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Dicarboximide fungicides, including captafol, captan, cyclohexylthiophthalimide, folpet, and procymidone, represent a distinct category of fungicides. 17β-Hydroxysteroid dehydrogenase 1 (17β-HSD1) catalyzes the conversion of estrone to estradiol in mammals. Yet, the impact of these fungicides on 17β-HSD1 activity remains unknown. In this study, we investigated their inhibition using human placental cytosols, rat and pig ovarian cytosols. Our observations revealed that dicarboximide fungicides significantly inhibited human 17β-HSD1 activity. Among them, captan showed the strongest potency, with its IC50 of 1.28 μM, whereas procymidone had an IC50 of 100.71 μM. However, both rat and pig 17β-HSD1 enzymes were less sensitive to the inhibition of these fungicides compared to the human enzyme, with captan displaying an IC50 of 5.65 μM for the rat enzyme and 7.36 μM for the pig enzyme. Correlation analysis indicated a positive correlation between IC50 values and LogP. Docking analysis revealed that these fungicides bound to cofactor or between the steroid and cofactor binding sites. The dithiothreitol treatment demonstrated that the formation of irreversible bonds between dicarboximide fungicides and the cysteine residues played a key role in the inhibition of 17β-HSD1 activity. In conclusion, dicarboximide fungicides inhibit 17β-HSD1 depending on lipophilicity, species, and cysteine residue interactions.

Background: The binding of endothelin-1 (ET-1) to endothelin type A receptor (ETAR) performs a critical action in pulmonary arterial smooth muscle cell (PASMC) proliferation leading to pulmonary vascular structural remodeling. More evidence showed that cystathionine γ-lyase (CSE)-catalyzed endogenous hydrogen sulfide (H2S) was involved in the pathogenesis of cardiovascular diseases. In this study, we aimed to explore the effect of endogenous H2S/CSE pathway on the ET-1/ETAR binding and its underlying mechanisms in the cellular and animal models of PASMC proliferation. Methods and results: Both live cell imaging and ligand-receptor assays revealed that H2S donor, NaHS, inhibited the binding of ET-1/ETAR in human PASMCs (HPASMCs) and HEK-293A cells, along with an inhibition of ET-1-activated HPASMC proliferation. While, an upregulated Ki-67 expression by the pulmonary arteries, a marked pulmonary artery structural remodeling, and an increased pulmonary artery pressure were observed in CSE knockout (CSE-KO) mice with a deficient H2S/CSE pathway compared with those in the wild type (WT) mice. Meanwhile, NaHS rescued the enhanced binding of ET-1 with ETAR and cell proliferation in the CSE-knockdowned HPASMCs. Moreover, the ETAR antagonist BQ123 blocked the enhanced proliferation of CSE-knockdowned HPASMCs. Mechanistically, ETAR persulfidation was reduced in the lung tissues of CSE-KO mice compared to that in WT mice, which could be reversed by NaHS treatment. Similarly, NaHS persulfidated ETAR in HPASMCs and HEK-293A cells. Whereas a thiol reductant dithiothreitol (DTT) reversed the H2S-induced ETAR persulfidation and further blocked the H2S-inhibited binding of ET-1/ETAR and HPASMC proliferation. Furthermore, the mutation of ETAR at cysteine (Cys) 69 abolished the persulfidation of ETAR by H2S, and subsequently blocked the H2S-suppressed ET-1/ETAR binding and HPASMC proliferation. Conclusion: Endogenous H2S persulfidated ETAR at Cys69 to inhibit the binding of ET-1 to ETAR, subsequently suppressed PASMC proliferation, and antagonized pulmonary vascular structural remodeling.

The challenge of understanding the interaction between trace elements and microbial life is critical for assessing environmental and ecological impacts. Nevertheless, cysteine (Cys), a low molecular weight thiol substance prevalent in the ecosystem, is able to influence the fate of certain trace elements, which increases the complexity of the interaction between trace elements and microorganisms. Therefore, we chose Cys, selenite and the model methanogenic archaeon Methanosarcina acetivorans C2A as research targets, and comprehensively explored the intricate role of Cys in modulating the biological effects of selenite on M. acetivorans C2A in terms of population growth, methane production and oxidative stress. Our results demonstrate that Cys significantly exacerbates the inhibitory effects of selenite on growth and methane production in M. acetivorans C2A. This increased toxicity is linked to heightened membrane permeability and oxidative stress, with a marked upregulation in reactive oxygen species and changes in NADPH levels. Transcriptomic analysis reveals alterations in genes associated with transmembrane transport and methanogenesis. Intriguingly, we also observed a potential interaction between selenite and phosphate transmembrane transporters, suggesting a novel pathway for selenite entry into cells. These findings highlight the complex interplay between trace elements and microbial processes, with significant implications for understanding environmental risks and developing remediation strategies.

Impacts of co-cold extrusion (≤50 °C) of whey protein isolate (WPI) and cysteine (Cys, 0, 20, 40, 60, 80 and 100 mmol/L) on its physicochemical, in vitro digestion and rheological properties were investigated. As Cys concentration increased, the emulsifying properties and in vitro digestibility of co-extruded WPI-Cys products showed an increasing trend. Specifically, when Cys reached 100 mmol/L, surface hydrophobicity, emulsification activity index (EAI), emulsification stability index (ESI) and in vitro stomach digestibility of the co-extruded WPI-Cys products increased by 205.07%, 77.51%, 193.95% and 71.81% compared with WPI, respectively. Principal component analysis (PCA) results further indicated that co-extruded WPI-Cys at a concentration of 100 mmol/L had the best functional properties. In addition, co-extruded WPI-Cys exhibited the strongest Péclet number (Pe) value and apparent viscosity at a Cys concentration of 100 mmol/L among all samples. Therefore, co-extrusion would be an effective method for modifying WPI, providing whey protein-based ingredients with excellent functional properties for food processing.

Background: In animals, dietary sulfur amino acid restriction (SAAR) improves metabolic health, possibly mediated by altering sulfur amino acid metabolism and enhanced anti-obesogenic processes in adipose tissue. Aim: To assess the effects of SAAR over time on the plasma and urine SAA-related metabolites (sulfurome) in humans with overweight and obesity, and explore whether such changes were associated with body weight, body fat and adipose tissue gene expression. Methods: Fifty-nine subjects were randomly allocated to SAAR (∼2 g SAA, n = 31) or a control diet (∼5.6 g SAA, n = 28) consisting of plant-based whole-foods and supplemented with capsules to titrate contents of SAA. Sulfurome metabolites in plasma and urine at baseline, 4 and 8 weeks were measured using HPLC and LC-MS/MS. mRNA-sequencing of subcutaneous white adipose tissue (scWAT) was performed to assess changes in gene expression. Data were analyzed with mixed model regression. Principal component analyses (PCA) were performed on the sulfurome data to identify potential signatures characterizing the response to SAAR. Results: SAAR led to marked decrease of the main urinary excretion product sulfate (p

Summary: The inherent ability of melanoma cells to alter the differentiation-associated transcriptional repertoire to evade treatment and facilitate metastatic spread is well accepted and has been termed phenotypic switching. However, how these facets of cellular behavior are controlled remains largely elusive. Here, we show that cysteine availability, whether from lysosomes (CTNS-dependent) or exogenously derived (SLC7A11-dependent or as N-acetylcysteine), controls melanoma differentiation-associated pathways by acting on the melanocyte master regulator MITF. Functional data indicate that low cysteine availability reduces MITF levels and impairs lysosome functions, which affects tumor ferroptosis sensitivity but improves metastatic spread in vivo. Mechanistically, cysteine-restrictive conditions reduce acetyl-CoA levels to decrease p300-mediated H3K27 acetylation at the melanocyte-restricted MITF promoter, thus forming a cysteine feedforward regulation that controls MITF levels and downstream lysosome functions. These findings collectively suggest that cysteine homeostasis governs melanoma differentiation by maintaining MITF levels and lysosome functions, which protect against ferroptosis and limit metastatic spread.

Maillard reaction products (MRPs) derived from rice protein isolate hydrolysate and D-xylose, with or without L-cysteine, were developed as a potential meat flavoring. The combined impact of temperature (80–140 °C) and cysteine on fundamental physicochemical characteristics, antioxidant activity, and flavor of MRPs were investigated through assessments of pH, color, UV–visible spectra, fluorescence spectra, free amino acids, volatile compounds, E-nose, E-tongue, and sensory evaluation. Results suggested that increasing temperature would reduce pH, deepen color, promote volatile compounds formation, and reduce the overall umami and bitterness. Cysteine addition contributed to the color inhibition, enhancement of DPPH radical-scavenging activity and reducing power, improvement in mouthfulness and continuity, reduction of bitterness, and the formation of sulfur compounds responsible for meaty flavor. Overall, MRPs prepared at 120 °C with cysteine addition could be utilized as a potential meat flavoring with the highest antioxidant activity and relatively high mouthfulness, continuity, umami, meaty aroma, and relatively low bitterness.

ABSTRACT: Despite the acknowledged significance of nutrition in bone development, effects of methionine (Met) and cysteine (Cys) on bone quality remain under-researched, particularly during Eimeria challenge. We investigated the effects of different supplemental Met to Cys ratios (MCR) on bone quality of broilers under Eimeria challenge. A total of 720 fourteen-day old Cobb500 broilers were allocated into a 5 × 2 factorial arrangement. Five diets with Met and Cys supplemented at MCR of 100:0, 75:25, 50:50, 25:75, and 0:100 were fed to the birds with or without Eimeria challenge. Body composition was measured by dual energy x-ray absorptiometry, and the femur bone characteristics were assessed by microtomography. Data were analyzed by two-way ANOVA and orthogonal polynomial contrast. The results reaffirmed the detrimental effects of Eimeria challenge on bone quality. On 9 d post inoculation (DPI), significant interaction effects were found for whole body bone mineral content (BMC), lean tissue weight, and body weight (P < 0.05); in the nonchallenged group (NCG), these parameters linearly decreased as MCR decreased (P < 0.05). In the challenged group (CG), body weight and lean tissue weight were unaffected by MCR, and BMC linearly increased as MCR decreased (P < 0.05). For the cortical bone of femoral metaphysis on 6 DPI, bone mineral density (BMD) linearly increased as MCR decreased (P < 0.05). Bone volume to tissue volume ratio (BV/TV) in the CG linearly increased as MCR decreased (P < 0.05). On 9 DPI, BMC and TV linearly increased as MCR decreased (P < 0.05) in the NCG. BMD and BV/TV changed quadratically as MCR decreased (P < 0.05). For the trabecular bone of femoral metaphysis on 9 DPI, BV/TV, and trabecular number linearly increased as MCR decreased (P < 0.05) in the NCG. For the femoral diaphysis, BV, TV, BMC on 6 DPI, and BMD on 9 DPI linearly increased as MCR decreased (P < 0.05). In conclusion, this study showed that both Eimeria challenge and varying supplemental MCR could influence bone quality of broilers.

Ferroptosis is inhibited by glutathione peroxidase 4 (GPX4), an antioxidant enzyme that uses reduced glutathione (GSH) as a cofactor to detoxify lipid hydroperoxides. As a selenoprotein, the core function of GPX4 is the thiol-dependent redox reaction. In addition to GSH, other small molecules such as cysteine and homocysteine also contain thiols; yet, whether GPX4 can exploit cysteine and homocysteine to directly detoxify lipid hydroperoxides and inhibit ferroptosis has not been addressed. In this study, we found that cysteine and homocysteine inhibit ferroptosis in a GPX4-dependent manner. However, cysteine inhibits ferroptosis independent of GSH synthesis, and homocysteine inhibits ferroptosis through non-cysteine and non-GSH pathway. Furthermore, we used molecular docking and GPX4 activity analysis to study the binding patterns and affinity between GPX4 and GSH, cysteine, and homocysteine. We found that besides GSH, cysteine and homocysteine are also able to serve as substrates for GPX4 though the affinities of GPX4 with cysteine and homocysteine are lower than that with GSH. Importantly, GPX family and the GSH synthetase pathway might be asynchronously evolved. When GSH synthetase is absent, for example in Flexibacter, the fGPX exhibits higher affinity with cysteine and homocysteine than GSH. Taken together, the present study provided the understanding of the role of thiol-dependent redox systems in protecting cells from ferroptosis and propose that GSH might be a substitute for cysteine or homocysteine to be used as a cofactor for GPX4 during the evolution of aerobic metabolism.

ABSTRACT: A study was conducted to investigate effects of different methionine (Met) to cysteine (Cys) supplementation ratios (MCR) on growth performance, oxidative status, intestinal health, immune responses, and methionine metabolism in broilers under Eimeria challenge. A total of 720 male Cobb500 broilers (14-day-old) were allocated in a 2 × 5 factorial arrangement (5 diets, with or without challenge) with 6 replicates per treatment. The total sulfur amino acid concentrations were consistent across treatments meeting the breeder's recommendation, only MCR varied. The diets were labeled as MET100; MET75; MET50; MET25; and MET0, representing MCR of 100:0; 75:25; 50:50; 25:75; and 0:100, respectively. Data were analyzed by 2-way ANOVA and orthogonal polynomial contrast. Growth performance declined linearly or quadratically as MCR decreased (P < 0.01). On 6-day postinoculation (DPI), interaction effects (P < 0.01) were found; BW and body weight gain were lower in MET0 compared to the other treatments in the nonchallenged groups, whereas not in the challenged groups. On 6 and 9 DPI, serum total antioxidant capacity linearly decreased as MCR decreased (P < 0.05). Hepatic activities of glutathione peroxidase on 6 DPI and superoxide dismutase on 9 DPI changed quadratically as MCR decreased (P < 0.05). The digestibility of Met linearly decreased whereas the digestibility of Cys linearly increased as MCR decreased. The ileal crypt depth linearly decreased as MCR decreased (P < 0.01) on 6 DPI. The expression of transforming growth factor beta on 6 and 9 DPI, tumor necrotic factor alpha and interleukin 10 on 9 DPI changed quadratically as MCR decreased (P < 0.05). Eimeria challenge increased expression of Met adenosyltransferase and cystathionine gamma-lyase, whereas decreasing the expression of other Met metabolism genes (P < 0.01) on 6 DPI. Expression of Met metabolism genes linearly increased as MCR decreased (P < 0.05). In conclusion, different Met to Cys supplementation ratios exerted linearly or quadratically effects on the growth performance, oxidative status, intestinal health, and metabolism of Met in broiler chickens under Eimeria infection.

In the present study, we demonstrated that the presence of cysteine could remarkably enhance the degradation of atrazine by Fe3O4/persulfate system. The results of electron paramagnetic resonance (EPR) spectra confirmed the combination of cysteine and Fe3O4 exhibited much higher activity on activation of persulfate to generate more SO4•- and •OH than Fe3O4 alone. At pH of 3.0, SO4•- and •OH contributed to about 58.2 % and 41.8 % of atrazine removal respectively, while •OH gradually dominated the oxidation of atrazine from neutral condition to alkaline condition. The co-existing Cl− and HCO3− could quench SO4•-, resulting in the inhibition of atrazine degradation. The presence of low natural organic matters (NOM) concentration (0–2 mg L−1) could enhance the atrazine removal, and high concentration (>5 mg L−1) of NOM restrained the atrazine degradation. During the Cysteine/Fe3O4/Persulfate process, cysteine served as a complexing reagent and reductant. Through acidolysis and complexation, Fe3O4 could release dissolved and surface bound Fe2+, both of which contributed to the activation of persulfate together. Meanwhile, cysteine was not rapidly consumed due to a regeneration process, which was beneficial for maintaining Fe2+/Fe3+ cycle and constantly accelerating the activation of persulfate for atrazine degradation. The reused Fe3O4 and cysteine in the Cysteine/Fe3O4/Persulfate process exhibited high stability for the atrazine degradation after three cycles. The degradation pathway of atrazine included alkylic-oxidation, dealkylation, dechlorination-hydroxylation processes. The present study indicates the novel Cysteine/Fe3O4/Persulfate process might be a high potential for treatment of organic polluted water.

Cysteine (Cys) is essential for numerous physiological processes and play a vital role in disease diagnostics, underscoring the necessity for effective and sensitive detection techniques for Cys. In this research, we introduce an ultrasensitive colorimetric approach based on oxidase-like Mn3O4 nanozymes. The nanozymes were synthesized through a straightforward wet chemical method and displayed a flower-like globular morphology, showing remarkable oxidase-like catalytic performance with a low Km value of 0.1896 mM and a high Vmax of 2.8 × 10⁻⁷ M s⁻¹. By leveraging the outstanding catalytic activities of Mn3O4 nanozymes and optimizing the TMB concentration, we developed a novel and sensitive colorimetric sensor for Cys detection. Experimental results indicated a detection range for Cys between 0.5 and 45 μM, with a lower limit of detection (LOD) of 0.02636 μM. This established method for detecting Cys reveals significant promise for the advancement of effective sensing systems.

Oxidative stress is biochemically complex. Like primary colours, specific reactive oxygen species (ROS) and antioxidant inputs can be mixed to create unique “shades” of oxidative stress. Even a minimal redox module comprised of just 12 (ROS & antioxidant) inputs and 3 outputs (oxidative damage, cysteine-dependent redox-regulation, or both) yields over half a million “shades” of oxidative stress. The present paper proposes the novel hypothesis that: state-specific shades of oxidative stress, such as a discrete disease, are associated with distinct tell-tale cysteine oxidation patterns. The patterns are encoded by many parameters, from the identity of the oxidised proteins, the cysteine oxidation type, and magnitude. The hypothesis is conceptually grounded in distinct ROS and antioxidant inputs coalescing to produce unique cysteine oxidation outputs. And considers the potential biological significance of the holistic cysteine oxidation outputs. The literature supports the existence of state-specific cysteine oxidation patterns. Measuring and manipulating these patterns offer promising avenues for advancing oxidative stress research. The pattern inspired hypothesis provides a framework for understanding the complex biochemical nature of state-specific oxidative stress.

Cysteine (Cys), one of the important participants in protecting cells from oxidative stress, is closely associated with the occurrence and development of various diseases. Moreover, cell viscosity as a pivotal microenvironmental parameter has recently attracted increasing attention due to its dominant role in governing intracellular signal transduction and diffusion of reactive metabolites. Thus, simultaneous detection of Cys and viscosity is imperative for investigating their pathophysiological functions and cross-link. Herein we present a mitochondria-targetable dual-channel fluorescence probe ABDSP by grafting the acrylate modified pyridinium unit to dimethylaminobenzene. Whilst the probe is a seemingly simple, it could simultaneously discriminate Cys and viscosity in a fashion of distinguishable signals. Furthermore, the probe was successfully employed for visualizing mitochondrial Cys and viscosity, and probe into their cross-link during acetaminophen-induced hepatotoxicity.

The excessive inflammatory response of macrophages plays a vital role in the pathogenesis of various diseases. The dynamic metabolic alterations in macrophages, including amino acid metabolism, are known to orchestrate their inflammatory phenotype. To explore a new metabolic pathway that regulates the inflammatory response, we examined metabolome changes in mouse peritoneal macrophages (PMs) in response to lipopolysaccharide (LPS) and found a coordinated increase of cysteine and its related metabolites, suggesting an enhanced demand for cysteine during the inflammatory response. Because Slc7a11, which encodes a cystine transporter xCT, was remarkably upregulated upon the pro-inflammatory challenge and found to serve as a major channel of cysteine supply, we examined the inflammatory behavior of Slc7a11 knockout PMs (xCT-KO PMs) to clarify an impact of the increased cysteine demand on inflammation. The xCT-KO PMs exhibited a prolonged upregulation of pro-inflammatory genes, which was recapitulated by cystine depletion in the culture media of wild-type PMs, suggesting that cysteine facilitates the resolution of inflammation. Detailed analysis of the sulfur metabolome revealed that supersulfides, such as cysteine persulfide, were increased in PMs in response to LPS, which was abolished in xCT-KO PMs. Supplementation of N-acetylcysteine tetrasulfide (NAC-S2), a supersulfide donor, attenuated the pro-inflammatory gene expression in xCT-KO PMs. Thus, activated macrophages increase cystine uptake via xCT and produce supersulfides, creating a negative feedback loop to limit excessive inflammation. Our study highlights the finely tuned regulation of macrophage inflammatory response by sulfur metabolism.

Cysteine, an early sulfur-containing compound in plants, is of significant importance in sulfur metabolism. CYS encodes cysteine synthetase that further catalyzes cysteine synthesis. In this investigation, CYS genes, identified from genome-wide analysis of Gossypium hirsutum bioinformatically, led to the discovery of GhCYS2 as the pivotal gene responsible for Cd2+ response. The silencing of GhCYS2 through virus-induced gene silencing (VIGS) rendered plants highly susceptible to Cd2+ stress. Silencing GhCYS2 in plants resulted in diminished levels of cysteine and glutathione while leading to the accumulation of MDA and ROS within cells, thereby impeding the regular process of photosynthesis. Consequently, the stomatal aperture of leaves decreased, epidermal cells underwent distortion and deformation, intercellular connections are dramatically disrupted, and fissures manifested between cells. Ultimately, these detrimental effected culminating in plant wilting and a substantial reduction in biomass. The association established between Cd2+ and cysteine in this investigation offered a valuable reference point for further inquiry into the functional and regulatory mechanisms of cysteine synthesis genes.

In the U.S., alcohol-associated liver disease (ALD) impacts millions of people and is a major healthcare burden. While the pathology of ALD is unmistakable, the molecular mechanisms underlying ethanol hepatotoxicity are not fully understood. Hepatic ethanol metabolism is intimately linked with alterations in extracellular and intracellular metabolic processes, specifically oxidation/reduction reactions. The xenobiotic detoxification of ethanol leads to significant disruptions in glycolysis, β-oxidation, and the TCA cycle, as well as oxidative stress. Perturbation of these regulatory networks impacts the redox status of critical regulatory protein thiols throughout the cell. Integrating these key concepts, our goal was to apply a cutting-edge approach toward understanding mechanisms of ethanol metabolism in disrupting hepatic thiol redox signaling. Utilizing a chronic murine model of ALD, we applied a cysteine targeted click chemistry enrichment coupled with quantitative nano HPLC-MS/MS to assess the thiol redox proteome. Our strategy reveals that ethanol metabolism largely reduces the cysteine proteome, with 593 cysteine residues significantly reduced and 8 significantly oxidized cysteines. Ingenuity Pathway Analysis demonstrates that ethanol metabolism reduces specific cysteines throughout ethanol metabolism (Adh1, Cat, Aldh2), antioxidant pathways (Prx1, Mgst1, Gsr), as well as many other biochemical pathways. Interestingly, a sequence motif analysis of reduced cysteines showed a correlation for hydrophilic, charged amino acids lysine or glutamic acid nearby. Further research is needed to determine how a reduced cysteine proteome impacts individual protein activity across these protein targets and pathways. Additionally, understanding how a complex array of cysteine-targeted post-translational modifications (e.g., S–NO, S-GSH, S–OH) are integrated to regulate redox signaling and control throughout the cell is key to the development of redox-centric therapeutic agents targeted to ameliorate the progression of ALD.

Background: Glycerol is a substrate for gluconeogenesis and fatty acid esterification in the liver, processes which are upregulated in obesity and may contribute to excess fat accumulation. Glycine and glutamate, in addition to cysteine, are components of glutathione, the major antioxidant in the liver. In principle, glycerol could be incorporated into glutathione via the TCA cycle or 3-phosphoglycerate, but it is unknown whether glycerol contributes to hepatic de novo glutathione biosynthesis. Methods: Glycerol metabolism to hepatic metabolic products including glutathione was examined in the liver from adolescents undergoing bariatric surgery. Participants received oral [U–13C3]glycerol (50 mg/kg) prior to surgery and liver tissue (0.2–0.7g) was obtained during surgery. Glutathione, amino acids, and other water-soluble metabolites were extracted from the liver tissue and isotopomers were quantified with nuclear magnetic resonance spectroscopy. Results: Data were collected from 8 participants (2 male, 6 female; age 17.1 years [range 14–19]; BMI 47.4 kg/m2 [range 41.3–63.3]). The concentrations of free glutamate, cysteine, and glycine were similar among participants, and so were the fractions of 13C-labeled glutamate and glycine derived from [U–13C3]glycerol. The signals from all component amino acids of glutathione - glutamate, cysteine and glycine - were strong and analyzed to obtain the relative concentrations of the antioxidant in the liver. The signals from glutathione containing [13C2]glycine or [13C2]glutamate derived from the [U–13C3]glycerol drink were readily detected, and 13C-labelling patterns in the moieties were consistent with the patterns in corresponding free amino acids from the de novo glutathione synthesis pathway. The newly synthesized glutathione with [U–13C3]glycerol trended to be lower in obese adolescents with liver pathology. Conclusions: This is the first report of glycerol incorporation into glutathione through glycine or glutamate metabolism in human liver. This could represent a compensatory mechanism to increase glutathione in the setting of excess glycerol delivery to the liver.

It has been demonstrated that the results caused by abnormal levels of Cysteine (Cys) are not negligible in biological processes. Furthermore, mitochondria are one of the most important organelles, which not only produce cellular energy, but also participate in signal transmission, growth, death, and other biological processes. Therefore, developing an efficient fluorescent probe targeting mitochondria to detect Cys in cells is useful for clinic pathological diagnosis. In this report, a near-infrared (NIR) fluorescent probe LAA based on link-anthocyanin as fluorophore and acrylate as identification groups has been prepared. Link-anthocyanin, as an NIR-emitting fluorophore, showed favorable water solubility and strong fluorescence intensity. Acrylate, as a recognition site, exhibited intense sensitivity and selectivity to Cys. In addition, LAA had a high tendency to localize to mitochondria due to the lipophilic positive ion in the link-anthocyanin. Under the excitation of 560 nm, the probe LAA could detect Cys with high selectivity and low detection limit (37 nM) at the emission of 670 nm, which belonged to the NIR range and was beneficial for biological application. Furthermore, LAA exhibited great biocompatibility and low cytotoxicity, which could monitor Cys in mitochondria of living cells in real-time.

The effect of cysteine concentration on the viscosity of soy protein isolate (SPI)-based film-forming solution (FFS) and physicochemical properties of SPI films was investigated. The apparent viscosity of FFS decreased after adding 1 mmol/L cysteine but did not change after adding 2–8 mmol/L cysteine. After treatment with 1 mmol/L cysteine, the film solubility decreased from 70.40% to 57.60%, but the other physical properties did not change. The water vapor permeability and contact angle of SPI films increased as cysteine concentration increased from 4 mmol/L to 8 mmol/L, whereas the film elongation at break decreased. Based on scanning electron microscopy and X-ray diffraction results, cysteine crystallization could be aggregated on the surface of SPI films treated with 4 or 8 mmol/L cysteine. In conclusion, pretreatment with approximately 2 mmol/L cysteine could reduce the viscosity of SPI-based FFS, but did not change the physicochemical properties of SPI films.

ABSTRACT: Previous research has showed that nonproteolytic Levilactobacillus brevis 145 (L) in coculture with Streptococcus thermophilus 1275 (S), not Lactobacillus delbrueckii ssp. bulgaricus (Lbu), was able to produce γ-aminobutyric acid (GABA) during milk fermentation in the presence of monosodium glutamate (MSG). It was assumed that differences of casein hydrolysis patterns between Strep. thermophilus 1275 and L. bulgaricus caused the phenomenon. Moreover, the GABA content was low and residual MSG was high in SL-fermented milk. In our research, comparison of peptide profiles determined by liquid chromatography/tandem mass spectrometry showed that αS2-casein, β-casein, and κ-casein degradation by L. bulgaricus and Strep. thermophilus varied. Importantly, the peptide number in the L and Lbu coculture group increased compared with the Lbu monoculture group, whereas the peptide number in the SL coculture group decreased in comparison with S monoculture group, suggesting that L. bulgaricus was not able to provide peptides for the growth of Lb. brevis 145. Furthermore, we found that after supplementation with cysteine (50 mg/L) during milk fermentation by SL, 10 g/L MSG was converted into 4.8 g/L GABA with a minimum level of residual MSG, viable cell counts of Lb. brevis and lactic acid production were increased, and the casein hydrolysis pattern was not influenced. Moreover, sulfhydryl group-containing chemicals including cystine, reduced glutathione, and oxidized glutathione showed effects similar to that of cysteine in improving GABA production. Finally, when L. bulgaricus YIB2 was combined with SL, supplementation of cysteine was also able to significantly improve GABA production.

NF-E2-related factor 2 (NRF2) plays a crucial role in the maintenance of cellular homeostasis by regulating various enzymes and proteins that are involved in the redox reactions utilizing sulfur. While substantial impacts of NRF2 on mitochondrial activity have been described, the precise mechanism by which NRF2 regulates mitochondrial function is still not fully understood. Here, we demonstrated that NRF2 increased intracellular persulfides by upregulating the cystine transporter xCT encoded by Slc7a11, a well-known NRF2 target gene. Persulfides have been shown to play an important role in mitochondrial function. Supplementation with glutathione trisulfide (GSSSG), which is a form of persulfide, elevated the mitochondrial membrane potential (MMP), increased the oxygen consumption rate (OCR) and promoted ATP production. Persulfide-mediated mitochondrial activation was shown to require the mitochondrial sulfur oxidation pathway, especially sulfide quinone oxidoreductase (SQOR). Consistently, NRF2-mediated mitochondrial activation was also dependent on SQOR activity. This study clarified that the facilitation of persulfide production and sulfur metabolism in mitochondria by increasing cysteine availability is one of the mechanisms for NRF2-dependent mitochondrial activation.

Here, we present a simple disulfide linked probe HTP for rapid detection of Cu 2+ ions, which was prepared by a condensation reaction between 7-diethylamino-4-hydroxycoumarin aldehyde and 2-aminothiophenol. The disulfide linked probe HTP was characterized using 1 H NMR, 13 C NMR, and HRMS spectroscopic analysis and confirmed by single crystal X-ray diffraction analysis. The photophysical behavior of HTP in various solvents (non-polar to polar) was studied and HTP displayed aggregation induced emission (AIE) characteristics in CH 3 CN-water mixtures (0-99 %). Upon binding with Cu 2+ ions, emission enhancement occurs along with color changes from weak green to intense yellow emission in CH 3 CN/Tris-HCl buffer (20 μM, 9:1, 10 mM Tris HCl Buffer, pH = 7.4). Detection limit for Cu 2+ ions was found to be 0.97 nM which is lower than the recommended tolerance limit by the WHO and the association constant 0.42 × 10 8  M -1 was obtained using B-H plot. Furthermore, the stoichiometric ratio 1:1 was confirmed by Job's plot, 1 H NMR, mass spectral analysis and DFT calculations were supported the formation of HTP-Cu 2+ complex. The reversibility of HTP with Cu 2+ ions was achieved by cysteine with detection limit and association constant value of 1.64 µM and 0.15 × 10 7  M -1 respectively. The reversible sensing nature of HTP with Cu 2+ /cysteine was further applied for constructing a molecular logic gate (INHIBIT) and practical applications such as paper strips, cotton swabs and real water analysis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

This study synthesized a 4,4-difluoro-2,6-di (1-octyl, 5-esteryluridine)-8-(3,4,5-tri (ethanoxy) phenyl) fluoroboron dipyrrole (BODIPY-A) near-infrared fluorescent probe based on 4,4-difluoro-2,6-diiodio-8-(3,4,5-tri (ethanoxy) phenyl) fluoroboron dipyrrole and 1-octanyl, 5-carboxyuracil. The emission wavelength of BODIPY-A probe is 672 nm, located in the near-infrared region, and it possesses benefits such as deep tissue penetration, low background self-fluorescence, and minimal light damage. The BODIPY-A probe exhibits a good turn-on fluorescence response to Cys through nucleophilic substitution reaction with cysteine (Cys), and can eliminate interference from homocysteine (Hcy) and glutathione (GSH). The BODIPY-A probe has been applied to the detection of Cys, with a linear range and detection limit of 0-90 μM and 0.3 μM, respectively. The BODIPY-A probe was applied to analyze the serum samples, achieving an absolute recovery rate ranging from 95 %-99 % and a relative standard deviation (RSD) of 0.031 %-0.371 %. Research has shown that the BODIPY-A probe has the hope to be used for sensitive detection of Cys in the human body., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Cysteine, as a vital endogenous small molecule mercaptan, plays a crucial role in various physiological processes. The high sensitivity and selectivity of fluorescent probes provide a method to monitor cysteine, which is helpful to understand the mechanism of cysteine in physiological processes more comprehensively. However, the detection of cysteine can be interfered by other small molecule biothiols. Therefore, the design of fluorescent probe based on the structural characteristics and reactivity of cysteine has become research focus currently. Given the biological compatibilities, biological targets, the metabolic pathway of 3-hydoxythalidomide, and its unique fluorescent properties, herein, we have designed a chemodosimeter, 2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl acrylate, for the detection of cysteine based on a tandem reaction of thiol-ene click chemistry and aminolysis involving 3-hydroxythalidomide as a parent compound. Experimental data exhibited that the probe showed unique selectivity and sensitivity for cysteine over other amino acid and biothiols. In addition, the fluorescent intensity at 511 nm increased linearly as a function of cysteine concentration in the range of 0-6 × 10 -7 M (regression factor, R 2  = 0.999), with a limit of detection of 6.1 nM. The sensing mechanism was confirmed through 1 H NMR titration and density functional theory calculations. Additionally, the probe was also successfully utilized for the detection of cysteine in sewage and for bioimaging in HeLa cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Ca 2+ /calmodulin-dependent protein kinase II α (CaMKIIα) "autonomous" activation induced by Thr286 phosphorylation has a crucial role in synaptic plasticity. Previous studies showed that in Alzheimer's disease brain, CaMKIIα autophosphorylation at Thr286 is reduced while the level of cysteine-oxidized CAMKIIα is elevated. We performed tryptic mapping of the oxidized CaMKIIα and discovered the formation of a disulfide between the N-terminal Cys6 and the regulatory domain Cys280. The apparent pK a values of Cys6 and Cys280 are 7.1 and 7.7, respectively, lower than the 8.5 for free Cys. The low apparent pK a of Cys6 facilitates the oxidation of its thiol to the sulfenic acid at physiological pH. The thiolate of Cys280 can then attack the sulfenic acid to form a disulfide. Using an antibody against phosphorylated Thr286, we showed that disulfide formation prevents Thr286 phosphorylation. CaMKIIα autonomous activation induced by disulfide formation is much lower than the autonomous activation induced by phosphorylation. The decreased autonomous activation may contribute to the synaptic impairment of Alzheimer's disease. We also generated a CaMKIIα mutant in which Cys6 was mutated to Ser6. This mutation prevented disulfide formation and restored autonomous activation induced by phosphorylation. Our findings provide insight into the mechanistic details of CaMKIIα autonomous activation induced by disulfide formation that may contribute to the impairment of long-term potentiation in Alzheimer's disease., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 Elsevier Inc. All rights reserved.)

L-Cysteine (Cys) and glutathione (GSH) are closely related biological species that widely exist in food and living cells. To simultaneously detect Cys and GSH from different emission channels, we developed a fluorescent probe (BDP-NBD) based on near-infrared BODIPY and 7-nitrobenzofurazan (NBD). Upon nucleophilic substitution reaction with GSH, BDP-NBD generated an emission band at 713 nm, which can be used to determine GSH (0-100 μM) with a low detection limit (34 nM). Different from GSH, BDP-NBD underwent a nucleophilic substitution-rearrangement reaction with Cys, affording two emission bands at 550 nm and 713 nm, respectively. BDP-NBD was successfully employed to quantify Cys and GSH in various food samples with good recoveries (86.6%-104.6%). Besides, BDP-NBD can image Cys and GSH in living cells from two emission channels. Therefore, this work developed a tool for the simultaneous determination of Cys and GSH in both food and living cells so as to ensure food safety and human health., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Due to the importance of Cys, the development of methods to detect Cys has become a hot research topic. In the present work, we constructed a novel near-infrared fluorescent probe BP-PTC, which specifically binds Cys with a mitochondria-targeting property, using a synthetic benzopyran salt as a chromophore attached to a phenyl thioformate group. The specific response to Cys is achieved based on the addition-cyclisation-elimination reaction process of Cys with phenyl thioformate, allowing it to exhibit fluorescence emission at 670 nm. In addition, the fluorescence intensity of BP-PTC shows a good linear relationship with Cys in the concentration range of 0 ∼ 28 μΜ. A series of characterization and property tests have demonstrated that BP-PTC has good selectivity and sensitivity for Cys and can be detected by "naked eyes". BP-PTC has been successfully used for the detection of endogenous and exogenous Cys in vivo., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Accurately detecting cysteine (Cys) in vivo is crucial for diagnosing Cys-related diseases. A novel ratiometric fluorescent probe featuring dual near-infrared emission is developed in this study for the in vivo ratio imaging of Cys. The probe comprises a hemicyanine organic small-molecule dye (HCy-CYS) with specific Cys recognition capabilities covalently coupled with carbon dots (CDs) synthesized using glutathione (GSH) as the carbon source (GCDs), forming a unique composite nanofluorescent probe (GCDs@CYS). The probe undergoes a specific reaction with acrylate upon the addition of Cys, converting HCy-CYS to HCy-OH. Consequently, the GCD fluorescence intensity at 685 nm gradually decreases, whereas that of HCy-OH at 720 nm progressively increases, yielding a ratiometric fluorescence signal. Notably, both emission wavelengths of the probe exceed 650 nm, thereby effectively mitigating the interference from background signals during cellular and in vivo imaging. Furthermore, the probe demonstrates high specificity for Cys, enabling its differentiation from homocysteine and GSH. The Cys concentration and fluorescence ratiometric intensity exhibit a strong linear correlation at 10-150 μM with a detection limit of 0.95 μM. These results indicate that the ratiometric fluorescent probe can serve as a valuable tool for monitoring Cys-related physiological or pathological processes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025. Published by Elsevier B.V.)

At least two of the six methionine (Met) residues in dehaloperoxidase (DHP) are shown to act as electron donors in both autoreduction and protein-heme crosslinking. Autoreduction observed in the two isozymes, DHP-A and DHP-B, is explained by the high heme reduction potential and an endogenous source of electrons from methionine (Met) or cysteine (Cys). This study provides evidence of a connection to protein-heme crosslinking that occurs when DHP is activated by H 2 O 2 in competition with substrate oxidation and autoreduction. The autoreduction yields of DHP-A and DHP-B are comparable and both are inversely proportional to DHP concentration. Both isoenzymes show an anti-cooperative effect on autoreduction kinetics associated with protein dimerization. Despite the presence of five tyrosine (Tyr) amino acids in DHP-A and four Tyr in DHP-B, the mass spectral evidence does not support a Tyr-heme or interprotein Tyr-Tyr crosslinking event as observed in some mammalian myoglobins. LC-MS and tandem MS/MS studies revealed three amino acids that were involved in the heme-protein crosslink, Cys73, Met63 and Met64. Cys73 facilitates dimer formation in DHP-A which also appears to slow the rate of autoreduction, but is not involved in covalent protein-heme crosslinking. Based on mutational studies, Met63 and 64 are involved in both covalent heme crosslinking and autoreduction. Proton-coupled electron transfer and crosslinking by Met to the heme may serve to regulate DHP function and protect it from uncontrolled oxidative damage., Competing Interests: Declaration of competing interest The authors collectively have no financial or other outside interests to declare. The research and authorship is entirely funded by public sources and there is no intellectual property or commercial application of the research., (Copyright © 2024. Published by Elsevier B.V.)

Cysteine (Cys) is involved in many physiological processes. It's challenging to detect Cys selectively as it has similar chemical structure with other biothiols such as homocysteine (Hcy) and glutathione (GSH). In this work, a novel fluorescence probe toward mitochondrial cysteine, HPXI-6C, has been developed by employing carbonate as a new recognizing unit and hemicyanine as a chromophore. HPXI-6C exhibits a high selectivity to Cys over hydrogen sulfide, homocysteine and glutathione. The limit of detection toward Cys was determined to be 42 nM. HPXI-6C can localize in mitochondria and produce strong fluorescence peaked at 725 nm in response to Cys in tumor cells. The uptake and generation pathways of Cys in acetaminophen hepatotoxicity cells was revealed by using HPXI-6C. HPXI-6C has been successfully applied in imaging of Cys in drug-induced liver injury in vivo. The research demonstrated that HPXI-6C is powerful in monitoring Cys and is conducive to the early diagnosis of drug-induced liver injury diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In this work, a near-infrared hepatocyte-targeting fluorescence probe TCF-Gal-Cys was developed. The TCF-Gal-Cys exhibited a low detection limit, good sensitivity and selectivity toward Cys. The responsive mechanism of TCF-Gal-Cys was proposed that the acrylate of TCF-Gal-Cys was subsequently attacked by the thiol group and the amino group of Cys, releasing a strong near-infrared fluorescent group. TCF-Gal-Cys displayed a good hepatocyte-targeting capacity and could specifically distinguish hepatocytes from A549, Hela, SGC-7901 cells because the galactose group of TCF-Gal-Cys can be recognized by HepG2 cells overexpressing ASGPR. The TCF-Gal-Cys has achieved excellently imaging performance to Cys in the zebrafish, so TCF-Gal-Cys has potential to be an effective tool to in real time monitor Cys-related diseases in vitro and in vivo., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The quantification of cysteine (Cys) levels in the organisms holds paramount significance in biological research and disease diagnosis, which can give the correlation between abnormal Cys levels and diseases. In this study, two fluorescent probes, designated as DEA-OH and DEA-AC, featuring a coumarin-aurone backbone specifically engineered for Cys detection, were meticulously designed and synthesized. The diethylamino coumarin-aurone probe DEA-OH and the acrylate-substituted probe DEA-AC demonstrated remarkable sensitivity in detecting cysteine by means of copper displacement (DEA-OH) and acrylate hydrolysis mechanisms (DEA-AC) with fluorescence detection limits of 7.25 μM and 1.65 μM, respectively. Moreover, the fluorescence peak wavelength of the two probes displayed a linear relationship with solvent polarity in the E T (30) range of 30-65 kcal•mol -1 , indicating the potential for monitoring changes in environmental polarity within this E T (30) range. The outstanding attributes exhibited by DEA-AC including superior photostability, remarkable selectivity, and swift response (kinetic rate constant: 0.00747 s -1 ), coupled with the exceptional anti-interference ability, have significantly broadened its scope of applications, for example detecting alterations in Cys within biological systems., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Excess reactive oxide species (ROS) is a direct factor in myocardial injury death, thus anti-oxidant therapy is a necessary measure to prevent rapid death of cardiomyocyte cell. Cysteine (Cys) is a potent antioxidant but easily become instability because of the hyperactivity. Therefore, in order to protect the the stability of Cys, we according to the mitochondria are the main sites of ROS production, utilized the loading and ROS scavenging capacity of mesoporous polydopamine (mPDA) constructed a nanosystem targeting mitochondria with effectively ROS elimination capability by loading cysteine (Cys-mPDA@TPP). The mesoporous structure of mPDA effectively inhibited the advance reaction and hyperactivity of Cys, thus effectively improving its stability that reached the double-collaborative treatment excess ROS. In particular, Cys-mPDA@TPP achieved directly reacting with ROS in mitochondria under the targeting of triphenylphosphine (TPP), not only enhancing the elimination efficiency of ROS, but also preventing mitochondrial dysfunction of monocyte-macrophage. Furthermore, with double-collaborative ROS elimination, Cys-mPDA@TPP effectively prevent the damage of cardiomyocyte cell through inhibiting macrophage inflammatory response. Therefore, this study provides a new therapeutic strategy for myocardial inflammatory injury., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Protein disulfide isomerases (PDIs) catalyze redox reactions that reduce, oxidize, or isomerize disulfide bonds and act as chaperones of proteins as they fold. The characteristic features of PDIs are the presence of one or more catalytic thioredoxin (TRX)-like domains harboring typical CXXC catalytic motifs responsible for redox reactions, as well as non-catalytic TRX-like domain. As increasing attention is paid to oxidative post-translational modifications of cysteines (Cys ox-PTMs) with the recognition that they control cellular signaling, strategies to identify sites of Cys ox-PTM by redox proteomics have been optimized. Exploration of an available Cys redoxome dataset supported by modeled structure provided arguments for the existence of an additional non-catalytic thiol-disulfide motif, distinct from those contained in the TRX type patterns, typical of PDIAs. Further structural analysis of PDIA3 and 6 allows us to consider the possibility that this hypothesis could be extended to other members of PDI. These elements invite future studies to decipher the exact role of these non-catalytic thiol-disulfide motifs in the functions of PDIs. Strategies that would allow to validate this hypothesis are discussed.

Maillard reaction products (MRPs) with roasted/broth flavors were prepared and analyzed for the resulting flavor differences. The identification of volatile compounds in MRPs was carried out by GC-MS and GC × GC-ToF-MS. A total of 88 compounds were identified by GC-MS; 130 compounds were identified by GC × GC-ToF-MS, especially acids and ketones were identified. Principal component analysis (PCA) was used to visualize the volatile compounds, and the roasted/broth flavors were differentiated. The contents and types of pyrazines were more in roasted flavors; thiol sulfides and thiophenes were more in broth flavors. All in all, the differences in volatile compounds producing roasted/broth flavors were studied through the cysteine-xylose-glutamate Maillard reaction system, which provided a theoretical basis for the future use of Maillard reaction to simulate meat flavor.

Aging is a natural process in organisms with its underlying mechanisms remaining unknown. Brain aging is accompanied by cognitive deficits and movement disorders, which signify the importance of elaborating its main mechanisms. In this study, oxidative stress biomarkers for lipid peroxidation and thiol redox state were assessed in different brain areas in male mice, categorised in respect to aging as young (three months), middle (eleven months) and elder aged (twenty-three months). Senescence was associated with an increase of lipid peroxidation and a decrease of reduced and oxidized glutathione. In some brain areas, reduced cysteine and oxidized protein thiols were increased with aging. Results support the theory that aging is associated with oxidative stress in the brain of mice and provide an insight in the biochemical aspect of aging in reference to thiol redox status as a potential marker for aging.

A novel class of curcumin-derived fluorescent probes was designed. This kind of probe introduces easy leaving groups methylsulfonyl and phenylsulfonyl respectively to achieve the detection effect through the nucleophilic attack of amino acids. At the same time, BF2 group is introduced to increase the emission wavelength of the probe. Probes 4 and 5 can respond quickly with amino acids, but can specifically recognize Cys. In UV detection, the maximum absorption wavelength of the probes can be blue-shifted by 81 nm with the addition of Cys and still show a strong fluorescence signal. The detection limits for compounds 4 and 5 were determined to be 0.40 μM and 0.87 μM, respectively, with a goodness-of-fit of 0.99. In addition, a rapid response of the probe to Cys could be observed with the naked eye within 1 min. These results provide a new method for rapid detection of Cys; And this kind of probe has the drug structure of curcumin, which can provide ideas for the design of drug-probe.

Background: Hydrogen sulfide (H2S), a gaseous signaling molecule that impacts multiple physiological processes including aging, is produced via select mammalian enzymes and enteric sulfur-reducing bacteria. H2S research is limited by the lack of an accurate internal standard-containing assay for its quantitation in biological matrices. Methods: After synthesizing [34S]H2S and developing sample preparation protocols that avoid sulfide contamination with the addition of thiol-containing standards or reducing reagents, we developed a stable isotope-dilution high performance liquid chromatography tandem-mass spectrometry (LC-MS/MS) method for the simultaneous quantification of Total H2S and other abundant thiols (cysteine, homocysteine, glutathione, glutamylcysteine, cysteinylglycine) in biological matrices, conducted a 20-day analytical validation/normal range study, and then both analyzed circulating Total H2S and thiols in plasma from 400 subjects, and within 20 volunteers before and after antibiotic-induced suppression of gut microbiota. Results: Using the new assay, all analytes showed minimal interference, no carryover, and excellent intra- and inter-day reproducibility (≤7.6%, and ≤12.7%, respectively), linearity (r2 > 0.997), recovery (90.9%–110%) and stability (90.0%–100.5%). Only circulating Total H2S levels showed significant age-associated reductions in both males and females (p

Cysteine (Cys) plays a critical role in various biological processes, including protein synthesis, cellular signaling, and antioxidant defense. However, precise detection of Cys in biological systems remains challenging due to interference from similar thiols such as homocysteine (Hcy) and glutathione (GSH). In this study, we report the synthesis and bioimaging of a novel ratio-type fluorescent probe based on the benzoBODIPY fluorophore, designed for the ratiometric detection of Cys. The probe operates through an intramolecular charge transfer (ICT) mechanism, where the reaction with Cys triggers a substitution reaction with 4-mercaptopyridine, followed by a Smiles rearrangement. This results in a shift from red to yellow-green fluorescence, providing a sensitive and specific method for the quantitative detection of Cys. The probe demonstrates excellent selectivity, with significantly lower responses to Hcy and GSH, and has been successfully applied in bioimaging experiments in HeLa cells and zebrafish models, highlighting its potential for diagnosing and treating Cys-related diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

S-nitrosylation is a redox post-translational modification widely recognized to play an important role in cellular signaling as it can modulate protein function and conformation. At the physiological level, nitrosoglutathione (GSNO) is considered the major physiological NO-releasing compound due to its ability to transfer the NO moiety to protein thiols but the structural determinants regulating its redox specificity are not fully elucidated. In this study, we employed photosynthetic glyceraldehyde-3-phosphate dehydrogenase from Chlamydomonas reinhardtii (CrGAPA) to investigate the molecular mechanisms underlying GSNO-dependent thiol oxidation. We first observed that GSNO causes reversible enzyme inhibition by inducing S-nitrosylation. While the cofactor NADP+ partially protects the enzyme from GSNO-mediated S-nitrosylation, protein inhibition is not observed in the presence of the substrate 1,3-bisphosphoglycerate, indicating that the S-nitrosylation of the catalytic Cys149 is responsible for CrGAPA inactivation. The crystal structures of CrGAPA in complex with NADP+ and NAD+ reveal a general structural similarity with other photosynthetic GAPDH. Starting from the 3D structure, we carried out molecular dynamics simulations to identify the protein residues involved in GSNO binding. The reaction mechanism of GSNO with CrGAPA Cys149 was investigated by quantum mechanical/molecular mechanical calculations, which permitted to disclose the relative contribution of protein residues in modulating the activation barrier of the trans-nitrosylation reaction. Based on our findings, we provide functional and structural insights into the response of CrGAPA to GSNO-dependent regulation, possibly expanding the mechanistic features to other protein cysteines susceptible to be oxidatively modified by GSNO.

Although listed as endocrine disruptor compounds, atrazine (ATZ) is still used in large quantities in agricultural production. Here, alfalfa seedling was cultivated in hydroponic media to investigate the toxic effects of ATZ on alfalfa and accumulation of ATZ in tissues of different plant parts. Alfalfa had a strong upward translocation ability to ATZ. The stress response of alfalfa under ATZ stress was studied using metabolomic and transcriptomic techniques. S-adenosylmethionine, glutathione, 3-mercaptopyruvic acid, ornithine, and aminopropylcadaverine were significantly increased by ATZ in pathways mtr00270 and mtr00480. Several genes of cysteine synthase and spermidine synthase were significantly up-regulated by ATZ induction. They may be markers and genes with potential physiological functions of alfalfa in response to ATZ stress. In addition, using high resolution mass spectrometry, a total of five ATZ metabolites secreted from alfalfa roots were detected. Among them, acetylated deisopropylated ATZ was discovered for the first time. Hydroxylated ATZ and acetylated deethylated ATZ were more readily excreted by the root system. This study not only provides potential genes for the construction of engineering plants to remediate ATZ-contaminated soil, but also provides monitoring objects for the ecological research of ATZ metabolites.

Poly 1,8-Diaminonaphtahlene/cysteine (poly 1,8-DAN/Cys) combined with carbon black (CB) nanoparticles are proposed as an excellent sensor for the detection of nitrite ions. To design the electrocatalyst, a simple approach consisting on drop-casting method was applied to disperse carbon black on the surface of glassy carbon electrode, followed by the immobilization of cysteine on the surface of CB nanoparticles. The electrochemical polymerization of 1,8-Diaminonaphthalene was conducted in acidic medium by using cyclic voltammetry. The prepared hybrid material was denoted poly 1,8-DAN /Cys/CB. Several methods were used to characterize the structural and electrochemical behavior of the reported hybrid material including Fourier transform infrared spectroscopy (FTIR), Scanning electron microscopy (SEM), cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), amperometry and differential pulse voltammetry (DPV). The prepared electrode displayed an outstanding electroactivity towards nitrite ions reflected by an enhancement in the intensity of the current and a decrease of the charge transfer resistance. Poly 1,8-DAN/Cys/CB displayed an excellent sensing performance towards the detection of nitrite with a very low detection limit of 0.25 µM. Two linear ranges of 1–40 µM and 20–210 µM when using amperometry and differential pulse voltammetry (DPV) were obtained respectively. This work highlights the simple preparation of a polymeric film rich in amine and thiol groups for nitrite detection.

The effects of Auranofin (AF) on protein expression and protein oxidation in A2780 cancer cells were investigated through a strategy based on simultaneous expression proteomics and redox proteomics determinations. Bioinformatics analysis of the proteomics data supports the view that the most critical cellular changes elicited by AF treatment consist of thioredoxin reductase inhibition, alteration of the cell redox state, impairment of the mitochondrial functions, metabolic changes associated with conversion to a glycolytic phenotype, induction of ER stress. The occurrence of the above cellular changes was extensively validated by performing direct biochemical assays. Our data are consistent with the concept that AF produces its effects through a multitarget mechanism that mainly affects the redox metabolism and the mitochondrial functions and results into severe ER stress. Results are discussed in the context of the current mechanistic knowledge existing on AF.

Atomic-resolution protein structural models are prerequisites for many downstream activities like structure-function studies or structure-based drug discovery. Unfortunately, this data is often unavailable for some of the most interesting and therapeutically important proteins. Thus, computational tools for building native-like structural models from less-than-ideal experimental data are needed. To this end, interaction homology exploits the character, strength and loci of the sets of interactions that define a structure. Each residue type has its own limited set of backbone angle-dependent interaction motifs, as defined by their environments. In this work, we characterize the interactions of serine, cysteine and S-bridged cysteine in terms of 3D hydropathic environment maps. As a result, we explore several intriguing questions. Are the environments different between the isosteric serine and cysteine residues? Do some environments promote the formation of cystine S–S bonds? With the increasing availability of structural data for water-insoluble membrane proteins, are there environmental differences for these residues between soluble and membrane proteins? The environments surrounding serine and cysteine residues are dramatically different: serine residues are about 50% solvent exposed, while cysteines are only 10% exposed; the latter are more involved in hydrophobic interactions although there are backbone angle-dependent differences. Our analysis suggests that one driving force for –S–S– bond formation is a rather substantial increase in burial and hydrophobic interactions in cystines. Serine and cysteine become less and more, respectively, solvent-exposed in membrane proteins. 3D hydropathic environment maps are an evolving structure analysis tool showing promise as elements in a new protein structure prediction paradigm.

Unraveling the role of VDAC3 within living cells is challenging and still requires a definitive answer. Unlike VDAC1 and VDAC2, the outer mitochondrial membrane porin 3 exhibits unique biophysical features that suggest unknown cellular functions. Electrophysiological studies on VDAC3 carrying selective cysteine mutations and mass spectrometry data about the redox state of such sulfur containing amino acids are consistent with a putative involvement of isoform 3 in mitochondrial ROS homeostasis. Here, we thoroughly examined this issue and provided for the first time direct evidence of the role of VDAC3 in cellular response to oxidative stress. Depletion of isoform 3 but not isoform 1 significantly exacerbated the cytotoxicity of redox cyclers such as menadione and paraquat, and respiratory complex I inhibitors like rotenone, promoting uncontrolled accumulation of mitochondrial free radicals. High-resolution respirometry of transiently transfected HAP1-ΔVDAC3 cells expressing the wild type or the cysteine-null mutant VDAC3 protein, unequivocally confirmed that VDAC3 cysteines are indispensable for protein ability to counteract ROS-induced oxidative stress.

Cysteine (Cys) can serve as a biomarker to indicate diseases or disorders, and its chiral sensing has attracted increasing attention. Herein, we established an ultrasound-facilitated chiral sensing method for Cys using 4-chloro-7-nitro-1,2,3-benzoxadiazole (NBD-Cl) and electronic circular dichroism (ECD) spectroscopy. The formation of chiral disulfide bonds induced degenerate exciton coupling between two NBD chromophores, resulting in intense Cotton effects (CEs) of the sensing product. The anisotropy factor (g) was linearly correlated with the enantiomeric excess of Cys across the visible region (400–500 nm), and other natural amino acids or biothiols did not interfere with the detection. This ultrasound-promoted efficient and specific chiral sensing method of Cys has potential for application in the diagnosis of related diseases.

Flaxseed derived Maillard reaction products (MRPs) have typical meaty flavor, but there is no report on comparison of their amino acids and peptides reactivity. The peptides and amino acids of flaxseed protein hydrolysates were separately collected by G-15 gel chromatography. Taste dilution analysis (TDA) showed that peptides-MRPs had high umami, mouthfulness, and continuity enhancement. Further, LC-MS/MS revealed that flaxseed protein hydrolysates consumed 41 peptides after Maillard reaction. Particularly, DLSFIP (Asp-Leu-Ser-Phe-Ile-Pro) and ELPGSP (Glu-Leu-Pro-Gly-Ser-Pro) accounted for 42.22% and 20.41% of total consumption, respectively. Aroma extract dilution analysis (AEDA) indicated that formation of sulfur-containing flavors was dependent on cysteine, while peptides were more reactive than amino acids for nitrogen-containing heterocycles. On the other hand, 11 flavor compounds with flavor dilution (FD) ≥ 64 were identified for flaxseed derived MRPs, such as 2-methylthiophene, 2-methyl-3-furanthiol, furfural, 2-furfurylthiol, 3-thiophenethiol, thieno[3,2-b] thiophene, 2,5-thiophenedicarboxaldehyde, 2-methylthieno[2,3-b] thiophene, 1-(2-methyl-3-furylthio)-ethanethiol, 2-methylthieno[3,2-b] thiophene, and bis(2-methyl-3-furyl)-disulfide. In addition, we further demonstrated the flavors formation mechanism of flaxseed derived MRPs.

Background: Cysteine (Cys) is the major intracellular thiol and plays a key role in human pathology. Furthermore, endogenous sulfur dioxide (SO 2 ) is produced in mammals. Abnormal levels of SO 2 are commonly associated with a variety of respiratory, cardiovascular, and neurological diseases. Therefore, given their important role in life activities, it is significant to construct a fluorescent probe that can detection between Cys and SO 2 ., Results: We have designed and synthesized a two-site fluorescent probe CUM with coumarin derivative and benzaldehyde molecules, which can detect and differentiate between Cys and SO 2 through dual excitation wavelengths. Its carbon-carbon double bond reacts with Cys and undergoes a nucleophilic reaction, emitting green fluorescence at 520 nm, while SO 3 2- reacts with benzaldehyde molecules in the probe CUM and undergoes a blue fluorescence at 460 nm. SO 3 2- reacts with the benzaldehyde molecule of probe CUM and fluoresces blue at 460 nm. Thus, the probe CUM with two reaction sites can distinguish between Cys and SO 2 and shows good selectivity and fast reaction speed. In addition, we successfully utilized probe CUM to image Cys and SO 2 in human breast cancer cells (MDA-MB-231)., Significance: This work provides an effective method for the molecular design of coumarin-based fluorescent probes. Probe CUM as a promising and reliable tool for the meticulous discrimination and quantification of Cys and SO 2 in diverse biological matrices, thereby opening up new avenues for various biological systems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Heavy metals, including Hg 2+ , Cr 6+ and Cd 2+ , have always been a major issue in environmental pollution, leading to abnormal changes in the levels of biologically active molecules including Cys in plants, seriously affecting all aspects of the growth and development of plants. This makes it essential to develop a simple and practical method to study the potential impact of heavy metals on plants. In this paper, our research group has developed near-infrared fluorescent probe WRM-S, which has the advantages of fast response, sensitivity to Cys, and successfully applying it to cells and zebrafish. Moreover, it combined the close relationship between heavy metal stress on plants and Cys, using Cys as the detection target, monitoring the internal environment changes of two plants under Hg 2+ , Cr 6+ , and Cd 2+ stress in the environment, and then conducting 3D imaging. The results indicated that the probe has strong penetration ability in plant tissues, and revealed abnormal changes in plant Cys levels caused by heavy metal stress-induced cellular oxidative stress or cytotoxicity. Thus, the in-situ imaging detection of this probe provides a direction for the physiological dynamics research of plant environmental stress., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)