Metagenomic and metatranscriptomic analyses reveal that biobed systems can enrich for antibiotic resistance and genetic mobility genes.

Antibiotic resistance gene pollution in the environment has been identified as a potential contributor to the global issue of antibiotic resistance prevalence, creating a need to identify and characterize environmental reservoirs for antibiotic resistance genes. Because many polluted environments have been shown to contain elevated levels of antibiotic resistance genes, agriculturally based pesticide bioremediation systems called 'biobeds' could serve as environmental reservoirs for antibiotic resistance genes, although this has never been extensively explored. Metagenomic and metatranscriptomic analyses of an on-farm biobed system sampled before and after a season of pesticide use demonstrated that in situ pesticide applications applied to biobeds can enrich for multidrug, sulphonamide, aminoglycoside and beta-lactam resistance genes. Additionally, this study demonstrated an enrichment for genes associated with gene mobilization, such as genes involved in horizontal gene transfer and plasmid mobility, as well as transposons and integrases.

Platelet-derived TLT-1 is a prognostic indicator in ALI/ARDS and prevents tissue damage in the lungs in a mouse model.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) affect >200 000 individuals yearly with a 40% mortality rate. Although platelets are implicated in the progression of ALI/ARDS, their exact role remains undefined. Triggering receptor expressed in myeloid cells (TREM)-like transcript 1 (TLT-1) is found on platelets, binds fibrinogen, and mediates clot formation. We hypothesized that platelets use TLT-1 to manage the progression of ALI/ARDS. Here we retrospectively measure plasma levels of soluble TLT-1 (sTLT-1) from the ARDS Network clinical trial and show that patients whose sTLT-1 levels were >1200 pg/mL had nearly twice the mortality risk as those with <1200 pg/mL (P < .001). After correcting for confounding factors such as creatinine levels, Acute Physiology And Chronic Health Evaluation III scores, age, platelet counts, and ventilation volume, sTLT-1 remains significant, suggesting that sTLT-1 is an independent prognostic factor (P < .0001). These data point to a role for TLT-1 during the progression of ALI/ARDS. We use a murine lipopolysaccharide-induced ALI model and demonstrate increased alveolar bleeding, aberrant neutrophil transmigration and accumulation associated with decreased fibrinogen deposition, and increased pulmonary tissue damage in the absence of TLT-1. The loss of TLT-1 resulted in an increased proportion of platelet-neutrophil conjugates (43.73 ± 24.75% vs 8.92 ± 2.4% in wild-type mice), which correlated with increased neutrophil death. Infusion of sTLT-1 restores normal fibrinogen deposition and reduces pulmonary hemorrhage by 40% (P ≤ .001) and tissue damage by 25% (P ≤ .001) in vivo. Our findings suggest that TLT-1 uses fibrinogen to govern the transition between inflammation and hemostasis and facilitate controlled leukocyte transmigration during the progression of ARDS.

Identifying the core bacterial and fungal communities within four agricultural biobeds used for the treatment of pesticide rinsates.

AIMS: This study evaluated the variability of bacterial and fungal communities within unique pesticide remediation biobeds. METHODS AND RESULTS: Four biobeds receiving different applied pesticide rinsates, were sampled throughout an operational season to determine pesticide removal efficacy and microbial communities. Biomixture samples collected from different biobed depths, were subjected to Illumina sequencing of the 16S rRNA (bacteria) and ITS2 (fungi) genes. Pesticide removal rates for all biobeds averaged 99%, with microbial community analysis revealing biobeds shared 60-70% of the most abundant bacterial and fungal orders, respectively. Though biobed depth did not greatly impact microbial community profile or diversity, bacterial and fungal taxa profiles between biobeds notably diverge at levels of genera and OTU. Biobed bacterial communities exhibited greater diversity than fungal communities between and within all biobeds. CONCLUSIONS: Biobeds receiving variable pesticide rinsates share a 'core' microbial community, exhibiting greater bacterial diversity relative to fungal diversity. Pesticide exposure increased bacterial diversity throughout the biobeds, while fungal diversity was variable, meriting further understanding of fungicide application to biobed fungal community stability. SIGNIFICANCE AND IMPACT OF THE STUDY: Biobeds achieve high treatment efficacy of unique pesticide rinsates, regardless of differentiation of specific genera in response to specific compounds; supporting biobeds as a robust engineered system for pesticide rinsates bioremediation.

Successful implementation of an Enhanced Recovery After Surgery program shortens length of stay and improves postoperative pain, and bowel and bladder function after colorectal surgery.

BACKGROUND: Despite international data indicating that Enhanced Recovery After Surgery (ERAS) programs, which combine evidence-based perioperative strategies, expedite recovery after surgery, few centers have successfully adopted this approach within the U.S. We describe the implementation and efficacy of an ERAS program for colorectal abdominal surgery in a tertiary teaching center in the U.S. METHODS: We used a multi-modal and continuously evolving approach to implement an ERAS program among all patients undergoing colorectal abdominal surgery at a single hospital at the University of California, San Francisco. 279 patients who participated in the Enhanced Recovery after Surgery program were compared to 245 previous patients who underwent surgery prior to implementation of the program. Primary end points were length of stay and readmission rates. Secondary end points included postoperative pain scores, opioid consumption, postoperative nausea and vomiting, length of urinary catheterization, and time to first solid meal. RESULTS: ERAS decreased both median total hospital length of stay (6.4 to 4.4 days) and post-procedure length of stay (6.0 to 4.1 days). 30-day all-cause readmission rates decreased from 21 to 9.4 %. Pain scores improved on postoperative day 0 (3.2 to 2.1) and day 1 (3.2 to 2.6) despite decreased opioid. Median time to first solid meal decreased from 4.7 to 2.7 days and duration of urinary catheterization decreased from 74 to 46 h. Similar improvements were observed in all other secondary end points. CONCLUSIONS: These results confirm that a multidisciplinary, iterative, team-based approach is associated with a reduction in hospital stay and an acceleration in recovery without increasing readmission rates.

Characterization of a mobile and multiple resistance plasmid isolated from swine manure and its detection in soil after manure application.

AIMS: To isolate and characterize multiple antibiotic resistance plasmids found in swine manure and test for plasmid-associated genetic markers in soil following manure application to an agricultural field. METHODS AND RESULTS: Plasmids were isolated from an erythromycin enrichment culture that used liquid swine manure as an inoculant. Plasmids were transformed into Escherichia coli DH10ß for subsequent characterization. We isolated and DNA sequenced a 22 102-bp plasmid (pMC2) that confers macrolide, and tetracycline resistances, and carries genes predicted to code for mercury and chromium resistance. Conjugation experiments using an pRP4 derivative as a helper plasmid confirm that pMC2 has a functional mobilization unit. PCR was used to detect genetic elements found on pMC2 in DNA extracted from manure amended soil. CONCLUSIONS: The pMC2 plasmid has a tetracycline-resistant core and has acquired additional resistance genes by insertion of an accessory region (12 762 bp) containing macrolide, mercury and chromium resistance genes, which was inserted between the truncated DDE motifs within the Tn903/IS102 mobile element. SIGNIFICANCE AND IMPACT OF THE STUDY: Liquid swine manure used for manure spreading contains multiple antibiotic resistance plasmids that can be detected in soil following manure application.

Quantitative real-time PCR assays for sensitive detection of Canada goose-specific fecal pollution in water sources.

Canada geese (Branta canadensis) are prevalent in North America and may contribute to fecal pollution of water systems where they congregate. This work provides two novel real-time PCR assays (CGOF1-Bac and CGOF2-Bac) allowing for the specific and sensitive detection of Bacteroides 16S rRNA gene markers present within Canada goose feces.

Discrete change in volatile anesthetic sensitivity in mice with inactivated tandem pore potassium ion channel TRESK.

BACKGROUND: We investigated the role of tandem pore potassium ion channel (K2P) TRESK in neurobehavioral function and volatile anesthetic sensitivity in genetically modified mice. METHODS: Exon III of the mouse TRESK gene locus was deleted by homologous recombination using a targeting vector. The genotype of bred mice (wild type, knockout, or heterozygote) was determined using polymerase chain reaction. Morphologic and behavioral evaluations of TRESK knockout mice were compared with wild-type littermates. Sensitivity of bred mice to isoflurane, halothane, sevoflurane, and desflurane were studied by determining the minimum alveolar concentration preventing movement to tail clamping in 50% of each genotype. RESULTS: With the exception of decreased number of inactive periods and increased thermal pain sensitivity (20% decrease in latency with hot plate test), TRESK knockout mice had healthy development and behavior. TRESK knockout mice showed a statistically significant 8% increase in isoflurane minimum alveolar concentration compared with wild-type littermates. Sensitivity to other volatile anesthetics was not significantly different. Spontaneous mortality of TRESK knockout mice after initial anesthesia testing was nearly threefold higher than that of wild-type littermates. CONCLUSIONS: TRESK alone is not critical for baseline central nervous system function but may contribute to the action of volatile anesthetics. The inhomogeneous change in anesthetic sensitivity corroborates findings in other K2P knockout mice and supports the theory that the mechanism of volatile anesthetic action involves multiple targets. Although it was not shown in this study, a compensatory effect by other K2P channels may also contribute to these observations.

Establishment of the dorso-ventral axis in Xenopus embryos is presaged by early asymmetries in beta-catenin that are modulated by the Wnt signaling pathway.

Eggs of Xenopus laevis undergo a postfertilization cortical rotation that specifies the position of the dorso-ventral axis and activates a transplantable dorsal-determining activity in dorsal blastomeres by the 32-cell stage. There have heretofore been no reported dorso-ventral asymmetries in endogenous signaling proteins that may be involved in this dorsal-determining activity during early cleavage stages. We focused on beta-catenin as a candidate for an asymmetrically localized dorsal-determining factor since it is both necessary and sufficient for dorsal axis formation. We report that beta-catenin displays greater cytoplasmic accumulation on the future dorsal side of the Xenopus embryo by the two-cell stage. This asymmetry persists and increases through early cleavage stages, with beta-catenin accumulating in dorsal but not ventral nuclei by the 16- to 32-cell stages. We then investigated which potential signaling factors and pathways are capable of modulating the steady-state levels of endogenous beta-catenin. Steady-state levels and nuclear accumulation of beta-catenin increased in response to ectopic Xenopus Wnt-8 (Xwnt-8) and to the inhibition of glycogen synthase kinase-3, whereas neither Xwnt-5A, BVg1, nor noggin increased beta-catenin levels before the mid-blastula stage. As greater levels and nuclear accumulation of beta-catenin on the future dorsal side of the embryo correlate with the induction of specific dorsal genes, our data suggest that early asymmetries in beta-catenin presage and may specify dorso-ventral differences in gene expression and cell fate. Our data further support the hypothesis that these dorso-ventral differences in beta-catenin arise in response to the postfertilization activation of a signaling pathway that involves Xenopus glycogen synthase kinase-3.

Interaction among GSK-3, GBP, axin, and APC in Xenopus axis specification.

Glycogen synthase kinase 3 (GSK-3) is a constitutively active kinase that negatively regulates its substrates, one of which is beta-catenin, a downstream effector of the Wnt signaling pathway that is required for dorsal-ventral axis specification in the Xenopus embryo. GSK-3 activity is regulated through the opposing activities of multiple proteins. Axin, GSK-3, and beta-catenin form a complex that promotes the GSK-3-mediated phosphorylation and subsequent degradation of beta-catenin. Adenomatous polyposis coli (APC) joins the complex and downregulates beta-catenin in mammalian cells, but its role in Xenopus is less clear. In contrast, GBP, which is required for axis formation in Xenopus, binds and inhibits GSK-3. We show here that GSK-3 binding protein (GBP) inhibits GSK-3, in part, by preventing Axin from binding GSK-3. Similarly, we present evidence that a dominant-negative GSK-3 mutant, which causes the same effects as GBP, keeps endogenous GSK-3 from binding to Axin. We show that GBP also functions by preventing the GSK-3-mediated phosphorylation of a protein substrate without eliminating its catalytic activity. Finally, we show that the previously demonstrated axis-inducing property of overexpressed APC is attributable to its ability to stabilize cytoplasmic beta-catenin levels, demonstrating that APC is impinging upon the canonical Wnt pathway in this model system. These results contribute to our growing understanding of how GSK-3 regulation in the early embryo leads to regional differences in beta-catenin levels and establishment of the dorsal axis.

Unusual topogenic sequence directs prion protein biogenesis.

Biosynthetic studies of the prion protein (PrP) have shown that two forms of different topology can be generated from the same pool of nascent chains in cell-free translation systems supplemented with microsomal membranes. A transmembrane form is the predominant product generated in wheat germ (WG) extracts, whereas a completely translocated (secretory) form is the major product synthesized in rabbit reticulocyte lysates (RRL). An unusual topogenic sequence within PrP is now shown to direct this system-dependent difference. The actions of this topogenic sequence were independent of on-going translation and could be conferred to heterologous proteins by the engineering of a discrete set of codons. System-dependent topology conferred by addition of RRL to WG translation products suggests that this sequence interacts with one or more cytosolic factors.

Assessment of the microbial quality of irrigation water in a prairie watershed.

AIMS: To assess levels of faecal contamination in the Qu'Appelle River (Saskatchewan, Canada) and its suitability for irrigation, by using the Colilert-18/Quanti-Tray technology. METHODS AND RESULTS: Various sites located along the Qu'Appelle River were sampled weekly from May to August 2005-2007. A total of 594 freshwater samples were collected and analysed for enumeration of Escherichia coli using the Colilert-18. The false-positive rate for E. coli detection using Colilert-18 was at most 1.5%. Throughout the irrigation period (June to August), up to 85% of the water samples collected from one of the irrigation water-pumping sites exceeded the recommended limit of 100 CFU per 100 ml. Spikes in E. coli counts were generally concomitant with the sudden rise in river flows. A sub-sample of confirmed E. coli isolates were typed by randomly amplified polymorphic DNA (RAPD). RAPD analysis revealed a high degree of genetic diversity among E. coli isolates. A significant association between RAPD patterns and the month of E. coli isolation was demonstrated. CONCLUSIONS: Colilert-18 provides an effective means for assessing microbial quality of irrigation water. SIGNIFICANCE AND IMPACT OF THE STUDY: Qu'Appelle River is subject to variability of faecal contamination during irrigation times and monitoring throughout irrigation season is important for ensuring safe production practices.

Knockout of the gene encoding the K(2P) channel KCNK7 does not alter volatile anesthetic sensitivity.

The molecular site of action for volatile anesthetics remains unknown despite many years of study. Members of the K(2P) potassium channel family, whose currents are potentiated by volatile anesthetics have emerged as possible anesthetic targets. In fact, a mouse model in which the gene for TREK-1 (KCNK2) has been inactivated shows resistance to volatile anesthetics. In this study we tested whether inactivation of another member of this ion channel family, KCNK7, in a knockout mouse displayed altered sensitivity to the anesthetizing effect of volatile anesthetics. KCNK7 knockout mice were produced by standard gene inactivation methods. Heterozygous breeding pairs produced animals that were homozygous, heterozygous or wild-type for the inactivated gene. Knockout animals were tested for movement in response to noxious stimulus (tail clamp) under varying concentrations of isoflurane, halothane, and desflurane to define the minimum alveolar concentration (MAC) preventing movement. Mice homozygous for inactivated KCNK7 were viable and indistinguishable in weight, general development and behavior from heterozygotes or wild-type littermates. Knockout mice (KCNK7-/-) displayed no difference in MAC for the three volatile anesthetics compared to heterozygous (+/-) or wild-type (+/+) littermates. Because inactivation of KCNK7 does not alter MAC, KCNK7 may play only a minor role in normal CNS function or may have had its function compensated for by other inhibitory mechanisms. Additional studies with transgenic animals will help define the overall role of the K(2P) channels in normal neurophysiology and in volatile anesthetic mechanisms.

Reference intervals for stool calprotectin in preterm neonates and their utility for the diagnosis of necrotizing enterocolitis.

OBJECTIVE: Calprotectin is an antimicrobial protein found in stool when released by granulocytes. We sought to create stool calprotectin reference ranges in preterm neonates and to evaluate whether levels exceeding the upper reference interval are diagnostic for necrotizing enterocolitis (NEC). STUDY DESIGN: Stool calprotectin was measured in premature neonates without gastrointestinal pathology to create reference intervals. For comparison, levels from infants undergoing "rule out NEC" evaluations were plotted on these reference intervals. RESULTS: Stool calprotectin reference intervals were created according to gestational age at birth and corrected gestational age. Levels during "rule out NEC" evaluations were more often above the upper reference interval with NEC vs. those without NEC. CONCLUSIONS: Stools from preterm neonates have a higher range of calprotectin than stools from healthy term neonates. In evaluating preterm neonates for NEC with stool calprotectin, a calprotectin upper reference interval that incorporates corrected gestational age best predicts the diagnosis of NEC.

Chirality in anesthesia II: stereoselective modulation of ion channel function by secondary alcohol enantiomers.

Chirality has been proposed as a means for distinguishing relevant from irrelevant molecular targets of action, but the sensitivity and specificity of this test is unknown for volatile anesthetics. We applied enantiomers of two chiral anesthetic alcohols (2-butanol and 2-pentanol) that are enantioselective for the minimum alveolar concentration (MAC) preventing movement in 50% of animals and one (2-hexanol) that was not to frog oocytes. Each oocyte expressed one of three anesthetic-sensitive ion channels: a Twik-related-spinal cord K+ (TRESK) channel, a gamma-amino butyric acid type A (GABA(A)) receptor and an N-methyl-d-aspartate (NMDA) receptor. Using voltage-clamp techniques, we found that 2-butanol was not enantioselective for any channel (e.g., 16 mM 2-butanol R(-) and S(-) enantiomers decreased current through an NMDA receptors by 44% +/- 3% [mean +/- se] and 37% +/- 4%, respectively); 2-pentanol was enantioselective for one channel (the GABA(A) receptor, the enantiomers increasing current by 277% +/- 20% and 141% +/- 30%); 2-hexanol was enantioselective for both GABA(A) and NMDA receptors (e.g., decreasing current through the NMDA receptor by 19% +/- 3% and 43% +/- 5%). We calculated the sensitivity and specificity of chirality as a test of anesthetic relevance under two scenarios: 1) all three channels were relevant mediators of MAC and 2) no channel was a mediator of MAC. These sensitivities and specificities were poor because there is no consistent correspondence between receptor and whole animal results. We recommend that enantioselectivity not be used as a test of relevance for inhaled anesthetic targets.

The ventilatory stimulant doxapram inhibits TASK tandem pore (K2P) potassium channel function but does not affect minimum alveolar anesthetic concentration.

TWIK-related acid-sensitive K(+)-1 (TASK-1 [KCNK3]) and TASK-3 (KCNK9) are tandem pore (K(2P)) potassium (K) channel subunits expressed in carotid bodies and the brainstem. Acidic pH values and hypoxia inhibit TASK-1 and TASK-3 channel function, and halothane enhances this function. These channels have putative roles in ventilatory regulation and volatile anesthetic mechanisms. Doxapram stimulates ventilation through an effect on carotid bodies, and we hypothesized that stimulation might result from inhibition of TASK-1 or TASK-3 K channel function. To address this, we expressed TASK-1, TASK-3, TASK-1/TASK-3 heterodimeric, and TASK-1/TASK-3 chimeric K channels in Xenopus oocytes and studied the effects of doxapram on their function. Doxapram inhibited TASK-1 (half-maximal effective concentration [EC50], 410 nM), TASK-3 (EC50, 37 microM), and TASK-1/TASK-3 heterodimeric channel function (EC50, 9 microM). Chimera studies suggested that the carboxy terminus of TASK-1 is important for doxapram inhibition. Other K2P channels required significantly larger concentrations for inhibition. To test the role of TASK-1 and TASK-3 in halothane-induced immobility, the minimum alveolar anesthetic concentration for halothane was determined and found unchanged in rats receiving doxapram by IV infusion. Our data indicate that TASK-1 and TASK-3 do not play a role in mediating the immobility produced by halothane, although they are plausible molecular targets for the ventilatory effects of doxapram.

Flavone acetic acid (NSC 347512)-induced DNA damage in Glasgow osteogenic sarcoma in vivo.

Flavone acetic acid (FAA) is a new antitumor agent with broad activity against transplantable solid tumors of mice but with only scant or no activity against leukemias and lymphomas. The technique of alkaline elution was used to study DNA lesions in s.c. implanted Glasgow osteogenic sarcoma in C57BL/6 x DBA/2 F1 mice treated i.v. with FAA. At efficacious dosages (235 and 200 mg/kg), FAA produced extensive single strand breakage. Formation of single strand breaks was dependent on time of assay after exposure to FAA with only minimal damage occurring prior to 5 h posttreatment. Apparently Glasgow osteogenic sarcoma had no capacity to repair single strand breaks for at least 45 h after drug administration. Thus, FAA differs in its mechanism from other scission agents (e.g., VP-16). Neither interstrand cross-links nor DNA-protein cross-links were detected. DNA single strand breaks did not occur in the bone marrow cells or in the unresponsive P388 leukemia cells at dosages causing extensive DNA damage in solid tumor cells.

Elevated fecal calprotectin levels during necrotizing enterocolitis are associated with activated neutrophils extruding neutrophil extracellular traps.

OBJECTIVE: Neonates with necrotizing enterocolitis (NEC) have higher calprotectin levels in stool than do healthy neonates. However, it is not known whether high stool calprotectin at the onset of bowel symptoms identifies neonates who truly have NEC vs other bowel disorders. STUDY DESIGN: Neonates were eligible for this study when an x-ray was ordered to 'rule-out NEC'. Stool calprotectin was quantified at that time and in a follow-up stool. Each episode was later categorized as NEC or not NEC. The location of calprotectin in the bowel was determined by immunohistochemistry. RESULTS: Neonates with NEC had higher initial and follow-up stool calprotectin levels than did neonates without NEC. Calprotectin in bowel from neonates with NEC was within neutrophil extracellular traps (NETs). CONCLUSION: At the onset of signs concerning for NEC, fecal calprotectin is likely to be higher in neonates with NEC. Calprotectin in their stools is exported from neutrophils via NETs.

Decreased cell wall digestibility in canola transformed with chimeric tyrosine decarboxylase genes from opium poppy

Tyrosine decarboxylase (TYDC) is a common plant enzyme involved in the biosynthesis of numerous secondary metabolites, including hydroxycinnamic acid amides. Although a definite function has not yet been determined, amides have been proposed to form a physical barrier against pathogens because they are usually found as integral cell wall components. Canola (Brassica napus) was independently transformed with chimeric genes (35S::TYDC1 and 35S::TYDC2) under the transcriptional control of the cauliflower mosaic virus 35S promoter, and encoding two TYDC isoforms from opium poppy (Papaver somniferum). All T0 plants displayed a suppressed level of wild-type TYDC activity, and transgene mRNAs were not detected. Silencing of 35S::TYDC1 was overcome in the T1 progeny of self-pollinated T0 plants, since high levels of TYDC1 mRNAs were detected, and TYDC activity increased up to 4-fold compared with wild-type levels. However, TYDC1 mRNA levels decreased in T2 plants and were not detected in the T3 progeny. TYDC activity also gradually declined in T2 and T3 plants to nearly wild-type levels. In contrast, silencing of 35S::TYDC2 was maintained through four consecutive generations. T1 plants with a 3- to 4-fold increase in wild-type TYDC activity showed a 30% decrease in cellular tyrosine pools and a 2-fold increase in cell wall-bound tyramine compared with wild-type plants. An increase in cell wall-bound aromatic compounds was also detected in these T1 plants by ultraviolet autofluorescence microscopy. The relative digestibility of cell walls measured by protoplast release efficiency was inversely related to the level of TYDC activity.

Protective effects of TASK-3 (KCNK9) and related 2P K channels during cellular stress.

Tandem pore domain (or 2P) K channels form a recently isolated family of channels that are responsible for background K currents in excitable tissues. Previous studies have indicated that 2P K channel activity produces membrane hyperpolarization, which may offer protection from cellular insults. To study the effect of these channels in neuroprotection, we overexpressed pH-sensitive 2P K channels by transfecting the partially transformed C8 cell line with these channels. Tandem pore weak inward rectifier K channel (TWIK)-related acid-sensitive K channel 3 (TASK-3, KCNK9) as well as other pH sensitive 2P K channels (TASK-1 and TASK-2) enhanced cell viability by inhibiting the activation of intracellular apoptosis pathways. To explore the cellular basis for this protection in a more complex cellular environment, we infected cultured hippocampal slices with Sindbis virus constructs containing the coding sequences of these channels. Expression of TASK-3 throughout the hippocampal structure afforded neurons within the dentate and CA1 regions significant protection from an oxygen-glucose deprivation (OGD) injury. Neuroprotection within TASK-3 expressing slices was also enhanced by incubation with isoflurane. These results confirm a protective physiologic capability of TASK-3 and related 2P K channels, and suggest agents that enhance their activity, such as volatile anesthetics may intensify these protective effects.

Two-pore domain potassium channels: new sites of local anesthetic action and toxicity.

Potassium (K+) channels form the largest family of ion channels with more than 70 such genes identified in the human genome. They are organized in 3 superfamilies according to their predicted membrane topology: (1) subunits with 6 membrane-spanning segments and 1-pore domain, (2) subunits with 2 membrane-spanning segments and 1-pore domain, and (3) subunits with 4 membrane-spanning segments and 2-pore domains arrayed in a tandem position. The last family has most recently been identified and comprises the so-called 2-pore domain potassium (K2P) channels, believed responsible for background or leak K+ currents. Despite their recent discovery, interest in them is growing rapidly with more than 270 references in the literature reported (www.ipmc.cnrs.fr/~duprat/2p/ref2p.htm#2P, accessed October 30, 2004). K2P channels are widely expressed in the central nervous system and are involved in the control of the resting membrane potential and the firing pattern of excitable cells. This article will therefore review recent findings on actions of local anesthetics with respect to 2P channels. It begins with an overview of the role of background K+ channels in neuronal excitability and nerve conduction and is followed by a description of the K2P channel family including experimental evidence for the contribution of K2P channels to the mechanism of action and toxicity of local anesthetics.