Tim J. Vickers

Seeking Health Information Online: Does Wikipedia Matter?

OBJECTIVE To determine the significance of the English Wikipedia as a source of online health information. DESIGN The authors measured Wikipedias ranking on general Internet search engines by entering keywords from MedlinePlus, NHS Direct Online, and the National Organization of Rare Diseases as queries into search engine optimization software. We assessed whether article quality influenced this ranking. The authors tested whether traffic to Wikipedia coincided with epidemiological trends and news of emerging health concerns, and how it compares to MedlinePlus. MEASUREMENTS Cumulative incidence and average position of Wikipedia compared to other Web sites among the first 20 results on general Internet search engines (Google, Google UK, Yahoo, and MSN, and page view statistics for selected Wikipedia articles and MedlinePlus pages. RESULTS Wikipedia ranked among the first ten results in 71-85% of search engines and keywords tested. Wikipedia surpassed MedlinePlus and NHS Direct Online (except for queries from the latter on Google UK), and ranked higher with quality articles. Wikipedia ranked highest for rare diseases, although its incidence in several categories decreased. Page views increased parallel to the occurrence of 20 seasonal disorders and news of three emerging health concerns. Wikipedia articles were viewed more often than MedlinePlus Topic (p = 0.001) but for MedlinePlus Encyclopedia pages, the trend was not significant (p = 0.07-0.10). CONCLUSIONS Based on its search engine ranking and page view statistics, the English Wikipedia is a prominent source of online health information compared to the other online health information providers studied.

Wikipedia: a key tool for global public health promotion.

The Internet has become an important health information resource for patients and the general public. Wikipedia, a collaboratively written Web-based encyclopedia, has become the dominant online reference work. It is usually among the top results of search engine queries, including when medical information is sought. Since April 2004, editors have formed a group called WikiProject Medicine to coordinate and discuss the English-language Wikipedia’s medical content. This paper, written by members of the WikiProject Medicine, discusses the intricacies, strengths, and weaknesses of Wikipedia as a source of health information and compares it with other medical wikis. Medical professionals, their societies, patient groups, and institutions can help improve Wikipedia’s health-related entries. Several examples of partnerships already show that there is enthusiasm to strengthen Wikipedia’s biomedical content. Given its unique global reach, we believe its possibilities for use as a tool for worldwide health promotion are underestimated. We invite the medical community to join in editing Wikipedia, with the goal of providing people with free access to reliable, understandable, and up-to-date health information.

Roles of Trypanothione S-Transferase and Tryparedoxin Peroxidase in Resistance to Antimonials

ABSTRACT The clinical value of antimonial drugs, the mainstay therapy for leishmaniasis, is now threatened by the emergence of acquired drug resistance, and a comprehensive understanding of the underlying mechanisms is required. Using the model organism Leishmania tarentolae, we have examined the role of trypanothione S-transferase (TST) in trivalent antimony [Sb(III)] resistance. TST has S-transferase activity with substrates such as chlorodinitrobenzene as well as peroxidase activity with alkyl and aryl hydroperoxides but not with hydrogen peroxide. Although S-transferase activity and TST protein levels were unchanged in Sb(III)-sensitive and -resistant lines, rates of metabolism of hydrogen peroxide, t-butyl hydroperoxide, and cumene hydroperoxide were significantly increased. Elevated peroxidase activities were shown to be both trypanothione and tryparedoxin dependent and were associated with the overexpression of classical tryparedoxin peroxidase (TryP) in the cytosol of L. tarentolae. The role of TryP in Sb(III) resistance was verified by overexpression of the recombinant Leishmania major protein in Sb(III)-sensitive promastigotes. An approximate twofold increase in the level of TryP activity in this transgenic cell line was accompanied by a significant decrease in sensitivity to Sb(III) (twofold; P < 0.001). Overexpression of an enzymatically inactive TryP failed to result in Sb(III) resistance. This indicates that TryP-dependent resistance is not due to sequestration of Sb(III) and suggests that enhanced antioxidant defenses may well be a key feature of mechanisms of clinical resistance to antimonial drugs.

A role for tetrahydrofolates in the metabolism of iron-sulfur clusters in all domains of life

Iron-sulfur (Fe/S) cluster enzymes are crucial to life. Their assembly requires a suite of proteins, some of which are specific for particular subsets of Fe/S enzymes. One such protein is yeast Iba57p, which aconitase and certain radical S-adenosylmethionine enzymes require for activity. Iba57p homologs occur in all domains of life; they belong to the COG0354 protein family and are structurally similar to various folate-dependent enzymes. We therefore investigated the possible relationship between folates and Fe/S cluster enzymes using the Escherichia coli Iba57p homolog, YgfZ. NMR analysis confirmed that purified YgfZ showed stereoselective folate binding. Inactivating ygfZ reduced the activities of the Fe/S tRNA modification enzyme MiaB and certain other Fe/S enzymes, although not aconitase. When successive steps in folate biosynthesis were ablated, ∆folE (lacking pterins and folates) and ∆folP (lacking folates) mutants mimicked the ∆ygfZ mutant in having low MiaB activities, whereas ∆folE ∆thyA mutants supplemented with 5-formyltetrahydrofolate (lacking pterins and depleted in dihydrofolate) and ∆gcvP ∆glyA mutants (lacking one-carbon tetrahydrofolates) had intermediate MiaB activities. These data indicate that YgfZ requires a folate, most probably tetrahydrofolate. Importantly, the ∆ygfZ mutant was hypersensitive to oxidative stress and grew poorly on minimal media. COG0354 genes of bacterial, archaeal, fungal, protistan, animal, or plant origin complemented one or both of these growth phenotypes as well as the MiaB activity phenotype. Comparative genomic analysis indicated widespread functional associations between COG0354 proteins and Fe/S cluster metabolism. Thus COG0354 proteins have an ancient, conserved, folate-dependent function in the activity of certain Fe/S cluster enzymes.

EatA, an Immunogenic Protective Antigen of Enterotoxigenic Escherichia coli, Degrades Intestinal Mucin

ABSTRACT Enterotoxigenic Escherichia coli (ETEC) is a major cause of morbidity and mortality due to infectious diarrhea in developing countries for which there is presently no effective vaccine. A central challenge in ETEC vaccinology has been the identification of conserved surface antigens to formulate a broadly protective vaccine. Here, we demonstrate that EatA, an immunogenic secreted serine protease of ETEC, contributes to virulence by degrading MUC2, the major protein present in the small intestinal mucous layer, and that removal of this barrier in vitro accelerates toxin access to the enterocyte surface. In addition, we demonstrate that vaccination with the recombinant secreted passenger domain of EatA (rEatAp) elicits high titers of antibody and is protective against intestinal infection with ETEC. These findings may have significant implications for development of both subunit and live-attenuated vaccines against ETEC and other enteric pathogens, including Shigella flexneri, that express similar proteins.

Specificity of the Trypanothione-Dependent Leishmania Major Glyoxalase I: Structure and Biochemical Comparison with the Human Enzyme.

Trypanothione replaces glutathione in defence against cellular damage caused by oxidants, xenobiotics and methylglyoxal in the trypanosomatid parasites, which cause trypanosomiasis and leishmaniasis. In Leishmania major, the first step in methylglyoxal detoxification is performed by a trypanothione‐dependent glyoxalase I (GLO1) containing a nickel cofactor; all other characterized eukaryotic glyoxalases use zinc. In kinetic studies L. major and human enzymes were active with methylglyoxal derivatives of several thiols, but showed opposite substrate selectivities: N1‐glutathionylspermidine hemithioacetal is 40‐fold better with L. major GLO1, whereas glutathione hemithioacetal is 300‐fold better with human GLO1. Similarly, S‐4‐bromobenzylglutathionylspermidine is a 24‐fold more potent linear competitive inhibitor of L. major than human GLO1 (Kis of 0.54 µM and 12.6 µM, respectively), whereas S‐4‐bromobenzylglutathione is > 4000‐fold more active against human than L. major GLO1 (Kis of 0.13 µM and > 500 µM respectively). The crystal structure of L. major GLO1 reveals differences in active site architecture to both human GLO1 and the nickel‐dependent Escherichia coli GLO1, including increased negative charge and hydrophobic character and truncation of a loop that may regulate catalysis in the human enzyme. These differences correlate with the differential binding of glutathione and trypanothione‐based substrates, and thus offer a route to the rational design of L. major‐specific GLO1 inhibitors.

Trypanothione-dependent glyoxalase I in Trypanosoma cruzi

The glyoxalase system, comprizing glyoxalase I and glyoxalase II, is a ubiquitous pathway that detoxifies highly reactive aldehydes, such as methylglyoxal, using glutathione as a cofactor. Recent studies of Leishmania major glyoxalase I and Trypanosoma brucei glyoxalase II have revealed a unique dependence upon the trypanosomatid thiol trypanothione as a cofactor. This difference suggests that the trypanothione-dependent glyoxalase system may be an attractive target for rational drug design against the trypanosomatid parasites. Here we describe the cloning, expression and kinetic characterization of glyoxalase I from Trypanosoma cruzi. Like L. major glyoxalase I, recombinant T. cruzi glyoxalase I showed a preference for nickel as its metal cofactor. In contrast with the L. major enzyme, T. cruzi glyoxalase I was far less fast-idious in its choice of metal cofactor efficiently utilizing cobalt, manganese and zinc. T. cruzi glyoxalase I isomerized hemithio-acetal adducts of trypanothione more than 2400 times more efficiently than glutathione adducts, with the methylglyoxal adducts 2-3-fold better substrates than the equivalent phenylglyoxal adducts. However, glutathionylspermidine hemithioacetal adducts were most efficiently isomerized and the glutathionylspermidine-based inhibitor S-4-bromobenzylglutathionylspermidine was found to be a potent linear competitive inhibitor of the T. cruzi enzyme with a K(i) of 5.4+/-0.6 microM. Prediction algorithms, combined with subcellular fractionation, suggest that T. cruzi glyoxalase I localizes not only to the cytosol but also the mitochondria of T. cruzi epimastigotes. The contrasting substrate specificities of human and trypanosomatid glyoxalase enzymes, confirmed in the present study, suggest that the glyoxalase system may be an attractive target for anti-trypanosomal chemotherapy.

Methylene tetrahydrofolate dehydrogenase/cyclohydrolase and the synthesis of 10-CHO-THF are essential in Leishmania major

10‐Formyl tetrahydrofolate (10‐CHO‐THF) is a key metabolite in C1 carbon metabolism, arising through the action of formate‐tetrahydrofolate ligase (FTL) and/or 5,10‐methenyltetrahydrofolate cyclohydrolase/5,10‐methylene tetrahydrofolate dehydrogenase (DHCH). Leishmania major possesses single DHCH1 and FTL genes encoding exclusively cytosolic proteins, unlike other organisms where isoforms occur in the mitochondrion as well. Recombinant DHCH1 showed typical NADP+‐dependent methylene tetrahydrofolate DH and 5,10‐methenyltetrahydrofolate CH activities, and the DH activity was potently inhibited by a substrate analogue 5,10‐CO‐THF (Ki 105 nM), as was Leishmania growth (EC50 1.1 μM). Previous studies showed null ftl‐ mutants were normal, raising the possibility that loss of the purine synthetic pathway had rendered 10‐CHO‐THF dispensable in evolution. We were unable to generate dhch1‐ null mutants by gene replacement, despite using a wide spectrum of nutritional supplements expected to bypass DHCH function. We applied an improved method for testing essential genes in Leishmania, based on segregational loss of episomal complementing genes rather than transfection; analysis of ∼1400 events without successful loss of DHCH1 again established its requirement. Lastly, we employed ‘genetic metabolite complementation’ using ectopically expressed FTL as an alternative source of 10‐CHO‐THF; now dhch1‐ null parasites were readily obtained. These data establish a requirement for 10‐CHO‐THF metabolism in L. major, and provide genetic and pharmacological validation of DHCH as a target for chemotherapy, in this and potentially other protozoan parasites.

Biochemical and genetic analysis of methylenetetrahydrofolate reductase in Leishmania metabolism and virulence.

Methylenetetrahydrofolate reductase (MTHFR; EC 1.5.1.20) is the sole enzyme responsible for generation of 5-methyltetrahydrofolate, which is required for methionine synthesis and provision of methyl groups via S-adenosylmethionine. Genome analysis showed that Leishmania species, unlike Trypanosoma brucei and Trypanosoma cruzi, contain genes encoding MTHFR and two distinct methionine synthases. Leishmania MTHFR differed from those in other eukaryotes by the absence of a C-terminal regulatory domain. L. major MTHFR was expressed in yeast and recombinant enzyme was produced in Escherichia coli. MTHFR was not inhibited by S-adenosylmethionine and, uniquely among folate-metabolizing enzymes, showed dual-cofactor specificity with NADH and NADPH under physiological conditions. MTHFR null mutants (mthfr–) lacked 5-methyltetrahydrofolate, the most abundant intracellular folate, and could not utilize exogenous homocysteine for growth. Under conditions of methionine limitation mthfr– mutant cells grew poorly, whereas their growth was normal in standard culture media. Neither in vitro MTHFR activity nor the growth of mthfr– mutants or MTHFR overexpressors were differentially affected by antifolates known to inhibit parasite growth via targets beyond dihydrofolate reductase and pteridine reductase 1. In a mouse model of infection mthfr– mutants showed good infectivity and virulence, indicating that sufficient methionine is available within the parasitophorous vacuole to meet the needs of the parasite.

PTR1-dependent synthesis of tetrahydrobiopterin contributes to oxidant susceptibility in the trypanosomatid protozoan parasite Leishmania major

Leishmania must survive oxidative stress, but lack many classical antioxidant enzymes and rely heavily on trypanothione-dependent pathways. We used forward genetic screens to recover loci mediating oxidant resistance via overexpression in Leishmania major, which identified pteridine reductase 1 (PTR1). Comparisons of isogenic lines showed ptr1− null mutants were 18-fold more sensitive to H2O2 than PTR1-overproducing lines, and significant three- to fivefold differences were seen with a broad panel of oxidant-inducing agents. The toxicities of simple nitric oxide generators and other drug classes (except antifolates) were unaffected by PTR1 levels. H2O2 susceptibility could be modulated by exogenous biopterin but not folate, in a PTR1- but not dihydrofolate reductase-dependent manner, implicating H4B metabolism specifically. Neither H2O2 consumption nor the level of intracellular oxidative stress was affected by PTR1 levels. Coupled with the fact that reduced pteridines are at least 100-fold less abundant than cellular thiols, these data argue strongly that reduced pteridines act through a mechanism other than scavenging. The ability of unconjugated pteridines to counter oxidative stress has implications to infectivity and response to chemotherapy. Since the intracellular pteridine levels of Leishmania can be readily manipulated, these organisms offer a powerful setting for the dissection of pteridine-dependent oxidant susceptibility in higher eukaryotes.