high stability and favorable reproducibility, and can be used to assess AMP residue levels in various real water samples (milk, tap water, river water), indicating its promising application in environmental antibiotic detection.

Simultaneous SERS Sensing of Cysteine and Homocysteine in Blood Based on the CBT-Cys Click Reaction: Toward Precisive Diagnosis of Schizophrenia.

At present, there is a lack of sufficiently specific laboratory diagnostic indicators for schizophrenia. Serum homocysteine (Hcy) levels have been found to be related to schizophrenia. Cysteine (Cys) is a demethylation product in the metabolism of Hcy, and they always coexist with highly similar structures in vivo. There are few reports on the use of Cys as a diagnostic biomarker for schizophrenia in collaboration with Hcy, mainly because the rapid, economical, accurate, and high-throughput simultaneous detection of Cys and Hcy in serum is highly challenging. Herein, a click reaction-based surface-enhanced Raman spectroscopy (SERS) sensor was developed for simultaneous and selective detection of Cys and Hcy. Through the efficient and specific CBT-Cys click reaction between the probe containing cyan benzothiazole and Cys/Hcy, the tiny methylene difference between the molecular structures of Cys and Hcy was converted into the difference between the ring skeletons of the corresponding products that could be identified by plasmonic silver nanoparticle enhanced molecular fingerprint spectroscopy to realize discriminative detection. Furthermore, the SERS sensor was successfully applied to the detection in related patient serum samples, and it was found that the combined analysis of Cys and Hcy can improve the diagnostic accuracy of schizophrenia compared to a single indicator.

Reversible Dual Fluorescence-Lifetime Imaging of Mitochondrial GSH and Microviscosity: Real-Time Evaluation of Ferroptosis Status.

Ferroptosis, as a new form of regulated cell death, is implicated in various physiological and pathological processes. Developing a single probe for an independent analysis of multiple analytes related to ferroptosis can provide more accurate information and simplify the detection procedures, but it faces great challenges. In this work, we develop a fluorescent probe for the simultaneous detection of GSH through ratiometric fluorescence response and microviscosity via a fluorescence lifetime model. Based on the reversible Michael addition reaction between GSH and unsaturated C═C bond, the probe responds reversibly to GSH with a ratiometric fluorescence variation and a fast response time (t1/2 = 4.7 s). At the same time, the probe is sensitive to environmental viscosity by changing its fluorescence lifetimes. The probe was applied to monitor the drug-induced ferroptosis process through both the classical Xc-/GSH/GPX4- and DHODH-mediated defense mechanisms. We hope that the probe will provide a useful molecular tool for the real-time live-cell imaging of GSH dynamics, which is benefit to unveiling related physiological and pathological processes.

Dual-mode optical biosensor based on multi-functional DNA structures for detecting bioactive small molecules.

Semiconducting polymer dots and hemin-functionalized DNA nanoflowers with excellent peroxidase-like activity and high fluorescent brightness are prepared for fluorescent/colorimetric dual-mode sensing of dopamine and glutathione as low as nM and µM, respectively. This biosensor is readily applied to the analysis of complicated biological samples with high selectivity and accuracy, which opens up promising prospects in clinical applications.

Protein intake affects erythrocyte glutathione synthesis in healthy adults aged ≥60 years in a repeated-measures trial.

BACKGROUND: Protein recommendations for older adults are based on nitrogen balance data from young adults. Physiological studies using the indicator amino acid oxidation method suggest they need 30% to 50% more protein than current recommendations. We herein present glutathione (GSH) as a physiological estimate of protein adequacy in older adults. OBJECTIVES: The objective was to measure GSH kinetics in response to varying protein intakes in a repeated-measures design in healthy adults aged ≥60 y using the precursor-product method. METHODS: Sixteen healthy older adults (n = 8 male and n = 8 female; body mass index ≤30 kg/m2) were studied. Each received 4 of 6 protein intakes in random order (0.66, 0.8, 0.9, 1.1, 1.3 and 1.5 g⋅kg-1⋅d-1). At each intake level, participants underwent isotope infusion studies of 7 h duration following a 3-d adaptation to the test level of protein. On the fourth day, GSH fractional (FSR) and absolute synthesis (ASR) rates were quantified by measuring the incorporation of U-[13C2-15N]glycine into GSH at isotopic steady state. A mixed-effect change-point regression model was used to determine a breakpoint in FSR and ASR. Secondary outcomes included plasma concentrations of oxidative stress markers, homocysteine, 5-L-oxoproline (5-OP), and urinary sulfate. The effect of secondary outcomes on GSH kinetics was analyzed using a joint linear mixed-effect model and Tukey's post hoc test. RESULTS: A protein intake of 1.08 g⋅kg-1⋅d-1 (95% confidence interval [CI]: 0.83, 1.32; Rm2 = 0.207; Rc2 = 0.671; P < 0.001) maximized GSH FSR. There was no effect of protein intake on concentrations of erythrocyte GSH, plasma homocysteine, oxidative stress markers, or 5-OP (P > 0.05). Protein intake had a positive effect on urinary sulfate excretion (P < 0.0001). CONCLUSION: A protein intake of 1.08 g⋅kg-1⋅d-1 from a high-quality protein maximized GSH synthesis in adults ≥60 y. This lends support to data suggesting a requirement higher than the current recommendation. This study was registered at clinicaltrials.gov as NCT02971046.

Electrochemical detection of glutathione based on accelerated CRISPR/Cas12a trans-cleavage with MnO2 nanosheets.

The CRISPR/Cas12a system is accelerated by glutathione-mediated reduction of MnO2 nanosheets. By monitoring the trans-cleavage of the DNA probe, an electrochemical method for glutathione assay is fabricated, with the detection limit of 3.5 pM. It provides a promising tool for plasma analysis with satisfactory performance, indicating the broad application prospects of this glutathione assay.

Synergism Variation between intracellular Glutathione, phycocyanin and SOD in microalgae by carbon quantum dot fluorescence.

Based on the use of CQDs as fluorescent probe and covalent coupling method to detect biological molecules with amino groups, to deeply analysis and detect the metabolism of Microcystis aeruginosa. The metabolic changes of carboxyl biomolecules in Microcystis aeruginosa were analyzed by covalent coupling method, including GSH, phycocyanin and SOD enzyme. The changes of GSH content and its correlation between phycocyanin, SOD were analyzed. The content of phycocyanin and SOD reached the maximum on the 65th day, and GSH was more sensitive to the growth and metabolism of microalgae. GSH plays an important role in reducing the external oxidative damage of microalgae cells. The synthesis of glutathione (GSH), GSH/GSSG mutual transformation, the production of phytochelating peptide (PC), the ASA-GSH cycle, and other physiological processes are interconnected. These interactions are crucial for preserving the antioxidant properties of microalgae and regulating redox-sensitive signal transduction.

Optimization of extraction and development of an LC-HRMS method to quantify glutathione and glutathione disulfide in white wine lees and yeast derivatives.

The antioxidant capacity of wine depends on its quality and aging potential. Aging on lees can improve this capacity thanks to the release of glutathione (GSH), as can the addition of yeast derivatives (YD). Therefore, the GSH potential of wine lees (WL) and YD requires investigation. We propose an optimized method to extract and quantify GSH from WL and YD. First, a method was developed to detect and quantify GSH and glutathione disulfide (GSSG) using LC-HRMS. Second, Box-Behnken response surface methodologies (RSM) were applied to both matrices. Results showed that the main parameter affecting GSH extraction efficiency was ethanol concentration. Quantitation of various samples revealed GSH concentrations of up to 900 µg/g for WL and 40 mg/g for YD. To our knowledge, the absolute quantitation of GSH/GSSG in these matrices has not been reported until now.

[Metabolic Stress of Red Blood Cells Induces Hemoglobin Glutathionylation].

Metabolic stress caused by a lack of glucose significantly affects the state of red blood cells, where glycolysis is the main pathway for the production of ATP. Hypoglycemia can be both physiological (occurring during fasting and heavy physical exertion) and pathological (accompanying a number of diseases, such as diabetes mellitus). In this study, we have characterized the state of isolated erythrocytes under metabolic stress caused by the absence of glucose. It was established that 24 h of incubation of the erythrocytes in a glucose-free medium to simulate blood plasma led to a two-fold decrease in the ATP level into them. The cell size, as well as intracellular sodium concentration increased. These findings could be the result of a disruption in ion transporter functioning because of a decrease in the ATP level. The calcium level remained unchanged. With a lack of glucose in the medium of isolated erythrocytes, there was no increase in ROS and a significant change in the level of nitric oxide, while the level of the main low-molecular weight thiol of cells, glutathione (GSH) decreased by almost 2 times. It was found that the metabolic stress of isolated red blood cells induced hemoglobin glutathionylation despite the absence of ROS growth. The cause was the lack of ATP, which led to a decrease in the level of GSH because of the inhibition of its synthesis and, probably, due to a decrease in the NADPH level required for glutathione (GSSG) reduction and protein deglutathionylation. Thus, erythrocyte metabolic stress induced hemoglobin glutathionylation, which is not associated with an increase in ROS. This may have an important physiological significance, since glutathionylation of hemoglobin changes its affinity for oxygen.