Antimalarial and antitumour activities of the steroidal quinone-methide celastrol and its combinations with artemiside, artemisone and methylene blue.

Artemisinin, isolated from the traditional Chinese medicinal plant qing hao (Artemisia annua) and its derivatives are used for treatment of malaria. With treatment failures now being recorded for the derivatives and companion drugs used in artemisinin combination therapies new drug combinations are urgently required. The amino-artemisinins artemiside and artemisone display optimal efficacies in vitro against asexual and sexual blood stages of the malaria parasite Plasmodium falciparum and are active against tumour cell lines. In continuing the evolution of combinations of the amino-artemisinins with new drugs, we examine the triterpenoid quinone methide celastrol isolated from the traditional Chinese medicinal plant léi gong téng (Tripterygium wilfordii). This compound is redox active, and has attracted considerable attention because of potent biological activities against manifold targets. We report that celastrol displays good IC50 activities ranging from 0.50-0.82 µM against drug-sensitive and resistant asexual blood stage Pf, and 1.16 and 0.28 µM respectively against immature and late stage Pf NF54 gametocytes. The combinations of celastrol with each of artemisone and methylene blue against asexual blood stage Pf are additive. Given that celastrol displays promising antitumour properties, we examined its activities alone and in combinations with amino-artemisinins against human liver HepG2 and other cell lines. IC50 values of the amino-artemisinins and celastrol against HepG2 cancer cells ranged from 0.55-0.94 µM. Whereas the amino-artemisinins displayed notable selectivities (SI > 171) with respect to normal human hepatocytes, in contrast, celastrol displayed no selectivity (SI < 1). The combinations of celastrol with artemiside or artemisone against HepG2 cells are synergistic. Given the promise of celastrol, judiciously designed formulations or structural modifications are recommended for mitigating its toxicity.

Comparison of the Accuracy and Completeness of Records of Serious Vascular Events in Routinely Collected Data vs Clinical Trial-Adjudicated Direct Follow-up Data in the UK: Secondary Analysis of the ASCEND Randomized Clinical Trial.

Importance: Routinely collected data could substantially decrease the cost of conducting trials. Objective: To assess the accuracy and completeness of UK routine data for ascertaining serious vascular events (SVEs) compared with adjudicated follow-up data. Design, Setting, and Participants: This was a secondary analysis of a randomized clinical trial. From June 24, 2005, to July 28, 2011, the ASCEND (A Study of Cardiovascular Events in Diabetes) primary prevention trial used mail-based methods to randomize people with diabetes without evidence of atherosclerotic vascular disease using a 2 × 2 factorial design to aspirin and/or ω-fatty acids vs matching placebo in the UK. Direct participant mail-based follow-up was the main source of outcome data, with more than 90% of the primary outcome events undergoing adjudication. Follow-up was completed on July 31, 2017. In parallel, more than 99% of participants were linked to routinely collected hospital admission and death registry data (ie, routine data), enabling post hoc randomized comparisons of different sources of outcome data (conducted from September 1, 2018, to October 1, 2021). Interventions: Random allocation to 100 mg of aspirin once daily vs matching placebo and separately to 1 g of ω-3 fatty acids once daily vs placebo. Main Outcomes and Measures: The primary outcome consisted of SVEs (a composite of nonfatal myocardial infarction, ischemic stroke, transient ischemic attack [TIA], or vascular death, excluding hemorrhagic stroke). Results: A total of 15 480 participants were randomized (mean [SD] age, 63 [9] years; 9684 [62.6%] men) and followed up for a mean (SD) of 7.4 (1.8) years. For SVEs, agreement between adjudicated direct follow-up and routine data sources was strong (1401 vs 1127 events; κ = 0.78 [95% CI, 0.76-0.80]; sensitivity, 72.0% [95% CI, 69.7%-74.4%]; specificity, 99.2% [95% CI, 99.0%-99.3%]), and sensitivity improved for SVEs excluding transient ischemic attack (1129 vs 1026 events; sensitivity, 80.6% [95% CI, 78.3%-82.9%]). Rate ratios for the aspirin-randomized comparison for adjudicated direct follow-up vs follow-up solely through routine data alone were 0.88 (95% CI, 0.79-0.97) vs 0.91 (95% CI, 0.81-1.02) for the primary outcome and 0.92 (95% CI, 0.82-1.03) vs 0.91 (95% CI, 0.80-1.02) for SVEs excluding TIA. Results were similar for the ω-3 fatty acid comparison, and adjudication did not seem to markedly change rate ratios. Conclusions and Relevance: Post hoc analyses of the ASCEND trial suggest that routinely collected hospital admission and death registry data in the UK could be used as the sole method of follow-up for myocardial infarction, ischemic stroke resulting in hospitalization, vascular death, and arterial revascularization in primary prevention cardiovascular trials, without the need for verification by clinical adjudication.

Streamlined mail-based methods for large randomised trials: lessons learnt from the ASCEND study.

Reliable assessment of the effects of an intervention usually requires large randomised trials but such studies are becoming increasingly complex and costly to run. 'Streamlined' trials are needed in which every aspect of the trial design and conduct is simplified, retaining only those elements needed to answer the research question and ensure the safety of the individual participants. In this review we discuss how the trial 'A Study of Cardiovascular Events iN Diabetes' (ASCEND) was streamlined. The study included a two-by-two factorial design: it assessed the effects of low-dose aspirin and, separately, supplementation with n-3 fatty acids on serious vascular events in 15,480 people with diabetes but no overt cardiovascular disease. Other key streamlined design features, such as mail-based recruitment and follow-up, mainly by post, with no in-person visits and use of a run-in period, are also described. We go on to discuss the success of the study and other studies that have employed a similar mail-based approach, and the type of clinical trials that are suitable for mail-based design. Finally, we consider the limitations of the study, and how these could be circumvented in future studies. ASCEND randomised large numbers of eligible participants, achieved good adherence rates and almost complete follow-up at a fraction of the cost of traditional clinic-based trials. Such studies are necessary if researchers are to address the important clinical questions most relevant to improving health.

Removal of Al, Ga, As, V and Mo from alkaline wastewater using pilot-scale constructed wetlands.

The study was initiated to evaluate constructed wetland technology as a method for treating alkaline (pH 8.0-8.6) drainage high in Al, Mo, V, As and Ga originating from bauxite residue storage areas. Pilot-scale horizontal flow constructed wetlands were operated over a 40-week period using three filter materials (granitic gravel, bauxite and alum water treatment sludge), and half of the wetlands were planted with Phragmites australis and the other half left unplanted. Gravel was the least effective medium for removing the target elements, while of the two active media, water treatment sludge was more effective than bauxite. Plants removed only small amounts of elements into their above- and below-ground dry matter (0.4-4.9% of that added). Nonetheless, the presence of plants greatly increased the effectiveness of all three media since their presence decreased effluent pH values by 0.5-1.3 pH units and that of the filter media by 0.4 pH units. Removal of elements followed the order Al > Ga > V > As > Mo. For planted wetlands, total elemental removal ranged from 18 to 98% for gravel, 80 to 99% for bauxite, and 93 to 99% for water treatment sludge. The lowest removal was for Mo (ranging from 18% for gravel to 93% for water treatment sludge) and the highest for Al (ranging from 98% in gravel to 99% in water treatment sludge). A sequential fractionation scheme for As, V and Mo on filter material at the end of the experiment showed that for bauxite and water treatment sludge, V and As were concentrated in the NaOH extractable fraction while Mo was concentrated in the less strongly adsorbed NaHCO3 extractable fraction. It was concluded that a constructed wetland with water treatment sludge as an active filter material is an effective technology for removal of the target elements from the alkali drainage.

The evaluation of the anti-cancer drug elesclomol that forms a redox-active copper chelate as a potential anti-tubercular drug.

The observations that the innate immune system employs copper to eliminate bacterial infection and that resistance to copper enhances virulence of Mycobacterium tuberculosis (Mtb) prompted us to examine the effects the anti-cancer agent elesclomol on Mtb. As a bis-thionohydrazide, elesclomol chelates with copper to form a copper complex in situ that via redox cycling of the metal ion greatly enhances oxidative stress in tumour cells. Here, we demonstrate that elesclomol is relatively potent against Mtb H37Rv with minimum inhibitory concentration of 10 µM (4 mg/L) and against multidrug resistant clinical isolates of Mtb, displays additive interactions with known tuberculosis drugs such as isoniazid and ethambutol, and a synergistic interaction with rifampicin. Controlled supplementation of elesclomol with copper in culture medium increased Mtb sensitivity by >65 fold. Overall, the activities of elesclomol in principle indicate the possibility of repurposing elesclomol or designing new thionohydrazides as potential drugs for use against Mtb. © 2019 IUBMB Life, 71(5):532-538, 2019.

Growth of Rhodes grass and leaching of ions from seawater neutralized bauxite residues after amendment with gypsum and organic wastes.

A 32-week leaching column study was carried out in the greenhouse to investigate the effects of incorporation of gypsum, cattle manure, biosolids, gypsum plus cattle manure and gypsum plus biosolids into the topsoil layer (0-10 cm) on growth of Rhodes grass, and on root distribution and chemical and microbial properties in the topsoil and subsoil (10-30 cm) layers of seawater neutralized bauxite residues. The columns were leached for a period of 8 weeks prior to sowing Rhodes grass and during that time the bulk of the salts accumulated during seawater neutralization were leached out. The main cation leached was Na+ and the main balancing anions were Cl- and SO42-. During this period the pH of leachates rose from 7 to 8 up to 9-10. At the end of the study, exchangeable Na and pH were lowered in the surface horizon by all treatments with a combination of gypsum plus organic amendments having the greatest effect. The latter treatments also caused a significant decrease in pH, extractable Al and exchangeable Na in the subsoil. Rhodes grass dry matter production followed the order Control < gypsum < cattle manure = gypsum plus cattle manure < biosolids = gypsum plus biosolids. Growth of roots into the subsoil layer was inhibited in the Control and gypsum treatments but when organic amendments were applied, 50% or more of root dry matter was recovered in the subsoil layer. It was concluded that incorporating a combination of gypsum and organic matter into the surface soil is an effective strategy for revegetation of bauxite residue.

Competitive adsorption and desorption of arsenate, vanadate, and molybdate onto the low-cost adsorbent materials alum water treatment sludge and bauxite.

When low-cost adsorbents are being used to remove contaminant ions (e.g. arsenate, vanadate, and molybdate) from wastewater, competitive adsorption/desorption are central processes determining their removal efficiency. Competitive adsorption of As, V, and Mo was investigated using equimolar oxyanion concentrations in single, binary, and tertiary combinations in adsorption isotherm and pH envelope studies while desorption of previously adsorbed oxyanions was examined in solutions containing single and binary oxyanion combinations. The low-cost adsorbent materials used were alum water treatment sludge (amorphous hydroxy-Al) and bauxite ore (crystalline Al oxides). Adsorption isotherm and pH envelope studies showed that Mo had only a small effect in decreasing adsorption of As and V but V and As had substantial and similar effects in reducing adsorption of the other. As had a greater effect than V in reducing adsorption of Mo and it was concluded that the affinity of oxyanions for the surfaces of water treatment sludge and bauxite followed the order As > V >> Mo. In 0.3 M NaCl electrolyte, desorption of previously adsorbed oxyanions amounted to 0.3-3.4% for V and As, and 11-20% for Mo. As had approximately four times greater effect than Mo in increasing desorption of V while V had about three times the effect of Mo in increasing desorption of As. Thus, the order of oxyanions in inducing desorption of the other oxyanions (i.e. As on V and As) was the same as that for adsorption selectivity: As > V >> Mo. Water treatment sludge was a more effective adsorbent than bauxite because it had a greater adsorption capacity for all three anions and, in addition, they were held more strongly so desorption in the background electrolyte was proportionately less. It was concluded that at similar molar concentrations, arsenate would tend to reduce adsorption of vanadate as well as displace vanadate already held on adsorbent surfaces while both anions will compete effectively with molybdate. The limiting factor for simultaneous removal of As, V, and Mo from multielement solutions by adsorption will therefore be the removal of Mo.

Effects of n-3 Fatty Acid Supplements in Diabetes Mellitus.

BACKGROUND: Increased intake of n-3 fatty acids has been associated with a reduced risk of cardiovascular disease in observational studies, but this finding has not been confirmed in randomized trials. It remains unclear whether n-3 (also called omega-3) fatty acid supplementation has cardiovascular benefit in patients with diabetes mellitus. METHODS: We randomly assigned 15,480 patients with diabetes but without evidence of atherosclerotic cardiovascular disease to receive 1-g capsules containing either n-3 fatty acids (fatty acid group) or matching placebo (olive oil) daily. The primary outcome was a first serious vascular event (i.e., nonfatal myocardial infarction or stroke, transient ischemic attack, or vascular death, excluding confirmed intracranial hemorrhage). The secondary outcome was a first serious vascular event or any arterial revascularization. RESULTS: During a mean follow-up of 7.4 years (adherence rate, 76%), a serious vascular event occurred in 689 patients (8.9%) in the fatty acid group and in 712 (9.2%) in the placebo group (rate ratio, 0.97; 95% confidence interval [CI], 0.87 to 1.08; P=0.55). The composite outcome of a serious vascular event or revascularization occurred in 882 patients (11.4%) and 887 patients (11.5%), respectively (rate ratio, 1.00; 95% CI, 0.91 to 1.09). Death from any cause occurred in 752 patients (9.7%) in the fatty acid group and in 788 (10.2%) in the placebo group (rate ratio, 0.95; 95% CI, 0.86 to 1.05). There were no significant between-group differences in the rates of nonfatal serious adverse events. CONCLUSIONS: Among patients with diabetes without evidence of cardiovascular disease, there was no significant difference in the risk of serious vascular events between those who were assigned to receive n-3 fatty acid supplementation and those who were assigned to receive placebo. (Funded by the British Heart Foundation and others; Current Controlled Trials number, ISRCTN60635500 ; ClinicalTrials.gov number, NCT00135226 .).

Properties of seawater neutralized bauxite residues and changes in chemical, physical and microbial properties induced by additions of gypsum and organic matter.

Seawater neutralization is a technique that can be used to treat bauxite residue prior to its storage but, as yet, no attempts have been made to revegetate it. Seawater neutralized bauxite residue was found to have a pH1:5 of 9.3 and to be highly saline (EC1.5 16.5 dS m-1). After leaching pH1:5 rose to 9.7 and the residue was still highly sodic (ESP = 64-69%). Addition of 1% gypsum, prior to leaching, arrested this increase in pH while with 5% gypsum addition the pH1:5 was lowered to 8.9. Addition of 5% gypsum also reduced ESP to 38% and increased watercress germination in the residue from 58% in control treatments to 88%. The major ions in leachates were Na+ and Cl- and gypsum application increased the quantities of Na+, Ca2+ and SO42- leached. Addition of 6% biosolids or 6% poultry manure added exchangeable cations to the mud and lowered ESP by 5-11%.The EC was 2.8-3.7 (mean 3.1) times higher and pH 0.2-0.7 (mean 0.43) units lower in saturation paste compared with 1:5 soil:water extracts. Addition of amendments had only small effects on physical properties. While organic C content was increased more by biosolids than poultry manure addition the reverse was the case for soluble organic C, microbial biomass C and basal respiration. It was concluded that although seawater neutralization initially lowers the pH of bauxite residues it is unlikely to increase the ease with which they can be revegetated.

Increased leaching and addition of amendments improve the properties of seawater-neutralized bauxite residue as a growth medium.

Laboratory and greenhouse experiments were carried out to investigate the chemical, physical, and microbial properties of seawater-neutralized bauxite residues and the effects of additional leaching (1 pore volume of deionized water versus an additional 6 pore volumes) and amendment with gypsum (5%) and/or cattle manure (6%) on its properties and on the growth of Rhodes grass (Chloris gayana). Additional leaching resulted in a decrease in EC, exchangeable Na, SAR, and ESP. For unamended control treatments, additional leaching induced a rise in pHSE from 8.5 to 9.6 and pH1:5 from 9.1 to 10.1 due to dissolution of residual alkalinity. Addition of gypsum arrested this pH increase resulting in a final pHSE of 7.5 and pH1:5 of 8.8. In control treatments, additional leaching resulted in a pronounced decrease in Rhodes grass yields. However, in gypsum and cattle manure-amended treatments, it led to substantial yield increases and decreases in tissue Al and Na concentrations and increased K/Na ratios. Upon drying for the first time, bauxite residue was shown to contract and form a solid massive structure. The aggregates formed from crushing this material were water stable (as measured by wet sieving). Additions of cattle manure or gypsum to residue aggregates did not affect pore size distribution. Addition of cattle manure increased organic C and microbial biomass C content and basal respiration rate while additional leaching increased basal respiration and metabolic quotient. It was concluded that a combination of drying and crushing the residue, amending it with gypsum and organic manure followed by extensive leaching results in the formation of a medium that supports plant growth.

Absorptive and Secretory Transport of Selected Artemisinin Derivatives Across Caco-2 Cell Monolayers.

BACKGROUND: Malaria continues to be a major health concern and affects more than 200 million people a year. Drugs currently used for treatment of malaria are increasingly rendered ineffectual by the ongoing emergence of parasite resistance. For any new drugs, however, knowledge of their membrane permeability is an essential pre-requisite for eventual use. Treatment failure and emergence of resistance can occur as a result of reduced availability of the drug at the desired site of action. Cellbased permeability assays such as Caco-2 cell monolayers serve as a model for predicting drug absorption and efflux, and provide an estimate of drug bioavailability. OBJECTIVE: Here we have studied the bi-directional transport of new anti-malarial compounds, artemisone and artemiside, as well as reference compounds, namely the known anti-malarial drug artemether, and caffeine and atenolol. METHODS: The Caco-2 cell monolayer model was used to assess the membrane permeation properties of these compounds, and to identify if they are subject to P-gp associated efflux, in the presence and absence of verapamil. The effect of piperine on the transport of the compounds that were identified to be P-gp substrates was also assessed. Samples withdrawn from the acceptor chambers at pre-determined time intervals were analysed by means of high-performance liquid chromatography (HPLC). RESULTS: Transport results in terms of the absorptive direction revealed that artemisone and artemether had low absorption rates relative to the reference compounds. It was further demonstrated that artemisone is slightly effluxed, and although both artemether and artemiside were susceptible to P-gp mediated efflux, it appears that other efflux proteins may also be involved. CONCLUSION: The low permeability of anti-malarial drugs must be borne in mind during development of effective dosage regimens of new drugs.

ASCEND: A Study of Cardiovascular Events iN Diabetes: Characteristics of a randomized trial of aspirin and of omega-3 fatty acid supplementation in 15,480 people with diabetes.

OBJECTIVES: The use of aspirin for the secondary prevention of cardiovascular disease (CVD) is firmly established, and the proportional reductions in heart attacks and strokes appear to be similar in people with and without diabetes. Uncertainty remains about the role of antiplatelet treatments for primary prevention of CVD, and guidelines vary in their recommendations. It has also been hypothesized that long-term aspirin can prevent gastro-intestinal and other cancers. Observational studies suggest associations between higher intakes of omega-3 fatty acids (FA) and lower rates of CVD, but there is no large-scale randomized evidence to support using prophylactic omega-3 FA supplementation in primary prevention. ASCEND is a randomized trial assessing whether 100 mg daily aspirin safely prevents CVD and cancer in patients with diabetes without known arterial disease. It is also assessing whether supplementation with 1 g omega-3 FA daily prevents CVD. This paper describes the methods and baseline characteristics of the randomized participants. METHODS AND RESULTS: Between 2005 and 2011, using mail-based methods, 15,480 people with diabetes were randomized to aspirin versus placebo and, in a factorial design, to omega-3 FA supplementation versus placebo. Blood and urine samples were collected to allow baseline stratification by biochemical prognostic variables (e.g. HbA1c, blood lipids). Follow-up is for a median of at least 7 years. CONCLUSIONS: Demonstrating that prophylactic aspirin safely reduces the risk of CVD or cancer in the primary prevention setting, or that omega-3 FA supplementation prevents CVD, would be relevant to hundreds of millions of people worldwide who are currently not receiving such therapies. The results of ASCEND will be reported in 2018.

Cost-effective recruitment methods for a large randomised trial in people with diabetes: A Study of Cardiovascular Events iN Diabetes (ASCEND).

BACKGROUND: Clinical trials require cost-effective methods for identifying, randomising, and following large numbers of people in order to generate reliable evidence. ASCEND (A Study of Cardiovascular Events iN Diabetes) is a randomised '2 × 2 factorial design' study of aspirin and omega-3 fatty acid supplements for the primary prevention of cardiovascular events in people with diabetes; this study used central disease registers and a mail-based approach to identify, randomise, and follow 15,000 people. In collaboration with UK consultants and general practitioners (GPs), researchers identified potentially eligible people with diabetes from centrally held registers (e.g. for retinopathy screening) and GP-held disease registers. Permission was obtained under section 251 of the National Health Service Act 2006 (previously section 60 of the NHS act 2001) to allow invitation letters to be generated centrally in the name of the holder of the register. In addition, with the collaboration of the National Institutes for Health Research (NIHR) Diabetes and Primary Care Research Networks (DRN and PCRN), general practices sent pre-assembled invitation packs to people with a diagnosis of diabetes. Invitation packs included a cover letter, screening questionnaire (with consent form), information leaflet, and a Freepost envelope. Eligible patients entered a 2-month, pre-randomisation, run-in phase on placebo tablets and were only randomised if they completed a randomisation form and remained willing and eligible at the end of the run-in. Follow-up is ongoing, using mail-based approaches that are being supplemented by central registry data. RESULTS: Information on approximately 600,000 people listed on 58 centrally held diabetes registers was obtained, and 300,188 potentially eligible patients were invited to join the study. In addition, 785 GP practices mailed invitations to 120,875 patients. A further 2,340 potential study participants were identified via other routes. In total, 423,403 people with diabetes were invited to take part; 26,462 entered the 2-month, pre-randomisation, run-in phase; and 15,480 were randomised. CONCLUSION: If sufficient numbers of potentially eligible patients can be identified centrally and the trial treatments do not require participants to attend clinics, the recruitment and follow-up of patients by mail is feasible and cost-effective. Wider use of these methods could allow more, large, randomised trials to be undertaken successfully and cost-effectively. TRIAL REGISTRATION: Current Controlled Trials, ISRCTN60635500 , registered on 14 July 2005.

Considerations on the mechanism of action of artemisinin antimalarials: part 1--the 'carbon radical' and 'heme' hypotheses.

The isolation of artemisinin from the traditional medicinal herb qing hao (Artemisia annua), its characterization as a peroxide and preparation of the derivatives dihydroartemisinin, artemether and artesunate in the 1970s and 1980s by Chinese scientists under the umbrella of Project 523 collectively represents one of the great events in medicine in the latter third of the 20(th) Century. Artemisinins have become the most important component of chemotherapy of malaria: although used initially in monotherapy, they are now used in combination therapies or ACTs with longer half-life quinolines or arylmethanols. Nevertheless, the recent emergence of artemisinin-tolerant strains of the malaria parasite as reflected in increased clearance times of parasitaemia in patients treated with ACTs represents the greatest threat to control of malaria since resistance to chloroquine was first reported over 55 years ago. Importantly, the event brings into sharp focus the realization that relatively little is precisely understood, as opposed to widely assumed, for the mechanism of drug action of artemisinins and their synthetic peroxide analogues. Thus, we review here their antimalarial activities, the use of artemisinins in combination therapies, drug-drug interactions with the quinolines and arylmethanols, and metabolism of the artemisinins and synthetic peroxides. The mechanism of action of quinolines and arylmethanols, in particular their ability to induce redistribution of heme into the parasite cytosol, is also highlighted. This collective information is then used as a counterpoint to screen the validity of two of the prevailing hypotheses of drug action of artemisinins and synthetic peroxides, namely i. 'the C-radical hypothesis' wherein the peroxide undergoes 'bioactivation' by ferrous iron to generate C-radicals that are held to be the cytotoxic agents and ii. the 'heme hypothesis' wherein ferrous heme may generate either the same type of 'cytotoxic' C-radical, or the peroxide forms heme adducts that apparently inherit the exquisite cytotoxicities of the parent peroxide in one way or another. In a subsequent review, we screen the third and fourth hypotheses: the SERCA hypothesis wherein artemisinins modulate operation of the malaria parasite sarcoendo plasmic reticulum calcium pump SERCA Ca(2+)-ATPase ATP6 and the co-factor hypothesis wherein artemisinins act as oxidant drugs through rapidly oxidizing reduced conjugates of flavin cofactors, or those of flavin cofactor precursors such as riboflavin, and other susceptible endogenous substrates that play a role in maintaining intraparasitic redox homeostasis. For the C-radical hypothesis, details of in vitro chemical studies in the context of established chemistry of C-radicals and their ability to react with radical trapping agents such as nitroso compounds, cyclic nitrones, persistent nitroxyl radicals and atmospheric oxygen (dioxygen) are summarized. Overall, there is no correlation between antimalarial activities and abilities of the derived C-radicals to react with trapping agents in a chemical flask. This applies in particular to the reactions of C-radicals from artemisinins and steroidal tetraoxanes with the trapping agents vis-a-vis those from adamantyl capped systems. In an intraparasitic medium, it is not possible to intercept C-radicals either through use of a vast excess of a nitroxyl radical or dioxygen. The lack of correlation of antimalarial activities also applies to the Fe(2+)-mediated decomposition of artemisinins and synthetic peroxides, where literature data taken as indicating otherwise are critically assessed. The antagonism to antimalarial activities of artemisinins exerted by desferrioxamine (DFO) and related Fe(3+)-chelating agents is due to formation of stable chelates with bioavailable Fe(3+) that shuts down redox cycling through Fe(2+) and the subsequent generation of reactive oxygen species (ROS) via the Fenton reaction. The generation of ROS by Fe(2+) complements the action of artemisinins, to be discussed in Part 2; there is no need to posit a reaction of Fe(2+) with the artemisinins to account for their antimalarial activity. The ability of artemisinins and synthetic peroxides to elicit membrane damage is examined in the light of established processes of autoxidation. The oxidant character of the intraparasitic environment is incompatible with the reducing conditions required for generation of C-radicals, and in contrast to the expectation raised by the C-radical hypothesis, and indeed by the heme hypothesis outlined below, antimalarial activities of artemisinins are enhanced under higher partial pressures of dioxygen. Structure-activity data from a wide variety of artemisinins and synthetic peroxides cannot be accommodated within the bounds of the C-radical hypothesis. Finally, the antimalarial Cradical construct sharply contrasts with that of the potently antitumour-active ene-diyne antibiotics such as neocarzinostatin. In an iron-free process, these compounds generate highly reactive aryl C-radicals that abstract H atoms from deoxyribose units in DNA to generate alkyl C-radicals. The last do react with dioxygen in a normal intracellular environment to initiate DNA strand cleavage. Overall, it must be concluded that the C-radical hypothesis as the basis for antimalarial activities of artemisinins and synthetic peroxides is untenable. Heme has been intensively studied as an 'activator' of artemisinins and other antimalarial peroxides, and indeed the hypothesis seemingly has become firmly embedded in the underlying brickwork of the scientific edifice. The locus of activity of the peroxides interacting with the heme is considered to be the parasite digestive vacuole. The basis for the nanomolar activities of artemisinins and synthetic peroxides is variously ascribed to heme-Fe(2+)-mediated generation of C-radicals from the peroxides, formation of heme-artemisinin adducts that are held either to engage in redox cycling with concomitant generation of ROS or to inhibit formation of hemozoin. In the last case, just like the aminoquinolines and arylmethanols, the peroxides are not the active agents, but exert their parasiticidal effects through allowing the build-up of free heme-Fe(3+), the ultimate cytotoxic entity. We assess the literature relating to generation of heme by hemoglobin digestion, and the stage at which this process becomes significant in the intraerythrocytic parasite. The claims of production of heme and conversion into hemozoin occurring in a lipid environment may have to be put aside based on recent literature data that indicates crystallization of hemozoin must take place an aqueous interface; association of lipids with the heme/hemozoin is likely to be a reflection of attractive van der Waals interactions involving the hydrophobic surface of the heme or hemozoin aggregates. In addition, the observation leading to the claim that hemozoin manufacture commences at the mid-ring stage cannot be independently verified. That the quinoline and arylmethanol antimalarials have essentially no activities on the ring stage parasites and exert greatest efficacy at the trophozoite stage where heme production is maximal is consistent with this. Conversely, artemisinins, and indeed redox active drugs such as methylene blue and others, are highly active against early ring stage parasites. Thus, there is a prominent disconnect between stage specificities of artemisinins vis-a-vis those of 4-aminoquinolines and arylmethanols suggesting that heme is not the target of the former class of drug. Further, the ability of the Fe(3+) chelate DFO to antagonize antimalarial activities of artemisinins, but not the activities of 4-aminoquinolines, cannot be explained by involvement of heme as a target for artemisinins. We critically examine the basis for formation of products obtained from reaction of heme with artemisinins and synthetic peroxides under conditions ranging from biomimetic - reactions employing catalytic reagents under aqueous or semi-aqueous conditions - to those conducted under highly reducing and eminently artificial conditions, usually in the solvent dimethyl sulfoxide (DMSO) that both forms well characterized complexes with heme-Fe(2+) and actually assists in driving single electron transfer processes. It is noted that alkylated products tend to form in high yields under the last conditions, and this aspect is readily explained. Irrespective of product yields obtained under various conditions, an overarching correlation between facility of the reaction of the peroxide with heme and their antimalarial activities does not exist. The is underscored by the reproducible outcomes of reactions conducted under biomimetic conditions indicating adducts cannot form in physiologically meaningful concentrations and that heme is a recalcitrant reaction partner to artemisinins in general. Again, as in the case of the C-radical hypothesis, structure-activity data from a wide variety of artemisinins and synthetic peroxides is difficult to reconcile with the heme hypothesis. This applies in particular to dimeric and trimeric artemisinin derivatives where the ascribing of biological activity to reactions of the derived radicals or to the vastly encumbered artemisinin-heme adducts is physically unrealistic. Finally, the facile metabolism and induction of metabolism of the current clinically used artemisinins by members of the CYP superfamily - heme proteins that require an intimate interaction of the heme with the artemisinin for metabolism to occur - is incompatible with the oft-cited proclivity of the peroxide to associate via complex formation with heme as a prelude to its 'activation' as an antimalarial agent within the malaria parasite. (ABSTRACT TRUNCATED)

Influence of organic waste and residue mud additions on chemical, physical and microbial properties of bauxite residue sand.

BACKGROUND, AIM AND SCOPE: In an alumina refinery, bauxite ore is treated with sodium hydroxide at high temperatures and pressures and for every tone of alumina produced, about 2 tones of alkaline, saline bauxite processing waste is also produced. At Alcoa, a dry stacking system of disposal is used, and it is the sand fraction of the processing waste that is rehabilitated. There is little information available regarding the most appropriate amendments to add to the processing sand to aid in revegetation. The purpose of this study was to investigate how the addition of organic wastes (biosolids and poultry manure), in the presence or absence of added residue mud, would affect the properties of the residue sand and its suitability for revegetation. MATERIALS AND METHODS: Samples of freshly deposited residue sand were collected from Alcoa's Kwinana refinery. Samples were treated with phosphogypsum (2% v/v), incubated, and leached. A laboratory experiment was then set up in which the two organic wastes were applied at 0 or the equivalent to 60 tones ha(-1) in combination with residue mud added at rates of 0%, 10% and 20% v/v. Samples were incubated for 8 weeks, after which, key chemical, physical and microbial properties of the residue sand were measured along with seed germination. RESULTS AND DISCUSSION: Additions of residue mud increased exchangeable Na(+), ESP and the pH, and HCO (3) (-) and Na(+) concentrations in saturation paste extracts. Additions of biosolids and poultry manure increased concentrations of extractable P, NH (4) (+) , K, Mg, Cu, Zn, Mn and Fe. Addition of residue mud, in combination with organic wastes, caused a marked decrease in macroporosity and a concomitant increase in mesoporosity, available water holding capacity and the quantity of water held at field capacity. With increasing residue mud additions, the percentage of sample present as sand particles (<1 mm diameter) decreased, and the percentage present in aggregated form (>2 mm diameter) increased; greatest aggregation occurred where a combination of residue mud and poultry manure were added. Stability of aggregates, as measured by wet sieving, was greatest where poultry manure was applied. Although total organic C and soluble organic C were greater in biosolids than poultry manure treatments, the reverse was the case for microbial biomass C and basal respiration. In the biosolids and poultry manure treatments, increasing residue mud additions tended to increase soluble C, microbial biomass C and basal respiration. Germination index of watercress was highest in control samples and reduced by additions of biosolids and poultry manure which was attributed to the high EC and possibly high extractable P and NH (4) (+) . CONCLUSIONS: The concurrent addition of residue mud and organic wastes can improve chemical, microbial and particularly physical properties of residue sand. Future research should include neutralisation of the mud (e.g. with gypsum) and subsequent leaching to remove salts originating from both the mud and organic wastes.

Facile oxidation of leucomethylene blue and dihydroflavins by artemisinins: relationship with flavoenzyme function and antimalarial mechanism of action.

The antimalarial drug methylene blue (MB) affects the redox behaviour of parasite flavin-dependent disulfide reductases such as glutathione reductase (GR) that control oxidative stress in the malaria parasite. The reduced flavin adenine dinucleotide cofactor FADH(2) initiates reduction to leucomethylene blue (LMB), which is oxidised by oxygen to generate reactive oxygen species (ROS) and MB. MB then acts as a subversive substrate for NADPH normally required to regenerate FADH(2) for enzyme function. The synergism between MB and the peroxidic antimalarial artemisinin derivative artesunate suggests that artemisinins have a complementary mode of action. We find that artemisinins are transformed by LMB generated from MB and ascorbic acid (AA) or N-benzyldihydronicotinamide (BNAH) in situ in aqueous buffer at physiological pH into single electron transfer (SET) rearrangement products or two-electron reduction products, the latter of which dominates with BNAH. Neither AA nor BNAH alone affects the artemisinins. The AA-MB SET reactions are enhanced under aerobic conditions, and the major products obtained here are structurally closely related to one such product already reported to form in an intracellular medium. A ketyl arising via SET with the artemisinin is invoked to explain their formation. Dihydroflavins generated from riboflavin (RF) and FAD by pretreatment with sodium dithionite are rapidly oxidised by artemisinin to the parent flavins. When catalytic amounts of RF, FAD, and other flavins are reduced in situ by excess BNAH or NAD(P)H in the presence of the artemisinins in the aqueous buffer, they are rapidly oxidised to the parent flavins with concomitant formation of two-electron reduction products from the artemisinins; regeneration of the reduced flavin by excess reductant maintains a catalytic cycle until the artemisinin is consumed. In preliminary experiments, we show that NADPH consumption in yeast GR with redox behaviour similar to that of parasite GR is enhanced by artemisinins, especially under aerobic conditions. Recombinant human GR is not affected. Artemisinins thus may act as antimalarial drugs by perturbing the redox balance within the malaria parasite, both by oxidising FADH(2) in parasite GR or other parasite flavoenzymes, and by initiating autoxidation of the dihydroflavin by oxygen with generation of ROS. Reduction of the artemisinin is proposed to occur via hydride transfer from LMB or the dihydroflavin to O1 of the peroxide. This hitherto unrecorded reactivity profile conforms with known structure-activity relationships of artemisinins, is consistent with their known ability to generate ROS in vivo, and explains the synergism between artemisinins and redox-active antimalarial drugs such as MB and doxorubicin. As the artemisinins appear to be relatively inert towards human GR, a putative model that accounts for the selective potency of artemisinins towards the malaria parasite also becomes apparent. Decisively, ferrous iron or carbon-centered free radicals cannot be involved, and the reactivity described herein reconciles disparate observations that are incompatible with the ferrous iron-carbon radical hypothesis for antimalarial mechanism of action. Finally, the urgent enquiry into the emerging resistance of the malaria parasite to artemisinins may now in one part address the possibilities either of structural changes taking place in parasite flavoenzymes that render the flavin cofactor less accessible to artemisinins or of an enhancement in the ability to use intra-erythrocytic human disulfide reductases required for maintenance of parasite redox balance.

Effects of homocysteine-lowering with folic acid plus vitamin B12 vs placebo on mortality and major morbidity in myocardial infarction survivors: a randomized trial.

CONTEXT: Blood homocysteine levels are positively associated with cardiovascular disease, but it is uncertain whether the association is causal. OBJECTIVE: To assess the effects of reducing homocysteine levels with folic acid and vitamin B(12) on vascular and nonvascular outcomes. DESIGN, SETTING, AND PATIENTS: Double-blind randomized controlled trial of 12,064 survivors of myocardial infarction in secondary care hospitals in the United Kingdom between 1998 and 2008. INTERVENTIONS: 2 mg folic acid plus 1 mg vitamin B(12) daily vs matching placebo. MAIN OUTCOME MEASURES: First major vascular event, defined as major coronary event (coronary death, myocardial infarction, or coronary revascularization), fatal or nonfatal stroke, or noncoronary revascularization. RESULTS: Allocation to the study vitamins reduced homocysteine by a mean of 3.8 micromol/L (28%). During 6.7 years of follow-up, major vascular events occurred in 1537 of 6033 participants (25.5%) allocated folic acid plus vitamin B(12) vs 1493 of 6031 participants (24.8%) allocated placebo (risk ratio [RR], 1.04; 95% confidence interval [CI], 0.97-1.12; P = .28). There were no apparent effects on major coronary events (vitamins, 1229 [20.4%], vs placebo, 1185 [19.6%]; RR, 1.05; 95% CI, 0.97-1.13), stroke (vitamins, 269 [4.5%], vs placebo, 265 [4.4%]; RR, 1.02; 95% CI, 0.86-1.21), or noncoronary revascularizations (vitamins, 178 [3.0%], vs placebo, 152 [2.5%]; RR, 1.18; 95% CI, 0.95-1.46). Nor were there significant differences in the numbers of deaths attributed to vascular causes (vitamins, 578 [9.6%], vs placebo, 559 [9.3%]) or nonvascular causes (vitamins, 405 [6.7%], vs placebo, 392 [6.5%]) or in the incidence of any cancer (vitamins, 678 [11.2%], vs placebo, 639 [10.6%]). CONCLUSION: Substantial long-term reductions in blood homocysteine levels with folic acid and vitamin B(12) supplementation did not have beneficial effects on vascular outcomes but were also not associated with adverse effects on cancer incidence. TRIAL REGISTRATION: isrctn.org Identifier: ISRCTN74348595.

Artemisinins: their growing importance in medicine.

Artemisinins are derived from extracts of sweet wormwood (Artemisia annua) and are well established for the treatment of malaria, including highly drug-resistant strains. Their efficacy also extends to phylogenetically unrelated parasitic infections such as schistosomiasis. More recently, they have also shown potent and broad anticancer properties in cell lines and animal models. In this review, we discuss recent advances in defining the role of artemisinins in medicine, with particular focus on their controversial mechanisms of action. This safe and cheap drug class that saves lives at risk from malaria can also have important potential in oncology.