The role of metals in hypothiocyanite resistance in Escherichia coli.

The innate immune system employs a variety of antimicrobial oxidants to control and kill host-associated bacteria. Hypothiocyanite/hypothiocyanous acid (-OSCN/HOSCN) is one such antimicrobial oxidant that is synthesized by lactoperoxidase, myeloperoxidase, and eosinophil peroxidase at sites throughout the human body. HOSCN has potent antibacterial activity while being largely non-toxic toward human cells. The molecular mechanisms by which bacteria sense and defend themselves against HOSCN have only recently begun to be elaborated, notably by the discovery of bacterial HOSCN reductase (RclA), an HOSCN-degrading enzyme widely conserved among bacteria that live on epithelial surfaces. In this paper, I show that Ni2+ sensitizes Escherichia coli to HOSCN by inhibiting glutathione reductase and that inorganic polyphosphate protects E. coli against this effect, probably by chelating Ni2+ ions. I also found that RclA is very sensitive to inhibition by Cu2+ and Zn2+, metals that are accumulated to high levels by innate immune cells, and that, surprisingly, thioredoxin and thioredoxin reductase are not involved in HOSCN stress resistance in E. coli. These results advance our understanding of the contribution of different oxidative stress responses and redox buffering pathways to HOSCN resistance in E. coli and illustrate important interactions between metal ions and the enzymes bacteria use to defend themselves against oxidative stress. IMPORTANCE: Hypothiocyanite (HOSCN) is an antimicrobial oxidant produced by the innate immune system. The molecular mechanisms by which host-associated bacteria defend themselves against HOSCN have only recently begun to be understood. The results in this paper are significant because they show that the low molecular weight thiol glutathione and enzyme glutathione reductase are critical components of the Escherichia coli HOSCN response, working by a mechanism distinct from that of the HOSCN-specific defenses provided by the RclA, RclB, and RclC proteins and that metal ions (including nickel, copper, and zinc) may impact the ability of bacteria to resist HOSCN by inhibiting specific defensive enzymes (e.g., glutathione reductase or RclA).

Sonic hedgehog signaling promotes angiogenesis of endothelial progenitor cells to improve pressure ulcers healing by PI3K/AKT/eNOS signaling.

BACKGROUND: Pressure ulcer is a severe disease in the paralyzed and aging populations. Endothelial progenitor cells (EPCs) are able to regulate ulcer healing by modulating angiogenesis, but the molecular mechanism is still obscure. Sonic hedgehog (SHH) signaling contributes to angiogenesis in various diseases and has been identified to modulate EPCs function. Here, we aimed to explore the significance of SHH signaling in EPCs function during pressure ulcers. METHODS: The EPCs were isolated and characterized by the expression of DiI-acLDL and bind fluorescein iso-thiocyanate UEA-1. Cell proliferation was detected by cell counting kit 8 (CCK-8). The DiI-acLDL and bind fluorescein iso-thiocyanate UEA-1 were analyzed by immunofluorescent analysis. The angiogenesis of EPCs was analyzed by tube formation assay. The pressure ulcers rat model was constructed, the wound injury was analyzed by H&E staining and angiogenesis was analyzed by the accumulation of CD31 based on immunofluorescent analysis. RESULTS: The expression of patched-1 and Gli-1 was enhanced by SHH activator SAG but reduced by SHH inhibitor cyclopamine in the EPCsThe PI3K, Akt, eNOS expression and the Akt phosphorylation were induced by SAG, while the treatment of cyclopamine presented a reversed result. The proliferation and migration of EPCs were enhanced by SAG but repressed by cyclopamine or PI3K/AKT/eNOS signaling inhibitor Y294002, in which the co-treatment of Y294002 could reverse the effect of SAG. CONCLUSIONS: Thus, we found that SHH signaling activated angiogenesis properties of EPCs to improve pressure ulcers healing by PI3K/AKT/eNOS signaling. SHH signaling may serve as the potential target for attenuating pressure ulcers.

Synthesis and antitumor activity of erysolin and its metabolites.

Erysolin and its two metabolites which were found in blood, ERY-GSH and ERY-NAC, were synthesized by alkylation, amination, isothiocyanation and oxidation reactions from 1-bromo-4-chlorobutane and sodium methyl mercaptide. The reaction temperature, time, feed ratios and purification method were also optimized. The synthesis method was simple, green, safe and low-cost. Erysolin, ERY-GSH and ERY-NAC showed good antitumor activities against MCF-7, HeLa, HepG2, A549 and SW480 cells, which suggested that the antitumor mechanism of erysolin can also be clarified from its metabolites in addition to itself.

Implications of the fate of hydrogen sulfide derived from assimilation of thiocyanate in rice plants.

Thiocyanate (SCN-) is a sulfur-containing pollutant, which is frequently detected in irrigation water and has negative effects on plant growth and crop yields. Uptake and assimilation of exogenous SCN- in rice plants was evident, in which two metabolic pathways, carbonyl sulfide (COS) and cyanate (CNO), are activated. Hydrogen sulfide (H2S) is an important concomitant derived from detoxification of exogenous SCN- in rice plants, which may cause coupling action on the endogenous source of H2S from sulfur metabolism. Since H2S has dual regulatory effects, the fate of H2S derived from assimilation of SCN- in plants is critical for clarifying the inclusiveness of H2S in various physiological activities. In fact, application of exogenous H2S not only positively changed the root phenotype traits of SCN--treated seedlings, but also effectively mitigated the toxic effects of SCN- in rice seedlings by stimulating the process of the PSII repair cycle. In this study, it is tempting to analyze and clarify the flux of the concomitant production of H2S from assimilation of exogenous SCN- into the innate pool, which may function in signaling regulation and other physiological processes in rice plants. This study would update our understanding of the fate of H2S derived from assimilation of SCN- in plants and provide new insights into the affirmative actions of H2S in direct proximity to SCN- exposure.

PR-619, a General Inhibitor of Deubiquitylating Enzymes, Diminishes Cisplatin Resistance in Urothelial Carcinoma Cells through the Suppression of c-Myc: An In Vitro and In Vivo Study.

Cisplatin-based chemotherapy is the standard treatment for bladder urothelial carcinoma (UC). Most patients experience chemoresistance, the primary cause of treatment failure, which leads to disease relapse. The underlying mechanism of chemoresistance involves reduced apoptosis. In this study, we investigated the antitumor effect of the deubiquitylating enzyme inhibitor PR-619 in cisplatin-resistant bladder UC. Deubiquitinase (ubiquitin-specific protease 14 (USP14) and USP21) immunohistochemical staining demonstrated that deubiquitination is related to chemoresistance in patients with metastatic UC and may be a target for overcoming chemoresistance. Cytotoxicity and apoptosis were assessed using fluorescence-activated flow cytometry and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium assay, and PR-619 was found to enhance the cytotoxic and apoptotic effects of cisplatin in cisplatin-resistant T24/R cells. Mitigated cisplatin chemoresistance was associated with the concurrent suppression of c-Myc expression in T24/R cells. Moreover, the expression of c-Myc was upregulated in human bladder UC specimens from patients with chemoresistance. Experiments in a xenograft nude mouse model confirmed that PR-619 enhanced the antitumor effects of cisplatin. These results are promising for the development of therapeutic strategies to prevent UC chemoresistance through the combined use of chemotherapeutic agents/deubiquitination inhibitors (PR-619) by targeting the c-Myc pathway.

MKP-1 modulates ubiquitination/phosphorylation of TLR signaling.

Ubiquitination and phosphorylation are reversible posttranslational protein modifications regulating physiological and pathological processes. MAPK phosphatase (MKP)-1 regulates innate and adaptive immunity. The multifaceted roles of MKP-1 were attributed to dephosphorylation of p38 and JNK MAPKs. We show that the lack of MKP-1 modulates the landscape of ubiquitin ligases and deubiquitinase enzymes (DUBs). MKP-1-/- showed an aberrant regulation of several DUBs and increased expression of proteins and genes involved in IL-1/TLR signaling upstream of MAPK, including IL-1R1, IRAK1, TRAF6, phosphorylated TAK1, and an increased K63 polyubiquitination on TRAF6. Increased K63 polyubiquitination on TRAF6 was associated with an enhanced phosphorylated form of A20. Among abundant DUBs, ubiquitin-specific protease-13 (USP13), which cleaves polyubiquitin-chains on client proteins, was substantially enhanced in murine MKP-1-deficient BMDMs. An inhibitor of USP13 decreased the K63 polyubiquitination on TRAF6, TAK1 phosphorylation, IL-1ß, and TNF-α induction in response to LPS in BMDMs. Our data show for the first time that MKP-1 modulates the ligase activity of TRAF6 through modulation of specific DUBs.

Lepidium graminifolium L.: Glucosinolate Profile and Antiproliferative Potential of Volatile Isolates.

Glucosinolates (GSLs) from Lepidium graminifolium L. were analyzed qualitatively and quantitatively by their desulfo-counterparts using UHPLC-DAD-MS/MS technique and by their volatile breakdown products-isothiocyanates (ITCs) using GC-MS analysis. Thirteen GSLs were identified with arylaliphatic as the major ones in the following order: 3-hydroxybenzyl GSL (glucolepigramin, 7), benzyl GSL (glucotropaeolin, 9), 3,4,5-trimethoxybenzyl GSL (11), 3-methoxybenzyl GSL (glucolimnanthin, 12), 4-hydroxy-3,5-dimethoxybenzyl GSL (3,5-dimethoxysinalbin, 8), 4-hydroxybenzyl GSL (glucosinalbin, 6), 3,4-dimethoxybenzyl GSL (10) and 2-phenylethyl GSL (gluconasturtiin, 13). GSL breakdown products obtained by hydrodistillation (HD) and CH2Cl2 extraction after hydrolysis by myrosinase for 24 h (EXT) as well as benzyl ITC were tested for their cytotoxic activity using MTT assay. Generally, EXT showed noticeable antiproliferative activity against human bladder cancer cell line UM-UC-3 and human glioblastoma cell line LN229, and can be considered as moderately active, while IC50 of benzyl ITC was 12.3 µg/mL, which can be considered as highly active.

Synergistic Interaction between 5-FU and an Analog of Sulforaphane-2-Oxohexyl Isothiocyanate-In an In Vitro Colon Cancer Model.

Combination therapy is based on the beneficial effects of pharmacodynamic interaction (synergistic or additive) between combined drugs or substances. A considerable group of candidates for combined treatments are natural compounds (e.g., isothiocyanates) and their analogs, which are tested in combination with anticancer drugs. We tested the anticancer effect of the combined treatment of isothiocyanate 2-oxohexyl isothiocyanate and 5-fluorouracil in colon and prostate cancer cell lines. The type of interaction was described using the Chou-Talalay method. The cytostatic and cytotoxic activities of the most promising combined treatments were investigated. In conclusion, we showed that combined treatment with 5-fluorouracil and 2-oxohexyl isothiocyanate acted synergistically in colon cancer. This activity is dependent on the cytostatic properties of the tested compounds and leads to the intensification of their individual cytotoxic activity. The apoptotic process is considered to be the main mechanism of cytotoxicity in this combined treatment.

Site-Specific Conversion of Cysteine in a Protein to Dehydroalanine Using 2-Nitro-5-thiocyanatobenzoic Acid.

Dehydroalanine exists natively in certain proteins and can also be chemically made from the protein cysteine. As a strong Michael acceptor, dehydroalanine in proteins has been explored to undergo reactions with different thiolate reagents for making close analogues of post-translational modifications (PTMs), including a variety of lysine PTMs. The chemical reagent 2-nitro-5-thiocyanatobenzoic acid (NTCB) selectively modifies cysteine to form S-cyano-cysteine, in which the S-Cß bond is highly polarized. We explored the labile nature of this bond for triggering E2 elimination to generate dehydroalanine. Our results indicated that when cysteine is at the flexible C-terminal end of a protein, the dehydroalanine formation is highly effective. We produced ubiquitin and ubiquitin-like proteins with a C-terminal dehydroalanine residue with high yields. When cysteine is located at an internal region of a protein, the efficiency of the reaction varies with mainly hydrolysis products observed. Dehydroalanine in proteins such as ubiquitin and ubiquitin-like proteins can serve as probes for studying pathways involving ubiquitin and ubiquitin-like proteins and it is also a starting point to generate proteins with many PTM analogues; therefore, we believe that this NTCB-triggered dehydroalanine formation method will find broad applications in studying ubiquitin and ubiquitin-like protein pathways and the functional annotation of many PTMs in proteins such as histones.

Inhibition of deubiquitination by PR-619 induces apoptosis and autophagy via ubi-protein aggregation-activated ER stress in oesophageal squamous cell carcinoma.

OBJECTIVES: Targeting the deubiquitinases (DUBs) has become a promising avenue for anti-cancer drug development. However, the effect and mechanism of pan-DUB inhibitor, PR-619, on oesophageal squamous cell carcinoma (ESCC) cells remain to be investigated. MATERIALS AND METHODS: The effect of PR-619 on ESCC cell growth and cell cycle was evaluated by CCK-8 and PI staining. Annexin V-FITC/PI double staining was performed to detect apoptosis. LC3 immunofluorescence and acridine orange staining were applied to examine autophagy. Intercellular Ca2+ concentration was monitored by Fluo-3AM fluorescence. The accumulation of ubi-proteins and the expression of the endoplasmic reticulum (ER) stress-related protein and CaMKKß-AMPK signalling were determined by immunoblotting. RESULTS: PR-619 could inhibit ESCC cell growth and induce G2/M cell cycle arrest by downregulating cyclin B1 and upregulating p21. Meanwhile, PR-619 led to the accumulation of ubiquitylated proteins, induced ER stress and triggered apoptosis by the ATF4-Noxa axis. Moreover, the ER stress increased cytoplasmic Ca2+ and then stimulated autophagy through Ca2+ -CaMKKß-AMPK signalling pathway. Ubiquitin E1 inhibitor, PYR-41, could reduce the accumulation of ubi-proteins and alleviate ER stress, G2/M cell cycle arrest, apoptosis and autophagy in PR-619-treated ESCC cells. Furthermore, blocking autophagy by chloroquine or bafilomycin A1 enhanced the cell growth inhibition effect and apoptosis induced by PR-619. CONCLUSIONS: Our findings reveal an unrecognized mechanism for the cytotoxic effects of general DUBs inhibitor (PR-619) and imply that targeting DUBs may be a potential anti-ESCC strategy.

Response of Pseudomonas aeruginosa to the Innate Immune System-Derived Oxidants Hypochlorous Acid and Hypothiocyanous Acid.

Pseudomonas aeruginosa is a significant nosocomial pathogen and is associated with lung infections in cystic fibrosis (CF). Once established, P. aeruginosa infections persist and are rarely eradicated despite host immune cells producing antimicrobial oxidants, including hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). There is limited knowledge as to how P. aeruginosa senses, responds to, and protects itself against HOCl and HOSCN and the contribution of such responses to its success as a CF pathogen. To investigate the P. aeruginosa response to these oxidants, we screened 707 transposon mutants, with mutations in regulatory genes, for altered growth following HOCl exposure. We identified regulators of antibiotic resistance, methionine biosynthesis, catabolite repression, and PA14_07340, the homologue of the Escherichia coli HOCl-sensor RclR (30% identical), which are required for protection against HOCl. We have shown that RclR (PA14_07340) protects specifically against HOCl and HOSCN stress and responds to both oxidants by upregulating the expression of a putative peroxiredoxin, rclX (PA14_07355). Transcriptional analysis revealed that while there was specificity in the response to HOCl (231 genes upregulated) and HOSCN (105 genes upregulated), there was considerable overlap, with 74 genes upregulated by both oxidants. These included genes encoding the type 3 secretion system, sulfur and taurine transport, and the MexEF-OprN efflux pump. RclR coordinates part of the response to both oxidants, including upregulation of pyocyanin biosynthesis genes, and, in the presence of HOSCN, downregulation of chaperone genes. These data indicate that the P. aeruginosa response to HOCl and HOSCN is multifaceted, with RclR playing an essential role.IMPORTANCE The bacterial pathogen Pseudomonas aeruginosa causes devastating infections in immunocompromised hosts, including chronic lung infections in cystic fibrosis patients. To combat infection, the host's immune system produces the antimicrobial oxidants hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). Little is known about how P. aeruginosa responds to and survives attack from these oxidants. To address this, we carried out two approaches: a mutant screen and transcriptional study. We identified the P. aeruginosa transcriptional regulator, RclR, which responds specifically to HOCl and HOSCN stress and is essential for protection against both oxidants. We uncovered a link between the P. aeruginosa transcriptional response to these oxidants and physiological processes associated with pathogenicity, including antibiotic resistance and the type 3 secretion system.

Role of thiocyanate in the modulation of myeloperoxidase-derived oxidant induced damage to macrophages.

Myeloperoxidase (MPO) is a vital component of the innate immune system, which produces the potent oxidant hypochlorous acid (HOCl) to kill invading pathogens. However, an overproduction of HOCl during chronic inflammatory conditions causes damage to host cells, which promotes disease, including atherosclerosis. As such, there is increasing interest in the use of thiocyanate (SCN-) therapeutically to decrease inflammatory disease, as SCN- is the favoured substrate for MPO, and a potent competitive inhibitor of HOCl formation. Use of SCN- by MPO forms hypothiocyanous acid (HOSCN), which can be less damaging to mammalian cells. In this study, we examined the ability of SCN- to modulate damage to macrophages induced by HOCl, which is relevant to lesion formation in atherosclerosis. Addition of SCN- prevented HOCl-mediated cell death, altered the extent and nature of thiol oxidation and the phosphorylation of mitogen activated protein kinases. These changes were dependent on the concentration of SCN- and were observed in some cases, at a sub-stoichiometric ratio of SCN-: HOCl. Co-treatment with SCN- also modulated HOCl-induced perturbations in the expression of various antioxidant and inflammatory genes. In general, the data reflect the conversion of HOCl to HOSCN, which can induce reversible modifications that are repairable by cells. However, our data also highlight the ability of HOSCN to increase pro-inflammatory gene expression and cytokine/chemokine release, which may be relevant to the use of SCN- therapeutically in atherosclerosis. Overall, this study provides further insight into the cellular pathways by which SCN- could exert protective effects on supplementation to decrease the development of chronic inflammatory diseases, such as atherosclerosis.

Ruthenium Bipyridyl Dithiocyanate Complex Exerted Adjuvant Activity on the Activated Mammalian Macrophages in vitro.

A cell's function can be regulated through its mechanism, and there has been a growing body of literature on how immune cells' metabolism shapes its overall immune response. Manipulation of the cells metabolic activity through a biocompatible material would present new venues to the field of medicine. These agents are known as immunomodulatory and immunostimulatory reagents. They can either stimulate the immune response in a disease case where the immune response is lacking the strength or they can determine the nature and strength of the immune response as an immunomodulator according to our needs to cope with certain disorders. In our recent studies, we have been examining different kinds of materials on the macrophages in order to delineate their immunostimulatory or immunomodulatory potentials. Ruthenium-based materials have gathered our attention due to their ability to get involved into the electron mobility processes in the solar cells. In line with our expectations, probably by interfering the electron transport processes of the macrophages, ruthenium bipyridyl dithiocyanate complex had a stark immunomodulatory function on the LPS-activated mammalian macrophages in vitro. Our results support that it can be utilized as an adjuvant in the new generation vaccines.

Deubiquitinases Maintain Protein Homeostasis and Survival of Cancer Cells upon Glutathione Depletion.

Cells are subjected to oxidative stress during the initiation and progression of tumors, and this imposes selective pressure for cancer cells to adapt mechanisms to tolerate these conditions. Here, we examined the dependency of cancer cells on glutathione (GSH), the most abundant cellular antioxidant. While cancer cell lines displayed a broad range of sensitivities to inhibition of GSH synthesis, the majority were resistant to GSH depletion. To identify cellular pathways required for this resistance, we carried out genetic and pharmacologic screens. Both approaches revealed that inhibition of deubiquitinating enzymes (DUBs) sensitizes cancer cells to GSH depletion. Inhibition of GSH synthesis, in combination with DUB inhibition, led to an accumulation of polyubiquitinated proteins, induction of proteotoxic stress, and cell death. These results indicate that depletion of GSH renders cancer cells dependent on DUB activity to maintain protein homeostasis and cell viability and reveal a potentially exploitable vulnerability for cancer therapy.

Anticancer properties of erucin, an H2 S-releasing isothiocyanate, on human pancreatic adenocarcinoma cells (AsPC-1).

Plants of the Brassicaceae family are well-known for containing the glucosinolate myrosinase system, which is able to release isothiocyanates after plant biotic and abiotic lesions. Erucin (ERU; 1-isothiocyanato-4-(methylthio)-butane), an isothiocyanate particularly abundant in arugula (Eruca sativa Mill., Eruca vesicaria L., etc.), derives from the hydrolysis of the glucosinolate glucoerucin by the enzyme myrosinase. Many other natural isothiocyanates influence cancer cells and, in particular, induce antiproliferative effects at relatively high concentrations. Similar antiproliferative effects have also been shown by the newly emerging gasotransmitter hydrogen sulfide (H2 S) and by H2 S-releasing compounds. In a previous study, our group demonstrated that isothiocyanates release H2 S in biological environments. In this work, we demonstrated the H2 S-donor properties of ERU in pancreatic adenocarcinoma cells (AsPC-1) and delineated its profile as a chemopreventive or anticancer agent. Indeed, ERU showed significant antiproliferative effects: ERU inhibited AsPC-1 cell viability at relatively high concentrations (30-100 µM). Moreover, ERU inhibited cell migration, altered the AsPC-1 cell cycle, and exhibited proapoptotic effects. Finally, ERU inhibited ERK1/2 phosphorylation. This mechanism is particularly important in AsPC-1 cells because they are characterized by a mutation in KRAS that determines KRAS hyperactivation followed by MAP-kinase hyperphosphorylation, which plays a pivotal role in pancreatic cancer proliferation, growth, and survival.

In Vitro Evaluation of Sulforaphane and a Natural Analog as Potent Inducers of 5-Fluorouracil Anticancer Activity.

Isothiocyanates (R-NCS) are sulphur-containing phytochemicals. The main source are plants of the Brassicaceae family. The best known plant-derived isothiocyanate is sulforaphane that has exhibited anticancer activity in both in vivo and in vitro studies. Recent attempts to expand their use in cancer therapy involve combining them with standard chemotherapeutics in order to increase their therapeutic efficacy. The aim of this paper is to determine the impact of sulforaphane and its natural analog alyssin on the anticancer activity of the well-known anticancer drug 5-fluorouracil. The type of drug-drug interactions was determined in prostate and colon cancer cell lines. Confocal microscopy, western blot and flow cytometry methods were employed to determine the mechanism of cytotoxic and cytostatic action of the combinations. The study revealed that additive or synergistic interactions were observed between 5-fluorouracil and both isothiocyanates, which enhanced the anticancer activity of 5-fluorouracil, particularly in colon cancer cell lines. An increased cytostatic effect was observed in case of alyssin while for sulforaphane the synergistic interaction with 5-fluorouracil involved an intensification of apoptotic cell death.

Remodeling the endoplasmic reticulum proteostasis network restores proteostasis of pathogenic GABAA receptors.

Biogenesis of membrane proteins is controlled by the protein homeostasis (proteostasis) network. We have been focusing on protein quality control of γ-aminobutyric acid type A (GABAA) receptors, the major inhibitory neurotransmitter-gated ion channels in mammalian central nervous system. Proteostasis deficiency in GABAA receptors causes loss of their surface expression and thus function on the plasma membrane, leading to epilepsy and other neurological diseases. One well-characterized example is the A322D mutation in the α1 subunit that causes its extensive misfolding and expedited degradation in the endoplasmic reticulum (ER), resulting in autosomal dominant juvenile myoclonic epilepsy. We aimed to correct misfolding of the α1(A322D) subunits in the ER as an approach to restore their functional surface expression. Here, we showed that application of BIX, a specific, potent ER resident HSP70 family protein BiP activator, significantly increases the surface expression of the mutant receptors in human HEK293T cells and neuronal SH-SY5Y cells. BIX attenuates the degradation of α1(A322D) and enhances their forward trafficking and function. Furthermore, because BiP is one major target of the two unfolded protein response (UPR) pathways: ATF6 and IRE1, we continued to demonstrate that modest activations of the ATF6 pathway and IRE1 pathway genetically enhance the plasma membrane trafficking of the α1(A322D) protein in HEK293T cells. Our results underlie the potential of regulating the ER proteostasis network to correct loss-of-function protein conformational diseases.

Ruthenium pyridyl thiocyanate complex increased the production of pro-inflammatory TNFα and IL1ß cytokines by the LPS stimulated mammalian macrophages in vitro.

Every cell in our body depends on the electron transport processes in order to generate energy and function properly. Being able to regulate the metabolic activity of a cell would enable us altering its function and eventually lead us to a desired biological outcome at the cellular level and more desirably at a systemic level. Immunomodulatory or immunostimulatory molecules have been focus of the recent studies in order to regulate or boost the activities of the immune system cells and suppress or eliminate the disease conditions such as cancer, autoimmune reactions, inflammatory disorders as well as infections. In our study we used a ruthenium pyridyl thiocyanate complex, K330, to examine its effect on the activity of the innate immune system cells, macrophages in vitro. K330 was our candidate due to its application in the solar cells. Especially, due to its ability to get involved in electron transfer systems we hypothesized that it could change the activity of the immune system cells at cellular level, possibly by interfering the electron transfer reactions of the cells. Our results support our hypothesis since K330 lead to a significant increase in TNFα and IL1ß cytokine production levels by LPS stimulated macrophages compared to only LPS treated control groups. Based on our in vitro results, K330 can also be utilized as an adjuvant candidate in vaccinations where the antigen itself is not sufficient to generate a proper immune response.

Histone H2A monoubiquitylation and p38-MAPKs regulate immediate-early gene-like reactivation of latent retrovirus HTLV-1.

It is not understood how the human T cell leukemia virus human T-lymphotropic virus-1 (HTLV-1), a retrovirus, regulates the in vivo balance between transcriptional latency and reactivation. The HTLV-1 proviral plus-strand is typically transcriptionally silent in freshly isolated peripheral blood mononuclear cells from infected individuals, but after short-term ex vivo culture, there is a strong, spontaneous burst of proviral plus-strand transcription. Here, we demonstrate that proviral reactivation in freshly isolated, naturally infected primary CD4+ T cells has 3 key attributes characteristic of an immediate-early gene. Plus-strand transcription is p38-MAPK dependent and is not inhibited by protein synthesis inhibitors. Ubiquitylation of histone H2A (H2AK119ub1), a signature of polycomb repressive complex-1 (PRC1), is enriched at the latent HTLV-1 provirus, and immediate-early proviral reactivation is associated with rapid deubiquitylation of H2A at the provirus. Inhibition of deubiquitylation by the deubiquitinase (DUB) inhibitor PR619 reverses H2AK119ub1 depletion and strongly inhibits plus-strand transcription. We conclude that the HTLV-1 proviral plus-strand is regulated with characteristics of a cellular immediate-early gene, with a PRC1-dependent bivalent promoter sensitive to p38-MAPK signaling. Finally, we compare the epigenetic signatures of p38-MAPK inhibition, DUB inhibition, and glucose deprivation at the HTLV-1 provirus, and we show that these pathways act as independent checkpoints regulating proviral reactivation from latency.

Activity of hypothiocyanite and lactoferrin (ALX-009) against respiratory cystic fibrosis pathogens in sputum.

Objectives: To determine the antimicrobial activity of ALX-009, a combination of bovine lactoferrin and hypothiocyanite, in sputum against Pseudomonas aeruginosa and Burkholderia cepacia complex (Bcc), key pathogens causing infection in the lungs of cystic fibrosis (CF) patients. Methods: The antimicrobial activity of ALX-009 against clinical respiratory P. aeruginosa isolates was determined by time-kill assay. Sputum from CF patients was treated with ALX-009, either alone or in combination with tobramycin, and the effect on P. aeruginosa, Bcc and total sputum density was determined. Results: Time-kill assay indicated that ALX-009 was bactericidal at 24 h against 4/4 P. aeruginosa isolates under aerobic conditions, and against 3/4 isolates under anaerobic conditions. ALX-009 was also bactericidal against P. aeruginosa in sputum samples at 6 h (n = 22/24 samples) and 24 h (n = 14/24 samples), and demonstrated significantly greater activity than tobramycin at both timepoints. Activity against Bcc in sputum samples (n = 9) was also demonstrated, but the magnitude of change in Bcc density was less than for P. aeruginosa. To determine the effect of treating sputum with two doses of ALX-009, similar to current regimens for inhaled antibiotics, aliquots of a further 10 sputum samples positive for P. aeruginosa were treated with one (t = 0 h) or two doses (t = 0 h, t = 12 h) of ALX-009; treatment with two doses resulted in bactericidal activity in 7/10 samples at 34 h compared with only 3/10 samples when treatment was with one dose. Conclusions: ALX-009 demonstrates promise as a novel antimicrobial that could be used to decrease P. aeruginosa density in the lungs of people with CF.