Detecting 16S rRNA Methyltransferases in Enterobacteriaceae by Use of Arbekacin.

16S rRNA methyltransferases confer resistance to most aminoglycosides, but discriminating their activity from that of aminoglycoside-modifying enzymes (AMEs) is challenging using phenotypic methods. We demonstrate that arbekacin, an aminoglycoside refractory to most AMEs, can rapidly detect 16S methyltransferase activity in Enterobacteriaceae with high specificity using the standard disk susceptibility test.

Genetic knockdown of a single organic anion transporter alters the expression of functionally related genes in Malpighian tubules of Drosophila melanogaster.

Insects excrete a wide variety of toxins via the Malpighian (renal) tubules. Previous studies have implicated three transporters in the secretion of the organic anion (OA) methotrexate (MTX) by the Drosophila Malpighian tubule: Drosophila multidrug resistance-associated protein (dMRP, CG6214), a multidrug efflux transporter (MET, CG30344), and an organic anion transporting polypeptide 58Dc (OATP58Dc, CG3380). RNA interference (RNAi) knockdown and P-element insertion mutation of single OA transporter genes were used to evaluate the importance of these three putative transporters in the secretion of MTX by the Malpighian tubules of Drosophila melanogaster. A major finding is that genetic knockdown of a single OA transporter gene leads to reductions in the expression of at least one other OA transporter gene and in secretion of MTX by Malpighian tubules isolated from flies reared on a standard diet. The pattern of changes indicates that decreases in MTX secretion do not correspond to decreases in dMRP expression in all of the RNAi lines. Genetic knockdown of a single OA transporter gene also alters the extent of upregulation of multiple OA transporter genes in the tubules in response to dietary MTX. Knockdown of dMRP is associated with a decrease in MET expression but an increase in OATP expression when flies are reared on MTX-enriched diet. Our results indicate that dMRP and MET are not the dominant MTX transporters in the tubules when flies are reared on MTX-enriched diets. At least one additional transporter, and possibly OATP, are required for MTX secretion. The implications of our results for studies using genetic knockdown techniques to identify OA transporters in whole tissues such as Malpighian tubules are discussed.

Effects of genetic knock-down of organic anion transporter genes on secretion of fluorescent organic ions by Malpighian tubules of Drosophila melanogaster.

An earlier study has shown that RNAi knock-down of a single organic anion transporter (OAT) gene in the principal cells of Drosophila Malpighian tubules is associated with reductions in the expression of multiple, functionally related genes. In this study, we measured the rates of secretion of four fluorescent ions by tubules isolated from flies expressing targeted RNAi knock-down of specific OAT genes. Droplets secreted by isolated tubules set up in the Ramsay assay were collected in optically flat capillary tubes and the concentrations of fluorescent ions were determined by confocal laser scanning microscopy. Reductions in the expression of organic anion (OA) transporting polypeptide 58Dc (OATP; CG3380) were associated with reduced secretion of the OAs fluorescein and Texas Red. Reduction in the expression of Drosophila multidrug resistance associated protein (dMRP; CG6214) was correlated with reduced secretion of the P-glycoprotein substrate daunorubicin. Secretion of the organic cation quinacrine was unaffected by reduced expression of OATP, dMRP, or a multidrug efflux transporter (MET; CG30344). The results highlight the difficulties of assigning a rate-limiting role in transport of a specific OA to a single membrane transporter.

Interactions between detoxification mechanisms and excretion in Malpighian tubules of Drosophila melanogaster.

Insects have long been known to excrete toxins via the Malpighian (renal) tubules. In addition, exposure to natural or synthetic toxins is commonly associated with increases in the activity of detoxification enzymes such as the P450 monoxygenases (P450s) and the glutathione-S-transferases (GSTs). We examined the links between mechanisms for detoxification and excretion in adult Drosophila melanogaster using functional assays and measurements of changes in gene expression by quantitative reverse transcriptase PCR in response to dietary exposure to compounds known to alter activity or gene expression of P450s and GSTs. Dietary exposure to phenol, which alters gene expression for multiple GSTs after seven to 10 generations, was also associated with an increase (more than twofold) in secretion of the organic anion methotrexate (MTX) by isolated tubules. Dietary exposure to the insecticide synergist piperonyl butoxide (PBO) was associated with reduced expression of two P450 genes (Cyp4e2, Cyp4p1) and two GST genes (GstD1, GstD5) in the tubules, as well as increased expression of Cyp12d1 and GstE1. Thin layer chromatographic analysis of fluid secreted by isolated tubules indicated that dietary exposure to PBO resulted in increased levels of an MTX metabolite. In addition, exposure to PBO altered the expression of transporter genes in the tubules, including a Drosophila multidrug resistance-associated protein, and was associated with a 73% increase in MTX secretion by isolated tubules. The results suggest that exposure of Drosophila to toxins evokes a coordinated response by the Malpighian tubules, involving both alterations in detoxification pathways as well as enhanced transport.

Effects of acute or chronic exposure to dietary organic anions on secretion of methotrexate and salicylate by Malpighian tubules of Drosophila melanogaster larvae.

The effects of dietary exposure to organic anions on the physiology of isolated Malpighian tubules and on tubule gene expression were examined using larvae of Drosophila melanogaster. Acute (24 h) or chronic (7 d) exposure to type I organic anions (fluorescein or salicylate) was associated with increased fluid secretion rates and increased fluxes of both salicylate and the type II organic anion methotrexate. By contrast, chronic exposure to dietary methotrexate was associated with increased fluid secretion rate and increased flux of methotrexate, but not salicylate. Exposure to methotrexate in the diet resulted in increases in the expression of a multidrug efflux transporter gene (MET; CG30344) in the Malpighian tubules. There were also increases in expression of genes for either a Drosophila multidrug resistance-associated protein (dMRP; CG6214) or an organic anion transporting polypeptide (OATP; CG3380), depending on the concentration of methotrexate in the diet. Exposure to salicylate in the diet was associated with an increase in expression of dMRP and with decreases of MET and OATP. Exposure to dietary salicylate or methotrexate was also associated with different patterns of expression of heat shock protein genes. The results suggest that exposure to specific type I or type II organic anions has multiple effects and results not only in increased organic anion transport but also in increased rates of inorganic ion transport, which drives osmotically-obliged fluid secretion. Increased fluid secretion may enhance secretion of organic anions by eliminating diffusive backflux from the tubule lumen to the hemolymph.

Cervical artery dissections: Factors that influence causation determination in litigated cases.

In litigated cases, the suspected causes of cervical artery dissections (CADs) are a source of considerable debate among experts. In this study, we sought to examine the factors influencing court decisions and discover how Canadian tribunals analyzed and arbitrated conflicting expert opinions in CAD cases. Cases for this review were identified through searches of the Canadian CANLII database. First, the results of this study show that there is no standardized methodology to assist health care personnel in the processing and interpretation of data in individual cases of CAD. This leads to wide ranges of personal interpretations and opinions which may confuse tribunals. Of concern is the implication of treating physicians who may not have the objectivity to act as expert witnesses when one of their patients is engaged in a legal proceeding.

A jurisdictional review of the legislation governing informed consent by chiropractors across Canada.

The objective of this jurisdictional review is to provide summary information pertaining to the regulation of chiropractors in Canadian provinces and territories on the topic of informed consent. Our review shows that two provinces have legislated for all healthcare professions: Ontario and Prince Edward Island. Two chiropractic regulatory bodies (New Brunswick and, Newfoundland and Labrador) have adopted the Canadian Chiropractic Association Code of Conduct. All chiropractic regulatory bodies of the other provinces and Yukon have adopted their own specific dispositions regarding informed consent. Chiropractors in Prince Edward Island, Saskatchewan and Québec must obtain informed consent in writing.

L'objectif de cette étude juridictionnelle est de fournir des informations sommaires portant sur la réglementation des chiropraticiens dans les provinces et territoires canadiens en ce qui concerne le consentement éclairé. Notre étude montre que deux provinces (l'Ontario et l'Île-du-Prince-Édouard) ont légiféré sur toutes les professions de la santé. Deux organismes de réglementation chiropratique (le Nouveau-Brunswick et, Terre-Neuve-et-Labrador) ont adopté le Code de conduite de l'Association chiropratique canadienne. Tous les organismes de réglementation de chiropratique des autres provinces et le Yukon ont adopté leurs propres dispositions spécifiques concernant le consentement éclairé. Les chiropraticiens de l'Île-du-Prince-Édouard, de la Saskatchewan et du Québec doivent obtenir le consentement éclairé par écrit.

Physiological and molecular characterization of methotrexate transport by Malpighian tubules of adult Drosophila melanogaster.

A radioisotope tracer technique and quantitative PCR were used to study the mechanisms and regulation of transepithelial transport of the type II organic anion methotrexate (MTX) by the Malpighian tubules of Drosophila melanogaster. Transport of MTX was saturable and Na(+)-independent; the kinetic parameters J(max) and K(t) were 437fmolmin(-1) and 23.5microM, respectively. The transport of MTX was competitively inhibited by phenol red and probenecid; non-competitively inhibited by salicylate, verapamil and MK-571; and uncompetitively inhibited by Texas Red. Dietary exposure to 0.1mM MTX led to dramatic increases in gene expression for several members of the ABC family of transporters in both the Malpighian tubules and the gut. Our results suggest that multiple transporters are upregulated in response to dietary exposure to MTX. Increased levels of the protein products which may result from expression of these genes may enhance elimination of toxic compounds such as MTX or its metabolites.