Reactive oxygen species (ROS), generated as a by-product of mitochondrial oxidative phosphorylation, are particularly damaging to the genome of skeletal muscle because of their high oxygen consumption. Proliferating myoblasts play a key role during muscle regeneration by undergoing myogenic differentiation to fuse and restore damaged muscle. This process is severely impaired during aging and in muscular dystrophies. In this study, we investigated the role of oxidatively damaged DNA and its repair in the mitochondrial genome of proliferating skeletal muscle progenitor myoblasts cells and their terminally differentiated product, myotubes. Using the C2C12 cell line as a well-established model for skeletal muscle differentiation, we show that myoblasts are highly sensitive to ROS-mediated DNA damage, particularly in the mitochondrial genome, due to deficiency in 5' end processing at the DNA strand breaks. Ectopic expression of the mitochondrial-specific 5' exonuclease, EXOG, a key DNA base excision/single strand break repair (BER/SSBR) enzyme, in myoblasts but not in myotubes, improves the cell's resistance to oxidative challenge. We linked loss of myoblast viability by activation of apoptosis with deficiency in the repair of the mitochondrial genome. Moreover, the process of myoblast differentiation increases mitochondrial biogenesis and the level of total glutathione. We speculate that our data may provide a mechanistic explanation for depletion of proliferating muscle precursor cells during the development of sarcopenia, and skeletal muscle dystrophies.

Uveal melanoma (UM) is the most common primary intraocular malignancy with very poor prognosis. Conventional chemotherapy only rarely prolongs the survival, therefore patients require novel treatment modalities. The discovery of specific receptors for hypothalamic hormones on cancer cells has led to the development of radiolabeled and cytotoxic hormone analogs.

The positive role of PARP1 in regulation of various nuclear DNA transactions is well established. Although a mitochondrial localization of PARP1 has been suggested, its role in the maintenance of the mitochondrial DNA is currently unknown. Here we investigated the role of PARP1 in the repair of the mitochondrial DNA in the baseline and oxidative stress conditions. We used wild-type A549 cells or cells depleted of PARP1. Our data show that intra-mitochondrial PARP1 interacts with a key mitochondrial-specific DNA base excision repair (BER) enzymes, namely EXOG and DNA polymerase gamma (Polγ), which under oxidative stress become poly(ADP-ribose)lated (PARylated). Interaction between mitochondrial BER enzymes was significantly affected in the presence of PARP1. Moreover, the repair of the oxidative-induced damage to the mitochondrial DNA in PARP1-depleted cells was found to be more robust compared to control counterpart. In addition, mitochondrial biogenesis was enhanced in PARP1-depleted cells, including mitochondrial DNA copy number and mitochondrial membrane potential. This observation was further confirmed by analysis of lung tissue isolated from WT and PARP1 KO mice. In summary, we conclude that mitochondrial PARP1, in opposite to nuclear PARP1, exerts a negative effect on several mitochondrial-specific transactions including the repair of the mitochondrial DNA.

We hypothesized that an in vitro, stretch-based model of neural injury may be useful to identify compounds that decrease the cellular damage in neurotrauma.

3-mercaptopyruvate sulfurtransferase (3-MST) is an enzyme capable of synthesizing hydrogen sulfide (H2S) and polysulfides. In spite of its ubiquitous presence in mammalian cells, very few studies have investigated its contribution to homeostasis and disease development, thus the role of 3-MST remains largely unexplored. Here, we present a clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) induced 3-mst mutant zebrafish line, which will allow the study of 3-MST's role in several biological processes. The 3-mst zebrafish orthologue was identified using a bioinformatic approach and verified by its ability to produce H2S in the presence of 3-mercaptopyruvate (3-MP). Its expression pattern was analyzed during zebrafish early development, indicating predominantly an expression in the heart and central nervous system. As expected, no detectable levels of 3-Mst protein were observed in homozygous mutant larvae. In line with this, H2S levels were reduced in 3-mst-/- zebrafish. Although the mutants showed no obvious morphological deficiencies, they exhibited increased lethality under oxidative stress conditions. The elevated levels of reactive oxygen species, detected following 3-mst deletion, are likely to drive this phenotype. In line with the increased ROS, we observed accelerated fin regenerative capacity in 3-mst deficient zebrafish. Overall, we provide evidence for the expression of 3-mst in zebrafish, confirm its important role in redox homeostasis and indicate the enzyme's possible involvement in the regeneration processes.

Hydrogen sulfide (H2S) is an endogenous gaseous biological mediator, which regulates, among others, the oxidative balance of cells under normal physiological conditions, as well as in various diseases. Several previous studies have reported that H2S attenuates inflammatory mediator production. In this study, we investigated the role of H2S in chromatin modulation in an in vitro model of lipopolysaccharide (LPS)-induced inflammation and evaluated its effects on inflammatory cytokine production. Tamm-Horsfall protein 1 (THP-1) differentiated macrophages were pre-treated with sodium hydrosulfide (NaHS) (an H2S donor) at 0.01, 0.1, 0.5 or 1 mM for 30 min. To stimulate cytokine production, the cells were challenged with bacterial LPS (1 µg/ml) for 1, 4, 8 or 24 h. Histone H3 acetylation was analyzed by chromatin immunoprecipitation (ChIP), cytokine production was measured by ELISA and histone deacetylase (HDAC) activity was analyzed using a standard biochemical assay. H2S inhibited the production of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in a concentration-dependent manner; it was most effective at the two highest concentrations used. This effect was associated with a decrease in histone H3 acetylation at the IL-6 and TNF-α promoters in the cells exposed to H2S or H2S + LPS. The findings of the present study suggest that H2S suppresses histone acetylation, which, in turn, inhibits chromatin openness, leading to a decrease in the gene transcription of various pro-inflammatory cytokines. Therefore, this mechanism may contribute to the previously demonstrated anti-inflammatory effects of H2S and various H2S donors.

Burn injury is associated with a loss of gut barrier function, resulting in systemic dissemination of gut-derived bacteria and their products. The bacterial protein and TLR5 agonist, flagellin, induces non-specific innate immune responses. Because we detected flagellin in the serum of burn patients, we investigated whether gut-derived flagellin was a primary or secondary contributor to intestinal dysfunction and systemic inflammation following burn injury. The apical surface of polarized human intestinal epithelial cells (IECs), Caco-2BBe, were exposed to 50 or 500 ng of purified flagellin and 1 x 105 of an intestinal E. coli (EC) isolate as follows: 1) flagellin added 30 min prior to EC, 2) flagellin and EC added simultaneously, or 3) EC added 30 min prior to flagellin. Our results showed that luminal flagellin and EC modulated each other's biological actions, which influenced their ability to induce basolateral secretion of inflammatory cytokines and subsequent translocation of bacteria and their products. A low dose of flagellin accompanied by an enteric EC in the lumen, tempered inflammation in a dose- and time-dependent manner. However, higher doses of flagellin acted synergistically with EC to induce both intestinal and systemic inflammation that compromised barrier integrity, increasing systemic inflammation following burn injury, a process we have termed flagellemia. In a murine model of burn injury we found that oral gavage of flagellin (1 μg/mouse) significantly affected the gut microbiome after burn injury. In these mice, flagellin disseminated out of the intestine into the serum and to distal organs (mesenteric lymph nodes and lungs) where it induced secretion of monocyte chemoattractant protein (MCP-1) and CXCL1/KC (mouse equivalent of human IL-8) at 24 and 48h post-burn. Our results illustrated that gut-derived flagellin alone or accompanied by a non-pathogenic enteric EC strain can function as an initiator of luminal and systemic inflammation following burn injury.

Cystathionine-β-synthase (CBS) has been recently identified as a drug target for several forms of cancer. Currently no potent and selective CBS inhibitors are available. Using a composite collection of 8871 clinically used drugs and well-annotated pharmacological compounds (including the LOPAC library, the FDA Approved Drug Library, the NIH Clinical Collection, the New Prestwick Chemical Library, the US Drug Collection, the International Drug Collection, the 'Killer Plates' collection and a small custom collection of PLP-dependent enzyme inhibitors), we conducted an in vitro screen in order to identify inhibitors for CBS using a primary 7-azido-4-methylcoumarin (AzMc) screen to detect CBS-derived hydrogen sulfide (H2S) production. Initial hits were subjected to counterscreens using the methylene blue assay (a secondary assay to measure H2S production) and were assessed for their ability to quench the H2S signal produced by the H2S donor compound GYY4137. Four compounds, hexachlorophene, tannic acid, aurintricarboxylic acid and benserazide showed concentration-dependent CBS inhibitory actions without scavenging H2S released from GYY4137, identifying them as direct CBS inhibitors. Hexachlorophene (IC50: ∼60μM), tannic acid (IC50: ∼40μM) and benserazide (IC50: ∼30μM) were less potent CBS inhibitors than the two reference compounds AOAA (IC50: ∼3μM) and NSC67078 (IC50: ∼1μM), while aurintricarboxylic acid (IC50: ∼3μM) was equipotent with AOAA. The second reference compound NSC67078 not only inhibited the CBS-induced AzMC fluorescence signal (IC50: ∼1μM), but also inhibited with the GYY4137-induced AzMC fluorescence signal with (IC50 of ∼6μM) indicative of scavenging/non-specific effects. Hexachlorophene (IC50: ∼6μM), tannic acid (IC50: ∼20μM), benserazide (IC50: ∼20μM), and NSC67078 (IC50: ∼0.3μM) inhibited HCT116 colon cancer cells proliferation with greater potency than AOAA (IC50: ∼300μM). In contrast, although a CBS inhibitor in the cell-free assay, aurintricarboxylic acid failed to inhibit HCT116 proliferation at lower concentrations, and stimulated cell proliferation at 300μM. Copper-containing compounds present in the libraries, were also found to be potent inhibitors of recombinant CBS; however this activity was due to the CBS inhibitory effect of copper ions themselves. However, copper ions, up to 300μM, did not inhibit HCT116 cell proliferation. Benserazide was only a weak inhibitor of the activity of the other H2S-generating enzymes CSE and 3-MST activity (16% and 35% inhibition at 100μM, respectively) in vitro. Benserazide suppressed HCT116 mitochondrial function and inhibited proliferation of the high CBS-expressing colon cancer cell line HT29, but not the low CBS-expressing line, LoVo. The major benserazide metabolite 2,3,4-trihydroxybenzylhydrazine also inhibited CBS activity and suppressed HCT116 cell proliferation in vitro. In an in vivo study of nude mice bearing human colon cancer cell xenografts, benserazide (50mg/kg/days.q.) prevented tumor growth. In silico docking simulations showed that benserazide binds in the active site of the enzyme and reacts with the PLP cofactor by forming reversible but kinetically stable Schiff base-like adducts with the formyl moiety of pyridoxal. We conclude that benserazide inhibits CBS activity and suppresses colon cancer cell proliferation and bioenergetics in vitro, and tumor growth in vivo. Further pharmacokinetic, pharmacodynamic and preclinical animal studies are necessary to evaluate the potential of repurposing benserazide for the treatment of colorectal cancers.

The PARP inhibitor olaparib has recently been approved for human use for the therapy of cancer. Considering the role of PARP in critical illness, we tested the effect of olaparib in a murine model of burn injury, in order to begin exploring the feasibility of repurposing olaparib for the therapy of burn patients.

Pretreatment with low doses of LPS (lipopolysaccharide, bacterial endotoxin) reduces the pro-inflammatory response to a subsequent higher LPS dose, a phenomenon known as endotoxin tolerance. Moreover, hydrogen sulfide (H2S), an endogenous gaseous mediator (gasotransmitter) can exert anti-inflammatory effects. Here we investigated the potential role of H2S in the development of LPS tolerance. THP1 differentiated macrophages were pretreated with the H2S donor NaHS (1 mM) or the H2S biosynthesis inhibitor aminooxyacetic acid (AOAA, 1 mM).

Hydrogen sulfide (H2S) has been proposed to exert pro- as well as anti-inflammatory effects in various models of critical illness. In this study, we compared bacterial lipopolysaccharide (LPS)‑induced changes in inflammatory mediator production, indices of multiple organ injury and survival in wild‑type (WT) mice and in mice with reduced expression of one of the three H2S‑producing enzymes, cystathionine-γ-lyase (CSE), cystathionine-β-synthase (CBS) or 3-mercaptopyruvate sulfurtransferase (3MST). Mice were injected intraperitoneally (i.p.) with LPS (10 mg/kg). After 6 h, the animals were sacrificed, blood and organs were collected and the following parameters were evaluated: blood urea nitrogen (BUN) levels in blood, myeloperoxidase (MPO) and malondialdehyde (MDA) in the lung, cytokine levels in plasma and the expression of the three H2S‑producing enzymes (CBS, CSE and 3MST) in the spleen, lung, liver and kidney. LPS induced a tissue‑dependent upregulation of some of the H2S‑producing enzymes in WT mice (upregulation of CBS in the spleen, upregulation of 3MST in the liver and upregulation of CBS, CSE and 3MST in the lung). Moreover, LPS impaired glomerular function, as evidenced by increased BUN levels. Renal impairment was comparable in the CSE‑/‑ and Δ3MST mice after LPS challenge; however, it was attenuated in the CBS+/‑ mice. MPO levels (an index of neutrophil infiltration) and MDA levels (an index of oxidative stress) in lung homogenates were significantly increased in response to LPS; these effects were similar in the WT, CBS+/‑, CSE‑/‑ and Δ3MST mice; however, the MDA levels tended to be lower in the CBS+/‑ and CSE‑/‑ mice. LPS induced significant increases in the plasma levels of multiple cytokines [tumor necrosis factor (TNF)α, interleukin (IL)‑1β, IL‑6, IL‑10, IL‑12 and interferon (IFN)γ] in plasma; TNFα, IL‑10 and IL‑12 levels tended to be lower in all three groups of animals expressing lower levels of H2S‑producing enzymes. The survival rates after the LPS challenge did not show any significant differences between the four animal groups tested. Thus, the findings of this study indicate that a deficiency in 3MST does not significantly affect endotoxemia, while a deficiency in CBS or CSE slightly ameliorates the outcome of LPS-induced endotoxemia in vivo.

Hydrogen sulfide (H2S) is a gasotransmitter produced from L-cysteine through the enzymatic action of cystathionine-γ-lyase (CSE) and/or cystathionine-β-synthase. D-Penicillamine is the d isomer of a dimethylated cysteine and has been used for the treatment of rheumatoid arthritis. AsD-penicillamine is structurally very similar to cysteine, we have investigated whether D-penicillamine, as a cysteine analogue, has an effect on the H2 S pathway.

Hydrogen sulfide (H₂S) is a signalling molecule that belongs to the gasotransmitter family. Two major sources for endogenous enzymatic production of H₂S are cystathionine β synthase (CBS) and cystathionine γ lyase (CSE). In the present study, we examined the selectivity of commonly used pharmacological inhibitors of H₂S biosynthesis towards CSE and CBS.

Uveal melanoma (UM) is the most common malignant tumor of the eye. Recently, we have established that 46% of UM specimens express LHRH receptors. This finding supports the idea of a LHRH receptor-targeted therapy of UM patients. Cytotoxic analog of LHRH, AEZS-108 exhibits effective anti-cancer activity in LHRH-receptor positive cancers. AEZS-108 is a hybrid molecule, composed of a synthetic peptide carrier and the cytotoxic doxorubicin (DOX). In the present study, we investigated AEZS-108 induced cytotoxicity and the altered mRNA expression profile of regulatory factors related to angiogenesis and metastasis in LHRH receptor positive OCM3 cells. Our results show that AEZS-108 upregulates the expression of MASPIN/SERPINB5 tumor suppressor gene, which is downregulated in normal uvea and UM specimens independently from the LHRH receptor-ligand interaction. AEZS-108 also substantially downregulates hypoxia-inducible factor 1 alpha (HIF1A) expression. In order to investigate the mechanism of the induction of MASPIN by AEZS-108, OCM3 cells were treated with free DOX, D-Lys6 LHRH analog, or AEZS-108. qRT- PCR analysis revealed in OCM3 cells that AEZS-108 is a more potent inducer of MASPIN than free DOX. In conclusion, we show for the first time that AEZS-108 has a major impact in the regulation of angiogenesis thus plays a potential role in tumor suppression. Taken together, our results support the development of novel therapeutic strategies for UM focusing on LHRH receptors.