Increasing evidence has revealed that hydrogen sulfide (H2S) has beneficial effects in the treatment of various cardiovascular diseases. However, whether H2S can attenuate the development of dilated cardiomyopathy (DCM) remains unclear. In this study, we generated a rat model of DCM induced by doxorubicin and investigated the protective effects of H2S against DCM. Cardiac structure and function were analyzed by two-dimensional echocardiography. Oxidative stress was evaluated by measuring malondialdehyde, superoxide dismutase, glutathione peroxidase and reactive oxygen species. Cardiomyocyte apoptosis was assessed by flow cytometry following Annexin V/PI staining. Our results showed that exogenous administration of H2S could improve left ventricular structure and function in DCM rats. H2S was found to suppress doxorubicin-induced oxidative stress by activating the Nrf2 pathway and upregulating the expression of antioxidant proteins NQO1 and GCLM. Moreover, H2S was also found to inhibit doxorubicin-induced cardiomyocyte apoptosis by activating the PI3K/Akt signaling pathway. In conclusion, our study demonstrates that H2S protects against doxorubicin-induced DCM via attenuation of oxidative stress and apoptosis.

This work investigates the role of hydrogen sulfide (H2S) in the browning and regulating the antioxidant defensive system in fresh-cut Chinese water chestnuts. The samples were fumigated with 0, 10, and 15 μl L-1 of H2S and stored at 10°C for 8 days. The results indicated that the H2S treatment significantly inhibited the browning of fresh-cut Chinese water chestnuts, reduced superoxide anion ( O 2 · - ) production rate and H2O2 content accumulation, promoted the increase of total phenol content, and enhanced activities of catalase (CAT), superoxide dismutase (SOD), ascorbate peroxidase (APX), and glutathione reductase (GR) (P < 0.05). On the other hand, phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO), and peroxidase (POD) activities remained at a low level in the H2S treatment (P < 0.05). This result suggested that H2S treatment might be a promising approach to inhibit browning and prolong the shelf life by enhancing oxidation resistance and inhibiting browning enzyme activity of fresh-cut Chinese water chestnuts during storage. Among them, the 15 μl L-1 H2S treatment had the best effect on fresh-cut Chinese water chestnuts.

The protein-folding mechanism remains a major puzzle in life science. Purified soluble activation-induced cytidine deaminase (AID) is one of the most difficult proteins to obtain. Starting from inclusion bodies containing a C-terminally truncated version of AID (residues 1-153; AID153), an optimized in vitro folding procedure was derived to obtain large amounts of AID153, which led to crystals with good quality and to final structural determination. Interestingly, it was found that the final refolding yield of the protein is proline residue-dependent. The difference in the distribution of cis and trans configurations of proline residues in the protein after complete denaturation is a major determining factor of the final yield. A point mutation of one of four proline residues to an asparagine led to a near-doubling of the yield of refolded protein after complete denaturation. It was concluded that the driving force behind protein folding could not overcome the cis-to-trans proline isomerization, or vice versa, during the protein-folding process. Furthermore, it was found that successful refolding of proteins optimally occurs at high pH values, which may mimic protein folding in vivo. It was found that high pH values could induce the polarization of peptide bonds, which may trigger the formation of protein secondary structures through hydrogen bonds. It is proposed that a hydrophobic environment coupled with negative charges is essential for protein folding. Combined with our earlier discoveries on protein-unfolding mechanisms, it is proposed that hydrogen bonds are a primary driving force for de novo protein folding.

Cervical cancer is considered one of the diseases with the highest mortality among women and with limited treatment options. Hydrogen (H2) inhalation has been reported to have a variety of tumor‑suppressive effects, but the exact mechanism remains unclear. In the present study, HeLa cervical cancer cells and HaCaT keratinocytes treated with H2, and a HeLa xenograft mouse model subjected to H2 inhalation were established. TUNEL, Cell Counting Kit‑8 and Ki67 staining assays were used to detect cell apoptosis and proliferation. Oxidative stress was determined according to the levels of reactive oxygen species, malondialdehyde and superoxide dismutase. Tumor growth was recorded every 3 days, and the excised tumors were stained with hematoxylin and eosin. High‑throughput RNA sequencing and subsequent Gene Ontology (GO) enrichment analysis were performed in HeLa‑treated and un‑treated HeLa cells. The expression of hypoxia‑inducible factor (HIF)‑1α and NF‑κB p65 was verified by western blotting, immunohistochemistry and reverse transcription‑quantitative PCR. The results revealed an increased apoptosis rate, and reduced cell proliferation and oxidative stress in H2‑treated HeLa cells but not in HaCaT cells. Similarly, decreased tumor growth and cell proliferation, and enhanced cell apoptosis were observed in H2‑treated HeLa tumors. RNA sequencing and GO analysis suggest that downregulated HIF1A (HIF‑1α mRNA) and RelA (NF‑κB p65) levels, and reduced NF‑κB signaling were associated with the antitumor effect of H2. Finally, decreased HIF‑1α and NF‑κB p65 expression both at the transcriptional and translational levels were observed in H2‑treated HeLa cells and in HeLa‑derived tumors. In conclusion, the present study reveals a novel mechanism of H2 against cervical cancer, which may serve as a potential therapeutic target in clinical practice.

As an antioxidant, hydrogen (H2) can selectively react with the highly toxic hydroxyl radical (·OH) in tumor cells to break the balance of reactive oxygen species (ROS) and cause oxidative stress. However, due to the high diffusibility and storage difficulty of hydrogen, it is impossible to achieve long-term release at the tumor site, which highly limited their therapeutic effect.

Hyperhomocysteinemia (Hhcy) is implicated in certain retinal neurovascular diseases, although whether it is causative remains uncertain. In isolated ganglion cells (GCs), mild Hhcy induces profound death, whereas retinal phenotypes in Hhcy mice caused by mutations in remethylation (methylene tetrahydrofolatereductase [Mthfr+/-]) or transsulfuration pathways (cystathionine β-synthase [Cbs+/-]) demonstrate mild GC loss and mild vasculopathy. The current work investigated compensation in vivo of one pathway for the other, and, because the transsulfuration pathway yields cysteine necessary for formation of glutathione (GSH), taurine, and hydrogen sulfide (H2S), they were analyzed also.

Hydrogen gas is neuroprotective in cerebral ischemia animal models. In this study, we tested the neuroprotective effects of hydrogen saline, which is safe and easy to use clinically, in a rat model of middle cerebral artery occlusion (MCAO). Sprague-Dawley male rats weighting 250-280 g were divided into sham, MCAO plus hydrogen saline and MCAO groups, and subjected to 90-min ischemia followed by 24 h of reperfusion. Hydrogen saline was injected intraperitoneally at 1 ml/100 g body weight. Infarct volume and brain water content were evaluated at different time points after reperfusion. Oxidative stress, inflammation, and apoptotic cell death markers were measured. Hydrogen saline significantly reduced the infarct volume and edema and improved the neurological function, when it was administered at 0, 3 and 6 h after reperfusion. Hydrogen saline decreased 8-hydroxyl-2'-deoxyguanosine (8-OHdG), reduced malondidehyde, interleukin-1β, tumor necrosis factor-α, and suppressed caspase 3 activity in the ischemic brain. These findings demonstrated hydrogen saline is neuroprotective when administered within 6 h after ischemia. Because hydrogen saline is safe and easy to use, it has clinical potentials to reduce neurological injuries.

Rare earth elements have excellent catalytic effects on improving hydrogen storage properties of the Mg2Ni-based alloys. This study used a small amount of Y to substitute Mg partially in Mg2Ni0.9Co0.1 and characterized and discussed the effects of Y on the solidification and de-/hydrogenation behaviors. The Mg2-xYxNi0.9Co0.1 (x = 0, 0.2, 0.3, and 0.4) hydrogen storage alloys were prepared using a metallurgy method. The phase composition of the alloys was studied using X-ray diffraction (XRD). Additionally, their microstructure and chemical composition were studied using scanning electron microscopy and energy-dispersive X-ray spectroscopy, respectively. The hydrogen absorption and desorption properties of the alloys were studied using pressure-composition isotherms and differential scanning calorimetric (DSC) measurements. The structure of the as-cast Mg2Ni0.9Co0.1 alloy was composed of the peritectic Mg2Ni, eutectic Mg-Mg2Ni, and a small amount of pre-precipitated Mg-Ni-Co ternary phases, and was converted into the Mg2NiH4, Mg2Ni0.9Co0.1H4, and MgH2 phases after hydrogen absorption. Furthermore, the XRD patterns of the alloys showed the MgYNi4 phase and a trace amount of the Y2O3 phase along with the Mg and Mg2Ni phases after the addition of Y. After hydrogen absorption, the phase of the alloys was composed of the Mg2NiH4, MgH2, MgYNi4, YH3, Y2O3, and Mg2NiH0.3 phases. With the increase of Y addition, the area ratios of the peritectic Mg2Ni matrix phase in the Mg2-xYxNi0.9Co0.1 (x = 0, 0.2, 0.3, and 0.4) alloys gradually decreased until they disappeared. However, the eutectic structure gradually increased, and the microstructures of the alloys were obviously refined. The addition of Y improves the activation performance of the alloys. The alloy only needed one cycle of de-/hydrogenation to complete the activation for x = 0.4. The DSC curves showed that the initial dehydrogenation temperatures of Mg2Ni0.9Co0.1 and Mg1.8Y0.2Ni0.9Co0.1 were 200 and 156 °C, respectively. The desorption activation energies of the hydrides of the Mg2Ni0.9Co0.1 and Mg1.8Y0.2Ni0.9Co0.1 alloys calculated using the Kissinger method were 94.7 and 56.5 kJ/mol, respectively. Moreover, the addition of Y reduced the initial desorption temperature of the alloys and improved their kinetic properties.

Oxidative damage is a major cause of lung diseases, including pulmonary fibrosis. Laminarin is a kind of polysaccharide extracted from brown algae and plays vital roles in various biological processes. However, the functions and mechanisms of laminarin in pulmonary oxidative damage are poorly understood. This study aimed at investigating the protective effect of laminarin against pulmonary oxidative damage and underlying mechanisms. Human lung fibroblasts MRC-5 cells were treated with hydrogen peroxide to induce oxidative damage. Laminarin treatment was performed before or after hydrogen peroxide treatment, and then major indexes of oxidative damage, including superoxide dismutase (SOD), malondialdehyde (MDA), reduced glutathione (GSH) and catalase (CAT), were quantified by biochemical assays. The expression of oxidation-related factor, nuclear factor erythroid 2 like 2 (NRF2) was analyzed by qPCR, Western blot and immunofluorescence assay. NRF2 knockdown and overexpression were performed by cell transfection to reveal possible mechanisms. Results showed that laminarin treatment of 0.020 mg/mL for 24 h, especially the pre-treatment, could significantly relieve changes in SOD, MDA, GSH and CAT that were altered by hydrogen peroxide, and promote NRF2 mRNA (P < 0.001). NRF2 protein was also elevated by laminarin, and nuclear translocation was observed. Factors in NRF2 signaling pathways, including KEAP1, NQO1, GCLC and HO1, were all regulated by laminarin. Roles of NRF2 were tested, suggesting that NRF2 regulated the concentration of SOD, MDA, GSH and CAT, suppressed KEAP1, and promoted NQO1, GCLC and HO1. These findings suggested the protective role of laminarin against pulmonary oxidative damage, which might involve the regulation of NRF2 signaling pathways. This study provided information for the clinical application of laminarin to pulmonary diseases like pulmonary fibrosis.

Probiotics are widely used for protection against stress-induced intestinal dysfunction. Oxidative stress plays a critical role in gastrointestinal disorders. It is established that probiotics alleviate oxidative stress; however, the mechanism of action has not been elucidated. We developed an in vitro intestinal porcine epithelial cells (IPEC-J2) model of oxidative stress to explore the antioxidant effect and potential mode of action of Lactobacillus plantarum ZLP001. The IPEC-J2 cells were preincubated with and without L. plantarum ZLP001 for 3 h and then exposed to hydrogen peroxide (H2O2) for 4 h. Pretreatment with L. plantarum ZLP001 protected IPEC-J2 cells against H2O2-induced oxidative damage as indicated by cell viability assays and significantly alleviated apoptosis elicited by H2O2. L. plantarum ZLP001 pretreatment decreased reactive oxygen species production and the cellular malondialdehyde concentration and increased the mitochondrial membrane potential compared with H2O2 treatment alone, suggesting that L. plantarum ZLP001 promotes the maintenance of redox homeostasis in the cells. Furthermore, L. plantarum ZLP001 regulated the expression and generation of some antioxidant enzymes, thereby activating the antioxidant defense system. Treatment with L. plantarum ZLP001 led to nuclear erythroid 2-related factor 2 (Nrf2) enrichment in the nucleus compared with H2O2 treatment alone. Knockdown of Nrf2 significantly weakened the alleviating effect of L. plantarum ZLP001 on antioxidant stress in IPEC-J2 cells, suggesting that Nrf2 is involved in the antioxidative effect of L. plantarum ZLP001. Collectively, these results indicate that L. plantarum ZLP001 is a promising probiotic bacterium that can potentially alleviate oxidative stress.

Hydrogen gas is recently proven to have anti-oxidative and anti-inflammation effects on ischemia-reperfusion injury. However, the efficacy of hydrogen therapy is limited by the efficiency of hydrogen storage, targeted delivery, and controlled release. In this study, H2 -PFOB nanoemulsions (NEs) is developed with high hydrogen loading capacity for targeted ischemic myocardium precision therapy. The hydrogen-carrying capacity of H2 -PFOB NEs is determined by gas chromatography and microelectrode methods. Positive uptake of H2 -PFOB NEs in ischemia-reperfusion myocardium and the influence of hydrogen on 19 F-MR signal are quantitatively visualized using a 9.4T MR imaging system. The biological therapeutic effects of H2 -PFOB NEs are examined on a myocardial ischemia-reperfusion injury mouse model. The results illustrated that the developed H2 -PFOB NEs can efficaciously achieve specific infiltration into ischemic myocardium and exhibit excellent antioxidant and anti-inflammatory properties on myocardial ischemia-reperfusion injury, which can be dynamically visualized by 19 F-MR imaging system. Moreover, hydrogen burst release induced by low-intensity focused ultrasound (LIFU) irradiation further promotes the therapeutic effect of H2 -PFOB NEs with a favorable biosafety profile. In this study, the potential therapeutic effects of H2 -PFOB NEs is fully unfolded, which may hold great potential for future hydrogen-based precision therapeutic applications tailored to ischemia-reperfusion injury.

Intracellular ribonucleotide (RN) and deoxyribonucleotide (dRN) pool sizes are critical for the fidelity of DNA synthesis. They are likely to be severely perturbed by many factors which disrupt the integrity and stability of DNA, leading to DNA damage. Exogenously supplied nucleosides are able to increase the deoxynucleoside triphosphate pools, then reverse the DNA damage, and decrease the oncogene-induced transformation dramatically. In this study, the impact of thymidine on the hydrogen peroxide (H2O2)-induced DNA damage was investigated in HepG2 liver cancer cells. From the result of the comet assay, the tail length of cells in the thymidine 600 μM + H2O2 1.0 mM group was dramatically decreased from 42.1 ± 10.8 to 21.9 ± 2.4 μm compared to that exposed with 1.0 mM H2O2 (p < 0.05), suggesting that pretreatment of thymidine reduced the DNA damage of HepG2 cells. Although the RN and dRN contents decreased in the damage group, most of them presented increasing tendency when pretreated with thymidine, especially the key metabolites dCTP, which was mainly related with the decline in the rate of DNA synthesis. The restoration also showed a significant G0/G1 phase arrest of cell cycle progression from 44.6 ± 2.2 to 56.6 ± 0.4% after pretreated with thymidine (p < 0.05). In conclusion, our data demonstrated that the pretreatment with thymidine had a potential protective ability against oxidative damage for DNA in HepG2 cells through the perturbation of RN and dRN pools as well as cell cycle arrest, which should provide new insights into the molecular basis of preventing H2O2-induced oxidative DNA damage in mammalian cells.

The grand challenge in the development of atomically dispersed metallic catalysts is their low metal-atom loading density, uncontrollable localization and ambiguous interactions with supports, posing difficulty in maximizing their catalytic performance. Here, we achieve an interface catalyst consisting of atomic cobalt array covalently bound to distorted 1T MoS2 nanosheets (SA Co-D 1T MoS2). The phase of MoS2 transforming from 2H to D-1T, induced by strain from lattice mismatch and formation of Co-S covalent bond between Co and MoS2 during the assembly, is found to be essential to form the highly active single-atom array catalyst. SA Co-D 1T MoS2 achieves Pt-like activity toward HER and high long-term stability. Active-site blocking experiment together with density functional theory (DFT) calculations reveal that the superior catalytic behaviour is associated with an ensemble effect via the synergy of Co adatom and S of the D-1T MoS2 support by tuning hydrogen binding mode at the interface.

Hydrogen sulfide (H2S) has recently been identified as an endogenous gaseous signaling molecule. The aim of the present study was to investigate the effect of H2S on high glucose- (HG-) induced ADAM17 expression and sFlt-1 production in 3T3-L1 adipocytes. Firstly, we found that HG DMEM upregulated the expression of ADAM17 and production of sFlt-1 in 3T3-L1 adipocytes. Knocking down ADAM17 attenuated the effect of high glucose on sFlt-1 production in adipocytes. HG decreased the expression of CSE and 3-MST, as well as the endogenous H2S production. Furthermore, knocking down CSE and 3-MST significantly increased ADAM17 expression and sFlt-1 production. The addition of exogenous H2S through the administration of sodium hydrosulfide (NaHS) inhibited HG-induced upregulation of ADAM17 expression and sFlt-1 production. In conclusion, decreased expression of CSE and 3-MST and the subsequent decrease in H2S production contribute to high glucose-induced sFlt-1 production via activating ADAM17 in adipocytes. Exogenous H2S donor NaHS has a potential therapeutic value for diabetic vascular complications.

Guard cells shrink in response to drought stress and abscisic acid (ABA) signaling, thereby reducing stomatal aperture. Hydrogen peroxide (H2O2) is an important signaling molecule acting to induce stomatal closure. As yet, the molecular basis of control over the level of H2O2 in the guard cells remains largely unknown. Here, the leucine-rich repeat (LRR)-receptor-like kinase (RLK) protein HSL3 has been shown to have the ability to negatively regulate stomatal closure by modulating the level of H2O2 in the guard cells. HSL3 was markedly up-regulated by treating plants with either ABA or H2O2, as well as by dehydration. In the loss-of-function hsl3 mutant, both stomatal closure and the activation of anion currents proved to be hypersensitive to ABA treatment, and the mutant was more tolerant than the wild type to moisture deficit; the overexpression of HSL3 had the opposite effect. In the hsl3 mutant, the transcription of NADPH oxidase gene RbohF involved in H2O2 production showed marked up-regulation, as well as the level of catalase activity was weakly inducible by ABA, allowing H2O2 to accumulate in the guard cells. HSL3 was concluded to participate in the regulation of the response to moisture deficit through ABA-induced stomatal closure triggered by the accumulation of H2O2 in the guard cells.

Hydrogen-bonded organic frameworks (HOFs), as an emerging porous material, have attracted increasing research interest in fluorescence sensing due to their inherent fluorescence emission units with unique physicochemical properties. Herein, based on the organic building block 3,3',5,5'-tetrakis-(4-carboxyphenyl)-1,1'-biphenyl (H4TCBP), the porous material HOF-TCBP was successfully synthesized using hydrogen bond self-assembly in a DMF solution. The fluorescence properties of the HOF-TCBP solution showed that when the concentration was high, excimers were easily formed, the PL emission was red-shifted, and the fluorescence intensity became weaker. HOF-TCBP showed good sensitivity and selectivity to metal ions Fe3+, Cr3+, and anion Cr2O72-. In addition, HOF-TCBP can serve as a label-free fluorescent sensor material for the sensitive and selective detection of dopamine (DA). HOF-based DA sensing is actually easy, low-cost, simple to operate, and highly selective for many potential interfering substances, and it has been successfully applied to the detection of DA in biological samples with satisfactory recoveries (101.1-104.9%). To our knowledge, this is the first report of HOF materials for efficient detection of the neurotransmitter dopamine in biological fluids. In short, this work widely broadens the application of HOF materials as fluorescent sensors for the sensing of ions and biological disease markers.

Oxidative stress contributes to intestinal dysfunction. Plant extracts can have antioxidant action; however, the specific phytogenic active ingredients and their potential mechanisms are not well known. We screened 845 phytogenic compounds using a porcine epithelial cell (IPEC-J2) oxidative stress model to identify oxidative-stress-alleviating compounds. Calycosin and deoxyshikonin were evaluated for their ability to alleviate H2O2-induced oxidative stress by measuring their effects on malondialdehyde (MDA) accumulation, reactive oxygen species (ROS) generation, apoptosis, mitochondrial membrane potential (MMP), and antioxidant defense. Nrf2 pathway activation and the effect of Nrf2 knockdown on the antioxidative effects of hit compounds were investigated. Calycosin protected IPEC-J2 cells against H2O2-induced oxidative damage, likely by improving the cellular redox state and upregulating antioxidant defense via the Nrf2-Keap1 pathway. Deoxyshikonin alleviated the H2O2-induced decrease in cell viability, ROS production, and MMP reduction, but had no significant effect on MDA accumulation and apoptosis. Nrf2 knockdown did not weaken the effect of deoxyshikonin in improving cell viability, but it weakened its effect in suppressing ROS production. These results indicate that the mechanisms of action of natural compounds differ. The newly identified phytogenic compounds can be developed as novel antioxidant agents to alleviate intestinal oxidative stress in animals.

Camellia nitidissima Chi (CNC) is a famous medicinal and edible plant with the name of "Tea for Longevity" in Guangxi province of China. In present study, we determined the protective effect of extract from CNC leaves on H2O2-induced cell injury and its underlying mechanisms in human neuroblastoma (SH-SY5Y) cells. The ethyl acetate fraction of CNC leaves (CLE, 50-200 μg/ml) treatment significantly increased the cell viability of H2O2-treated SH-SY5Y cells and reduced the leakage of LDH in a reversed "U"-shape manner. It was confirmed by Hoechst 33,342 staining that CLE attenuated H2O2-induced apoptosis in SH-SY5Y cells. The CLE (100 and 150 μg/ml) treatment significantly relieved H2O2-induced oxidative stress by decreasing intracellular ROS level, and increasing the activities of superoxide dismutase (SOD) and catalase (CAT). Western blot analysis demonstrated that the CLE treatment reserved H2O2-induced decrease of pCREB (Ser133) expression, and its downstream protein BDNF. In addition, 37 phenolic compounds in CLE were identified by UPLC-TOF MS/MS, and the main active phytochemicals seemed to be catechins, quercetin, kaempferol, and their derivatives. In conclusion, the data analysis showed that the neuroprotective effect of CNC leaves might be achieved via synergistically boosting endogenous antioxidant defenses and neurotrophic signaling pathway. These results suggest that CNC leaves are valuable resources for functional foods and beverages.

This study investigated the protective effect and the molecular mechanism of piceatannol on hydrogen peroxide (H2O2)-induced retinal pigment epithelium cell (ARPE-19) damage. Piceatannol treatment significantly inhibited H2O2-induced RPE cell death and reactive oxygen species (ROS) generation by 64.4% and 75.0%, respectively. Results of flow cytometry showed that H2O2-induced ARPE-19 cells apoptosis was ameliorated by piceatannol supplementation, along with decreased relative protein expressions of Bax/Bcl-2, Cleave-Caspase-3, and Cleave-PARP. Moreover, piceatannol treatment induced NF-E2-related factor 2 (Nrf2) signaling activation, which was evidenced by increased transcription of anti-oxidant genes, glutamate-cysteine ligase catalytic subunit (GCLc), SOD, and HO-1. Knockdown of Nrf2 through targeted siRNA alleviated piceatannol-mediated HO-1 transcription, and significantly abolished piceatannol-mediated cytoprotection. LY294002 (PI3K inhibitor) dramatically blocked piceatannol-mediated increasing of Nrf2 nuclear translocation, HO-1 expression, and cytoprotective activity, indicating the involvement of PI3K/Akt pathway in the cytoprotective effect of piceatannol. The results from this suggest the potential of piceatannol in reducing the risk of age-related macular degeneration.

Corn bran is a major agro-industrial byproduct from corn starch processing. It contains abundant arabinoxylan that can be converted into value-added chemicals via biotechnology. Corn bran arabinoxylan (CBAX) is one of the most recalcitrant xylans for enzymatic degradation due to its particular heterogeneous nature. The present study aimed to investigate the capability of the filamentous fungus Penicillium parvum 4-14 to enzymatically saccharify CBAX and reveal the fungal carbohydrate-active enzyme (CAZyme) repertoire by genome sequencing and secretome analysis.