In Situ Detection of Hydrogen Sulfide in 3D-Cultured, Live Prostate Cancer Cells Using a Paper-Integrated Analytical Device

In this study, a paper-integrated analytical device that combined a paper-based colorimetric assay with a paper-based cell culture platform was developed for the in situ detection of hydrogen sulfide (H2S) in three-dimensional (3D)-cultured, live prostate cancer cells. Two kinds of paper substrates were fabricated using a simple wax-printing methodology to form the cell culture and detection zones, respectively. LNCaP cells were seeded directly on the paper substrate and grown in the paper-integrated analytical device. The cell viability and H2S production of LNCaP cells were assessed using a simple water-soluble tetrazolium salt colorimetric assay and H2S-sensing paper, respectively. The H2S-sensing paper showed good sensitivity (sensitivity: 6.12 blue channel intensity/μM H2S, R2 = 0.994) and a limit of quantification of 1.08 μM. As a result, we successfully measured changes in endogenous H2S production in 3D-cultured, live LNCaP cells within the paper-integrated analytical device while varying the duration of incubation and substrate concentration (L-cysteine). This paper-integrated analytical device can provide a simple and effective method to investigate H2S signaling pathways and drug screening in a 3D culture model.

Roles of a Cysteine Desulfhydrase LCD1 in Regulating Leaf Senescence in Tomato

Hydrogen sulfide (H2S), a novel gasotransmitter in both mammals and plants, plays important roles in plant development and stress responses. Leaf senescence represents the final stage of leaf development. The role of H2S-producing enzyme L-cysteine desulfhydrase in regulating tomato leaf senescence is still unknown. In the present study, the effect of an L-cysteine desulfhydrase LCD1 on leaf senescence in tomato was explored by physiological analysis. LCD1 mutation caused earlier leaf senescence, whereas LCD1 overexpression significantly delayed leaf senescence compared with the wild type in 10-week tomato seedlings. Moreover, LCD1 overexpression was found to delay dark-induced senescence in detached tomato leaves, and the lcd1 mutant showed accelerated senescence. An increasing trend of H2S production was observed in leaves during storage in darkness, while LCD1 deletion reduced H2S production and LCD1 overexpression produced more H2S compared with the wild-type control. Further investigations showed that LCD1 overexpression delayed dark-triggered chlorophyll degradation and reactive oxygen species (ROS) accumulation in detached tomato leaves, and the increase in the expression of chlorophyll degradation genes NYC1, PAO, PPH, SGR1, and senescence-associated genes (SAGs) during senescence was attenuated by LCD1 overexpression, whereas lcd1 mutants showed enhanced senescence-related parameters. Moreover, a correlation analysis indicated that chlorophyll content was negatively correlated with H2O2 and malondialdehyde (MDA) content, and also negatively correlated with the expression of chlorophyll degradation-related genes and SAGs. Therefore, these findings increase our understanding of the physiological functions of the H2S-generating enzyme LCD1 in regulating leaf senescence in tomato.

Transcriptomics Reveals the ERF2-bHLH2-CML5 Module Responses to H2S and ROS in Postharvest Calcium Deficiency Apples

Calcium deficiency usually causes accelerated quality deterioration in postharvest fruit, whereas the underlining mechanism is still unclear. Here, we report that calcium deficiency induced the development of bitter pit on the surface of apple peels compared with the healthy appearance in control apples during postharvest storage. Physiological analysis indicates that calcium-deficient peels contained higher levels of superoxide anion (O2•−), malondialdehyde (MDA), total phenol, flavonoid contents and polyphenol oxidase (PPO) activity, and reduced calcium, H2S production, anthocyanin, soluble protein content, and peroxidase (POD) activity compared with those in calcium-sufficient peels. The principal component analysis (PCA) results show that calcium content, ROS, and H2S production were the main factors between calcium-deficient and calcium-sufficient apple peels. Transcriptome data indicated that four calmodulin-like proteins (CMLs), seven AP2/ERFs, and three bHLHs transcripts were significantly differentially expressed in calcium-deficient apple peels. RT-qPCR and correlation analyses further revealed that CML5 expression was significantly positively correlated with the expression of ERF2/17, bHLH2, and H2S production related genes. In addition, transcriptional co-activation of CML5 by ERF2 and bHLH2 was demonstrated by apple transient expression assays and dual-luciferase reporter system experiments. Therefore, these findings provide a basis for studying the molecular mechanism of postharvest quality decline in calcium-deficient apples and the potential interaction between Ca2+ and endogenous H2S.

Persulfidation of Nitrate Reductase 2 Is Involved in L-Cysteine Desulfhydrase-Regulated Rice Drought Tolerance

Hydrogen sulfide (H2S) is an important signaling molecule that regulates diverse cellular signaling pathways through persulfidation. Our previous study revealed that H2S is involved in the improvement of rice drought tolerance. However, the corresponding enzymatic sources of H2S and its regulatory mechanism in response to drought stress are not clear. Here, we cloned and characterized a putative L-cysteine desulfhydrase (LCD) gene in rice, which encodes a protein possessing H2S-producing activity and was named OsLCD1. Overexpression of OsLCD1 results in enhanced H2S production, persulfidation of total soluble protein, and confers rice drought tolerance. Further, we found that nitrate reductase (NR) activity was decreased under drought stress, and the inhibition of NR activity was controlled by endogenous H2S production. Persulfidation of NIA2, an NR isoform responsible for the main NR activity, led to a decrease in total NR activity in rice. Furthermore, drought stress-triggered inhibition of NR activity and persulfidation of NIA2 was intensified in the OsLCD1 overexpression line. Phenotypical and molecular analysis revealed that mutation of NIA2 enhanced rice drought tolerance by activating the expression of genes encoding antioxidant enzymes and ABA-responsive genes. Taken together, our results showed the role of OsLCD1 in modulating H2S production and provided insight into H2S-regulated persulfidation of NIA2 in the control of rice drought stress.

Mill Scale Addition to Reduce Hydrogen Sulfide Production in Anaerobic Digestion

Direct addition of sulfur-reducing agents during anaerobic digestion (AD) is very effective in controlling hydrogen sulfide (H2S) content in biogas, although one major problem is the high operational cost due to the large amount of chemicals used. The objective of this study was to remove H2S using a waste mill scale (MS) as a sulfur-reducing agent. To evaluate its feasibility, MS was added to AD fed with food waste (FW) at concentrations between 0 and 160 g MS/kg total chemical oxygen demand (TCOD) during the batch test, and the experimental results were compared to those of the batch test with the addition of iron chloride (FeCl3). Both FeCl3 and MS played an important role as electro-conductive materials in improving methane productivity by promoting direct interspecies electron transfer. An increase in H2S removal efficiency was observed with increases in both materials. In total, 30%, 60%, and 90% of H2S production based on the maximum sulfur in the form of H2S (control) was 3.7, 9.4, and 23.8 g FeCl3/kg TCOD and 13.3, 34.1, and 86.2 g MS/kg TCOD, respectively. This finding indicates that MS can be used as a sulfur-reducing agent substitute for H2S removal in AD fed with FW.

312 Effects of Bismuth Subsalicylate and Different Sulfate Concentrations on in vitro Ruminal Fermentation Using a Warm-season Perennial Hay Substrate

A randomized complete block design was used to evaluate the effects of bismuth subsalicylate (BSS) on in vitro ruminal fermentation with differing concentrations of sulfate. In vitro fermentation consisted of 50 mL of a 4:1 buffer:ruminal fluid inoculum and 0.7 g (pre-dehydrated) of substrate [WW-B Dahl bluestem hay (Bothriochloa bladhii)] incubated for 48 h (39oC). Treatments were arranged as a 3 × 4 factorial with concentration of sulfate (0.2, 2.9, or 5.6 g sulfate/L buffer) and BSS (0.0, 0.165, 0.330, or 0.495% substrate DM) as the main factors. In vitro organic matter digestibility (IVOMD), and CH4, H2S, and total gas production (TGP) were measured. Data were analyzed using the MIXED procedure of SAS with the fixed effects of BSS, sulfate, and their interaction. Incubation day (block) was considered a random effect. The average of 2 bottles within day was considered experimental unit. A BSS × sulfate interaction was observed for TGP (P = 0.040) and H2S production (P < 0.001), where BSS had a larger negative impact on TGP and production of H2S with greater concentrations of sulfate. A linear effect (P < 0.001) of sulfate was observed for CH4 production per gram of incubated OM, where CH4 was decreased as sulfate concentration increased. A quadratic effect of sulfate was observed for IVOMD (P = 0.010) and pH (P = 0.009). Production of H2S linearly decreased (P = 0.001) as BSS concentration increased. The addition of BSS to in vitro incubations did not affect (P > 0.10) any other variables measured. Bismuth subsalicylate does not appear to have negative effects on in vitro fermentation parameters while decreasing H2S production; however, elevated concentrations of sulfate in the buffer appears to have negative impacts on fermentation. Further in vivo research is warranted to support BSS supplementation to cattle with high dietary sulfate.

Development of a New Assay for Measuring H2S Production during Alcoholic Fermentation: Application to the Evaluation of the Main Factors Impacting H2S Production by Three Saccharomyces cerevisiae Wine Strains

Hydrogen sulfide (H2S) is the main volatile sulfur compound produced by Saccharomyces cerevisiae during alcoholic fermentation and its overproduction leads to poor wine sensory profiles. Several factors modulate H2S production and winemakers and researchers require an easy quantitative tool to quantify their impact. In this work, we developed a new sensitive method for the evaluation of total H2S production during alcoholic fermentation using a metal trap and a fluorescent probe. With this method, we evaluated the combined impact of three major factors influencing sulfide production by wine yeast during alcoholic fermentation: assimilable nitrogen, sulfur dioxide and strain, using a full factorial experimental design. All three factors significantly impacted H2S production, with variations according to strains. This method enables large experimental designs for the better understanding of sulfide production by yeasts during fermentation.

H2O2-Mediated Oxidative Stress Enhances Cystathionine γ-Lyase-Derived H2S Synthesis via a Sulfenic Acid Intermediate

Hydrogen sulfide (H2S), which is generated mainly by cystathionine γ-lyase (CSE) in the cardiovascular system, plays a pivotal role in a wide range of physiological and pathological processes. However, the regulatory mechanism of the CSE/H2S system is poorly understood. Herein, we show that oxidation induces the disulfide bond formation between Cys252 and Cys255 in the CXXC motif, thus stimulating the H2S-producing activity of CSE. The activity of oxidized CSE is approximately 2.5 fold greater than that of the reduced enzyme. Molecular dynamics and molecular docking suggest that the disulfide bond formation induces the conformational change in the active site of CSE and consequently increases the affinity of the enzyme for the substrate L-cysteine. Mass spectrometry and mutagenesis studies further established that the residue Cys255 is crucial for oxidation sensing. Oxidative stress-mediated sulfenylation of Cys255 leads to a sulfenic acid intermediate that spontaneously forms an intramolecular disulfide bond with the vicinal thiol group of Cys252. Moreover, we demonstrate that exogenous hydrogen peroxide (H2O2) and endogenous H2O2 triggered by vascular endothelial growth factor (VEGF) promote cellular H2S production through the enhancement of CSE activity under oxidative stress conditions. By contrast, incubation with H2O2 or VEGF did not significantly enhance cellular H2S production in the presence of PEG-catalase, an enzymatic cell-permeable H2O2 scavenger with high H2O2 specificity. Taken together, we report a new posttranslational modification of CSE that provides a molecular mechanism for H2O2/H2S crosstalk in cells under oxidative stress.

The Gasotransmitter Hydrogen Sulfide (H2S) Prevents Pathologic Calcification (PC) in Cartilage

Pathologic calcification (PC) is a painful and disabling condition whereby calcium-containing crystals deposit in tissues that do not physiologically calcify: cartilage, tendons, muscle, vessels and skin. In cartilage, compression and inflammation triggered by PC leads to cartilage degradation typical of osteoarthritis (OA). The PC process is poorly understood and treatments able to target the underlying mechanisms of the disease are lacking. Here we show a crucial role of the gasotransmitter hydrogen sulfide (H2S) and, in particular, of the H2S-producing enzyme cystathionine γ-lyase (CSE), in regulating PC in cartilage. Cse deficiency (Cse KO mice) exacerbated calcification in both surgically-induced (menisectomy) and spontaneous (aging) murine models of cartilage PC, and augmented PC was closely associated with cartilage degradation (OA). On the contrary, Cse overexpression (Cse tg mice) protected from these features. In vitro, Cse KO chondrocytes showed increased calcification, potentially via enhanced alkaline phosphatase (Alpl) expression and activity and increased IL-6 production. The opposite results were obtained in Cse tg chondrocytes. In cartilage samples from patients with OA, CSE expression inversely correlated with the degree of tissue calcification and disease severity. Increased cartilage degradation in murine and human tissues lacking or expressing low CSE levels may be accounted for by dysregulated catabolism. We found higher levels of matrix-degrading metalloproteases Mmp-3 and -13 in Cse KO chondrocytes, whereas the opposite results were obtained in Cse tg cells. Finally, by high-throughput screening, we identified a novel small molecule CSE positive allosteric modulator (PAM), and demonstrated that it was able to increase cellular H2S production, and decrease murine and human chondrocyte calcification and IL-6 secretion. Together, these data implicate impaired CSE-dependent H2S production by chondrocytes in the etiology of cartilage PC and worsening of secondary outcomes (OA). In this context, enhancing CSE expression and/or activity in chondrocytes could represent a potential strategy to inhibit PC.