Mitochondria-targeted hydrogen sulfide donor reduces fatty liver and obesity in mice fed a high fat diet by inhibiting de novo lipogenesis and inflammation via mTOR/SREBP-1 and NF-κB signaling pathways.

Metabolic diseases that include obesity and metabolic-associated fatty liver disease (MAFLD) are a rapidly growing worldwide public health problem. The pathogenesis of MAFLD includes abnormally increased lipogenesis, chronic inflammation, and mitochondrial dysfunction. Mounting evidence suggests that hydrogen sulfide (H2S) is an important player in the liver, regulating lipid metabolism and mitochondrial function. However, direct delivery of H2S to mitochondria has not been investigated as a therapeutic strategy in obesity-related metabolic disorders. Therefore, our aim was to comprehensively evaluate the influence of prolonged treatment with a mitochondria sulfide delivery molecule (AP39) on the development of fatty liver and obesity in a high fat diet (HFD) fed mice. Our results demonstrated that AP39 reduced hepatic steatosis in HFD-fed mice, which was corresponded with decreased triglyceride content. Furthermore, treatment with AP39 downregulated pathways related to biosynthesis of unsaturated fatty acids, lipoprotein assembly and PPAR signaling. It also led to a decrease in hepatic de novo lipogenesis by downregulating mTOR/SREBP-1/SCD1 pathway. Moreover, AP39 administration alleviated obesity in HFD-fed mice, which was reflected by reduced weight of mice and adipose tissue, decreased leptin levels in the plasma and upregulated expression of adipose triglyceride lipase in epididymal white adipose tissue (eWAT). Finally, AP39 reduced inflammation in the liver and eWAT measured as the expression of proinflammatory markers (Il1b, Il6, Tnf, Mcp1), which was due to downregulated mTOR/NF-κB pathway. Taken together, mitochondria-targeted sulfide delivery molecules could potentially provide a novel therapeutic approach to the treatment/prevention of obesity-related metabolic disorders.

Combination treatment of mannose and GalNAc conjugated small interfering RNA protects against lethal Marburg virus infection.

Marburg virus (MARV) infection results in severe viral hemorrhagic fever with mortalities up to 90%, and there is a pressing need for effective therapies. Here, we established a small interfering RNA (siRNA) conjugate platform that enabled successful subcutaneous delivery of siRNAs targeting the MARV nucleoprotein. We identified a hexavalent mannose ligand with high affinity to macrophages and dendritic cells, which are key cellular targets of MARV infection. This ligand enabled successful siRNA conjugate delivery to macrophages both in vitro and in vivo. The delivered hexa-mannose-siRNA conjugates rendered substantial target gene silencing in macrophages when supported by a mannose functionalized endosome release polymer. This hexa-mannose-siRNA conjugate was further evaluated alongside our hepatocyte-targeting GalNAc-siRNA conjugate, to expand targeting of infected liver cells. In MARV-Angola-infected guinea pigs, these platforms offered limited survival benefit when used as individual agents. However, in combination, they achieved up to 100% protection when dosed 24 h post infection. This novel approach, using two different ligands to simultaneously deliver siRNA to multiple cell types relevant to infection, provides a convenient subcutaneous route of administration for treating infection by these dangerous pathogens. The mannose conjugate platform has potential application to other diseases involving macrophages and dendritic cells.

Ligand conjugate SAR and enhanced delivery in NHP.

N-Acetylgalactosamine (GalNAc) conjugated short interfering RNAs (siRNAs) are a leading RNA interference (RNAi) platform allowing targeted inhibition of disease-causing genes in hepatocytes. More than a decade of development has recently resulted in the first approvals for this class of drugs. While substantial effort has been made to improve nucleic acid modification patterns for better payload stability and efficacy, relatively little attention has been given to the GalNAc targeting ligand. In addition, the lack of an intrinsic endosomal release mechanism has limited potency. Here, we report a stepwise analysis of the structure activity relationships (SAR) of the components comprising these targeting ligands. We show that there is relatively little difference in biological performance between bi-, tri-, and tetravalent ligand structures while identifying other features that affect their biological activity more significantly. Further, we demonstrate that subcutaneous co-administration of a GalNAc-functionalized, pH responsive endosomal release agent markedly improved the activity and duration of effect for siRNA conjugates, without compromising tolerability, in non-human primates. These findings could address a significant bottleneck for future siRNA ligand conjugate development.

Experimental investigation of a combinational iron chelating protoporphyrin IX prodrug for fluorescence detection and photodynamic therapy.

Photodynamic therapy (PDT) is an oxygen-dependent, light-activated, and locally destructive drug treatment of cancer. Protoporphyrin IX (PpIX)-induced PDT exploits cancer cells' own innate heme biosynthesis to hyper-accumulate the naturally fluorescent and photoactive precursor to heme, PpIX. This occurs as a result of administering heme precursors (e.g., aminolevulinic acid; ALA) because the final step of the pathway (the insertion of ferrous iron into PpIX by ferrochelatase to form heme) is relatively slow. Separate administration of an iron chelating agent has previously been demonstrated to significantly improve dermatological PpIX-PDT by further limiting heme production. A newly synthesized combinational iron chelating PpIX prodrug (AP2-18) has been assessed experimentally in cultured primary human cells of bladder and dermatological origin, as an alternative photosensitizing agent to ALA or its methyl or hexyl esters (MAL and HAL respectively) for photodetection/PDT. Findings indicated that the technique of iron chelation (either through the separate administration of the established hydroxypyridinone iron chelator CP94 or the just as effective combined AP2-18) did not enhance either PpIX fluorescence or PDT-induced (neutral red assessed) cell death in human primary normal and malignant bladder cells. However, 500 µM AP2-18 significantly increased PpIX accumulation and produced a trend of increased cell death within epithelial squamous carcinoma cells. PpIX accumulation destabilized the actin cytoskeleton in bladder cancer cells prior to PDT and resulted in caspase-3 cleavage/early apoptosis afterwards. AP2-18 iron chelation should continue to be investigated for the enhancement of dermatological PpIX-PDT applications but not bladder photodetection/PDT.

Anti-tumour necrosis factor treatment for the prevention of ischaemic events in patients with deficiency of adenosine deaminase 2 (DADA2).

OBJECTIVE: To evaluate the impact of anti-Tumour Necrosis Factor-α (anti-TNF) treatment on the occurrence of vasculitic ischaemic events in patients with deficiency of adenosine deaminase 2 (DADA2). METHODS: A retrospective analysis of DADA2 patients referred from six centres to Great Ormond Street Hospital for Children was conducted. Ischaemic events, vasculitic disease activity, biochemical, immunological, and radiological features were compared, before and after anti-TNF treatment. RESULTS: A total of 31 patients with genetically confirmed DADA2 were included in the study. The median duration of active disease activity prior to anti-TNF treatment was 73 months (inter-quartile range [IQR] 27.5-133.5 months). Twenty seven/31 patients received anti-TNF treatment for a median of 32 months (IQR 12.0-71.5 months). The median event rate of central nervous system (CNS) and non-CNS ischemic events before anti-TNF treatment was 2.37 per 100 patient-months (IQR 1.25-3.63); compared with 0.00 per 100 patient-months (IQR 0.0-0.0) post-treatment (p< 0.0001). Paediatric vasculitis activity score (PVAS) was also significantly reduced: median score of 20/63 (IQR 13.0-25.8/63) pre-treatment vs. 2/63 (IQR 0.0-3.8/63) following anti-TNF treatment (p< 0.0001), with mild livedoid rash being the main persisting feature. Anti-TNF treatment was not effective for severe immunodeficiency or bone marrow failure, which required haematopoietic stem cell transplantation (HSCT). CONCLUSION: Anti-TNF treatment significantly reduced the incidence of ischaemic events and other vasculitic manifestations of DADA2, but was not effective for immunodeficiency or bone marrow failure.

Cardiovascular Effects and Molecular Mechanisms of Bisphenol A and Its Metabolite MBP in Zebrafish.

The plastic monomer bisphenol A (BPA) is one of the highest production volume chemicals in the world and is frequently detected in wildlife and humans, particularly children. BPA has been associated with numerous adverse health outcomes relating to its estrogenic and other hormonal properties, but direct causal links are unclear in humans and animal models. Here we simulated measured (1×) and predicted worst-case (10× ) maximum fetal exposures for BPA, or equivalent concentrations of its metabolite MBP, using fluorescent reporter embryo-larval zebrafish, capable of quantifying Estrogen Response Element (ERE) activation throughout the body. Heart valves were primary sites for ERE activation by BPA and MBP, and transcriptomic analysis of microdissected heart tissues showed that both chemicals targeted several molecular pathways constituting biomarkers for calcific aortic valve disease (CAVD), including extra-cellular matrix (ECM) alteration. ECM collagen deficiency and impact on heart valve structural integrity were confirmed by histopathology for high-level MBP exposure, and structural defects (abnormal curvature) of the atrio-ventricular valves corresponded with impaired cardiovascular function (reduced ventricular beat rate and blood flow). Our results are the first to demonstrate plausible mechanistic links between ERE activation in the heart valves by BPA's reactive metabolite MBP and the development of valvular-cardiovascular disease states.

Estrogenic Mechanisms and Cardiac Responses Following Early Life Exposure to Bisphenol A (BPA) and Its Metabolite 4-Methyl-2,4-bis( p-hydroxyphenyl)pent-1-ene (MBP) in Zebrafish.

Environmental exposure to Bisphenol A (BPA) has been associated with a range of adverse health effects, including on the cardiovascular system in humans. Lack of agreement on its mechanism(s) of action likely stem from comparisons between in vivo and in vitro test systems and potential multiple effects pathways. In rodents, in vivo, metabolic activation of BPA produces 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP), which is reported to be up to 1000 times more potent as an estrogen than BPA. We investigated the estrogenic effects and estrogen receptor signaling pathway(s) of BPA and MBP following early life exposure using a transgenic, estrogen responsive (ERE-TG) zebrafish and a targeted morpholino approach to knockdown the three fish estrogen receptor (ER) subtypes. The functional consequences of BPA exposure on the cardiovascular system of zebrafish larvae were also examined. The heart atrioventricular valves and the bulbus arteriosus were primary target tissues for both BPA and MBP in the ERE-TG zebrafish, and MBP was approximately 1000-fold more potent than BPA as an estrogen in these tissues. Estrogen receptor knockdown with morpholinos indicated that the estrogenic responses in the heart for both BPA and MBP were mediated via an estrogen receptor 1 (esr1) dependent pathway. At the highest BPA concentration tested (2500 µg/L), alterations in the atrial:ventricular beat ratio indicated a functional impact on the heart of 5 days post fertilization (dpf) larvae, and there was also a significantly reduced heart rate in these larvae at 14 dpf. Our findings indicate that some of the reported adverse effects on heart function associated with BPA exposure (in mammals) may act through an estrogenic mechanism, but that fish are unlikely to be susceptible to adverse effects on heart development for environmentally relevant exposures.

An experimental investigation of a novel iron chelating protoporphyrin IX prodrug for the enhancement of photodynamic therapy.

OBJECTIVES: Non-melanoma skin cancers are the most frequently occurring type of cancer worldwide. They can be effectively treated using topical dermatological photodynamic therapy (PDT) employing protoporphyrin IX (PpIX) as the active photosensitising agent as long as the disease remains superficial. Novel iron chelating agents are being investigated to enhance the effectiveness and extend the applications of this treatment modality, as limiting free iron increases the accumulation of PpIX available for light activation and thus cell kill. METHODS: Human lung fibroblasts (MRC-5) and epithelial squamous carcinoma (A431) cells were treated with PpIX precursors (aminolaevulinic acid [ALA] or methyl-aminolevulinate [MAL]) with or without the separate hydroxypyridinone iron chelating agent (CP94) or alternatively, the new combined iron chelator and PpIX producing agent, AP2-18. PpIX fluorescence was monitored hourly for 6 hours prior to irradiation. PDT effectiveness was then assessed the following day using the lactate dehydrogenase and neutral red assays. RESULTS: Generally, iron chelation achieved via CP94 or AP2-18 administration significantly increased PpIX fluorescence. ALA was more effective as a PpIX-prodrug than MAL in A431 cells, corresponding with the lower PpIX accumulation observed with the latter congener in this cell type. Addition of either iron chelating agent consistently increased PpIX accumulation but did not always convey an extra beneficial effect on PpIX-PDT cell kill when using the already highly effective higher dose of ALA. However, these adjuvants were highly beneficial in the skin cancer cells when compared with MAL administration alone. AP2-18 was also at least as effective as CP94 + ALA/MAL co-administration throughout and significantly better than CP94 supplementation at increasing PpIX fluorescence in MRC5 cells as well as at lower doses where PpIX accumulation was observed to be more limited. CONCLUSIONS: PpIX fluorescence levels, as well as PDT cell kill effects on irradiation can be significantly increased by pyridinone iron chelation, either via the addition of CP94 to the administration of a PpIX precursor or alternatively via the newly synthesized combined PpIX prodrug and siderophore, AP2-18. The effect of the latter compound appears to be at least equivalent to, if not better than, the separate administration of its constituent parts, particularly when employing MAL to destroy skin cancer cells. AP2-18 therefore warrants further detailed analysis, as it may have the potential to improve dermatological PDT outcomes in applications currently requiring enhancement. Lasers Surg. Med. 50:552-565, 2018. © 2018 The Authors. Lasers in Surgery and Medicine Published by Wiley Periodicals, Inc.

Effect of anticipatory stress on placebo alcohol consumption in a bar laboratory.

BACKGROUND: Only one laboratory study has examined the relation between stress and alcohol use in an ecologically valid drinking context. In that prior study, drinking was measured after the stressful situation. OBJECTIVE: To examine the effect of an anticipatory stressor, and trait social anxiety on "alcohol" consumption in a bar laboratory. METHODS: College students aged 18 and older (N = 127) in same-sex groups of two or three participants took part in a study that ostensibly examined alcohol's effect on language fluency. Using a between-subjects design, participants were randomly assigned to a stress or control condition. Participants in the stress condition anticipated giving a stressful speech for the fluency procedure, whereas those in the control group anticipated a nonstressful activity. Before the alleged fluency task, groups could order and consume mixed drinks ad lib in a bar laboratory. No beverages actually contained alcohol, but we used a validated procedure to ensure that participants included in these analyses were deceived. Primary analyses were performed with a hierarchical linear model (HLM) due to a substantial group/modeling effect. RESULTS: Counter to expectations, participants in the control group consumed more placebo alcohol than those in the stress condition. This main effect was moderated by past 3 months' drinks per week, such that the effect was attenuated (or reversed) among heavy drinkers. No main or interaction effects were observed for trait social anxiety. CONCLUSIONS: Some stressors (i.e., those invoking performance anxiety) may decrease consumption. People with higher levels of alcohol involvement might be especially motivated to drink for tension reduction purposes.

A Novel Approach for Streamlining Delivery of Brief Motivational Interventions to Mandated College Students: Using Group and Individual Sessions Matched to Level of Risk.

BACKGROUND: Mandated college students (those in violation of a campus alcohol policy) are heterogenous with respect to alcohol consumption. Thus, when universities consider required treatment for mandated students, one promising option is to match treatment intensity according to level of alcohol involvement. The present study evaluates such an approach with minimal resources. METHODS: Mandated students (N = 285) were required to complete a baseline assessment. Participants identified as high-risk (5+ past month alcohol problems AND 2+ past month heavy drinking episodes) received a one session individual Brief Motivational Intervention (I-BMI) whereas those identified as low-risk (all others) received a one session group Brief Motivational Intervention (G-BMI). I-BMI and G-BMI sessions were delivered by doctoral students in Clinical Psychology. Follow-up assessments were collected 1 month post-intervention (response rate = 73%). RESULTS: The vast majority of students complied with their requirement. Participants assigned to I-BMI reported an 82% reduction in drinks per week, a 58% reduction in heavy episodic drinking, and a 74% reduction in alcohol-related problems at a 1 month follow-up. Participants assigned to G-BMI reported a 61% reduction in drinks per week and a 42% reduction in alcohol-related problems at follow-up relative to baseline. Conclusion/Importance: We demonstrate that matching intervention intensity on baseline alcohol involvement with mandated students is feasible and associated with short-term reductions in alcohol use and consequences. Universities may wish to consider the procedure outlined here as a way of allocating more resources to those who drink at problematic levels.

Infection-Related Death among Persons with Refractory Juvenile Idiopathic Arthritis.

Severe infections are emerging as major risk factors for death among children with juvenile idiopathic arthritis (JIA). In particular, children with refractory JIA treated with long-term, multiple, and often combined immunosuppressive and antiinflammatory agents, including the new biological disease-modifying antirheumatic drugs (DMARDs), are at increased risk for severe infections and death. We investigated 4 persons with JIA who died during 1994-2013, three of overwhelming central venous catheter-related bacterial sepsis caused by coagulase-negative Staphylococus or α-hemolytic Streptococcus infection and 1 of disseminated adenovirus and Epstein-Barr virus infection). All 4 had active JIA refractory to long-term therapy with multiple and combined conventional and biological DMARDs. Two died while receiving high-dose systemic corticosteroids, methotrexate, and after recent exposure to anti-tumor necrosis factor-α biological DMARDs, and 2 during hematopoietic stem cell transplantation procedure. Reporting all cases of severe infections and especially deaths in these children is of paramount importance for accurate surveillance.

Protein kinase C is essential for viability of the rice blast fungus Magnaporthe oryzae.

Protein kinase C constitutes a family of serine-threonine kinases found in all eukaryotes and implicated in a wide range of cellular functions, including regulation of cell growth, cellular differentiation and immunity. Here, we present three independent lines of evidence which indicate that protein kinase C is essential for viability of Magnaporthe oryzae. First, all attempts to generate a target deletion of PKC1, the single copy protein kinase C-encoding gene, proved unsuccessful. Secondly, conditional gene silencing of PKC1 by RNA interference led to severely reduced growth of the fungus, which was reversed by targeted deletion of the Dicer2-encoding gene, MDL2. Finally, selective kinase inhibition of protein kinase C by targeted allelic replacement with an analogue-sensitive PKC1(AS) allele led to specific loss of fungal viability in the presence of the PP1 inhibitor. Global transcriptional profiling following selective PKC inhibition identified significant changes in gene expression associated with cell wall re-modelling, autophagy, signal transduction and secondary metabolism. When considered together, these results suggest protein kinase C is essential for growth and development of M. oryzae with extensive downstream targets in addition to the cell integrity pathway. Targeting protein kinase C signalling may therefore prove an effective means of controlling rice blast disease.

Involvement of Potassium Channels and Calcium-Independent Mechanisms in Hydrogen Sulfide-Induced Relaxation of Rat Mesenteric Small Arteries.

Endogenous hydrogen sulfide (H2S) is involved in the regulation of vascular tone. We hypothesized that the lowering of calcium and opening of potassium (K) channels as well as calcium-independent mechanisms are involved in H2S-induced relaxation in rat mesenteric small arteries. Amperometric recordings revealed that free [H2S] after addition to closed tubes of sodium hydrosulfide (NaHS), Na2S, and GYY4137 [P-(4-methoxyphenyl)-P-4-morpholinyl-phosphinodithioic acid] were, respectively, 14%, 17%, and 1% of added amount. The compounds caused equipotent relaxations in isometric myographs, but based on the measured free [H2S], GYY4137 caused more relaxation in relation to released free H2S than NaHS and Na2S in rat mesenteric small arteries. Simultaneous measurements of [H2S] and tension showed that 15 µM of free H2S caused 61% relaxation in superior mesenteric arteries. Simultaneous measurements of smooth muscle calcium and tension revealed that NaHS lowered calcium and caused relaxation of NE-contracted arteries, while high extracellular potassium reduced NaHS relaxation without corresponding calcium changes. In NE-contracted arteries, NaHS (1 mM) lowered the phosphorylation of myosin light chain, while phosphorylation of myosin phosphatase target subunit 1 remained unchanged. Protein kinase A and G, inhibitors of guanylate cyclase, failed to reduce NaHS relaxation, whereas blockers of voltage-gated KV7 channels inhibited NaHS relaxation, and blockers of mitochondrial complex I and III abolished NaHS relaxation. Our findings suggest that low micromolar concentrations of free H2S open K channels followed by lowering of smooth muscle calcium, and by another mechanism involving mitochondrial complex I and III leads to uncoupling of force, and hence vasodilation.

Cardioprotection by H2S Donors: Nitric Oxide-Dependent and ­Independent Mechanisms.

Hydrogen sulfide (H2S) is a signaling molecule with protective effects in the cardiovascular system. To harness the therapeutic potential of H2S, a number of donors have been developed. The present study compares the cardioprotective actions of representative H2S donors from different classes and studies their mechanisms of action in myocardial injury in vitro and in vivo. Exposure of cardiomyocytes to H2O2 led to significant cytotoxicity, which was inhibited by sodium sulfide (Na2S), thiovaline (TV), GYY4137 [morpholin-4-ium 4 methoxyphenyl(morpholino) phosphinodithioate], and AP39 [(10-oxo-10-(4-(3-thioxo-3H-1,2-dithiol5yl)phenoxy)decyl) triphenylphospho-nium bromide]. Inhibition of nitric oxide (NO) synthesis prevented the cytoprotective effects of Na2S and TV, but not GYY4137 and AP39, against H2O2-induced cardiomyocyte injury. Mice subjected to left anterior descending coronary ligation were protected from ischemia-reperfusion injury by the H2S donors tested. Inhibition of nitric oxide synthase (NOS) in vivo blocked only the beneficial effect of Na2S. Moreover, Na2S, but not AP39, administration enhanced the phosphorylation of endothelial NOS and vasodilator-associated phosphoprotein. Both Na2S and AP39 reduced infarct size in mice lacking cyclophilin-D (CypD), a modulator of the mitochondrial permeability transition pore (PTP). Nevertheless, only AP39 displayed a direct effect on mitochondria by increasing the mitochondrial Ca(2+) retention capacity, which is evidence of decreased propensity to undergo permeability transition. We conclude that although all the H2S donors we tested limited infarct size, the pathways involved were not conserved. Na2S had no direct effects on PTP opening, and its action was nitric oxide dependent. In contrast, the cardioprotection exhibited by AP39 could result from a direct inhibitory effect on PTP acting at a site different than CypD.

GYY4137, a Slow-Releasing Hydrogen Sulfide Donor, Ameliorates Renal Damage Associated with Chronic Obstructive Uropathy.

PURPOSE: Chronic obstructive uropathy can cause irreversible kidney injury, atrophy and inflammation, which can ultimately lead to fibrosis. Epithelial-mesenchymal transition is a key trigger of fibrosis that is caused by up-regulation of TGF-ß1 (transforming growth factor-ß1) and ANGII (angiotensin II). H2S is an endogenously produced gasotransmitter with cytoprotective properties. We sought to elucidate the effects of the slow-releasing H2S donor GYY4137 on chronic ureteral obstruction and evaluate the potential mechanisms. MATERIALS AND METHODS: Following unilateral ureteral obstruction male Lewis rats were given daily intraperitoneal administration of phosphate buffered saline vehicle (obstruction group) or phosphate buffered saline plus 200 µmol/kg GYY4137 (obstruction plus GYY4137 group) for 30 days. Urine and serum samples were collected to determine physiological parameters of renal function and injury. Kidneys were removed on postoperative day 30 to evaluate histopathology and protein expression. Epithelial-mesenchymal transition in LLC-PK1 pig kidney epithelial cells was induced with TGF-ß1 and treated with GYY4137 to evaluate potential mechanisms via in vitro scratch wound assays. RESULTS: H2S treatment decreased serum creatinine and the urine protein-to-creatinine excretion ratio after unilateral ureteral obstruction. In addition, H2S mitigated cortical loss, inflammatory damage and tubulointerstitial fibrosis. Tissues showed decreased expression of epithelial-mesenchymal transition markers upon H2S treatment. Epithelial-mesenchymal transition progression in LLC-PK1 was alleviated upon in vitro administration of GYY4137. CONCLUSIONS: To our knowledge our findings demonstrate for the first time the protective effects of H2S in chronic obstructive uropathy. This may represent a potential therapeutic solution to ameliorate renal damage and improve the clinical outcomes of urinary obstruction.

Pharmacological postconditioning against myocardial infarction with a slow-releasing hydrogen sulfide donor, GYY4137.

Exogenous hydrogen sulfide (H2S) protects against myocardial ischemia/reperfusion injury but the mechanism of action is unclear. The present study investigated the effect of GYY4137, a slow-releasing H2S donor, on myocardial infarction given specifically at reperfusion and the signalling pathway involved. Thiobutabarbital-anesthetised rats were subjected to 30min of left coronary artery occlusion and 2h reperfusion. Infarct size was assessed by tetrazolium staining. In the first study, animals randomly received either no treatment or GYY4137 (26.6, 133 or 266µmolkg(-1)) by intravenous injection 10min before reperfusion. In a second series, involvement of PI3K and NO signalling were interrogated by concomitant administration of LY294002 or L-NAME respectively and the effects on the phosphorylation of Akt, eNOS, GSK-3ß and ERK1/2 during early reperfusion were assessed by immunoblotting. GYY4137 266µmolkg(-1) significantly limited infarct size by 47% compared to control hearts (P<0.01). In GYY4137-treated hearts, phosphorylation of Akt, eNOS and GSK-3ß was increased 2.8, 2.2 and 2.2 fold respectively at early reperfusion. Co-administration of L-NAME and GYY4137 attenuated the cardioprotection afforded by GYY4137, associated with attenuated phosphorylation of eNOS. LY294002 totally abrogated the infarct-limiting effect of GYY4137 and inhibited Akt, eNOS and GSK-3ß phosphorylation. These data are the first to demonstrate that GYY4137 protects the heart against lethal reperfusion injury through activation of PI3K/Akt signalling, with partial dependency on NO signalling and inhibition of GSK-3ß during early reperfusion. H2S-based therapeutic approaches may have value as adjuncts to reperfusion in the treatment of acute myocardial infarction.

The novel mitochondria-targeted hydrogen sulfide (H2S) donors AP123 and AP39 protect against hyperglycemic injury in microvascular endothelial cells in vitro.

The development of diabetic vascular complications is initiated, at least in part, by mitochondrial reactive oxygen species (ROS) production in endothelial cells. Hyperglycemia induces superoxide production in the mitochondria and initiates changes in the mitochondrial membrane potential that leads to mitochondrial dysfunction. Hydrogen sulfide (H2S) supplementation has been shown to reduce the mitochondrial oxidant production and shows efficacy against diabetic vascular damage in vivo. However, the half-life of H2S is very short and it is not specific for the mitochondria. We have therefore evaluated two novel mitochondria-targeted anethole dithiolethione and hydroxythiobenzamide H2S donors (AP39 and AP123 respectively) at preventing hyperglycemia-induced oxidative stress and metabolic changes in microvascular endothelial cells in vitro. Hyperglycemia (HG) induced significant increase in the activity of the citric acid cycle and led to elevated mitochondrial membrane potential. Mitochondrial oxidant production was increased and the mitochondrial electron transport decreased in hyperglycemic cells. AP39 and AP123 (30-300nM) decreased HG-induced hyperpolarisation of the mitochondrial membrane and inhibited the mitochondrial oxidant production. Both H2S donors (30-300nM) increased the electron transport at respiratory complex III and improved the cellular metabolism. Targeting H2S to mitochondria retained the cytoprotective effect of H2S against glucose-induced damage in endothelial cells suggesting that the molecular target of H2S action is within the mitochondria. Mitochondrial targeting of H2S also induced >1000-fold increase in the potency of H2S against hyperglycemia-induced injury. The high potency and long-lasting effect elicited by these H2S donors strongly suggests that these compounds could be useful against diabetic vascular complications.

The Effectiveness of Message Framing and Temporal Context on College Student Alcohol Use and Problems: A Selective E-Mail Intervention.

AIMS: Only one study has examined message framing on college drinking, but did so in a laboratory setting among a general sample of college students. The current study was designed to: (a) compare the efficacy of emailed interventions differing by message framing and temporal context on alcohol involvement among heavy drinking college students and (b) examine need for cognition (NFC), consideration of future consequences (CFC) and self-efficacy as putative moderators. METHODS: Hazardous drinking college students (N = 220) were randomly assigned to conditions in a 2 (Frame: gain vs. loss) × 2 (Temporal Context: long-term vs. short-term consequences) factorial design. Participants received four emails on heavy drinking consequences phrased in a manner consistent with their condition. After each message, participants were given a manipulation check. Participants were sent a 1-month follow-up assessment. Primary outcome measures were heavy episodic drinking (HED) and alcohol-related problems. We hypothesized two main effects (less alcohol consumption in the gain-frame and short-term condition), qualified by a Frame × Temporal Context interaction with substantially less alcohol involvement in the gain-frame/short-term condition. RESULTS: There was very little study attrition (96.4% completed follow-up survey, 93.2-99.5% completed manipulation checks), and strong effects were observed for the manipulations. A 2 × 2 ANCOVA, controlling for baseline alcohol involvement, revealed no consistent main effects or interactions on either outcome. No moderation was observed for any putative moderator. CONCLUSIONS: These results do not replicate prior laboratory-based research. The null findings may be attributed to the heavy drinking sample or electronic means of message delivery.

Primary hypertrophic osteoarthropathy: ultrasound and MRI findings.

Primary hypertrophic osteoarthropathy is a rare genetic disorder related to failures in prostaglandin metabolism. Patients present with joint pain, limb enlargement, skin thickening and finger clubbing. Radiographs show characteristic periosteal reaction and thickening along the long bones. We present MRI and US findings in a child with the condition. Ultrasound showed echogenic tissue surrounding the long bones, presumably reflecting oedema and inflammatory tissue. Doppler sonograms demonstrated increased vascularity on the surface of some superficial bony structures.