Role of efflux pumps in the antibiotic resistance of bacteria embedded in a biofilm.

PubMed

Soto, Sara M

2013-04-01

Biofilms are complex microbial associations anchored to abiotic or biotic surfaces, embedded in extracellular matrix produced by the biofilms themselves where they interact with each other and the environment. One of the main properties of biofilms is their capacity to be more resistant to antimicrobial agents than planktonic cells. Efflux pumps have been reported as one of the mechanisms responsible for the antimicrobial resistance in biofilm structures. Evidence of the role of efflux pump in biofilm resistance has been found in several microorganisms such as Pseudomonas aeruginosa, Escherichia coli and Candida albicans. However, in spite of the studies on the importance of efflux pumps in biofilm growth and about their relevance in antimicrobial resistance forming biofilm, the exact role of these efflux systems has not been determined as yet.

Evaluation of the tannic acid inhibitory effect against the NorA efflux pump of Staphylococcus aureus.

PubMed

Tintino, Saulo R; Oliveira-Tintino, Cícera D M; Campina, Fábia F; Silva, Raimundo L P; Costa, Maria do S; Menezes, Irwin R A; Calixto-Júnior, João T; Siqueira-Junior, José P; Coutinho, Henrique D M; Leal-Balbino, Tereza C; Balbino, Valdir Q

2016-08-01

During the early periods of antibiotic usage, bacterial infections were considered tamed. However, widespread antibiotic use has promoted the emergence of antibiotic-resistant pathogens, including multidrug resistant strains. Active efflux is a mechanism for bacterial resistance to inhibitory substances, known simply as drug efflux pumps. The bacterium Staphylococcus aureus is an important pathogenic bacterium responsible for an array of infections. The NorA efflux pump has been shown to be responsible for moderate fluoroquinolone resistance of S. aureus. The inhibition of the efflux pump was assayed using a sub-inhibitory concentration of standard efflux pump inhibitors and tannic acid (MIC/8), where its capacity to decrease the MIC of Ethidium bromide (EtBr) and antibiotics due to the possible inhibitory effect of these substances was observed. The MICs of EtBr and antibiotics were significantly reduced in the presence of tannic acid, indicating the inhibitory effect of this agent against the efflux pumps of both strains causing a three-fold reduction of the MIC when compared with the control. These results indicate the possible usage of tannic acid as an adjuvant in antibiotic therapy against multidrug resistant bacteria (MDR). Copyright © 2016 Elsevier Ltd. All rights reserved.

A Simple Method for Assessment of MDR Bacteria for Over-Expressed Efflux Pumps

PubMed Central

Martins, Marta; McCusker, Matthew P; Viveiros, Miguel; Couto, Isabel; Fanning, Séamus; Pagès, Jean-Marie; Amaral, Leonard

2013-01-01

It is known that bacteria showing a multi-drug resistance phenotype use several mechanisms to overcome the action of antibiotics. As a result, this phenotype can be a result of several mechanisms or a combination of thereof. The main mechanisms of antibiotic resistance are: mutations in target genes (such as DNA gyrase and topoisomerase IV); over-expression of efflux pumps; changes in the cell envelope; down regulation of membrane porins, and modified lipopolysaccharide component of the outer cell membrane (in the case of Gram-negative bacteria). In addition, adaptation to the environment, such as quorum sensing and biofilm formation can also contribute to bacterial persistence. Due to the rapid emergence and spread of bacterial isolates showing resistance to several classes of antibiotics, methods that can rapidly and efficiently identify isolates whose resistance is due to active efflux have been developed. However, there is still a need for faster and more accurate methodologies. Conventional methods that evaluate bacterial efflux pump activity in liquid systems are available. However, these methods usually use common efflux pump substrates, such as ethidium bromide or radioactive antibiotics and therefore, require specialized instrumentation, which is not available in all laboratories. In this review, we will report the results obtained with the Ethidium Bromide-agar Cartwheel method. This is an easy, instrument-free, agar based method that has been modified to afford the simultaneous evaluation of as many as twelve bacterial strains. Due to its simplicity it can be applied to large collections of bacteria to rapidly screen for multi-drug resistant isolates that show an over-expression of their efflux systems. The principle of the method is simple and relies on the ability of the bacteria to expel a fluorescent molecule that is substrate for most efflux pumps, ethidium bromide. In this approach, the higher the concentration of ethidium bromide required to

An allosteric transport mechanism for the AcrAB-TolC multidrug efflux pump

PubMed Central

Wang, Zhao; Fan, Guizhen; Hryc, Corey F; Blaza, James N; Serysheva, Irina I; Schmid, Michael F; Chiu, Wah; Luisi, Ben F; Du, Dijun

2017-01-01

Bacterial efflux pumps confer multidrug resistance by transporting diverse antibiotics from the cell. In Gram-negative bacteria, some of these pumps form multi-protein assemblies that span the cell envelope. Here, we report the near-atomic resolution cryoEM structures of the Escherichia coli AcrAB-TolC multidrug efflux pump in resting and drug transport states, revealing a quaternary structural switch that allosterically couples and synchronizes initial ligand binding with channel opening. Within the transport-activated state, the channel remains open even though the pump cycles through three distinct conformations. Collectively, our data provide a dynamic mechanism for the assembly and operation of the AcrAB-TolC pump. DOI: http://dx.doi.org/10.7554/eLife.24905.001 PMID:28355133

Dynamics of Intact MexAB-OprM Efflux Pump: Focusing on the MexA-OprM Interface

DOE PAGES

Lopez, Cesar A.; Travers, Timothy; Pos, Klaas M.; ...

2017-11-28

Antibiotic efflux is one of the most critical mechanisms leading to bacterial multidrug resistance. Antibiotics are effluxed out of the bacterial cell by a tripartite efflux pump, a complex machinery comprised of outer membrane, periplasmic adaptor, and inner membrane protein components. Understanding the mechanism of efflux pump assembly and its dynamics could facilitate discovery of novel approaches to counteract antibiotic resistance in bacteria. We built here an intact atomistic model of the Pseudomonas aeruginosa MexAB-OprM pump in a Gram-negative membrane model that contained both inner and outer membranes separated by a periplasmic space. All-atom molecular dynamics (MD) simulations confirm thatmore » the fully assembled pump is stable in the microsecond timescale. Using a combination of all-atom and coarse-grained MD simulations and sequence covariation analysis, we characterized the interface between MexA and OprM in the context of the entire efflux pump. These analyses suggest a plausible mechanism by which OprM is activated via opening of its periplasmic aperture through a concerted interaction with MexA.« less

Dynamics of Intact MexAB-OprM Efflux Pump: Focusing on the MexA-OprM Interface

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lopez, Cesar A.; Travers, Timothy; Pos, Klaas M.

Antibiotic efflux is one of the most critical mechanisms leading to bacterial multidrug resistance. Antibiotics are effluxed out of the bacterial cell by a tripartite efflux pump, a complex machinery comprised of outer membrane, periplasmic adaptor, and inner membrane protein components. Understanding the mechanism of efflux pump assembly and its dynamics could facilitate discovery of novel approaches to counteract antibiotic resistance in bacteria. We built here an intact atomistic model of the Pseudomonas aeruginosa MexAB-OprM pump in a Gram-negative membrane model that contained both inner and outer membranes separated by a periplasmic space. All-atom molecular dynamics (MD) simulations confirm thatmore » the fully assembled pump is stable in the microsecond timescale. Using a combination of all-atom and coarse-grained MD simulations and sequence covariation analysis, we characterized the interface between MexA and OprM in the context of the entire efflux pump. These analyses suggest a plausible mechanism by which OprM is activated via opening of its periplasmic aperture through a concerted interaction with MexA.« less

Microbial Efflux Pump Inhibition: Tactics and Strategies

PubMed Central

Tegos, George P.; Haynes, Mark; Strouse, J. Jacob; Khan, Mohiuddin Md. T.; Bologa, Cristian G.; Oprea, Tudor I.; Sklar, Larry A.

2013-01-01

Traditional antimicrobials are increasingly suffering from the emergence of multidrug resistance among pathogenic microorganisms. To overcome these deficiencies, a range of novel approaches to control microbial infections are under investigation as potential alternative treatments. Multidrug efflux is a key target of these efforts. Efflux mechanisms are broadly recognized as major components of resistance to many classes of chemotherapeutic agents as well as antimicrobials. Efflux occurs due to the activity of membrane transporter proteins widely known as Multidrug Efflux Systems (MES). They are implicated in a variety of physiological roles other than efflux and identifying natural substrates and inhibitors is an active and expanding research discipline. One plausible alternative is the combination of conventional antimicrobial agents/antibiotics with small molecules that block MES known as multidrug efflux pump inhibitors (EPIs). An array of approaches in academic and industrial research settings, varying from high-throughput screening (HTS) ventures to bioassay guided purification and determination, have yielded a number of promising EPIs in a series of pathogenic systems. This synergistic discovery platform has been exploited in translational directions beyond the potentiation of conventional antimicrobial treatments. This venture attempts to highlight different tactical elements of this platform, identifying the need for highly informative and comprehensive EPI-discovery strategies. Advances in assay development genomics, proteomics as well as the accumulation of bioactivity and structural information regarding MES facilitates the basis for a new discovery era. This platform is expanding drastically. A combination of chemogenomics and chemoinformatics approaches will integrate data mining with virtual and physical HTS ventures and populate the chemical-biological interface with a plethora of novel chemotypes. This comprehensive step will expedite the

The ins and outs of RND efflux pumps in Escherichia coli.

PubMed

Anes, João; McCusker, Matthew P; Fanning, Séamus; Martins, Marta

2015-01-01

Infectious diseases remain one of the principal causes of morbidity and mortality in the world. Relevant authorities including the WHO and CDC have expressed serious concern regarding the continued increase in the development of multidrug resistance among bacteria. They have also reaffirmed the urgent need for investment in the discovery and development of new antibiotics and therapeutic approaches to treat multidrug resistant (MDR) bacteria. The extensive use of antimicrobial compounds in diverse environments, including farming and healthcare, has been identified as one of the main causes for the emergence of MDR bacteria. Induced selective pressure has led bacteria to develop new strategies of defense against these chemicals. Bacteria can accomplish this by several mechanisms, including enzymatic inactivation of the target compound; decreased cell permeability; target protection and/or overproduction; altered target site/enzyme and increased efflux due to over-expression of efflux pumps. Efflux pumps can be specific for a single substrate or can confer resistance to multiple antimicrobials by facilitating the extrusion of a broad range of compounds including antibiotics, heavy metals, biocides and others, from the bacterial cell. To overcome antimicrobial resistance caused by active efflux, efforts are required to better understand the fundamentals of drug efflux mechanisms. There is also a need to elucidate how these mechanisms are regulated and how they respond upon exposure to antimicrobials. Understanding these will allow the development of combined therapies using efflux inhibitors together with antibiotics to act on Gram-negative bacteria, such as the emerging globally disseminated MDR pathogen Escherichia coli ST131 (O25:H4). This review will summarize the current knowledge on resistance-nodulation-cell division efflux mechanisms in E. coli, a bacteria responsible for community and hospital-acquired infections, as well as foodborne outbreaks worldwide.

Xenon for tunnelling analysis of the efflux pump component OprN.

PubMed

Ntsogo Enguéné, Yvette Véronique; Phan, Gilles; Garnier, Cyril; Ducruix, Arnaud; Prangé, Thierry; Broutin, Isabelle

2017-01-01

Tripartite efflux pumps are among the main actors responsible for antibiotics resistance in Gram-negative bacteria. In the last two decades, structural studies gave crucial information about the assembly interfaces and the mechanistic motions. Thus rigidifying the assembly seems to be an interesting way to hamper the drug efflux. In this context, xenon is a suitable probe for checking whether small ligands could act as conformational lockers by targeting hydrophobic cavities. Here we focus on OprN, the outer membrane channel of the MexEF efflux pump from Pseudomonas aeruginosa. After exposing OprN crystals to xenon gas pressure, 14 binding sites were observed using X-ray crystallography. These binding sites were unambiguously characterized in hydrophobic cavities of OprN. The major site is observed in the sensitive iris-like region gating the channel at the periplasmic side, built by the three key-residues Leu 405, Asp 109, and Arg 412. This arrangement defines along the tunnel axis a strong hydrophobic/polar gradient able to enhance the passive efflux mechanism of OprN. The other xenon atoms reveal strategic hydrophobic regions of the channel scaffold to target, with the aim to freeze the dynamic movements responsible of the open/close conformational equilibrium in OprN.

Xenon for tunnelling analysis of the efflux pump component OprN

PubMed Central

Garnier, Cyril; Ducruix, Arnaud; Broutin, Isabelle

2017-01-01

Tripartite efflux pumps are among the main actors responsible for antibiotics resistance in Gram-negative bacteria. In the last two decades, structural studies gave crucial information about the assembly interfaces and the mechanistic motions. Thus rigidifying the assembly seems to be an interesting way to hamper the drug efflux. In this context, xenon is a suitable probe for checking whether small ligands could act as conformational lockers by targeting hydrophobic cavities. Here we focus on OprN, the outer membrane channel of the MexEF efflux pump from Pseudomonas aeruginosa. After exposing OprN crystals to xenon gas pressure, 14 binding sites were observed using X-ray crystallography. These binding sites were unambiguously characterized in hydrophobic cavities of OprN. The major site is observed in the sensitive iris-like region gating the channel at the periplasmic side, built by the three key-residues Leu 405, Asp 109, and Arg 412. This arrangement defines along the tunnel axis a strong hydrophobic/polar gradient able to enhance the passive efflux mechanism of OprN. The other xenon atoms reveal strategic hydrophobic regions of the channel scaffold to target, with the aim to freeze the dynamic movements responsible of the open/close conformational equilibrium in OprN. PMID:28886086

Identification and evolution of drug efflux pump in clinical Enterobacter aerogenes strains isolated in 1995 and 2003.

PubMed

Chevalier, Jacqueline; Mulfinger, Céline; Garnotel, Eric; Nicolas, Pierre; Davin-Régli, Anne; Pagès, Jean-Marie

2008-09-12

The high mortality impact of infectious diseases will increase due to accelerated evolution of antibiotic resistance in important human pathogens. Development of antibiotic resistance is a evolutionary process inducing the erosion of the effectiveness of our arsenal of antibiotics. Resistance is not necessarily limited to a single class of antibacterial agents but may affect many unrelated compounds; this is termed 'multidrug resistance' (MDR). The major mechanism of MDR is the active expulsion of drugs by bacterial pumps; the treatment of gram negative bacterial infections is compromised due to resistance mechanisms including the expression of efflux pumps that actively expel various usual antibiotics (beta-lactams, quinolones, ...). Enterobacter aerogenes has emerged among Enterobacteriaceae associated hospital infections during the last twenty years due to its faculty of adaptation to antibiotic stresses. Clinical isolates of E. aerogenes belonging to two strain collections isolated in 1995 and 2003 respectively, were screened to assess the involvement of efflux pumps in antibiotic resistance. Drug susceptibility assays were performed on all bacterial isolates and an efflux pump inhibitor (PAbetaN) previously characterized allowed to decipher the role of efflux in the resistance. Accumulation of labelled chloramphenicol was monitored in the presence of an energy poison to determine the involvement of active efflux on the antibiotic intracellular concentrations. The presence of the PAbetaN-susceptible efflux system was also identified in resistant E. aerogenes strains. For the first time a noticeable increase in clinical isolates containing an efflux mechanism susceptible to pump inhibitor is report within an 8 year period. After the emergence of extended spectrum beta-lactamases in E. aerogenes and the recent characterisation of porin mutations in clinical isolates, this study describing an increase in inhibitor-susceptible efflux throws light on a new step in

Identification and Evolution of Drug Efflux Pump in Clinical Enterobacter aerogenes Strains Isolated in 1995 and 2003

PubMed Central

Garnotel, Eric; Nicolas, Pierre; Davin-Régli, Anne; Pagès, Jean-Marie

2008-01-01

Background The high mortality impact of infectious diseases will increase due to accelerated evolution of antibiotic resistance in important human pathogens. Development of antibiotic resistance is a evolutionary process inducing the erosion of the effectiveness of our arsenal of antibiotics. Resistance is not necessarily limited to a single class of antibacterial agents but may affect many unrelated compounds; this is termed ‘multidrug resistance’ (MDR). The major mechanism of MDR is the active expulsion of drugs by bacterial pumps; the treatment of Gram negative bacterial infections is compromised due to resistance mechanisms including the expression of efflux pumps that actively expel various usual antibiotics (ß-lactams, quinolones, …). Methodology/Principal Findings Enterobacter aerogenes has emerged among Enterobacteriaceae associated hospital infections during the last twenty years due to its faculty of adaptation to antibiotic stresses. Clinical isolates of E. aerogenes belonging to two strain collections isolated in 1995 and 2003 respectively, were screened to assess the involvement of efflux pumps in antibiotic resistance. Drug susceptibility assays were performed on all bacterial isolates and an efflux pump inhibitor (PAßN) previously characterized allowed to decipher the role of efflux in the resistance. Accumulation of labelled chloramphenicol was monitored in the presence of an energy poison to determine the involvement of active efflux on the antibiotic intracellular concentrations. The presence of the PAßN-susceptible efflux system was also identified in resistant E. aerogenes strains. Conclusions/Significance For the first time a noticeable increase in clinical isolates containing an efflux mechanism susceptible to pump inhibitor is report within an 8 year period. After the emergence of extended spectrum ß-lactamases in E. aerogenes and the recent characterisation of porin mutations in clinical isolates, this study describing an increase

Use and engineering of efflux pumps for the export of olefins in microbes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mukhopadhyay, Aindrila

2016-07-14

The scope of the project is to investigate efflux pump systems in engineered host microorganisms, such as E. coli, and develop a pump engineered to export a target compound. To initiate the project in coordination with other TOTAL driven projects, the first target compound to be studied was 1-hexene. However, we were investigating other chemicals as Styrene. The main goal of the project was to generate a set of optimized efflux pump systems for microorganisms (E. coli and Streptomyces or other host) engineered to contain biosynthetic pathways to export large titers of target compounds that are toxic (or accumulate andmore » push back biosynthesis) to the host cell. An optimized microbial host will utilize specific and efficient cell wall located pumps to extrude harmful target compounds and enable greater production of these compounds.« less

Stress Introduction Rate Alters the Benefit of AcrAB-TolC Efflux Pumps.

PubMed

Langevin, Ariel M; Dunlop, Mary J

2018-01-01

Stress tolerance studies are typically conducted in an all-or-none fashion. However, in realistic settings-such as in clinical or metabolic engineering applications-cells may encounter stresses at different rates. Therefore, how cells tolerate stress may depend on its rate of appearance. To address this, we studied how the rate of stress introduction affects bacterial stress tolerance by focusing on a key stress response mechanism. Efflux pumps, such as AcrAB-TolC of Escherichia coli , are membrane transporters well known for the ability to export a wide variety of substrates, including antibiotics, signaling molecules, and biofuels. Although efflux pumps improve stress tolerance, pump overexpression can result in a substantial fitness cost to the cells. We hypothesized that the ideal pump expression level would involve a rate-dependent trade-off between the benefit of pumps and the cost of their expression. To test this, we evaluated the benefit of the AcrAB-TolC pump under different rates of stress introduction, including a step, a fast ramp, and a gradual ramp. Using two chemically diverse stresses, the antibiotic chloramphenicol and the jet biofuel precursor pinene, we assessed the benefit provided by the pumps. A mathematical model describing these effects predicted the benefit as a function of the rate of stress introduction. Our findings demonstrate that as the rate of introduction is lowered, stress response mechanisms provide a disproportionate benefit to pump-containing strains, allowing cells to survive beyond the original inhibitory concentrations. IMPORTANCE Efflux pumps are ubiquitous in nature and provide stress tolerance in the cells of species ranging from bacteria to mammals. Understanding how pumps provide tolerance has far-reaching implications for diverse fields, from medicine to biotechnology. Here, we investigated how the rate of stressor appearance impacts tolerance. We focused on two distinct substrates of AcrAB-TolC efflux pumps, the

Multidrug Efflux Pumps from Enterobacteriaceae, Vibrio cholerae and Staphylococcus aureus Bacterial Food Pathogens

PubMed Central

Andersen, Jody L.; He, Gui-Xin; Kakarla, Prathusha; KC, Ranjana; Kumar, Sanath; Lakra, Wazir Singh; Mukherjee, Mun Mun; Ranaweera, Indrika; Shrestha, Ugina; Tran, Thuy; Varela, Manuel F.

2015-01-01

Foodborne illnesses caused by bacterial microorganisms are common worldwide and constitute a serious public health concern. In particular, microorganisms belonging to the Enterobacteriaceae and Vibrionaceae families of Gram-negative bacteria, and to the Staphylococcus genus of Gram-positive bacteria are important causative agents of food poisoning and infection in the gastrointestinal tract of humans. Recently, variants of these bacteria have developed resistance to medically important chemotherapeutic agents. Multidrug resistant Escherichia coli, Salmonella enterica, Vibrio cholerae, Enterobacter spp., and Staphylococcus aureus are becoming increasingly recalcitrant to clinical treatment in human patients. Of the various bacterial resistance mechanisms against antimicrobial agents, multidrug efflux pumps comprise a major cause of multiple drug resistance. These multidrug efflux pump systems reside in the biological membrane of the bacteria and actively extrude antimicrobial agents from bacterial cells. This review article summarizes the evolution of these bacterial drug efflux pump systems from a molecular biological standpoint and provides a framework for future work aimed at reducing the conditions that foster dissemination of these multidrug resistant causative agents through human populations. PMID:25635914

Multidrug efflux pumps from Enterobacteriaceae, Vibrio cholerae and Staphylococcus aureus bacterial food pathogens.

PubMed

Andersen, Jody L; He, Gui-Xin; Kakarla, Prathusha; K C, Ranjana; Kumar, Sanath; Lakra, Wazir Singh; Mukherjee, Mun Mun; Ranaweera, Indrika; Shrestha, Ugina; Tran, Thuy; Varela, Manuel F

2015-01-28

Foodborne illnesses caused by bacterial microorganisms are common worldwide and constitute a serious public health concern. In particular, microorganisms belonging to the Enterobacteriaceae and Vibrionaceae families of Gram-negative bacteria, and to the Staphylococcus genus of Gram-positive bacteria are important causative agents of food poisoning and infection in the gastrointestinal tract of humans. Recently, variants of these bacteria have developed resistance to medically important chemotherapeutic agents. Multidrug resistant Escherichia coli, Salmonella enterica, Vibrio cholerae, Enterobacter spp., and Staphylococcus aureus are becoming increasingly recalcitrant to clinical treatment in human patients. Of the various bacterial resistance mechanisms against antimicrobial agents, multidrug efflux pumps comprise a major cause of multiple drug resistance. These multidrug efflux pump systems reside in the biological membrane of the bacteria and actively extrude antimicrobial agents from bacterial cells. This review article summarizes the evolution of these bacterial drug efflux pump systems from a molecular biological standpoint and provides a framework for future work aimed at reducing the conditions that foster dissemination of these multidrug resistant causative agents through human populations.

Involvement of the Efflux Pumps in Chloramphenicol Selected Strains of Burkholderia thailandensis: Proteomic and Mechanistic Evidence

PubMed Central

Biot, Fabrice V.; Valade, Eric; Garnotel, Eric; Chevalier, Jacqueline; Villard, Claude; Thibault, François M.; Vidal, Dominique R.; Pagès, Jean-Marie

2011-01-01

Burkholderia is a bacterial genus comprising several pathogenic species, including two species highly pathogenic for humans, B. pseudomallei and B. mallei. B. thailandensis is a weakly pathogenic species closely related to both B. pseudomallei and B. mallei. It is used as a study model. These bacteria are able to exhibit multiple resistance mechanisms towards various families of antibiotics. By sequentially plating B. thailandensis wild type strains on chloramphenicol we obtained several resistant variants. This chloramphenicol-induced resistance was associated with resistance against structurally unrelated antibiotics including quinolones and tetracyclines. We functionally and proteomically demonstrate that this multidrug resistance phenotype, identified in chloramphenicol-resistant variants, is associated with the overexpression of two different efflux pumps. These efflux pumps are able to expel antibiotics from several families, including chloramphenicol, quinolones, tetracyclines, trimethoprim and some β-lactams, and present a partial susceptibility to efflux pump inhibitors. It is thus possible that Burkholderia species can develop such adaptive resistance mechanisms in response to antibiotic pressure resulting in emergence of multidrug resistant strains. Antibiotics known to easily induce overexpression of these efflux pumps should be used with discernment in the treatment of Burkholderia infections. PMID:21347382

Evaluation of a series of 2-napthamide derivatives as inhibitors of the drug efflux pump AcrB for the reversal of antimicrobial resistance.

PubMed

Wang, Yinhu; Mowla, Rumana; Guo, Liwei; Ogunniyi, Abiodun D; Rahman, Taufiq; De Barros Lopes, Miguel A; Ma, Shutao; Venter, Henrietta

2017-02-15

Drug efflux pumps confer multidrug resistance to dangerous pathogens which makes these pumps important drug targets. We have synthesised a novel series of compounds based on a 2-naphthamide pharmacore aimed at inhibiting the efflux pumps from Gram-negative bacteria. The archeatypical transporter AcrB from Escherichia coli was used as model efflux pump as AcrB is widely conserved throughout Gram-negative organisms. The compounds were tested for their antibacterial action, ability to potentiate the action of antibiotics and for their ability to inhibit Nile Red efflux by AcrB. None of the compounds were antimicrobial against E. coli wild type cells. Most of the compounds were able to inhibit Nile Red efflux indicating that they are substrates of the AcrB efflux pump. Three compounds were able to synergise with antibiotics and reverse resistance in the resistant phenotype. Compound A3, 4-(isopentyloxy)-2-naphthamide, reduced the MICs of erythromycin and chloramphenicol to the MIC levels of the drug sensitive strain that lacks an efflux pump. A3 had no effect on the MIC of the non-substrate rifampicin indicating that this compound acts specifically through the AcrB efflux pump. A3 also does not act through non-specific mechanisms such as outer membrane or inner membrane permeabilisation and is not cytotoxic against mammalian cell lines. Therefore, we have designed and synthesised a novel chemical compound with great potential to further optimisation as inhibitor of drug efflux pumps. Copyright © 2017 Elsevier Ltd. All rights reserved.

An alternative physiological role for the EmhABC efflux pump in Pseudomonas fluorescens cLP6a

PubMed Central

2011-01-01

Background Efflux pumps belonging to the resistance-nodulation-division (RND) superfamily in bacteria are involved in antibiotic resistance and solvent tolerance but have an unknown physiological role. EmhABC, a RND-type efflux pump in Pseudomonas fluorescens strain cLP6a, extrudes hydrophobic antibiotics, dyes and polycyclic aromatic hydrocarbons including phenanthrene. The effects of physico-chemical factors such as temperature or antibiotics on the activity and expression of EmhABC were determined in order to deduce its physiological role(s) in strain cLP6a in comparison to the emhB disruptant strain, cLP6a-1. Results Efflux assays conducted with 14C-phenanthrene showed that EmhABC activity is affected by incubation temperature. Increased phenanthrene efflux was measured in cLP6a cells grown at 10°C and decreased efflux was observed at 35°C compared with cells grown at the optimum temperature of 28°C. Membrane fatty acids in cLP6a cells were substantially altered by changes in growth temperature and in the presence of tetracycline. Changed membrane fatty acids and increased membrane permeability were associated with ~30-fold increased expression of emhABC in cLP6a cells grown at 35°C, and with increased extracellular free fatty acids. Growth of P. fluorescens cLP6a at supra-optimal temperature was enhanced by the presence of EmhABC compared to strain cLP6a-1. Conclusions Combined, these observations suggest that the EmhABC efflux pump may be involved in the management of membrane stress effects such as those due to unfavourable incubation temperatures. Efflux of fatty acids replaced as a result of membrane damage or phospholipid turnover may be the primary physiological role of the EmhABC efflux pump in P. fluorescens cLP6a. PMID:22085438

Efflux pump induction by quaternary ammonium compounds and fluoroquinolone resistance in bacteria.

PubMed

Buffet-Bataillon, Sylvie; Tattevin, Pierre; Maillard, Jean-Yves; Bonnaure-Mallet, Martine; Jolivet-Gougeon, Anne

2016-01-01

Biocides, primarily those containing quaternary ammonium compounds (QAC), are heavily used in hospital environments and various industries (e.g., food, water, cosmetic). To date, little attention has been paid to potential implications of QAC use in the emergence of antibiotic resistance, especially fluoroquinolone-resistant bacteria in patients and in the environment. QAC-induced overexpression of efflux pumps can lead to: cross resistance with fluoroquinolones mediated by multidrug efflux pumps; stress response facilitating mutation in the Quinolone Resistance Determining Region; and biofilm formation increasing the risk of transfer of mobile genetic elements carrying fluoroquinolone or QAC resistance determinants. By following the European Biocidal Product Regulation, manufacturers of QAC are required to ensure that their QAC-based biocidal products are safe and will not contribute to emerging bacterial resistance.

Characterization of the Serratia marcescens SdeCDE multidrug efflux pump studied via gene knockout mutagenesis.

PubMed

Begic, Sanela; Worobec, Elizabeth A

2008-05-01

Serratia marcescens is an important nosocomial agent having high antibiotic resistance. A major mechanism for S. marcescens antibiotic resistance is active efflux. To ascertain the substrate specificity of the S. marcescens SdeCDE efflux pump, we constructed pump gene deletion mutants. sdeCDE knockout strains showed no change in antibiotic susceptibility in comparison with the parental strains for any of the substrates, with the exception of novobiocin. In addition, novobiocin was the only antibiotic to be accumulated by sdeCDE-deficient strains. Based on the substrates used in our study, we conclude that SdeCDE is a Resistance-Nodulation-Cell Division family pump with limited substrate specificity.

Contribution of putative efflux pump genes to isoniazid resistance in clinical isolates of Mycobacterium tuberculosis.

PubMed

Narang, Anshika; Giri, Astha; Gupta, Shraddha; Garima, Kushal; Bose, Mridula; Varma-Basil, Mandira

2017-01-01

Isoniazid (INH) resistance in Mycobacterium tuberculosis has been mainly attributed to mutations in katG (64%) and inhA (19%). However, 20%-30% resistance to INH cannot be explained by mutations alone. Hence, other mechanisms besides mutations may play a significant role in providing drug resistance. Here, we explored the role of 24 putative efflux pump genes conferring INH-resistance in M. tuberculosis. Real-time expression profiling of the efflux pump genes was performed in five INH-susceptible and six high-level INH-resistant clinical isolates of M. tuberculosis exposed to the drug. Isolates were also analyzed for mutations in katG and inhA. Four high-level INH-resistant isolates (minimum inhibitory concentration [MIC] ≥2.5 mg/L) with mutations at codon 315 (AGC-ACC) of katG showed upregulation of one of the efflux genes Rv1634, Rv0849, efpA, or p55. Another high-level INH-resistant isolate (MIC 1.5 mg/L), with no mutations at katG or inhA overexpressed 8/24 efflux genes, namely, Rv1273c, Rv0194, Rv1634, Rv1250, Rv3823c, Rv0507, jefA, and p55. Five of these, namely, Rv0194, Rv1634, Rv1250, Rv0507, and p55 were induced only in resistant isolates. The high number of efflux genes overexpressed in an INH-resistant isolate with no known INH resistance associated mutations, suggests a role for efflux pumps in resistance to this antituberculous agent, with the role of Rv0194 and Rv0507 in INH resistance being reported for the first time.

Importance of Real-Time Assays To Distinguish Multidrug Efflux Pump-Inhibiting and Outer Membrane-Destabilizing Activities in Escherichia coli.

PubMed

Misra, Rajeev; Morrison, Keith D; Cho, Hyun Jae; Khuu, Thanh

2015-08-01

The constitutively expressed AcrAB multidrug efflux system of Escherichia coli shows a high degree of homology with the normally silent AcrEF system. Exposure of a strain with acrAB deleted to antibiotic selection pressure frequently leads to the insertion sequence-mediated activation of the homologous AcrEF system. In this study, we used strains constitutively expressing either AcrAB or AcrEF from their normal chromosomal locations to resolve a controversy about whether phenylalanylarginine β-naphthylamide (PAβN) inhibits the activities of AcrAB and AcrEF and/or acts synergistically with antibiotics by destabilizing the outer membrane permeability barrier. Real-time efflux assays allowed a clear distinction between the efflux pump-inhibiting activity of PAβN and the outer membrane-destabilizing action of polymyxin B nonapeptide (PMXBN). When added in equal amounts, PAβN, but not PMXBN, strongly inhibited the efflux activities of both AcrAB and AcrEF pumps. In contrast, when outer membrane destabilization was assessed by the nitrocefin hydrolysis assay, PMXBN exerted a much greater damaging effect than PAβN. Strong action of PAβN in inhibiting efflux activity compared to its weak action in destabilizing the outer membrane permeability barrier suggests that PAβN acts mainly by inhibiting efflux pumps. We concluded that at low concentrations, PAβN acts specifically as an inhibitor of both AcrAB and AcrEF efflux pumps; however, at high concentrations, PAβN in the efflux-proficient background not only inhibits efflux pump activity but also destabilizes the membrane. The effects of PAβN on membrane integrity are compounded in cells unable to extrude PAβN. The increase in multidrug-resistant bacterial pathogens at an alarming rate has accelerated the need for implementation of better antimicrobial stewardship, discovery of new antibiotics, and deeper understanding of the mechanism of drug resistance. The work carried out in this study highlights the importance

Novel structural analogues of piperine as inhibitors of the NorA efflux pump of Staphylococcus aureus.

PubMed

Kumar, Ashwani; Khan, Inshad Ali; Koul, Surrinder; Koul, Jawahir Lal; Taneja, Subhash Chandra; Ali, Intzar; Ali, Furqan; Sharma, Sandeep; Mirza, Zahid Mehmood; Kumar, Manoj; Sangwan, Pyare Lal; Gupta, Pankaj; Thota, Niranjan; Qazi, Ghulam Nabi

2008-06-01

Evaluation of novel synthetic analogues of piperine as inhibitors of multidrug efflux pump NorA of Staphylococcus aureus. A library of piperine-derived compounds was evaluated for their potential to inhibit ethidium bromide efflux in NorA-overexpressing S. aureus SA 1199B. The active compounds were then individually combined with ciprofloxacin to study the potentiation of ciprofloxacin's activity. Based on the efflux inhibition assay, a library of 200 compounds was screened. Three piperine analogues, namely SK-20, SK-56 and SK-29, were found to be the most potent inhibitors of the NorA efflux pump. These inhibitors acted in a synergistic manner with ciprofloxacin, by substantially increasing its activity against both NorA-overexpressing and wild-type S. aureus isolates. These analogues were 2- to 4-fold more potent than piperine at a significantly lower minimal effective concentration. Furthermore, these inhibitors also significantly suppressed the in vitro emergence of ciprofloxacin-resistant S. aureus. A newly identified class of compounds derived from a natural amide, piperine, is more potent than the parent molecule in potentiating the activity of ciprofloxacin through the inhibition of the NorA efflux pump. These molecules may prove useful in augmenting the antibacterial activities of fluoroquinolones in a clinical setting.

Macrolide resistance in Legionella pneumophila: the role of LpeAB efflux pump.

PubMed

Massip, Clémence; Descours, Ghislaine; Ginevra, Christophe; Doublet, Patricia; Jarraud, Sophie; Gilbert, Christophe

2017-05-01

A previous study on 12 in vitro -selected azithromycin-resistant Legionella pneumophila lineages showed that ribosomal mutations were major macrolide resistance determinants. In addition to these mechanisms that have been well described in many species, mutations upstream of lpeAB operon, homologous to acrAB in Escherichia coli , were identified in two lineages. In this study, we investigated the role of LpeAB and of these mutations in macrolide resistance of L. pneumophila . The role of LpeAB was studied by testing the antibiotic susceptibility of WT, deleted and complemented L. pneumophila Paris strains. Translational fusion experiments using GFP as a reporter were conducted to investigate the consequences of the mutations observed in the upstream sequence of lpeAB operon. We demonstrated the involvement of LpeAB in an efflux pump responsible for a macrolide-specific reduced susceptibility of L. pneumophila Paris strain. Mutations in the upstream sequence of lpeAB operon were associated with an increased protein expression. Increased expression was also observed under sub-inhibitory macrolide concentrations in strains with both mutated and WT promoting regions. LpeAB are components of an efflux pump, which is a macrolide resistance determinant in L. pneumophila Paris strain. Mutations observed in the upstream sequence of lpeAB operon in resistant lineages led to an overexpression of this efflux pump. Sub-inhibitory concentrations of macrolides themselves participated in upregulating this efflux and could constitute a first step in the acquisition of a high macrolide resistance level. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Inactivation of a putative efflux pump (LmrB) in Streptococcus mutans results in altered biofilm structure and increased exopolysaccharide synthesis: implications for biofilm resistance.

PubMed

Liu, Jia; Zhang, Jianying; Guo, Lihong; Zhao, Wei; Hu, Xiaoli; Wei, Xi

2017-07-01

Efflux pumps are a mechanism associated with biofilm formation and resistance. There is limited information regarding efflux pumps in Streptococcus mutans, a major pathogen in dental caries. The aim of this study was to investigate potential roles of a putative efflux pump (LmrB) in S. mutans biofilm formation and susceptibility. Upon lmrB inactivation and antimicrobial exposure, the biofilm structure and expression of other efflux pumps were examined using confocal laser scanning microscopy (CLSM) and qRT-PCR. lmrB inactivation resulted in biofilm structural changes, increased EPS formation and EPS-related gene transcription (p < 0.05), but no improvement in susceptibility was observed. The expression of most efflux pump genes increased upon lmrB inactivation when exposed to antimicrobials (p < 0.05), suggesting a feedback mechanism that activated the transcription of other efflux pumps to compensate for the loss of lmrB. These observations imply that sole inactivation of lmrB is not an effective solution to control biofilms.

Inhibitors of bacterial multidrug efflux pumps potentiate antimicrobial photoinactivation.

PubMed

Tegos, George P; Masago, Kayo; Aziz, Fatima; Higginbotham, Andrew; Stermitz, Frank R; Hamblin, Michael R

2008-09-01

Antimicrobial photodynamic inactivation (APDI) combines a nontoxic photoactivatable dye or photosensitizer (PS) with harmless visible light to generate singlet oxygen and reactive oxygen species that kill microbial cells. Cationic phenothiazinium dyes, such as toluidine blue O (TBO), are the only PS used clinically for APDI, and we recently reported that this class of PS are substrates of multidrug efflux pumps in both gram-positive and gram-negative bacteria. We now report that APDI can be significantly potentiated by combining the PS with an efflux pump inhibitor (EPI). Killing of Staphylococcus aureus mediated by TBO and red light is greatly increased by coincubation with known inhibitors of the major facilitator pump (NorA): the diphenyl urea INF271, reserpine, 5'-methoxyhydnocarpin, and the polyacylated neohesperidoside, ADH7. The potentiation effect is greatest in the case of S. aureus mutants that overexpress NorA and least in NorA null cells. Addition of the EPI before TBO has a bigger effect than addition of the EPI after TBO. Cellular uptake of TBO is increased by EPI. EPI increased photodynamic inactivation killing mediated by other phenothiazinium dyes, such as methylene blue and dimethylmethylene blue, but not that mediated by nonphenothiazinium PS, such as Rose Bengal and benzoporphyrin derivative. Killing of Pseudomonas aeruginosa mediated by TBO and light was also potentiated by the resistance nodulation division pump (MexAB-OprM) inhibitor phenylalanine-arginine beta-naphthylamide but to a lesser extent than for S. aureus. These data suggest that EPI could be used in combination with phenothiazinium salts and light to enhance their antimicrobial effect against localized infections.

Fluoroquinolone resistance of Serratia marcescens: involvement of a proton gradient-dependent efflux pump.

PubMed

Kumar, Ayush; Worobec, Elizabeth A

2002-10-01

To determine the presence of a proton gradient-dependent efflux of fluoroquinolone drugs in Serratia marcescens. Thirteen clinical isolates of S. marcescens were screened for resistance to four fluoroquinolones: ofloxacin, ciprofloxacin, norfloxacin and nalidixic acid by determining MICs. The presence of a proton gradient-dependent efflux mechanism was assessed using ethidium bromide accumulation assays. Drug accumulation studies for norfloxacin, ciprofloxacin and ofloxacin were performed to determine the drug specificity of efflux. Western transfer of cellular proteins, followed by immunodetection using anti-AcrA (Escherichia coli) antibodies were used to demonstrate the presence of a resistance-nodulation-cell division (RND) pump protein. PCR was used to identify a RND pump-encoding gene using primers for two conserved motifs within inner membrane components of RND proteins. A mutant strain of S. marcescens, UOC-67WL, was isolated by culturing the wild-type strain in the presence of ciprofloxacin in T-soy media and was subjected to the same studies as described above for the clinical isolates. Ethidium bromide accumulation assays confirmed the presence of a proton gradient-dependent efflux mechanism in S. marcescens. One clinical isolate, T-861, and the mutant strain, UOC-67WL, were found to efflux ciprofloxacin and ofloxacin. Western immunoblot results confirmed overexpression of an AcrA-like protein in T-861 and UOC-67WL. Sequencing of the PCR product showed the presence of a mexF-like gene, which is overexpressed in nfxC mutants of Pseudomonas aeruginosa. This study reports the presence of a proton gradient-dependent efflux mechanism in S. marcescens.

NorA efflux pump inhibitory activity of coumarins from Mesua ferrea.

PubMed

Roy, Somendu K; Kumari, Neela; Pahwa, Sonika; Agrahari, Udai C; Bhutani, Kamlesh K; Jachak, Sanjay M; Nandanwar, Hemraj

2013-10-01

The purpose of this investigation was to study the modulator and efflux pump inhibitor activity of coumarins isolated from Mesua ferrea against clinical strains as well as NorA-over expressed strain of Staphylococcus aureus 1199B. Seven coumarins were tested for modulator activity using ethidium bromide (EtBr) as a substrate. Compounds 1, 4-7 modulated the MIC of EtBr by ≥ 2 fold against wild type clinical strains of S. aureus 1199 and S. aureus 1199B, whereas compounds 4-7 modulated the MIC of EtBr by ≥ 16 fold against MRSA 831. Compounds 1, 4-7 also reduced the MIC of norfloxacin by ≥ 8 fold against S. aureus 1199B, and 4-6 reduced the MIC of norfloxacin by ≥ 8 fold against MRSA 831 at half of their MICs. Inhibition of EtBr efflux by NorA-overproducing S. aureus 1199B and MRSA 831 confirmed the role of compounds 4-6 as NorA efflux pump inhibitors (EPI). Dose-dependent activity at sub-inhibitory concentration (6.25 μg/mL) suggested that compounds 4 and 5 are promising EPI compared to verapamil against 1199B and MRSA 831 strains. © 2013.

First identification of boronic species as novel potential inhibitors of the Staphylococcus aureus NorA efflux pump.

PubMed

Fontaine, Fanny; Hequet, Arnaud; Voisin-Chiret, Anne-Sophie; Bouillon, Alexandre; Lesnard, Aurélien; Cresteil, Thierry; Jolivalt, Claude; Rault, Sylvain

2014-03-27

Overexpression of efflux pumps is an important mechanism of bacterial resistance that results in the extrusion of antimicrobial agents outside the bacterial cell. Inhibition of such pumps appears to be a promising strategy that could restore the potency of existing antibiotics. The NorA efflux pump of Staphylococcus aureus confers resistance to a wide range of unrelated substrates, such as hydrophilic fluoroquinolones, leading to a multidrug-resistance phenotype. In this work, approximately 150 heterocyclic boronic species were evaluated for their activity against susceptible and resistant strains of S. aureus. Twenty-four hit compounds, although inactive when tested alone, were found to potentiate ciprofloxacin activity by a 4-fold increase at concentrations ranging from 0.5 to 8 μg/mL against S. aureus 1199B, which overexpresses NorA. Boron-free analogues showed no biological activity, thus revealing that the boron atom is crucial for biological activity. This work describes the first reported efflux pump inhibitory activity of boronic acid derivatives.

Ligand Promiscuity between the Efflux Pumps Human P-Glycoprotein and S. aureus NorA.

PubMed

Brincat, Jean Pierre; Broccatelli, Fabio; Sabatini, Stefano; Frosini, Maria; Neri, Annalisa; Kaatz, Glenn W; Cruciani, Gabriele; Carosati, Emanuele

2012-03-08

Thirty-two diverse compounds were evaluated for their ability to inhibit both Pgp-mediated efflux in mouse T-lymphoma L5178 MDR1 and NorA-mediated efflux in S. aureus SA-1199B. Only four compounds were strong inhibitors of both efflux pumps. Three compounds were found to inhibit Pgp exclusively and strongly, while seven compounds inhibited only NorA. These results demonstrate that Pgp and NorA inhibitors do not necessarily overlap, opening the way to safer therapeutic use of effective NorA inhibitors.

Chalcone inhibitors of the NorA efflux pump in Staphylococcus aureus whole cells and enriched everted membrane vesicles.

PubMed

Holler, Jes Gitz; Slotved, Hans-Christian; Mølgaard, Per; Olsen, Carl Erik; Christensen, Søren Brøgger

2012-07-15

A library of 117 chalcones was screened for efflux pump inhibitory (EPI) activity against NorA mediated ethidium bromide efflux. Five of the chalcones (5-7, 9, and 10) were active and two chalcones (9 and 10) were equipotent to reserpine with IC(50)-values of 9.0 and 7.7 μM, respectively. Twenty chalcones were subsequently proved to be inhibitors of the NorA efflux pump in everted membrane vesicles. Compounds 5, 7, and 9 synergistically increased the effect of ciprofloxacin on Staphylococcus aureus. Our results suggest that chalcones might be developed into drugs for overcoming multidrug resistance based on efflux transporters of microorganisms. Copyright © 2012 Elsevier Ltd. All rights reserved.

Influence of intestinal efflux pumps on the absorption and transport of furosemide

PubMed Central

Al-Mohizea, Abdullah M.

2010-01-01

Purpose Furosemide is a commonly used diuretic which is used in the treatment of edema, congestive heart failure, hypertension and renal failure. Its absorption exhibits inter- and intra-subject variability that can be attributed to many factors including the intestinal efflux pumps such as the P-glycoprotein (P-gp). This study was done due to the great disagreement between what is published in the literature regarding the influence of P-gp on furosemide and at the same time due to the importance of this drug in the treatment of different conditions as described above. In addition, an investigation of the effect of two of the commonly used pharmaceutical excipients (hydroxypropyl β-cyclodextrin [HPβCD] and Tween 80) and also a P-gp inhibitor (verapamil hydrochloride) on the intestinal absorption of this drug were also done. Methods The study utilized the everted intestinal sacs technique to investigate both the effect of the efflux transporter (P-gp) on furosemide absorption and also the effect of the chosen excipients. Results The absorption of furosemide was significantly influenced by the P-gp as confirmed by the everted vis the non-everted sacs together with the verapamil study in which the transport of furosemide was inhibited by verapamil. In addition, Tween 80 was also shown to inhibit the P-gp pump whereas the HPβCD did not significantly influence the efflux of furosemide in this study. Conclusions P-glycoprotein and some of the used excipients in the formulation play a very important role in the transport of furosemide and other drugs. Thus excipients that affect the activity of P-gp should be avoided when formulating drugs that are substrate for the P-gp or other efflux pumps. PMID:23960725

Vitamin K3 Induces the Expression of the Stenotrophomonas maltophilia SmeVWX Multidrug Efflux Pump.

PubMed

Blanco, P; Corona, F; Sánchez, M B; Martínez, J L

2017-05-01

Stenotrophomonas maltophilia is an opportunistic pathogen with increasing prevalence, which is able to cause infections in immunocompromised patients or in those with a previous pathology. The treatment of the infections caused by this bacterium is often complicated due to the several intrinsic antibiotic resistance mechanisms that it presents. Multidrug efflux pumps are among the best-studied mechanisms of S. maltophilia antibiotic resistance. Some of these efflux pumps have a basal expression level but, in general, their expression is often low and only reaches high levels when the local regulator is mutated or bacteria are in the presence of an effector. In the current work, we have developed a yellow fluorescent protein (YFP)-based sensor with the aim to identify effectors able to trigger the expression of SmeVWX, an efflux pump that confers resistance to quinolones, chloramphenicol, and tetracycline when it is expressed at high levels. With this purpose in mind, we tested a variety of different compounds and analyzed the fluorescence signal given by the expression of YFP under the control of the smeVWX promoter. Among the tested compounds, vitamin K 3 , which is a compound belonging to the 2-methyl-1,4-naphthoquinone family, is produced by plants in defense against infection, and has increasing importance in human therapy, was able to induce the expression of the SmeVWX efflux pump. In addition, a decrease in the susceptibility of S. maltophilia to ofloxacin and chloramphenicol was observed in the presence of vitamin K 3 , in both wild-type and smeW -deficient strains. Copyright © 2017 American Society for Microbiology.

From phenothiazine to 3-phenyl-1,4-benzothiazine derivatives as inhibitors of the Staphylococcus aureus NorA multidrug efflux pump.

PubMed

Sabatini, Stefano; Kaatz, Glenn W; Rossolini, Gian Maria; Brandini, David; Fravolini, Arnaldo

2008-07-24

Overexpression of efflux pumps is an important mechanism by which bacteria evade effects of substrate antimicrobial agents and inhibition of such pumps is a promising strategy to circumvent this resistance mechanism. NorA is a Staphylococcus aureus multidrug efflux pump, the activity of which confers decreased susceptibility to many structurally unrelated agents, including fluoroquinolones, resulting in a multidrug resistant (MDR) phenotype. In this work, a series of 1,4-benzothiazine derivatives were designed and synthesized as a minimized structural template of phenothiazine MDR efflux pump inhibitors (EPIs) in an effort to identify more potent S. aureus NorA EPIs. Almost all derivatives evaluated showed good activity in combination with ciprofloxacin against S. aureus ATCC 25923; some were capable of completely restoring ciprofloxacin activity in a norA-overexpressing strain (SA-K2378). Compounds 6k and 7j displayed good activity against SA-1199B, a strain that also overexpresses norA, in an ethidium bromide (EtBr) efflux inhibition assay.

Antimicrobial and Efflux Pump Inhibitory Activity of Caffeoylquinic Acids from Artemisia absinthium against Gram-Positive Pathogenic Bacteria

PubMed Central

Fiamegos, Yiannis C.; Kastritis, Panagiotis L.; Exarchou, Vassiliki; Han, Haley; Bonvin, Alexandre M. J. J.; Vervoort, Jacques; Lewis, Kim; Hamblin, Michael R.; Tegos, George P.

2011-01-01

Background Traditional antibiotics are increasingly suffering from the emergence of multidrug resistance amongst pathogenic bacteria leading to a range of novel approaches to control microbial infections being investigated as potential alternative treatments. One plausible antimicrobial alternative could be the combination of conventional antimicrobial agents/antibiotics with small molecules which block multidrug efflux systems known as efflux pump inhibitors. Bioassay-driven purification and structural determination of compounds from plant sources have yielded a number of pump inhibitors which acted against gram positive bacteria. Methodology/Principal Findings In this study we report the identification and characterization of 4′,5′-O-dicaffeoylquinic acid (4′,5′-ODCQA) from Artemisia absinthium as a pump inhibitor with a potential of targeting efflux systems in a wide panel of Gram-positive human pathogenic bacteria. Separation and identification of phenolic compounds (chlorogenic acid, 3′,5′-ODCQA, 4′,5′-ODCQA) was based on hyphenated chromatographic techniques such as liquid chromatography with post column solid-phase extraction coupled with nuclear magnetic resonance spectroscopy and mass spectroscopy. Microbial susceptibility testing and potentiation of well know pump substrates revealed at least two active compounds; chlorogenic acid with weak antimicrobial activity and 4′,5′-ODCQA with pump inhibitory activity whereas 3′,5′-ODCQA was ineffective. These intitial findings were further validated with checkerboard, berberine accumulation efflux assays using efflux-related phenotypes and clinical isolates as well as molecular modeling methodology. Conclusions/Significance These techniques facilitated the direct analysis of the active components from plant extracts, as well as dramatically reduced the time needed to analyze the compounds, without the need for prior isolation. The calculated energetics of the docking poses supported the

An efflux pump (MexAB-OprM) of Pseudomonas aeruginosa is associated with antibacterial activity of Epigallocatechin-3-gallate (EGCG).

PubMed

Kanagaratnam, Rashmi; Sheikh, Rida; Alharbi, Fahad; Kwon, Dong H

2017-12-01

Pseudomonas aeruginosa is a notorious multidrug resistant nosocomial pathogen. An efflux pump (MexAB-OprM) is the main contributor to the multidrug resistance in clinical isolates of P. aeruginosa. Epigallocatechin-3-gallate (EGCG), a polyphenolic compound extracted from green tea, exhibits antibacterial activity. It is unclear that molecular details of the antibacterial activity of EGCG, EGCG-effect on antibiotic susceptibility, and clinical relevance of EGCG in bacteria. This study aimed to determine the roles of the efflux pump and an efflux pump inhibitor (phenylalanine-arginine β-naphthylamide; PAβN) in the antibacterial activity of EGCG and the EGCG-effect on antibiotic susceptibility. Twenty-two multidrug resistant clinical isolates of P. aeruginosa and a wild type P. aeruginosa PAO1 were used to determine antibacterial activity of EGCG and EGCG-effect on antibiotic susceptibility. An efflux pump (MexAB-OPrM) mutant strain, its complemented strain carrying an intact mexAB-oprM, and their parental strain were used to determine roles of MexAB-OprM in the antibacterial activity of EGCG and EGCG-mediated antibiotic susceptibility. PAβN was also used to evaluate EGCG as a possible efflux pump inhibitor. EGCG inhibited cellular growth and killed 100% of cells at 64-512 µg/ml and at 256-1024 µg/ml, respectively, in all tested 22 clinical isolates including the wild type strain. A subinhibitory concentration of EGCG significantly enhanced susceptibility to antibiotics, unexceptionally to chloramphenicol and tetracyclines (≥4-fold) of the clinical isolates. Both the antibacterial activity of EGCG and the EGCG-mediated antibiotic susceptibility were enhanced more in the efflux pump mutant strain (mexB::Gm) than the parental strain, suggesting additionally accumulated-EGCG produced the more antibacterial activity in the mutant strain. EGCG was synergistically interacted with PAβN with enhancing susceptibility to all tested antibiotics (up to >500-fold) at

Efflux-Mediated Drug Resistance in Bacteria: an Update

PubMed Central

Li, Xian-Zhi; Nikaido, Hiroshi

2010-01-01

Drug efflux pumps play a key role in drug resistance and also serve other functions in bacteria. There has been a growing list of multidrug and drug-specific efflux pumps characterized from bacteria of human, animal, plant and environmental origins. These pumps are mostly encoded on the chromosome although they can also be plasmid-encoded. A previous article (Li X-Z and Nikaido H, Drugs, 2004; 64[2]: 159–204) had provided a comprehensive review regarding efflux-mediated drug resistance in bacteria. In the past five years, significant progress has been achieved in further understanding of drug resistance-related efflux transporters and this review focuses on the latest studies in this field since 2003. This has been demonstrated in multiple aspects that include but are not limited to: further molecular and biochemical characterization of the known drug efflux pumps and identification of novel drug efflux pumps; structural elucidation of the transport mechanisms of drug transporters; regulatory mechanisms of drug efflux pumps; determining the role of the drug efflux pumps in other functions such as stress responses, virulence and cell communication; and development of efflux pump inhibitors. Overall, the multifaceted implications of drug efflux transporters warrant novel strategies to combat multidrug resistance in bacteria. PMID:19678712

Efflux Pumps Are Involved in the Defense of Gram-Negative Bacteria against the Natural Products Isobavachalcone and Diospyrone ▿

PubMed Central

Kuete, Victor; Ngameni, Bathélémy; Tangmouo, Jean G.; Bolla, Jean-Michel; Alibert-Franco, Sandrine; Ngadjui, Bonaventure T.; Pagès, Jean-Marie

2010-01-01

The activities of two naturally occurring compounds, isobavachalcone and diospyrone, against documented strains and multidrug-resistant (MDR) Gram-negative bacterial isolates were evaluated. The results indicated that the two compounds exhibited intrinsic antibacterial activity against several Gram-negative bacteria, and their activities were significantly improved in the presence of an efflux pump inhibitor (MIC values decreased to below 10 μg/ml). In addition, the activities of isobavachalcone and diospyrone against various strains exhibiting deletions of the major efflux pump components (AcrAB, TolC) were significantly increased. The overall results indicate that isobavachalcone and diospyrone could be candidates for the development of new drugs against MDR strains and that their use in combination with efflux pump inhibitors reinforces their activity. PMID:20160051

Efflux pumps as antimicrobial resistance mechanisms.

PubMed

Poole, Keith

2007-01-01

Antibiotic resistance continues to hamper antimicrobial chemotherapy of infectious disease, and while biocide resistance outside of the laboratory is as yet unrealized, in vitro and in vivo episodes of reduced biocide susceptibility are not uncommon. Efflux mechanisms, both drug-specific and multidrug, are important determinants of intrinsic and/or acquired resistance to these antimicrobials in important human pathogens. Multidrug efflux mechanisms are generally chromosome-encoded, with their expression typically resultant from mutations in regulatory genes, while drug-specific efflux mechanisms are encoded by mobile genetic elements whose acquisition is sufficient for resistance. While it has been suggested that drug-specific efflux systems originated from efflux determinants of self-protection in antibiotic-producing Actinomycetes, chromosomal multidrug efflux determinants, at least in Gram-negative bacteria, are appreciated as having an intended housekeeping function unrelated to drug export and resistance. Thus, it will be important to elucidate the intended natural function of these efflux mechanisms in order, for example, to anticipate environmental conditions or circumstances that might promote their expression and, so, compromise antimicrobial chemotherapy. Given the clinical significance of antimicrobial exporters, it is clear that efflux must be considered in formulating strategies for treatment of drug-resistant infections, both in the development of new agents, for example, less impacted by efflux or in targeting efflux directly with efflux inhibitors.

Plasmid-Encoded Tetracycline Efflux Pump Protein Alters Bacterial Stress Responses and Ecological Fitness of Acinetobacter oleivorans

PubMed Central

Hong, Hyerim; Jung, Jaejoon; Park, Woojun

2014-01-01

Acquisition of the extracellular tetracycline (TC) resistance plasmid pAST2 affected host gene expression and phenotype in the oil-degrading soil bacterium, Acinetobacter oleivorans DR1. Whole-transcriptome profiling of DR1 cells harboring pAST2 revealed that all the plasmid genes were highly expressed under TC conditions, and the expression levels of many host chromosomal genes were modulated by the presence of pAST2. The host energy burden imposed by replication of pAST2 led to (i) lowered ATP concentrations, (ii) downregulated expression of many genes involved in cellular growth, and (iii) reduced growth rate. Interestingly, some phenotypes were restored by deleting the plasmid-encoded efflux pump gene tetH, suggesting that the membrane integrity changes resulting from the incorporation of efflux pump proteins also resulted in altered host response under the tested conditions. Alteration of membrane integrity by tetH deletion was shown by measuring permeability of fluorescent probe and membrane hydrophobicity. The presence of the plasmid conferred peroxide and superoxide resistance to cells, but only peroxide resistance was diminished by tetH gene deletion, suggesting that the plasmid-encoded membrane-bound efflux pump protein provided peroxide resistance. The downregulation of fimbriae-related genes presumably led to reduced swimming motility, but this phenotype was recovered by tetH gene deletion. Our data suggest that not only the plasmid replication burden, but also its encoded efflux pump protein altered host chromosomal gene expression and phenotype, which also alters the ecological fitness of the host in the environment. PMID:25229538

Plasmid-encoded tetracycline efflux pump protein alters bacterial stress responses and ecological fitness of Acinetobacter oleivorans.

PubMed

Hong, Hyerim; Jung, Jaejoon; Park, Woojun

2014-01-01

Acquisition of the extracellular tetracycline (TC) resistance plasmid pAST2 affected host gene expression and phenotype in the oil-degrading soil bacterium, Acinetobacter oleivorans DR1. Whole-transcriptome profiling of DR1 cells harboring pAST2 revealed that all the plasmid genes were highly expressed under TC conditions, and the expression levels of many host chromosomal genes were modulated by the presence of pAST2. The host energy burden imposed by replication of pAST2 led to (i) lowered ATP concentrations, (ii) downregulated expression of many genes involved in cellular growth, and (iii) reduced growth rate. Interestingly, some phenotypes were restored by deleting the plasmid-encoded efflux pump gene tetH, suggesting that the membrane integrity changes resulting from the incorporation of efflux pump proteins also resulted in altered host response under the tested conditions. Alteration of membrane integrity by tetH deletion was shown by measuring permeability of fluorescent probe and membrane hydrophobicity. The presence of the plasmid conferred peroxide and superoxide resistance to cells, but only peroxide resistance was diminished by tetH gene deletion, suggesting that the plasmid-encoded membrane-bound efflux pump protein provided peroxide resistance. The downregulation of fimbriae-related genes presumably led to reduced swimming motility, but this phenotype was recovered by tetH gene deletion. Our data suggest that not only the plasmid replication burden, but also its encoded efflux pump protein altered host chromosomal gene expression and phenotype, which also alters the ecological fitness of the host in the environment.

Evaluation of expression of NorA efflux pump in ciprofloxacin resistant Staphylococcus aureus against hexahydroquinoline derivative by real-time PCR.

PubMed

Pourmand, Mohammad Reza; Yousefi, Masoud; Salami, Seyed Alireza; Amini, Mohsen

2014-01-01

Staphylococcus aureus causes a wide variety of infections worldwide. Methicillin-resistant S. aureus is one of most common causes of nosocomial and community acquired infections. The fluoroquinolones are an important class of antibiotics that used to treat infections caused by S. aureus. Today, a significant increase in the rate of ciprofloxacin resistance in methicillin-resistant S. aureus strains is concerning. The norA efflux pump is considered as contributors to antibiotic resistance. Here, we aimed to evaluate the expression of norA efflux pump in the presence of hexahydroquinoline derivative in methicillin and ciprofloxacin resistant S. aureus. We were determined minimum inhibitory concentration of ciprofloxacin and hexahydroquinoline derivative and their combination by broth microdilution method against ciprofloxacin resistant S. aureus. The expression of the norA efflux pump gene was evaluated by quantitative Real-time PCR. This study showed that minimum inhibitory concentrations of ciprofloxacin in the presence of hexahydroquinoline derivative in comparison to ciprofloxacin were decreased. Quantitative Real-time PCR identified the increased expression of norA efflux pump gene in methicillin and ciprofloxacin resistant S. aureus strain. The increased expression of norA efflux pump gene may have resulted in the effort of S. aureus to survive. The results showed that the hexahydroquinoline derivative enhanced the antibacterial effect of ciprofloxacin against methicillin and ciprofloxacin resistant S. aureus. Therefore, the derivatives may be used as inhibitors of antibiotic resistance for combination therapy.

Structure-Activity Relationships of a Novel Pyranopyridine Series of Gram-negative Bacterial Efflux Pump Inhibitors

PubMed Central

Nguyen, Son T.; Kwasny, Steven M.; Ding, Xiaoyuan; Cardinale, Steven C.; McCarthy, Courtney T.; Kim, Hong-Suk; Nikaido, Hiroshi; Peet, Norton P.; Williams, John D.; Bowlin, Terry L.; Opperman, Timothy J.

2015-01-01

Recently we described a novel pyranopyridine inhibitor (MBX2319) of RND-type efflux pumps of the Enterobacteriaceae. MBX2319 (3,3-dimethyl-5-cyano-8-morpholino-6-(phenethylthio)-3,4-dihydro-1H-pyrano[3,4-c]pyridine) is structurally distinct from other known Gram-negative efflux pump inhibitors (EPIs), such as 1-(1-naphthylmethyl)-piperazine (NMP), phenylalanylarginine-β-naphthylamide (PAβN), D13-9001, and the pyridopyrimidine derivatives. Here, we report the synthesis and biological evaluation of 60 new analogs of MBX2319 that were designed to probe the structure activity relationships (SARs) of the pyranopyridine scaffold. The results of these studies produced a molecular activity map of the scaffold, which identifies regions that are critical to efflux inhibitory activities and those that can be modified to improve potency, metabolic stability and solubility. Several compounds, such as 22d–f, 22i and 22k, are significantly more effective than MBX2319 at potentiating the antibacterial activity of levofloxacin and piperacillin against Escherichia coli. PMID:25818767

Tracking metal ions through a Cu/Ag efflux pump assigns the functional roles of the periplasmic proteins.

PubMed

Chacón, Kelly N; Mealman, Tiffany D; McEvoy, Megan M; Blackburn, Ninian J

2014-10-28

Copper is an essential nutrient for all aerobic organisms but is toxic in excess. At the host-pathogen interface, macrophages respond to bacterial infection by copper-dependent killing mechanisms, whereas the invading bacteria are thought to counter with an up-regulation of copper transporters and efflux pumps. The tripartite efflux pump CusCBA and its metallochaperone CusF are vital to the detoxification of copper and silver ions in the periplasm of Escherichia coli. However, the mechanism of efflux by this complex, which requires the activation of the inner membrane pump CusA, is poorly understood. Here, we use selenomethionine (SeM) active site labels in a series of biological X-ray absorption studies at the selenium, copper, and silver edges to establish a "switch" role for the membrane fusion protein CusB. We determine that metal-bound CusB is required for activation of cuprous ion transfer from CusF directly to a site in the CusA antiporter, showing for the first time (to our knowledge) the in vitro activation of the Cus efflux pump. This metal-binding site of CusA is unlike that observed in the crystal structures of the CusA protein and is composed of one oxygen and two sulfur ligands. Our results suggest that metal transfer occurs between CusF and apo-CusB, and that, when metal-loaded, CusB plays a role in the regulation of metal ion transfer from CusF to CusA in the periplasm.

Antibacterial and efflux pump inhibitors of thymol and carvacrol against food-borne pathogens.

PubMed

Miladi, Hanene; Zmantar, Tarek; Chaabouni, Yassine; Fedhila, Kais; Bakhrouf, Amina; Mahdouani, Kacem; Chaieb, Kamel

2016-10-01

In this study thymol (THY) and carvacrol (CAR), two monoterpenic phenol produced by various aromatic plants, was tested for their antibacterial and efflux pump inhibitors potencies against a panel of clinical and foodborne pathogenes. Our results demonstrated a substantial susceptibility of the tested bacteria toward THY and CAR. Especially, THY displayed a strong inhibitory activity (MIC's values ranged from 32 to 64 μg/mL) against the majority of the tested strains compared to CAR. Moreover, a significant reduction in MIC's of TET and benzalkonium chloride (QAC) were noticed when tested in combinations with THY and CAR. Their synergic effect was more significant in the case of THY which resulted a reduction of MIC's values of TET (2-8 fold) and QAC (2-8 fold). We noted also that THY and CAR inhibited the ethidium bromide (EtBr) cell efflux in a concentration-dependent manner. The rate of EtBr accumulation in food-borne pathogen was enhanced with THY and CAR (0, 250 and 500 μg/mL). The lowest concentration causing 50% of EtBr efflux inhibition (IC 50) was noticed in Salmonella enteritidis (1129) at 150 μg/mL of THY and 190 μg/mL of CAR respectively. These findings indicate that THY and CAR may serve as potential sources of efflux pump inhibitor in food-borne pathogens. Copyright © 2016 Elsevier Ltd. All rights reserved.

Aminoguanidine hydrazones (AGH's) as modulators of norfloxacin resistance in Staphylococcus aureus that overexpress NorA efflux pump.

PubMed

Dantas, Natalina; de Aquino, Thiago Mendonça; de Araújo-Júnior, João Xavier; da Silva-Júnior, Edeildo; Gomes, Ednaldo Almeida; Gomes, Antoniel Augusto Severo; Siqueira-Júnior, José Pinto; Mendonça Junior, Francisco Jaime Bezerra

2018-01-25

One of the promising fields for improving the effectiveness of antimicrobial agents is their combination with efflux pump inhibitors (EPIs), which besides expanding the use of existing antibiotics. The goal of this research was to evaluate a series of aminoguanidine hydrazones (AGH's, 1-19) as antibacterial agents and NorA efflux pump inhibitors in Staphylococcus aureus strain SA-1199B. Molecular modeling and docking studies were also performed in order to explain at the molecular level the interactions of the compounds with the generated NorA efflux pump model. The MICs of the antibiotic and ethidium bromide were determined by microdilution assay in absence or presence of a subinhibitory concentration of aminoguanidine hydrazones and macrophages viability was determined through MTT assay. Bioinformatic software Swiss-Model and AutoDock 4.2 were used to perform modeling and docking studies, respectively. As results, all AGH's were able to potentiate the action for the antibiotic norfloxacin, causing MIC's reduction of 16-fold and 32-fold to ethidium bromide. In the cell viability test, the concentration of 10 μg/mL showed better results than 90% and the concentration of 1000 μg/mL showed the lowest viability, reaching a maximum of 50% for the analyzed aminoguanidine hydrazones. Molecular docking studies showed that both norfloxacin and derivative 13 were recognized by the same binding site of NorA pump, suggesting a competitive mechanism. The present work demonstrated for the first time that AGH derivatives have potential to be putative inhibitors of NorA efflux pump, showing a promising activity as an antibacterial drug development. Copyright © 2017 Elsevier B.V. All rights reserved.

The MerR-like regulator BrlR confers biofilm tolerance by activating multidrug efflux pumps in Pseudomonas aeruginosa biofilms.

PubMed

Liao, Julie; Schurr, Michael J; Sauer, Karin

2013-08-01

A defining characteristic of biofilms is antibiotic tolerance that can be up to 1,000-fold greater than that of planktonic cells. In Pseudomonas aeruginosa, biofilm tolerance to antimicrobial agents requires the biofilm-specific MerR-type transcriptional regulator BrlR. However, the mechanism by which BrlR mediates biofilm tolerance has not been elucidated. Genome-wide transcriptional profiling indicated that brlR was required for maximal expression of genes associated with antibiotic resistance, in particular those encoding the multidrug efflux pumps MexAB-OprM and MexEF-OprN. Chromatin immunoprecipitation (ChIP) analysis revealed a direct regulation of these genes by BrlR, with DNA binding assays confirming BrlR binding to the promoter regions of the mexAB-oprM and mexEF-oprN operons. Quantitative reverse transcriptase PCR (qRT-PCR) analysis further indicated BrlR to be an activator of mexAB-oprM and mexEF-oprN gene expression. Moreover, immunoblot analysis confirmed increased MexA abundance in cells overexpressing brlR. Inactivation of both efflux pumps rendered biofilms significantly more susceptible to five different classes of antibiotics by affecting MIC but not the recalcitrance of biofilms to killing by bactericidal agents. Overexpression of either efflux pump in a ΔbrlR strain partly restored tolerance of ΔbrlR biofilms to antibiotics. Expression of brlR in mutant biofilms lacking both efflux pumps partly restored antimicrobial tolerance of biofilms to wild-type levels. Our results indicate that BrlR acts as an activator of multidrug efflux pumps to confer tolerance to P. aeruginosa biofilms and to resist the action of antimicrobial agents.

Determination of the concentration of potential efflux pump inhibitors, pheophorbide a and pyropheophorbide a, in the feces of animals by fluorescence spectroscopy.

PubMed

Barnes, Charles A; Rasmussen, Sharon L; Petrich, Jacob W; Rasmussen, Mark A

2012-10-24

Efflux pumps are vital bacterial components, and research has demonstrated that some plant compounds such as pheophorbide a (php) possess efflux pump inhibitor (EPI) activity. This study determined the quantity of php present in feces as an indicator of EPI activity. Feces were collected from different species of animals fed a variety of feeds. The chlorophyll metabolites php and pyropheophorbide a (pyp) were determined using fluorescense spectroscopy. The average concentrations [μg/g dry matter (DM) feces] of pyp/php in feces were as follows: guinea pig, 180; goat, 150; rabbit, 150; dairy cow, 120; feedlot cattle, 60; rat, <1; pig, <1; chicken, <1. These data indicate that animals consuming "green" diets will excrete feces with concentrations of php/pyp that exceed levels demonstrated to be inhibitory to bacterial efflux pumps (0.5 μg/mL). The natural presence EPIs in the gastrointestinal tract may modulate the activity of microbial efflux pumps and exert selection pressure upon resident microbial populations.

Mechanism of action of tetrandrine, a natural inhibitor of Candida albicans drug efflux pumps.

PubMed

Zhang, Hong; Gao, Aili; Li, Fengxia; Zhang, Gehua; Ho, Hon In; Liao, Wanqing

2009-05-01

Synergistic effects have previously been observed for a natural compound, tetrandrine (TET), with fluconazole (FLC) in vitro and in the treatment of Candida albicans-infected mice. To investigate the mechanisms of these synergistic effects, 16 strains of C. albicans from the same parent but with different FLC sensitivities were examined using flow cytometry and fluorescent spectrophotometry. Rhodamine 123 (Rh123)-positive cells and intracellular Rh123 fluorescence intensity were determined in accumulation/efflux experiments involving no or a noncytotoxic dose of TET. Total RNA extracted from each strain was used to compare the expressions of drug efflux pump genes in FLC-susceptible, -susceptible dose-dependent, and -resistant strains before and 24 h after TET administration. Accumulation experiments determined that mean percentages of Rh123-positive cells were 26.65% (TET-free) and 70.99% (TET 30 microg/ml), and mean respective intracellular Rh123 fluorescence intensities were 11.34 and 18.00. Efflux experiments showed that percentages of Rh123-positive cells were 1.79% (TET free) and 42.57% (TET 30 microg/ml), respectively, and respective mean intracellular Rh123 fluorescence intensities were 0.74 and 2.19. Differences in MDR1, FLU1, CDR1, and CDR2 expression levels in the absence of TET were statistically significant (p<0.05) between FLC-susceptible, -susceptible dose-dependent, and -resistant strains. Compared with TET-free conditions, 24 h TET-treated strains showed statistically different (p<0.05) expression of MDR1 (FLC-resistant strain), FLU1 (FLC-susceptible dose-dependent and -resistant strains), and CDR1 and CDR2 (FLC-susceptible, -susceptible dose-dependent, and -resistant strains). Thus TET can inhibit the C. albicans drug efflux system and reduce drug efflux. Its mechanism of action is related to the inhibition of expression of the drug efflux pump genes MDR1, FLU1, CDR1, and CDR2.

Tracking metal ions through a Cu/Ag efflux pump assigns the functional roles of the periplasmic proteins

DOE PAGES

Chacon, Kelly N.; Mealman, Tiffany D.; McEvoy, Megan M.; ...

2014-10-13

Copper is an essential nutrient for all aerobic organisms but is toxic in excess. At the host–pathogen interface, macrophages respond to bacterial infection by copper-dependent killing mechanisms, whereas the invading bacteria are thought to counter with an up-regulation of copper transporters and efflux pumps. The tripartite efflux pump CusCBA and its metallochaperone CusF are vital to the detoxification of copper and silver ions in the periplasm of Escherichia coli. However, the mechanism of efflux by this complex, which requires the activation of the inner membrane pump CusA, is poorly understood. In this paper, we use selenomethionine (SeM) active site labelsmore » in a series of biological X-ray absorption studies at the selenium, copper, and silver edges to establish a “switch” role for the membrane fusion protein CusB. We determine that metal-bound CusB is required for activation of cuprous ion transfer from CusF directly to a site in the CusA antiporter, showing for the first time (to our knowledge) the in vitro activation of the Cus efflux pump. This metal-binding site of CusA is unlike that observed in the crystal structures of the CusA protein and is composed of one oxygen and two sulfur ligands. Finally, our results suggest that metal transfer occurs between CusF and apo-CusB, and that, when metal-loaded, CusB plays a role in the regulation of metal ion transfer from CusF to CusA in the periplasm.« less

The AcrAB-TolC pump is involved in macrolide resistance but not in telithromycin efflux in Enterobacter aerogenes and Escherichia coli.

PubMed

Chollet, Renaud; Chevalier, Jacqueline; Bryskier, André; Pagès, Jean-Marie

2004-09-01

The role of the AcrAB-TolC pump in macrolide and ketolide susceptibility in Escherichia coli and Enterobacter aerogenes was studied. Efflux pump inhibitor restored erythromycin, clarithromycin, and telithromycin susceptibilities to multidrug-resistant isolates. No modification of telithromycin accumulation was detected in E. aerogenes acrAB or tolC derivatives compared to that in the parental strain. Two independent efflux pumps, inhibited by phenylalanine arginine beta-naphthylamide, expel macrolides and telithromycin in E. aerogenes.

Homology modeling, molecular dynamics, and virtual screening of NorA efflux pump inhibitors of Staphylococcus aureus

PubMed Central

Bhaskar, Baki Vijaya; Babu, Tirumalasetty Muni Chandra; Reddy, Netala Vasudeva; Rajendra, Wudayagiri

2016-01-01

Emerging drug resistance in clinical isolates of Staphylococcus aureus might be implicated to the overexpression of NorA efflux pump which is capable of extruding numerous structurally diverse compounds. However, NorA efflux pump is considered as a potential drug target for the development of efflux pump inhibitors. In the present study, NorA model was constructed based on the crystal structure of glycerol-3-phosphate transporter (PDBID: 1PW4). Molecular dynamics (MD) simulation was performed using NAMD2.7 for NorA which is embedded in the hydrated lipid bilayer. Structural design of NorA unveils amino (N)- and carboxyl (C)-terminal domains which are connected by long cytoplasmic loop. N and C domains are composed of six transmembrane α-helices (TM) which exhibits pseudo-twofold symmetry and possess voluminous substrate binding cavity between TM helices. Molecular docking of reserpine, totarol, ferruginol, salvin, thioxanthene, phenothiazine, omeprazole, verapamil, nalidixic acid, ciprofloxacin, levofloxacin, and acridine to NorA found that all the molecules were bound at the large hydrophobic cleft and indicated significant interactions with the key residues. In addition, structure-based virtual screening was employed which indicates that 14 potent novel lead molecules such as CID58685302, CID58685367, CID5799283, CID5578487, CID60028372, ZINC12196383, ZINC72140751, ZINC72137843, ZINC39227983, ZINC43742707, ZINC12196375, ZINC66166948, ZINC39228014, and ZINC14616160 have highest binding affinity for NorA. These lead molecules displayed considerable pharmacological properties as evidenced by Lipinski rule of five and prophecy of toxicity risk assessment. Thus, the present study will be helpful in designing and synthesis of a novel class of NorA efflux pump inhibitors that restore the susceptibilities of drug compounds. PMID:27757014

Homology modeling, molecular dynamics, and virtual screening of NorA efflux pump inhibitors of Staphylococcus aureus.

PubMed

Bhaskar, Baki Vijaya; Babu, Tirumalasetty Muni Chandra; Reddy, Netala Vasudeva; Rajendra, Wudayagiri

2016-01-01

Emerging drug resistance in clinical isolates of Staphylococcus aureus might be implicated to the overexpression of NorA efflux pump which is capable of extruding numerous structurally diverse compounds. However, NorA efflux pump is considered as a potential drug target for the development of efflux pump inhibitors. In the present study, NorA model was constructed based on the crystal structure of glycerol-3-phosphate transporter (PDBID: 1PW4). Molecular dynamics (MD) simulation was performed using NAMD2.7 for NorA which is embedded in the hydrated lipid bilayer. Structural design of NorA unveils amino (N)- and carboxyl (C)-terminal domains which are connected by long cytoplasmic loop. N and C domains are composed of six transmembrane α-helices (TM) which exhibits pseudo-twofold symmetry and possess voluminous substrate binding cavity between TM helices. Molecular docking of reserpine, totarol, ferruginol, salvin, thioxanthene, phenothiazine, omeprazole, verapamil, nalidixic acid, ciprofloxacin, levofloxacin, and acridine to NorA found that all the molecules were bound at the large hydrophobic cleft and indicated significant interactions with the key residues. In addition, structure-based virtual screening was employed which indicates that 14 potent novel lead molecules such as CID58685302, CID58685367, CID5799283, CID5578487, CID60028372, ZINC12196383, ZINC72140751, ZINC72137843, ZINC39227983, ZINC43742707, ZINC12196375, ZINC66166948, ZINC39228014, and ZINC14616160 have highest binding affinity for NorA. These lead molecules displayed considerable pharmacological properties as evidenced by Lipinski rule of five and prophecy of toxicity risk assessment. Thus, the present study will be helpful in designing and synthesis of a novel class of NorA efflux pump inhibitors that restore the susceptibilities of drug compounds.

Plagiochin E, a botanic-derived phenolic compound, reverses fungal resistance to fluconazole relating to the efflux pump.

PubMed

Guo, X-L; Leng, P; Yang, Y; Yu, L-G; Lou, H-X

2008-03-01

In this study, we investigated the effect of plagiochin E (PLE), a botanic-derived phenolic natural product, on reversal of fungal resistance to fluconazole (FLC) in vitro and the related mechanism. A synergistic action of PLE and FLC was observed in the FLC-resistant Candida albicans strains and was evaluated using the fractional inhibited concentration index. The effect of PLE on FLC intracellular uptake was investigated in FLC-resistant C. albicans cells by liquid chromatography-tandem mass spectrometry, and the effect on efflux drug pump was assessed by measuring the efflux of Rhodamine 123 (Rh123). PLE significantly inhibited the efflux, but not the absorption, of Rh123 in FLC-resistant strains in phosphate-buffered saline with 5% glucose. Overexpression of the multidrug-resistance gene CDR1 in FLC-resistant C. albicans isolates was detected, and the introduction of PLE to the cells showed a significant reduction of the CDR1 expression in those FLC-resistant isolates. These findings indicate that PLE could reverse the fungal resistant to FLC by inhibiting the efflux of FLC from C. albicans, and this effect may be related to the efflux pump. These results indicate that the combination of PLE and FLC may provide an approach for the clinical therapy of fungus infection induced by FLC-resistant strains.

The role of drug efflux pumps in Malassezia pachydermatis and Malassezia furfur defence against azoles.

PubMed

Iatta, Roberta; Puttilli, Maria Rita; Immediato, Davide; Otranto, Domenico; Cafarchia, Claudia

2017-03-01

This study aims to evaluate the effect of efflux pump modulators (EPMs) on the minimal inhibitory concentration (MIC) of fluconazole (FLZ) and voriconazole (VOR) in Malassezia furfur and Malassezia pachydermatis. The in vitro efficacy of azoles, in combination with EPMs (ie haloperidol-HAL, promethazine-PTZ and cyclosporine A-CYS), against 21 M. furfur from bloodstream infection patients and 14 M. pachydermatis from the skin of dogs with dermatitis, was assessed using a broth microdilution chequerboard analysis. Data were analysed using the model-fractional inhibitory concentration index (FICI) method. The MIC of FLZ and VOR of Malassezia spp. decreased in the presence of sub-inhibitory concentrations of HAL and/or PTZ. The synergic effect was observed only in strains with FLZ MIC≥128 μg/mL for M. furfur, FLZ MIC≥64 μg/mL for M. pachydermatis and VOR MIC≥4 μg/mL in both Malassezia spp. These results suggest that the drug efflux pumps are involved as defence mechanisms to azole drugs in Malassezia yeast. The synergism might be related to an increased expression of efflux pump genes, eventually resulting in azole resistance phenomena. Finally, the above FLZ and VOR MIC values might be considered the cut-off to discriminate susceptible and resistant strains. © 2016 Blackwell Verlag GmbH.

Contribution of AcrAB efflux pump to ciprofloxacin resistance in Klebsiella pneumoniae isolated from burn patients.

PubMed

Pakzad, Iraj; Zayyen Karin, Maasoume; Taherikalani, Morovat; Boustanshenas, Mina; Lari, Abdolaziz Rastegar

2013-01-01

Resistance to fluoroquinolones has been recently increased among bacterial strains isolated from outpatients. Multidrug-resistant K. pneumoniae is one of the major organisms isolated from burn patients and the AcrAB efflux pump is the principal pump contributing to the intrinsic resistance in K. pneumoniae against multiple antimicrobial agents including ciprofloxacin and other fluoroquinolones. Fifty-two K. pneumoniae isolated from burn patients in Shahid Motahari hospital and confirmed by conventional biochemical tests. Antimicrobial susceptibility testing was done according to CLSI 2011 guidelines, to determine the antimicrobial resistance pattern of isolates. AcrA gene was detected among ciprofloxacin-resistant isolates by PCR assay. MICs to ciprofloxacin were measured with and without carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Forty out of the 52 K. pneumoniae isolated from burn patients in Shahid Motahari hospital were resistant to ciprofloxacin according to breakpoint of CLSI guideline. PCR assay for acrA gene demonstrated that all ciprofloxacin-resistant isolates harbored acrA gene coding the membrane fusion protein AcrA and is a part of AcrAB efflux system. Among these isolates, 19 strains (47.5%) showed 2 to 32 fold reduction in MICs after using CCCP as an efflux pump inhibitor. The other 21 strains (52.5%) showed no disparity in MICs before and after using CCCP. In conclusion, the AcrAB efflux system is one of the principal mechanisms contribute in ciprofloxacin resistance among K. pneumoniae isolates but there are some other mechanisms interfere with ciprofloxacin resistance such as mutation in target proteins of DNA gyrase of topoisomerase IV enzymes.

Evolution from a natural flavones nucleus to obtain 2-(4-Propoxyphenyl)quinoline derivatives as potent inhibitors of the S. aureus NorA efflux pump.

PubMed

Sabatini, Stefano; Gosetto, Francesca; Manfroni, Giuseppe; Tabarrini, Oriana; Kaatz, Glenn W; Patel, Diixa; Cecchetti, Violetta

2011-08-25

Overexpression of efflux pumps is an important mechanism by which bacteria evade the effects of substrate antimicrobial agents. Inhibition of such pumps is a promising strategy to circumvent this resistance mechanism. NorA is a Staphylococcus aureus efflux pump that confers reduced susceptibility to many structurally unrelated agents, including fluoroquinolones, resulting in a multidrug resistant phenotype. In this work, a series of 2-phenyl-4(1H)-quinolone and 2-phenyl-4-hydroxyquinoline derivatives, obtained by modifying the flavone nucleus of known efflux pump inhibitors (EPIs), were synthesized in an effort to identify more potent S. aureus NorA EPIs. The 2-phenyl-4-hydroxyquinoline derivatives 28f and 29f display potent EPI activity against SA-1199B, a strain that overexpresses norA, in an ethidium bromide efflux inhibition assay. The same compounds, in combination with ciprofloxacin, were able to completely restore its antibacterial activity against both S. aureus SA-K2378 and SA-1199B, norA-overexpressing strains. © 2011 American Chemical Society

Whole genome and transcriptome analyses of environmental antibiotic sensitive and multi-resistant Pseudomonas aeruginosa isolates exposed to waste water and tap water

PubMed Central

Schwartz, Thomas; Armant, Olivier; Bretschneider, Nancy; Hahn, Alexander; Kirchen, Silke; Seifert, Martin; Dötsch, Andreas

2015-01-01

The fitness of sensitive and resistant Pseudomonas aeruginosa in different aquatic environments depends on genetic capacities and transcriptional regulation. Therefore, an antibiotic-sensitive isolate PA30 and a multi-resistant isolate PA49 originating from waste waters were compared via whole genome and transcriptome Illumina sequencing after exposure to municipal waste water and tap water. A number of different genomic islands (e.g. PAGIs, PAPIs) were identified in the two environmental isolates beside the highly conserved core genome. Exposure to tap water and waste water exhibited similar transcriptional impacts on several gene clusters (antibiotic and metal resistance, genetic mobile elements, efflux pumps) in both environmental P. aeruginosa isolates. The MexCD-OprJ efflux pump was overexpressed in PA49 in response to waste water. The expression of resistance genes, genetic mobile elements in PA49 was independent from the water matrix. Consistently, the antibiotic sensitive strain PA30 did not show any difference in expression of the intrinsic resistance determinants and genetic mobile elements. Thus, the exposure of both isolates to polluted waste water and oligotrophic tap water resulted in similar expression profiles of mentioned genes. However, changes in environmental milieus resulted in rather unspecific transcriptional responses than selected and stimuli-specific gene regulation. PMID:25186059

Quantifying the Evolutionary Conservation of Genes Encoding Multidrug Efflux Pumps in the ESKAPE Pathogens To Identify Antimicrobial Drug Targets.

PubMed

Brooks, Lauren E; Ul-Hasan, Sabah; Chan, Benjamin K; Sistrom, Mark J

2018-01-01

Increasing rates of antibiotic-resistant bacterial infection are one of the most pressing contemporary global health concerns. The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) have been identified as the leading global cause of multidrug-resistant bacterial infections, and overexpression of multidrug efflux (MEX) transport systems has been identified as one of the most critical mechanisms facilitating the evolution of multidrug resistance in ESKAPE pathogens. Despite efforts to develop efflux pump inhibitors to combat antibiotic resistance, the need persists to identify additional targets for future investigations. We evaluated evolutionary pressures on 110 MEX-encoding genes from all annotated ESKAPE organism genomes. We identify several MEX genes under stabilizing selection-representing targets which can facilitate broad-spectrum treatments with evolutionary constraints limiting the potential emergence of escape mutants. We also examine MEX systems being evaluated as drug targets, demonstrating that divergent selection may underlie some of the problems encountered in the development of effective treatments-specifically in relation to the NorA system in S. aureus. This study provides a comprehensive evolutionary context to efflux in the ESKAPE pathogens, which will provide critical context to the evaluation of efflux systems as antibiotic targets. IMPORTANCE Increasing rates of antibiotic-resistant bacterial infection are one of the most pressing contemporary global health concerns. The ESKAPE pathogen group represents the leading cause of these infections, and upregulation of efflux pump expression is a significant mechanism of resistance in these pathogens. This has resulted in substantial interest in the development of efflux pump inhibitors to combat antibiotic-resistant infections; however, no widespread treatments have been developed to date

Quantifying the Evolutionary Conservation of Genes Encoding Multidrug Efflux Pumps in the ESKAPE Pathogens To Identify Antimicrobial Drug Targets

PubMed Central

Ul-Hasan, Sabah; Chan, Benjamin K.; Sistrom, Mark J.

2018-01-01

ABSTRACT Increasing rates of antibiotic-resistant bacterial infection are one of the most pressing contemporary global health concerns. The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) have been identified as the leading global cause of multidrug-resistant bacterial infections, and overexpression of multidrug efflux (MEX) transport systems has been identified as one of the most critical mechanisms facilitating the evolution of multidrug resistance in ESKAPE pathogens. Despite efforts to develop efflux pump inhibitors to combat antibiotic resistance, the need persists to identify additional targets for future investigations. We evaluated evolutionary pressures on 110 MEX-encoding genes from all annotated ESKAPE organism genomes. We identify several MEX genes under stabilizing selection—representing targets which can facilitate broad-spectrum treatments with evolutionary constraints limiting the potential emergence of escape mutants. We also examine MEX systems being evaluated as drug targets, demonstrating that divergent selection may underlie some of the problems encountered in the development of effective treatments—specifically in relation to the NorA system in S. aureus. This study provides a comprehensive evolutionary context to efflux in the ESKAPE pathogens, which will provide critical context to the evaluation of efflux systems as antibiotic targets. IMPORTANCE Increasing rates of antibiotic-resistant bacterial infection are one of the most pressing contemporary global health concerns. The ESKAPE pathogen group represents the leading cause of these infections, and upregulation of efflux pump expression is a significant mechanism of resistance in these pathogens. This has resulted in substantial interest in the development of efflux pump inhibitors to combat antibiotic-resistant infections; however, no widespread treatments have been

Frequency of efflux pump genes mediating ciprofloxacin and antiseptic resistance in methicillin-resistant Staphylococcus aureus isolates.

PubMed

Hassanzadeh, Sepideh; Mashhadi, Rahil; Yousefi, Masoud; Askari, Emran; Saniei, Maryam; Pourmand, Mohammad Reza

2017-10-01

Efflux pumps are well known as a key role to fluoroquinolone resistance in methicillin-resistant Staphylococcus aureus (MRSA). In this study, among 60 clinical MRSA isolates, 42 isolates (70%) were resistant to ciprofloxacin. MRSA were isolated to detect efflux genes including norA, norB, norC, mepA, sepA, mdeA, qacA/B and smr. Isolates subjected to PCR detection and DNA sequence analysis for these genes. PCR detection showed that 42 isolates (70%) contained at least one efflux pump gene. Among ciprofloxacin-resistant isolates, mdeA and qacA/B genes were found with the highest (61.7%) and lowest (3.3%) frequency, respectively. We also observed that the highest minimum inhibitory concentrations of ciprofloxacin in the presence of mdeA+mepA+norA-C+sepA+smr combination. This type of combination may have the greatest impact on resistance to ciprofloxacin. Finally, compared to previous studies, our study demonstrates that prevalence of ciprofloxacin resistance has been increasing among MRSA clinical isolates. Copyright © 2017 Elsevier Ltd. All rights reserved.

Random Mutagenesis of the Multidrug Transporter AcrB from Escherichia coli for Identification of Putative Target Residues of Efflux Pump Inhibitors

PubMed Central

Kohler, Samay; Buck, Annika; Dambacher, Christine; König, Armin; Bohnert, Jürgen A.; Kern, Winfried V.

2014-01-01

Efflux is an important mechanism of bacterial multidrug resistance (MDR), and the inhibition of MDR pumps by efflux pump inhibitors (EPIs) could be a promising strategy to overcome MDR. 1-(1-Naphthylmethyl)-piperazine (NMP) and phenylalanine-arginine-β-naphthylamide (PAβN) are model EPIs with activity in various Gram-negative bacteria expressing AcrB, the major efflux pump of Escherichia coli, or similar homologous pumps of the resistance-nodulation-cell division class. The aim of the present study was to generate E. coli AcrB mutants resistant to the inhibitory action of the two model EPIs and to identify putative EPI target residues in order to better understand mechanisms of pump inhibition. Using an in vitro random mutagenesis approach focusing on the periplasmic domain of AcrB, we identified the double mutation G141D N282Y, which substantially compromised the synergistic activity of NMP with linezolid, was associated with similar intracellular linezolid concentrations in the presence and absence of NMP, and did not impair the intrinsic MICs of various pump substrates and dye accumulation. We propose that these mutations near the outer face of the distal substrate binding pocket reduce NMP trapping. Other residues found to be relevant for efflux inhibition by NMP were G288 and A279, but mutations at these sites also changed the susceptibility to several pump substrates. Unlike with NMP, we were unable to generate AcrB periplasmic domain mutants with resistance or partial resistance to the EPI activity of PAβN, which is consistent with the modes of action of PAβN differing from those of NMP. PMID:25182653

Contribution of Efflux Pumps, Porins, and β-Lactamases to Multidrug Resistance in Clinical Isolates of Acinetobacter baumannii

PubMed Central

Rumbo, C.; Gato, E.; López, M.; Ruiz de Alegría, C.; Fernández-Cuenca, F.; Martínez-Martínez, L.; Vila, J.; Pachón, J.; Cisneros, J. M.; Rodríguez-Baño, J.; Pascual, A.

2013-01-01

We investigated the mechanisms of resistance to carbapenems, aminoglycosides, glycylcyclines, tetracyclines, and quinolones in 90 multiresistant clinical strains of Acinetobacter baumannii isolated from two genetically unrelated A. baumannii clones: clone PFGE-ROC-1 (53 strains producing the OXA-58 β-lactamase enzyme and 18 strains with the OXA-24 β-lactamase) and clone PFGE-HUI-1 (19 strains susceptible to carbapenems). We used real-time reverse transcriptase PCR to correlate antimicrobial resistance (MICs) with expression of genes encoding chromosomal β-lactamases (AmpC and OXA-51), porins (OmpA, CarO, Omp33, Dcap-like, OprB, Omp25, OprC, OprD, and OmpW), and proteins integral to six efflux systems (AdeABC, AdeIJK, AdeFGH, CraA, AbeM, and AmvA). Overexpression of the AdeABC system (level of expression relative to that by A. baumannii ATCC 17978, 30- to 45-fold) was significantly associated with resistance to tigecycline, minocycline, and gentamicin and other biological functions. However, hyperexpression of the AdeIJK efflux pump (level of expression relative to that by A. baumannii ATCC 17978, 8- to 10-fold) was significantly associated only with resistance to tigecycline and minocycline (to which the TetB efflux system also contributed). TetB and TetA(39) efflux pumps were detected in clinical strains and were associated with resistance to tetracyclines and doxycycline. The absence of the AdeABC system and the lack of expression of other mechanisms suggest that tigecycline-resistant strains of the PFGE-HUI-1 clone may be associated with a novel resistance-nodulation-cell efflux pump (decreased MICs in the presence of the inhibitor Phe-Arg β-naphthylamide dihydrochloride) and the TetA(39) system. PMID:23939894

All0809/8/7 is a DevBCA-like ABC-type efflux pump required for diazotrophic growth in Anabaena sp. PCC 7120.

PubMed

Staron, Peter; Maldener, Iris

2012-10-01

Efflux pumps export a wide variety of proteinaceous and non-proteinaceous substrates across the Gram-negative cell wall. For the filamentous cyanobacterium Anabaena sp. strain PCC 7120, the ATP-driven glycolipid efflux pump DevBCA-TolC has been shown to be crucial for the differentiation of N(2)-fixing heterocysts from photosynthetically active vegetative cells. In this study, a homologous system was described. All0809/8/7-TolC form a typical ATP-driven efflux pump as shown by surface plasmon resonance. This putative exporter is also involved in diazotrophic growth of Anabaena sp. PCC 7120. A mutant in all0809 encoding the periplasmic membrane fusion protein of the pump was not able to grow without combined nitrogen. Although heterocysts of this mutant were not distinguishable from those of the wild-type in light and electron micrographs, they were impaired in providing the microoxic environment necessary for N(2) fixation. RT-PCR of all0809 transcripts and localization studies on All0807-GFP revealed that All0809/8/7 was initially downregulated during heterocyst maturation and upregulated at later stages of heterocyst formation in all cells of the filament. A substrate of the efflux pump could not be identified in ATP hydrolysis assays. We discuss a role for All0809/8/7-TolC in maintaining the continuous periplasm and how this would be of special importance for heterocyst differentiation.

Capsaicin, a novel inhibitor of the NorA efflux pump, reduces the intracellular invasion of Staphylococcus aureus.

PubMed

Kalia, Nitin Pal; Mahajan, Priya; Mehra, Rukmankesh; Nargotra, Amit; Sharma, Jai Parkash; Koul, Surrinder; Khan, Inshad Ali

2012-10-01

To delineate the role of capsaicin (8-methyl-N-vanillyl-6-nonenamide) as an inhibitor of the NorA efflux pump and its impact on invasion of macrophages by Staphylococcus aureus. Capsaicin in combination with ciprofloxacin was tested for activity against S. aureus SA-1199B (NorA overproducing), SA-1199 (wild-type) and SA-K1758 (norA knockout). The role of NorA in the intracellular invasion of S. aureus and the ability of capsaicin to inhibit this invasion was established in J774 macrophage cell lines. The three-dimensional structure of NorA was predicted using an in silico approach and docking studies of capsaicin were performed. Capsaicin significantly reduced the MIC of ciprofloxacin for S. aureus SA-1199 and SA-1199B. Furthermore, capsaicin also extended the post-antibiotic effect of ciprofloxacin by 1.1 h at MIC concentration. There was a decrease in mutation prevention concentration of ciprofloxacin when combined with capsaicin. Inhibition of ethidium bromide efflux by NorA-overproducing S. aureus SA-1199B confirmed the role of capsaicin as a NorA efflux pump inhibitor (EPI). The most significant finding of this study was the ability of capsaicin to reduce the intracellular invasion of S. aureus SA-1199B (NorA overproducing) in J774 macrophage cell lines by 2 log(10). This study, for the first time, has shown that capsaicin, a novel EPI, not only inhibits the NorA efflux pump of S. aureus but also reduces the invasiveness of S. aureus, thereby reducing its virulence.

In vivo evaluation of anionic thiolated polymers as oral delivery systems for efflux pump inhibition.

PubMed

Palmberger, Thomas F; Laffleur, Flavia; Greindl, Melanie; Bernkop-Schnürch, Andreas

2015-08-01

Recently, the cationic polymer thiolated chitosan has been reported to modulate drug absorption by inhibition of intestinal efflux pumps. The objective of this study was to evaluate in vitro and in vivo whether thiolated anionic biopolymers also show an efflux pump inhibitory effect in order to improve intestinal transcellular drug uptake. Therefore, three thiomers have been synthesized due covalent attachment of cysteine to various polymer backbones: pectin-cysteine (pect-cys), carboxymethylcellulose-cysteine (CMC-cys) and alginate-cysteine (alg-cys). In vitro, the permeation enhancing properties of these thiomers and their corresponding unmodified polymers have been evaluated on rat small intestine in Ussing-type chambers, using sulforhodamine 101 (SR-101) as MRP2 model substrate. In comparison to buffer only, SR-101 transport in presence of pect-cys, CMC-cys and alg-cys was improved 1.5-fold, 1.8-fold and 3.0-fold, respectively. Due to the comparatively best in vitro performance of thiolated alginate, it has been chosen for in vivo studies: a SR-101 solution containing 4% (w/v) alg-cys led to an AUC0 ≥ 12 of SR-101 of 109 ng ml(-1)h in rats representing a 3.8-fold improvement in comparison to a SR-101 buffer solution. Unmodified alginate improved the AUC0 ≥ 12 of SR-101 by a factor of 1.9. These findings suggest thiolated alginate as promising auxiliary agent for drugs being anionic efflux pump substrates, since the oral bioavailability of a MRP2 substrate could be significantly improved. Copyright © 2015 Elsevier B.V. All rights reserved.

Efflux as a mechanism of antimicrobial drug resistance in clinical relevant microorganisms: the role of efflux inhibitors.

PubMed

Willers, Clarissa; Wentzel, Johannes Frederik; du Plessis, Lissinda Hester; Gouws, Chrisna; Hamman, Josias Hendrik

2017-01-01

Microbial resistance against antibiotics is a serious threat to the effective treatment of infectious diseases. Several mechanisms exist through which microorganisms can develop resistance against antimicrobial drugs, of which the overexpression of genes to produce efflux pumps is a major concern. Several efflux transporters have been identified in microorganisms, which infer resistance against specific antibiotics and even multidrug resistance. Areas covered: This paper focuses on microbial resistance against antibiotics by means of the mechanism of efflux and gives a critical overview of studies conducted to overcome this problem by combining efflux pump inhibitors with antibiotics. Information was obtained from a literature search done with MEDLINE, Pubmed, Scopus, ScienceDirect, OneSearch and EBSCO host. Expert opinion: Efflux as a mechanism of multidrug resistance has presented a platform for improved efficacy against resistant microorganisms by co-administration of efflux pump inhibitors with antimicrobial agents. Although proof of concept has been shown for this approach with in vitro experiments, further research is needed to develop more potent inhibitors with low toxicity which is clinically effective.

Amino acid amides of piperic acid (PA) and 4-ethylpiperic acid (EPA) as NorA efflux pump inhibitors of Staphylococcus aureus.

PubMed

Wani, Naiem Ahmad; Singh, Samsher; Farooq, Saleem; Shankar, Sudha; Koul, Surrinder; Khan, Inshad Ali; Rai, Rajkishor

2016-09-01

A total of eighteen piperic acid (PA) and 4-ethylpiperic acid (EPA) amides (C1-C18) with α-, β- and γ-amino acids were synthesized, characterized and evaluated for their efflux pump inhibitory activity against ciprofloxacin resistant Staphylococcus aureus. The amides were screened against NorA overexpressing S. aureus SA-1199B and wild type S. aureus SA-1199 using ethidium bromide as NorA efflux pump substrate. EPI C6 was found to be most potent and reduced the MIC of ciprofloxacin by 16 fold followed by C18 which showed 4 fold reduction of MIC. Ethidium bromide efflux inhibition and accumulation assay proved these compounds as NorA inhibitors. Copyright © 2016 Elsevier Ltd. All rights reserved.

Whole genome and transcriptome analyses of environmental antibiotic sensitive and multi-resistant Pseudomonas aeruginosa isolates exposed to waste water and tap water.

PubMed

Schwartz, Thomas; Armant, Olivier; Bretschneider, Nancy; Hahn, Alexander; Kirchen, Silke; Seifert, Martin; Dötsch, Andreas

2015-01-01

The fitness of sensitive and resistant Pseudomonas aeruginosa in different aquatic environments depends on genetic capacities and transcriptional regulation. Therefore, an antibiotic-sensitive isolate PA30 and a multi-resistant isolate PA49 originating from waste waters were compared via whole genome and transcriptome Illumina sequencing after exposure to municipal waste water and tap water. A number of different genomic islands (e.g. PAGIs, PAPIs) were identified in the two environmental isolates beside the highly conserved core genome. Exposure to tap water and waste water exhibited similar transcriptional impacts on several gene clusters (antibiotic and metal resistance, genetic mobile elements, efflux pumps) in both environmental P. aeruginosa isolates. The MexCD-OprJ efflux pump was overexpressed in PA49 in response to waste water. The expression of resistance genes, genetic mobile elements in PA49 was independent from the water matrix. Consistently, the antibiotic sensitive strain PA30 did not show any difference in expression of the intrinsic resistance determinants and genetic mobile elements. Thus, the exposure of both isolates to polluted waste water and oligotrophic tap water resulted in similar expression profiles of mentioned genes. However, changes in environmental milieus resulted in rather unspecific transcriptional responses than selected and stimuli-specific gene regulation. © 2014 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Structure of the periplasmic adaptor protein from a major facilitator superfamily (MFS) multidrug efflux pump.

PubMed

Hinchliffe, Philip; Greene, Nicholas P; Paterson, Neil G; Crow, Allister; Hughes, Colin; Koronakis, Vassilis

2014-08-25

Periplasmic adaptor proteins are key components of bacterial tripartite efflux pumps. The 2.85 Å resolution structure of an MFS (major facilitator superfamily) pump adaptor, Aquifex aeolicus EmrA, shows linearly arranged α-helical coiled-coil, lipoyl, and β-barrel domains, but lacks the fourth membrane-proximal domain shown in other pumps to interact with the inner membrane transporter. The adaptor α-hairpin, which binds outer membrane TolC, is exceptionally long at 127 Å, and the β-barrel contains a conserved disordered loop. The structure extends the view of adaptors as flexible, modular components that mediate diverse pump assembly, and suggests that in MFS tripartite pumps a hexamer of adaptors could provide a periplasmic seal. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

Dithiazole thione derivative as competitive NorA efflux pump inhibitor to curtail multi drug resistant clinical isolate of MRSA in a zebrafish infection model.

PubMed

Lowrence, Rene Christena; Raman, Thiagarajan; Makala, Himesh V; Ulaganathan, Venkatasubramanian; Subramaniapillai, Selva Ganesan; Kuppuswamy, Ashok Ayyappa; Mani, Anisha; Chittoor Neelakantan, Sundaresan; Nagarajan, Saisubramanian

2016-11-01

Multi drug resistant (MDR) pathogens pose a serious threat to public health since they can easily render most potent drugs ineffective. Efflux pump inhibitors (EPI) can be used to counter the MDR phenotypes arising due to increased efflux. In the present study, a series of dithiazole thione derivatives were synthesized and checked for its antibacterial and efflux pump inhibitory (EPI) activity. Among 10 dithiazole thione derivatives, real-time efflux studies revealed that seven compounds were potent EPIs relative to CCCP. Zebrafish toxicity studies identified four non-toxic putative EPIs. Both DTT3 and DTT9 perturbed membrane potential and DTT6 was haemolytic. Among DTT6 and DTT10, the latter was less toxic as evidenced by histopathology studies. Since DTT10 was non-haemolytic, did not affect the membrane potential, and was least toxic, it was chosen further for in vivo study, wherein DTT10 potentiated effect of ciprofloxacin against clinical strain of MRSA and reduced bacterial burden in muscle and skin tissue of infected zebrafish by ~ 1.7 and 2.5 log fold respectively. Gene expression profiling of major efflux transport proteins by qPCR revealed that clinical isolate of MRSA, in the absence of antibiotic, upregulated NorA, NorB and MepA pump, whereas it downregulates NorC and MgrA relative to wild-type strain of Staphylococcus aureus. In vitro studies with NorA mutant strains and substrate profiling revealed that at higher concentrations DTT10 is likely to function as a competitive inhibitor of NorA efflux protein in S. aureus, whereas at lower concentrations it might inhibit ciprofloxacin efflux through NorB and MepA as implied by docking studies. A novel non-toxic, non-haemolytic dithiazole thione derivative (DTT10) was identified as a potent competitive inhibitor of NorA efflux pump in S. aureus using in silico, in vitro and in vivo studies. This study also underscores the importance of using zebrafish infection model to screen and evaluate putative EPI for

The Impact of Efflux Pump Inhibitors on the Activity of Selected Non-Antibiotic Medicinal Products against Gram-Negative Bacteria.

PubMed

Laudy, Agnieszka E; Kulińska, Ewa; Tyski, Stefan

2017-01-11

The potential role of non-antibiotic medicinal products in the treatment of multidrug-resistant Gram-negative bacteria has recently been investigated. It is highly likely that the presence of efflux pumps may be one of the reasons for the weak activity of non-antibiotics, as in the case of some non-steroidal anti-inflammatory drugs (NSAIDs), against Gram-negative rods. The activity of eight drugs of potential non-antibiotic activity, active substance standards, and relevant medicinal products were analysed with and without of efflux pump inhibitors against 180 strains of five Gram-negative rod species by minimum inhibitory concentration (MIC) value determination in the presence of 1 mM MgSO₄. Furthermore, the influence of non-antibiotics on the susceptibility of clinical strains to quinolones with or without PAβN (Phe-Arg-β-naphthylamide) was investigated. The impacts of PAβN on the susceptibility of bacteria to non-antibiotics suggests that amitriptyline, alendronate, nicergoline, and ticlopidine are substrates of efflux pumps in Gram-negative rods. Amitriptyline/Amitriptylinum showed the highest direct antibacterial activity, with MICs ranging 100-800 mg/L against all studied species. Significant decreases in the MIC values of other active substances (acyclovir, atorvastatin, and famotidine) tested with pump inhibitors were not observed. The investigated non-antibiotic medicinal products did not alter the MICs of quinolones in the absence and in the presence of PAβN to the studied clinical strains of five groups of species.

The putative drug efflux systems of the Bacillus cereus group

PubMed Central

Elbourne, Liam D. H.; Vörös, Aniko; Kroeger, Jasmin K.; Simm, Roger; Tourasse, Nicolas J.; Finke, Sarah; Henderson, Peter J. F.; Økstad, Ole Andreas; Paulsen, Ian T.; Kolstø, Anne-Brit

2017-01-01

The Bacillus cereus group of bacteria includes seven closely related species, three of which, B. anthracis, B. cereus and B. thuringiensis, are pathogens of humans, animals and/or insects. Preliminary investigations into the transport capabilities of different bacterial lineages suggested that genes encoding putative efflux systems were unusually abundant in the B. cereus group compared to other bacteria. To explore the drug efflux potential of the B. cereus group all putative efflux systems were identified in the genomes of prototypical strains of B. cereus, B. anthracis and B. thuringiensis using our Transporter Automated Annotation Pipeline. More than 90 putative drug efflux systems were found within each of these strains, accounting for up to 2.7% of their protein coding potential. Comparative analyses demonstrated that the efflux systems are highly conserved between these species; 70–80% of the putative efflux pumps were shared between all three strains studied. Furthermore, 82% of the putative efflux system proteins encoded by the prototypical B. cereus strain ATCC 14579 (type strain) were found to be conserved in at least 80% of 169 B. cereus group strains that have high quality genome sequences available. However, only a handful of these efflux pumps have been functionally characterized. Deletion of individual efflux pump genes from B. cereus typically had little impact to drug resistance phenotypes or the general fitness of the strains, possibly because of the large numbers of alternative efflux systems that may have overlapping substrate specificities. Therefore, to gain insight into the possible transport functions of efflux systems in B. cereus, we undertook large-scale qRT-PCR analyses of efflux pump gene expression following drug shocks and other stress treatments. Clustering of gene expression changes identified several groups of similarly regulated systems that may have overlapping drug resistance functions. In this article we review current

The "racemic approach" in the evaluation of the enantiomeric NorA efflux pump inhibition activity of 2-phenylquinoline derivatives.

PubMed

Carotti, Andrea; Ianni, Federica; Sabatini, Stefano; Di Michele, Alessandro; Sardella, Roccaldo; Kaatz, Glenn W; Lindner, Wolfgang; Cecchetti, Violetta; Natalini, Benedetto

2016-09-10

Among the mechanisms adopted by bacteria, efflux pumps (EPs) have been recognized as being significantly involved in contributing to resistance to commonly used antibacterial agents. However, little is known about their three-dimensional structures or the steric requirements for their inhibition. Lack of such knowledge includes NorA, one of the most studied Staphylococcus aureus EPs. In the present study, the use of two commercialized Cinchona alkaloid-based zwitterionic chiral stationary phases allowed the enantioseparation of four 2-((2-(4-propoxyphenyl)quinolin-4-yl)oxy)alkylamines 1-4 previously found to be potent S. aureus NorA efflux pump inhibitors when tested as racemates. In the identified optimal polar-ionic conditions (MeOH/THF/H2O-49/49/2 (v/v/v)+25mM formic acid, 12.5mM diethylamine), repeated consecutive injections of 1 allowed the isolation of sufficient amount of its enantiomers (2.6mg and 2.8mg, for (R)-1 and (S)-1, respectively) and then to evaluate their ability to inhibit the S. aureus NorA efflux pump. The biological evaluation highlighted the main contribution of the (R)-1 enantiomer to both the EtBr efflux inhibition and synergistic effect with against SA-1199B (norA+/A116E GrlA) respect to the racemate activity. The comparison between the experimental electronic circular dichroism and the time-dependent density functional theory calculations spectra of the two isolated enantiomeric fractions allowed for all compounds a clear and easy assignment of the enantiomeric elution order. Copyright © 2016 Elsevier B.V. All rights reserved.

The efficiency of the heat pump water heater, during DHW tapping cycle

NASA Astrophysics Data System (ADS)

Gużda, Arkadiusz; Szmolke, Norbert

2017-10-01

This paper discusses one of the most effective systems for domestic hot water (DHW) production based on air-source heat pump with an integrated tank. The operating principle of the heat pump is described in detail. Moreover, there is an account of experimental set-up and results of the measurements. In the experimental part, measurements were conducted with the aim of determining the energy parameters and measures of the economic efficiency related to the presented solution. The measurements that were conducted are based on the tapping cycle that is similar to the recommended one in EN-16147 standard. The efficiency of the air source heat pump during the duration of the experiment was 2.43. In the end of paper, authors conducted a simplified ecological analysis in order to determine the influence of operation of air-source heat pump with integrated tank on the environment. Moreover the compression with the different source of energy (gas boiler with closed combustion chamber and boiler fired by the coal) was conducted. The heat pump is the ecological friendly source of the energy.

Boronic species as promising inhibitors of the Staphylococcus aureus NorA efflux pump: study of 6-substituted pyridine-3-boronic acid derivatives.

PubMed

Fontaine, Fanny; Héquet, Arnaud; Voisin-Chiret, Anne-Sophie; Bouillon, Alexandre; Lesnard, Aurélien; Cresteil, Thierry; Jolivalt, Claude; Rault, Sylvain

2015-05-05

In response to the extensive use of antibiotics, bacteria have evolved numerous mechanisms of defense against antimicrobial agents. Among them, extrusion of the antimicrobial agents outside the bacterial cell through efflux pumps is a major cause of concern. At first limited to one or few structurally-related antibiotics, bacterial resistance have then progressed towards cross-resistance between different classes of antibiotics, leading to multidrug-resistant microorganisms. Emergence of these pathogens requires development of novel therapeutic strategies and inhibition of efflux pumps appears to be a promising strategy that could restore the potency of existing antibiotics. NorA is the most studied chromosomal efflux pump of Staphylococcus aureus; it is known to be implied in resistance of Methicillin-resistant S. aureus (MRSA) strains against a wide range of unrelated substrates, including hydrophilic fluoroquinolones. Starting from 6-benzyloxypyridine-3-boronic acid I that we previously identified as a potential inhibitor of the NorA efflux pump against the NorA-overexpressing S. aureus 1199B strain (SA1199B), we describe here the synthesis and biological evaluation of a series of 6-(aryl)alkoxypyridine-3-boronic acids. 6-(3-Phenylpropoxy)pyridine-3-boronic acid 3i and 6-(4-phenylbutoxy)pyridine-3-boronic acid 3j were found to potentiate ciprofloxacin activity by a 4-fold increase compared to the parent compound I. In addition, it has been shown that both compounds promote Ethidium Bromide (EtBr) accumulation in SA1199B, thus corroborating their potential mode of action as NorA inhibitors. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Binding site feature description of 2-substituted benzothiazoles as potential AcrAB-TolC efflux pump inhibitors in E. coli.

PubMed

Yilmaz, S; Altinkanat-Gelmez, G; Bolelli, K; Guneser-Merdan, D; Ufuk Over-Hasdemir, M; Aki-Yalcin, E; Yalcin, I

2015-01-01

The resistance-nodulation-division (RND) family efflux pumps are important in the antibiotic resistance of Gram-negative bacteria. However, although a number of bacterial RND efflux pump inhibitors have been developed, there has been no clinically available RND efflux pump inhibitor to date. A set of BSN-coded 2-substituted benzothiazoles were tested alone and in combinations with ciprofloxacin (CIP) against the AcrAB-TolC overexpressor Escherichia coli AG102 clinical strain. The results indicated that the BSN compounds did not show intrinsic antimicrobial activity when tested alone. However, when used in combinations with CIP, a reversal in the antibacterial activity of CIP with up to 10-fold better MIC values was observed. In order to describe the binding site features of these BSN compounds with AcrB, docking studies were performed using the CDocker method. The performed docking poses and the calculated binding energy scores revealed that the tested compounds BSN-006, BSN-023, and BSN-004 showed significant binding interactions with the phenylalanine-rich region in the distal binding site of the AcrB binding monomer. Moreover, the tested compounds BSN-006 and BSN-023 possessed stronger binding energies than CIP, verifying that BSN compounds are acting as the putative substrates of AcrB.

Inhibition of the NorA efflux pump of Staphylococcus aureus by synthetic riparins.

PubMed

Costa, L M; de Macedo, E V; Oliveira, F A A; Ferreira, J H L; Gutierrez, S J C; Peláez, W J; Lima, F C A; de Siqueira Júnior, J P; Coutinho, H D M; Kaatz, G W; de Freitas, R M; Barreto, H M

2016-11-01

The goal of this study was to increase knowledge about the antimicrobial activity of some synthetic Riparin-derived compounds, alone or in combination with fluoroquinolone antibiotics, against a strain of Staphylococcus aureus resistant to fluoroquinolone by way of overexpression of the NorA efflux pump. Microdilution tests showed that Riparins A and B did not show any significant antibacterial activity against Staph. aureus strains. On the other hand, the intrinsic antibacterial activity increased with increasing lipophilicity of the compounds, in the following order: Riparin-D (MIC 256 μg ml -1 ; Log P 2·95) < Riparin-C (MIC 102 μg ml -1 ; Log P 3·22) < Riparin-E (MIC 16 μg ml -1 ; Log P 3·57). The addition of all riparins to growth media at subinhibitory concentrations caused an increase in the antibacterial activity of antibiotics against the NorA-overexpressing test strain. Riparin-B, which has two methoxyl groups at the phenethyl moiety, showed the best modulatory effect. Riparin-E is a good anti-staphylococci agent, while Riparin-B functions as a NorA efflux pump inhibitor. Our data suggest the possibility of using Riparin-B in combination with norfloxacin or ciprofloxacin for therapy of infections caused by multi-drug resistant Staph. aureus. © 2016 The Society for Applied Microbiology.

Re-evolution of the 2-phenylquinolines: ligand-based design, synthesis, and biological evaluation of a potent new class of Staphylococcus aureus NorA efflux pump inhibitors to combat antimicrobial resistance.

PubMed

Sabatini, Stefano; Gosetto, Francesca; Iraci, Nunzio; Barreca, Maria Letizia; Massari, Serena; Sancineto, Luca; Manfroni, Giuseppe; Tabarrini, Oriana; Dimovska, Mirjana; Kaatz, Glenn W; Cecchetti, Violetta

2013-06-27

Overexpression of efflux pumps is an important mechanism by which bacteria evade the effects of antimicrobial agents that are substrates. NorA is a Staphylococcus aureus efflux pump that confers reduced susceptibility to many structurally unrelated agents, including fluoroquinolones, biocides, and dyes, resulting in a multidrug resistant (MDR) phenotype. In this work, a series of 2-phenylquinoline derivatives was designed by means of ligand-based pharmacophore modeling in an attempt to identify improved S. aureus NorA efflux pump inhibitors (EPIs). Most of the 2-phenylquinoline derivatives displayed potent EPI activity against the norA overexpressing strain SA-1199B. The antibacterial activity of ciprofloxacin, when used in combination with some of the synthesized compounds, was completely restored in SA-1199B and SA-K2378, a strain overexpressing norA from a multicopy plasmid. Compounds 3m and 3q also showed potent synergistic activity with the ethidium bromide dye in a strain overexpressing the MepA MDR efflux pump.

Identification of Natural Compound Inhibitors for Multidrug Efflux Pumps of Escherichia coli and Pseudomonas aeruginosa Using In Silico High-Throughput Virtual Screening and In Vitro Validation

PubMed Central

Aparna, Vasudevan; Dineshkumar, Kesavan; Mohanalakshmi, Narasumani; Velmurugan, Devadasan; Hopper, Waheeta

2014-01-01

Pseudomonas aeruginosa and Escherichia coli are resistant to wide range of antibiotics rendering the treatment of infections very difficult. A main mechanism attributed to the resistance is the function of efflux pumps. MexAB-OprM and AcrAB-TolC are the tripartite efflux pump assemblies, responsible for multidrug resistance in P. aeruginosa and E. coli respectively. Substrates that are more susceptible for efflux are predicted to have a common pharmacophore feature map. In this study, a new criterion of excluding compounds with efflux substrate-like features was used, thereby refining the selection process and enriching the inhibitor identification process. An in-house database of phytochemicals was created and screened using high-throughput virtual screening against AcrB and MexB proteins and filtered by matching with the common pharmacophore models (AADHR, ADHNR, AAHNR, AADHN, AADNR, AAADN, AAADR, AAANR, AAAHN, AAADD and AAADH) generated using known efflux substrates. Phytochemical hits that matched with any one or more of the efflux substrate models were excluded from the study. Hits that do not have features similar to the efflux substrate models were docked using XP docking against the AcrB and MexB proteins. The best hits of the XP docking were validated by checkerboard synergy assay and ethidium bromide accumulation assay for their efflux inhibition potency. Lanatoside C and diadzein were filtered based on the synergistic potential and validated for their efflux inhibition potency using ethidium bromide accumulation study. These compounds exhibited the ability to increase the accumulation of ethidium bromide inside the bacterial cell as evidenced by these increase in fluorescence in the presence of the compounds. With this good correlation between in silico screening and positive efflux inhibitory activity in vitro, the two compounds, lanatoside C and diadzein could be promising efflux pump inhibitors and effective to use in combination therapy against drug

Identification of natural compound inhibitors for multidrug efflux pumps of Escherichia coli and Pseudomonas aeruginosa using in silico high-throughput virtual screening and in vitro validation.

PubMed

Aparna, Vasudevan; Dineshkumar, Kesavan; Mohanalakshmi, Narasumani; Velmurugan, Devadasan; Hopper, Waheeta

2014-01-01

Pseudomonas aeruginosa and Escherichia coli are resistant to wide range of antibiotics rendering the treatment of infections very difficult. A main mechanism attributed to the resistance is the function of efflux pumps. MexAB-OprM and AcrAB-TolC are the tripartite efflux pump assemblies, responsible for multidrug resistance in P. aeruginosa and E. coli respectively. Substrates that are more susceptible for efflux are predicted to have a common pharmacophore feature map. In this study, a new criterion of excluding compounds with efflux substrate-like features was used, thereby refining the selection process and enriching the inhibitor identification process. An in-house database of phytochemicals was created and screened using high-throughput virtual screening against AcrB and MexB proteins and filtered by matching with the common pharmacophore models (AADHR, ADHNR, AAHNR, AADHN, AADNR, AAADN, AAADR, AAANR, AAAHN, AAADD and AAADH) generated using known efflux substrates. Phytochemical hits that matched with any one or more of the efflux substrate models were excluded from the study. Hits that do not have features similar to the efflux substrate models were docked using XP docking against the AcrB and MexB proteins. The best hits of the XP docking were validated by checkerboard synergy assay and ethidium bromide accumulation assay for their efflux inhibition potency. Lanatoside C and diadzein were filtered based on the synergistic potential and validated for their efflux inhibition potency using ethidium bromide accumulation study. These compounds exhibited the ability to increase the accumulation of ethidium bromide inside the bacterial cell as evidenced by these increase in fluorescence in the presence of the compounds. With this good correlation between in silico screening and positive efflux inhibitory activity in vitro, the two compounds, lanatoside C and diadzein could be promising efflux pump inhibitors and effective to use in combination therapy against drug

Virtual screening for novel Staphylococcus Aureus NorA efflux pump inhibitors from natural products.

PubMed

Thai, Khac-Minh; Ngo, Trieu-Du; Phan, Thien-Vy; Tran, Thanh-Dao; Nguyen, Ngoc-Vinh; Nguyen, Thien-Hai; Le, Minh-Tri

2015-01-01

NorA is a member of the Major Facilitator Superfamily (MFS) drug efflux pumps that have been shown to mediate antibiotic resistance in Staphylococcus aureus (SA). In this study, QSAR analysis, virtual screening and molecular docking were implemented in an effort to discover novel SA NorA efflux pump inhibitors. Originally, a set of 47 structurally diverse compounds compiled from the literature was used to develop linear QSAR models and another set of 15 different compounds were chosen for extra validation. The final model which was estimated by statistical values for the full data set (n = 45, Q(2) = 0.80, RMSE = 0.20) and for the external test set (n = 15, R(2) = 0.60, |res|max = 0.75, |res|min = 0.02) was applied on the collection of 182 flavonoides and the traditional Chinese medicine (TCM) database to screen for novel NorA inhibitors. Finally, 33 lead compounds that met the Lipinski's rules of five/three and had good predicted pIC50 values from in silico screening process were employed to analyze the binding ability by docking studies on NorA homology model in place of its unavailable crystal structures at two active sites, the central channel and the Walker B.

Relevant role of efflux pumps in high levels of rifaximin resistance in Escherichia coli clinical isolates.

PubMed

Gomes, C; Ruiz, L; Pons, M J; Ochoa, T J; Ruiz, J

2013-09-01

Enteropathogens have shown a high level of resistance against commonly used antibacterial drugs in Peru and it is necessary to explore alternative treatments. The aim of this study was to analyse the in vitro activity of rifaximin against diarrhoeagenic and commensal Escherichia coli in children less than 2 years of age. The minimal inhibitory concentration (MIC) to rifampicin and rifaximin was determined for 210 strains in the presence and absence of phenyl-arginine-β-naphthylamide (PAβN) and the mechanisms of resistance were investigated. The MIC levels ranged between 8 and >256 mg/litre and the predominant mechanism of resistance to rifaximin was the efflux pumps inhibited by PAβN in 95.2% of the isolates. The present MIC values are higher than those observed in other studies. Efflux pumps inhibited by PAβN were the cause of the rifaximin resistance in the majority of cases and suggest the presence of an environmental selective pressure. Consequently, rifaximin should be used with caution in the treatment of diarrhoea in Peru.

Expression of Sme efflux pumps and multilocus sequence typing in clinical isolates of Stenotrophomonas maltophilia.

PubMed

Cho, Hye Hyun; Sung, Ji Youn; Kwon, Kye Chul; Koo, Sun Hoe

2012-01-01

Stenotrophomonas maltophilia has emerged as an important opportunistic pathogen, which causes infections that are often difficult to manage because of the inherent resistance of the pathogen to a variety of antimicrobial agents. In this study, we analyzed the expressions of smeABC and smeDEF and their correlation with antimicrobial susceptibility. We also evaluated the genetic relatedness and epidemiological links among 33 isolates of S. maltophilia. In total, 33 S. maltophilia strains were isolated from patients in a tertiary hospital in Daejeon. Minimum inhibitory concentrations (MICs) of 11 antimicrobial agents were determined by using agar dilution method and E-test (BioMérieux, France). Real-time PCR analysis was performed to evaluate the expression of the Sme efflux systems in the S. maltophilia isolates. Additionally, an epidemiological investigation was performed using multilocus sequence typing (MLST) assays. The findings of susceptibility testing showed that the majority of the S. maltophilia isolates were resistant to β-lactams and aminoglycosides. Twenty-one clinical isolates overexpressed smeABC and showed high resistance to ciprofloxacin. Moreover, a high degree of genetic diversity was observed among the S. maltophilia isolates; 3 sequence types (STs) and 23 allelic profiles were observed. The smeABC efflux pump was associated with multidrug resistance in clinical isolates of S. maltophilia. In particular, smeABC efflux pumps appear to perform an important role in ciprofloxacin resistance of S. maltophilia. The MLST scheme for S. maltophilia represents a discriminatory typing method with stable markers and is appropriate for studying population structures.

Response to Bile Salts in Clinical Strains of Acinetobacter baumannii Lacking the AdeABC Efflux Pump: Virulence Associated with Quorum Sensing

PubMed Central

López, Maria; Blasco, Lucia; Gato, Eva; Perez, Astrid; Fernández-Garcia, Laura; Martínez-Martinez, Luis; Fernández-Cuenca, Felipe; Rodríguez-Baño, Jesús; Pascual, Alvaro; Bou, German; Tomás, Maria

2017-01-01

Introduction: Acinetobacter baumannii is an opportunistic nosocomial pathogen associated with multiple infections. This pathogen usually colonizes (first stage of microbial infection) host tissues that are in contact with the external environment. As one of the sites of entry in human hosts is the gastrointestinal tract, the pathogen must be capable of tolerating bile salts. However, studies analyzing the molecular characteristics involved in the response to bile salts in clinical strains of A. baumannii are scarce. Material and Methods: Microbiological and transcriptional studies (arrays and RT-PCR) in the response to bile salts were carried out in isogenic (A. baumanni ΔadeB ATCC 17978 and A. baumannii ΔadeL ATCC 17978) and clinical strains from clone ST79/PFGE-HUI-1 which is characterized by lacking the AdeABC efflux pump and by overexpression the AdeFGH efflux pump. Results and Discussion: In presence of bile salts, in addition to the glutamate/aspartate transporter were found overexpressed in A. baumannii ΔadeB ATCC 17978, the virulence factors (surface motility, biofilm, and Type VI Secretion System) which are associated with activation of the Quorum Sensing system. Overexpression of these factors was confirmed in clinical strains of clone ST79/PFGE-HUI-1. Conclusions: This the first study about the adaptive response to bile salts investigating the molecular and microbiological characteristics in response to bile salts of an isogenic model of A. baumannii ATCC 17978 and clinical isolates of A. baumannii (clinical strains of ST79/PFGE-HUI-1) lacking the main RND efflux pump (AdeABC). Clinical isolates of A. baumannii lacking the AdeABC efflux pump (clone ST79/PFGE-HUI-1) displayed a new clinical profile (increased invasiveness) possibly associated with the response to stress conditions (such as the presence of bile salts). PMID:28536672

Multidrug Efflux Systems in Microaerobic and Anaerobic Bacteria

PubMed Central

Xu, Zeling; Yan, Aixin

2015-01-01

Active drug efflux constitutes an important mechanism of antibiotic and multidrug resistance in bacteria. Understanding the distribution, expression, and physiological functions of multidrug efflux pumps, especially under physiologically and clinically relevant conditions of the pathogens, is the key to combat drug resistance. In animal hosts, most wounded, infected and inflamed tissues display low oxygen tensions. In this article, we summarize research development on multidrug efflux pumps in the medicinally relevant microaerobic and anaerobic pathogens and their implications in the effort to combat drug-resistant infections. PMID:27025630

The Challenge of Efflux-Mediated Antibiotic Resistance in Gram-Negative Bacteria

PubMed Central

Plésiat, Patrick

2015-01-01

SUMMARY The global emergence of multidrug-resistant Gram-negative bacteria is a growing threat to antibiotic therapy. The chromosomally encoded drug efflux mechanisms that are ubiquitous in these bacteria greatly contribute to antibiotic resistance and present a major challenge for antibiotic development. Multidrug pumps, particularly those represented by the clinically relevant AcrAB-TolC and Mex pumps of the resistance-nodulation-division (RND) superfamily, not only mediate intrinsic and acquired multidrug resistance (MDR) but also are involved in other functions, including the bacterial stress response and pathogenicity. Additionally, efflux pumps interact synergistically with other resistance mechanisms (e.g., with the outer membrane permeability barrier) to increase resistance levels. Since the discovery of RND pumps in the early 1990s, remarkable scientific and technological advances have allowed for an in-depth understanding of the structural and biochemical basis, substrate profiles, molecular regulation, and inhibition of MDR pumps. However, the development of clinically useful efflux pump inhibitors and/or new antibiotics that can bypass pump effects continues to be a challenge. Plasmid-borne efflux pump genes (including those for RND pumps) have increasingly been identified. This article highlights the recent progress obtained for organisms of clinical significance, together with methodological considerations for the characterization of MDR pumps. PMID:25788514

Water-mediated interactions enable smooth substrate transport in a bacterial efflux pump.

PubMed

Vargiu, Attilio Vittorio; Ramaswamy, Venkata Krishnan; Malvacio, Ivana; Malloci, Giuliano; Kleinekathöfer, Ulrich; Ruggerone, Paolo

2018-04-01

Efflux pumps of the Resistance-Nodulation-cell Division superfamily confer multi-drug resistance to Gram-negative bacteria. The most-studied polyspecific transporter belonging to this class is the inner-membrane trimeric antiporter AcrB of Escherichia coli. In previous studies, a functional rotation mechanism was proposed for its functioning, according to which the three monomers undergo concerted conformational changes facilitating the extrusion of substrates. However, the molecular determinants and the energetics of this mechanism still remain unknown, so its feasibility must be proven mechanistically. A computational protocol able to mimic the functional rotation mechanism in AcrB was developed. By using multi-bias molecular dynamics simulations we characterized the translocation of the substrate doxorubicin driven by conformational changes of the protein. In addition, we estimated for the first time the free energy profile associated to this process. We provided a molecular view of the process in agreement with experimental data. Moreover, we showed that the conformational changes occurring in AcrB enable the formation of a layer of structured waters on the internal surface of the transport channel. This water layer, in turn, allows for a fairly constant hydration of the substrate, facilitating its diffusion over a smooth free energy profile. Our findings reveal a new molecular mechanism of polyspecific transport whereby water contributes by screening potentially strong substrate-protein interactions. We provided a mechanistic understanding of a fundamental process related to multi-drug transport. Our results can help rationalizing the behavior of other polyspecific transporters and designing compounds avoiding extrusion or inhibitors of efflux pumps. Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.

Regulation of the aceI multidrug efflux pump gene in Acinetobacter baumannii.

PubMed

Liu, Qi; Hassan, Karl A; Ashwood, Heather E; Gamage, Hasinika K A H; Li, Liping; Mabbutt, Bridget C; Paulsen, Ian T

2018-06-01

To investigate the function of AceR, a putative transcriptional regulator of the chlorhexidine efflux pump gene aceI in Acinetobacter baumannii. Chlorhexidine susceptibility and chlorhexidine induction of aceI gene expression were determined by MIC and quantitative real-time PCR, respectively, in A. baumannii WT and ΔaceR mutant strains. Recombinant AceR was prepared as both a full-length protein and as a truncated protein, AceR (86-299), i.e. AceRt, which has the DNA-binding domain deleted. The binding interaction of the purified AceR protein and its putative operator region was investigated by electrophoretic mobility shift assays and DNase I footprinting assays. The binding of AceRt with its putative ligand chlorhexidine was examined using surface plasmon resonance and tryptophan fluorescence quenching assays. MIC determination assays indicated that the ΔaceI and ΔaceR mutant strains both showed lower resistance to chlorhexidine than the parental strain. Chlorhexidine-induced expression of aceI was abolished in a ΔaceR background. Electrophoretic mobility shift assays and DNase I footprinting assays demonstrated chlorhexidine-stimulated binding of AceR with two sites upstream of the putative aceI promoter. Surface plasmon resonance and tryptophan fluorescence quenching assays suggested that the purified ligand-binding domain of the AceR protein was able to bind with chlorhexidine with high affinity. This study provides strong evidence that AceR is an activator of aceI gene expression when challenged with chlorhexidine. This study is the first characterization, to our knowledge, of a regulator controlling expression of a PACE family multidrug efflux pump.

Hyper Accumulation of Arsenic in Mutants of Ochrobactrum tritici Silenced for Arsenite Efflux Pumps

PubMed Central

Piedade, Ana Paula; Morais, Paula V.

2015-01-01

Ochrobactrum tritici SCII24T is a highly As-resistant bacterium, with two previously described arsenic resistance operons, ars1 and ars2. Among a large number of genes, these operons contain the arsB and Acr3 genes that encode the arsenite efflux pumps responsible for arsenic resistance. Exploring the genome of O. tritici SCII24T, an additional putative operon (ars3) was identified and revealed the presence of the Acr3_2 gene that encodes for an arsenite efflux protein but which came to prove to not be required for full As resistance. The genes encoding for arsenite efflux pumps, identified in this strain, were inactivated to develop microbial accumulators of arsenic as new tools for bioremediation. Six different mutants were produced, studied and three were more useful as biotools. O. tritici wild type and the Acr3-mutants showed the highest resistance to As(III), being able to grow up to 50 mM of arsenite. On the other hand, arsB-mutants were not able to grow at concentrations higher than 1 mM As(III), and were the most As(III) sensitive mutants. In the presence of 1 mM As(III), the strain with arsB and Acr3_1 mutated showed the highest intracellular arsenic concentration (up to 17 ng(As)/mg protein), while in assays with 5 mM As(III), the single arsB-mutant was able to accumulate the highest concentration of arsenic (up to 10 ng(As)/mg protein). Therefore, arsB is the main gene responsible for arsenite resistance in O. tritici. However, both genes arsB and Acr3_1 play a crucial role in the resistance mechanism, depending on the arsenite concentration in the medium. In conclusion, at moderate arsenite concentrations, the double arsB- and Acr3_1-mutant exhibited a great ability to accumulate arsenite and can be seen as a promising bioremediation tool for environmental arsenic detoxification. PMID:26132104

In vitro transport activity of the fully assembled MexAB-OprM efflux pump from Pseudomonas aeruginosa

NASA Astrophysics Data System (ADS)

Verchère, Alice; Dezi, Manuela; Adrien, Vladimir; Broutin, Isabelle; Picard, Martin

2015-04-01

Antibiotic resistance is a major public health issue and many bacteria responsible for human infections have now developed a variety of antibiotic resistance mechanisms. For instance, Pseudomonas aeruginosa, a disease-causing Gram-negative bacteria, is now resistant to almost every class of antibiotics. Much of this resistance is attributable to multidrug efflux pumps, which are tripartite membrane protein complexes that span both membranes and actively expel antibiotics. Here we report an in vitro procedure to monitor transport by the tripartite MexAB-OprM pump. By combining proteoliposomes containing the MexAB and OprM portions of the complex, we are able to assay energy-dependent substrate translocation in a system that mimics the dual-membrane architecture of Gram-negative bacteria. This assay facilitates the study of pump transport dynamics and could be used to screen pump inhibitors with potential clinical use in restoring therapeutic activity of old antibiotics.

Role of the cell envelope stress regulators BaeR and CpxR in control of RND-type multidrug efflux pumps and transcriptional cross talk with exopolysaccharide synthesis in Erwinia amylovora.

PubMed

Pletzer, Daniel; Stahl, Antje; Oja, Anna Elisabeth; Weingart, Helge

2015-08-01

The purpose of this study was to identify the role of the cell envelope stress-sensing systems BaeSR and CpxARP in regulation of multidrug efflux and exopolysaccharide synthesis in Erwinia amylovora. We have previously reported that BaeR activates transcription of the RND-type efflux pumps AcrD and MdtABC. In this study, we found that a cpxR-deficient mutant was highly susceptible to β-lactams, aminoglycosides and lincomycin, whereas a baeR mutant showed no change in antimicrobial sensitivity. However, overexpression of BaeR in a mutant lacking the major RND pump AcrB increased resistance of E. amylovora to several compounds that are not substrates of AcrD or MdtABC. Furthermore, we observed that overexpression of BaeR significantly increased amylovoran production. Moreover, the expression of RND-type efflux pumps was changed in regulatory mutants of exopolysaccharide production. Our data suggest that BaeSR and CpxARP regulate additional mechanisms, beside efflux, which are responsible for antimicrobial resistance of E. amylovora.

Metabolic Compensation of Fitness Costs Is a General Outcome for Antibiotic-Resistant Pseudomonas aeruginosa Mutants Overexpressing Efflux Pumps.

PubMed

Olivares Pacheco, Jorge; Alvarez-Ortega, Carolina; Alcalde Rico, Manuel; Martínez, José Luis

2017-07-25

It is generally assumed that the acquisition of antibiotic resistance is associated with a fitness cost. We have shown that overexpression of the MexEF-OprN efflux pump does not decrease the fitness of a resistant Pseudomonas aeruginosa strain compared to its wild-type counterpart. This lack of fitness cost was associated with a metabolic rewiring that includes increased expression of the anaerobic nitrate respiratory chain when cells are growing under fully aerobic conditions. It was not clear whether this metabolic compensation was exclusive to strains overexpressing MexEF-OprN or if it extended to other resistant strains that overexpress similar systems. To answer this question, we studied a set of P. aeruginosa mutants that independently overexpress the MexAB-OprM, MexCD-OprJ, or MexXY efflux pumps. We observed increased expression of the anaerobic nitrate respiratory chain in all cases, with a concomitant increase in NO 3 consumption and NO production. These efflux pumps are proton/substrate antiporters, and their overexpression may lead to intracellular H + accumulation, which may in turn offset the pH homeostasis. Indeed, all studied mutants showed a decrease in intracellular pH under anaerobic conditions. The fastest way to eliminate the excess of protons is by increasing oxygen consumption, a feature also displayed by all analyzed mutants. Taken together, our results support metabolic rewiring as a general mechanism to avoid the fitness costs derived from overexpression of P. aeruginosa multidrug efflux pumps. The development of drugs that block this metabolic "reaccommodation" might help in reducing the persistence and spread of antibiotic resistance elements among bacterial populations. IMPORTANCE It is widely accepted that the acquisition of resistance confers a fitness cost in such a way that in the absence of antibiotics, resistant populations will be outcompeted by susceptible ones. Based on this assumption, antibiotic cycling regimes have been

Development of an accumulation assay and evaluation of the effects of efflux pump inhibitors on the retention of chlorhexidine digluconate in Pseudomonas aeruginosa and Staphylococcus aureus.

PubMed

Mombeshora, Molly; Mukanganyama, Stanley

2017-07-26

Chlorhexidine digluconate (CHG) is used as a disinfectant. The emergence of pathogens resistant to the biocide raises health concern. Information on specific efflux mechanisms utilised by bacteria to confer reduced susceptibility to the biocide, may be used to develop ways of preventing the efflux of the biocide from nosocomial pathogens resulting in higher disinfection activity. The aim of the study was to evaluate the role of ATP-binding cassette transporters on the transport of CHG in bacteria. Clinical strains of Pseudomonas aeruginosa, Staphylococcus aureus and their respective laboratory strains ATCC 27853 and ATCC 9144 were used for susceptibility tests. The minimum inhibitory concentration (MIC) of CHG with or without an efflux pump inhibitor [reserpine or carbonyl cyanide m-chlorophenylhydrazone (CCCP)] was determined using the broth microdilution method. A spectrophotometric method to quantify the amount of chlorhexidine in a sample was developed, validated and used to quantify CHG within P. aeruginosa and S. aureus cells. In the presence of reserpine, the MIC of CHG against the clinical strains of P. aeruginosa and S. aureus decreased from 6.3 to 3.2 µg/ml but showed no change against both ATCC isolates. The MIC of CHG in the presence of CCCP for both strains of P. aeruginosa remained unchanged but showed a reduction for both isolates of S. aureus. The suitability of the spectrophotometric method developed for quantifying the amount of CHG accumulated in microbial cells was validated and used successfully to quantify CHG accumulated within bacterial cells. The spectrophotometric determination of CHG within microbial cells may be used to quantify CHG in microbial cells. Only the clinical strain of P. aeruginosa showed significant efflux of CHG suggesting the participation of efflux transporters in the pumping out of CHG from this isolate. The use of efflux pump inhibitors together with the biocide may be explored to preventing the efflux of the biocide

Attaching NorA efflux pump inhibitors to methylene blue enhances antimicrobial photodynamic inactivation of Escherichia coli and Acinetobacter baumannii in vitro and in vivo.

PubMed

Rineh, Ardeshir; Bremner, John B; Hamblin, Michael R; Ball, Anthony R; Tegos, George P; Kelso, Michael J

2018-02-24

Resistance of bacteria to antibiotics is a public health concern worldwide due to the increasing failure of standard antibiotic therapies. Antimicrobial photodynamic inactivation (aPDI) is a promising non-antibiotic alternative for treating localized bacterial infections that uses non-toxic photosensitizers and harmless visible light to produce reactive oxygen species and kill microbes. Phenothiazinium photosensitizers like methylene blue (MB) and toluidine blue O are hydrophobic cations that are naturally expelled from bacterial cells by multidrug efflux pumps, which reduces their effectiveness. We recently reported the discovery of a NorA efflux pump inhibitor-methylene blue (EPI-MB) hybrid compound INF55-(Ac)en-MB that shows enhanced photodynamic inactivation of the Gram-positive bacterium methicillin-resistant Staphylococcus aureus (MRSA) relative to MB, both in vitro and in vivo. Here, we report the surprising observation that INF55-(Ac)en-MB and two related hybrids bearing the NorA efflux pump inhibitors INF55 and INF271 also show enhanced aPDI activity in vitro (relative to MB) against the Gram-negative bacteria Escherichia coli and Acinetobacter baumannii, despite neither species expressing the NorA pump. Two of the hybrids showed superior effects to MB in murine aPDI infection models. The findings motivate wider exploration of aPDI with EPI-MB hybrids against Gram-negative pathogens and more detailed studies into the molecular mechanisms underpinning their activity. Copyright © 2018 Elsevier Ltd. All rights reserved.

Kinetic Parameters of Efflux of Penicillins by the Multidrug Efflux Transporter AcrAB-TolC of Escherichia coli▿

PubMed Central

Lim, Siew Ping; Nikaido, Hiroshi

2010-01-01

The multidrug efflux transporter AcrAB-TolC is known to pump out a diverse range of antibiotics, including β-lactams. However, the kinetic constants of the efflux process, needed for the quantitative understanding of resistance, were not available until those accompanying the efflux of some cephalosporins were recently determined by combining efflux with the hydrolysis of drugs by the periplasmic β-lactamase. In the present study we extended this approach to the study of a wide range of penicillins, from ampicillin and penicillin V to ureidopenicillins and isoxazolylpenicillins, by combining efflux with hydrolysis with the OXA-7 penicillinase. We found that the penicillins had a much stronger apparent affinity to AcrB and higher maximum rates of efflux than the cephalosporins. All penicillins showed strong positive cooperativity kinetics for export. The kinetic constants obtained were validated, as the MICs theoretically predicted on the basis of efflux and hydrolysis kinetics were remarkably similar to the observed MICs (except for the isoxazolylpenicillins). Surprisingly, however, the efflux kinetics of cloxacillin, for example, whose MIC decreased 512-fold in Escherichia coli upon the genetic deletion of the acrB gene, were quite similar to those of ampicillin, whose MIC decreased only 2-fold with the same treatment. Analysis of this phenomenon showed that the extensive decrease in the MIC for the acrB mutant is primarily due to the low permeation of the drug and that comparison of the MICs between the parent and the acrB strains is a very poor measure of the ability of AcrB to pump a drug out. PMID:20160052

1-(1H-indol-3-yl)ethanamine derivatives as potent Staphylococcus aureus NorA efflux pump inhibitors.

PubMed

Hequet, Arnaud; Burchak, Olga N; Jeanty, Matthieu; Guinchard, Xavier; Le Pihive, Emmanuelle; Maigre, Laure; Bouhours, Pascale; Schneider, Dominique; Maurin, Max; Paris, Jean-Marc; Denis, Jean-Noël; Jolivalt, Claude

2014-07-01

The synthesis of 37 1-(1H-indol-3-yl)ethanamine derivatives, including 12 new compounds, was achieved through a series of simple and efficient chemical modifications. These indole derivatives displayed modest or no intrinsic anti-staphylococcal activity. By contrast, several of the compounds restored, in a concentration-dependent manner, the antibacterial activity of ciprofloxacin against Staphylococcus aureus strains that were resistant to fluoroquinolones due to overexpression of the NorA efflux pump. Structure-activity relationships studies revealed that the indolic aldonitrones halogenated at position 5 of the indole core were the most efficient inhibitors of the S. aureus NorA efflux pump. Among the compounds, (Z)-N-benzylidene-2-(tert-butoxycarbonylamino)-1-(5-iodo-1H-indol-3-yl)ethanamine oxide led to a fourfold decrease of the ciprofloxacin minimum inhibitory concentration against the SA-1199B strain when used at a concentration of 0.5 mg L(-1) . To the best of our knowledge, this activity is the highest reported to date for an indolic NorA inhibitor. In addition, a new antibacterial compound, tert-butyl (2-(3-hydroxyureido)-2-(1H-indol-3-yl)ethyl)carbamate, which is not toxic for human cells, was also found. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Gene expression analysis of the SdeAB multidrug efflux pump in antibiotic-resistant clinical isolates of Serratia marcescens.

PubMed

Dalvi, S D; Worobec, E A

2012-01-01

Many isolates of Serratia marcescens, a well-known opportunistic pathogen, can be multidrug resistant. Fluoroquinolones are among the most important groups of antibiotics used for treatment of these organisms. However, fluoroquinolone resistance among S. marcescens isolates is fast increasing. Drug extrusion through efflux pumps like SdeAB/ HasF is one of the major mechanisms of resistance to fluoroquinolones. This study was carried out to analyze, through gene expression analysis of sdeB, the relative contribution of this mechanism toward fluoroquinolone resistance in clinical isolates of Serratia. Total RNA from 45 clinical isolates of S. marcescens was isolated. Quantitative real-time RT PCR was performed on the extracted RNA to study the gene expression of sdeB and was normalized to the sdeB expression in the standard strain of S. marcescens. Of the 45 isolates analyzed, sdeB expression was found to be elevated in 20 isolates (44%). Of these 20 isolates, eight (40%) were fully resistant to at least one of the fluoroquinolones studied. Conversely, of the 20 isolates that over-expressed sdeB, 12 (60%) were fully sensitive to all fluoroquinolones tested. Drug efflux pumps are an important means of fluoroquinolone resistance among clinically important species of Serratia. The expression of these pumps can be up-regulated in the presence of antibiotics and have the potential for changing the phenotype from sensitive to resistant, thus contributing to therapeutic failures.

Inhibition of the essential oil from Chenopodium ambrosioides L. and α-terpinene on the NorA efflux-pump of Staphylococcus aureus.

PubMed

de Morais Oliveira-Tintino, Cícera Datiane; Tintino, Saulo Relison; Limaverde, Paulo W; Figueredo, Fernando G; Campina, Fábia F; da Cunha, Francisco A B; da Costa, Roger H S; Pereira, Pedro Silvino; Lima, Luciene F; de Matos, Yedda M L S; Coutinho, Henrique Douglas Melo; Siqueira-Júnior, José P; Balbino, Valdir Q; da Silva, Teresinha Gonçalves

2018-10-01

This study was carried out to test the essential oil from C. ambrosioides leaves and its main constituent, α-Terpinene, in an antibacterial activity assay. As well, it was evaluated ability reduce resistance to norfloxacin and ethidium bromide was compared the Staphylococcus aureus 1199B whith 1199 wild type strain. The MIC of the C. ambrosioides essential oil and α-Terpinene were determined by microdilution method. The MIC of the essential oil and α-Terpinene presented a value ≥ 1024 μg/mL. However, when associated with antibacterials, the essential oil from C. ambrosioides leaves significantly reduced the MIC of antibiotics and ethidium bromide, characterizing an efflux pump inhibition. The C. ambrosioides essential oil, despite having no direct antibacterial activity against the S. aureus 1199B strain, showed a potentiating action when associated with antibacterial agents, this being attributed to an inhibition of efflux pumps. Copyright © 2018 Elsevier Ltd. All rights reserved.

Modulation of the multidrug efflux pump EmrD-3 from Vibrio cholerae by Allium sativum extract and the bioactive agent allyl sulfide plus synergistic enhancement of antimicrobial susceptibility by A. sativum extract.

PubMed

Bruns, Merissa M; Kakarla, Prathusha; Floyd, Jared T; Mukherjee, Mun Mun; Ponce, Robert C; Garcia, John A; Ranaweera, Indrika; Sanford, Leslie M; Hernandez, Alberto J; Willmon, T Mark; Tolson, Grace L; Varela, Manuel F

2017-10-01

The causative agent of cholera, Vibrio cholerae, is a public health concern. Multidrug-resistant V. cholerae variants may reduce chemotherapeutic efficacies of severe cholera. We previously reported that the multidrug efflux pump EmrD-3 from V. cholerae confers resistance to multiple structurally distinct antimicrobials. Medicinal plant compounds are potential candidates for EmrD-3 efflux pump modulation. The antibacterial activities of garlic Allium sativum, although poorly understood, predicts that a main bioactive component, allyl sulfide, modulates EmrD-3 efflux. Thus, we tested whether A. sativum extract acts in synergy with antimicrobials and that a main bioactive component allyl sulfide inhibits EmrD-3 efflux. We found that A. sativum extract and allyl sulfide inhibited ethidium bromide efflux in cells harboring EmrD-3 and that A. sativum lowered the MICs of multiple antibacterials. We conclude that A. sativum and allyl sulfide inhibit EmrD-3 and that A. sativum extract synergistically enhances antibacterial agents.

Efflux drug transporters at the forefront of antimicrobial resistance.

PubMed

Rahman, Tahmina; Yarnall, Benjamin; Doyle, Declan A

2017-10-01

Bacterial antibiotic resistance is rapidly becoming a major world health consideration. To combat antibiotics, microorganisms employ their pre-existing defence mechanisms that existed long before man's discovery of antibiotics. Bacteria utilise levels of protection that range from gene upregulation, mutations, adaptive resistance, and production of resistant phenotypes (persisters) to communal behaviour, as in swarming and the ultimate defence of a biofilm. A major part of all of these responses involves the use of antibiotic efflux transporters. At the single cell level, it is becoming apparent that the use of efflux pumps is the first line of defence against an antibiotic, as these pumps decrease the intracellular level of antibiotic while the cell activates the various other levels of protection. This frontline of defence involves a coordinated network of efflux transporters. In the future, inhibition of this efflux transporter network, as a target for novel antibiotic therapy, will require the isolation and then biochemical/biophysical characterisation of each pump against all known and new antibiotics. This depth of knowledge is required so that we can fully understand and tackle the mechanisms of developing antimicrobial resistance.

Improved Potency of Indole-Based NorA Efflux Pump Inhibitors: From Serendipity toward Rational Design and Development.

PubMed

Buonerba, Federica; Lepri, Susan; Goracci, Laura; Schindler, Bryan D; Seo, Susan M; Kaatz, Glenn W; Cruciani, Gabriele

2017-01-12

The NorA efflux pump is a potential drug target for reversal of resistance to selected antibacterial agents, and recently we described indole-based inhibitor candidates. Herein we report a second class of inhibitors derived from them but with significant differences in shape and size. In particular, compounds 13 and 14 are very potent inhibitors in that they demonstrated the lowest IC 50 values (2 μM) ever observed among all indole-based compounds we have evaluated.

Clonality, outer-membrane proteins profile and efflux pump in KPC- producing Enterobacter sp. in Brazil.

PubMed

Rosa, Juliana Ferraz; Rizek, Camila; Marchi, Ana Paula; Guimaraes, Thais; Miranda, Lourdes; Carrilho, Claudia; Levin, Anna S; Costa, Silvia F

2017-03-17

Carbapenems resistance in Enterobacter spp. has increased in the last decade, few studies, however, described the mechanisms of resistance in this bacterium. This study evaluated clonality and mechanisms of carbapenems resistance in clinical isolates of Enterobacter spp. identified in three hospitals in Brazil (Hospital A, B and C) over 7-year. Antibiotics sensitivity, pulsed-field gel electrophoresis (PFGE), PCR for carbapenemase and efflux pump genes were performed for all carbapenems-resistant isolates. Outer-membrane protein (OMP) was evaluated based on PFGE profile. A total of 130 isolates of Enterobacter spp were analyzed, 44/105 (41, 9%) E. aerogenes and 8/25 (32,0%) E. cloacae were resistant to carbapenems. All isolates were susceptible to fosfomycin, polymyxin B and tigecycline. KPC was present in 88.6% of E. aerogenes and in all E. cloacae resistant to carbapenems. The carbapenems-resistant E. aerogenes identified in hospital A belonged to six clones, however, a predominant clone was identified in this hospital over the study period. There is a predominant clone in Hospital B and Hospital C as well. The mechanisms of resistance to carbapenems differ among subtypes. Most of the isolates co-harbored blaKPC, blaTEM and /or blaCTX associated with decreased or lost of 35-36KDa and or 39 KDa OMP. The efflux pump AcrAB-TolC gene was only identified in carbapenems-resistant E. cloacae. There was a predominant clone in each hospital suggesting that cross-transmission of carbapenems-resistant Enterobacter spp. was frequent. The isolates presented multiple mechanisms of resistance to carbapenems including OMP alteration.

Impact of anatase and rutile titanium dioxide nanoparticles on uptake carriers and efflux pumps in Caco-2 gut epithelial cells

NASA Astrophysics Data System (ADS)

Dorier, M.; Brun, E.; Veronesi, G.; Barreau, F.; Pernet-Gallay, K.; Desvergne, C.; Rabilloud, T.; Carapito, C.; Herlin-Boime, N.; Carrière, M.

2015-04-01

TiO2 microparticles are widely used in food products, where they are added as a white food colouring agent. This food additive contains a significant amount of nanoscale particles; still the impact of TiO2 nanoparticles (TiO2-NPs) on gut cells is poorly documented. Our study aimed at evaluating the impact of rutile and anatase TiO2-NPs on the main functions of enterocytes, i.e. nutrient absorption driven by solute-liquid carriers (SLC transporters) and protection against other xenobiotics driven by efflux pumps from the ATP-binding cassette (ABC) family. We show that acute exposure of Caco-2 cells to both anatase (12 nm) and rutile (20 nm) TiO2-NPs induce early upregulation of a battery of efflux pumps and nutrient transporters. In addition they cause overproduction of reactive oxygen species and misbalance redox repair systems, without inducing cell mortality or DNA damage. Taken together, these data suggest that TiO2-NPs may increase the functionality of gut epithelial cells, particularly their property to form a protective barrier against exogenous toxicants and to absorb nutrients.TiO2 microparticles are widely used in food products, where they are added as a white food colouring agent. This food additive contains a significant amount of nanoscale particles; still the impact of TiO2 nanoparticles (TiO2-NPs) on gut cells is poorly documented. Our study aimed at evaluating the impact of rutile and anatase TiO2-NPs on the main functions of enterocytes, i.e. nutrient absorption driven by solute-liquid carriers (SLC transporters) and protection against other xenobiotics driven by efflux pumps from the ATP-binding cassette (ABC) family. We show that acute exposure of Caco-2 cells to both anatase (12 nm) and rutile (20 nm) TiO2-NPs induce early upregulation of a battery of efflux pumps and nutrient transporters. In addition they cause overproduction of reactive oxygen species and misbalance redox repair systems, without inducing cell mortality or DNA damage. Taken

Characterization and regulation of the resistance-nodulation-cell division-type multidrug efflux pumps MdtABC and MdtUVW from the fire blight pathogen Erwinia amylovora.

PubMed

Pletzer, Daniel; Weingart, Helge

2014-07-11

The Gram-negative bacterium Erwinia amylovora is the causal agent of the devastating disease fire blight in rosaceous plants such as apple, pear, quince, raspberry, and cotoneaster. In order to survive and multiply in a host, microbes must be able to circumvent the toxic effects of antimicrobial plant compounds, such as flavonoids and tannins. E. amylovora uses multidrug efflux transporters that recognize and actively export toxic compounds out of the cells. Here, two heterotrimeric resistance-nodulation-cell division (RND)-type multidrug efflux pumps, MdtABC and MdtUVW, from E. amylovora were identified. These RND systems are unusual in that they contain two different RND proteins forming a functional pump. To find the substrate specificities of the two efflux systems, we overexpressed the transporters in a hypersensitive mutant lacking the major RND pump AcrB. Both transporters mediated resistance to several flavonoids, fusidic acid and novobiocin. Additionally, MdtABC mediated resistance towards josamycin, bile salts and silver nitrate, and MdtUVW towards clotrimazole. The ability of the mdtABC- and mdtUVW-deficient mutants to multiply in apple rootstock was reduced. Quantitative RT-PCR analyses revealed that the expression of the transporter genes was induced during infection of apple rootstock. The polyphenolic plant compound tannin, as well as the heavy metal salt tungstate was found to induce the expression of mdtABC. Finally, the expression of the mdtABC genes was shown to be regulated by BaeR, the response regulator of the two-component system BaeSR, a cell envelope stress response system that controls the adaptive responses to changes in the environment. The expression of MdtABC and MdtUVW is induced during growth of E. amylovora in planta. We identified the plant polyphenol tannin as inducer of mdtABC expression. The reduced ability of the mdtABC- and mdtUVW-deficient mutants to multiply in apple rootstock suggests that the efflux pumps are involved in

Characterization and regulation of the Resistance-Nodulation-Cell Division-type multidrug efflux pumps MdtABC and MdtUVW from the fire blight pathogen Erwinia amylovora

PubMed Central

2014-01-01

Background The Gram-negative bacterium Erwinia amylovora is the causal agent of the devastating disease fire blight in rosaceous plants such as apple, pear, quince, raspberry, and cotoneaster. In order to survive and multiply in a host, microbes must be able to circumvent the toxic effects of antimicrobial plant compounds, such as flavonoids and tannins. E. amylovora uses multidrug efflux transporters that recognize and actively export toxic compounds out of the cells. Here, two heterotrimeric resistance-nodulation-cell division (RND)-type multidrug efflux pumps, MdtABC and MdtUVW, from E. amylovora were identified. These RND systems are unusual in that they contain two different RND proteins forming a functional pump. Results To find the substrate specificities of the two efflux systems, we overexpressed the transporters in a hypersensitive mutant lacking the major RND pump AcrB. Both transporters mediated resistance to several flavonoids, fusidic acid and novobiocin. Additionally, MdtABC mediated resistance towards josamycin, bile salts and silver nitrate, and MdtUVW towards clotrimazole. The ability of the mdtABC- and mdtUVW-deficient mutants to multiply in apple rootstock was reduced. Quantitative RT-PCR analyses revealed that the expression of the transporter genes was induced during infection of apple rootstock. The polyphenolic plant compound tannin, as well as the heavy metal salt tungstate was found to induce the expression of mdtABC. Finally, the expression of the mdtABC genes was shown to be regulated by BaeR, the response regulator of the two-component system BaeSR, a cell envelope stress response system that controls the adaptive responses to changes in the environment. Conclusions The expression of MdtABC and MdtUVW is induced during growth of E. amylovora in planta. We identified the plant polyphenol tannin as inducer of mdtABC expression. The reduced ability of the mdtABC- and mdtUVW-deficient mutants to multiply in apple rootstock suggests that the

The PerR-Regulated P1B-4-Type ATPase (PmtA) Acts as a Ferrous Iron Efflux Pump in Streptococcus pyogenes.

PubMed

Turner, Andrew G; Ong, Cheryl-Lynn Y; Djoko, Karrera Y; West, Nicholas P; Davies, Mark R; McEwan, Alastair G; Walker, Mark J

2017-06-01

Streptococcus pyogenes (group A Streptococcus [GAS]) is an obligate human pathogen responsible for a broad spectrum of human disease. GAS has a requirement for metal homeostasis within the human host and, as such, tightly modulates metal uptake and efflux during infection. Metal acquisition systems are required to combat metal sequestration by the host, while metal efflux systems are essential to protect against metal overload poisoning. Here, we investigated the function of PmtA ( P erR-regulated m etal t ransporter A ), a P 1B-4 -type ATPase efflux pump, in invasive GAS M1T1 strain 5448. We reveal that PmtA functions as a ferrous iron [Fe(II)] efflux system. In the presence of high Fe(II) concentrations, the 5448Δ pmtA deletion mutant exhibited diminished growth and accumulated 5-fold-higher levels of intracellular Fe(II) than did the wild type and the complemented mutant. The 5448Δ pmtA deletion mutant also showed enhanced susceptibility to killing by the Fe-dependent antibiotic streptonigrin as well as increased sensitivity to hydrogen peroxide and superoxide. We suggest that the PerR-mediated control of Fe(II) efflux by PmtA is important for bacterial defense against oxidative stress. PmtA represents an exemplar for an Fe(II) efflux system in a host-adapted Gram-positive bacterial pathogen. Copyright © 2017 American Society for Microbiology.

Efflux-Mediated Resistance to Tigecycline (GAR-936) in Pseudomonas aeruginosa PAO1

PubMed Central

Dean, Charles R.; Visalli, Melissa A.; Projan, Steven J.; Sum, Phaik-Eng; Bradford, Patricia A.

2003-01-01

Pseudomonas aeruginosa strains are less susceptible to tigecycline (previously GAR-936; MIC, 8 μg/ml) than many other bacteria (P. J. Petersen, N. V. Jacobus, W. J. Weiss, P. E. Sum, and R. T. Testa, Antimicrob. Agents Chemother. 43:738-744, 1999). To elucidate the mechanism of resistance to tigecycline, P. aeruginosa PAO1 strains defective in the MexAB-OprM and/or MexXY (OprM) efflux pumps were tested for susceptibility to tigecycline. Increased susceptibility to tigecycline (MIC, 0.5 to 1 μg/ml) was specifically associated with loss of MexXY. Transcription of mexX and mexY was also responsive to exposure of cells to tigecycline. To test for the emergence of compensatory efflux pumps in the absence of MexXY-OprM, mutants lacking MexXY-OprM were plated on medium containing tigecycline at 4 or 6 μg/ml. Resistant mutants were readily recovered, and these also had decreased susceptibility to several other antibiotics, suggesting efflux pump recruitment. One representative carbenicillin-resistant strain overexpressed OprM, the outer membrane channel component of the MexAB-OprM efflux pump. The mexAB-oprM repressor gene, mexR, from this strain contained a 15-bp in-frame deletion. Two representative chloramphenicol-resistant strains showed expression of an outer membrane protein slightly larger than OprM. The mexCD-OprJ repressor gene, nfxB, from these mutants contained a 327-bp in-frame deletion and an IS element insertion, respectively. Together, these data indicated drug efflux mediated by MexCD-OprJ. The MICs of the narrower-spectrum semisynthetic tetracyclines doxycycline and minocycline increased more substantially than did those of tigecycline and other glycylcyclines against the MexAB-OprM- and MexCD-OprJ-overexpressing mutant strains. This suggests that glycylcyclines, although they are subject to efflux from P. aeruginosa, are generally inferior substrates for P. aeruginosa efflux pumps than are narrower-spectrum tetracyclines. PMID:12604529

Impact of anatase and rutile titanium dioxide nanoparticles on uptake carriers and efflux pumps in Caco-2 gut epithelial cells.

PubMed

Dorier, M; Brun, E; Veronesi, G; Barreau, F; Pernet-Gallay, K; Desvergne, C; Rabilloud, T; Carapito, C; Herlin-Boime, N; Carrière, M

2015-04-28

TiO2 microparticles are widely used in food products, where they are added as a white food colouring agent. This food additive contains a significant amount of nanoscale particles; still the impact of TiO2 nanoparticles (TiO2-NPs) on gut cells is poorly documented. Our study aimed at evaluating the impact of rutile and anatase TiO2-NPs on the main functions of enterocytes, i.e. nutrient absorption driven by solute-liquid carriers (SLC transporters) and protection against other xenobiotics driven by efflux pumps from the ATP-binding cassette (ABC) family. We show that acute exposure of Caco-2 cells to both anatase (12 nm) and rutile (20 nm) TiO2-NPs induce early upregulation of a battery of efflux pumps and nutrient transporters. In addition they cause overproduction of reactive oxygen species and misbalance redox repair systems, without inducing cell mortality or DNA damage. Taken together, these data suggest that TiO2-NPs may increase the functionality of gut epithelial cells, particularly their property to form a protective barrier against exogenous toxicants and to absorb nutrients.

Tripartite assembly of RND multidrug efflux pumps

NASA Astrophysics Data System (ADS)

Daury, Laetitia; Orange, François; Taveau, Jean-Christophe; Verchère, Alice; Monlezun, Laura; Gounou, Céline; Marreddy, Ravi K. R.; Picard, Martin; Broutin, Isabelle; Pos, Klaas M.; Lambert, Olivier

2016-02-01

Tripartite multidrug efflux systems of Gram-negative bacteria are composed of an inner membrane transporter, an outer membrane channel and a periplasmic adaptor protein. They are assumed to form ducts inside the periplasm facilitating drug exit across the outer membrane. Here we present the reconstitution of native Pseudomonas aeruginosa MexAB-OprM and Escherichia coli AcrAB-TolC tripartite Resistance Nodulation and cell Division (RND) efflux systems in a lipid nanodisc system. Single-particle analysis by electron microscopy reveals the inner and outer membrane protein components linked together via the periplasmic adaptor protein. This intrinsic ability of the native components to self-assemble also leads to the formation of a stable interspecies AcrA-MexB-TolC complex suggesting a common mechanism of tripartite assembly. Projection structures of all three complexes emphasize the role of the periplasmic adaptor protein as part of the exit duct with no physical interaction between the inner and outer membrane components.

Antibacterial Efficacy of Polysaccharide Capped Silver Nanoparticles Is Not Compromised by AcrAB-TolC Efflux Pump

PubMed Central

Mishra, Mitali; Kumar, Satish; Majhi, Rakesh K.; Goswami, Luna; Goswami, Chandan; Mohapatra, Harapriya

2018-01-01

Antibacterial therapy is of paramount importance in treatment of several acute and chronic infectious diseases caused by pathogens. Over the years extensive use and misuse of antimicrobial agents has led to emergence of multidrug resistant (MDR) and extensive drug resistant (XDR) pathogens. This drastic escalation in resistant phenotype has limited the efficacy of available therapeutic options. Thus, the need of the hour is to look for alternative therapeutic approaches to mitigate healthcare concerns caused due to MDR bacterial infections. Nanoparticles have gathered much attention as potential candidates for antibacterial therapy. Equipped with advantages of, wide spectrum bactericidal activity at very low dosage, inhibitor of biofilm formation and ease of permeability, nanoparticles have been considered as leading therapeutic candidates to curtail infections resulting from MDR bacteria. However, substrate non-specificity of efflux pumps, particularly those belonging to resistance nodulation division super family, have been reported to reduce efficacy of many potent antibacterial therapeutic drugs. Previously, we had reported antibacterial activity of polysaccharide-capped silver nanoparticles (AgNPs) toward MDR bacteria. We showed that AgNPs inhibits biofilm formation and alters expression of cytoskeletal proteins FtsZ and FtsA, with minimal cytotoxicity toward mammalian cells. In the present study, we report no reduction in antibacterial efficacy of silver nanoparticles in presence of AcrAB-TolC efflux pump proteins. Antibacterial tests were performed according to CLSI macrobroth dilution method, which revealed that both silver nanoparticles exhibited bactericidal activity at very low concentrations. Further, immunoblotting results indicated that both the nanoparticles modulate the transporter AcrB protein expression. However, expression of the membrane fusion protein AcrA did show a significant increase after exposure to AgNPs. Our results indicate that both

Effects of chlorophyll-derived efflux pump inhibitor pheophorbide a and pyropheophorbide a on growth and macrolide antibiotic resistance of indicator and anaerobic swine manure bacteria

USDA-ARS?s Scientific Manuscript database

Natural plant compounds, such as the chlorophyll a catabolites pheophorbide a (php) and pyropheophorbide a (pyp), are potentially active in the gastrointestinal tracts and wastes of livestock as antimicrobial resistance-modifying agents through inhibition of bacterial efflux pumps. To investigate w...

Tetracycline Resistance and Presence of Tetracycline Resistance Determinants tet(V) and tap in Rapidly Growing Mycobacteria from Agricultural Soils and Clinical Isolates

PubMed Central

Kyselková, Martina; Chron̂áková, Alica; Volná, Lucie; Nêmec, Jan; Ulmann, Vít; Scharfen, Josef; Elhottová, Dana

2012-01-01

Rapidly growing mycobacteria (RGM) inhabit soil and water but certain strains represent a health risk for human and animals. Both clinical and soil RGM may be under selection pressure for resistance to tetracycline (TET) antibiotics, since tetracyclines are administrated to humans and farm animals, and TET residues enter soil through manuring; however, resistance to TET and the presence of TET-resistance genes have been assessed only in clinical isolates. We were therefore interested in comparing soil and clinical RGM in terms of TET resistance and the presence of TET-resistance genes. We used 44 RGM from grasslands with different exposure to animal manure, and 38 clinical RGM from Czech hospitals. There was no difference between the clinical and soil isolates in TET resistance, with >50% resistant isolates in both groups. otr(A), otr(B), tet(K), tet(L) or tet(M) were not detected in any soil or clinical isolate. In contrast, most isolates harbored tet(V) and tap, both encoding mycobacterial efflux pumps, including species where these genes have never been evidenced before. The phylogeny of tet(V) correlated with isolates’ BOX-PCR profiles, suggesting that this gene evolved along with mycobacterial genomes as a part of the intrinsic resistome. In certain cases, tet(V) and/or tap were found in TET-sensitive isolates, or inversely, were not found in resistant strains. Concluding, intrinsic efflux pumps may be more important for TET resistance than horizontally transferred genes in both soil and clinical RGM. Their simple presence, however, does not attest to resistance, and therefore their diversity, function and expression merit further research. PMID:22673307

Novel Piperazine Arylideneimidazolones Inhibit the AcrAB-TolC Pump in Escherichia coli and Simultaneously Act as Fluorescent Membrane Probes in a Combined Real-Time Influx and Efflux Assay.

PubMed

Bohnert, Jürgen A; Schuster, Sabine; Kern, Winfried V; Karcz, Tadeusz; Olejarz, Agnieszka; Kaczor, Aneta; Handzlik, Jadwiga; Kieć-Kononowicz, Katarzyna

2016-04-01

In this study, we tested five compounds belonging to a novel series of piperazine arylideneimidazolones for the ability to inhibit the AcrAB-TolC efflux pump. The biphenylmethylene derivative (BM-19) and the fluorenylmethylene derivative (BM-38) were found to possess the strongest efflux pump inhibitor (EPI) activities in the AcrAB-TolC-overproducingEscherichia colistrain 3-AG100, whereas BM-9, BM-27, and BM-36 had no activity at concentrations of up to 50 μM in a Nile red efflux assay. MIC microdilution assays demonstrated that BM-19 at 1/4 MIC (intrinsic MIC, 200 μM) was able to reduce the MICs of levofloxacin, oxacillin, linezolid, and clarithromycin 8-fold. BM-38 at 1/4 MIC (intrinsic MIC, 100 μM) was able to reduce only the MICs of oxacillin and linezolid (2-fold). Both compounds markedly reduced the MIC of rifampin (BM-19, 32-fold; and BM-38, 4-fold), which is suggestive of permeabilization of the outer membrane as an additional mechanism of action. Nitrocefin hydrolysis assays demonstrated that in addition to their EPI activity, both compounds were in fact weak permeabilizers of the outer membrane. Moreover, it was found that BM-19, BM-27, BM-36, and BM-38 acted as near-infrared-emitting fluorescent membrane probes, which allowed for their use in a combined influx and efflux assay and thus for tracking of the transport of an EPI across the outer membrane by an efflux pump in real time. The EPIs BM-38 and BM-19 displayed the most rapid influx of all compounds, whereas BM-27, which did not act as an EPI, showed the slowest influx. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Enhancement of antibiotic activity by efflux inhibitors against multidrug resistant Mycobacterium tuberculosis clinical isolates from Brazil

PubMed Central

Coelho, Tatiane; Machado, Diana; Couto, Isabel; Maschmann, Raquel; Ramos, Daniela; von Groll, Andrea; Rossetti, Maria L.; Silva, Pedro A.; Viveiros, Miguel

2015-01-01

Drug resistant tuberculosis continues to increase and new approaches for its treatment are necessary. The identification of M. tuberculosis clinical isolates presenting efflux as part of their resistant phenotype has a major impact in tuberculosis treatment. In this work, we used a checkerboard procedure combined with the tetrazolium microplate-based assay (TEMA) to study single combinations between antituberculosis drugs and efflux inhibitors (EIs) against multidrug resistant M. tuberculosis clinical isolates using the fully susceptible strain H37Rv as reference. Efflux activity was studied on a real-time basis by a fluorometric method that uses ethidium bromide as efflux substrate. Quantification of efflux pump genes mRNA transcriptional levels were performed by RT-qPCR. The fractional inhibitory concentrations (FIC) indicated synergistic activity for the interactions between isoniazid, rifampicin, amikacin, ofloxacin, and ethidium bromide plus the EIs verapamil, thioridazine and chlorpromazine. The FICs ranged from 0.25, indicating a four-fold reduction on the MICs, to 0.015, 64-fold reduction. The detection of active efflux by real-time fluorometry showed that all strains presented intrinsic efflux activity that contributes to the overall resistance which can be inhibited in the presence of the EIs. The quantification of the mRNA levels of the most important efflux pump genes on these strains shows that they are intrinsically predisposed to expel toxic compounds as the exposure to subinhibitory concentrations of antibiotics were not necessary to increase the pump mRNA levels when compared with the non-exposed counterpart. The results obtained in this study confirm that the intrinsic efflux activity contributes to the overall resistance in multidrug resistant clinical isolates of M. tuberculosis and that the inhibition of efflux pumps by the EIs can enhance the clinical effect of antibiotics that are their substrates. PMID:25972842

Studies on 2-phenylquinoline Staphylococcus aureus NorA efflux pump inhibitors: New insights on the C-6 position.

PubMed

Felicetti, Tommaso; Cannalire, Rolando; Nizi, Maria Giulia; Tabarrini, Oriana; Massari, Serena; Barreca, Maria Letizia; Manfroni, Giuseppe; Schindler, Bryan D; Cecchetti, Violetta; Kaatz, Glenn W; Sabatini, Stefano

2018-06-06

The alarming and rapid spread of antimicrobial resistance among bacteria represents a high risk for global health. Targeting factors involved in resistance to restore the activity of failing antibiotics is a promising strategy to overcome this urgent medical need. Efflux pump inhibitors are able to increase antibiotic concentrations in bacteria, thus they can be considered true antimicrobial resistance breakers. In this work, continuing our studies on inhibitors of the Staphylococcus aureus NorA pump, we designed, synthesized and biologically evaluated novel 2-phenylquinoline derivatives starting from our hits 1 and 2. Two of the synthesized compounds (6 and 7) bearing a C-6 benzyloxy group showed the best NorA inhibition activity, thereby providing an excellent starting point to direct future chemical optimizations. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Gallic acid-based indanone derivative interacts synergistically with tetracycline by inhibiting efflux pump in multidrug resistant E. coli.

PubMed

Dwivedi, Gaurav Raj; Tiwari, Nimisha; Singh, Aastha; Kumar, Akhil; Roy, Sudeep; Negi, Arvind Singh; Pal, Anirban; Chanda, Debabrata; Sharma, Ashok; Darokar, Mahendra P

2016-03-01

The purpose of the present study was to study the synergy potential of gallic acid-based derivatives in combination with conventional antibiotics using multidrug resistant cultures of Escherichia coli. Gallic acid-based derivatives significantly reduced the MIC of tetracycline against multidrug resistant clinical isolate of E. coli. The best representative, 3-(3',4,'5'-trimethoxyphenyl)-4,5,6-trimethoxyindanone-1, an indanone derivative of gallic acid, was observed to inhibit ethidium bromide efflux and ATPase which was also supported by in silico docking. This derivative extended the post-antibiotic effect and decreased the mutation prevention concentration of tetracycline. This derivative in combination with TET was able to reduce the concentration of TNFα up to 18-fold in Swiss albino mice. This derivative was nontoxic and well tolerated up to 300 mg/kg dose in subacute oral toxicity study in mice. This is the first report of gallic acid-based indanone derivative as drug resistance reversal agent acting through ATP-dependent efflux pump inhibition.

Effects of chlorophyll-derived efflux pump inhibitor pheophorbide a and pyropheophorbide a on erythromycin resistance of Staphylococcus aureus, Enterococcus faecalis, Salmonella Typhimurium and Escherichia coli

USDA-ARS?s Scientific Manuscript database

The purpose of this study was to validate the hypothesis that pheophorbide a and pyropheophorbide a reduce erythromycin resistance of reference strains of facultative anaerobic bacteria with multidrug or macrolide efflux pumps, as indicative of their effect on bacteria indigenous to anaerobic swine ...

Temporal Interplay between Efflux Pumps and Target Mutations in Development of Antibiotic Resistance in Escherichia coli

PubMed Central

Singh, Renu; Swick, Michelle C.; Ledesma, Kimberly R.; Yang, Zhen; Hu, Ming; Zechiedrich, Lynn

2012-01-01

The emergence of resistance presents a debilitating change in the management of infectious diseases. Currently, the temporal relationship and interplay between various mechanisms of drug resistance are not well understood. A thorough understanding of the resistance development process is needed to facilitate rational design of countermeasure strategies. Using an in vitro hollow-fiber infection model that simulates human drug treatment, we examined the appearance of efflux pump (acrAB) overexpression and target topoisomerase gene (gyrA and parC) mutations over time in the emergence of quinolone resistance in Escherichia coli. Drug-resistant isolates recovered early (24 h) had 2- to 8-fold elevation in the MIC due to acrAB overexpression, but no point mutations were noted. In contrast, high-level (≥64× MIC) resistant isolates with target site mutations (gyrA S83L with or without parC E84K) were selected more readily after 120 h, and regression of acrAB overexpression was observed at 240 h. Using a similar dosing selection pressure, the emergence of levofloxacin resistance was delayed in a strain with acrAB deleted compared to the isogenic parent. The role of efflux pumps in bacterial resistance development may have been underappreciated. Our data revealed the interplay between two mechanisms of quinolone resistance and provided a new mechanistic framework in the development of high-level resistance. Early low-level levofloxacin resistance conferred by acrAB overexpression preceded and facilitated high-level resistance development mediated by target site mutation(s). If this interpretation is correct, then these findings represent a paradigm shift in the way quinolone resistance is thought to develop. PMID:22232279

In vivo evaluation of thiolated poly(acrylic acid) as a drug absorption modulator for MRP2 efflux pump substrates.

PubMed

Greindl, Melanie; Föger, Florian; Hombach, Juliane; Bernkop-Schnürch, Andreas

2009-08-01

Recently, several polymers have been reported to modulate drug absorption by inhibition of intestinal efflux pumps such as multidrug resistance proteins (MRPs) and P-glycoprotein (P-gp). The aim of the present study was to evaluate the efficiency of thiolated poly(acrylic acid) (PAA-Cys) to act as a drug absorption modulator for MRP2 efflux pump substrates in vivo, using sulforhodamine 101 as representative MRP2 substrate. In vitro, the permeation-enhancing effect of unmodified PAA and PAA(250)-Cys(,) displaying 580 micromol free thiol groups per gram polymer, was evaluated by using freshly excised rat intestinal mucosa mounted in Ussing-type chambers. In comparison to that of the buffer control, the sulforhodamine 101 transport in the presence of 0.5% unmodified PAA(250) and 0.5% (w/v) PAA(250)-Cys was 1.3- and 4.0-fold improved, respectively. In vivo, sulforhodamine 101 solutions containing 4% (w/v) unmodified PAA(250) or 4% (w/v) thiolated PAA(250) were orally given to rats. The PAA(250)-Cys solution increased the area under the plasma concentration-time curve (AUC(0-12)) of sulforhodamine 101 3.8-fold in comparison to control and 2.2-fold in comparison to unmodified PAA(250). This in vivo study revealed that PAA(250)-Cys significantly increased the oral bioavailability of MRP2 substrate sulforhodamine 101.

Hydroxypropyl-sulfobutyl-β-cyclodextrin improves the oral bioavailability of edaravone by modulating drug efflux pump of enterocytes.

PubMed

Rong, Wen-Ting; Lu, Ya-Peng; Tao, Qing; Guo, Miao; Lu, Yu; Ren, Yong; Yu, Shu-Qin

2014-02-01

The objective of the study was to evaluate the effect of hydroxypropyl-sulfobutyl-β-cyclodextrin (HP-SBE-βCD) on the bioavailability and intestinal absorption of edaravone, and identify its mechanism of action. We devised HP-SBE-βCD as a carrier and modulator of P-glycoprotein (Pgp) efflux pump, and edaravone as a model drug, and prepared edaravone/HP-SBE-βCD inclusion complex. HP-SBE-βCD improved the water solubility and enhanced the bioavailability of edaravone by 10.3-fold in rats. Then, in situ single-pass intestinal perfusion showed that HP-SBE-βCD had an effect of improving the permeability and inhibiting the efflux of edaravone. Furthermore, the effects of HP-SBE-βCD on Pgp were achieved through interfering with the lipid raft and depleting the cholesterol of enterocytes membrane. From the results, we presented the novel mechanisms. First, edaravone/HP-SBE-βCD had a lower release from the inclusion compound to protect edaravone from the low pH of the stomach. Then, HP-SBE-βCD modulated the membrane microenvironment of intestinal absorption epithelial cells. At last, the result was that HP-SBE-βCD enhanced the absorption of edaravone by interfering with Pgp. In conclusion, HP-SBE-βCD improves the bioavailability of drug not only because of its enhancing water solubility of the drug, but also because it modulates the Pgp-mediated efflux from enterocytes. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association.

Assembly and Channel Opening of Outer Membrane Protein in Tripartite Drug Efflux Pumps of Gram-negative Bacteria*

PubMed Central

Xu, Yongbin; Moeller, Arne; Jun, So-Young; Le, Minho; Yoon, Bo-Young; Kim, Jin-Sik; Lee, Kangseok; Ha, Nam-Chul

2012-01-01

Gram-negative bacteria are capable of expelling diverse xenobiotic substances from within the cell by use of three-component efflux pumps in which the energy-activated inner membrane transporter is connected to the outer membrane channel protein via the membrane fusion protein. In this work, we describe the crystal structure of the membrane fusion protein MexA from the Pseudomonas aeruginosa MexAB-OprM pump in the hexameric ring arrangement. Electron microscopy study on the chimeric complex of MexA and the outer membrane protein OprM reveals that MexA makes a tip-to-tip interaction with OprM, which suggests a docking model for MexA and OprM. This docking model agrees well with genetic results and depicts detailed interactions. Opening of the OprM channel is accompanied by the simultaneous exposure of a protein structure resembling a six-bladed cogwheel, which intermeshes with the complementary cogwheel structure in the MexA hexamer. Taken together, we suggest an assembly and channel opening model for the MexAB-OprM pump. This study provides a better understanding of multidrug resistance in Gram-negative bacteria. PMID:22308040

Reversal of the Drug Binding Pocket Defects of the AcrB Multidrug Efflux Pump Protein of Escherichia coli

PubMed Central

Soparkar, Ketaki; Kinana, Alfred D.; Weeks, Jon W.; Morrison, Keith D.; Nikaido, Hiroshi

2015-01-01

ABSTRACT The AcrB protein of Escherichia coli, together with TolC and AcrA, forms a contiguous envelope conduit for the capture and extrusion of diverse antibiotics and cellular metabolites. In this study, we sought to expand our knowledge of AcrB by conducting genetic and functional analyses. We began with an AcrB mutant bearing an F610A substitution in the drug binding pocket and obtained second-site substitutions that overcame the antibiotic hypersusceptibility phenotype conferred by the F610A mutation. Five of the seven unique single amino acid substitutions—Y49S, V127A, V127G, D153E, and G288C—mapped in the periplasmic porter domain of AcrB, with the D153E and G288C mutations mapping near and at the distal drug binding pocket, respectively. The other two substitutions—F453C and L486W—were mapped to transmembrane (TM) helices 5 and 6, respectively. The nitrocefin efflux kinetics data suggested that all periplasmic suppressors significantly restored nitrocefin binding affinity impaired by the F610A mutation. Surprisingly, despite increasing MICs of tested antibiotics and the efflux of N-phenyl-1-naphthylamine, the TM suppressors did not improve the nitrocefin efflux kinetics. These data suggest that the periplasmic substitutions act by influencing drug binding affinities for the distal binding pocket, whereas the TM substitutions may indirectly affect the conformational dynamics of the drug binding domain. IMPORTANCE The AcrB protein and its homologues confer multidrug resistance in many important human bacterial pathogens. A greater understanding of how these efflux pump proteins function will lead to the development of effective inhibitors against them. The research presented in this paper investigates drug binding pocket mutants of AcrB through the isolation and characterization of intragenic suppressor mutations that overcome the drug susceptibility phenotype of mutations affecting the drug binding pocket. The data reveal a remarkable structure

Lack of AcrB Efflux Function Confers Loss of Virulence on Salmonella enterica Serovar Typhimurium

PubMed Central

Wang-Kan, Xuan; Chirullo, Barbara; Betts, Jonathan; La Ragione, Roberto M.; Ivens, Alasdair; Ricci, Vito; Opperman, Timothy J.

2017-01-01

ABSTRACT AcrAB-TolC is the paradigm resistance-nodulation-division (RND) multidrug resistance efflux system in Gram-negative bacteria, with AcrB being the pump protein in this complex. We constructed a nonfunctional AcrB mutant by replacing D408, a highly conserved residue essential for proton translocation. Western blotting confirmed that the AcrB D408A mutant had the same native level of expression of AcrB as the parental strain. The mutant had no growth deficiencies in rich or minimal medium. However, compared with wild-type SL1344, the mutant had increased accumulation of Hoechst 33342 dye and decreased efflux of ethidium bromide and was multidrug hypersusceptible. The D408A mutant was attenuated in vivo in mouse and Galleria mellonella models and showed significantly reduced invasion into intestinal epithelial cells and macrophages in vitro. A dose-dependent inhibition of invasion was also observed when two different efflux pump inhibitors were added to the wild-type strain during infection of epithelial cells. RNA sequencing (RNA-seq) revealed downregulation of bacterial factors necessary for infection, including those in the Salmonella pathogenicity islands 1, 2, and 4; quorum sensing genes; and phoPQ. Several general stress response genes were upregulated, probably due to retention of noxious molecules inside the bacterium. Unlike loss of AcrB protein, loss of efflux function did not induce overexpression of other RND efflux pumps. Our data suggest that gene deletion mutants are unsuitable for studying membrane transporters and, importantly, that inhibitors of AcrB efflux function will not induce expression of other RND pumps. PMID:28720734

Functional Relationships between the AcrA Hairpin Tip Region and the TolC Aperture Tip Region for the Formation of the Bacterial Tripartite Efflux Pump AcrAB-TolC ▿ † ‡

PubMed Central

Kim, Hong-Man; Xu, Yongbin; Lee, Minho; Piao, Shunfu; Sim, Se-Hoon; Ha, Nam-Chul; Lee, Kangseok

2010-01-01

Tripartite efflux pumps found in Gram-negative bacteria are involved in antibiotic resistance and toxic-protein secretion. In this study, we show, using site-directed mutational analyses, that the conserved residues located in the tip region of the α-hairpin of the membrane fusion protein (MFP) AcrA play an essential role in the action of the tripartite efflux pump AcrAB-TolC. In addition, we provide in vivo functional data showing that both the length and the amino acid sequence of the α-hairpin of AcrA can be flexible for the formation of a functional AcrAB-TolC pump. Genetic-complementation experiments further indicated functional interrelationships between the AcrA hairpin tip region and the TolC aperture tip region. Our findings may offer a molecular basis for understanding the multidrug resistance of pathogenic bacteria. PMID:20581201

Mode of action of the 2-phenylquinoline efflux inhibitor PQQ4R against Escherichia coli

PubMed Central

Machado, Diana; Fernandes, Laura; Costa, Sofia S.; Cannalire, Rolando; Manfroni, Giuseppe; Tabarrini, Oriana; Couto, Isabel; Sabatini, Stefano

2017-01-01

Efflux pump inhibitors are of great interest since their use as adjuvants of bacterial chemotherapy can increase the intracellular concentrations of the antibiotics and assist in the battle against the rising of antibiotic-resistant bacteria. In this work, we have described the mode of action of the 2-phenylquinoline efflux inhibitor (4-(2-(piperazin-1-yl)ethoxy)-2-(4-propoxyphenyl) quinolone – PQQ4R), against Escherichia coli, by studding its efflux inhibitory ability, its synergistic activity in combination with antibiotics, and compared its effects with the inhibitors phenyl-arginine-β-naphthylamide (PAβN) and chlorpromazine (CPZ). The results showed that PQQ4R acts synergistically, in a concentration dependent manner, with antibiotics known to be subject to efflux in E. coli reducing their MIC in correlation with the inhibition of their efflux. Real-time fluorometry assays demonstrated that PQQ4R at sub-inhibitory concentrations promote the intracellular accumulation of ethidium bromide inhibiting its efflux similarly to PAβN or CPZ, well-known and described efflux pump inhibitors for Gram-negative bacteria and whose clinical usage is limited by their levels of toxicity at clinical and bacteriological effective concentrations. The time-kill studies showed that PQQ4R, at bactericidal concentrations, has a rapid antimicrobial activity associated with a fast decrease of the intracellular ATP levels. The results also indicated that the mode of action of PQQ4R involves the destabilization of the E. coli inner membrane potential and ATP production impairment, ultimately leading to efflux pump inhibition by interference with the energy required by the efflux systems. At bactericidal concentrations, membrane permeabilization increases and finally ATP is totally depleted leading to cell death. Since drug resistance mediated by the activity of efflux pumps depends largely on the proton motive force (PMF), dissipaters of PMF such as PQQ4R, can be regarded as future

Novel inhibitory activity of the Staphylococcus aureus NorA efflux pump by a kaempferol rhamnoside isolated from Persea lingue Nees.

PubMed

Holler, Jes Gitz; Christensen, S Brøgger; Slotved, Hans-Christian; Rasmussen, Hasse B; Gúzman, Alfonso; Olsen, Carl-Erik; Petersen, Bent; Mølgaard, Per

2012-05-01

To isolate a plant-derived compound with efflux inhibitory activity towards the NorA transporter of Staphylococcus aureus. Bioassay-guided isolation was used, with inhibition of ethidium bromide efflux via NorA as a guide. Characterization of activity was carried out using MIC determination and potentiation studies of a fluoroquinolone antibiotic in combination with the isolated compound. Everted membrane vesicles of Escherichia coli cells enriched with NorA were prepared to study efflux inhibitory activity in an isolated manner. The ethanolic extract of Persea lingue was subjected to bioassay-guided fractionation and led to the isolation of the known compound kaempferol-3-O-α-L-(2,4-bis-E-p-coumaroyl)rhamnoside (compound 1). Evaluation of the dose-response relationship of compound 1 showed that ethidium bromide efflux was inhibited, with an IC(50) value of 2 μM. The positive control, reserpine, was found to have an IC(50) value of 9 μM. Compound 1 also inhibited NorA in enriched everted membrane vesicles of E. coli. Potentiation studies revealed that compound 1 at 1.56 mg/L synergistically increased the antimicrobial activity of ciprofloxacin 8-fold against a NorA overexpresser, and the synergistic activity was exerted at a fourth of the concentration necessary for reserpine. Compound 1 was not found to exert a synergistic effect on ciprofloxacin against a norA deletion mutant. The 2,3-coumaroyl isomer of compound 1 has been shown previously not to cause acute toxicity in mice at 20 mg/kg/day. Our results show that compound 1 acts through inhibition of the NorA efflux pump. Combination of compound 1 with subinhibitory concentrations of ciprofloxacin renders a wild-type more susceptible and a NorA overexpresser S. aureus susceptible.

Sequence- and structure-based computational analyses of Gram-negative tripartite efflux pumps in the context of bacterial membranes

DOE PAGES

Travers, Timothy; Wang, Katherine J.; Lopez, Cesar A.; ...

2018-02-09

Gram-negative multidrug resistance currently presents a serious threat to public health with infections effectively rendered untreatable. Multiple molecular mechanisms exist that cause antibiotic resistance and in addition, the last three decades have seen slowing rates of new drug development. In this paper, we summarize the use of various computational techniques for investigating the mechanisms of multidrug resistance mediated by Gram-negative tripartite efflux pumps and membranes. Recent work in our lab combines data-driven sequence and structure analyses to study the interactions and dynamics of these bacterial components. Computational studies can complement experimental methodologies for gaining crucial insights into combatting multidrug resistance.

Sequence- and structure-based computational analyses of Gram-negative tripartite efflux pumps in the context of bacterial membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Travers, Timothy; Wang, Katherine J.; Lopez, Cesar A.

Gram-negative multidrug resistance currently presents a serious threat to public health with infections effectively rendered untreatable. Multiple molecular mechanisms exist that cause antibiotic resistance and in addition, the last three decades have seen slowing rates of new drug development. In this paper, we summarize the use of various computational techniques for investigating the mechanisms of multidrug resistance mediated by Gram-negative tripartite efflux pumps and membranes. Recent work in our lab combines data-driven sequence and structure analyses to study the interactions and dynamics of these bacterial components. Computational studies can complement experimental methodologies for gaining crucial insights into combatting multidrug resistance.

Efflux systems in bacteria and their metabolic engineering applications.

PubMed

Jones, Christopher M; Hernández Lozada, Néstor J; Pfleger, Brian F

2015-11-01

The production of valuable chemicals from metabolically engineered microbes can be limited by excretion from the cell. Efflux is often overlooked as a bottleneck in metabolic pathways, despite its impact on alleviating feedback inhibition and product toxicity. In the past, it has been assumed that endogenous efflux pumps and membrane porins can accommodate product efflux rates; however, there are an increasing number of examples wherein overexpressing efflux systems is required to improve metabolite production. In this review, we highlight specific examples from the literature where metabolite export has been studied to identify unknown transporters, increase tolerance to metabolites, and improve the production capabilities of engineered bacteria. The review focuses on the export of a broad spectrum of valuable chemicals including amino acids, sugars, flavins, biofuels, and solvents. The combined set of examples supports the hypothesis that efflux systems can be identified and engineered to confer export capabilities on industrially relevant microbes.

Contribution of efflux to colistin heteroresistance in a multidrug resistant Acinetobacter baumannii clinical isolate.

PubMed

Machado, Diana; Antunes, Jéssica; Simões, Ana; Perdigão, João; Couto, Isabel; McCusker, Matthew; Martins, Marta; Portugal, Isabel; Pacheco, Teresa; Batista, Judite; Toscano, Cristina; Viveiros, Miguel

2018-06-01

The mechanisms underlying colistin heteroresistance in Acinetobacter baumannii are not fully understood. Here, we investigated the role of efflux in colistin-heteroresistant populations of a multidrug-resistant (MDR) A. baumannii clinical isolate. Three colistin-resistant A. baumannii strain variants isolated from the same clinical sample were studied for the presence of heteroresistance to colistin by drug susceptibility testing, genotyping and drug resistance target mutation analysis. The existence of active efflux was studied by synergism assays with efflux inhibitors, real-time efflux activity measurements and analysis of the mRNA transcriptional levels of selected efflux pump genes in response to colistin. All of the strain variants belong to the ST218, clonal complex 92, international clonal lineage II. Different colistin susceptibility levels were observed among the three strain variants, indicating that colistin-heteroresistant subpopulations were being selected upon exposure to colistin. No mutations were found in the genes lpxACD and pmrAB, which are associated with colistin resistance. The results showed the existence of synergistic interactions between efflux inhibitors and colistin and ethidium bromide. Real-time efflux assays demonstrated that the three strain variants had increased efflux activity that could be inhibited in the presence of the inhibitors. The efflux pump genes adeB, adeJ, adeG, craA, amvA, abeS and abeM were found to be overexpressed in the strain variants in response to colistin exposure. This study shows that efflux activity contributes to colistin heteroresistance in an MDR A. baumannii clinical isolate. The use of efflux inhibitors as adjuvants of the therapy can resensitize A. baumannii to colistin and prevent the emergence of drug resistance.

Interplay between Mutations and Efflux in Drug Resistant Clinical Isolates of Mycobacterium tuberculosis.

PubMed

Machado, Diana; Coelho, Tatiane S; Perdigão, João; Pereira, Catarina; Couto, Isabel; Portugal, Isabel; Maschmann, Raquel De Abreu; Ramos, Daniela F; von Groll, Andrea; Rossetti, Maria L R; Silva, Pedro A; Viveiros, Miguel

2017-01-01

Numerous studies show efflux as a universal bacterial mechanism contributing to antibiotic resistance and also that the activity of the antibiotics subject to efflux can be enhanced by the combined use of efflux inhibitors. Nevertheless, the contribution of efflux to the overall drug resistance levels of clinical isolates of Mycobacterium tuberculosis is poorly understood and still is ignored by many. Here, we evaluated the contribution of drug efflux plus target-gene mutations to the drug resistance levels in clinical isolates of M. tuberculosis . A panel of 17 M. tuberculosis clinical strains were characterized for drug resistance associated mutations and antibiotic profiles in the presence and absence of efflux inhibitors. The correlation between the effect of the efflux inhibitors and the resistance levels was assessed by quantitative drug susceptibility testing. The bacterial growth/survival vs. growth inhibition was analyzed through the comparison between the time of growth in the presence and absence of an inhibitor. For the same mutation conferring antibiotic resistance, different MICs were observed and the different resistance levels found could be reduced by efflux inhibitors. Although susceptibility was not restored, the results demonstrate the existence of a broad-spectrum synergistic interaction between antibiotics and efflux inhibitors. The existence of efflux activity was confirmed by real-time fluorometry. Moreover, the efflux pump genes mmr, mmpL7, Rv1258c, p55 , and efpA were shown to be overexpressed in the presence of antibiotics, demonstrating the contribution of these efflux pumps to the overall resistance phenotype of the M. tuberculosis clinical isolates studied, independently of the genotype of the strains. These results showed that the drug resistance levels of multi- and extensively-drug resistant M. tuberculosis clinical strains are a combination between drug efflux and the presence of target-gene mutations, a reality that is often

Homologs of the Acinetobacter baumannii AceI transporter represent a new family of bacterial multidrug efflux systems.

PubMed

Hassan, Karl A; Liu, Qi; Henderson, Peter J F; Paulsen, Ian T

2015-02-10

Multidrug efflux systems are a major cause of resistance to antimicrobials in bacteria, including those pathogenic to humans, animals, and plants. These proteins are ubiquitous in these pathogens, and five families of bacterial multidrug efflux systems have been identified to date. By using transcriptomic and biochemical analyses, we recently identified the novel AceI (Acinetobacter chlorhexidine efflux) protein from Acinetobacter baumannii that conferred resistance to the biocide chlorhexidine, via an active efflux mechanism. Proteins homologous to AceI are encoded in the genomes of many other bacterial species and are particularly prominent within proteobacterial lineages. In this study, we expressed 23 homologs of AceI and examined their resistance and/or transport profiles. MIC analyses demonstrated that, like AceI, many of the homologs conferred resistance to chlorhexidine. Many of the AceI homologs conferred resistance to additional biocides, including benzalkonium, dequalinium, proflavine, and acriflavine. We conducted fluorimetric transport assays using the AceI homolog from Vibrio parahaemolyticus and confirmed that resistance to both proflavine and acriflavine was mediated by an active efflux mechanism. These results show that this group of AceI homologs represent a new family of bacterial multidrug efflux pumps, which we have designated the proteobacterial antimicrobial compound efflux (PACE) family of transport proteins. Bacterial multidrug efflux pumps are an important class of resistance determinants that can be found in every bacterial genome sequenced to date. These transport proteins have important protective functions for the bacterial cell but are a significant problem in the clinical setting, since a single efflux system can mediate resistance to many structurally and mechanistically diverse antibiotics and biocides. In this study, we demonstrate that proteins related to the Acinetobacter baumannii AceI transporter are a new class of multidrug

Calcium Efflux Systems in Stress Signaling and Adaptation in Plants

PubMed Central

Bose, Jayakumar; Pottosin, Igor I.; Shabala, Stanislav S.; Palmgren, Michael G.; Shabala, Sergey

2011-01-01

Transient cytosolic calcium ([Ca2+]cyt) elevation is an ubiquitous denominator of the signaling network when plants are exposed to literally every known abiotic and biotic stress. These stress-induced [Ca2+]cyt elevations vary in magnitude, frequency, and shape, depending on the severity of the stress as well the type of stress experienced. This creates a unique stress-specific calcium “signature” that is then decoded by signal transduction networks. While most published papers have been focused predominantly on the role of Ca2+ influx mechanisms to shaping [Ca2+]cyt signatures, restoration of the basal [Ca2+]cyt levels is impossible without both cytosolic Ca2+ buffering and efficient Ca2+ efflux mechanisms removing excess Ca2+ from cytosol, to reload Ca2+ stores and to terminate Ca2+ signaling. This is the topic of the current review. The molecular identity of two major types of Ca2+ efflux systems, Ca2+-ATPase pumps and Ca2+/H+ exchangers, is described, and their regulatory modes are analyzed in detail. The spatial and temporal organization of calcium signaling networks is described, and the importance of existence of intracellular calcium microdomains is discussed. Experimental evidence for the role of Ca2+ efflux systems in plant responses to a range of abiotic and biotic factors is summarized. Contribution of Ca2+-ATPase pumps and Ca2+/H+ exchangers in shaping [Ca2+]cyt signatures is then modeled by using a four-component model (plasma- and endo-membrane-based Ca2+-permeable channels and efflux systems) taking into account the cytosolic Ca2+ buffering. It is concluded that physiologically relevant variations in the activity of Ca2+-ATPase pumps and Ca2+/H+ exchangers are sufficient to fully describe all the reported experimental evidence and determine the shape of [Ca2+]cyt signatures in response to environmental stimuli, emphasizing the crucial role these active efflux systems play in plant adaptive responses to environment. PMID:22639615

The BpeEF-OprC Efflux Pump Is Responsible for Widespread Trimethoprim Resistance in Clinical and Environmental Burkholderia pseudomallei Isolates

PubMed Central

Podnecky, Nicole L.; Wuthiekanun, Vanaporn; Peacock, Sharon J.

2013-01-01

Trimethoprim-sulfamethoxazole (co-trimoxazole) is the primary drug used for oral eradication therapy of Burkholderia pseudomallei infections (melioidosis). Here, we demonstrate that trimethoprim resistance is widespread in clinical and environmental isolates from northeast Thailand and northern Australia. This resistance was shown to be due to BpeEF-OprC efflux pump expression. No dihydrofolate reductase target mutations were involved, although frequent insertion of ISBma2 was noted within the putative folA transcriptional terminator. All isolates tested remained susceptible to trimethoprim-sulfamethoxazole, suggesting that resistance to trimethoprim alone in these strains probably does not affect the efficacy of co-trimoxazole therapy. PMID:23817379

Cytotoxicity and Efflux Pump Inhibition Induced by Molybdenum Disulfide and Boron Nitride Nanomaterials with Sheetlike Structure.

PubMed

Liu, Su; Shen, Zhuoyan; Wu, Bing; Yu, Yue; Hou, Hui; Zhang, Xu-Xiang; Ren, Hong-Qiang

2017-09-19

Sheetlike molybdenum disulfide (MoS 2 ) and boron nitride (BN) nanomaterials have attracted attention in the past few years due to their unique material properties. However, information on adverse effects and their underlying mechanisms for sheetlike MoS 2 and BN nanomaterials is rare. In this study, cytotoxicities of sheetlike MoS 2 and BN nanomaterials on human hepatoma HepG2 cells were systematically investigated at different toxic end points. Results showed that MoS 2 and BN nanomaterials decreased cell viability at 30 μg/mL and induced adverse effects on intracellular ROS generation (≥2 μg/mL), mitochondrial depolarization (≥4 μg/mL), and membrane integrity (≥8 μg/mL for MoS 2 and ≥2 μg/mL for BN). Furthermore, this study first found that low exposure concentrations (0.2-2 μg/mL) of MoS 2 and BN nanomaterials could increase plasma membrane fluidity and inhibit transmembrane ATP binding cassette (ABC) efflux transporter activity, which make both nanomaterials act as a chemosensitizer (increasing arsenic toxicity). Damage to plasma membrane and release of soluble Mo or B species might be two reasons that both nanomaterials inhibit efflux pump activities. This study provides a systematic understanding of the cytotoxicity of sheetlike MoS 2 and BN nanomaterials at different exposure levels, which is important for their safe use.

The role of the Serratia marcescens SdeAB multidrug efflux pump and TolC homologue in fluoroquinolone resistance studied via gene-knockout mutagenesis.

PubMed

Begic, Sanela; Worobec, Elizabeth A

2008-02-01

Serratia marcescens is a prominent opportunistic nosocomial pathogen resistant to several classes of antibiotics. The major mechanism for fluoroquinolone resistance in various Gram-negative pathogens is active efflux. Our group previously identified SdeAB, a resistance-nodulation-cell division (RND) efflux pump complex, and a TolC-like outer-membrane protein (HasF), which together mediate energy-dependent fluoroquinolone efflux. In addition, a regulatory protein-encoding gene in the upstream region of sdeAB was identified (sdeR) and found to be 40 % homologous to MarA, an Escherichia coli transcriptional regulator. To provide conclusive evidence as to the role of these components in S. marcescens, sdeB, hasF and sdeR deletion mutants were constructed. Suicide vectors were created and introduced via triparental mating into S. marcescens UOC-67 (wild-type) and, for sdeB and hasF, T-861 (clinical isolate). We have analysed these genetically altered strains using minimal inhibitory concentration (MIC) assays for a wide range of compounds (fluoroquinolones, SDS, novobiocin, ethidium bromide and chloramphenicol). Intracellular accumulation of a variety of fluoroquinolones was measured fluorospectroscopically. The sdeB, hasF and sdeR knockout strains were consistently more susceptible to antibiotics than the parent strains, with the sdeB/hasF double knockout strain showing the highest susceptibility. A marked increase in fluoroquinolone (ciprofloxacin) accumulation was observed for strains deficient in either the sdeB or hasF genes when compared to the parental strains, with the highest ciprofloxacin accumulation observed for the sdeB/hasF double knockout. Antibiotic accumulation assays for the sdeB knockout mutant strains performed in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP), a proton-motive-force inhibitor, demonstrated that SdeAB-mediated efflux is proton-motive-force dependent. Due to the comparable susceptibility of the sdeB and the has

Efflux inhibitor suppresses Streptococcus mutans virulence properties.

PubMed

Zeng, Huihui; Liu, Jia; Ling, Junqi

2017-04-01

It is well established that efflux pumps play important roles in bacterial pathogenicity and efflux inhibitors (EIs) have been proved to be effective in suppressing bacterial virulence properties. However, little is known regarding the EI of Streptococcus mutans, a well-known caries-inducing bacterium. In this study, we identified the EI of S. mutans through ethidium bromide efflux assay and investigated how EI affected S. mutans virulence regarding the cariogenicity and stress response. Results indicated that reserpine, the identified EI, suppressed acid tolerance, mutacin production and transformation efficiency of S. mutans, and modified biofilm architecture and extracellular polysaccharide distribution. Suppressed glycosyltransferase activity was also noted after reserpine exposure. The data from quantitative real-time-PCR demonstrated that reserpine significantly altered the expression profile of quorum-sensing and virulence-associated genes. These findings suggest that reserpine represents a promising adjunct anticariogenic agent in that it suppresses virulence properties of S. mutans. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Structure and function of Neisseria gonorrhoeae MtrF illuminates a class of antimetabolite efflux pumps

DOE PAGES

Su, Chih -Chia; Bolla, Jani  Reddy; Kumar, Nitin; ...

2015-04-01

Neisseria gonorrhoeae is an obligate human pathogen and the causative agent of the sexually transmitted disease gonorrhea. The control of this disease has been compromised by the increasing proportion of infections due to antibiotic-resistant strains, which are growing at an alarming rate. N. gonorrhoeae MtrF is an integral membrane protein that belongs to the AbgT family of transporters for which no structural information is available. Here, we describe the crystal structure of MtrF, revealing a dimeric molecule with architecture distinct from all other families of transporters. MtrF is a bowl-shaped dimer with a solvent-filled basin extending from the cytoplasm tomore » halfway across the membrane bilayer. Each subunit of the transporter contains nine transmembrane helices and two hairpins, posing a plausible pathway for substrate transport. A combination of the crystal structure and biochemical functional assays suggests that MtrF is an antibiotic efflux pump mediating bacterial resistance to sulfonamide antimetabolite drugs.« less

Structure and function of Neisseria gonorrhoeae MtrF illuminates a class of antimetabolite efflux pumps

PubMed Central

Su, Chih-Chia; Bolla, Jani Reddy; Kumar, Nitin; Radhakrishnan, Abhijith; Long, Feng; Delmar, Jared A.; Chou, Tsung-Han; Rajashankar, Kanagalaghatta R.; Shafer, William M.; Yu, Edward W.

2015-01-01

SUMMARY Neisseria gonorrhoeae is an obligate human pathogen and the causative agent of the sexually-transmitted disease gonorrhea. The control of this disease has been compromised by the increasing proportion of infections due to antibiotic-resistant strains, which are growing at an alarming rate. N. gonorrhoeae MtrF is an integral membrane protein, which belongs to the AbgT family of transporters for which no structural information is available. Here we describe the crystal structure of MtrF, revealing a dimeric molecule with architecture distinct from all other families of transporters. MtrF is a bowl-shaped dimer with a solvent-filled basin extending from the cytoplasm to halfway across the membrane bilayer. Each subunit of the transporter contains nine transmembrane helices and two hairpins, posing a plausible pathway for substrate transport. A combination of the crystal structure and biochemical functional assays suggests that MtrF is an antibiotic efflux pump, mediating bacterial resistance to sulfonamide antimetabolite drugs. PMID:25818299

PigZ, a TetR/AcrR family repressor, modulates secondary metabolism via the expression of a putative four-component resistance-nodulation-cell-division efflux pump, ZrpADBC, in Serratia sp. ATCC 39006.

PubMed

Gristwood, Tamzin; Fineran, Peter C; Everson, Lee; Salmond, George P C

2008-07-01

The Gram-negative enterobacterium, Serratia sp. ATCC 39006 synthesizes several secondary metabolites, including prodigiosin (Pig) and a carbapenem antibiotic (Car). A complex hierarchical network of regulatory proteins control Pig and Car production. In this study we characterize a TetR family regulator, PigZ, which represses transcription of a divergently transcribed putative resistance-nodulation-cell-division (RND) efflux pump, encoded by zrp (PigZ repressed pump) ADBC, via direct binding to the zrpA-pigZ intergenic region. Unusually, this putative RND pump contains two predicted membrane fusion proteins (MFPs), ZrpA and ZrpD. A mutation in pigZ resulted in multiple phenotypic changes, including exoenzyme production, motility and differential regulation of Pig and Car production. A polar suppressor mutation, within zrpA, restored all tested phenotypes to parental strain levels, indicating that the changes observed are due to the increase in expression of ZrpADBC in the absence of the repressor, PigZ. Genomic deletions of zrpA and zrpD indicate that the MFP ZrpD, but not ZrpA, is essential for activity of the putative pump. Bioinformatic analysis revealed that putative RND efflux pumps encoding two MFP components are not uncommon, particularly among plant-associated, Gram-negative bacteria. In addition, based on phylogenetic analysis, we propose that these pairs of MFPs consist of two distinct subtypes.

Synthesis of omeprazole analogues and evaluation of these as potential inhibitors of the multidrug efflux pump NorA of Staphylococcus aureus.

PubMed

Vidaillac, Céline; Guillon, Jean; Arpin, Corinne; Forfar-Bares, Isabelle; Ba, Boubakar B; Grellet, Jean; Moreau, Stéphane; Caignard, Daniel-Henri; Jarry, Christian; Quentin, Claudine

2007-03-01

A series of 11 pyrrolo[1,2-a]quinoxaline derivatives, 1a to 1k, sharing structural analogies with omeprazole, a eukaryotic efflux pump inhibitor (EPI) used as an antiulcer agent, was synthesized. Their inhibitory effect was evaluated using Staphylococcus aureus strain SA-1199B overexpressing NorA. By determinations of the MIC of norfloxacin in the presence of these EPIs devoid of intrinsic antibacterial activity and used at 128 microg/ml, and by the checkerboard method, compound 1e (MIC decrease, 16-fold; fractional inhibitory concentration index [SigmaFIC], 0.18) appeared to be more active than compounds 1b to 1d, reserpine, and omeprazole (MIC decrease, eightfold; SigmaFIC, 0.31), followed by compounds 1a and 1f (MIC decrease, fourfold; SigmaFIC, 0.37) and 1g to 1k (MIC decrease, twofold; SigmaFIC, 0.50 to 0.56). By time-kill curves combining norfloxacin (1/4 MIC) and the most efficient EPIs (128 microg/ml), compound 1e persistently restored the bactericidal activity of norfloxacin (inoculum reduction, 3 log(10) CFU/ml at 8 and 24 h), compound 1f led to a delayed but progressive decrease in the number of viable cells, and compounds 1b to 1d and omeprazole acted synergistically (inoculum reduction, 3 log(10) CFU/ml at 8 h but further regrowth), while compound 1a and reserpine slightly enhanced norfloxacin activity. The bacterial uptake of norfloxacin monitored by high-performance liquid chromatography confirmed that compounds 1a to 1f increased antibiotic accumulation, as did reserpine and omeprazole. Since these EPIs did not disturb the Deltapsi and DeltapH, they might directly interact with the pump. A structure-activity relationships study identified the benzimidazole nucleus of omeprazole as the main structural element involved in efflux pump inhibition and highlighted the critical role of the chlorine substituents in the stability and efficiency of compounds 1e to 1f. However, further pharmacomodulation is required to obtain therapeutically applicable

Indole-Induced Activities of β-Lactamase and Efflux Pump Confer Ampicillin Resistance in Pseudomonas putida KT2440

PubMed Central

Kim, Jisun; Shin, Bora; Park, Chulwoo; Park, Woojun

2017-01-01

Indole, which is widespread in microbial communities, has received attention because of its effects on bacterial physiology. Pseudomonas putida and Pseudomonas aeruginosa can acquire ampicillin (Amp) resistance during growth on indole-Amp agar. Transcriptome, mutant, and inhibitor studies have suggested that Amp resistance induced by indole can be attributed to increased gene expression of ttgAB encoding two genes of RND-type multidrug efflux operons and an ampC encoding β-lactamase. Expression, enzyme activities, and mutational analyses indicated that AmpC β-lactamase is important for acquiring Amp resistance of P. putida in the presence of indole. Here, we show, for the first time, that volatile indole increased Amp-resistant cells. Consistent with results of the volatile indole assay, a low concentration of indole in liquid culture promoted growth initially, but led to mutagenesis after indole was depleted, which could not be observed at high indole concentrations. Interestingly, ttgAB and ampC gene expression levels correlate with the concentration of indole, which might explain the low number of Amp-mutated cells in high indole concentrations. The expression levels of genes involved in mutagenesis, namely rpoS, recA, and mutS, were also modulated by indole. Our data indicates that indole reduces Amp-induced heterogeneity by promoting expression of TtgABC or MexAB-OprM efflux pumps and the indole-induced β-lactamase in P. putida and P. aeruginosa. PMID:28352264

Characterization of AcrD, a resistance-nodulation-cell division-type multidrug efflux pump from the fire blight pathogen Erwinia amylovora.

PubMed

Pletzer, Daniel; Weingart, Helge

2014-01-21

Multidrug efflux pumps are membrane translocases that have the ability to extrude a variety of structurally unrelated compounds from the cell. AcrD, a resistance-nodulation-cell division (RND) transporter, was shown to be involved in efflux of highly hydrophilic aminoglycosides and a limited number of amphiphilic compounds in E. coli. Here, a homologue of AcrD in the plant pathogen and causal agent of fire blight disease Erwinia amylovora was identified. The substrate specificity of AcrD was studied by overexpression of the corresponding gene from a high-copy plasmid in E. amylovora Ea1189-3, which is hypersensitive to many drugs due to a deficiency of the major multidrug pump AcrB. AcrD mediated resistance to several amphiphilic compounds including clotrimazole and luteolin, two compounds hitherto not described as substrates of AcrD in enterobacteria. However, AcrD was not able to expel aminoglycosides. An acrD mutant exhibited full virulence on apple rootstock and immature pear fruits. RT-PCR analysis revealed an induction of acrD expression in infected apple tissue but not on pear fruits. Moreover, a direct binding of BaeR, the response regulator of the two-component regulatory system BaeSR, to the acrD promoter was observed as has already been shown in other enterobacteria. AcrD from E. amylovora is involved in resistance to a limited number of amphiphilic compounds, but in contrast to AcrD of E. coli, it is not involved in resistance to aminoglycosides. The expression of acrD was up-regulated by addition of the substrates deoxycholate, naringenin, tetracycline and zinc. AcrD appears to be regulated by the BaeSR two-component system, an envelope stress signal transduction pathway.

Characterization of AcrD, a Resistance-Nodulation-Cell Division-type multidrug efflux pump from the fire blight pathogen Erwinia amylovora

PubMed Central

2014-01-01

Background Multidrug efflux pumps are membrane translocases that have the ability to extrude a variety of structurally unrelated compounds from the cell. AcrD, a resistance-nodulation-cell division (RND) transporter, was shown to be involved in efflux of highly hydrophilic aminoglycosides and a limited number of amphiphilic compounds in E. coli. Here, a homologue of AcrD in the plant pathogen and causal agent of fire blight disease Erwinia amylovora was identified. Results The substrate specificity of AcrD was studied by overexpression of the corresponding gene from a high-copy plasmid in E. amylovora Ea1189-3, which is hypersensitive to many drugs due to a deficiency of the major multidrug pump AcrB. AcrD mediated resistance to several amphiphilic compounds including clotrimazole and luteolin, two compounds hitherto not described as substrates of AcrD in enterobacteria. However, AcrD was not able to expel aminoglycosides. An acrD mutant exhibited full virulence on apple rootstock and immature pear fruits. RT-PCR analysis revealed an induction of acrD expression in infected apple tissue but not on pear fruits. Moreover, a direct binding of BaeR, the response regulator of the two-component regulatory system BaeSR, to the acrD promoter was observed as has already been shown in other enterobacteria. Conclusions AcrD from E. amylovora is involved in resistance to a limited number of amphiphilic compounds, but in contrast to AcrD of E. coli, it is not involved in resistance to aminoglycosides. The expression of acrD was up-regulated by addition of the substrates deoxycholate, naringenin, tetracycline and zinc. AcrD appears to be regulated by the BaeSR two-component system, an envelope stress signal transduction pathway. PMID:24443882

Reversal of efflux mediated antifungal resistance underlies synergistic activity of two monoterpenes with fluconazole.

PubMed

Ahmad, Aijaz; Khan, Amber; Manzoor, Nikhat

2013-01-23

Thymol (THY) and carvacrol (CARV), the principal chemical components of thyme oil have long been known for their wide use in medicine due to antimicrobial and disinfectant properties. This study, however, draws attention to a possible synergistic antifungal effect of these monoterpenes with azole antimycotic-fluconazole. Resistance to azoles in Candida albicans involves over-expression of efflux-pump genes MDR1, CDR1, CDR2 or mutations and over-expression of target gene ERG11. The inhibition of drug efflux pumps is considered a feasible strategy to overcome clinical antifungal resistance. To put forward this approach, we investigated the combination effects of these monoterpenes and FLC against 38 clinically obtained FLC-sensitive, and eleven FLC-resistant Candida isolates. Synergism was observed with combinations of THY-FLC and CARV-FLC evaluated by checkerboard microdilution method and nature of the interactions was calculated by FICI. In addition, antifungal activity was assessed using agar-diffusion and time-kill curves. The drug efflux activity was determined using two dyes, Rhodamine6G (R6G) and fluorescent Hoechst 33342. No significant differences were observed in dye uptakes between FLC-susceptible and resistant isolates, incubated in glucose free buffer. However, a significantly higher efflux was recorded in FLC-resistant isolates when glucose was added. Both monoterpenes inhibited efflux by 70-90%, showing their high potency to block drug transporter pumps. Significant differences, in the expression levels of CDR1 and MDR1, induced by monoterpenes revealed reversal of FLC-resistance. The selectively fungicidal characteristics and ability to restore FLC susceptibility in resistant isolates signify a promising candidature of THY and CARV as antifungal agents in combinational treatments for candidiasis. Copyright © 2012 Elsevier B.V. All rights reserved.

An ATP-driven efflux pump is a novel pathogenicity factor in rice blast disease.

PubMed Central

Urban, M; Bhargava, T; Hamer, J E

1999-01-01

Cells tolerate exposure to cytotoxic compounds through the action of ATP-driven efflux pumps belonging to the ATP-binding cassette (ABC) superfamily of membrane transporters. Phytopathogenic fungi encounter toxic environments during plant invasion as a result of the plant defense response. Here we demonstrate the requirement for an ABC transporter during host infection by the fungal plant pathogen Magnaporthe grisea. The ABC1 gene was identified in an insertional mutagenesis screen for pathogenicity mutants. The ABC1 insertional mutant and a gene-replacement mutant arrest growth and die shortly after penetrating either rice or barley epidermal cells. The ABC1-encoded protein is similar to yeast ABC transporters implicated in multidrug resistance, and ABC1 gene transcripts are inducible by toxic drugs and a rice phytoalexin. However, abc1 mutants are not hypersensitive to antifungal compounds. The non-pathogenic, insertional mutation in ABC1 occurs in the promoter region and dramatically reduces transcript induction by metabolic poisons. These data strongly suggest that M.grisea requires the up-regulation of specific ABC transporters for pathogenesis; most likely to protect itself against plant defense mechanisms. PMID:9927411

Exploring the contribution of efflux on the resistance to fluoroquinolones in clinical isolates of Staphylococcus aureus

PubMed Central

2011-01-01

Background Antimicrobial resistance mediated by efflux systems is still poorly characterized in Staphylococcus aureus, despite the description of several efflux pumps (EPs) for this bacterium. In this work we used several methodologies to characterize the efflux activity of 52 S. aureus isolates resistant to ciprofloxacin collected in a hospital in Lisbon, Portugal, in order to understand the role played by these systems in the resistance to fluoroquinolones. Results Augmented efflux activity was detected in 12 out of 52 isolates and correlated with increased resistance to fluoroquinolones. Addition of efflux inhibitors did not result in the full reversion of the fluoroquinolone resistance phenotype, yet it implied a significant decrease in the resistance levels, regardless of the type(s) of mutation(s) found in the quinolone-resistance determining region of grlA and gyrA genes, which accounted for the remaining resistance that was not efflux-mediated. Expression analysis of the genes coding for the main efflux pumps revealed increased expression only in the presence of inducing agents. Moreover, it showed that not only different substrates can trigger expression of different EP genes, but also that the same substrate can promote a variable response, according to its concentration. We also found isolates belonging to the same clonal type that showed different responses towards drug exposure, thus evidencing that highly related clinical isolates may diverge in the efflux-mediated response to noxious agents. The data gathered by real-time fluorometric and RT-qPCR assays suggest that S. aureus clinical isolates may be primed to efflux antimicrobial compounds. Conclusions The results obtained in this work do not exclude the importance of mutations in resistance to fluoroquinolones in S. aureus, yet they underline the contribution of efflux systems for the emergence of high-level resistance. All together, the results presented in this study show the potential role

NorM, an Erwinia amylovora Multidrug Efflux Pump Involved in In Vitro Competition with Other Epiphytic Bacteria

PubMed Central

Burse, Antje; Weingart, Helge; Ullrich, Matthias S.

2004-01-01

Blossoms are important sites of infection for Erwinia amylovora, the causal agent of fire blight of rosaceous plants. Before entering the tissue, the pathogen colonizes the stigmatic surface and has to compete for space and nutrient resources within the epiphytic community. Several epiphytes are capable of synthesizing antibiotics with which they antagonize phytopathogenic bacteria. Here, we report that a multidrug efflux transporter, designated NorM, of E. amylovora confers tolerance to the toxin(s) produced by epiphytic bacteria cocolonizing plant blossoms. According to sequence comparisons, the single-component efflux pump NorM is a member of the multidrug and toxic compound extrusion protein family. The corresponding gene is widely distributed among E. amylovora strains and related plant-associated bacteria. NorM mediated resistance to the hydrophobic cationic compounds norfloxacin, ethidium bromide, and berberine. A norM mutant was constructed and exhibited full virulence on apple rootstock MM 106. However, it was susceptible to antibiotics produced by epiphytes isolated from apple and quince blossoms. The epiphytes were identified as Pantoea agglomerans by 16S rRNA analysis and were isolated from one-third of all trees examined. The promoter activity of norM was twofold greater at 18°C than at 28°C. The lower temperature seems to be beneficial for host infection because of the availability of moisture necessary for movement of the pathogen to the infection sites. Thus, E. amylovora might employ NorM for successful competition with other epiphytic microbes to reach high population densities, particularly at a lower temperature. PMID:14766544

Effects of L-arabinose efflux on λ Red recombination-mediated gene knockout in multiple-antimicrobial-resistant Salmonella enterica serovar Choleraesuis.

PubMed

Liao, Shi-Wei; Lee, Jen-Jie; Ptak, Christopher P; Wu, Ying-Chen; Hsuan, Shih-Ling; Kuo, Chih-Jung; Chen, Ter-Hsin

2018-03-01

In this study, six swine-derived multiple-antimicrobial-resistant (MAR) strains of Salmonella Choleraesuis (S. Choleraesuis) were demonstrated to possess higher efflux pump activity than the wild-type (WT). L-Arabinose, a common inducer for gene expression, modulated S. Choleraesuis efflux pump activity in a dose-dependent manner. At low L-arabinose concentrations, increasing L-arabinose led to a corresponding increase in fluorophore efflux, while at higher L-arabinose concentrations, increasing L-arabinose decreased fluorophore efflux activity. The WT S. Choleraesuis that lacks TolC (ΔtolC), an efflux protein associated with bacterial antibiotic resistance and virulence, was demonstrated to possess a significantly reduced ability to extrude L-arabinose. Further, due to the rapid export of L-arabinose, an efficient method for recombination-mediated gene knockout, the L-arabinose-inducible bacteriophage λ Red recombinase system, has a reduced recombination frequency (~ 12.5%) in clinically isolated MAR Salmonella strains. An increased recombination frequency (up to 60%) can be achieved using a higher concentration of L-arabinose (fivefold) for genetic manipulation and functional analysis for MAR Salmonella using the λ Red system. The study suggests that L-arabinose serves not only as an inducer of the TolC-dependent efflux system but also acts as a competitive substrate of the efflux system. In addition, understanding the TolC-dependent efflux of L-arabinose should facilitate the optimization of L-arabinose induction in strains with high efflux activity.

Effect of biofilm formation by clinical isolates of Helicobacter pylori on the efflux-mediated resistance to commonly used antibiotics.

PubMed

Attaran, Bahareh; Falsafi, Tahereh; Ghorbanmehr, Nassim

2017-02-21

To evaluate the role of biofilm formation on the resistance of Helicobacter pylori ( H. pylori ) to commonly prescribed antibiotics, the expression rates of resistance genes in biofilm-forming and planktonic cells were compared. A collection of 33 H. pylori isolates from children and adult patients with chronic infection were taken for the present study. The isolates were screened for biofilm formation ability, as well as for polymerase chain reaction (PCR) reaction with HP1165 and hp1165 efflux pump genes. Susceptibilities of the selected strains to antibiotic and differences between susceptibilities of planktonic and biofilm-forming cell populations were determined. Quantitative real-time PCR (qPCR) analysis was performed using 16S rRNA gene as a H. pylori -specific primer, and two efflux pumps-specific primers, hp1165 and hefA . The strains were resistant to amoxicillin, metronidazole, and erythromycin, except for one strain, but they were all susceptible to tetracycline. Minimum bactericidal concentrations of antibiotics in the biofilm-forming cells were significantly higher than those of planktonic cells. qPCR demonstrated that the expression of efflux pump genes was significantly higher in the biofilm-forming cells as compared to the planktonic ones. The present work demonstrated an association between H. pylori biofilm formation and decreased susceptibility to all the antibiotics tested. This decreased susceptibility to antibiotics was associated with enhanced functional activity of two efflux pumps: hp1165 and hefA .

N,N'-disubstituted cinnamamide derivatives potentiate ciprofloxacin activity against overexpressing NorA efflux pump Staphylococcus aureus 1199B strains.

PubMed

Radix, Sylvie; Jordheim, Anne Doléans; Rocheblave, Luc; N'Digo, Serge; Prignon, Anne-Laure; Commun, Carine; Michalet, Serge; Dijoux-Franca, Marie-Geneviève; Mularoni, Angélique; Walchshofer, Nadia

2018-04-25

A multi-step procedure has been described which afforded satisfactory yields of N,N'-disubstituted cinnamamides derived from N-Boc-protected amino acids (Boc-Gly, Boc-Val, Boc-Phe). The key step of this synthesis was a regioselective RedAl reduction of an amide function in presence of a carbamate group. Next, these cinnamamides were evaluated in co-admnistration with ciprofloxacin as efflux pump inhibitors against two S. aureus strains, NorA overexpressing SA1199B and wild type SA1199. In parallel, their intrinsic toxicity was appreciated on human lung fibroblast MRC5 cells. Therefore, the cinnamamide combining both carbamate and indol-3-yl groups, was found to be the most active and one of the less toxic EPI and constituted a promising hit. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Attaching the NorA Efflux Pump Inhibitor INF55 to Methylene Blue Enhances Antimicrobial Photodynamic Inactivation of Methicillin-Resistant Staphylococcus aureus in Vitro and in Vivo.

PubMed

Rineh, Ardeshir; Dolla, Naveen K; Ball, Anthony R; Magana, Maria; Bremner, John B; Hamblin, Michael R; Tegos, George P; Kelso, Michael J

2017-10-13

Antimicrobial photodynamic inactivation (aPDI) uses photosensitizers (PSs) and harmless visible light to generate reactive oxygen species (ROS) and kill microbes. Multidrug efflux systems can moderate the phototoxic effects of PSs by expelling the compounds from cells. We hypothesized that increasing intracellular concentrations of PSs by inhibiting efflux with a covalently attached efflux pump inhibitor (EPI) would enhance bacterial cell phototoxicity and reduce exposure of neighboring host cells to damaging ROS. In this study, we tested the hypothesis by linking NorA EPIs to methylene blue (MB) and examining the photoantimicrobial activity of the EPI-MB hybrids against the human pathogen methicillin-resistant Staphylococcus aureus (MRSA). Photochemical/photophysical and in vitro microbiological evaluation of 16 hybrids carrying four different NorA EPIs attached to MB via four linker types identified INF55-(Ac)en-MB 12 as a lead. Compound 12 showed increased uptake into S. aureus cells and enhanced aPDI activity and wound healing effects (relative to MB) in a murine model of an abrasion wound infected by MRSA. The study supports a new approach for treating localized multidrug-resistant MRSA infections and paves the way for wider exploration of the EPI-PS hybrid strategy in aPDI.

Drug Tolerance in Replicating Mycobacteria Mediated by a Macrophage-Induced Efflux Mechanism

PubMed Central

Adams, Kristin N.; Takaki, Kevin; Connolly, Lynn E.; Wiedenhoft, Heather; Winglee, Kathryn; Humbert, Olivier; Edelstein, Paul H.; Cosma, Christine L.; Ramakrishnan, Lalita

2011-01-01

SUMMARY Treatment of tuberculosis, a complex granulomatous disease, requires long-term multidrug therapy to overcome tolerance, an epigenetic drug resistance that is widely attributed to nonreplicating bacterial subpopulations. Here, we deploy Mycobacterium marinum-infected zebrafish larvae for in vivo characterization of antitubercular drug activity and tolerance. We describe the existence of multi-drug tolerant organisms that arise within days of infection, are enriched in the replicating intracellular population, and are amplified and disseminated by the tuberculous granuloma. Bacterial efflux pumps that are required for intracellular growth mediate this macrophage-induced tolerance. This newly discovered tolerant population also develops when Mycobacterium tuberculosis infects cultured macrophages, suggesting that it contributes to the burden of drug tolerance in human tuberculosis. Efflux pump inhibitors like verapamil reduce this tolerance. Thus, the addition of this currently approved drug, or more specific inhibitors, to standard antitubercular therapy may shorten the duration of curative treatment. PMID:21376383

Overproduction of active efflux pump and variations of OprD dominate in imipenem-resistant Pseudomonas aeruginosa isolated from patients with bloodstream infections in Taiwan.

PubMed

Kao, Cheng-Yen; Chen, Shu-Sheng; Hung, Kuei-Hsiang; Wu, Hsiu-Mei; Hsueh, Po-Ren; Yan, Jing-Jou; Wu, Jiunn-Jong

2016-06-13

The emergence of imipenem-resistant Pseudomonas aeruginosa (IRPA) has become a great concern worldwide. The aim of this study was to investigate resistance mechanisms associated with bloodstream isolated IRPA strains in Taiwan. A total of 78 non-duplicated IRPA isolates were isolated from patients with bloodstream infection. The average prevalence of imipenem-resistance in those isolates was 5.9 % during a 10-year longitudinal surveillance in Taiwan. PFGE results showed high clonal diversity among the 78 isolates. VIM-2, VIM-3, OXA-10, and OXA-17 β-lactamases were identified in 2 (2.6 %), 3 (3.8 %), 2 (2.6 %), and 1 (1.3 %) isolates, respectively. Active efflux pumps, AmpC β-lactamase overproduction, and extended-spectrum AmpC cephalosporinases (ESACs) were found in 58 (74.4 %), 25 (32.1 %) and 15 (19.2 %) of IRPA isolates, respectively. oprD mutations with amino acid substitution, shortened putative loop L7, premature stop codon caused by point mutation, frameshift by nucleotide insertion or deletion, and interruption by insertion sequence were found in 19 (24.4 %), 18 (23.1 %), 15 (19.2 %), 14 (17.9 %), and 10 (12.8 %) of isolates, respectively. This study suggests that alterations in the OprD protein and having an active efflux pump are the main mechanisms associated with bloodstream isolated IRPA. Overproduction of AmpC, ESACs, and the presence of VIM- and OXA-type β-lactamases play additional roles in reduced susceptibility to imipenem in P. aeruginosa isolates in Taiwan.

The Pseudomonas aeruginosa efflux pump MexGHI-OpmD transports a natural phenazine that controls gene expression and biofilm development

PubMed Central

Sakhtah, Hassan; Koyama, Leslie; Zhang, Yihan; Morales, Diana K.; Fields, Blanche L.; Price-Whelan, Alexa; Hogan, Deborah A.; Shepard, Kenneth; Dietrich, Lars E. P.

2016-01-01

Redox-cycling compounds, including endogenously produced phenazine antibiotics, induce expression of the efflux pump MexGHI-OpmD in the opportunistic pathogen Pseudomonas aeruginosa. Previous studies of P. aeruginosa virulence, physiology, and biofilm development have focused on the blue phenazine pyocyanin and the yellow phenazine-1-carboxylic acid (PCA). In P. aeruginosa phenazine biosynthesis, conversion of PCA to pyocyanin is presumed to proceed through the intermediate 5-methylphenazine-1-carboxylate (5-Me-PCA), a reactive compound that has eluded detection in most laboratory samples. Here, we apply electrochemical methods to directly detect 5-Me-PCA and find that it is transported by MexGHI-OpmD in P. aeruginosa strain PA14 planktonic and biofilm cells. We also show that 5-Me-PCA is sufficient to fully induce MexGHI-OpmD expression and that it is required for wild-type colony biofilm morphogenesis. These physiological effects are consistent with the high redox potential of 5-Me-PCA, which distinguishes it from other well-studied P. aeruginosa phenazines. Our observations highlight the importance of this compound, which was previously overlooked due to the challenges associated with its detection, in the context of P. aeruginosa gene expression and multicellular behavior. This study constitutes a unique demonstration of efflux-based self-resistance, controlled by a simple circuit, in a Gram-negative pathogen. PMID:27274079

Use of a combined effect model approach for discriminating between ABCB1- and ABCC1-type efflux activities in native bivalve gill tissue

DOE Office of Scientific and Technical Information (OSTI.GOV)

Faria, Melissa; CESAM & Departamento de Biologia, Universidade de Aveiro, 3810-193 Aveiro; Pavlichenko, Vasiliy

Aquatic organisms, such as bivalves, employ ATP binding cassette (ABC) transporters for efflux of potentially toxic chemicals. Anthropogenic water contaminants can, as chemosensitizers, disrupt efflux transporter function enabling other, putatively toxic compounds to enter the organism. Applying rapid amplification of cDNA ends (RACE) PCR we identified complete cDNAs encoding ABCB1- and ABCC1-type transporter homologs from zebra mussel providing the molecular basis for expression of both transporter types in zebra mussel gills. Further, efflux activities of both transporter types in gills were indicated with dye accumulation assays where efflux of the dye calcein-am was sensitive to both ABCB1- (reversin 205, verapamil)more » and ABCC1- (MK571) type specific inhibitors. The assumption that different inhibitors targeted different efflux pump types was confirmed when comparing measured effects of binary inhibitor compound mixtures in dye accumulation assays with predictions from mixture effect models. Effects by the MK571/reversin 205 mixture corresponded better with independent action, whereas reversin 205/verapamil joint effects were better predicted by the concentration addition model indicating different and equal targets, respectively. The binary mixture approach was further applied to identify the efflux pump type targeted by environmentally relevant chemosensitizing compounds. Pentachlorophenol and musk ketone, which were selected after a pre-screen of twelve compounds that previously had been identified as chemosensitizers, showed mixture effects that corresponded better with concentration addition when combined with reversine 205 but with independent action predictions when combined with MK571 indicating targeting of an ABCB1-type efflux pump by these compounds. - Highlights: • Sequences and function of ABC efflux transporters in bivalve gills were explored. • Full length Dreissena polymorpha abcb1 and abcc1 cDNA sequences were identified. • A

Study on the association between drug‑resistance and gene mutations of the active efflux pump acrAB‑tolC gene and its regulatory genes.

PubMed

Ma, Quan-Ping; Su, Liang; Liu, Jing-Wen; Yao, Ming-Xiao; Yuan, Guang-Ying

2018-06-01

The aim of the present study was to investigate the correlation between the multi‑drug resistance of Shigella flexneri and the drug‑resistant gene cassette carried by integrons; in the meanwhile, to detect the associations between drug‑resistance and gene mutations of the active efflux pump acrAB‑tolC gene and its regulatory genes, including marOR, acrR and soxS. A total of 158 isolates were isolated from the stool samples of 1,026 children with diarrhoea aged 14 years old between May 2012 and October 2015 in Henan. The K‑B method was applied for the determination of drug resistance of Shigella flexneri, and polymerase chain reaction amplification was used for class 1, 2 and 3 integrase genes. Enzyme digestion and sequence analysis were performed for the variable regions of positive strains. Based on the drug sensitivity assessment, multi‑drug resistant strains that were resistant to five or more antibiotics, and sensitive strains were selected for amplification. Their active efflux pump genes, acrA and acrB, and regulatory genes, marOR, acrR and soxS, were selected for sequencing. The results revealed that 91.1% of the 158 strains were multi‑resistant to ampicillin, chloramphenicol, tetracycline and streptomycin, and 69.6% of the strains were multi‑resistant to sulfamethoxazole/trimethoprim. The resistance to ceftazidime, ciprofloxacin and levofloxacin was <32.9%. All strains (100%) were sensitive to cefoxitin, cefoperazone/sulbactam and imipenem. The rate of the class 1 integron positivity was 91.9% (144/158). Among these class 1 integron‑positive strains, 18 strains exhibited the resistance gene cassette dfrV in the variable region of the strain, four strains exhibited dfrA17‑aadA5 in the variable region and 140 strains exhibited blaOXA‑30‑aadA1 in the variable region. Four strains showed no resistance gene in the variable regions. The rate of class 2 integron positivity was 86.1% (136/158), and all positive strains harboured the

High-level tetracycline resistance mediated by efflux pumps Tet(A) and Tet(A)-1 with two start codons.

PubMed

Wang, Weixia; Guo, Qinglan; Xu, Xiaogang; Sheng, Zi-ke; Ye, Xinyu; Wang, Minggui

2014-11-01

Efflux is the most common mechanism of tetracycline resistance. Class A tetracycline efflux pumps, which often have high prevalence in Enterobacteriaceae, are encoded by tet(A) and tet(A)-1 genes. These genes have two potential start codons, GTG and ATG, located upstream of the genes. The purpose of this study was to determine the start codon(s) of the class A tetracycline resistance (tet) determinants tet(A) and tet(A)-1, and the tetracycline resistance level they mediated. Conjugation, transformation and cloning experiments were performed and the genetic environment of tet(A)-1 was analysed. The start codons in class A tet determinants were investigated by site-directed mutagenesis of ATG and GTG, the putative translation initiation codons. High-level tetracycline resistance was transferred from the clinical strain of Klebsiella pneumoniae 10-148 containing tet(A)-1 plasmid pHS27 to Escherichia coli J53 by conjugation. The transformants harbouring recombinant plasmids that carried tet(A) or tet(A)-1 exhibited tetracycline MICs of 256-512 µg ml(-1), with or without tetR(A). Once the ATG was mutated to a non-start codon, the tetracycline MICs were not changed, while the tetracycline MICs decreased from 512 to 64 µg ml(-1) following GTG mutation, and to ≤4 µg ml(-1) following mutation of both GTG and ATG. It was presumed that class A tet determinants had two start codons, which are the primary start codon GTG and secondary start codon ATG. Accordingly, two putative promoters were predicted. In conclusion, class A tet determinants can confer high-level tetracycline resistance and have two start codons. © 2014 The Authors.

Efflux pump-deficient mutants as a platform to search for microbes that produce antibiotics

PubMed Central

Molina-Santiago, Carlos; Udaondo, Zulema; Daddaoua, Abdelali; Roca, Amalia; Martín, Jesús; Pérez-Victoria, Ignacio; Reyes, Fernando; Ramos, Juan-Luis

2015-01-01

Pseudomonas putida DOT-T1E-18 is a strain deficient in the major antibiotic efflux pump (TtgABC) that exhibits an overall increased susceptibility to a wide range of drugs when compared with the wild-type strain. We used this strain as a platform to search for microbes able to produce antibiotics that inhibit growth. A collection of 2400 isolates from soil, sediments and water was generated and a drop assay developed to identify, via growth inhibition halos, strains that prevent the growth of DOT-T1E-18 on solid Luria–Bertani plates. In this study, 35 different isolates that produced known and unknown antibiotics were identified. The most potent inhibitor of DOT-T1E-18 growth was an isolate named 250J that, through multi-locus sequence analysis, was identified as a Pseudomonas sp. strain. Culture supernatants of 250J contain four different xantholysins that prevent growth of Gram-positive bacteria, Gram-negative and fungi. Two of the xantholysins were produced in higher concentrations and purified. Xantholysin A was effective against Bacillus, Lysinibacillus and Rhodococcus strains, and the effect against these microbes was enhanced when used in combination with other antibiotics such as ampicillin, gentamicin and kanamycin. Xantholysin C was also efficient against Gram-positive bacteria and showed an interesting antimicrobial effect against Pseudomonas strains, and a synergistic inhibitory effect with ampicillin, chloramphenicol and gentamicin. PMID:26059350

Genome analysis of urease positive Serratia marcescens, co-producing SRT-2 and AAC(6')-Ic with multidrug efflux pumps for antimicrobial resistance.

PubMed

Srinivasan, Vijaya Bharathi; Rajamohan, Govindan

2018-04-05

In this study, we present the genome sequence of Serratia marcescens SM03, recovered from a human gut in India. The final assembly consists of 26 scaffolds (4620 coding DNA sequences, 5.08 Mb, 59.6% G + C ratio) and 79 tRNA genes. Analysis identified novel genes associated with lactose utilization, virulence, P-loop GTPases involved in urease production, CFA/I fimbriae apparatus and Yersinia - type CRISPR proteins. Antibiotic susceptibility testing indicated drug tolerant phenotype and inhibition assays demonstrated involvement of extrusion in resistance. Presence of enzymes SRT-2, AAC(6')-Ic, with additional Ybh transporter and EamA-like efflux pumps signifies the genetic plasticity observed in S. marcescens SM03. Copyright © 2018 Elsevier Inc. All rights reserved.

Contribution of Resistance-Nodulation-Division Efflux Pump Operon smeU1-V-W-U2-X to Multidrug Resistance of Stenotrophomonas maltophilia ▿

PubMed Central

Chen, Chao-Hsien; Huang, Chiang-Ching; Chung, Tsao-Chuen; Hu, Rouh-Mei; Huang, Yi-Wei; Yang, Tsuey-Ching

2011-01-01

KJ09C, a multidrug-resistant mutant of Stenotrophomonas maltophilia KJ, was generated by in vitro selection with chloramphenicol. The multidrug-resistant phenotype of KJ09C was attributed to overexpression of a resistance nodulation division (RND)-type efflux system encoded by an operon consisting of five genes: smeU1, smeV, smeW, smeU2, and smeX. Proteins encoded by smeV, smeW, and smeX were similar to the membrane fusion protein, RND transporter, and outer membrane protein, respectively, of known RND-type systems. The proteins encoded by smeU1 and smeU2 were found to belong to the family of short-chain dehydrogenases/reductases. Mutant KJ09C exhibited increased resistance to chloramphenicol, quinolones, and tetracyclines and susceptibility to aminoglycosides; susceptibility to β-lactams and erythromycin was not affected. The expression of the smeU1-V-W-U2-X operon was regulated by the divergently transcribed LysR-type regulator gene smeRv. Overexpression of the SmeVWX pump contributed to the acquired resistance to chloramphenicol, quinolones, and tetracyclines. Inactivation of smeV and smeW completely abolished the activity of the SmeVWX pump, whereas inactivation of smeX alone decreased the activity of the SmeVWX pump. The enhanced aminoglycoside susceptibility observed in KJ09C resulted from SmeX overexpression. PMID:21930878

Role of EfrAB efflux pump in biocide tolerance and antibiotic resistance of Enterococcus faecalis and Enterococcus faecium isolated from traditional fermented foods and the effect of EDTA as EfrAB inhibitor.

PubMed

Lavilla Lerma, Leyre; Benomar, Nabil; Valenzuela, Antonio Sánchez; Casado Muñoz, María del Carmen; Gálvez, Antonio; Abriouel, Hikmate

2014-12-01

Enterococcus faecalis and Enterococcus faecium isolated from various traditional fermented foods of both animal and vegetable origins have shown multidrug resistance to several antibiotics and tolerance to biocides. Reduced susceptibility was intra and inter-species dependent and was due to specific and unspecific mechanisms such as efflux pumps. EfrAB, a heterodimeric ABC transporter efflux pump, was detected in 100% of multidrug resistant (MDR) E. faecalis strains and only in 12% of MDR E. faecium strains. EfrAB expression was induced by half of minimum inhibitory concentration (MIC) of gentamicin, streptomycin and chloramphenicol. However, expression of efrA and efrB genes was highly dependent on the strain tested and on the antimicrobial used. Our results indicated that 3 mM EDTA highly reduced the MICs of almost all drugs tested. Nevertheless, the higher reductions (>8 folds) were obtained with gentamicin, streptomycin, chlorhexidine and triclosan. Reductions of MICs were correlated with down-regulation of EfrAB expression (10-140 folds) in all three MDR enterococci strains. This is the first report describing the role of EfrAB in the efflux of antibiotics and biocides which reflect also the importance of EfrAB in multidrug resistance in enterococci. EDTA used at low concentration as food preservative could be one of the best choices to prevent spread of multidrug resistant enterococci throughout food chain by decreasing EfrAB expression. EfrAB could be an attractive target not only in enterococci present in food matrix but also those causing infections as well by using EDTA as therapeutic agent in combination with low doses of antibiotics. Copyright © 2014 Elsevier Ltd. All rights reserved.

The drug efflux pump Pgp1 in pro-inflammatory lymphocytes is a target for novel treatment strategies in COPD.

PubMed

Hodge, Greg; Holmes, Mark; Jersmann, Hubertus; Reynolds, Paul N; Hodge, Sandra

2013-06-03

Pro-inflammatory/cytotoxic T cells (IFNγ, TNFα, granzyme B+) are increased in the peripheral circulation in COPD. NKT-like and NK cells are effector lymphocytes that we have also shown to be major sources of pro-inflammatory cytokines and granzymes. P-glycoprotein 1 (Pgp1) is a transmembrane efflux pump well characterised in drug resistant cancer cells. We hypothesized that Pgp1 would be increased in peripheral blood T, NKT-like and NK cells in patients with COPD, and that this would be accompanied by increased expression of IFNγ, TNFα and granzyme B. We further hypothesized that treatment with cyclosporine A, a Pgp1 inhibitor, would render cells more sensitive to treatment with corticosteroids. Pgp1, granzyme B, IFNγ and TNFα expression were measured in peripheral blood T, NK and NKT-like cells from COPD patients and control subjects (± cyclosporine A and prednisolone) following in vitro stimulation and results correlated with uptake of efflux dye Calcein-AM using flow cytometry. There was increased Pgp1 expression by peripheral blood T, NKT-like and NK cells co-expressing IFNγ, TNFα and granzyme B in COPD patients compared with controls (e.g. %IFNγ/Pgp1 T, NKT-like, NK for COPD (Control): 25(6), 54(27), 39(23)). There was an inverse correlation between Pgp1 expression and Calcein-AM uptake. Treatment with 2.5 ng/ml cylosporin A and10-6 M prednisolone resulted in synergistic inhibition of pro-inflammatory cytokines in Pgp1 + cells (p < 0.05 for all). Treatment strategies that target Pgp1 in T, NKT-like and NK cells may reduce systemic inflammatory mediators in COPD and improve patient morbidity.

Efflux pump-deficient mutants as a platform to search for microbes that produce antibiotics.

PubMed

Molina-Santiago, Carlos; Udaondo, Zulema; Daddaoua, Abdelali; Roca, Amalia; Martín, Jesús; Pérez-Victoria, Ignacio; Reyes, Fernando; Ramos, Juan-Luis

2015-07-01

Pseudomonas putida DOT-T1E-18 is a strain deficient in the major antibiotic efflux pump (TtgABC) that exhibits an overall increased susceptibility to a wide range of drugs when compared with the wild-type strain. We used this strain as a platform to search for microbes able to produce antibiotics that inhibit growth. A collection of 2400 isolates from soil, sediments and water was generated and a drop assay developed to identify, via growth inhibition halos, strains that prevent the growth of DOT-T1E-18 on solid Luria-Bertani plates. In this study, 35 different isolates that produced known and unknown antibiotics were identified. The most potent inhibitor of DOT-T1E-18 growth was an isolate named 250J that, through multi-locus sequence analysis, was identified as a Pseudomonas sp. strain. Culture supernatants of 250J contain four different xantholysins that prevent growth of Gram-positive bacteria, Gram-negative and fungi. Two of the xantholysins were produced in higher concentrations and purified. Xantholysin A was effective against Bacillus, Lysinibacillus and Rhodococcus strains, and the effect against these microbes was enhanced when used in combination with other antibiotics such as ampicillin, gentamicin and kanamycin. Xantholysin C was also efficient against Gram-positive bacteria and showed an interesting antimicrobial effect against Pseudomonas strains, and a synergistic inhibitory effect with ampicillin, chloramphenicol and gentamicin. © 2015 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Boeravinone B, A Novel Dual Inhibitor of NorA Bacterial Efflux Pump of Staphylococcus aureus and Human P-Glycoprotein, Reduces the Biofilm Formation and Intracellular Invasion of Bacteria.

PubMed

Singh, Samsher; Kalia, Nitin P; Joshi, Prashant; Kumar, Ajay; Sharma, Parduman R; Kumar, Ashok; Bharate, Sandip B; Khan, Inshad A

2017-01-01

This study elucidated the role of boeravinone B, a NorA multidrug efflux pump inhibitor, in biofilm inhibition. The effects of boeravinone B plus ciprofloxacin, a NorA substrate, were evaluated in NorA-overexpressing, wild-type, and knocked-out Staphylococcus aureus (SA-1199B, SA-1199, and SA-K1758, respectively). The mechanism of action was confirmed using the ethidium bromide accumulation and efflux assay. The role of boeravinone B as a human P -glycoprotein ( P -gp) inhibitor was examined in the LS-180 (colon cancer) cell line. Moreover, its role in the inhibition of biofilm formation and intracellular invasion of S. aureus in macrophages was studied. Boeravinone B reduced the minimum inhibitory concentration (MIC) of ciprofloxacin against S. aureus and its methicillin-resistant strains; the effect was stronger in SA-1199B. Furthermore, time-kill kinetics revealed that boeravinone B plus ciprofloxacin, at subinhibitory concentration (0.25 × MIC), is as equipotent as that at the MIC level. This combination also had a reduced mutation prevention concentration. Boeravinone B reduced the efflux of ethidium bromide and increased the accumulation, thus strengthening the role as a NorA inhibitor. Biofilm formation was reduced by four-eightfold of the minimal biofilm inhibitory concentration of ciprofloxacin, effectively preventing bacterial entry into macrophages. Boeravinone B effectively inhibited P -gp with half maximal inhibitory concentration (IC 50 ) of 64.85 μM. The study concluded that boeravinone B not only inhibits the NorA-mediated efflux of fluoroquinolones but also considerably inhibits the biofilm formation of S. aureus. Its P -gp inhibition activity demonstrates its potential as a bioavailability and bioefficacy enhancer.

Boeravinone B, A Novel Dual Inhibitor of NorA Bacterial Efflux Pump of Staphylococcus aureus and Human P-Glycoprotein, Reduces the Biofilm Formation and Intracellular Invasion of Bacteria

PubMed Central

Singh, Samsher; Kalia, Nitin P.; Joshi, Prashant; Kumar, Ajay; Sharma, Parduman R.; Kumar, Ashok; Bharate, Sandip B.; Khan, Inshad A.

2017-01-01

This study elucidated the role of boeravinone B, a NorA multidrug efflux pump inhibitor, in biofilm inhibition. The effects of boeravinone B plus ciprofloxacin, a NorA substrate, were evaluated in NorA-overexpressing, wild-type, and knocked-out Staphylococcus aureus (SA-1199B, SA-1199, and SA-K1758, respectively). The mechanism of action was confirmed using the ethidium bromide accumulation and efflux assay. The role of boeravinone B as a human P-glycoprotein (P-gp) inhibitor was examined in the LS-180 (colon cancer) cell line. Moreover, its role in the inhibition of biofilm formation and intracellular invasion of S. aureus in macrophages was studied. Boeravinone B reduced the minimum inhibitory concentration (MIC) of ciprofloxacin against S. aureus and its methicillin-resistant strains; the effect was stronger in SA-1199B. Furthermore, time–kill kinetics revealed that boeravinone B plus ciprofloxacin, at subinhibitory concentration (0.25 × MIC), is as equipotent as that at the MIC level. This combination also had a reduced mutation prevention concentration. Boeravinone B reduced the efflux of ethidium bromide and increased the accumulation, thus strengthening the role as a NorA inhibitor. Biofilm formation was reduced by four–eightfold of the minimal biofilm inhibitory concentration of ciprofloxacin, effectively preventing bacterial entry into macrophages. Boeravinone B effectively inhibited P-gp with half maximal inhibitory concentration (IC50) of 64.85 μM. The study concluded that boeravinone B not only inhibits the NorA-mediated efflux of fluoroquinolones but also considerably inhibits the biofilm formation of S. aureus. Its P-gp inhibition activity demonstrates its potential as a bioavailability and bioefficacy enhancer. PMID:29046665

Heat pumps in the PESAG supply district

NASA Astrophysics Data System (ADS)

Osterhus, A.

1980-04-01

The paper examines the feasibility of using large scale heat pumps in the PESAG (Paderborner Elektrizitaetswerk und Strassenbahn AG) power supply district. It is shown that due to favorable geological factors in the district which allow the tapping of ground water, the market share for heat pumps will increase steadily. Topics discussed include: calculation of electricity consumption, operating experiences with heat pumps in one- and two-family houses, heat pumps in multifamily houses, and industrially used systems.

A D-octapeptide drug efflux pump inhibitor acts synergistically with azoles in a murine oral candidiasis infection model.

PubMed

Hayama, Kazumi; Ishibashi, Hiroko; Ishijima, Sanae A; Niimi, Kyoko; Tansho, Shigeru; Ono, Yasuo; Monk, Brian C; Holmes, Ann R; Harding, David R K; Cannon, Richard D; Abe, Shigeru

2012-03-01

Clinical management of patients undergoing treatment of oropharyngeal candidiasis with azole antifungals can be impaired by azole resistance. High-level azole resistance is often caused by the overexpression of Candida albicans efflux pump Cdr1p. Inhibition of this pump therefore represents a target for combination therapies that reverse azole resistance. We assessed the therapeutic potential of the D-octapeptide derivative RC21v3, a Cdr1p inhibitor, in the treatment of murine oral candidiasis caused by either the azole-resistant C. albicans clinical isolate MML611 or its azole-susceptible parental strain MML610. RC21v3, fluconazole (FLC), or a combination of both drugs were administered orally to immunosuppressed ICR mice at 3, 24, and 27 h after oral inoculation with C. albicans. FLC protected the mice inoculated with MML610 from oral candidiasis, but was only partially effective in MML611-infected mice. The co-application of RC21v3 (0.02 μmol per dose) potentiated the therapeutic performance of FLC for mice infected with either strain. It caused a statistically significant decrease in C. albicans cfu isolated from the oral cavity of the infected mice and reduced oral lesions. RC21v3 also enhanced the therapeutic activity of itraconazole against MML611 infection. These results indicate that RC21v3 in combination with azoles has potential as a therapy against azole-resistant oral candidiasis. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

IP/sub 3/ stimulates CA/sup + +/ efflux from fusogenic carrot protoplasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rincon, M.; Boss, W.F.

1986-04-01

Polyphosphoinositide breakdown plays an important role in signal transduction in animal cells (Berridge and Irvine, 1984, Nature, 312:315). Upon stimulation, phospholipase C hydrolyzes phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate (IP/sub 3/) and diacylglycerol both of which act as cellular second messengers. IP/sub 3/ mobilizes Ca/sup + +/ from internal stores, hence the cytosolic free Ca/sup + +/ concentration increases and those physiological activities regulated by Ca/sup + +/ are stimulated. To test if plant cells also responded to IP/sub 3/, Ca/sup + +/ efflux studies were done with fusogenic carrot protoplasts released in EGTA. The protoplasts were preloaded with /sup 45/Ca/supmore » + +/ placed in a Ca/sup + +/-free medium, and efflux determined as /sup 45/Ca/sup + +/ loss from the protoplasts. IP/sub 3/ (10-20..mu..M) caused enhanced /sup 45/Ca/sup + +/ efflux and the response was sustained for at least 15 min. In plants, as in animals, the observed IP/sub 3/-enhanced /sup 45/Ca/sup + +/ efflux suggested that IP/sub 3/ released Ca/sup + +/ from internal stores, and the increased free cytosolic Ca/sup + +/ activated Ca/sup + +/ pumping mechanisms which restored the Ca/sup + +/ concentration in the cytosol to the normal level.« less

Haitian Tap-Taps

ERIC Educational Resources Information Center

Sterling, Joan

2011-01-01

In the small island country of Haiti, colorful taxis transport the natives to the market. Although the taxis may be crowded with people, goods, and even livestock, it is considered a luxury to ride rather than go on foot. The children's picture book, "Tap-Tap," is a wonderful introduction to the culture of this land. The name…

Anti-Campylobacter activity of resveratrol and an extract from waste Pinot noir grape skins and seeds, and resistance of Camp. jejuni planktonic and biofilm cells, mediated via the CmeABC efflux pump.

PubMed

Klančnik, A; Šikić Pogačar, M; Trošt, K; Tušek Žnidarič, M; Mozetič Vodopivec, B; Smole Možina, S

2017-01-01

To define anti-Campylobacter jejuni activity of an extract from waste skins and seeds of Pinot noir grapes (GSS), resveratrol and possible resistance mechanisms, and the influence of these on Camp. jejuni morphology. Using gene-specific knock-out Camp. jejuni mutants and an efflux pump inhibitor, we showed CmeABC as the most active efflux pump for extrusion across the outer membrane of GSS extract and resveratrol. Using polystyrene surface and pig small intestine epithelial (PSI) and human foetal small intestine (H4) cell lines, GSS extract shows an efficient inhibition of adhesion of Camp. jejuni to these abiotic and biotic surfaces. Low doses of GSS extract can inhibit Camp. jejuni adhesion to polystyrene surfaces and to PSI and H4 cells, and can thus modulate Camp. jejuni invasion and intracellular survival. An understanding of the activities of GSS extract and resveratrol as bacterial growth inhibitors and the specific mechanisms of cell accumulation is crucial for our understanding of Camp. jejuni resistance. GSS extract inhibition of Camp. jejuni adhesion to abiotic and biotic surfaces provides a further step towards the application of new innovative strategies to control Campylobacter contamination and infection via the food chain. © 2016 The Society for Applied Microbiology.

Microbial Efflux Systems and Inhibitors: Approaches to Drug Discovery and the Challenge of Clinical Implementation

PubMed Central

Kourtesi, Christina; Ball, Anthony R; Huang, Ying-Ying; Jachak, Sanjay M; Vera, D Mariano A; Khondkar, Proma; Gibbons, Simon; Hamblin, Michael R; Tegos, George P

2013-01-01

Conventional antimicrobials are increasingly ineffective due to the emergence of multidrug-resistance among pathogenic microorganisms. The need to overcome these deficiencies has triggered exploration for novel and unconventional approaches to controlling microbial infections. Multidrug efflux systems (MES) have been a profound obstacle in the successful deployment of antimicrobials. The discovery of small molecule efflux system blockers has been an active and rapidly expanding research discipline. A major theme in this platform involves efflux pump inhibitors (EPIs) from natural sources. The discovery methodologies and the available number of natural EPI-chemotypes are increasing. Advances in our understanding of microbial physiology have shed light on a series of pathways and phenotypes where the role of efflux systems is pivotal. Complementing existing antimicrobial discovery platforms such as photodynamic therapy (PDT) with efflux inhibition is a subject under investigation. This core information is a stepping stone in the challenge of highlighting an effective drug development path for EPIs since the puzzle of clinical implementation remains unsolved. This review summarizes advances in the path of EPI discovery, discusses potential avenues of EPI implementation and development, and underlines the need for highly informative and comprehensive translational approaches. PMID:23569468

Inhibitors of bacterial multidrug efflux pumps from the resin glycosides of Ipomoea murucoides.

PubMed

Chérigo, Lilia; Pereda-Miranda, Rogelio; Fragoso-Serrano, Mabel; Jacobo-Herrera, Nadia; Kaatz, Glenn W; Gibbons, Simon

2008-06-01

A reinvestigation of the CHCl 3-soluble extract from flowers of the Mexican medicinal arborescent morning glory, Ipomoea murucoides, through preparative-scale recycling HPLC, yielded six new pentasaccharides, murucoidins VI-XI (1- 6), as well as the known pescaprein III (7), stoloniferin I (8), and murucoidins I-V (9- 13). Their structures were characterized through the interpretation of their NMR spectroscopic and FABMS data. Compounds 1-6 were found to be macrolactones of three known glycosidic acids identified as simonic acids A and B, and operculinic acid A, with different fatty acids esterifying the same positions, C-2 on the second rhamnose unit and C-4 on the third rhamnose moiety. The lactonization site of the aglycone was placed at C-2 or C-3 of the second saccharide unit. The esterifying residues were composed of two short-chain fatty acids, 2-methylpropanoic and (2S)-methylbutyric acids, and two long-chain fatty acids, n-dodecanoic (lauric) acid and the new (8R)-(-)-8-hydroxydodecanoic acid. For the latter residue, its absolute configuration was determined by analysis of its Mosher ester derivatives. All members of the murucoidin series exerted a potentiation effect of norfloxacin against the NorA overexpressing Staphylococcus aureus strain SA-1199B by increasing the activity 4-fold (8 microg/mL from 32 microg/mL) at concentrations of 5-25 microg/mL. Stoloniferin I (8) enhanced norfloxacin activity 8-fold when incorporated at a concentration of 5 microg/mL. Therefore, this type of amphipathic oligosaccharide could be developed further to provide more potent inhibitors of this multidrug efflux pump.

Abdominal tap

MedlinePlus

Peritoneal tap; Paracentesis; Ascites - abdominal tap; Cirrhosis - abdominal tap; Malignant ascites - abdominal tap ... abdominal cavity ( most often cancer of the ovaries ) Cirrhosis of the liver Damaged bowel Heart disease Infection ...

Further evidence for a potassium-like action of lithium ions on sodium efflux in frog skeletal muscle

PubMed Central

Beaugé, L. A.; Ortiz, Olga

1972-01-01

1. The efflux of labelled sodium as well as net sodium and lithium changes were studied in aged high sodium sartorius muscles of the South American frog Leptodactilus ocelatus. 2. In the presence of 2·5 mM potassium in the media, the replacement of external sodium with lithium or magnesium resulted in an increase in sodium efflux. The magnitude of such increase was always larger in lithium. 3. With the absence of potassium in the media, the response of sodium efflux to replacement of external sodium varied with the cation used as a substitute. In lithium Ringer there was always a noticeable increase, whereas in magnesium there was always a marked reduction. The same results were observed when calcium was substituted for magnesium. 4. The replacement of 60 mM external sodium with sucrose did not prevent the stimulating effect of 5 mM potassium on sodium efflux, nor the inhibitory action of 10-4 M ouabain. This indicates that neither sucrose by itself, nor the lowering of the ionic strength, modified to an appreciable extent the function of the sodium pump. 5. Net sodium extrusion took place against an electrochemical gradient in potassium-free — 50 mM sodium — mM lithium Ringer. About 75% of this efflux was ouabain sensitive. 6. Muscles made both sodium and lithium rich and incubated in potassium-free — 60 mM sodium — 50 mM lithium Ringer also showed net sodium extrusion against an electrochemical gradient, which was 85% ouabain sensitive. This extrusion took place even under conditions where the changes in free energy favouring lithium entry were always lower than the changes in free energy opposing sodium going out. This indicates that a sodium-lithium exchange by a counter-transport process is unlikely. 7. External potassium reduced the ouabain sensitive lithium influx in muscles incubated in lithium Ringer. The values found were 5·90 ± 0·39 μ-mole/g.hr and 2·66 ± 0·43 μmole/g.hr in potassium-free and 15 mM potassium respectively. At the same

Efflux-mediated antimicrobial resistance.

PubMed

Poole, Keith

2005-07-01

Antibiotic resistance continues to plague antimicrobial chemotherapy of infectious disease. And while true biocide resistance is as yet unrealized, in vitro and in vivo episodes of reduced biocide susceptibility are common and the history of antibiotic resistance should not be ignored in the development and use of biocidal agents. Efflux mechanisms of resistance, both drug specific and multidrug, are important determinants of intrinsic and/or acquired resistance to these antimicrobials, with some accommodating both antibiotics and biocides. This latter raises the spectre (as yet generally unrealized) of biocide selection of multiple antibiotic-resistant organisms. Multidrug efflux mechanisms are broadly conserved in bacteria, are almost invariably chromosome-encoded and their expression in many instances results from mutations in regulatory genes. In contrast, drug-specific efflux mechanisms are generally encoded by plasmids and/or other mobile genetic elements (transposons, integrons) that carry additional resistance genes, and so their ready acquisition is compounded by their association with multidrug resistance. While there is some support for the latter efflux systems arising from efflux determinants of self-protection in antibiotic-producing Streptomyces spp. and, thus, intended as drug exporters, increasingly, chromosomal multidrug efflux determinants, at least in Gram-negative bacteria, appear not to be intended as drug exporters but as exporters with, perhaps, a variety of other roles in bacterial cells. Still, given the clinical significance of multidrug (and drug-specific) exporters, efflux must be considered in formulating strategies/approaches to treating drug-resistant infections, both in the development of new agents, for example, less impacted by efflux and in targeting efflux directly with efflux inhibitors.

[Molecular evolution of the sulphite efflux gene SSU1 in Saccharomyces cerevisiae].

PubMed

Peng, Li-Xin; Sun, Fei-Fei; Huang, Yan-Yan; Li, Zhen-Chong

2013-11-01

The SSU1 gene encoding a membrane sulfite pump is a main facilitator invovled in sulfite efflux. In Saccharomyce cerevisiae, various range of resistance to sulfite was observed among strains. To explore the evolution traits of SSU1 gene, the population data of S. cerevisiae were collected and analyzed. The phylogenetic analysis indicated that S. cerevisiae population can be classified into three sub-populations, and the positive selection was detected in population by McDonald-Kreitman test. The anaylsis of Ka/Ks ratios further showed that S. cerevisiae sub-population was undergoing positive selection. This finding was also supported by PAML branch model. Nine potential positive selection sites were predicted by branch-site model, and four sites exclusively belong to the sub-population under positive seletion. The data from ssulp protein structure demonstrated that three sites are substitutions between polar and hydrophobic amino acids, and only one site of substitutaion from basic amino acid to basic amino acid (345R/K). Because amino acid pKa values are crucial for sulfite pump to maintain their routine function, positive selection of these amino acid substitutions might affect sulfite efflux efficient.

Expression of multidrug resistance efflux pump gene norA is iron responsive in Staphylococcus aureus.

PubMed

Deng, Xin; Sun, Fei; Ji, Quanjiang; Liang, Haihua; Missiakas, Dominique; Lan, Lefu; He, Chuan

2012-04-01

Staphylococcus aureus utilizes efflux transporter NorA to pump out a wide range of structurally dissimilar drugs, conferring low-level multidrug resistance. The regulation of norA expression has yet to be fully understood although past studies have revealed that this gene is under the control of the global transcriptional regulator MgrA and the two-component system ArlRS. To identify additional regulators of norA, we screened a transposon library in strain Newman expressing the transcriptional fusion norA-lacZ for altered β-galactosidase activity. We identify a transposon insertion in fhuB, a gene that encodes a ferric hydroxamate uptake system permease, and propose that the norA transcription is iron responsive. In agreement with this observation, addition of FeCl(3) repressed the induction of norA-lacZ, suggesting that bacterial iron uptake plays an important role in regulating norA transcription. In addition, a fur (ferric uptake regulator) deletion exhibited compromised norA transcription and reduced resistance to quinolone compared to the wild-type strain, indicating that fur functions as a positive regulator of norA. A putative Fur box identified in the promoter region of norA was confirmed by electrophoretic mobility shift and DNase I footprint assays. Finally, by employing a siderophore secretion assay, we reveal that NorA may contribute to the export of siderophores. Collectively, our experiments uncover some novel interactions between cellular iron level and norA regulation in S. aureus.

Expression of Multidrug Resistance Efflux Pump Gene norA Is Iron Responsive in Staphylococcus aureus

PubMed Central

Deng, Xin; Sun, Fei; Ji, Quanjiang; Liang, Haihua; Missiakas, Dominique; Lan, Lefu

2012-01-01

Staphylococcus aureus utilizes efflux transporter NorA to pump out a wide range of structurally dissimilar drugs, conferring low-level multidrug resistance. The regulation of norA expression has yet to be fully understood although past studies have revealed that this gene is under the control of the global transcriptional regulator MgrA and the two-component system ArlRS. To identify additional regulators of norA, we screened a transposon library in strain Newman expressing the transcriptional fusion norA-lacZ for altered β-galactosidase activity. We identify a transposon insertion in fhuB, a gene that encodes a ferric hydroxamate uptake system permease, and propose that the norA transcription is iron responsive. In agreement with this observation, addition of FeCl3 repressed the induction of norA-lacZ, suggesting that bacterial iron uptake plays an important role in regulating norA transcription. In addition, a fur (ferric uptake regulator) deletion exhibited compromised norA transcription and reduced resistance to quinolone compared to the wild-type strain, indicating that fur functions as a positive regulator of norA. A putative Fur box identified in the promoter region of norA was confirmed by electrophoretic mobility shift and DNase I footprint assays. Finally, by employing a siderophore secretion assay, we reveal that NorA may contribute to the export of siderophores. Collectively, our experiments uncover some novel interactions between cellular iron level and norA regulation in S. aureus. PMID:22267518

Quantitative structure activity relationship (QSAR) of piperine analogs for bacterial NorA efflux pump inhibitors.

PubMed

Nargotra, Amit; Sharma, Sujata; Koul, Jawahir Lal; Sangwan, Pyare Lal; Khan, Inshad Ali; Kumar, Ashwani; Taneja, Subhash Chander; Koul, Surrinder

2009-10-01

Quantitative structure activity relationship (QSAR) analysis of piperine analogs as inhibitors of efflux pump NorA from Staphylococcus aureus has been performed in order to obtain a highly accurate model enabling prediction of inhibition of S. aureus NorA of new chemical entities from natural sources as well as synthetic ones. Algorithm based on genetic function approximation method of variable selection in Cerius2 was used to generate the model. Among several types of descriptors viz., topological, spatial, thermodynamic, information content and E-state indices that were considered in generating the QSAR model, three descriptors such as partial negative surface area of the compounds, area of the molecular shadow in the XZ plane and heat of formation of the molecules resulted in a statistically significant model with r(2)=0.962 and cross-validation parameter q(2)=0.917. The validation of the QSAR models was done by cross-validation, leave-25%-out and external test set prediction. The theoretical approach indicates that the increase in the exposed partial negative surface area increases the inhibitory activity of the compound against NorA whereas the area of the molecular shadow in the XZ plane is inversely proportional to the inhibitory activity. This model also explains the relationship of the heat of formation of the compound with the inhibitory activity. The model is not only able to predict the activity of new compounds but also explains the important regions in the molecules in quantitative manner.

In Vitro Selection of ramR and soxR Mutants Overexpressing Efflux Systems by Fluoroquinolones as Well as Cefoxitin in Klebsiella pneumoniae▿

PubMed Central

Bialek-Davenet, Suzanne; Marcon, Estelle; Leflon-Guibout, Véronique; Lavigne, Jean-Philippe; Bert, Frédéric; Moreau, Richard; Nicolas-Chanoine, Marie-Hélène

2011-01-01

The relationship between efflux system overexpression and cross-resistance to cefoxitin, quinolones, and chloramphenicol has recently been reported in Klebsiella pneumoniae. In 3 previously published clinical isolates and 17 in vitro mutants selected with cefoxitin or fluoroquinolones, mutations in the potential regulator genes of the AcrAB efflux pump (acrR, ramR, ramA, marR, marA, soxR, soxS, and rob) were searched, and their impacts on efflux-related antibiotic cross-resistance were assessed. All mutants but 1, and 2 clinical isolates, overexpressed acrB. No mutation was detected in the regulator genes studied among the clinical isolates and 8 of the mutants. For the 9 remaining mutants, a mutation was found in the ramR gene in 8 of them and in the soxR gene in the last one, resulting in overexpression of ramA and soxS, respectively. Transformation of the ramR mutants and the soxR mutant with the wild-type ramR and soxR genes, respectively, abolished overexpression of acrB and ramA in the ramR mutants and of soxS in the soxR mutant, as well as antibiotic cross-resistance. Resistance due to efflux system overexpression was demonstrated for 4 new antibiotics: cefuroxime, cefotaxime, ceftazidime, and ertapenem. This study shows that the ramR and soxR genes control the expression of efflux systems in K. pneumoniae and suggests the existence of efflux pumps other than AcrAB and of other loci involved in the regulation of AcrAB expression. PMID:21464248

An Arg-Gly-Asp peptide stimulates Ca2+ efflux from osteoclast precursors through a novel mechanism

NASA Technical Reports Server (NTRS)

Yamakawa, K.; Duncan, R.; Hruska, K. A.

1994-01-01

We examined the effect of a peptide containing the Arg-Gly-Asp (RGD) sequence on 45Ca2+ efflux from osteoclast precursors. 45Ca(2+)-loaded osteoclast precursors were treated with GRGDSP (170 microM) for 10 min after 30 min of basal perfusion with a bicarbonate-containing buffer. GRGDSP significantly increased fractional efflux of Ca2+ from treated cells compared with vehicle-treated cells (P < 0.01) or cells treated with up to 200 micrograms/ml of a control peptide containing GRGESP. The effect of RGD was sustained for 15 min after the peptide was removed from the perfusate, but control levels of Ca2+ efflux returned by 1 h. The Ca2+ efflux effect of GRGDSP was most likely due to activation of the plasma membrane Ca(2+)-adenosinetriphosphatase (Ca(2+)-ATPase) pump, as indicated by its inhibition with vanadate and a calmodulin antagonist, N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide, and the absence of an effect of Na+/Ca2+ exchange inhibition. An inhibitor of cyclic nucleotide-dependent protein kinases, N-[2-(methylamino)ethyl]-5-isoquinoline-sulfonamide (0.1 mM), failed to inhibit GRGDSP-stimulated Ca2+ efflux. However, genistein and herbimycin A, inhibitors of protein-tyrosine kinases, blocked Ca2+ efflux stimulated by GRGDSP. The results indicate that RGD sequences of matrix proteins may stimulate Ca2+ efflux from osteoclasts through activation of protein-tyrosine kinases and suggest that GRGDSP-stimulated Ca2+ efflux is mediated via the plasma membrane Ca(2+)-ATPase.

Lack of efflux mediated quinolone resistance in Salmonella enterica serovars Typhi and Paratyphi A

PubMed Central

Baucheron, Sylvie; Monchaux, Isabelle; Le Hello, Simon; Weill, François-Xavier; Cloeckaert, Axel

2014-01-01

Salmonella enterica serovars Typhi and Paratyphi A isolates from human patients in France displaying different levels of resistance to quinolones or fluoroquinolones were studied for resistance mechanisms to these antimicrobial agents. All resistant isolates carried either single or multiple target gene mutations (i.e., in gyrA, gyrB, or parC) correlating with the resistance levels observed. Active efflux, through upregulation of multipartite efflux systems, has also been previously reported as contributing mechanism for other serovars. Therefore, we investigated also the occurrence of non-target gene mutations in regulatory regions affecting efflux pump expression. However, no mutation was detected in these regions in both Typhi and Paratyphi isolates of this study. Besides, no overexpression of the major efflux systems was observed for these isolates. Nevertheless, a large deletion of 2334 bp was identified in the acrS-acrE region of all S. Typhi strains but which did not affect the resistance phenotype. As being specific to S. Typhi, this deletion could be used for specific molecular detection purposes. In conclusion, the different levels of quinolone or FQ resistance in both S. Typhi and S. Paratyphi A seem to rely only on target modifications. PMID:24478769

Influence of high-frequency ambient pressure pumping on carbon dioxide efflux from soil

Treesearch

Eugene S. Takle; William J. Massman; James R. Brandle; R. A. Schmidt; Xinhua Zhou; Irina V. Litvina; Rick Garcia; Geoffrey Doyle; Charles W. Rice

2004-01-01

We report measurements at 2Hz of pressure fluctuations at and beneath the soil in an agricultural field with dry soil and no vegetation. The objective of our study was to examine the possible role of pressure fluctuations produced by fluctuations in ambient wind on the efflux of CO2 at the soil surface.We observed that pressure fluctuations penetrate to 50 cm in the...

Rifaximin Resistance in Escherichia coli Associated with Inflammatory Bowel Disease Correlates with Prior Rifaximin Use, Mutations in rpoB, and Activity of Phe-Arg-β-Naphthylamide-Inhibitable Efflux Pumps

PubMed Central

Kothary, Vishesh; Scherl, Ellen J.; Bosworth, Brian; Jiang, Zhi-Dong; DuPont, Herbert L.; Harel, Josee

2013-01-01

Escherichia coli is implicated in the pathogenesis of inflammatory bowel disease (IBD). Rifaximin, a nonabsorbable derivative of rifampin effective against E. coli, improves symptoms in mild-to-moderate IBD. However, rifaximin resistance can develop in a single step in vitro. We examined the prevalence and mechanisms of rifaximin resistance in 62 strains of E. coli isolated from the ileal mucosa of 50 patients (19 with ileal Crohn's disease [L1+L3], 6 with colonic Crohn's disease [L2], 13 with ulcerative colitis [UC], 4 with symptomatic non-IBD diagnoses [NI], and 8 healthy [H]). Resistance (MIC > 1,024 mg/liter) was present in 12/48 IBD-associated ileal E. coli strains. Resistance correlated with prior rifaximin treatment (P < 0.00000001) but not with the presence of ileal inflammation (P = 0.73) or E. coli phylogroup. Mutations in a 1,057-bp region of rpoB, which encodes the bacterial target of rifaximin, were identified in 10/12 resistant strains versus 0/50 sensitive strains (P < 0.000000001) and consisted of seven amino acid substitutions. The efflux pump inhibitor Phe-Arg-β-naphthylamide (PAβN) lowered the MIC of 9/12 resistant strains 8- to 128-fold. Resistance was stable in the absence of rifaximin in 10/12 resistant strains after 30 passages. We conclude that IBD-associated ileal E. coli frequently manifest resistance to rifaximin that correlates with prior rifaximin use, amino acid substitutions in rpoB, and activity of PAβN-inhibitable efflux pumps, but not with the presence of ileal inflammation or E. coli phylogroup. These findings have significant implications for treatment trials targeting IBD-associated E. coli. PMID:23183443

Multidrug efflux pumps: the structures of prokaryotic ATP-binding cassette transporter efflux pumps and implications for our understanding of eukaryotic P-glycoproteins and homologues.

PubMed

Kerr, Ian D; Jones, Peter M; George, Anthony M

2010-02-01

One of the Holy Grails of ATP-binding cassette transporter research is a structural understanding of drug binding and transport in a eukaryotic multidrug resistance pump. These transporters are front-line mediators of drug resistance in cancers and represent an important therapeutic target in future chemotherapy. Although there has been intensive biochemical research into the human multidrug pumps, their 3D structure at atomic resolution remains unknown. The recent determination of the structure of a mouse P-glycoprotein at subatomic resolution is complemented by structures for a number of prokaryotic homologues. These structures have provided advances into our knowledge of the ATP-binding cassette exporter structure and mechanism, and have provided the template data for a number of homology modelling studies designed to reconcile biochemical data on these clinically important proteins.

The influence of TAP1 and TAP2 gene polymorphisms on TAP function and its inhibition by viral immune evasion proteins.

PubMed

Praest, P; Luteijn, R D; Brak-Boer, I G J; Lanfermeijer, J; Hoelen, H; Ijgosse, L; Costa, A I; Gorham, R D; Lebbink, R J; Wiertz, E J H J

2018-06-04

Herpesviruses encode numerous immune evasion molecules that interfere with the immune system, particularly with certain stages in the MHC class I antigen presentation pathway. In this pathway, the transporter associated with antigen processing (TAP) is a frequent target of viral immune evasion strategies. This ER-resident transporter is composed of the proteins TAP1 and TAP2, and plays a crucial role in the loading of viral peptides onto MHC class I molecules. Several variants of TAP1 and TAP2 occur in the human population, some of which are linked to autoimmune disorders and susceptibility to infections. Here, we assessed the influence of naturally occurring TAP variants on peptide transport and MHC class I expression. In addition, we tested the inhibitory capacity of three viral immune evasion proteins, the TAP inhibitors US6 from human cytomegalovirus, ICP47 from herpes simplex virus type 1 and BNLF2a from Epstein-Barr virus, for a series of TAP1 and TAP2 variants. Our results suggest that these TAP polymorphisms have no or limited effect on peptide transport or MHC class I expression. Furthermore, our study indicates that the herpesvirus-encoded TAP inhibitors target a broad spectrum of TAP variants; inhibition of TAP is not affected by the naturally occurring polymorphisms of TAP tested in this study. Our findings suggest that the long-term coevolution of herpesviruses and their host did not result in selection of inhibitor-resistant TAP variants in the human population. Copyright © 2018. Published by Elsevier Ltd.

Carpobrotus edulis methanol extract inhibits the MDR efflux pumps, enhances killing of phagocytosed S. aureus and promotes immune modulation.

PubMed

Ordway, Diane; Hohmann, Judit; Viveiros, Miguel; Viveiros, Antonio; Molnar, Joseph; Leandro, Clara; Arroz, Maria Jorge; Gracio, Maria Amelia; Amaral, Leonard

2003-05-01

Although alkaloids from the family Aizoaceae have anticancer activity, species of this family have received little attention. Because these alkaloids also exhibit properties normally associated with compounds that have activity at the level of the plasma membrane, a methanol extract of Carpobrotus edulis, a common plant found along the Portuguese coast, was studied for properties normally associated with plasma membrane active compounds. The results of this study show that the extract is non-toxic at concentrations that inhibit a verapamil sensitive efflux pump of L5178 mouse T cell lymphoma cell line thereby rendering these multi-drug resistant cells susceptible to anticancer drugs. These non-toxic concentrations also prime THP-1 human monocyte-derived macrophages to kill ingested Staphylococcus aureus and to promote the release of lymphokines associated with cellular immune functions. The extract also induces the proliferation of THP-1 cells within 1 day of exposure to quantities normally associated with phytohaemagglutinin. The potential role of the compound(s) isolated from this plant in cancer biology is intriguing and is currently under investigation. It is supposed that the resistance modifier and immunomodulatory effect of this plant extract can be exploited in the experimental chemotherapy of cancer and bacterial or viral infections. Copyright 2003 John Wiley & Sons, Ltd.

Structural basis for the blockade of MATE multidrug efflux pumps

DOE PAGES

Radchenko, Martha; Symersky, Jindrich; Nie, Rongxin; ...

2015-08-06

Multidrug and toxic compound extrusion (MATE) transporters underpin multidrug resistance by using the H + or Na + electrochemical gradient to extrude different drugs across cell membranes. MATE transporters can be further parsed into the DinF, NorM and eukaryotic subfamilies based on their amino-acid sequence similarity. Here we report the 3.0 Å resolution X-ray structures of a protonation-mimetic mutant of an H +-coupled DinF transporter, as well as of an H +-coupled DinF and a Na +-coupled NorM transporters in complexes with verapamil, a small-molecule pharmaceutical that inhibits MATE-mediated multidrug extrusion. Combining structure-inspired mutational and functional studies, we confirm themore » biological relevance of our crystal structures, reveal the mechanistic differences among MATE transporters, and suggest how verapamil inhibits MATE-mediated multidrug efflux. Our findings offer insights into how MATE transporters extrude chemically and structurally dissimilar drugs and could inform the design of new strategies for tackling multidrug resistance.« less

[Efflux systems in Serratia marcescens].

PubMed

Mardanova, A M; Bogomol'naia, L M; Romanova, Iu D; Sharipova, M R

2014-01-01

A widespread bacterium Serratia marcescens (family Enterobacteriaceae) is an opportunistic and exhibits multiple drug resistance. Active removal of antibiotics and other antimicrobials from pathogen and exhibits multiple drug resistance. Active removal of antibiotics and other antimicrobials from the cells by efflux systems is one of the mechanisms responsible for microbial resistance to these compounds. Among enterobacteria, efflux systems of Escherichia coli and Salmonella enterica var. Typhimurium have been studied most extensively. Few efflux systems that belong to different families have been reported for S. marcescens. In this review, we analyzed available literature about S. marcescens efflux systems and carried out the comparative analysis of the genes encoding the RND type systems in different Serratia species and in other enterobacteria. Bioinformatical analysis of the S. marcescens genome allowed us to identify the previously unknown efflux systems based on their homology with the relevant E. coli genes. Identification of additional efflux systems in S. marcescens genome will promote our understanding of physiology of these bacteria, will detect new molecular mechanisms of resistance and will reveal their resistance potential.

Design and Study of Efflux Function of EGFP Fused MexAB-OprM Membrane Transporter in Pseudomonas aeruginosa Using Fluorescence Spectroscopy

PubMed Central

Ding, Feng; Lee, Kerry J.; Vahedi-Faridi, Ardeschir; Yoneyama, Hiroshi; Osgood, Christopher J.; Xu, Xiao-Hong Nancy

2014-01-01

Multidrug membrane transporters (efflux pumps) can selectively extrude a variety of structurally and functionally diverse substrates (e.g., chemotoxics, antibiotics), leading to multidrug resistance (MDR) and ineffective treatment of a wide variety of diseases. In this study, we have designed and constructed fusion gene (egfp-mexB) of N-terminal mexB with C-terminal egfp, inserted it into a plasmid vector (pMMB67EH), and successfully expressed it in ΔMexB (MexB deletion) strain of Pseudomonas aeruginosa to create a new strain that expresses MexA-(EGFP-MexB)-OprM. We characterized the fusion gene using gel electrophoresis and DNA sequencing, and determined their expression in live cells by measuring the fluorescence of EGFP in single live cells using fluorescence microscopy. Efflux function of the new strain was studied by measuring its accumulation kinetics of ethidium bromide (EtBr, a pump substrate) using fluorescence spectroscopy, which was compared with the cells (WT, ΔMexM, ΔABM, and nalB1) with various expression levels of MexAB-OprM. The new strain shows 6-fold lower accumulation rates of EtBr (15 μM) than ΔABM, 4-fold lower than ΔMexB, but only 1.1-fold higher than WT. As EtBr concentration increases to 40 μM, the new strain has nearly the same accumulation rate of EtBr as ΔMexB, but 1.4-fold higher than WT. We observed the nearly same level of inhibitory effect of CCCP (carbonyl cyanide-m-chlorophenylhydrazone) on the efflux of EtBr by the new strain and WT. Antibiotic susceptibility study shows that the minimum inhibitory concentrations (MICs) of aztreonam (AZT) and chloramphenicol (CP) for the new strain are 6-fold or 3-fold lower than WT, respectively, and 2-fold higher than those of ΔMexB. Taken together, the results suggest that the fusion protein partially retains the efflux function of MexAB-OprM. Modeled structure of the fusion protein shows that the position and orientation of the N-terminal fused EGFP domain may either partially block

Transporters associated with antigen processing (TAP) in sea bass (Dicentrarchus labrax, L.): molecular cloning and characterization of TAP1 and TAP2.

PubMed

Pinto, Rute D; Pereira, Pedro J B; dos Santos, Nuno M S

2011-11-01

The transporters associated with antigen processing (TAP), play an important role in the MHC class I antigen presentation pathway. In this work, sea bass (Dicentrarchus labrax) TAP1 and TAP2 genes and transcripts were isolated and characterized. Only the TAP2 gene is structurally similar to its human orthologue. As other TAP molecules, sea bass TAP1 and TAP2 are formed by one N-terminal accessory domain, one core membrane-spanning domain and one canonical C-terminal nucleotide-binding domain. Homology modelling of the sea bass TAP dimer predicts that its quaternary structure is in accordance with that of other ABC transporters. Phylogenetic analysis segregates sea bass TAP1 and TAP2 into each subfamily cluster of transporters, placing them in the fish class and suggesting that the basic structure of these transport-associated proteins is evolutionarily conserved. Furthermore, the present data provides information that will enable more studies on the class I antigen presentation pathway in this important fish species. Copyright © 2011 Elsevier Ltd. All rights reserved.

Fullerene inhibits benzo(a)pyrene Efflux from Cyprinus carpio hepatocytes by affecting cell membrane fluidity and P-glycoprotein expression.

PubMed

Chen, Qiqing; Hu, Xialin; Wang, Rui; Yuan, Jin; Yin, Daqiang

2016-05-01

P-Glycoprotein (P-gp) can protect cells by pumping out toxic compounds, and has been found widely expressed in fish tissues. Here, we illustrate the P-gp efflux ability for benzo(a)pyrene (BaP) in the hepatocytes of common carp (Cyprinus carpio) after exposing to fullerene aqueous suspension (nC60). The results revealed that nC60 increased the membrane fluidity by decreasing the ratio of saturated to unsaturated fatty acids, and increased the cholesterol contents. These findings, combined with 10-38% and 70-75% down-regulation of P-gp mRNA and protein respectively, suggested that nC60 caused inhibition on P-gp efflux transport system. Therefore, we further investigated the cellular efflux ability for BaP. Results showed unequivocally that nC60 is a potent P-gp inhibitor. The retaining BaP amounts after efflux were elevated by 1.7-2.8 fold during the 10 day exposure. Meanwhile, 5mg/L humic acid (one of the important fractions of natural organic matter, which is ubiquitous in aquatic environment) alleviated the nC60 damage to hepatocytes in terms of oxidative damage, cholesterol increment, and P-gp content reduction; and finally attenuated the suppressed P-gp efflux ability. Collectively, this study provides the first evidence of nC60 toxicity to P-gp functionality in fish and illustrates the possible mechanism of the suppressed P-gp efflux ability for BaP. Copyright © 2016 Elsevier B.V. All rights reserved.

Kinetic control of TolC recruitment by multidrug efflux complexes.

PubMed

Tikhonova, Elena B; Dastidar, Vishakha; Rybenkov, Valentin V; Zgurskaya, Helen I

2009-09-22

In Gram-negative pathogens, multidrug efflux pumps that provide clinically significant levels of antibiotic resistance function as three-component complexes. They are composed of the inner membrane transporters belonging to one of three superfamilies of proteins, RND, ABC, or MF; periplasmic proteins belonging to the membrane fusion protein (MFP) family; and outer membrane channels exemplified by the Escherichia coli TolC. The three-component complexes span the entire two-membrane envelope of Gram-negative bacteria and expel toxic molecules from the cytoplasmic membrane to the medium. The architecture of these complexes is expected to vary significantly because of the structural diversity of the inner membrane transporters. How the three-component pumps are assembled, their architecture, and their dynamics remain unclear. In this study, we reconstituted interactions and compared binding kinetics of the E. coli TolC with AcrA, MacA, and EmrA, the periplasmic MFPs that function in multidrug efflux with transporters from the RND, ABC, and MF superfamilies, respectively. By using surface plasmon resonance, we demonstrate that TolC interactions with MFPs are highly dynamic and sensitive to pH. The affinity of TolC to MFPs decreases in the order MacA > EmrA > AcrA. We further show that MFPs are prone to oligomerization, but differ dramatically from each other in oligomerization kinetics and stability of oligomers. The propensity of MFPs to oligomerize correlates with the stability of MFP-TolC complexes and structural features of inner membrane transporters. We propose that recruitment of TolC by various MFPs is determined not only by kinetics of MFP-TolC interactions but also by oligomerization kinetics of MFPs and pH.

Use of juglone as antibacterial and potential efflux pump inhibitors in Staphylococcus aureus isolated from the oral cavity.

PubMed

Zmantar, Tarek; Miladi, Hanene; Kouidhi, Bochra; Chaabouni, Yassine; Ben Slama, Rihab; Bakhrouf, Amina; Mahdouani, Kacem; Chaieb, Kamel

2016-12-01

In this study the minimal inhibitory concentration (MICs) of tetracycline (Tet), erythromycin (Ery) and benzalkonium chloride (BC) in absence and in presence of a sub-MIC of juglone (Jug) were determined. In addition, the Ethidium bromide (EtBr) efflux assay was performed to assess the effect of Jug on EtBr cells accumulation. Our results showed a selective antimicrobial activity of Jug against the tested strains. A synergistic effect of Jug, drugs (Tet and Ery) and disinfectant (BC) was noticed with a reduction rate varied from 2 to 16-fold. In addition, the efflux of EtBr was inhibited depending on the Jug concentration. In the presence of Jug, a decrease in loss of EtBr from bacteria was observed. The concentration inducing 50 % of EtBr efflux inhibition after 15 min was about 182 μg ml -1 for S. aureus ATCC 25923, 236 μg ml -1 for S. aureus B193 and 195 μg ml -1  for S. aureus B456. It appears from this study that Jug may be used as a natural source for resistance-modifying activity in same bacteria. Copyright © 2016 Elsevier Ltd. All rights reserved.

Searching for Novel Inhibitors of the S. aureus NorA Efflux Pump: Synthesis and Biological Evaluation of the 3-Phenyl-1,4-benzothiazine Analogues.

PubMed

Felicetti, Tommaso; Cannalire, Rolando; Burali, Maria Sole; Massari, Serena; Manfroni, Giuseppe; Barreca, Maria Letizia; Tabarrini, Oriana; Schindler, Bryan D; Sabatini, Stefano; Kaatz, Glenn W; Cecchetti, Violetta

2017-08-22

Bacterial resistance to antimicrobial agents has become an increasingly serious health problem in recent years. Among the strategies by which resistance can be achieved, overexpression of efflux pumps such as NorA of Staphylococcus aureus leads to a sub-lethal concentration of the antibacterial agent at the active site that in turn may predispose the organism to the development of high-level target-based resistance. With an aim to improve both the chemical stability and potency of our previously reported 3-phenyl-1,4-benzothiazine NorA inhibitors, we replaced the benzothiazine core with different nuclei. None of the new synthesized compounds showed any appreciable intrinsic antibacterial activity, and, in particular, 2-(3,4-dimethoxyphenyl)quinoline (6 c) was able to decrease, in a concentration-dependent manner, the ciprofloxacin MIC against the norA-overexpressing strains S. aureus SA-K2378 (norA++) and SA-1199B (norA+/A116E GrlA). © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Polyamines cause plasma membrane depolarization, activate Ca2+-, and modulate H+-ATPase pump activity in pea roots.

PubMed

Pottosin, Igor; Velarde-Buendía, Ana María; Bose, Jayakumar; Fuglsang, Anja T; Shabala, Sergey

2014-06-01

Polyamines regulate a variety of cation and K(+) channels, but their potential effects on cation-transporting ATPases are underexplored. In this work, noninvasive microelectrode ion flux estimation and conventional microelectrode techniques were applied to study the effects of polyamines on Ca(2+) and H(+) transport and membrane potential in pea roots. Externally applied spermine or putrescine (1mM) equally activated eosin yellow (EY)-sensitive Ca(2+) pumping across the root epidermis and caused net H(+) influx or efflux. Proton influx induced by spermine was suppressed by EY, supporting the mechanism in which Ca(2+) pump imports 2 H(+) per each exported Ca(2+). Suppression of the Ca(2+) pump by EY diminished putrescine-induced net H(+) efflux instead of increasing it. Thus, activities of Ca(2+) and H(+) pumps were coupled, likely due to the H(+)-pump inhibition by intracellular Ca(2+). Additionally, spermine but not putrescine caused a direct inhibition of H(+) pumping in isolated plasma membrane vesicles. Spermine, spermidine, and putrescine (1mM) induced membrane depolarization by 70, 50, and 35 mV, respectively. Spermine-induced depolarization was abolished by cation transport blocker Gd(3+), was insensitive to anion channels' blocker niflumate, and was dependent on external Ca(2+). Further analysis showed that uptake of polyamines but not polyamine-induced cationic (K(+)+Ca(2+)+H(+)) fluxes were a main cause of membrane depolarization. Polyamine increase is a common component of plant stress responses. Activation of Ca(2+) efflux by polyamines and contrasting effects of polyamines on net H(+) fluxes and membrane potential can contribute to Ca(2+) signalling and modulate a variety of transport processes across the plasma membrane under stress. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

NASA/UK TAP

NASA Technical Reports Server (NTRS)

1987-01-01

The purpose of the Technology Applications Program (TAP) is to provide problem solving information and assistance to both the public and private sectors in the Commonwealth of Kentucky, with emphasis primarily in the public sector. The TAP accesses over 1200 online computer databases, including files from the U.S., Canada, Europe, and Australia. During the 1985 to 1986 contract period, TAP responded to 645 inquiries which resulted in an increase of 16 percent over the 1984 to 1985 contract period. The activities of TAP for the 1985 to 1986 contract period are summarized.

More than just tapping: index finger-tapping measures procedural learning in schizophrenia.

PubMed

Da Silva, Felipe N; Irani, Farzin; Richard, Jan; Brensinger, Colleen M; Bilker, Warren B; Gur, Raquel E; Gur, Ruben C

2012-05-01

Finger-tapping has been widely studied using behavioral and neuroimaging paradigms. Evidence supports the use of finger-tapping as an endophenotype in schizophrenia, but its relationship with motor procedural learning remains unexplored. To our knowledge, this study presents the first use of index finger-tapping to study procedural learning in individuals with schizophrenia or schizoaffective disorder (SCZ/SZA) as compared to healthy controls. A computerized index finger-tapping test was administered to 1169 SCZ/SZA patients (62% male, 88% right-handed), and 689 healthy controls (40% male, 93% right-handed). Number of taps per trial and learning slopes across trials for the dominant and non-dominant hands were examined for motor speed and procedural learning, respectively. Both healthy controls and SCZ/SZA patients demonstrated procedural learning for their dominant hand but not for their non-dominant hand. In addition, patients showed a greater capacity for procedural learning even though they demonstrated more variability in procedural learning compared to healthy controls. Left-handers of both groups performed better than right-handers and had less variability in mean number of taps between non-dominant and dominant hands. Males also had less variability in mean tap count between dominant and non-dominant hands than females. As expected, patients had a lower mean number of taps than healthy controls, males outperformed females and dominant-hand trials had more mean taps than non-dominant hand trials in both groups. The index finger-tapping test can measure both motor speed and procedural learning, and motor procedural learning may be intact in SCZ/SZA patients. Copyright © 2012 Elsevier B.V. All rights reserved.

Transcriptome analysis reveals that multidrug efflux genes are upregulated to protect Pseudomonas aeruginosa from pentachlorophenol stress.

PubMed

Muller, Jocelyn Fraga; Stevens, Ann M; Craig, Johanna; Love, Nancy G

2007-07-01

Through chemical contamination of natural environments, microbial communities are exposed to many different types of chemical stressors; however, research on whole-genome responses to this contaminant stress is limited. This study examined the transcriptome response of a common soil bacterium, Pseudomonas aeruginosa, to the common environmental contaminant pentachlorophenol (PCP). Cells were grown in chemostats at a low growth rate to obtain substrate-limited, steady-state, balanced-growth conditions. The PCP stress was administered as a continuous increase in concentration, and samples taken over time were examined for physiological function changes with whole-cell acetate uptake rates (WAURs) and cell viability and for gene expression changes by Affymetrix GeneChip technology and real-time reverse transcriptase PCR. Cell viability, measured by heterotrophic plate counts, showed a moderately steady decrease after exposure to the stressor, but WAURs did not change in response to PCP. In contrast to the physiological data, the microarray data showed significant changes in the expression of several genes. In particular, genes coding for multidrug efflux pumps, including MexAB-OprM, were strongly upregulated. The upregulation of these efflux pumps protected the cells from the potentially toxic effects of PCP, allowing the physiological whole-cell function to remain constant.

Mutations in MexB that affect the efflux of antibiotics with cytoplasmic targets.

PubMed

Ohene-Agyei, Thelma; Lea, Jon D; Venter, Henrietta

2012-08-01

Drug efflux pumps such as MexAB-OprM from Pseudomonas aeruginosa confer resistance to a wide range of chemically different compounds. Within the tripartite assembly, the inner membrane protein MexB is mainly responsible for substrate recognition. Recently, considerable advances have been made in elucidating the drug efflux pathway through the large periplasmic domains of resistance-nodulation-division (RND) transporters. However, little is known about the role of amino acids in other parts of the protein. We have investigated the role of two conserved phenylalanine residues that are aligned around the cytoplasmic side of the central cavity of MexB. The two conserved phenylalanine residues have been mutated to alanine residues (FAFA MexB). The interaction of the wild-type and mutant proteins with a variety of drugs from different classes was investigated by assays of cytotoxicity and drug transport. The FAFA mutation affected the efflux of compounds that have targets inside the cell, but antibiotics that act on cell wall synthesis and membrane probes were unaffected. Combined, our results indicate the presence of a hitherto unidentified cytoplasmic-binding site in RND drug transporters and enhance our understanding of the molecular mechanisms that govern drug resistance in Gram-negative pathogens. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Freely Chosen Index Finger Tapping Frequency Is Increased in Repeated Bouts of Tapping.

PubMed

Hansen, Ernst Albin; Ebbesen, Brian Duborg; Dalsgaard, Ane; Mora-Jensen, Mark Holten; Rasmussen, Jakob

2015-01-01

Healthy individuals (n = 40) performed index finger tapping at freely chosen frequency during repeated bouts and before and after near-maximal muscle action consisting of 3 intense flexions of the index finger metacarpal phalangeal joint. One experiment showed, unexpectedly, that a bout of tapping increased the tapping frequency in the subsequent bout. Thus, a cumulating increase of 8.2 ± 5.4% (p < .001) occurred across 4 bouts, which were all separated by 10 min rest periods. Follow-up experiments revealed that tapping frequency was still increased in consecutive bouts when rest periods were extended to 20 min. Besides, near-maximal muscle activation, followed by 5 min rest, did not affect the tapping frequency. In conclusion, freely chosen tapping frequency was increased in repeated bouts of tapping, which were separated by 10-20 min rest periods. The observed phenomenon is suggested to be termed repeated bout rate enhancement.

Structures of the TetR-like simocyclinone efflux pump repressor, SimR, and the mechanism of ligand-mediated derepression.

PubMed

Le, Tung B K; Stevenson, Clare E M; Fiedler, Hans-Peter; Maxwell, Anthony; Lawson, David M; Buttner, Mark J

2011-04-22

Simocyclinone D8 (SD8), a potent DNA gyrase inhibitor made by Streptomyces antibioticus, is exported from the producing organism by the SimX efflux pump. The expression of simX is under the control of SimR, a member of the TetR family of transcriptional regulators. SimR represses simX transcription by binding to operators in the intergenic region between simR and simX. Previously, we have shown that the mature antibiotic SD8 or its biosynthetic intermediate, simocyclinone C4, can dissociate SimR from its operators, leading to derepression of simX and export of SD8 from the cell. This provides a mechanism that couples the biosynthesis of the antibiotic to its export. Here, we report the crystal structures of SimR alone and in complex with either SD8 or simocyclinone C4. The ligand-binding pocket is unusual compared to those of other characterized TetR-family transcriptional regulators: the structures show an extensive ligand-binding pocket spanning both monomers in the functional dimeric unit, with the aminocoumarin moiety of SD8 buried in the protein core, while the angucyclic polyketide moiety is partially exposed to bulk solvent. Through comparisons of the structures, we postulate a derepression mechanism for SimR that invokes rigid-body motions of the subunits relative to one another, coupled with a putative locking mechanism to restrict further conformational change. Copyright © 2011 Elsevier Ltd. All rights reserved.

Expression of the CDR1 efflux pump in clinical Candida albicans isolates is controlled by a negative regulatory element

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gaur, Naseem Akhtar; Manoharlal, Raman; Saini, Preeti

2005-06-24

Resistance to azole antifungal drugs in clinical isolates of the human fungal pathogen Candida albicans is often caused by constitutive overexpression of the CDR1 gene, which encodes a multidrug efflux pump of the ABC transporter superfamily. To understand the relevance of a recently identified negative regulatory element (NRE) in the CDR1 promoter for the control of CDR1 expression in the clinical scenario, we investigated the effect of mutation or deletion of the NRE on CDR1 expression in two matched pairs of azole-sensitive and resistant clinical isolates of C. albicans. Expression of GFP or lacZ reporter genes from the wild typemore » CDR1 promoter was much higher in the azole-resistant C. albicans isolates than in the azole-susceptible isolates, reflecting the known differences in CDR1 expression in these strains. Deletion or mutation of the NRE resulted in enhanced reporter gene expression in azole-sensitive strains, but did not further increase the already high CDR1 promoter activity in the azole-resistant strains. In agreement with these findings, electrophoretic mobility shift assays showed a reduced binding to the NRE of nuclear extracts from the resistant C. albicans isolates as compared with extracts from the sensitive isolates. These results demonstrate that the NRE is involved in maintaining CDR1 expression at basal levels and that this repression is overcome in azole-resistant clinical C. albicans isolates, resulting in constitutive CDR1 overexpression and concomitant drug resistance.« less

K(+)- and HCO3(-)-dependent acid-base transport in squid giant axons. I. Base efflux

PubMed Central

1995-01-01

We used microelectrodes to monitor the recovery (i.e., decrease) of intracellular pH (pHi) after using internal dialysis to load squid giant axons with alkali to pHi values of 7.7, 8.0, or 8.3. The dialysis fluid (DF) contained 400 mM K+ but was free of Na+ and Cl-. The artificial seawater (ASW) lacked Na+, K+, and Cl-, thereby eliminating effects of known acid-base transporters on pHi. Under these conditions, halting dialysis unmasked a slow pHi decrease caused at least in part by acid-base transport we refer to as "base efflux." Replacing K+ in the DF with either NMDG+ or TEA+ significantly reduced base efflux and made membrane voltage (Vm) more positive. Base efflux in K(+)-dialyzed axons was stimulated by decreasing the pH of the ASW (pHo) from 8 to 7, implicating transport of acid or base. Although postdialysis acidifications also occurred in axons in which we replaced the K+ in the DF with Li+, Na+, Rb+, or Cs+, only with Rb+ was base efflux stimulated by low pHo. Thus, the base effluxes supported by K+ and Rb+ appear to be unrelated mechanistically to those observed with Li+, Na+, or Cs+. The combination of 437 mM K+ and 12 mM HCO3- in the ASW, which eliminates the gradient favoring a hypothetical K+/HCO3- efflux, blocked pHi recovery in K(+)-dialyzed axons. However, the pHi recovery was not blocked by the combination of 437 mM Na+, veratridine, and CO2/HCO3- in the ASW, a treatment that inverts electrochemical gradients for H+ and HCO3- and would favor passive H+ and HCO3- fluxes that would have alkalinized the axon. Similarly, the recovery was not blocked by K+ alone or HCO3- alone in the ASW, nor was it inhibited by the K-H pump blocker Sch28080 nor by the Na-H exchange inhibitors amiloride and hexamethyleneamiloride. Our data suggest that a major component of base efflux in alkali-loaded axons cannot be explained by metabolism, a H+ or HCO3- conductance, or by a K-H exchanger. However, this component could be mediated by a novel K/HCO3- cotransporter

On the physics of multidrug efflux through a biomolecular complex

NASA Astrophysics Data System (ADS)

Mishima, Hirokazu; Oshima, Hiraku; Yasuda, Satoshi; Amano, Ken-ichi; Kinoshita, Masahiro

2013-11-01

focused on the entropic component. We show that the entropically inserted solute can be released by a continuous variation of the vessel geometry which forms a time-dependent entropic force continuing to accelerate the solute motion to the exit. Solutes with a wide range of sizes are entropically released using the same vessel-geometry variation. The results obtained are fairly general and also applicable to the efflux pump protein AcrB and ATP-binding cassette transporter.

The properties of an improvised piston pump for the rapid delivery of intravenous fluids.

PubMed

Smart, C M; Primrose, C W; Peters, A L; Speirits, E J

2014-02-01

To maximise the effect of a small fluid load, it is occasionally desirable to bolus manually with multiple depressions of a large-capacity syringe. This is usually achieved by placing the syringe on the side port of a three-way tap. We modified this technique by placing two-one-way valves in line with the three-way tap, effectively creating a piston pump, the infusion rates via which we compared with those achieved by an inflatable pressure-infuser in a simulated resuscitation. Fluid flow was faster using the piston pump than with the pressure-infuser (mean (SD) time to infuse 2000 ml saline 0.9% via a 16-G cannula 352 (10) s vs 495 (19) s, respectively, p < 0.0001). The piston pump appears to have potential for both tight control of fluid delivery and major high-volume resuscitation. The lightweight nature of the pump and its lack of reliance on gravity may also make it suitable for the pre-hospital setting. © 2014 The Association of Anaesthetists of Great Britain and Ireland.

Efflux mediated adaptive and cross resistance to ciprofloxacin and benzalkonium chloride in Pseudomonas aeruginosa of dairy origin.

PubMed

Pagedar, Ankita; Singh, Jitender; Batish, Virender K

2011-06-01

The present study was undertaken to investigate the role of efflux pump activity (EPA) in conferring adaptive and cross resistances against ciprofloxacin (CF) and benzalkonium chloride (BC) in dairy isolates of Pseudomonas aeruginosa. Biofilm formation potential was correlated with development of adaptive resistance in originally resistant strains. Irrespective of parent strains's susceptibility, isolates developed substantial adaptive resistance against CF and BC. Significant difference was observed in ability of non resistant isolates to develop adaptive resistance against CF and BC (P < 0.02) and subsequent cross resistance. EPA was quantified using EtBr (Ethidium Bromide) model and its role was more prominent [confirmed by its inhibition using efflux pump inhibitor (EPI) 2,4-dinitrophenol (DNP)], in conferring adaptive resistance (P = 0.147) than cross resistance (P = 0.343). Reduction in adaptive resistances due to EPI was more evident in originally non resistant strains, which reaffirms EPA as probable mechanism of adaptive resistance. The present study perhaps first of its kind, suggests an active role of EPA in conferring adaptive and cross resistances in food related P. aeruginosa isolates and supports reverse hypothesis that antibiotic-resistant organisms eventually become tolerant to other antibacterial agents as well. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Computational analysis of structure-based interactions and ligand properties can predict efflux effects on antibiotics.

PubMed

Sarkar, Aurijit; Anderson, Kelcey C; Kellogg, Glen E

2012-06-01

AcrA-AcrB-TolC efflux pumps extrude drugs of multiple classes from bacterial cells and are a leading cause for antimicrobial resistance. Thus, they are of paramount interest to those engaged in antibiotic discovery. Accurate prediction of antibiotic efflux has been elusive, despite several studies aimed at this purpose. Minimum inhibitory concentration (MIC) ratios of 32 β-lactam antibiotics were collected from literature. 3-Dimensional Quantitative Structure-Activity Relationship on the β-lactam antibiotic structures revealed seemingly predictive models (q(2)=0.53), but the lack of a general superposition rule does not allow its use on antibiotics that lack the β-lactam moiety. Since MIC ratios must depend on interactions of antibiotics with lipid membranes and transport proteins during influx, capture and extrusion of antibiotics from the bacterial cell, descriptors representing these factors were calculated and used in building mathematical models that quantitatively classify antibiotics as having high/low efflux (>93% accuracy). Our models provide preliminary evidence that it is possible to predict the effects of antibiotic efflux if the passage of antibiotics into, and out of, bacterial cells is taken into account--something descriptor and field-based QSAR models cannot do. While the paucity of data in the public domain remains the limiting factor in such studies, these models show significant improvements in predictions over simple LogP-based regression models and should pave the path toward further work in this field. This method should also be extensible to other pharmacologically and biologically relevant transport proteins. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Levofloxacin Efflux and smeD in Clinical Isolates of Stenotrophomonas maltophilia.

PubMed

Chong, So Young; Lee, Kyungwon; Chung, Hae-Sun; Hong, Seong Geun; Suh, Younghee; Chong, Yunsop

2017-03-01

Trimethoprim-sulfamethoxazole is the first-line antimicrobial combination for Stenotrophomonas maltophilia infections. However, allergy or intolerance and increasing resistance limit the use of trimethoprim-sulfamethoxazole. Quinolones can be used as an alternative therapeutic option, but resistance can emerge rapidly during therapy. We analyzed the contribution of SmeABC and SmeDEF efflux pumps to levofloxacin resistance in clinical isolates of S. maltophilia. Nonduplicate clinical isolates of S. maltophilia were collected in 2010 from 11 university hospitals (n = 102). Fifty-five levofloxacin nonsusceptible (minimum inhibitory concentration [MIC] ≥4 μg/ml) and 47 susceptible (MIC ≤2 μg/ml) isolates were tested for efflux pump overexpression. Real-time reverse transcription-PCR was performed for amplification and quantification of smeB, smeC, smeD, and smeF mRNA. To determine which antimicrobials were affected by smeD overexpression, the growth rates of a levofloxacin-susceptible S. maltophilia isolate were compared by measuring absorbance of antimicrobial-supplemented Luria-Bertani broth (LB) cultures with or without triclosan. Significant relationships between sme gene overexpression and resistance were observed for smeD against levofloxacin, smeC and smeF against ceftazidime, and smeC against ticarcillin-clavulanate. The mean MICs of moxifloxacin and tigecycline did not significantly differ for isolates with or without overexpression of smeB, smeC, and smeF, but were significantly higher for isolates with smeD overexpression. The mean MICs of amikacin were significantly higher for smeC or smeF overexpressing isolates. Increased growth of a levofloxacin-susceptible isolate was observed in LB with 1/2 MIC levofloxacin in the presence of triclosan. These data suggest that the expression of smeD plays a role in levofloxacin resistance in S. maltophilia.

Finger-tapping motion analysis in cervical myelopathy by magnetic-sensor tapping device.

PubMed

Miwa, Toshitada; Hosono, Noboru; Mukai, Yoshihiro; Makino, Takahiro; Kandori, Akihiko; Fuji, Takeshi

2013-08-01

Case-control study. The purpose of this study is to determine finger motion of patients with cervical myelopathy during finger-tapping cycles. A major symptom of patients with compressive cervical myelopathy is finger clumsiness. Therefore, understanding finger motion is prerequisite in assessing the severity of myelopathy. The popular grip-and-release test evaluates only the number of motion cycles, which is insufficient to fully describe complex finger motion. Forty-three patients with cervical myelopathy and 41 healthy controls tapped their index fingers against their thumbs as rapidly as possible for 30 seconds and the motion was recorded by a magnetic-sensor coil attached to the nail surface. Output signals were stored in a computer, which automatically calculated tapping frequency, distance moved, ratio of opening/closing velocity and the SD of the tapping interval. The SD of the tapping interval was significantly greater and all other measures were significantly smaller in patients with cervical myelopathy, than in healthy controls. All indices significantly improved after surgical decompression of the cervical spine. Distance moved (Pearson correlation coefficient: r=0.590, P<0.001) and the SD of the tapping interval (r=-0.451; P=0.002) were significantly correlated with the Japanese Orthopedic Association score (neurological scale). The quantitative evaluation of finger paralysis was performed by this tapping device. Speed and regularity in repetitive motion of fingers were correlated with the severity of cervical myelopathy.

Molecular Evidence and Functional Expression of a Novel Drug Efflux pump (ABCC2) in Human Corneal Epithelium and Rabbit Cornea and its role in Ocular drug efflux

PubMed Central

Karla, Pradeep K.; Pal, Dhananjay; Quinn, Tim; Mitra, Ashim K.

2007-01-01

Cornea is considered as a major barrier for ocular drug delivery. Low ocular bioavailability of drugs has been attributed primarily to low permeability across corneal epithelium thus leading to sub-therapeutic concentrations of drug in the eye and treatment failure. The role of drug efflux proteins, particularly the Pglycoprotein in ocular drug bioavailability has been reported. The objective of this research was to determine whether human corneal epithelium expresses multi drug resistance associated proteins contributing to drug efflux by employing both cultured corneal cells and freshly excised rabbit cornea. SV40 HCEC and rPCEC were selected for in-vitro testing. SV40-HCEC and freshly excised rabbit corneas were utilized for transport studies. [3H]-cyclosporine-A and [14C]-erythromycin which are known substrates for ABCC2 and MK-571, a specific inhibitor for MRP were applied in this study. RT-PCR indicated a unique and distinct band at ∼272 bp corresponding to ABCC2 in HCEC, SV40-HCEC, rabbit cornea, rPCEC and MDCKII-MRP2 cells. Also RT-PCR indicated a unique band ∼181 bp for HCEC and SV40-HCEC. Immunoprecipitation followed by Western Blot analysis revealed a specific band at ∼190-kDa in membrane fraction of SV40-HCEC, MDCKII-MRP2 and no band with isotype control. Uptake of [3H]-cyclosporine-A and [14C]-erythromycin in the presence of MK-571 was significantly enhanced than control in both SV40 HCEC and rPCEC. Similarly a significant elevation in (A→B) permeability of [3H]-cyclosporine-A and [14C]-erythromycin was observed in the presence of MK-571 in SV40-HCEC. A→B transport of [3H]-cyclosporine-A was elevated in the presence of MK-571 in freshly excised rabbit cornea indicating potential role of this efflux transporter and high clinical significance of this finding. PMID:17156953

Multidrug efflux transporter, AcrB--the pumping mechanism.

PubMed

Murakami, Satoshi

2008-08-01

Resistance nodulation cell division (RND) transporters are one of the main causes of the bacterial multidrug resistance. They pump a wide range of antibiotics out of the cell by proton motive force. AcrB is the major RND transporter in Escherichia coli. Recently, the crystal structures of AcrB have been determined by different space groups. All these structures are consistent with asymmetric trimer. Each monomer has different conformation corresponding to one of the three functional states of the transport cycle. Transporting hydrophobic drug was bound in the periplasmic domain on one of the three monomers. The transport pathway with alternating access mechanism is located at the hydrophilic domain protruded into the periplasmic space while this mechanism of other transporter families like ATP binding cassette (ABC) and major facilitator superfamily (MFS) transporter is located in the membrane-embedded region. For the RND, protonation might also take place asymmetrically at the functionally important charged residues in the transmembrane (TM) region. The structures indicate that drugs are transported by a three-step functional rotation in which substrates undergo ordered binding change.

Self-Advancing Step-Tap Drills

NASA Technical Reports Server (NTRS)

Pettit, Donald R.; Camarda, Charles J.; Penner, Ronald K.; Franklin, Larry D.

2007-01-01

Self-advancing tool bits that are hybrids of drills and stepped taps make it possible to form threaded holes wider than about 1/2 in. (about 13 mm) without applying any more axial force than is necessary for forming narrower pilot holes. These self-advancing stepped-tap drills were invented for use by space-suited astronauts performing repairs on reinforced carbon/carbon space-shuttle leading edges during space walks, in which the ability to apply axial drilling forces is severely limited. Self-advancing stepped-tap drills could also be used on Earth for making wide holes without applying large axial forces. A self-advancing stepped-tap drill (see figure) includes several sections having progressively larger diameters, typically in increments between 0.030 and 0.060 in. (between about 0.8 and about 1.5 mm). The tip section, which is the narrowest, is a pilot drill bit that typically has a diameter between 1/8 and 3/16 in. (between about 3.2 and about 4.8 mm). The length of the pilot-drill section is chosen, according to the thickness of the object to be drilled and tapped, so that the pilot hole is completed before engagement of the first tap section. Provided that the cutting-edge geometry of the drill bit is optimized for the material to be drilled, only a relatively small axial force [typically of the order of a few pounds (of the order of 10 newtons)] must be applied during drilling of the pilot hole. Once the first tap section engages the pilot hole, it is no longer necessary for the drill operator to apply axial force: the thread engagement between the tap and the workpiece provides the axial force to advance the tool bit. Like the pilot-drill section, each tap section must be long enough to complete its hole before engagement of the next, slightly wider tap section. The precise values of the increments in diameter, the thread pitch, the rake angle of the tap cutting edge, and other geometric parameters of the tap sections must be chosen, in consideration of

Substrate Specificities of MexAB-OprM, MexCD-OprJ, and MexXY-OprM Efflux Pumps in Pseudomonas aeruginosa

PubMed Central

Masuda, Nobuhisa; Sakagawa, Eiko; Ohya, Satoshi; Gotoh, Naomasa; Tsujimoto, Hideto; Nishino, Takeshi

2000-01-01

To find the exact substrate specificities of three species of tripartite efflux systems of Pseudomonas aeruginosa, MexAB-OprM, MexCD-OprJ, and MexXY-OprM, we constructed a series of isogenic mutants, each of which constitutively overproduced one of the three efflux systems and lacked the other two, and their isogenic mutants, which lacked all these systems. Comparison of the susceptibilities of the constructed mutants to 52 antimicrobial agents belonging to various groups suggested the following substrate specificities. All of the efflux systems extrude a wide variety of antimicrobial agent groups, i.e., quinolones, macrolides, tetracyclines, lincomycin, chloramphenicol, most penicillins (all but carbenicillin and sulbenicillin), most cephems (all but cefsulodin and ceftazidime), meropenem, and S-4661, but none of them extrude polymyxin B or imipenem. Extrusion of aminoglycosides is specific to MexXY-OprM, and extrusion of a group of the β-lactams, i.e., carbenicillin, sulbenicillin, ceftazidime, moxalactam, and aztreonam, is specific to MexAB-OprM. Moreover, MexAB-OprM and MexCD-OprJ extrude novobiocin, cefsulodin, and flomoxef, while MexXY-OprM does not. These substrate specificities are distinct from those reported previously. PMID:11083635

Listeria monocytogenes CadC Regulates Cadmium Efflux and Fine-tunes Lipoprotein Localization to Escape the Host Immune Response and Promote Infection.

PubMed

Pombinho, Rita; Camejo, Ana; Vieira, Ana; Reis, Olga; Carvalho, Filipe; Almeida, Maria Teresa; Pinheiro, Jorge Campos; Sousa, Sandra; Cabanes, Didier

2017-05-01

Listeria monocytogenes is a major intracellular human foodborne bacterial pathogen. We previously revealed L. monocytogenes cadC as highly expressed during mouse infection. Here we show that L. monocytogenes CadC is a sequence-specific, DNA-binding and cadmium-dependent regulator of CadA, an efflux pump conferring cadmium resistance. CadC but not CadA is required for L. monocytogenes infection in vivo. Interestingly, CadC also directly represses lspB, a gene encoding a lipoprotein signal peptidase whose expression appears detrimental for infection. lspB overexpression promotes the release of the LpeA lipoprotein to the extracellular medium, inducing tumor necrosis factor α and interleukin 6 expression, thus impairing L. monocytogenes survival in macrophages. We propose that L. monocytogenes uses CadC to repress lspB expression during infection to avoid LpeA exposure to the host immune system, diminishing inflammatory cytokine expression and promoting intramacrophagic survival and virulence. CadC appears as the first metal efflux pump regulator repurposed during infection to fine-tune lipoprotein processing and host responses. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

Sodium efflux from voltage clamped squid giant axons.

PubMed Central

Landowne, D

1977-01-01

1. The efflux of radioactive sodium was measured from squid axons during simultaneous voltage clamp experiments such that it was possible to determine the efflux of sodium associated with a measured voltage clamp current. 2. The extra efflux of sodium associated with voltage clamp pulses increased linearly with the magnitude of the depolarization above 40 mV. A 100 mV pulse of sufficient duration to produce all of the sodium current increased the rate constant of efflux by about 10(-6). 3. Application of 100 nM tetrodotoxin eliminated the sodium current and the extra efflux of radioactive sodium. 4. Cooling the axon increased the extra efflux/voltage clamp pulse slightly with a Q10 of 1/1-1. On the same axons cooling increased the integral of the sodium current with a Q10 of 1/1-4. 5. Replacing external sodium with Tris, dextrose or Mg-mannitol reduced the extra efflux of sodium by about 50%. The inward sodium current was replaced with an outward current as expected. 6. Replacing external sodium with lithium also reduced the extra efflux by about 50% but the currents seen in lithium were slightly larger than those in sodium. 7. The effect of replacing external sodium was not voltage dependent. Cooling reduced the effect so that there was less reduction of efflux on switching to Tris ASW in the cold than in the warm. 8. The extra efflux of sodium into sodium-free ASW is approximately the same as the integral of the sodium current. Adding external sodium produces a deviation from the independence principle such that there is more exchange of sodium than predicted. Such a deviation from prediction was noted by Hodgkin & Huxley (1952c). 9. Using the equations of Hodgkin & Huxley (1952c) modified to include the deviation from independence reported in this paper and its temperature dependence, one can predict the temperature dependence of the sodium efflux associated with action potentials and obtain much better agreement than is possibly without these phenomena. 10

Coupling of remote alternating-access transport mechanisms for protons and substrates in the multidrug efflux pump AcrB

PubMed Central

Eicher, Thomas; Seeger, Markus A; Anselmi, Claudio; Zhou, Wenchang; Brandstätter, Lorenz; Verrey, François; Diederichs, Kay; Faraldo-Gómez, José D; Pos, Klaas M

2014-01-01

Membrane transporters of the RND superfamily confer multidrug resistance to pathogenic bacteria, and are essential for cholesterol metabolism and embryonic development in humans. We use high-resolution X-ray crystallography and computational methods to delineate the mechanism of the homotrimeric RND-type proton/drug antiporter AcrB, the active component of the major efflux system AcrAB-TolC in Escherichia coli, and one most complex and intriguing membrane transporters known to date. Analysis of wildtype AcrB and four functionally-inactive variants reveals an unprecedented mechanism that involves two remote alternating-access conformational cycles within each protomer, namely one for protons in the transmembrane region and another for drugs in the periplasmic domain, 50 Å apart. Each of these cycles entails two distinct types of collective motions of two structural repeats, coupled by flanking α-helices that project from the membrane. Moreover, we rationalize how the cross-talk among protomers across the trimerization interface might lead to a more kinetically efficient efflux system. DOI: http://dx.doi.org/10.7554/eLife.03145.001 PMID:25248080

Biomechanical loading on the upper extremity increases from single key tapping to directional tapping.

PubMed

Qin, Jin; Trudeau, Matthieu; Katz, Jeffrey N; Buchholz, Bryan; Dennerlein, Jack T

2011-08-01

Musculoskeletal disorders associated with computer use span the joints of the upper extremity. Computing typically involves tapping in multiple directions. Thus, we sought to describe the loading on the finger, wrist, elbow and shoulder joints in terms of kinematic and kinetic difference across single key switch tapping to directional tapping on multiple keys. An experiment with repeated measures design was conducted. Six subjects tapped with their right index finger on a stand-alone number keypad placed horizontally in three conditions: (1) on single key switch (the number key 5); (2) left and right on number key 4 and 6; (3) top and bottom on number key 8 and 2. A force-torque transducer underneath the keypad measured the fingertip force. An active-marker infrared motion analysis system measured the kinematics of the fingertip, hand, forearm, upper arm and torso. Joint moments for the metacarpophalangeal, wrist, elbow, and shoulder joints were estimated using inverse dynamics. Tapping in the top-bottom orientation introduced the largest biomechanical loading on the upper extremity especially for the proximal joint, followed by tapping in the left-right orientation, and the lowest loading was observed during single key switch tapping. Directional tapping on average increased the fingertip force, joint excursion, and peak-to-peak joint torque by 45%, 190% and 55%, respectively. Identifying the biomechanical loading patterns associated with these fundamental movements of keying improves the understanding of the risks of upper extremity musculoskeletal disorders for computer keyboard users. Copyright © 2010 Elsevier Ltd. All rights reserved.

TAPS for Pupils

ERIC Educational Resources Information Center

Earle, Sarah

2018-01-01

By placing the Focused Assessment approach within the TAPS pyramid framework, schools are beginning to find a number of ways in which learning in science can be enhanced for pupils. The quotations in this article provide examples of the ways in which science subject leaders (SSL) describe the impact of TAPS on their pupils.

Stereotypes for lever-tap operation.

PubMed

Chan, Alan H S; Tsang, Steve N H; Hoffmann, Errol R

2016-04-15

Lever-operated taps have become more popular and are commonly used in operating theatres, food preparation areas and where users have poor strength; however, there is very little data available for user expectations on tap operation. Thus, an experiment on dual lever-operated water tap (faucets) was conducted with the aim of for providing information for improved design. This study aims to compare different lever-tap designs and their stereotypes adopted by the end-user to operate them also to verify the stereotypes for increasing or decreasing the water flow. 240 participants were requested to rotate the lever tap to indicate direction for increasing and decreasing water flow with simulated hardware, using actual taps placed at the top of a simulated washbasin. Nine initial positions of the lever were used for increasing and decreasing flows, ranging from the ends of both levers facing outward from the bowl center to the ends of both levers facing inward. All levers operated in the horizontal plane. Strong stereotypes (greater than 80%) for several initial lever orientations were found for increasing water flow, especially when the initial lever end positions were facing outwards. However, for different initial positions at which participants were told that the water was flowing and the flow was to be decreased, no strong stereotypes existed. The stereotypes for increasing water flow of dual-lever taps were strong, whereas those for decreasing water flow were weak and hence the stereotype reversibility was also weak. In terms of user expectations, lever taps do not show any great advantage over cross-taps in terms of operator expectations for increasing and decreasing water flow.

49 CFR 192.151 - Tapping.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 3 2010-10-01 2010-10-01 false Tapping. 192.151 Section 192.151 Transportation... BY PIPELINE: MINIMUM FEDERAL SAFETY STANDARDS Design of Pipeline Components § 192.151 Tapping. (a) Each mechanical fitting used to make a hot tap must be designed for at least the operating pressure of...

Genomic Analysis of ATP Efflux in Saccharomyces cerevisiae

PubMed Central

Peters, Theodore W.; Miller, Aaron W.; Tourette, Cendrine; Agren, Hannah; Hubbard, Alan; Hughes, Robert E.

2015-01-01

Adenosine triphosphate (ATP) plays an important role as a primary molecule for the transfer of chemical energy to drive biological processes. ATP also functions as an extracellular signaling molecule in a diverse array of eukaryotic taxa in a conserved process known as purinergic signaling. Given the important roles of extracellular ATP in cell signaling, we sought to comprehensively elucidate the pathways and mechanisms governing ATP efflux from eukaryotic cells. Here, we present results of a genomic analysis of ATP efflux from Saccharomyces cerevisiae by measuring extracellular ATP levels in cultures of 4609 deletion mutants. This screen revealed key cellular processes that regulate extracellular ATP levels, including mitochondrial translation and vesicle sorting in the late endosome, indicating that ATP production and transport through vesicles are required for efflux. We also observed evidence for altered ATP efflux in strains deleted for genes involved in amino acid signaling, and mitochondrial retrograde signaling. Based on these results, we propose a model in which the retrograde signaling pathway potentiates amino acid signaling to promote mitochondrial respiration. This study advances our understanding of the mechanism of ATP secretion in eukaryotes and implicates TOR complex 1 (TORC1) and nutrient signaling pathways in the regulation of ATP efflux. These results will facilitate analysis of ATP efflux mechanisms in higher eukaryotes. PMID:26585826

Hand-pumps as reservoirs for microbial contamination of well water

PubMed Central

Ferguson, Andrew S.; Mailloux, Brian J.; Ahmed, Kazi M.; van Geen, Alexander; McKay, Larry D.; Culligan, Patricia J.

2018-01-01

The retention and release of total coliforms and Escherichia coli was investigated in hand-pumps removed from tubewells tapping a faecally contaminated aquifer in Matlab, Bangladesh, and from a new hand-pump deliberately spiked with E. coli. All hand-pumps were connected to reservoirs of sterile water and flushed. Faecal coliforms were observed in the discharge from all three of the previously used hand-pumps, at concentrations comparable to levels measured in discharge when they were attached to the tubewells. During daily flushing of one of the previously used hand-pumps, the concentration of total coliforms in the discharge remained relatively constant (≈103 MPN/100 mL). Concentrations of E. coli in the pump discharge declined over time, but E. coli was still detectable up to 29 days after the start of flushing. In the deliberately spiked hand-pump, E. coli was observed in the discharge over 125 days (t50 = 8 days) and found to attach preferentially to elastomeric materials within the hand-pump. Attempts to disinfect both the village and new hand-pumps using shock chlorination were shown to be unsuccessful. These results demonstrate that hand-pumps can act as persistent reservoirs for microbial indicator bacteria. This could potentially influence drinking water quality and bias testing of water quality. PMID:22048430

Functional implications of an intermeshing cogwheel-like interaction between TolC and MacA in the action of macrolide-specific efflux pump MacAB-TolC.

PubMed

Xu, Yongbin; Song, Saemee; Moeller, Arne; Kim, Nahee; Piao, Shunfu; Sim, Se-Hoon; Kang, Mooseok; Yu, Wookyung; Cho, Hyun-Soo; Chang, Iksoo; Lee, Kangseok; Ha, Nam-Chul

2011-04-15

Macrolide-specific efflux pump MacAB-TolC has been identified in diverse gram-negative bacteria including Escherichia coli. The inner membrane transporter MacB requires the outer membrane factor TolC and the periplasmic adaptor protein MacA to form a functional tripartite complex. In this study, we used a chimeric protein containing the tip region of the TolC α-barrel to investigate the role of the TolC α-barrel tip region with regard to its interaction with MacA. The chimeric protein formed a stable complex with MacA, and the complex formation was abolished by substitution at the functionally essential residues located at the MacA α-helical tip region. Electron microscopic study delineated that this complex was made by tip-to-tip interaction between the tip regions of the α-barrels of TolC and MacA, which correlated well with the TolC and MacA complex calculated by molecular dynamics. Taken together, our results demonstrate that the MacA hexamer interacts with TolC in a tip-to-tip manner, and implies the manner by which MacA induces opening of the TolC channel.

Functional Implications of an Intermeshing Cogwheel-like Interaction between TolC and MacA in the Action of Macrolide-specific Efflux Pump MacAB-TolC*

PubMed Central

Xu, Yongbin; Song, Saemee; Moeller, Arne; Kim, Nahee; Piao, Shunfu; Sim, Se-Hoon; Kang, Mooseok; Yu, Wookyung; Cho, Hyun-Soo; Chang, Iksoo; Lee, Kangseok; Ha, Nam-Chul

2011-01-01

Macrolide-specific efflux pump MacAB-TolC has been identified in diverse Gram-negative bacteria including Escherichia coli. The inner membrane transporter MacB requires the outer membrane factor TolC and the periplasmic adaptor protein MacA to form a functional tripartite complex. In this study, we used a chimeric protein containing the tip region of the TolC α-barrel to investigate the role of the TolC α-barrel tip region with regard to its interaction with MacA. The chimeric protein formed a stable complex with MacA, and the complex formation was abolished by substitution at the functionally essential residues located at the MacA α-helical tip region. Electron microscopic study delineated that this complex was made by tip-to-tip interaction between the tip regions of the α-barrels of TolC and MacA, which correlated well with the TolC and MacA complex calculated by molecular dynamics. Taken together, our results demonstrate that the MacA hexamer interacts with TolC in a tip-to-tip manner, and implies the manner by which MacA induces opening of the TolC channel. PMID:21325274

SatR Is a Repressor of Fluoroquinolone Efflux Pump SatAB

PubMed Central

Escudero, Jose Antonio; San Millan, Alvaro; Montero, Natalia; Gutierrez, Belen; Ovejero, Cristina Martinez; Carrilero, Laura

2013-01-01

Streptococcus suis is an emerging zoonotic agent responsible for high-mortality outbreaks among the human population in China. In this species, the ABC transporter SatAB mediates fluoroquinolone resistance when overexpressed. Here, we describe and characterize satR, an open reading frame (ORF) encoding a MarR superfamily regulator that acts as a repressor of satAB. satR is cotranscribed with satAB, and its interruption entails the overexpression of the pump, leading to a clinically relevant increase in resistance to fluoroquinolones. PMID:23650171

Investigation of formaldehyde pollution of tap water and rain water using a novel visual colorimetry.

PubMed

Murai, K; Okano, M; Kuramitz, H; Hata, N; Kawakami, T; Taguchi, S

2008-01-01

The pollution of tap water and rain water with formaldehyde in Toyama Pref., Japan was investigated by means of a simple, rapid and cost-effective visual colorimetry developed by us. The levels of formaldehyde in three tap waters from different sources of dams on mountainside and a well-water pumped in urban area in Toyama Pref. were lower than 0.01 mg L(-1) that was the detection limit of the colorimetry. On the other hand, rain waters were seriously polluted with formaldehyde. Rain waters were sampled from three different sites (urban area, top of hill and industrial area) in Toyama Pref. from autumn to winter in 2006. The levels of formaldehyde in the rain waters ranged from 0.07 to 0.30 mg L(-1). The analytical results by the visual colorimetry were in good agreement with those obtained by GC-MS method. It was confirmed that the colorimetry is excellent for practical use for the determination of formaldehyde. It must be concerned about the pollution of rainwater with formaldehyde, when rain water is applied for tap water and miscellaneous purpose. Copyright IWA Publishing 2008.

Spontaneous eye blinks are entrained by finger tapping.

PubMed

Cong, D-K; Sharikadze, M; Staude, G; Deubel, H; Wolf, W

2010-02-01

We studied the mutual cross-talk between spontaneous eye blinks and continuous, self-paced unimanual and bimanual tapping. Both types of motor activities were analyzed with regard to their time-structure in synchronization-continuation tapping tasks which involved different task instructions, namely "standard" finger tapping (Experiment 1), "strong" tapping (Experiment 2) requiring more forceful finger movements, and "impulse-like" tapping (Experiment 3) where upward-downward finger movements had to be very fast. In a further control condition (Experiment 4), tapping was omitted altogether. The results revealed a prominent entrainment of spontaneous blink behavior by the manual tapping, with bimanual tapping being more effective than unimanual tapping, and with the "strong" and "impulse-like" tapping showing the largest effects on blink timing. Conversely, we found no significant effects of the tapping on the timing of the eye blinks across all experiments. The findings suggest a functional overlap of the motor control structures responsible for voluntary, rhythmic finger movements and eye blinking behavior.

RBE4 cells are highly resistant to paraquat-induced cytotoxicity: studies on uptake and efflux mechanisms.

PubMed

Vilas-Boas, V; Silva, R; Guedes-de-Pinho, P; Carvalho, F; Bastos, M L; Remião, F

2014-09-01

Paraquat (PQ) is a widely used, highly toxic and non-selective contact herbicide, which has been associated with central neurotoxic effects, namely the development of Parkinson's disease, but whose effects to the blood-brain barrier (BBB) itself have rarely been studied. This work studied the mechanisms of PQ uptake and efflux in a rat's BBB cell model, the RBE4 cells. PQ is believed to enter cells using the basic or neutral amino acid or polyamine transport systems or through the choline-uptake system. In contrast, PQ efflux from cells is reported to be mediated by P-glycoprotein. Therefore, we evaluated PQ-induced cytotoxicity and the effect of some substrates/blockers of these transporters (such as arginine, L-valine, putrescine, hemicholinium-3 and GF120918) on such cytotoxicity. RBE4 cells were shown to be extremely resistant to PQ after 24 h of exposure; even at concentrations as high as 50 mM approximately 45% of the cells remained viable. Prolonging exposure until 48 h elicited significant cytotoxicity only for PQ concentrations above 5 mM. Although hemicholinium-3, a choline-uptake system inhibitor, significantly protected cells against PQ-induced toxicity, none of the effects were observed for arginine, L-valine or putrescine. Meanwhile, inhibiting the efflux pump P-glycoprotein using GF120918 significantly enhanced PQ-induced cytotoxicity. In conclusion, PQ used the choline-uptake system, instead of the transporters for the basic or neutral amino acids or for the polyamines, to enter RBE4 cells. P-glycoprotein extrudes PQ back to the extracellular medium. However, this efflux mechanism only partially explains the observed RBE4 resistance to PQ. Copyright © 2013 John Wiley & Sons, Ltd.

Efflux Of Nitrate From Hydroponically Grown Wheat

NASA Technical Reports Server (NTRS)

Huffaker, R. C.; Aslam, M.; Ward, M. R.

1992-01-01

Report describes experiments to measure influx, and efflux of nitrate from hydroponically grown wheat seedlings. Ratio between efflux and influx greater in darkness than in light; increased with concentration of nitrate in nutrient solution. On basis of experiments, authors suggest nutrient solution optimized at lowest possible concentration of nitrate.

A Perspective on Efflux Transport Proteins in the Liver

PubMed Central

Kock, K; Brouwer, K.L.R

2013-01-01

Detailed knowledge regarding the influence of hepatic transport proteins on drug disposition has advanced at a rapid pace over the past decade. Efflux transport proteins located in the basolateral and apical (canalicular) membranes of hepatocytes play an important role in the hepatic elimination of many endogenous and exogenous compounds, including drugs and metabolites. This review focuses on the role of these efflux transporters in hepatic drug excretion. The impact of these proteins as underlying factors for disease is highlighted, and the importance of hepatic efflux proteins in the efficacy and toxicity of drugs is discussed. In addition, a brief overview of methodology to evaluate the function of hepatic efflux transport proteins is provided. Current challenges in predicting the impact of altered efflux protein function on systemic, intestinal and hepatocyte exposure to drugs and metabolites are highlighted. PMID:22948894

Striatal norepinephrine efflux in l-DOPA-induced dyskinesia.

PubMed

Ostock, Corinne Y; Bhide, Nirmal; Goldenberg, Adam A; George, Jessica A; Bishop, Christopher

2018-03-01

l-DOPA remains the primary treatment for Parkinson's disease (PD). Unfortunately, its therapeutic benefits are compromised by the development of abnormal involuntary movements (AIMs) known as l-DOPA-induced dyskinesia (LID). The norepinephrine (NE) system originating in the locus coeruleus is profoundly affected in PD and known to influence dopamine (DA) signaling. However, the effect of noradrenergic loss on l-DOPA-induced striatal monoamine efflux and Parkinsonian motor behavior remains controversial and is frequently overlooked in traditional animal models of LID. Thus, the current study sought to determine whether degeneration of the DA and/or NE system(s) altered l-DOPA-induced striatal monoamine efflux in hemiparkinsonian rats with additional NE loss induced by the potent NE-toxin α DA beta hydroxylase (DBH)-saporin. Sham-, DA-, NE-, and dual DA + NE-lesioned rats were treated with l-DOPA (6 mg/kg, s.c.) for 2 weeks. Thereafter, l-DOPA-mediated striatal monoamine efflux was measured with in vivo microdialysis, and concurrent AIMs testing occurred to determine responsiveness to l-DOPA. Noradrenergic lesions exacerbated parkinsonian motor deficits but did not significantly alter LID expression or corresponding l-DOPA-induced striatal monoamine efflux. Interestingly, l-DOPA-induced striatal NE efflux rather than DA efflux, corresponded more closely with dyskinesia severity. Moreover, marked reductions in striatal NE tissue concentration did not appear to impact l-DOPA-induced striatal NE efflux. The current study implicates l-DOPA-induced striatal NE as an important factor in LID expression and demonstrates the importance of developing treatment strategies that co-modulate the NE and DA systems. Copyright © 2018 Elsevier Ltd. All rights reserved.

Two host-induced Ralstonia solanacearum genes, acrA and dinF, encode multidrug efflux pumps and contribute to bacterial wilt virulence.

PubMed

Brown, Darby G; Swanson, Jill K; Allen, Caitilyn

2007-05-01

Multidrug efflux pumps (MDRs) are hypothesized to protect pathogenic bacteria from toxic host defense compounds. We created mutations in the Ralstonia solanacearum acrA and dinF genes, which encode putative MDRs in the broad-host-range plant pathogen. Both mutations reduced the ability of R. solanacearum to grow in the presence of various toxic compounds, including antibiotics, phytoalexins, and detergents. Both acrAB and dinF mutants were significantly less virulent on the tomato plant than the wild-type strain. Complementation restored near-wild-type levels of virulence to both mutants. Addition of either dinF or acrAB to Escherichia coli MDR mutants KAM3 and KAM32 restored the resistance of these strains to several toxins, demonstrating that the R. solanacearum genes can function heterologously to complement known MDR mutations. Toxic and DNA-damaging compounds induced expression of acrA and dinF, as did growth in both susceptible and resistant tomato plants. Carbon limitation also increased expression of acrA and dinF, while the stress-related sigma factor RpoS was required at a high cell density (>10(7) CFU/ml) to obtain wild-type levels of acrA expression both in minimal medium and in planta. The type III secretion system regulator HrpB negatively regulated dinF expression in culture at high cell densities. Together, these results show that acrAB and dinF encode MDRs in R. solanacearum and that they contribute to the overall aggressiveness of this phytopathogen, probably by protecting the bacterium from the toxic effects of host antimicrobial compounds.

Lower extremity kinetics in tap dance.

PubMed

Mayers, Lester; Bronner, Shaw; Agraharasamakulam, Sujani; Ojofeitimi, Sheyi

2010-01-01

Tap dance is a unique performing art utilizing the lower extremities as percussion instruments. In a previous study these authors reported decreased injury prevalence among tap dancers compared to other dance and sports participants. No biomechanical analyses of tap dance exist to explain this finding. The purpose of the current pilot study was to provide a preliminary overview of normative peak kinetic and kinematic data, based on the hypothesis that tap dance generates relatively low ground reaction forces and joint forces and moments. Six professional tap dancers performed four common tap dance sequences that produced data captured by the use of a force platform and a five-camera motion analysis system. The mean vertical ground reaction force for all sequences was found to be 2.06+/-0.55 BW. Mean peak sagittal, frontal, and transverse plane joint moments (hip, knee, and ankle) ranged from 0.07 to 2.62 N.m/kg. These small ground reaction forces and joint forces and moments support our hypothesis, and may explain the relatively low injury incidence in tap dancers. Nevertheless, the analysis is highly complex, and other factors remain to be studied and clarified.

Montelukast is a potent and durable inhibitor of multidrug resistance protein 2 (MRP2)-mediated efflux of taxol and saquinavir

PubMed Central

Roy, Upal; Chakravarty, Geetika; Honer Zu Bentrup, Kerstin; Mondal, Debasis

2009-01-01

The ATP binding cassette (ABC)-transporters are energy dependent efflux pumps which regulate the pharmacokinetics of both anti-cancer chemotherapeutic agents, e.g. taxol, and of HIV-1 protease inhibitors (HPIs), e.g. saquinavir. Increased expression of several ABC-transporters, especially P-gp and MRP2, are observed in multidrug resistant (MDR) tumor cells and on HIV-1 infected lymphocytes. In addition, due to their apical expression on vascular endothelial barriers, both P-gp and MRP2 are of crucial importance towards dictating drug access into sequestered tissues. However, although a number of P-gp inhibitors are currently in clinical trials, possible inhibitors of MRP2 are not being thoroughly investigated. The experimental leukotriene receptor antagonist (LTRA), MK-571 is known to be a potent inhibitor of MRP transporters. Using the MRP2 over-expressing cell line, MDCKII-MRP2, we evaluated whether the clinically approved LTRAs, e.g. montelukast (Singulair™) and zafirlukast (Accolate™), can similarly suppress MRP2-mediated efflux. We compared the efficacy of increasing concentrations (20-100 μM) of MK-571, montelukast, and zafirlukast, in suppressing the efflux of calcein-AM, a fluorescent MRP substrate, and the radiolabeled [3H-] drugs, taxol and saquinavir. Montelukast was the most potent inhibitor (p<0.01) of MRP2-mediated efflux of all three substrates. Montelukast also increased (p<0.01) the duration of intracellular retention of both taxol and saquinavir. More than 50% of the drugs were retained in cells even after 90 mins post removal of montelukast from the medium. Our findings implicate that montelukast, a relatively safe anti-asthmatic agent, may be used as an adjunct therapy to suppress the efflux of taxol and saquinavir from MRP2 overexpressing cells. PMID:19952419

Self-advancing step-tap tool

NASA Technical Reports Server (NTRS)

Pettit, Donald R. (Inventor); Penner, Ronald K. (Inventor); Franklin, Larry D. (Inventor); Camarda, Charles J. (Inventor)

2008-01-01

Methods and tool for simultaneously forming a bore in a work piece and forming a series of threads in said bore. In an embodiment, the tool has a predetermined axial length, a proximal end, and a distal end, said tool comprising: a shank located at said proximal end; a pilot drill portion located at said distal end; and a mill portion intermediately disposed between said shank and said pilot drill portion. The mill portion is comprised of at least two drill-tap sections of predetermined axial lengths and at least one transition section of predetermined axial length, wherein each of said at least one transition section is sandwiched between a distinct set of two of said at least two drill-tap sections. The at least two drill-tap sections are formed of one or more drill-tap cutting teeth spirally increasing along said at least two drill-tap sections, wherein said tool is self-advanced in said work piece along said formed threads, and wherein said tool simultaneously forms said bore and said series of threads along a substantially similar longitudinal axis.

CO2 efflux from cleared mangrove peat.

PubMed

Lovelock, Catherine E; Ruess, Roger W; Feller, Ilka C

2011-01-01

CO(2) emissions from cleared mangrove areas may be substantial, increasing the costs of continued losses of these ecosystems, particularly in mangroves that have highly organic soils. We measured CO(2) efflux from mangrove soils that had been cleared for up to 20 years on the islands of Twin Cays, Belize. We also disturbed these cleared peat soils to assess what disturbance of soils after clearing may have on CO(2) efflux. CO(2) efflux from soils declines from time of clearing from ∼10,600 tonnes km(-2) year(-1) in the first year to 3000 tonnes km(2) year(-1) after 20 years since clearing. Disturbing peat leads to short term increases in CO(2) efflux (27 umol m(-2) s(-1)), but this had returned to baseline levels within 2 days. Deforesting mangroves that grow on peat soils results in CO(2) emissions that are comparable to rates estimated for peat collapse in other tropical ecosystems. Preventing deforestation presents an opportunity for countries to benefit from carbon payments for preservation of threatened carbon stocks.

Gaining Momentum, Losing Ground. Tapping America's Potential (TAP) Progress Report, 2008. Executive Summary

ERIC Educational Resources Information Center

Tapping America's Potential, 2008

2008-01-01

In July 2005, Business Roundtable and fifteen of America's most prominent business organizations--Tapping America's Potential, the TAP coalition--issued a report stating that "one of the pillars of American economic prosperity--U.S. scientific and technological superiority--is beginning to atrophy even as other nations are developing their own…

Development of classification models for identifying "true" P-glycoprotein (P-gp) inhibitors through inhibition, ATPase activation and monolayer efflux assays.

PubMed

Rapposelli, Simona; Coi, Alessio; Imbriani, Marcello; Bianucci, Anna Maria

2012-01-01

P-glycoprotein (P-gp) is an efflux pump involved in the protection of tissues of several organs by influencing xenobiotic disposition. P-gp plays a key role in multidrug resistance and in the progression of many neurodegenerative diseases. The development of new and more effective therapeutics targeting P-gp thus represents an intriguing challenge in drug discovery. P-gp inhibition may be considered as a valid approach to improve drug bioavailability as well as to overcome drug resistance to many kinds of tumours characterized by the over-expression of this protein. This study aims to develop classification models from a unique dataset of 59 compounds for which there were homogeneous experimental data on P-gp inhibition, ATPase activation and monolayer efflux. For each experiment, the dataset was split into a training and a test set comprising 39 and 20 molecules, respectively. Rational splitting was accomplished using a sphere-exclusion type algorithm. After a two-step (internal/external) validation, the best-performing classification models were used in a consensus predicting task for the identification of compounds named as "true" P-gp inhibitors, i.e., molecules able to inhibit P-gp without being effluxed by P-gp itself and simultaneously unable to activate the ATPase function.

Drinking of tap water is smart, but how do it better? - A tap water quality research

NASA Astrophysics Data System (ADS)

Mika, Anna; Sekuła, Klaudia; Dendys, Marta; Ptaszek, Weronika; Postawa, Adam

2018-02-01

Drinking tap water has recently become popular. It is a way to fight with the tons of garbage (disposable, plastic bottles). However, many people are afraid of water quality. The research was performed in December 2015 in Krakow, during one week. 56 samples were collected. The samples were taken in different times of the day and in the two types of building (old one with installation from the 80s and new one with installation built in past few years). Samples were taken by two qualified operators. The first sample was collected at the morning at 6 a.m., before anyone uses the tap. The second one after the tap was flushed and then the third one after 30 minutes stagnation. At the evening was taken one sample (after using the tap all day).The aim of the research was to check the quality of drinking water in the end-user. The results show that quality of tap water in Krakow is good, also in the end-user, but the concentration of chemical elements are changing during the flushing and using of the tap.

Traffic Aware Planner (TAP) Flight Evaluation

NASA Technical Reports Server (NTRS)

Maris, John M.; Haynes, Mark A.; Wing, David J.; Burke, Kelly A.; Henderson, Jeff; Woods, Sharon E.

2014-01-01

NASA's Traffic Aware Planner (TAP) is a cockpit decision support tool that has the potential to achieve significant fuel and time savings when it is embedded in the data-rich Next Generation Air Transportation System (NextGen) airspace. To address a key step towards the operational deployment of TAP and the NASA concept of Traffic Aware Strategic Aircrew Requests (TASAR), a system evaluation was conducted in a representative flight environment in November, 2013. Numerous challenges were overcome to achieve this goal, including the porting of the foundational Autonomous Operations Planner (AOP) software from its original simulation-based, avionics-embedded environment to an Electronic Flight Bag (EFB) platform. A flight-test aircraft was modified to host the EFB, the TAP application, an Automatic Dependent Surveillance Broadcast (ADS-B) processor, and a satellite broadband datalink. Nine Evaluation Pilots conducted 26 hours of TAP assessments using four route profiles in the complex eastern and north-eastern United States airspace. Extensive avionics and video data were collected, supplemented by comprehensive inflight and post-flight questionnaires. TAP was verified to function properly in the live avionics and ADS-B environment, characterized by recorded data dropouts, latency, and ADS-B message fluctuations. Twelve TAP-generated optimization requests were submitted to ATC, of which nine were approved, and all of which resulted in fuel and/or time savings. Analysis of subjective workload data indicated that pilot interaction with TAP during flight operations did not induce additional cognitive loading. Additionally, analyses of post-flight questionnaire data showed that the pilots perceived TAP to be useful, understandable, intuitive, and easy to use. All program objectives were met, and the next phase of TAP development and evaluations with partner airlines is in planning for 2015.

Assessment of leg muscle activity using toe tapping in patients with Parkinson's disease: comparison of two types of toe tapping.

PubMed

Taniguchi, Seira; Peper, Ferdinand; Shimokawa, Tetsuya

2018-05-01

[Purpose] This study investigates two types of toe tapping, i.e., "closed," with both feet on the floor, and "open," in which the foot does not touch the ground, and evaluates their usefulness in combination with monitoring of muscle activity during toe tapping. [Subjects and Methods] The study enrolled 11 patients with Parkinson's disease (PD) and 9 controls (Controls). The tibialis anterior (TA) and gastrocnemius (GS) muscle activity during toe tapping was measured using surface electromyography. [Results] In closed tapping, the minima in GS activation with the first tap was significantly higher in patients with PD than in Controls. In open tapping, the coefficient of variation (CV) of local maxima in TA activation was significantly higher in patients with PD than in Controls. In both types of tapping, the CV of extrema in GS activities increased with disease duration, but this may be due to the long-term administration of Levodopa, which itself tends to cause excessive GS activities. [Conclusion] Closed tapping is suitable for the assessment of GS activity and can detect excessive activities, which is observed as visible movement. Open tapping, on the other hand, is suitable for assessment of TA activity.

Timescale dependence of environmental controls on methane efflux from Poyang Hu, China

NASA Astrophysics Data System (ADS)

Liu, Lixiang; Xu, Ming; Li, Renqiang; Shao, Rui

2017-04-01

Lakes are an important natural source of CH4 to the atmosphere. However, the multi-seasonal CH4 efflux from lakes has been rarely studied. In this study, the CH4 efflux from Poyang Hu, the largest freshwater lake in China, was measured monthly over a 4-year period by using the floating-chamber technique. The mean annual CH4 efflux throughout the 4 years was 0.54 mmol m-2 day-1, ranging from 0.47 to 0.60 mmol m-2 day-1. The CH4 efflux had a high seasonal variation with an average summer (June to August) efflux of 1.34 mmol m-2 day-1 and winter (December to February) efflux of merely 0.18 mmol m-2 day-1. The efflux showed no apparent diel pattern, although most of the peak effluxes appeared in the late morning, from 10:00 to 12:00 CST (GMT + 8). Multivariate stepwise regression on a seasonal scale showed that environmental factors, such as sediment temperature, sediment total nitrogen content, dissolved oxygen, and total phosphorus content in the water, mainly regulated the CH4 efflux. However, the CH4 efflux only showed a strong positive linear correlation with wind speed within 1 day on a bihourly scale in the multivariate regression analyses but almost no correlation with wind speed on diurnal and seasonal scales.

Two Host-Induced Ralstonia solanacearum Genes, acrA and dinF, Encode Multidrug Efflux Pumps and Contribute to Bacterial Wilt Virulence▿ †

PubMed Central

Brown, Darby G.; Swanson, Jill K.; Allen, Caitilyn

2007-01-01

Multidrug efflux pumps (MDRs) are hypothesized to protect pathogenic bacteria from toxic host defense compounds. We created mutations in the Ralstonia solanacearum acrA and dinF genes, which encode putative MDRs in the broad-host-range plant pathogen. Both mutations reduced the ability of R. solanacearum to grow in the presence of various toxic compounds, including antibiotics, phytoalexins, and detergents. Both acrAB and dinF mutants were significantly less virulent on the tomato plant than the wild-type strain. Complementation restored near-wild-type levels of virulence to both mutants. Addition of either dinF or acrAB to Escherichia coli MDR mutants KAM3 and KAM32 restored the resistance of these strains to several toxins, demonstrating that the R. solanacearum genes can function heterologously to complement known MDR mutations. Toxic and DNA-damaging compounds induced expression of acrA and dinF, as did growth in both susceptible and resistant tomato plants. Carbon limitation also increased expression of acrA and dinF, while the stress-related sigma factor RpoS was required at a high cell density (>107 CFU/ml) to obtain wild-type levels of acrA expression both in minimal medium and in planta. The type III secretion system regulator HrpB negatively regulated dinF expression in culture at high cell densities. Together, these results show that acrAB and dinF encode MDRs in R. solanacearum and that they contribute to the overall aggressiveness of this phytopathogen, probably by protecting the bacterium from the toxic effects of host antimicrobial compounds. PMID:17337552

Identification and prevalence of RND family multidrug efflux pump oqxAB genes in Enterococci isolates from swine manure in China.

PubMed

Yuan, Li; Zhai, Ya-Jun; Wu, Hua; Sun, Hua-Run; He, Zhi-Pei; Wang, Ya-Bin; Pan, Yu-Shan; Kuang, Nan-Nan; Hu, Gong-Zheng

2018-06-01

The resistance/nodulation/cell division (RND) family multidrug efflux pump, OqxAB, has been identified as one of the leading mechanisms of plasmid-mediated quinolone resistance and has become increasingly prevalent among Enterobacteriaceae in recent years. However, oqxAB genes have not yet been reported in Enterococcus isolates. The aim of the present study was to identify the oqxAB genes and investigate their prevalence among Enterococcus from swine manure in China. The oqxAB genes were screened in 87 Enterococcus isolates by PCR. The transferability of the oqxAB genes in Enterococcus was determined by conjugation experiments. The genetic environment of oqxAB genes was investigated by cloning experiments, PCR mapping and sequencing. A high prevalence (86.2 %) of olaquindox resistance was observed in Enterococcus and 98.9 % isolates exhibited multidrug-resistance phenotypes. The occurrence of oqxA and oqxB in Enterococcus was also high (79.3 and 65.5 %, respectively). Sequence analysis of the cloned fragment indicated that the oqxAB cassette was linked to an incomplete Tn5 transposon containing aph(3')-IIa and flanked by IS26 [IS26-oqxAB-IS26-aph(3')-IIa]. The oqxAB-aph(3')-IIa-positive transconjugant or transformant showed resistance or reduced susceptibility to enrofloxacin, ciprofloxacin, olaquindox, mequindox, florfenicol, neomycin and kanamycin. This is the first time that the oqxAB genes have been identified in Enterococcus faecalis from swine manure. The genetic linkage of oqxAB-aph(3')-IIa in Enterococcus has not been described before. The high prevalence of oqxAB genes in Enterococcus suggests that it may constitute a reservoir for oqxAB genes and pose a potential threat to public health.

Glutathione Efflux and Cell Death

PubMed Central

2012-01-01

Abstract Significance: Glutathione (GSH) depletion is a central signaling event that regulates the activation of cell death pathways. GSH depletion is often taken as a marker of oxidative stress and thus, as a consequence of its antioxidant properties scavenging reactive species of both oxygen and nitrogen (ROS/RNS). Recent Advances: There is increasing evidence demonstrating that GSH loss is an active phenomenon regulating the redox signaling events modulating cell death activation and progression. Critical Issues: In this work, we review the role of GSH depletion by its efflux, as an important event regulating alterations in the cellular redox balance during cell death independent from oxidative stress and ROS/RNS formation. We discuss the mechanisms involved in GSH efflux during cell death progression and the redox signaling events by which GSH depletion regulates the activation of the cell death machinery. Future Directions: The evidence summarized here clearly places GSH transport as a central mechanism mediating redox signaling during cell death progression. Future studies should be directed toward identifying the molecular identity of GSH transporters mediating GSH extrusion during cell death, and addressing the lack of sensitive approaches to quantify GSH efflux. Antioxid. Redox Signal. 17, 1694–1713. PMID:22656858

PfeT, a P1B4 -type ATPase, effluxes ferrous iron and protects Bacillus subtilis against iron intoxication.

PubMed

Guan, Guohua; Pinochet-Barros, Azul; Gaballa, Ahmed; Patel, Sarju J; Argüello, José M; Helmann, John D

2015-11-01

Iron is an essential element for nearly all cells and limited iron availability often restricts growth. However, excess iron can also be deleterious, particularly when cells expressing high affinity iron uptake systems transition to iron rich environments. Bacillus subtilis expresses numerous iron importers, but iron efflux has not been reported. Here, we describe the B. subtilis PfeT protein (formerly YkvW/ZosA) as a P1B4 -type ATPase in the PerR regulon that serves as an Fe(II) efflux pump and protects cells against iron intoxication. Iron and manganese homeostasis in B. subtilis are closely intertwined: a pfeT mutant is iron sensitive, and this sensitivity can be suppressed by low levels of Mn(II). Conversely, a pfeT mutant is more resistant to Mn(II) overload. In vitro, the PfeT ATPase is activated by both Fe(II) and Co(II), although only Fe(II) efflux is physiologically relevant in wild-type cells, and null mutants accumulate elevated levels of intracellular iron. Genetic studies indicate that PfeT together with the ferric uptake repressor (Fur) cooperate to prevent iron intoxication, with iron sequestration by the MrgA mini-ferritin playing a secondary role. Protection against iron toxicity may also be a key role for related P1B4 -type ATPases previously implicated in bacterial pathogenesis. © 2015 John Wiley & Sons Ltd.

CO2 Efflux from Cleared Mangrove Peat

PubMed Central

Lovelock, Catherine E.; Ruess, Roger W.; Feller, Ilka C.

2011-01-01

Background CO2 emissions from cleared mangrove areas may be substantial, increasing the costs of continued losses of these ecosystems, particularly in mangroves that have highly organic soils. Methodology/Principal Findings We measured CO2 efflux from mangrove soils that had been cleared for up to 20 years on the islands of Twin Cays, Belize. We also disturbed these cleared peat soils to assess what disturbance of soils after clearing may have on CO2 efflux. CO2 efflux from soils declines from time of clearing from ∼10 600 tonnes km−2 year−1 in the first year to 3000 tonnes km2 year−1 after 20 years since clearing. Disturbing peat leads to short term increases in CO2 efflux (27 umol m−2 s−1), but this had returned to baseline levels within 2 days. Conclusions/Significance Deforesting mangroves that grow on peat soils results in CO2 emissions that are comparable to rates estimated for peat collapse in other tropical ecosystems. Preventing deforestation presents an opportunity for countries to benefit from carbon payments for preservation of threatened carbon stocks. PMID:21738628

ATP binding cassette G1-dependent cholesterol efflux during inflammation.

PubMed

de Beer, Maria C; Ji, Ailing; Jahangiri, Anisa; Vaughan, Ashley M; de Beer, Frederick C; van der Westhuyzen, Deneys R; Webb, Nancy R

2011-02-01

ATP binding cassette transporter G1 (ABCG1) mediates the transport of cellular cholesterol to HDL, and it plays a key role in maintaining macrophage cholesterol homeostasis. During inflammation, HDL undergoes substantial remodeling, acquiring lipid changes and serum amyloid A (SAA) as a major apolipoprotein. In the current study, we investigated whether remodeling of HDL that occurs during acute inflammation impacts ABCG1-dependent efflux. Our data indicate that lipid free SAA acts similarly to apolipoprotein A-I (apoA-I) in mediating sequential efflux from ABCA1 and ABCG1. Compared with normal mouse HDL, acute phase (AP) mouse HDL containing SAA exhibited a modest but significant 17% increase in ABCG1-dependent efflux. Interestingly, AP HDL isolated from mice lacking SAA (SAAKO mice) was even more effective in promoting ABCG1 efflux. Hydrolysis with Group IIA secretory phospholipase A(2) (sPLA(2)-IIA) significantly reduced the ability of AP HDL from SAAKO mice to serve as a substrate for ABCG1-mediated cholesterol transfer, indicating that phospholipid (PL) enrichment, and not the presence of SAA, is responsible for alterations in efflux. AP human HDL, which is not PL-enriched, was somewhat less effective in mediating ABCG1-dependent efflux compared with normal human HDL. Our data indicate that inflammatory remodeling of HDL impacts ABCG1-dependent efflux independent of SAA.

Reflex Augmentation of a Tap-Elicited Eyeblink: The Effects of Tone Frequency and Tap Intensity.

ERIC Educational Resources Information Center

Cohen, Michelle E.; And Others

1986-01-01

Describes two experiments that examined whether the amplitude of the human eyeblink by a mild tap between the eyebrows can be increased if a brief tone is presented simultaneously with the tap and how these effects change from newborn infants to adults. (HOD)

Kinematics of the tooth tapping movement.

PubMed

Widmalm, S E; Hedegård, B

1977-07-01

Electrical activity in the masseter muscles and tooth contact vibrations were recorded simultaneously from subjects tapping their teeth slowly into maximal intercuspidation and again with maximal frequency. High speed cinephotography was also used with four of the ten subjects. Three main parts could be distinguished on the obtained graphical representation of the tooth tapping movement: tooth contact phase (TCP), opening phase (OP) and closing phase (CP). Tapping frequency was increased by decreasing the jaw opening degree and the durations of TCP, OP and CP. The jaw velocity immediately before tooth contact, which may be of significance for the reflex response, was however not increased. The average jaw speed was nevertheless increased from 10 to 15 cm/s since the turning from OP to CP was more abrupt in high than in low frequency tapping. The duration of electrical activity after tooth contact was significantly shorter at tapping with high than with low frequency. The teeth maintained contact without detectable rebound between each open-close cycle. The OP started about 100 ms after the cessation of electrical activity both at low and high tapping frequency. The time between end of electrical activity and the start of a new OP was supposed to be dependent upon the relaxation time of the masseter muscles.

Ascorbic Acid Efflux from Human Brain Microvascular Pericytes: Role of Re-uptake

PubMed Central

May, James M.; Qu, Zhi-chao

2015-01-01

Microvascular pericytes take up ascorbic acid on the ascorbate transporter SVCT2. Intracellular ascorbate then protects the cells against apoptosis induced by culture at diabetic glucose concentrations. To investigate whether pericytes might also provide ascorbate to the underlying endothelial cells, we studied ascorbate efflux from human pericytes. When loaded with ascorbate to intracellular concentrations of 0.8–1.0 mM, almost two-thirds of intracellular ascorbate effluxed from the cells over 2 h. This efflux was opposed by ascorbate re-uptake from the medium, since preventing re-uptake by destroying extracellular ascorbate with ascorbate oxidase increased ascorbate loss even further. Ascorbate re-uptake occurred on the SVCT2, since its blockade by replacing medium sodium with choline, by the SVCT2 inhibitor sulfinpyrazone, or by extracellular ascorbate accelerated ascorbate loss from the cells. This was supported by finding that net efflux of radiolabeled ascorbate was increased by unlabeled extracellular ascorbate with a half-maximal effect in the range of the high affinity Km of the SVCT2. Intracellular ascorbate did not inhibit its efflux. To assess the mechanism of ascorbate efflux, known inhibitors of volume-regulated anion channels (VRACs) were tested. These potently inhibited ascorbate transport into cells on the SVCT2, but not its efflux. An exception was the anion transport inhibitor DIDS, which, despite inhibition of ascorbate uptake, also inhibited net efflux at 25–50 µM. These results suggest that ascorbate efflux from vascular pericytes occurs on a DIDS-inhibitable transporter or channel different from VRACs. Further, ascorbate efflux is opposed by re-uptake of ascorbate on the SVCT2, providing a potential regulatory mechanism. PMID:26340060

Metabolic targeting of lactate efflux by malignant glioma inhibits invasiveness and induces necrosis: an in vivo study.

PubMed

Colen, Chaim B; Shen, Yimin; Ghoddoussi, Farhad; Yu, Pingyang; Francis, Todd B; Koch, Brandon J; Monterey, Michael D; Galloway, Matthew P; Sloan, Andrew E; Mathupala, Saroj P

2011-07-01

Glioblastoma multiforme (GBM) are the most malignant among brain tumors. They are frequently refractory to chemotherapy and radiotherapy with mean patient survival of approximately 6 months, despite surgical intervention. The highly glycolytic nature of glioblastomas describes their propensity to metabolize glucose to lactic acid at an elevated rate. To survive, GBMs efflux lactic acid to the tumor microenvironment through transmembrane transporters denoted monocarboxylate transporters (MCTs). We hypothesized that inhibition of MCT function would impair the glycolytic metabolism and affect both glioma invasiveness and survival. We examined the effect on invasiveness with α-cyano-4-hydroxy-cinnamic acid (ACCA, 4CIN, CHCA), a small-molecule inhibitor of lactate transport, through Matrigel-based and organotypic (brain) slice culture invasive assays using U87-MG and U251-MG glioma cells. We then conducted studies in immunodeficient rats by stereotaxic intracranial implantation of the glioma cells followed by programmed orthotopic application of ACCA through osmotic pumps. Effect on the implanted tumor was monitored by small-animal magnetic resonance imaging. Our assays indicated that glioma invasion was markedly impaired when lactate efflux was inhibited. Convection-enhanced delivery of inhibitor to the tumor bed caused tumor necrosis, with 50% of the animals surviving beyond the experimental end points (3 months after inhibitor exhaustion). Most importantly, control animals did not display any adverse neurologic effects during orthotopic administration of ACCA to brain through programmed delivery. These results indicate the clinical potential of targeting lactate efflux in glioma through delivery of small-molecule inhibitors of MCTs either to the tumor bed or to the postsurgical resection cavity.

Evaluation of primary stability of self-tapping and non-self-tapping dental implants. A 12-week clinical study.

PubMed

Marković, Aleksa; Calvo-Guirado, José Luís; Lazić, Zoran; Gómez-Moreno, Gerardo; Ćalasan, Dejan; Guardia, Javier; Čolic, Snježana; Aguilar-Salvatierra, Antonio; Gačić, Bojan; Delgado-Ruiz, Rafael; Janjić, Bojan; Mišić, Tijana

2013-06-01

The aim of this study was to investigate the relationship between surgical techniques and implant macro-design (self-tapping/non-self-tapping) for the optimization of implant stability in the low-density bone present in the posterior maxilla using resonance frequency analysis (RFA). A total of 102 implants were studied. Fifty-six self-tapping BlueSkyBredent® (Bredent GmbH&Co.Kg®, Senden, Germany) and 56 non-self-tapping Standard Plus Straumann® (Institut Straumann AG®, Waldenburg, Switzerland) were placed in the posterior segment of the maxilla. Implants of both types were placed in sites prepared with either lateral bone-condensing or with bone-drilling techniques. Implant stability measurements were performed using RFA immediately after implant placement and weekly during a 12-week follow-up period. Both types of implants placed after bone condensing achieved significantly higher stability immediately after surgery, as well as during the entire 12-week observation period compared with those placed following bone drilling. After bone condensation, there were no significant differences in primary stability or in implant stability after the first week between both implant types. From 2 to 12 postoperative weeks, significantly higher stability was shown by self-tapping implants. After bone drilling, self-tapping implants achieved significantly higher stability than non-self-tapping implants during the entire follow-up period. The outcomes of the present study indicate that bone drilling is not an effective technique for improving implant stability and, following this technique, the use of self-tapping implants is highly recommended. Implant stability optimization in the soft bone can be achieved by lateral bone-condensing technique, regardless of implant macro-design. © 2011 Wiley Periodicals, Inc.

BmeRABC5 is a multidrug efflux system that can confer metronidazole resistance in Bacteroides fragilis.

PubMed

Pumbwe, Lilian; Chang, Abraham; Smith, Rachel L; Wexler, Hannah M

2007-01-01

The RND-family efflux pump gene bmeB5 was previously shown to be overexpressed in metronidazole-resistant laboratory mutants of Bacteroides fragilis. In the present study, we characterized the bmeABC5 genes and an upstream putative TetR-family regulator gene (bmeR5). bmeR5 (645 bp) was located 51 bp upstream of bmeA5 and encoded a 24.9-kDa protein. Deletant strains lacking bmeB5 or bmeR5 were constructed from a wild-type B. fragilis strain ADB77. Strain antimicrobial susceptibility was determined and gene expression was quantified. bmeR5 was overexpressed in Escherichia coli using a 6x-His tag system; BmeR5-His6 was isolated from inclusion bodies and its binding to bmeABC5 promoter regions was determined. BmeR5-His6 bound specifically to the bmeR5-bmeC5 intergenic region (IT1). Deletion of bmeR5 (ADB77DeltabmeR5) resulted in a significant (p < 0.05) increase in expression of bmeA5, bmeB5, and bmeC5, and > two-fold increase in minimum inhibitory concentrations (MICs) of ampicillin, cefoxitin, cefoperazone, ciprofloxacin, imipenem, metronidazole, ethidium bromide, and sodium dodecyl sulfate (SDS). MICs were reduced by the efflux pump inhibitor carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The MICs of ampicillin, cefoperazone, metronidazole, and SDS were reduced by approximately two-fold in ADB77DeltabmeB5. A multidrug (metronidazole)-resistant, nim-negative B. fragilis clinical isolate overexpressed bmeABC5 genes, had a G-->T point mutation in IT1, and significantly reduced binding to BmeR5-His6. These data demonstrate that BmeR5 is a local repressor of bmeABC5 expression and that mutations in IT1 can lead to a derepression and resistance to multiple antimicrobial agents, including metronidazole.

Comparative study of the active cadmium efflux systems operating at the plasma membrane and tonoplast of cucumber root cells.

PubMed

Migocka, Magdalena; Papierniak, Anna; Kosatka, Ewelina; Klobus, Grazyna

2011-10-01

The strategies developed by plants to avoid the toxicity of cadmium (Cd) and other heavy metals involve active sequestration of metals into the apoplast and vacuoles. The protein systems excluding heavy metals from the cell cytosol localize to the plasma membrane and tonoplast and are energized either by ATP or by the electrochemical gradient generated by H(+)-ATPase or by V-ATPase and pyrophosphatase (PPase), respectively. In this work, a comparative study on the contribution of both the plasma membrane and tonoplast in the active detoxification of plant cells after treatment with Cd was performed. The studies using plants treated and untreated with Cd reveal that both, H(+)-coupled and MgATP-driven efflux of Cd across plasma membranes and tonoplast is markedly stimulated in the presence of Cd in the environment. Previous studies on plasma-membrane localized H(+)-coupled Cd efflux together with the present data demonstrating tonoplast H(+)/Cd(2+) antiport activity suggest that H(+)-coupled secondary transport of Cd displays a lower affinity for Cd when compared with Cd primary pumps driven by MgATP. In addition, it is shown that MgATP-energized Cd efflux across both membranes is significantly enhanced by cysteine, dithiothreitol, and glutathione. These results suggest that Cd is excluded from the cytosol through an energy-dependent system as a free ion as well as a complexed form. Although both membranes contribute in the active exclusion of ionized and complexed Cd from the cytosol, the overall calculation of Cd accumulation in the everted plasma membranes and vacuolar vesicles suggests that the tonoplast and vacuole have a major function in Cd efflux from the cytosol in the roots of cucumber subjected to Cd stress.

V1-receptor mediated GSH efflux by vasopressin from rat hepatocytes.

PubMed

Sato, C; Liu, J H; Uchihara, M; Izumi, N; Yauchi, T; Sakaj, Y; Asahina, Y; Fukuma, T; Takano, T; Marumo, F

1992-01-01

Vasopression increases sinusoidal efflux of GSH in the perfused rat liver. The mechanism of this effect was studied in the perfused rat liver and in isolated rat hepatocytes. Vasopressin stimulated GSH efflux in both systems and a V1-receptor antagonist (OPC-21268) significantly inhibited the effect of vasopressin suggesting that vasopressin stimulates GSH efflux from rat hepatocytes via V1-receptor.

Cyclic AMP efflux inhibitors as potential therapeutic agents for leukemia.

PubMed

Perez, Dominique R; Smagley, Yelena; Garcia, Matthew; Carter, Mark B; Evangelisti, Annette; Matlawska-Wasowska, Ksenia; Winter, Stuart S; Sklar, Larry A; Chigaev, Alexandre

2016-06-07

Apoptotic evasion is a hallmark of cancer. We propose that some cancers may evade cell death by regulating 3'-5'-cyclic adenosine monophosphate (cAMP), which is associated with pro-apoptotic signaling. We hypothesize that leukemic cells possess mechanisms that efflux cAMP from the cytoplasm, thus protecting them from apoptosis. Accordingly, cAMP efflux inhibition should result in: cAMP accumulation, activation of cAMP-dependent downstream signaling, viability loss, and apoptosis. We developed a novel assay to assess cAMP efflux and performed screens to identify inhibitors. In an acute myeloid leukemia (AML) model, several identified compounds reduced cAMP efflux, appropriately modulated pathways that are responsive to cAMP elevation (cAMP-responsive element-binding protein phosphorylation, and deactivation of Very Late Antigen-4 integrin), and induced mitochondrial depolarization and caspase activation. Blocking adenylyl cyclase activity was sufficient to reduce effects of the most potent compounds. These compounds also decreased cAMP efflux and viability of B-lineage acute lymphoblastic leukemia (B-ALL) cell lines and primary patient samples, but not of normal primary peripheral blood mononuclear cells. Our data suggest that cAMP efflux is a functional feature that could be therapeutically targeted in leukemia. Furthermore, because some of the identified drugs are currently used for treating other illnesses, this work creates an opportunity for repurposing.

Tet38 Efflux Pump Affects Staphylococcus aureus Internalization by Epithelial Cells through Interaction with CD36 and Contributes to Bacterial Escape from Acidic and Nonacidic Phagolysosomes.

PubMed

Truong-Bolduc, Q C; Khan, N S; Vyas, J M; Hooper, D C

2017-02-01

We previously reported that the Tet38 efflux pump is involved in internalization of Staphylococcus aureus by A549 lung epithelial cells. A lack of tet38 reduced bacterial uptake by A549 cells to 36% of that of the parental strain RN6390. Using invasion assays coupled with confocal microscopy imaging, we studied the host cell receptor(s) responsible for bacterial uptake via interaction with Tet38. We also assessed the ability of S. aureus to survive following alkalinization of the phagolysosomes by chloroquine. Antibody to the scavenger receptor CD36 reduced the internalization of S. aureus RN6390 by A549 cells, but the dependence on CD36 was reduced in QT7 tet38, suggesting that an interaction between Tet38 and CD36 contributed to S. aureus internalization. Following fusion of the S. aureus-associated endosomes with lysosomes, alkalinization of the acidic environment with chloroquine led to a rapid increase in the number of S. aureus RN6390 bacteria in the cytosol, followed by a decrease shortly thereafter. This effect of chloroquine was not seen in the absence of intact Tet38 in mutant QT7. These data taken together suggest that Tet38 plays a role both in bacterial internalization via interaction with CD36 and in bacterial escape from the phagolysosomes. Copyright © 2017 American Society for Microbiology.

Tet38 Efflux Pump Affects Staphylococcus aureus Internalization by Epithelial Cells through Interaction with CD36 and Contributes to Bacterial Escape from Acidic and Nonacidic Phagolysosomes

PubMed Central

Truong-Bolduc, Q. C.; Khan, N. S.

2016-01-01

ABSTRACT We previously reported that the Tet38 efflux pump is involved in internalization of Staphylococcus aureus by A549 lung epithelial cells. A lack of tet38 reduced bacterial uptake by A549 cells to 36% of that of the parental strain RN6390. Using invasion assays coupled with confocal microscopy imaging, we studied the host cell receptor(s) responsible for bacterial uptake via interaction with Tet38. We also assessed the ability of S. aureus to survive following alkalinization of the phagolysosomes by chloroquine. Antibody to the scavenger receptor CD36 reduced the internalization of S. aureus RN6390 by A549 cells, but the dependence on CD36 was reduced in QT7 tet38, suggesting that an interaction between Tet38 and CD36 contributed to S. aureus internalization. Following fusion of the S. aureus-associated endosomes with lysosomes, alkalinization of the acidic environment with chloroquine led to a rapid increase in the number of S. aureus RN6390 bacteria in the cytosol, followed by a decrease shortly thereafter. This effect of chloroquine was not seen in the absence of intact Tet38 in mutant QT7. These data taken together suggest that Tet38 plays a role both in bacterial internalization via interaction with CD36 and in bacterial escape from the phagolysosomes. PMID:27956597

CO2 efflux from soils with seasonal water repellency

NASA Astrophysics Data System (ADS)

Urbanek, Emilia; Doerr, Stefan H.

2017-10-01

Soil carbon dioxide (CO2) emissions are strongly dependent on pore water distribution, which in turn can be modified by reduced wettability. Many soils around the world are affected by soil water repellency (SWR), which reduces infiltration and results in diverse moisture distribution. SWR is temporally variable and soils can change from wettable to water-repellent and vice versa throughout the year. Effects of SWR on soil carbon (C) dynamics, and specifically on CO2 efflux, have only been studied in a few laboratory experiments and hence remain poorly understood. Existing studies suggest soil respiration is reduced with increasing severity of SWR, but the responses of soil CO2 efflux to varying water distribution created by SWR are not yet known.Here we report on the first field-based study that tests whether SWR indeed reduces soil CO2 efflux, based on in situ measurements carried out over three consecutive years at a grassland and pine forest sites under the humid temperate climate of the UK.Soil CO2 efflux was indeed very low on occasions when soil exhibited consistently high SWR and low soil moisture following long dry spells. Low CO2 efflux was also observed when SWR was absent, in spring and late autumn when soil temperatures were low, but also in summer when SWR was reduced by frequent rainfall events. The highest CO2 efflux occurred not when soil was wettable, but when SWR, and thus soil moisture, was spatially patchy, a pattern observed for the majority of the measurement period. Patchiness of SWR is likely to have created zones with two different characteristics related to CO2 production and transport. Zones with wettable soil or low persistence of SWR with higher proportion of water-filled pores are expected to provide water with high nutrient concentration resulting in higher microbial activity and CO2 production. Soil zones with high SWR persistence, on the other hand, are dominated by air-filled pores with low microbial activity, but facilitating O2

Experimental Study on Environment Friendly Tap Hole Clay for Blast Furnace

NASA Astrophysics Data System (ADS)

Siva kumar, R.; Mohammed, Raffi; Srinivasa Rao, K.

2018-03-01

Blast furnace (BF) is the best possible route of iron production available. Blast furnace is a high pressure vessel where iron ore is melted and liquid iron is produced. The liquid iron is tapped through the hole in Blast Furnace called tap hole. The tapped liquid metal flowing through the tap hole is plugged using a clay called tap hole clay. Tap hole clay (THC) is a unshaped refractory used to plug the tap hole. The tap hole clay extruded through the tap hole using a gun. The tap hole clay is designed to expand and plug the tap hole. The tap hole filled with clay is drilled using drill bit and the hole made through the tap hole to tap the liquid metal accumulated inside the furnace. The number of plugging and drilling varies depending on the volume of the furnace. The tap hole clay need to have certain properties to avoid problems during plugging and drilling. In the present paper tap hole clay properties in industrial use was tested and studied. The problems were identified related to tap hole clay manufacturing. Experiments were conducted in lab scale to solve the identified problems. The present composition was modified with experimental results. The properties of the modified tap hole clay were found suitable and useful for blast furnace operation with lab scale experimental results.

Transportation Air Pollution Studies (TAPS) System

DOT National Transportation Integrated Search

1974-03-01

This report describes the Transportation Air Pollution Studies (TAPS) Data Base and the Software System which has been developed in association with it. : The TAPS Data Base will be used to store the transportation air pollution data (including emiss...

Ezetimibe Promotes Brush Border Membrane-to-Lumen Cholesterol Efflux in the Small Intestine

PubMed Central

Nakano, Takanari; Inoue, Ikuo; Takenaka, Yasuhiro; Ono, Hiraku; Katayama, Shigehiro; Awata, Takuya; Murakoshi, Takayuki

2016-01-01

Ezetimibe inhibits Niemann-Pick C1-like 1 (NPC1L1), an apical membrane cholesterol transporter of enterocytes, thereby reduces intestinal cholesterol absorption. This treatment also increases extrahepatic reverse cholesterol transport via an undefined mechanism. To explore this, we employed a trans-intestinal cholesterol efflux (TICE) assay, which directly detects circulation-to-intestinal lumen 3H-cholesterol transit in a cannulated jejunal segment, and found an increase of TICE by 45%. To examine whether such increase in efflux occurs at the intestinal brush border membrane(BBM)-level, we performed luminal perfusion assays, similar to TICE but the jejunal wall was labelled with orally-given 3H-cholesterol, and determined elevated BBM-to-lumen cholesterol efflux by 3.5-fold with ezetimibe. Such increased efflux probably promotes circulation-to-lumen cholesterol transit eventually; thus increases TICE. Next, we wondered how inhibition of NPC1L1, an influx transporter, resulted in increased efflux. When we traced orally-given 3H-cholesterol in mice, we found that lumen-to-BBM 3H-cholesterol transit was rapid and less sensitive to ezetimibe treatment. Comparison of the efflux and fractional cholesterol absorption revealed an inverse correlation, indicating the efflux as an opposite-regulatory factor for cholesterol absorption efficiency and counteracting to the naturally-occurring rapid cholesterol influx to the BBM. These suggest that the ezetimibe-stimulated increased efflux is crucial in reducing cholesterol absorption. Ezetimibe-induced increase in cholesterol efflux was approximately 2.5-fold greater in mice having endogenous ATP-binding cassette G5/G8 heterodimer, the major sterol efflux transporter of enterocytes, than the knockout counterparts, suggesting that the heterodimer confers additional rapid BBM-to-lumen cholesterol efflux in response to NPC1L1 inhibition. The observed framework for intestinal cholesterol fluxes may provide ways to modulate the flux

Ezetimibe Promotes Brush Border Membrane-to-Lumen Cholesterol Efflux in the Small Intestine.

PubMed

Nakano, Takanari; Inoue, Ikuo; Takenaka, Yasuhiro; Ono, Hiraku; Katayama, Shigehiro; Awata, Takuya; Murakoshi, Takayuki

2016-01-01

Ezetimibe inhibits Niemann-Pick C1-like 1 (NPC1L1), an apical membrane cholesterol transporter of enterocytes, thereby reduces intestinal cholesterol absorption. This treatment also increases extrahepatic reverse cholesterol transport via an undefined mechanism. To explore this, we employed a trans-intestinal cholesterol efflux (TICE) assay, which directly detects circulation-to-intestinal lumen 3H-cholesterol transit in a cannulated jejunal segment, and found an increase of TICE by 45%. To examine whether such increase in efflux occurs at the intestinal brush border membrane(BBM)-level, we performed luminal perfusion assays, similar to TICE but the jejunal wall was labelled with orally-given 3H-cholesterol, and determined elevated BBM-to-lumen cholesterol efflux by 3.5-fold with ezetimibe. Such increased efflux probably promotes circulation-to-lumen cholesterol transit eventually; thus increases TICE. Next, we wondered how inhibition of NPC1L1, an influx transporter, resulted in increased efflux. When we traced orally-given 3H-cholesterol in mice, we found that lumen-to-BBM 3H-cholesterol transit was rapid and less sensitive to ezetimibe treatment. Comparison of the efflux and fractional cholesterol absorption revealed an inverse correlation, indicating the efflux as an opposite-regulatory factor for cholesterol absorption efficiency and counteracting to the naturally-occurring rapid cholesterol influx to the BBM. These suggest that the ezetimibe-stimulated increased efflux is crucial in reducing cholesterol absorption. Ezetimibe-induced increase in cholesterol efflux was approximately 2.5-fold greater in mice having endogenous ATP-binding cassette G5/G8 heterodimer, the major sterol efflux transporter of enterocytes, than the knockout counterparts, suggesting that the heterodimer confers additional rapid BBM-to-lumen cholesterol efflux in response to NPC1L1 inhibition. The observed framework for intestinal cholesterol fluxes may provide ways to modulate the flux

α-Synuclein Regulates Neuronal Cholesterol Efflux.

PubMed

Hsiao, Jen-Hsiang T; Halliday, Glenda M; Kim, Woojin Scott

2017-10-19

α-Synuclein is a neuronal protein that is at the center of focus in understanding the etiology of a group of neurodegenerative diseases called α-synucleinopathies, which includes Parkinson's disease (PD). Despite much research, the exact physiological function of α-synuclein is still unclear. α-Synuclein has similar biophysical properties as apolipoproteins and other lipid-binding proteins and has a high affinity for cholesterol. These properties suggest a possible role for α-synuclein as a lipid acceptor mediating cholesterol efflux (the process of removing cholesterol out of cells). To test this concept, we "loaded" SK-N-SH neuronal cells with fluorescently-labelled cholesterol, applied exogenous α-synuclein, and measured the amount of cholesterol removed from the cells using a classic cholesterol efflux assay. We found that α-synuclein potently stimulated cholesterol efflux. We found that the process was dose and time dependent, and was saturable at 1.0 µg/mL of α-synuclein. It was also dependent on the transporter protein ABCA1 located on the plasma membrane. We reveal for the first time a novel role of α-synuclein that underscores its importance in neuronal cholesterol regulation, and identify novel therapeutic targets for controlling cellular cholesterol levels.

Metabolic Targeting of Lactate Efflux by Malignant Glioma Inhibits Invasiveness and Induces Necrosis: An In Vivo Study1

PubMed Central

Colen, Chaim B; Shen, Yimin; Ghoddoussi, Farhad; Yu, Pingyang; Francis, Todd B; Koch, Brandon J; Monterey, Michael D; Galloway, Matthew P; Sloan, Andrew E; Mathupala, Saroj P

2011-01-01

Glioblastoma multiforme (GBM) are the most malignant among brain tumors. They are frequently refractory to chemotherapy and radiotherapy with mean patient survival of approximately 6 months, despite surgical intervention. The highly glycolytic nature of glioblastomas describes their propensity to metabolize glucose to lactic acid at an elevated rate. To survive, GBMs efflux lactic acid to the tumor microenvironment through transmembrane transporters denoted monocarboxylate transporters (MCTs). We hypothesized that inhibition of MCT function would impair the glycolytic metabolism and affect both glioma invasiveness and survival. We examined the effect on invasiveness with α-cyano-4-hydroxy-cinnamic acid (ACCA, 4CIN, CHCA), a small-molecule inhibitor of lactate transport, through Matrigel-based and organotypic (brain) slice culture invasive assays using U87-MG and U251-MG glioma cells. We then conducted studies in immunodeficient rats by stereotaxic intracranial implantation of the glioma cells followed by programmed orthotopic application of ACCA through osmotic pumps. Effect on the implanted tumor was monitored by small-animal magnetic resonance imaging. Our assays indicated that glioma invasion was markedly impaired when lactate efflux was inhibited. Convection-enhanced delivery of inhibitor to the tumor bed caused tumor necrosis, with 50% of the animals surviving beyond the experimental end points (3 months after inhibitor exhaustion). Most importantly, control animals did not display any adverse neurologic effects during orthotopic administration of ACCA to brain through programmed delivery. These results indicate the clinical potential of targeting lactate efflux in glioma through delivery of small-molecule inhibitors of MCTs either to the tumor bed or to the postsurgical resection cavity. PMID:21750656

New saliva secretion model based on the expression of Na+-K+ pump and K+ channels in the apical membrane of parotid acinar cells.

PubMed

Almássy, János; Siguenza, Elias; Skaliczki, Marianna; Matesz, Klara; Sneyd, James; Yule, David I; Nánási, Péter P

2018-04-01

The plasma membrane of parotid acinar cells is functionally divided into apical and basolateral regions. According to the current model, fluid secretion is driven by transepithelial ion gradient, which facilitates water movement by osmosis into the acinar lumen from the interstitium. The osmotic gradient is created by the apical Cl - efflux and the subsequent paracellular Na + transport. In this model, the Na + -K + pump is located exclusively in the basolateral membrane and has essential role in salivary secretion, since the driving force for Cl - transport via basolateral Na + -K + -2Cl - cotransport is generated by the Na + -K + pump. In addition, the continuous electrochemical gradient for Cl - flow during acinar cell stimulation is maintained by the basolateral K + efflux. However, using a combination of single-cell electrophysiology and Ca 2+ -imaging, we demonstrate that photolysis of Ca 2+ close to the apical membrane of parotid acinar cells triggered significant K + current, indicating that a substantial amount of K + is secreted into the lumen during stimulation. Nevertheless, the K + content of the primary saliva is relatively low, suggesting that K + might be reabsorbed through the apical membrane. Therefore, we investigated the localization of Na + -K + pumps in acinar cells. We show that the pumps appear evenly distributed throughout the whole plasma membrane, including the apical pole of the cell. Based on these results, a new mathematical model of salivary fluid secretion is presented, where the pump reabsorbs K + from and secretes Na + to the lumen, which can partially supplement the paracellular Na + pathway.

Advanced analysis of finger-tapping performance: a preliminary study.

PubMed

Barut, Cağatay; Kızıltan, Erhan; Gelir, Ethem; Köktürk, Fürüzan

2013-06-01

The finger-tapping test is a commonly employed quantitative assessment tool used to measure motor performance in the upper extremities. This task is a complex motion that is affected by external stimuli, mood and health status. The complexity of this task is difficult to explain with a single average intertap-interval value (time difference between successive tappings) which only provides general information and neglects the temporal effects of the aforementioned factors. This study evaluated the time course of average intertap-interval values and the patterns of variation in both the right and left hands of right-handed subjects using a computer-based finger-tapping system. Cross sectional study. Thirty eight male individuals aged between 20 and 28 years (Mean±SD = 22.24±1.65) participated in the study. Participants were asked to perform single-finger-tapping test for 10 seconds of test period. Only the results of right-handed (RH) 35 participants were considered in this study. The test records the time of tapping and saves data as the time difference between successive tappings for further analysis. The average number of tappings and the temporal fluctuation patterns of the intertap-intervals were calculated and compared. The variations in the intertap-interval were evaluated with the best curve fit method. An average tapping speed or tapping rate can reliably be defined for a single-finger tapping test by analysing the graphically presented data of the number of tappings within the test period. However, a different presentation of the same data, namely the intertap-interval values, shows temporal variation as the number of tapping increases. Curve fitting applications indicate that the variation has a biphasic nature. The measures obtained in this study reflect the complex nature of the finger-tapping task and are suggested to provide reliable information regarding hand performance. Moreover, the equation reflects both the variations in and the general

Vaccination and the TAP-independent antigen processing pathways.

PubMed

López, Daniel; Lorente, Elena; Barriga, Alejandro; Johnstone, Carolina; Mir, Carmen

2013-09-01

The cytotoxic CD8(+) T lymphocyte-mediated cellular response is important for the elimination of virus-infected cells and requires the prior recognition of short viral peptide antigens previously translocated to the endoplasmic reticulum by the transporter associated with antigen processing (TAP). However, individuals with nonfunctional TAP complexes or infected cells with TAP molecules blocked by specific viral proteins, such as the cowpoxvirus, a component of the first source of early empirical vaccination against smallpox, are still able to present several HLA class I ligands generated by the TAP-independent antigen processing pathways to specific cytotoxic CD8(+) T lymphocytes. Currently, bioterrorism and emerging infectious diseases have renewed interest in poxviruses. Recent works that have identified HLA class I ligands and epitopes in virus-infected TAP-deficient cells have implications for the study of both the effectiveness of early empirical vaccination and the analysis of HLA class I antigen processing in TAP-deficient subjects.

What's Wrong with the Tap? Examining Perceptions of Tap Water and Bottled Water at Purdue University

NASA Astrophysics Data System (ADS)

Saylor, Amber; Prokopy, Linda Stalker; Amberg, Shannon

2011-09-01

The environmental impacts of bottled water prompted us to explore drinking water choices at Purdue University, located in West Lafayette, IN. A random sample of 2,045 Purdue University students, staff, and faculty was invited to participate in an online survey. The survey assessed current behaviors as well as perceived barriers and benefits to drinking tap water versus bottled water. 677 surveys were completed for a response rate of 33.1%. We then conducted qualitative interviews with a purposive sample of university undergraduates ( n = 21) to obtain contextual insights into the survey results and the beliefs of individuals with a variety of drinking water preferences. This study revealed that women drink disproportionately more bottled water then men while undergraduate students drink more than graduate students, staff and faculty. The study also uncovered a widespread belief that recycling eliminates the environmental impacts of bottled water. Important barriers to drinking tap water at Purdue include: perceived risks from tap water and the perceived safety of bottled water, preferring the taste of bottled water, and the convenience of drinking bottled water. The qualitative interviews revealed that drinking water choices can be influenced by several factors—especially whether individuals trust tap water to be clean—but involve varying levels of complexity. The implications of these results for social marketing strategies to promote tap water are discussed.

What's wrong with the tap? Examining perceptions of tap water and bottled water at Purdue University.

PubMed

Saylor, Amber; Prokopy, Linda Stalker; Amberg, Shannon

2011-09-01

The environmental impacts of bottled water prompted us to explore drinking water choices at Purdue University, located in West Lafayette, IN. A random sample of 2,045 Purdue University students, staff, and faculty was invited to participate in an online survey. The survey assessed current behaviors as well as perceived barriers and benefits to drinking tap water versus bottled water. 677 surveys were completed for a response rate of 33.1%. We then conducted qualitative interviews with a purposive sample of university undergraduates (n = 21) to obtain contextual insights into the survey results and the beliefs of individuals with a variety of drinking water preferences. This study revealed that women drink disproportionately more bottled water then men while undergraduate students drink more than graduate students, staff and faculty. The study also uncovered a widespread belief that recycling eliminates the environmental impacts of bottled water. Important barriers to drinking tap water at Purdue include: perceived risks from tap water and the perceived safety of bottled water, preferring the taste of bottled water, and the convenience of drinking bottled water. The qualitative interviews revealed that drinking water choices can be influenced by several factors-especially whether individuals trust tap water to be clean-but involve varying levels of complexity. The implications of these results for social marketing strategies to promote tap water are discussed.

INL’s TAP Systems

DOE Office of Scientific and Technical Information (OSTI.GOV)

None

2017-06-22

The starting point for many consumer products is the industrial manufacture of platform chemicals. The recent boom in domestic shale gas production makes it possible to envision a new resource for chemical manufacturing. Catalysts are the accelerants behind most industrial chemical reactions. A sophisticated research technique using a Temporal Analysis of Products (or TAP) reactor can now help. By shedding light on a catalyst’s fundamental step-by-step process, a TAP reactor can help chemists and chemical engineers understand why a new catalyst works better in the lab than in the chemical plant.

TAP polymorphism in patients with Behçet's disease.

PubMed Central

González-Escribano, M F; Morales, J; García-Lozano, J R; Castillo, M J; Sánchez-Román, J; Núñez-Roldán, A; Sánchez, B

1995-01-01

OBJECTIVE--To determine if susceptibility to Behçet's disease (BD) is associated with polymorphism of HLA-DRB1, HLA-DQB1, DQB1, and TAP1 and TAP2 genes. METHODS--Fifty eight Spanish BD patients and 116 ethnically matched unrelated healthy subjects were typed at the HLA-DRB1 and HLA-DQB1 loci using polymerase chain reaction/sequence specific oligotyping (PCR/SSO). TAP1 and TAP2 alleles were assigned using amplification refractory mutation system-PCR. RESULTS--TAP1C was absent in BD patients, but was found in 12.1% of control subjects (pcorr < 0.05; relative risk = 0.06). Additionally, a linkage disequilibrium between HLA-DQB1*0501 and TAP2B was observed in BD patients (delta = 0.095, pcorr < 0.02), but not in the control group (delta = -0.0031, p > 0.05). CONCLUSIONS--The complete absence of TAP1C alleles in BD patients may indicate that TAP1 polymorphism is not without some significance in the development of BD. Furthermore, the existence of a linkage disequilibrium between HLA-DQB1*0501 and TAP2B in our patients suggests that the gene conferring susceptibility for BD is inherited as an extended haplotype in the population studied. PMID:7794046

Florfenicol As a Modulator Enhancing Antimicrobial Activity: Example Using Combination with Thiamphenicol against Pasteurella multocida

PubMed Central

Wei, Chia-Fong; Shien, Jui-Hung; Chang, Shao-Kuang; Chou, Chi-Chung

2016-01-01

Synergistic effects between the same class of antibiotics are rarely reported. Our previous study found synergistic-like interaction between florfenicol (FFC) and thiamphenicol (TAP) against Staphylococcus aureus. Here, the enhanced antimicrobial activity was evaluated in 97 clinical isolates of both Gram-negative and Gram-positive bacteria. Susceptible strains were initially identified by checkerboard microdilution assay (fractional inhibitory concentration index [FICI] ≤ 0.625), followed by confirmation of synergism using the time-kill methodology (≥2 log10 CFU/ml reduction). In all, 43% of Pasteurella multocida tested were susceptible to the enhanced bactericidal effect. In chicken fowl cholera models, FFC and TAP combination at much lower dosage that is correspondent to their MIC deduction provided maximum protection in vivo. Furthermore, synergistic combination of FFC with oxytetracycline (OTC) against Pseudomonas aeruginosa in vitro was also demonstrated. Based on the enhanced uptake of TAP and OTC, FFC presumably elicits enhanced antimicrobial activity in an orderly manner through alteration of bacterial membrane permeability or efflux systems and subsequent increase of intracellular concentration of the antibiotics used in combination. Results of ethidium bromide accumulation assay and RNA-seq showed little evidence for the involvement of efflux pumps in the synergy but further investigation is required. This study suggests the potentiality of a novel combination regimen involving FFC as an initiating modulator effective against both Gram-positive and Gram-negative bacteria depending on the antibiotics that are combined. The observed improvement of bacteriostatic effect to bactericidal, and the extended effectiveness against FFC-resistant bacterial strains warrant further studies. PMID:27065961

Volume-activated trimethylamine oxide efflux in red blood cells of spiny dogfish (Squalus acanthias).

PubMed

Koomoa, D L; Musch, M W; MacLean, A V; Goldstein, L

2001-09-01

The aims of this study were to determine the pathway of swelling-activated trimethylamine oxide (TMAO) efflux and its regulation in spiny dogfish (Squalus acanthias) red blood cells and compare the characteristics of this efflux pathway with the volume-activated osmolyte (taurine) channel present in erythrocytes of fishes. The characteristics of the TMAO efflux pathway were similar to those of the taurine efflux pathway. The swelling-activated effluxes of both TMAO and taurine were significantly inhibited by known anion transport inhibitors (DIDS and niflumic acid) and by the general channel inhibitor quinine. Volume expansion by hypotonicity, ethylene glycol, and diethyl urea activated both TMAO and taurine effluxes similarly. Volume expansion by hypotonicity, ethylene glycol, and diethyl urea also stimulated the activity of tyrosine kinases p72syk and p56lyn, although the stimulations by the latter two treatments were less than by hypotonicity. The volume activations of both TMAO and taurine effluxes were inhibited by tyrosine kinase inhibitors, suggesting that activation of tyrosine kinases may play a role in activating the osmolyte effluxes. These results indicate that the volume-activated TMAO efflux occurs via the organic osmolyte (taurine) channel and may be regulated by the volume activation of tyrosine kinases.

Wood CO(2) efflux and foliar respiration for Eucalyptus in Hawaii and Brazil.

PubMed

Ryan, Michael G; Cavaleri, Molly A; Almeida, Auro C; Penchel, Ricardo; Senock, Randy S; Luiz Stape, José

2009-10-01

We measured CO(2) efflux from wood for Eucalyptus in Hawaii for 7 years and compared these measurements with those on three- and four-and-a-half-year-old Eucalyptus in Brazil. In Hawaii, CO(2) efflux from wood per unit biomass declined approximately 10x from age two to age five, twice as much as the decline in tree growth. The CO(2) efflux from wood in Brazil was 8-10x lower than that for comparable Hawaii trees with similar growth rates. Growth and maintenance respiration coefficients calculated from Hawaii wood CO(2) efflux declined with tree age and size (the growth coefficient declined from 0.4 mol C efflux mol C(-1) wood growth at age one to 0.1 mol C efflux mol C(-1) wood growth at age six; the maintenance coefficient from 0.006 to 0.001 micromol C (mol C biomass)(-1) s(-1) at 20 degrees C over the same time period). These results suggest interference with CO(2) efflux through bark that decouples CO(2) efflux from respiration. We also compared the biomass fractions and wood CO(2) efflux for the aboveground woody parts for 3- and 7-year-old trees in Hawaii to estimate how focusing measurements near the ground might bias the stand-level estimates of wood CO(2) efflux. Three-year-old Eucalyptus in Hawaii had a higher proportion of branches < 0.5 cm in diameter and a lower proportion of stem biomass than did 7-year-old trees. Biomass-specific CO(2) efflux measured at 1.4 m extrapolated to the tree could bias tree level estimates by approximately 50%, assuming no refixation from bark photosynthesis. However, the bias did not differ for the two tree sizes. Foliar respiration was identical per unit nitrogen for comparable treatments in Brazil and Hawaii (4.2 micromol C mol N(-1) s(-1) at 20 degrees C).

Analysis of sequence variation among smeDEF multi drug efflux pump genes and flanking DNA from defined 16S rRNA subgroups of clinical Stenotrophomonas maltophilia isolates.

PubMed

Gould, Virginia C; Okazaki, Aki; Howe, Robin A; Avison, Matthew B

2004-08-01

To determine the level of variation in the smeDEF efflux pump and smeT transcriptional regulator genes among three defined 16S rRNA sequence subgroups of clinical Stenotrophomonas maltophilia isolates. smeDEF sequencing used a PCR genome walking approach. Determination of the sequence surrounding smeDEF used a flanking primer PCR method and specific primers anchored in smeD or smeF together with random primers. smeDEF is chromosomal and located in the same position in the chromosome in all three subgroups of isolates. Flanking smeD is a gene, smeT, encoding a putative transcriptional repressor for smeDEF. Variation at these loci among the isolates is considerably lower (up to 10%) than at intrinsic beta-lactamase loci (up to 30%) in the same isolates, implying greater functional constraint. The smeD-smeT intergenic region contains a highly conserved section, which maps with previously predicted promoter/operator regions, and a hypervariable untranslated region, which can be used to subgroup clinical isolates. These data provide further evidence that it is possible to group clinical isolates of the inherently variable species, S. maltophilia, based on genotypic properties. Isolate D457, in which most work concerning smeDEF expression has been performed, does not fall into S. maltophilia subgroup A, which is the most typical.

49 CFR 192.627 - Tapping pipelines under pressure.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 3 2010-10-01 2010-10-01 false Tapping pipelines under pressure. 192.627 Section... NATURAL AND OTHER GAS BY PIPELINE: MINIMUM FEDERAL SAFETY STANDARDS Operations § 192.627 Tapping pipelines under pressure. Each tap made on a pipeline under pressure must be performed by a crew qualified to make...

Water use and time analysis in ablution from taps

NASA Astrophysics Data System (ADS)

Zaied, Roubi A.

2017-09-01

There is a lack of water resources and an extreme use of potable water in our Arab region. Ablution from taps was studied since it is a repeated daily activity that consumes more water. Five different tap types are investigated for water consumption fashions including traditional mixing tap and automatic tap. Analyzing 100 experimental observations revealed that 22.7-28.8 % of ablution water is used for washing of feet and the largest water waste occurs during washing of face portions. Moreover, 30-47 % amount of water consumed in ablution from taps is wasted which can be saved if tap releases water only at moments of need. The push-type tap is being spread recently especially in airports. If it is intended for use in ablution facilities, batch duration and volume must be tuned. When each batch is 0.25 L of water and lasts for 3 s, 3 L are sufficient for one complete ablution in average which means considerable saving. A cost-benefit model is proposed for using different tap types and an economic feasibility study is performed on a case study. This analysis can help us to design better ablution systems.

Vertical variations in wood CO2 efflux for live emergent trees in a Bornean tropical rainforest.

PubMed

Katayama, Ayumi; Kume, Tomonori; Komatsu, Hikaru; Ohashi, Mizue; Matsumoto, Kazuho; Ichihashi, Ryuji; Kumagai, Tomo'omi; Otsuki, Kyoichi

2014-05-01

Difficult access to 40-m-tall emergent trees in tropical rainforests has resulted in a lack of data related to vertical variations in wood CO2 efflux, even though significant variations in wood CO2 efflux are an important source of errors when estimating whole-tree total wood CO2 efflux. This study aimed to clarify vertical variations in wood CO2 efflux for emergent trees and to document the impact of the variations on the whole-tree estimates of stem and branch CO2 efflux. First, we measured wood CO2 efflux and factors related to tree morphology and environment for seven live emergent trees of two dipterocarp species at four to seven heights of up to ∼ 40 m for each tree using ladders and a crane. No systematic tendencies in vertical variations were observed for all the trees. Wood CO2 efflux was not affected by stem and air temperature, stem diameter, stem height or stem growth. The ratios of wood CO2 efflux at the treetop to that at breast height were larger in emergent trees with relatively smaller diameters at breast height. Second, we compared whole-tree stem CO2 efflux estimates using vertical measurements with those based on solely breast height measurements. We found similar whole-tree stem CO2 efflux estimates regardless of the patterns of vertical variations in CO2 efflux because the surface area in the canopy, where wood CO2 efflux often differed from that at breast height, was very small compared with that at low stem heights, resulting in little effect of the vertical variations on the estimate. Additionally, whole-tree branch CO2 efflux estimates using measured wood CO2 efflux in the canopy were considerably different from those measured using only breast height measurements. Uncertainties in wood CO2 efflux in the canopy did not cause any bias in stem CO2 efflux scaling, but affected branch CO2 efflux. © The Author 2014. Published by Oxford University Press. All rights reserved.

BIOMECHANICAL EVALUATION OF THE INFLUENCE OF CERVICAL SCREWS TAPPING AND DESIGN.

PubMed

Silva, Patricia; Rosa, Rodrigo César; Shimano, Antonio Carlos; Albuquerque de Paula, Francisco José; Volpon, José Batista; Aparecido Defino, Helton Luiz

2009-01-01

To assess if the screw design (self-drilling/self-tapping) and the pilot hole tapping could affect the insertion torque and screw pullout strength of the screw used in anterior fixation of the cervical spine. Forty self-tapping screws and 20 self-drilling screws were inserted into 10 models of artificial bone and 10 cervical vertebrae of sheep. The studied parameters were the insertion torque and pullout strength. The following groups were created: Group I-self-tapping screw insertion after pilot hole drilling and tapping; Group II-self-tapping screw insertion after pilot hole drilling without tapping; Group III-self-drilling screw insertion without drilling and tapping. In Groups I and II, the pilot hole had 14.0 mm in depth and was made with a 3mmn drill, while tapping was made with a 4mm tap. The insertion torque was measured and the pullout test was performed. The comparison between groups was made considering the mean insertion torque and the maximum mean pullout strength with the variance analysis (ANOVA; p≤ 0.05). Previous drilling and tapping of pilot hole significantly decreased the insertion torque and the pullout strength. The insertion torque and pullout strength of self-drilling screws were significantly higher when compared to self-tapping screws inserted after pilot hole tapping.

Tapping unsustainable groundwater stores for agricultural production in the High Plains Aquifer of Kansas, projections to 2110.

PubMed

Steward, David R; Bruss, Paul J; Yang, Xiaoying; Staggenborg, Scott A; Welch, Stephen M; Apley, Michael D

2013-09-10

Groundwater provides a reliable tap to sustain agricultural production, yet persistent aquifer depletion threatens future sustainability. The High Plains Aquifer supplies 30% of the nation's irrigated groundwater, and the Kansas portion supports the congressional district with the highest market value for agriculture in the nation. We project groundwater declines to assess when the study area might run out of water, and comprehensively forecast the impacts of reduced pumping on corn and cattle production. So far, 30% of the groundwater has been pumped and another 39% will be depleted over the next 50 y given existing trends. Recharge supplies 15% of current pumping and would take an average of 500-1,300 y to completely refill a depleted aquifer. Significant declines in the region's pumping rates will occur over the next 15-20 y given current trends, yet irrigated agricultural production might increase through 2040 because of projected increases in water use efficiencies in corn production. Water use reductions of 20% today would cut agricultural production to the levels of 15-20 y ago, the time of peak agricultural production would extend to the 2070s, and production beyond 2070 would significantly exceed that projected without reduced pumping. Scenarios evaluate incremental reductions of current pumping by 20-80%, the latter rate approaching natural recharge. Findings substantiate that saving more water today would result in increased net production due to projected future increases in crop water use efficiencies. Society has an opportunity now to make changes with tremendous implications for future sustainability and livability.

Tapping unsustainable groundwater stores for agricultural production in the High Plains Aquifer of Kansas, projections to 2110

PubMed Central

Steward, David R.; Bruss, Paul J.; Yang, Xiaoying; Staggenborg, Scott A.; Welch, Stephen M.; Apley, Michael D.

2013-01-01

Groundwater provides a reliable tap to sustain agricultural production, yet persistent aquifer depletion threatens future sustainability. The High Plains Aquifer supplies 30% of the nation’s irrigated groundwater, and the Kansas portion supports the congressional district with the highest market value for agriculture in the nation. We project groundwater declines to assess when the study area might run out of water, and comprehensively forecast the impacts of reduced pumping on corn and cattle production. So far, 30% of the groundwater has been pumped and another 39% will be depleted over the next 50 y given existing trends. Recharge supplies 15% of current pumping and would take an average of 500–1,300 y to completely refill a depleted aquifer. Significant declines in the region’s pumping rates will occur over the next 15–20 y given current trends, yet irrigated agricultural production might increase through 2040 because of projected increases in water use efficiencies in corn production. Water use reductions of 20% today would cut agricultural production to the levels of 15–20 y ago, the time of peak agricultural production would extend to the 2070s, and production beyond 2070 would significantly exceed that projected without reduced pumping. Scenarios evaluate incremental reductions of current pumping by 20–80%, the latter rate approaching natural recharge. Findings substantiate that saving more water today would result in increased net production due to projected future increases in crop water use efficiencies. Society has an opportunity now to make changes with tremendous implications for future sustainability and livability. PMID:23980153

C2 Arylated Benzo[b]thiophene Derivatives as Staphylococcus aureus NorA Efflux Pump Inhibitors.

PubMed

Liger, François; Bouhours, Pascale; Ganem-Elbaz, Carine; Jolivalt, Claude; Pellet-Rostaing, Stéphane; Popowycz, Florence; Paris, Jean-Marc; Lemaire, Marc

2016-02-04

An innovative and straightforward synthesis of second-generation 2-arylbenzo[b]thiophenes as structural analogues of INF55 and the first generation of our laboratory-made molecules was developed. The synthesis of C2-arylated benzo[b]thiophene derivatives was achieved through a method involving direct arylation, followed by simple structural modifications. Among the 34 compounds tested, two of them were potent NorA pump inhibitors, which led to a 16-fold decrease in the ciprofloxacin minimum inhibitory concentration (MIC) against the SA-1199B strain at concentrations of 0.25 and 0.5 μg mL(-1) (1 and 1.5 μm, respectively). This is a promising result relative to that obtained for reserpine (MIC=20 μg mL(-1)), a reference compound amongst NorA pump inhibitors. These molecules thus represent promising candidates to be used in combination with ciprofloxacin against fluoroquinolone-resistant strains. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Computerized measures of finger tapping: reliability, malingering and traumatic brain injury.

PubMed

Hubel, Kerry A; Yund, E William; Herron, Timothy J; Woods, David L

2013-01-01

We analyzed computerized finger tapping metrics in four experiments. Experiment 1 showed tapping-rate differences associated with hand dominance, digits, sex, and fatigue that replicated those seen in a previous, large-scale community sample. Experiment 2 revealed test-retest correlations (r = .91) that exceeded those reported in previous tapping studies. Experiment 3 investigated subjects simulating symptoms of traumatic brain injury (TBI); 62% of malingering subjects produced abnormally slow tapping rates. A tapping-rate malingering index, based on rate-independent tapping patterns, provided confirmatory evidence of malingering in 48% of the subjects with abnormal tapping rates. Experiment 4 compared tapping in 24 patients with mild TBI (mTBI) and a matched control group; mTBI patients showed slowed tapping without evidence of malingering. Computerized finger tapping measures are reliable measures of motor speed, useful in detecting subjects performing with suboptimal effort, and are sensitive to motor abnormalities following mTBI.

Transintestinal transport of the anti-inflammatory drug 4F and the modulation of transintestinal cholesterol efflux[S

PubMed Central

Meriwether, David; Sulaiman, Dawoud; Wagner, Alan; Grijalva, Victor; Kaji, Izumi; Williams, Kevin J.; Yu, Liqing; Fogelman, Spencer; Volpe, Carmen; Bensinger, Steven J.; Anantharamaiah, G. M.; Shechter, Ishaiahu; Fogelman, Alan M.; Reddy, Srinivasa T.

2016-01-01

The site and mechanism of action of the apoA-I mimetic peptide 4F are incompletely understood. Transintestinal cholesterol efflux (TICE) is a process involved in the clearance of excess cholesterol from the body. While TICE is responsible for at least 30% of the clearance of neutral sterols from the circulation into the intestinal lumen, few pharmacological agents have been identified that modulate this pathway. We show first that circulating 4F selectively targets the small intestine (SI) and that it is predominantly transported into the intestinal lumen. This transport of 4F into the SI lumen is transintestinal in nature, and it is modulated by TICE. We also show that circulating 4F increases reverse cholesterol transport from macrophages and cholesterol efflux from lipoproteins via the TICE pathway. We identify the cause of this modulation of TICE either as 4F being a cholesterol acceptor with respect to enterocytes, from which 4F enhances cholesterol efflux, or as 4F being an intestinal chaperone with respect to TICE. Our results assign a novel role for 4F as a modulator of the TICE pathway and suggest that the anti-inflammatory functions of 4F may be a partial consequence of the codependent intestinal transport of both 4F and cholesterol. PMID:27199144

PREDICTIVE MODELING OF ACOUSTIC SIGNALS FROM THERMOACOUSTIC POWER SENSORS (TAPS)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dumm, Christopher M.; Vipperman, Jeffrey S.

2016-06-30

Thermoacoustic Power Sensor (TAPS) technology offers the potential for self-powered, wireless measurement of nuclear reactor core operating conditions. TAPS are based on thermoacoustic engines, which harness thermal energy from fission reactions to generate acoustic waves by virtue of gas motion through a porous stack of thermally nonconductive material. TAPS can be placed in the core, where they generate acoustic waves whose frequency and amplitude are proportional to the local temperature and radiation flux, respectively. TAPS acoustic signals are not measured directly at the TAPS; rather, they propagate wirelessly from an individual TAPS through the reactor, and ultimately to a low-powermore » receiver network on the vessel’s exterior. In order to rely on TAPS as primary instrumentation, reactor-specific models which account for geometric/acoustic complexities in the signal propagation environment must be used to predict the amplitude and frequency of TAPS signals at receiver locations. The reactor state may then be derived by comparing receiver signals to the reference levels established by predictive modeling. In this paper, we develop and experimentally benchmark a methodology for predictive modeling of the signals generated by a TAPS system, with the intent of subsequently extending these efforts to modeling of TAPS in a liquid sodium environmen« less

A new tetracycline efflux gene, tet(40), is located in tandem with tet(O/32/O) in a human gut firmicute bacterium and in metagenomic library clones.

PubMed

Kazimierczak, Katarzyna A; Rincon, Marco T; Patterson, Andrea J; Martin, Jennifer C; Young, Pauline; Flint, Harry J; Scott, Karen P

2008-11-01

The bacterium Clostridium saccharolyticum K10, isolated from a fecal sample obtained from a healthy donor who had received long-term tetracycline therapy, was found to carry three tetracycline resistance genes: tet(W) and the mosaic tet(O/32/O), both conferring ribosome protection-type resistance, and a novel, closely linked efflux-type resistance gene designated tet(40). tet(40) encodes a predicted membrane-associated protein with 42% amino acid identity to tetA(P). Tetracycline did not accumulate in Escherichia coli cells expressing the Tet(40) efflux protein, and resistance to tetracycline was reduced when cells were incubated with an efflux pump inhibitor. E. coli cells carrying tet(40) had a 50% inhibitory concentration of tetracycline of 60 microg/ml. Analysis of a transconjugant from a mating between donor strain C. saccharolyticum K10 and the recipient human gut commensal bacterium Roseburia inulinivorans suggested that tet(O/32/O) and tet(40) were cotransferred on a mobile element. Sequence analysis of a 37-kb insert identified on the basis of tetracycline resistance from a metagenomic fosmid library again revealed a tandem arrangement of tet(O/32/O) and tet(40), flanked by regions with homology to parts of the VanG operon previously identified in Enterococcus faecalis. At least 10 of the metagenomic inserts that carried tet(O/32/O) also carried tet(40), suggesting that tet(40), although previously undetected, may be an abundant efflux gene.

Frankincense tapping reduces the carbohydrate storage of Boswellia trees.

PubMed

Mengistu, Tefera; Sterck, Frank J; Fetene, Masresha; Bongers, Frans

2013-06-01

Carbohydrates fixed by photosynthesis are stored in plant organs in the form of starch or sugars. Starch and sugars sum to the total non-structural carbohydrate pool (TNC) and may serve as intermediate pools between assimilation and utilization. We examined the impact of tapping on TNC concentrations in stem-wood, bark and root tissues of the frankincense tree (Boswellia papyrifera (Del.) Hochst) in two natural woodlands of Ethiopia. Two tapping treatments, one without tapping (control) and the other with tapping at 12 incisions, are applied on experimental trees. Trees are tapped in the leafless dry period, diminishing their carbon storage pools. If storage pools are not refilled by assimilation during the wet season, when crowns are in full leaf, tapping may deplete the carbon pool and weaken Boswellia trees. The highest soluble sugar concentrations were in the bark and the highest starch concentrations in the stem-wood. The stem-wood contains 12 times higher starch than soluble sugar concentrations. Hence, the highest TNC concentrations occurred in the stem-wood. Moreover, wood volume was larger than root or bark volumes and, as a result, more TNC was stored in the stem-wood. As predicted, tapping reduced the TNC concentrations and pool sizes in frankincense trees during the dry season. During the wet season, these carbon pools were gradually filled in tapped trees, but never to the size of non-tapped trees. We conclude that TNC is dynamic on a seasonal time scale and offers resilience against stress, highlighting its importance for tree carbon balance. But current resin tapping practices are intensive and may weaken Boswellia populations, jeopardizing future frankincense production.

Relationship between tap water hardness, magnesium, and calcium concentration and mortality due to ischemic heart disease or stroke in The Netherlands.

PubMed

Leurs, Lina J; Schouten, Leo J; Mons, Margreet N; Goldbohm, R Alexandra; van den Brandt, Piet A

2010-03-01

Conflicting results on the relationship between the hardness of drinking water and mortality related to ischemic heart disease (IHD) or stroke have been reported. We investigated the possible association between tap water calcium or magnesium concentration and total hardness and IHD mortality or stroke mortality. In 1986, a cohort of 120,852 men and women aged 5569 years provided detailed information on dietary and other lifestyle habits. Follow-up for mortality until 1996 was established by linking data from the Central Bureau of Genealogy and Statistics Netherlands. We calculated tap water hardness for each postal code using information obtained from all pumping stations in the Netherlands. Tap water hardness was categorized as soft [< 1.5 mmol/L calcium carbonate (CaCO3)], medium hard (1.62.0 mmol/L CaCO3), and hard (> 2.0 mmol/L CaCO3). The multivariate case-cohort analysis was based on 1,944 IHD mortality and 779 stroke mortality cases and 4,114 subcohort members. For both men and women, we observed no relationship between tap water hardness and IHD mortality [hard vs. soft water: hazard ratio (HR) = 1.03; 95% confidence interval (CI), 0.851.28 for men and HR = 0.93; 95% CI, 0.711.21 for women) and stroke mortality (hard vs. soft water HR = 0.90; 95% CI, 0.661.21 and HR = 0.86; 95% CI, 0.621.20, respectively). For men with the 20% lowest dietary magnesium intake, an inverse association was observed between tap water magnesium intake and stroke mortality (HR per 1 mg/L intake = 0.75; 95% CI, 0.610.91), whereas for women with the 20% lowest dietary magnesium intake, the opposite was observed. We found no evidence for an overall significant association between tap water hardness, magnesium or calcium concentrations, and IHD mortality or stroke mortality. More research is needed to investigate the effect of tap water magnesium on IHD mortality or stroke mortality in subjects with low dietary magnesium intake.

Astronomy on Tap: Public Outreach Events in Bars

NASA Astrophysics Data System (ADS)

Rice, E. L.; Levine, B. W.

2016-12-01

Astronomy on Tap public outreach events are as easy to organise or as elaborate as you would like them to be. In addition to communicating cutting-edge research and fundamental concepts to the public, Astronomy on Tap events showcase the passion, creativity and diversity of scientists, facilitate personal and meaningful interactions between scientists and the general public, and offer networking and professional development opportunities for scientists. Astronomy on Tap organisers provide a growing cadre of resources for starting similar events, which have so far taken place in twenty locations around the world, mainly in the United States but also in Canada, Chile, and Taiwan, reaching a total of almost 15 000 people. Through this reflection on the Astronomy on Tap project we invite you to consider whether you could adopt aspects of the Astronomy on Tap model for existing outreach programmes, or even organise a new satellite event in your location.

Centrifugal Pump Effect on Average Particle Diameter of Oil-Water Emulsion

NASA Astrophysics Data System (ADS)

Morozova, A.; Eskin, A.

2017-11-01

In this paper we review the process of oil-water emulsion particles fragmentation in a turbulent flow created by a centrifugal pump. We examined the influence of time necessary for oil-water emulsion preparation on the particle size of oil products and the dependence of a centrifugal pump emulsifying capacity on the initial emulsion dispersion. The investigated emulsion contained the brand fuel oil M-100 and tap water; it was sprayed with a nozzle in a gas-water flare. After preparation of the emulsion, the centrifugal pump was turned on and the emulsion samples were taken before and after the pump passing in 15, 30 and 45 minutes of spraying. To determine the effect the centrifugal pump has on the dispersion of the oil-water emulsion, the mean particle diameter of the emulsion particles was determined by the optical and microscopic method before and after the pump passing. A dispersion analysis of the particles contained in the emulsion was carried out by a laser diffraction analyzer. By analyzing the pictures of the emulsion samples, it was determined that after the centrifugal pump operation a particle size of oil products decreases. This result is also confirmed by the distribution of the obtained analyzer where the content of fine particles with a diameter less than 10 μm increased from 12% to 23%. In case of increasing emulsion preparation time, a particle size of petroleum products also decreases.

Inhibitory Effect of Flavonoids on the Efflux of N-Acetyl 5-Aminosalicylic Acid Intracellularly Formed in Caco-2 Cells

PubMed Central

Shin, Yoshimura; Kentaro, Kawano; Ryusuke, Matsumura; Narumi, Sugihara; Koji, Furuno

2009-01-01

N-acetyl 5-aminosalicylic acid (5-AcASA) that was intracellularly formed from 5-aminosalicylic acid (5-ASA) at 200 μM was discharged 5.3, 7.1, and 8.1-fold higher into the apical site than into the basolateral site during 1, 2, and 4-hour incubations, respectively, in Caco-2 cells grown in Transwells. The addition of flavonols (100 μM) such as fisetin and quercetin with 5-ASA remarkably decreased the apically directed efflux of 5-AcASA. When 5-ASA (200 μM) was added to Caco-2 cells grown in tissue culture dishes, the formation of 5-AcASA decreased, and, in addition, the formed 5-AcASA was found to be accumulated within the cells in the presence of such flavonols. Thus, the decrease in 5-AcASA efflux by such flavonols was attributed not only to the inhibition of N-acetyl-conjugation of 5-ASA but to the predominant cellular accumulation of 5-AcASA. Various flavonoids also had both of the effects with potencies that depend on their specific structures. The essential structure of flavonoids was an absence of a hydroxyl substitution at the C5 position on the A-ring of flavone structure for the inhibitory effect on the N-acetyl-conjugation of 5-ASA, and a presence of hydroxyl substitutions at the C3′ or C4′ position on the B-ring of flavone structure for the promoting effect on the cellular accumulation of 5-AcASA. Both the decrease in 5-AcASA apical efflux and the increase in 5-AcASA cellular accumulation were also caused by MK571 and indomethacin, inhibitors of MRPs, but not by quinidine, cyclosporin A, P-glycoprotein inhibitors, and mitoxantrone, a BCRP substrate. These results suggest that certain flavonoids suppress the apical efflux of 5-AcASA possibly by inhibiting MRPs pumps located on apical membranes in Caco-2 cells. PMID:19688110

Finger tapping ability in healthy elderly and young adults.

PubMed

Aoki, Tomoko; Fukuoka, Yoshiyuki

2010-03-01

The maximum isometric force production capacity of the fingers decreases with age. However, little information is available on age-related changes in dynamic motor capacity of individual fingers. The purpose of this study was to compare the dynamic motor function of individual fingers between elderly and young adults using rapid single-finger and double-finger tapping. Fourteen elderly and 14 young adults performed maximum frequency tapping by the index, middle, ring, or little finger (single-finger tapping) and with alternate movements of the index-middle, middle-ring, or ring-little finger-pair (double-finger tapping). The maximum pinch force between the thumb and each finger, tactile sensitivity of each fingertip, and time taken to complete a pegboard test were also measured. Compared with young subjects, the older subjects had significantly slower tapping rates in all fingers and finger-pairs in the tapping tasks. The age-related decline was also observed in the tactile sensitivities of all fingers and in the pegboard test. However, there was no group difference in the pinch force of any finger. The tapping rate of each finger did not correlate with the pinch force or tactile sensitivity for the corresponding finger in the elderly subjects. Maximum rate of finger tapping was lower in the elderly adults compared with the young adults. The decline of finger tapping ability in elderly adults seems to be less affected by their maximum force production capacities of the fingers as well as tactile sensitivities at the tips of the fingers.

Thermal Analyzer for Planetary Soil (TAPS): an in Situ Instrument for Mineral and Volatile-element Measurements

NASA Technical Reports Server (NTRS)

Gooding, J. L.; Ming, D. W.; Gruener, J. E.; Gibbons, F. L.; Allton, J. H.

1993-01-01

Thermal Analyzer for Planetary Soil (TAPS) offers a specific implementation for the generic thermal analyzer/evolved-gas analyzer (TA/EGA) function included in the Mars Environmental Survey (MESUR) strawman payload; applications to asteroids and comets are also possible. The baseline TAPS is a single-sample differential scanning calorimeter (DSC), backed by a capacitive-polymer humidity sensor, with an integrated sampling mechanism. After placement on a planetary surface, TAPS acquires 10-50 mg of soil or sediment and heats the sample from ambient temperature to 1000-1300 K. During heating, DSC data are taken for the solid and evolved gases are swept past the water sensor. Through ground based data analysis, multicomponent DSC data are deconvolved and correlated with the water release profile to quantitatively determine the types and relative proportions of volatile-bearing minerals such as clays and other hydrates, carbonates, and nitrates. The rapid-response humidity sensors also achieve quantitative analysis of total water. After conclusion of soil-analysis operations, the humidity sensors become available for meteorology. The baseline design fits within a circular-cylindrical volume less than 1000 cm(sup 3), occupies 1.2 kg mass, and consumes about 2 Whr of power per analysis. Enhanced designs would acquire and analyze multiple samples and employ additional microchemical sensors for analysis of CO2, SO2, NO(x), and other gaseous species. Atmospheric pumps are also being considered as alternatives to pressurized purge gas.

Thermal Analyzer for Planetary Soil (TAPS): an in situ instrument for mineral and volatile-element measurements

NASA Astrophysics Data System (ADS)

Gooding, J. L.; Ming, D. W.; Gruener, J. E.; Gibbons, F. L.; Allton, J. H.

Thermal Analyzer for Planetary Soil (TAPS) offers a specific implementation for the generic thermal analyzer/evolved-gas analyzer (TA/EGA) function included in the Mars Environmental Survey (MESUR) strawman payload; applications to asteroids and comets are also possible. The baseline TAPS is a single-sample differential scanning calorimeter (DSC), backed by a capacitive-polymer humidity sensor, with an integrated sampling mechanism. After placement on a planetary surface, TAPS acquires 10-50 mg of soil or sediment and heats the sample from ambient temperature to 1000-1300 K. During heating, DSC data are taken for the solid and evolved gases are swept past the water sensor. Through ground based data analysis, multicomponent DSC data are deconvolved and correlated with the water release profile to quantitatively determine the types and relative proportions of volatile-bearing minerals such as clays and other hydrates, carbonates, and nitrates. The rapid-response humidity sensors also achieve quantitative analysis of total water. After conclusion of soil-analysis operations, the humidity sensors become available for meteorology. The baseline design fits within a circular-cylindrical volume less than 1000 cm3, occupies 1.2 kg mass, and consumes about 2 Whr of power per analysis. Enhanced designs would acquire and analyze multiple samples and employ additional microchemical sensors for analysis of CO2, SO2, NO(x), and other gaseous species. Atmospheric pumps are also being considered as alternatives to pressurized purge gas.

Strong synergism of dexamethasone in combination with fluconazole against resistant Candida albicans mediated by inhibiting drug efflux and reducing virulence.

PubMed

Sun, Wenwen; Wang, Decai; Yu, Cuixiang; Huang, Xin; Li, Xiuyun; Sun, Shujuan

2017-09-01

Candida albicans is the most commonly isolated Candida spp. in the clinic and its resistance to fluconazole (FLC) has been emerging rapidly. Combination therapy may be a potentially effective approach to combat drug resistance. In this study, the combination antifungal effects of dexamethasone (DXM) and FLC against resistant C. albicans in vitro were assayed using minimum inhibitory concentrations (MICs), sessile MICs and time-kill curves. The in vivo efficacy of this drug combination was evaluated using a Galleria mellonella model by determining survival rate, fungal burden and histological damage. In addition, the impact of DXM on efflux pump activity was investigated using a rhodamine 6G assay. Expression of CDR1, CDR2 and MDR1 was determined by real-time quantitative PCR, and extracellular phospholipase activity was detected by the egg yolk agar method to reveal the potential synergistic mechanism. The results showed that DXM potentiates the antifungal effect of FLC against resistant C. albicans strains both in vitro and in vivo, and the synergistic mechanism is related to inhibiting the efflux of drugs and reducing the virulence of C. albicans. Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

Estimation method of finger tapping dynamics using simple magnetic detection system

NASA Astrophysics Data System (ADS)

Kandori, Akihiko; Sano, Yuko; Miyashita, Tsuyoshi; Okada, Yoshihisa; Irokawa, Masataka; Shima, Keisuke; Tsuji, Toshio; Yokoe, Masaru; Sakoda, Saburo

2010-05-01

We have developed the simple estimation method of a finger tapping dynamics model for investigating muscle resistance and stiffness during tapping movement in normal subjects. We measured finger tapping movements of 207 normal subjects using a magnetic finger tapping detection system. Each subject tapped two fingers in time with a metronome at 1, 2, 3, 4, and 5 Hz. The velocity and acceleration values for both the closing and opening tapping data were used to estimate a finger tapping dynamics model. Using the frequency response of the ratio of acceleration to velocity of the mechanical impedance parameters, we estimated the resistance (friction coefficient) and compliance (stiffness). We found two dynamics models for the maximum open position and tap position. In the maximum open position, the extensor muscle resistance was twice as high as the flexor muscle resistance and males had a higher spring constant. In the tap position, the flexor muscle resistance was much higher than the extensor muscle resistance. This indicates that the tapping dynamics in the maximum open position are controlled by the balance of extensor and flexor muscle friction resistances and the flexor stiffness, and the flexor friction resistance is the main component in the tap position. It can be concluded that our estimation method makes it possible to understand the tapping dynamics.

Estimation method of finger tapping dynamics using simple magnetic detection system.

PubMed

Kandori, Akihiko; Sano, Yuko; Miyashita, Tsuyoshi; Okada, Yoshihisa; Irokawa, Masataka; Shima, Keisuke; Tsuji, Toshio; Yokoe, Masaru; Sakoda, Saburo

2010-05-01

We have developed the simple estimation method of a finger tapping dynamics model for investigating muscle resistance and stiffness during tapping movement in normal subjects. We measured finger tapping movements of 207 normal subjects using a magnetic finger tapping detection system. Each subject tapped two fingers in time with a metronome at 1, 2, 3, 4, and 5 Hz. The velocity and acceleration values for both the closing and opening tapping data were used to estimate a finger tapping dynamics model. Using the frequency response of the ratio of acceleration to velocity of the mechanical impedance parameters, we estimated the resistance (friction coefficient) and compliance (stiffness). We found two dynamics models for the maximum open position and tap position. In the maximum open position, the extensor muscle resistance was twice as high as the flexor muscle resistance and males had a higher spring constant. In the tap position, the flexor muscle resistance was much higher than the extensor muscle resistance. This indicates that the tapping dynamics in the maximum open position are controlled by the balance of extensor and flexor muscle friction resistances and the flexor stiffness, and the flexor friction resistance is the main component in the tap position. It can be concluded that our estimation method makes it possible to understand the tapping dynamics.

Quantification of Finger-Tapping Angle Based on Wearable Sensors

PubMed Central

Djurić-Jovičić, Milica; Jovičić, Nenad S.; Roby-Brami, Agnes; Popović, Mirjana B.; Kostić, Vladimir S.; Djordjević, Antonije R.

2017-01-01

We propose a novel simple method for quantitative and qualitative finger-tapping assessment based on miniature inertial sensors (3D gyroscopes) placed on the thumb and index-finger. We propose a simplified description of the finger tapping by using a single angle, describing rotation around a dominant axis. The method was verified on twelve subjects, who performed various tapping tasks, mimicking impaired patterns. The obtained tapping angles were compared with results of a motion capture camera system, demonstrating excellent accuracy. The root-mean-square (RMS) error between the two sets of data is, on average, below 4°, and the intraclass correlation coefficient is, on average, greater than 0.972. Data obtained by the proposed method may be used together with scores from clinical tests to enable a better diagnostic. Along with hardware simplicity, this makes the proposed method a promising candidate for use in clinical practice. Furthermore, our definition of the tapping angle can be applied to all tapping assessment systems. PMID:28125051

Quantification of Finger-Tapping Angle Based on Wearable Sensors.

PubMed

Djurić-Jovičić, Milica; Jovičić, Nenad S; Roby-Brami, Agnes; Popović, Mirjana B; Kostić, Vladimir S; Djordjević, Antonije R

2017-01-25

We propose a novel simple method for quantitative and qualitative finger-tapping assessment based on miniature inertial sensors (3D gyroscopes) placed on the thumb and index-finger. We propose a simplified description of the finger tapping by using a single angle, describing rotation around a dominant axis. The method was verified on twelve subjects, who performed various tapping tasks, mimicking impaired patterns. The obtained tapping angles were compared with results of a motion capture camera system, demonstrating excellent accuracy. The root-mean-square (RMS) error between the two sets of data is, on average, below 4°, and the intraclass correlation coefficient is, on average, greater than 0.972. Data obtained by the proposed method may be used together with scores from clinical tests to enable a better diagnostic. Along with hardware simplicity, this makes the proposed method a promising candidate for use in clinical practice. Furthermore, our definition of the tapping angle can be applied to all tapping assessment systems.

Acid sphingomyelinase-deficient macrophages have defective cholesterol trafficking and efflux.

PubMed

Leventhal, A R; Chen, W; Tall, A R; Tabas, I

2001-11-30

Cholesterol efflux from macrophage foam cells, a key step in reverse cholesterol transport, requires trafficking of cholesterol from intracellular sites to the plasma membrane. Sphingomyelin is a cholesterol-binding molecule that transiently exists with cholesterol in endosomes and lysosomes but is rapidly hydrolyzed by lysosomal sphingomyelinase (L-SMase), a product of the acid sphingomyelinase (ASM) gene. We therefore hypothesized that sphingomyelin hydrolysis by L-SMase enables cholesterol efflux by preventing cholesterol sequestration by sphingomyelin. Macrophages from wild-type and ASM knockout mice were incubated with [(3)H]cholesteryl ester-labeled acetyl-LDL and then exposed to apolipoprotein A-I or high density lipoprotein. In both cases, [(3)H]cholesterol efflux was decreased substantially in the ASM knockout macrophages. Similar results were shown for ASM knockout macrophages labeled long-term with [(3)H]cholesterol added directly to medium, but not for those labeled for a short period, suggesting defective efflux from intracellular stores but not from the plasma membrane. Cholesterol trafficking to acyl-coenzyme A:cholesterol acyltransferase (ACAT) was also defective in ASM knockout macrophages. Using filipin to probe cholesterol in macrophages incubated with acetyl-LDL, we found there was modest staining in the plasma membrane of wild-type macrophages but bright, perinuclear fluorescence in ASM knockout macrophages. Last, when wild-type macrophages were incubated with excess sphingomyelin to "saturate" L-SMase, [(3)H]cholesterol efflux was decreased. Thus, sphingomyelin accumulation due to L-SMase deficiency leads to defective cholesterol trafficking and efflux, which we propose is due to sequestration of cholesterol by sphingomyelin and possibly other mechanisms. This model may explain the low plasma high density lipoprotein found in ASM-deficient humans and may implicate L-SMase deficiency and/or sphingomyelin enrichment of lipoproteins as novel

Effects of aging on control of timing and force of finger tapping.

PubMed

Sasaki, Hirokazu; Masumoto, Junya; Inui, Nobuyuki

2011-04-01

The present study examined whether the elderly produced a hastened or delayed tap with a negative or positive constant intertap interval error more frequently in self-paced tapping than in the stimulus-synchronized tapping for the 2 N target force at 2 or 4 Hz frequency. The analysis showed that, at both frequencies, the percentage of the delayed tap was larger in the self-paced tapping than in the stimulus-synchronized tapping, whereas the hastened tap showed the opposite result. At the 4 Hz frequency, all age groups had more variable intertap intervals during the self-paced tapping than during the stimulus-synchronized tapping, and the variability of the intertap intervals increased with age. Thus, although the increase in the frequency of delayed taps and variable intertap intervals in the self-paced tapping perhaps resulted from a dysfunction of movement timing in the basal ganglia with age, the decline in timing accuracy was somewhat improved by an auditory cue. The force variability of tapping at 4 Hz further increased with age, indicating an effect of aging on the control of force.

Fractionation of Sodium Efflux in Frog Sartorius Muscles by Strophanthidin and Removal of External Sodium

PubMed Central

Horowicz, P.; Taylor, J. W.; Waggoner, D. M.

1970-01-01

The influence of strophanthidin, ouabain, and the removal of external sodium on the sodium efflux from frog sartorius muscle was measured. In freshly dissected muscles strophanthidin and ouabain in maximally effective concentrations reduced the efflux of sodium by about 50%. Of the sodium efflux which is strophanthidin-insensitive about 75% is inhibited after complete replacement of external sodium by lithium. In the absence of strophanthidin replacement of external sodium by lithium, calcium, or magnesium produces an initial rise in the sodium efflux, followed by a fall in the efflux as the exposure of the muscles to sodium-free media is continued. When the muscles are exposed for prolonged periods in sodium-free media, the fraction of internal sodium lost per minute is higher when returned to normal Ringer fluid than it was initially. The activation of sodium efflux by external sodium after long periods in sodium-free solutions is partly strophanthidin-sensitive and partly strophanthidin-insensitive. The internal sodium concentration is an important factor in these effects. The effects of temperature on the sodium efflux were also measured. Above 7°C the Q 10 of both the strophanthidin-sensitive and strophanthidin-insensitive sodium efflux is about 2.0. Below 7°C the strophanthidin-insensitive sodium efflux has a Q 10 of about 7.4. PMID:5315424

Brain activity during bilateral rapid alternate finger tapping measured with magnetoencephalography

NASA Astrophysics Data System (ADS)

Fukuda, Hiroshi; Odagaki, Masato; Hiwaki, Osamu; Kodabashi, Atsushi; Fujimoto, Toshiro

2009-04-01

Using magnetoencephalography (MEG), brain regions involved in an alternate bimanual tapping task by index fingers triggered with spontaneous timing were investigated. The tapping mode in which both index fingers moved simultaneously was interlaced during the task. The groups of the alternate tapping (AL mode) and the simultaneous tapping (SI mode) were extracted from the successive alternating taps with a histogram of intervals between the right and left index fingers. MEG signals in each mode were averaged separately before and after the tapping initiation of the dominant index finger. The activities of the contralateral sensorimotor cortex before and after the tapping initiation in the AL mode were larger than that in the SI mode. The result indicates that the activity of the contralateral sensorimotor cortex depends on the degree of achievement in the difficult motor task such as the voluntary alternate tapping movements.

Molecular Components of Nitrate and Nitrite Efflux in Yeast

PubMed Central

Cabrera, Elisa; González-Montelongo, Rafaela; Giraldez, Teresa; de la Rosa, Diego Alvarez

2014-01-01

Some eukaryotes, such as plant and fungi, are capable of utilizing nitrate as the sole nitrogen source. Once transported into the cell, nitrate is reduced to ammonium by the consecutive action of nitrate and nitrite reductase. How nitrate assimilation is balanced with nitrate and nitrite efflux is unknown, as are the proteins involved. The nitrate assimilatory yeast Hansenula polymorpha was used as a model to dissect these efflux systems. We identified the sulfite transporters Ssu1 and Ssu2 as effective nitrate exporters, Ssu2 being quantitatively more important, and we characterize the Nar1 protein as a nitrate/nitrite exporter. The use of strains lacking either SSU2 or NAR1 along with the nitrate reductase gene YNR1 showed that nitrate reductase activity is not required for net nitrate uptake. Growth test experiments indicated that Ssu2 and Nar1 exporters allow yeast to cope with nitrite toxicity. We also have shown that the well-known Saccharomyces cerevisiae sulfite efflux permease Ssu1 is also able to excrete nitrite and nitrate. These results characterize for the first time essential components of the nitrate/nitrite efflux system and their impact on net nitrate uptake and its regulation. PMID:24363367

V-ATPase proton pumping activity is required for adult zebrafish appendage regeneration.

PubMed

Monteiro, Joana; Aires, Rita; Becker, Jörg D; Jacinto, António; Certal, Ana C; Rodríguez-León, Joaquín

2014-01-01

The activity of ion channels and transporters generates ion-specific fluxes that encode electrical and/or chemical signals with biological significance. Even though it is long known that some of those signals are crucial for regeneration, only in recent years the corresponding molecular sources started to be identified using mainly invertebrate or larval vertebrate models. We used adult zebrafish caudal fin as a model to investigate which and how ion transporters affect regeneration in an adult vertebrate model. Through the combined use of biophysical and molecular approaches, we show that V-ATPase activity contributes to a regeneration-specific H+ ef`flux. The onset and intensity of both V-ATPase expression and H+ efflux correlate with the different regeneration rate along the proximal-distal axis. Moreover, we show that V-ATPase inhibition impairs regeneration in adult vertebrate. Notably, the activity of this H+ pump is necessary for aldh1a2 and mkp3 expression, blastema cell proliferation and fin innervation. To the best of our knowledge, this is the first report on the role of V-ATPase during adult vertebrate regeneration.

V-ATPase Proton Pumping Activity Is Required for Adult Zebrafish Appendage Regeneration

PubMed Central

Monteiro, Joana; Aires, Rita; Becker, Jörg D.; Jacinto, António; Certal, Ana C.; Rodríguez-León, Joaquín

2014-01-01

The activity of ion channels and transporters generates ion-specific fluxes that encode electrical and/or chemical signals with biological significance. Even though it is long known that some of those signals are crucial for regeneration, only in recent years the corresponding molecular sources started to be identified using mainly invertebrate or larval vertebrate models. We used adult zebrafish caudal fin as a model to investigate which and how ion transporters affect regeneration in an adult vertebrate model. Through the combined use of biophysical and molecular approaches, we show that V-ATPase activity contributes to a regeneration-specific H+ ef`flux. The onset and intensity of both V-ATPase expression and H+ efflux correlate with the different regeneration rate along the proximal-distal axis. Moreover, we show that V-ATPase inhibition impairs regeneration in adult vertebrate. Notably, the activity of this H+ pump is necessary for aldh1a2 and mkp3 expression, blastema cell proliferation and fin innervation. To the best of our knowledge, this is the first report on the role of V-ATPase during adult vertebrate regeneration. PMID:24671205

Epigallocatechin gallate as a modulator of Campylobacter resistance to macrolide antibiotics.

PubMed

Kurinčič, Marija; Klančnik, Anja; Smole Možina, Sonja

2012-11-01

Comprehensive therapeutic use of macrolides in humans and animals is important in the selection of macrolide-resistant Campylobacter isolates. This study shows high co-resistance to erythromycin, azithromycin, clarithromycin, dirithromycin and tylosin, with contributions from the 23S rRNA gene and drug efflux systems. The CmeABC efflux pump plays an important role in reduced macrolide susceptibility, accompanied by contributions from the CmeDEF efflux pump and potentially a third efflux pump. To improve clinical performance of licensed antibiotics and chemotherapeutic agents, it is important to understand the factors in Campylobacter that affect susceptibility to macrolide antibiotics. Using mutants that lack the functional genes coding for the CmeB and CmeF efflux pump proteins and the CmeR transcriptional repressor, we show that these efflux pumps are potential targets for the development of therapeutic strategies that use a combination of a macrolide with an efflux pump inhibitor (EPI) to restore macrolide efficacy. The natural phenolic compound epigallocatechin gallate (EGCG) has good modulatory activity over the extrusion across the outer membrane of the macrolides tested, both in sensitive and resistant Campylobacter isolates. Comparing EGCG with known chemical EPIs, correlations in the effects on the particular macrolide antibiotics were seen. EGCG modifies Campylobacter multidrug efflux systems and thus could have an impact on restoring macrolide efficacy in resistant strains. Copyright © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Ascorbic acid efflux and re-uptake in endothelial cells: maintenance of intracellular ascorbate.

PubMed

May, James M; Qu, Zhi-chao

2009-05-01

Entry of vitamin C or ascorbate into most tissues requires its movement across the endothelial cell barrier of vessels. If trans-cellular ascorbate movement occurs, then it should be evident as ascorbate efflux from endothelial cells. Cultured EA.926 endothelial cells that had been loaded to about 3.5 mM intracellular ascorbate lost 70-80% of ascorbate to the medium over several hours at 37 degrees C via a non-saturable process that was insensitive to anion transport inhibitors and thiol reagents. Oxidation of this extracellular ascorbate by ascorbate oxidase or ferricyanide enhanced apparent ascorbate efflux, suggesting that efflux of the vitamin was countered in part by its re-uptake on ascorbate transporters. Although basal ascorbate efflux was not calcium-dependent, increased entry of calcium into the cells enhanced ascorbate release. These results support the hypothesis that ascorbate efflux reflects trans-endothelial cell ascorbate movement out of the blood vessel.

Mind the gap: non-biological processes contributing to soil CO2 efflux.

PubMed

Rey, Ana

2015-05-01

Widespread recognition of the importance of soil CO2 efflux as a major source of CO2 to the atmosphere has led to active research. A large soil respiration database and recent reviews have compiled data, methods, and current challenges. This study highlights some deficiencies for a proper understanding of soil CO2 efflux focusing on processes of soil CO2 production and transport that have not received enough attention in the current soil respiration literature. It has mostly been assumed that soil CO2 efflux is the result of biological processes (i.e. soil respiration), but recent studies demonstrate that pedochemical and geological processes, such as geothermal and volcanic CO2 degassing, are potentially important in some areas. Besides the microbial decomposition of litter, solar radiation is responsible for photodegradation or photochemical degradation of litter. Diffusion is considered to be the main mechanism of CO2 transport in the soil, but changes in atmospheric pressure and thermal convection may also be important mechanisms driving soil CO2 efflux greater than diffusion under certain conditions. Lateral fluxes of carbon as dissolved organic and inorganic carbon occur and may cause an underestimation of soil CO2 efflux. Traditionally soil CO2 efflux has been measured with accumulation chambers assuming that the main transport mechanism is diffusion. New techniques are available such as improved automated chambers, CO2 concentration profiles and isotopic techniques that may help to elucidate the sources of carbon from soils. We need to develop specific and standardized methods for different CO2 sources to quantify this flux on a global scale. Biogeochemical models should include biological and non-biological CO2 production processes before we can predict the response of soil CO2 efflux to climate change. Improving our understanding of the processes involved in soil CO2 efflux should be a research priority given the importance of this flux in the global

Genetic Variants in TAP Are Associated with High-Grade Cervical Neoplasia

PubMed Central

Einstein, Mark H.; Leanza, Suzanne; Chiu, Lydia G.; Schlecht, Nicolas F.; Goldberg, Gary L.; Steinberg, Bettie M.; Burk, Robert D.

2018-01-01

Purpose The transporter associated with antigen processing (TAP) is essential in assembling MHC-I proteins. Human papillomavirus (HPV) evades immune recognition by decreasing class I MHC cell surface expression through down-regulation of TAP1 levels. Consistent with heterogeneity in MHC expression is the individual variability in clearing detectable HPV infections. Genetic polymorphisms in TAP genes may affect protein structure, function, and the ability to clear HPV infection. Experimental Design Case-control study of women with cervical intraepithelial neoplasia (CIN) II or III (n = 114) and women without high-grade CIN (n = 366). Five nonsynonymous single nucleotide polymorphisms (SNP) in TAP1 and TAP2 were genotyped using DNA collected in cervicovaginal lavage samples using microsphere array technology (Luminex ×MAP). HPV typing was done using a PCR-based system with MY09/MY11 primers. TAP1 and TAP2 SNPs were validated by direct sequencing. Results Differences in allele distribution between women with high-grade cervical neoplasia and women without was seen for TAP1 I333V (P = 0.02) and TAP1 D637G (p = 0.01).The odds ratios (OR) for CIN III were significantly lower among carriers of the TAP1 I333V polymorphism (OR, 0.28; 95% confidence interval, 0.1-0.8), and TAP1 D637G polymorphism (OR, 0.27; 95% confidence interval, 0.1-0.7). These associations remained significant even after restricting the evaluation to women who were positive for high-risk HPV types. Conclusions In addition to the down-regulation of MHC-1 by oncogenic HPV, HPV pathogenesis might be facilitated by polymorphisms in the TAP proteins. Identifying TAP polymorphisms may potentially be used to identify women less susceptible to progression to high-grade CIN and cervical cancer. PMID:19188174

Abnormal maximal finger tapping in abstinent cannabis users.

PubMed

Flavel, Stanley C; White, Jason M; Todd, Gabrielle

2013-11-01

To investigate movement speed and rhythmicity in abstinent cannabis users, we hypothesized that abstinent cannabis users exhibit decreased maximal finger tapping frequency and increased variability of tapping compared with non-drug users. The study involved 10 healthy adult cannabis users and 10 age-matched and gender-matched controls with no history of illicit drug use. Subjects underwent a series of screening tests prior to participation. Subjects were then asked to tap a strain gauge as fast as possible with the index finger of their dominant hand (duration 5 s). The average intertap interval did not significantly differ between groups, but the coefficient of variation of the intertap interval was significantly greater in the cannabis group than in controls (p=0.011). The cannabis group also exhibited a slow tapping frequency at the beginning of the task. Rhythmicity of finger tapping is abnormal in individuals with a history of cannabis use. The abnormality appears to be long lasting and adds to the list of functional changes present in abstinent cannabis users. Copyright © 2013 John Wiley & Sons, Ltd.

Inside out: efflux of carbon dioxide from leaves represents more than leaf metabolism.

PubMed

Stutz, Samantha S; Anderson, Jeremiah; Zulick, Rachael; Hanson, David T

2017-05-17

High concentrations of inorganic carbon in the xylem, produced from root, stem, and branch respiration, travel via the transpiration stream and eventually exit the plant through distant tissues as CO2. Unlike previous studies that focused on the efflux of CO2 from roots and woody tissues, we focus on efflux from leaves and the potential effect on leaf respiration measurements. We labeled transported inorganic carbon, spanning reported xylem concentrations, with 13C and then manipulated transpiration rates in the dark in order to vary the rates of inorganic carbon supply to cut leaves from Brassica napus and Populus deltoides. We used tunable diode laser absorbance spectroscopy to directly measure the rate of gross 13CO2 efflux, derived from inorganic carbon supplied from outside of the leaf, relative to gross 12CO2 efflux generated from leaf cells. These experiemnts showed that 13CO2 efflux was dependent upon the rate of inorganic carbon supply to the leaf and the rate of transpiration. Our data show that the gross leaf efflux of xylem-transported CO2 is likely small in the dark when rates of transpiration are low. However, gross leaf efflux of xylem-transported CO2 could approach half the rate of leaf respiration in the light when transpiration rates and branch inorganic carbon concentrations are high, irrespective of the grossly different petiole morphologies in our experiment. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Fluorescent Trimethoprim Conjugate Probes To Assess Drug Accumulation in Wild Type and Mutant Escherichia coli

PubMed Central

2016-01-01

Reduced susceptibility to antimicrobials in Gram-negative bacteria may result from multiple resistance mechanisms, including increased efflux pump activity or reduced porin protein expression. Up-regulation of the efflux pump system is closely associated with multidrug resistance (MDR). To help investigate the role of efflux pumps on compound accumulation, a fluorescence-based assay was developed using fluorescent derivatives of trimethoprim (TMP), a broad-spectrum synthetic antibiotic that inhibits an intracellular target, dihydrofolate reductase (DHFR). Novel fluorescent TMP probes inhibited eDHFR activity with comparable potency to TMP, but did not kill or inhibit growth of wild type Escherichia coli. However, bactericidal activity was observed against an efflux pump deficient E. coli mutant strain (ΔtolC). A simple and quick fluorescence assay was developed to measure cellular accumulation of the TMP probe using either fluorescence spectroscopy or flow cytometry, with validation by LC-MS/MS. This fluorescence assay may provide a simple method to assess efflux pump activity with standard laboratory equipment. PMID:27737551

Effects of age, task, and frequency on variability of finger tapping.

PubMed

Sommervoll, Yngve; Ettema, Gertjan; Vereijken, Beatrix

2011-10-01

The goal was to assess whether prior studies might have overestimated performance variability in older adults in dual task conditions by relying on primary motor tasks that are not constant with aging. 30 younger and 31 older adults performed a bimanual tapping task at four different frequencies in isolation or concurrently with a secondary task. Results showed that performance of younger and older adults was not significantly different in performing the tapping task at all frequencies and with either secondary task, as indicated by mean tapping performance and low number of errors in the secondary tasks. Both groups showed increased variability as tapping frequency increased and with the presence of a secondary task. Tapping concurrently while reading words increased tapping variability more than tapping concurrently while naming colours. Although older participants' performances were overall more variable, no interaction effects with age were found and at the highest frequencies of tapping, younger and older participants did not differ in performance.

An ex vivo study of nitric oxide efflux from human erythrocytes in both genders.

PubMed

Duarte, Catarina; Napoleão, Patrícia; Freitas, Teresa; Saldanha, Carlota

2016-01-01

Acetylcholinesterase (AChE) is located on outer surface of erythrocyte membrane. Gender-related differences in erythrocyte AChE enzyme activity had been verified in young adults. It is also known that binding of acetylcholine (ACh) with AChE on erythrocyte membrane initiates a signal transduction mechanism that stimulates nitric oxide (NO) efflux. This ex vivo study was done to compare the amount of NO efflux obtained from erythrocytes of healthy donors in males and females. We included 66 gender age-matched healthy donors (40-60 years old). We performed quantification of erythrocyte NO efflux from erythrocytes and of the membrane AChE enzyme activity. There are no significant differences in NO efflux from erythrocytes between men and women. Regarding AChE enzyme activity values, in this range of age, no differences between genders were obtained. However, the values of AChE enzyme activity in the third quartile of NO efflux values were significantly higher (p < 0.05) in women than in men. The efflux of NO from erythrocyte of healthy humans did not change with gender. For the same range of values of NO efflux from erythrocytes, in both gender, it was verified higher values of AChE enzyme activity in women.

Ascorbic Acid Efflux and Re-uptake in Endothelial Cells: Maintenance of Intracellular Ascorbate

PubMed Central

May, James M.; Qu, Zhi-chao

2013-01-01

Entry of vitamin C or ascorbate into most tissues requires its movement across the endothelial cell barrier of vessels. If trans-cellular ascorbate movement occurs, then it should be evident as ascorbate efflux from endothelial cells. Cultured EA.926 endothelial cells that had been loaded to about 3.5 mM intracellular ascorbate lost 70–80% of ascorbate to the medium over several hours at 37 °C via a non-saturable process that was insensitive to anion transport inhibitors and thiol reagents. Oxidation of this extracellular ascorbate by ascorbate oxidase or ferricyanide enhanced apparent ascorbate efflux, suggesting that efflux of the vitamin was countered in part by its re-uptake on ascorbate transporters. Although basal ascorbate efflux was not calcium-dependent, increased entry of calcium into the cells enhanced ascorbate release. These results support the hypothesis that ascorbate efflux reflects trans-endothelial cell ascorbate movement out of the blood vessel. PMID:19148707

CO₂ efflux from shrimp ponds in Indonesia.

PubMed

Sidik, Frida; Lovelock, Catherine E

2013-01-01

The conversion of mangrove forest to aquaculture ponds has been increasing in recent decades. One of major concerns of this habitat loss is the release of stored 'blue' carbon from mangrove soils to the atmosphere. In this study, we assessed carbon dioxide (CO₂) efflux from soil in intensive shrimp ponds in Bali, Indonesia. We measured CO₂ efflux from the floors and walls of shrimp ponds. Rates of CO₂ efflux within shrimp ponds were 4.37 kg CO₂ m⁻² y⁻¹ from the walls and 1.60 kg CO₂ m⁻² y⁻¹ from the floors. Combining our findings with published data of aquaculture land use in Indonesia, we estimated that shrimp ponds in this region result in CO₂ emissions to the atmosphere between 5.76 and 13.95 Tg y⁻¹. The results indicate that conversion of mangrove forests to aquaculture ponds contributes to greenhouse gas emissions that are comparable to peat forest conversion to other land uses in Indonesia. Higher magnitudes of CO₂ emission may be released to atmosphere where ponds are constructed in newly cleared mangrove forests. This study indicates the need for incentives that can meet the target of aquaculture industry without expanding the converted mangrove areas, which will lead to increased CO₂ released to atmosphere.

The Transfer Achievement Program (TAP): Information Packet.

ERIC Educational Resources Information Center

Segura, Armando; Noseworthy, Victoria

Santa Barbara City College (California) created the Transfer Achievement Program (TAP) to deliver an integrated and cohesive set of services to underrepresented students to help increase their transfer rate to four-year institutions. TAP provides students with a developmental map of transfer-related activities through the use of the Transfer Task…

Fixture For Drilling And Tapping A Curved Workpiece

NASA Technical Reports Server (NTRS)

Espinosa, P. S.; Lockyer, R. T.

1992-01-01

Simple fixture guides drilling and tapping of holes in prescribed locations and orientations on workpiece having curved surface. Tool conceived for use in reworking complexly curved helicopter blades made of composite materials. Fixture is block of rigid foam with epoxy filler, custom-fitted to surface contour, containing bushings and sleeves at drilling and tapping sites. Bushings changed, so taps and drills of various sizes accommodated. In use, fixture secured to surface by hold-down bolts extending through sleeves and into threads in substrate.

Association of Cholesterol Efflux Capacity With Clinical Features of Metabolic Syndrome: Relevance to Atherosclerosis.

PubMed

Gall, Julie; Frisdal, Eric; Bittar, Randa; Le Goff, Wilfried; Bruckert, Eric; Lesnik, Philippe; Guerin, Maryse; Giral, Philippe

2016-11-23

The contribution of high-density lipoprotein to cardiovascular benefit is closely linked to its role in the cellular cholesterol efflux process; however, various clinical and biochemical variables are known to modulate the overall cholesterol efflux process. The aim of this study was to evaluate the extent to which clinical and biological anomalies associated with the establishment of the metabolic syndrome modulate cholesterol efflux capacity and contribute to development of atherosclerosis. This study involved patients (n=1202) displaying atherogenic dyslipidemia in primary prevention who were referred to our prevention center. Among these patients, 25% presented at least 3 criteria of the metabolic syndrome, as defined by the National Cholesterol Education Program Adult Treatment Panel III. We measured the capacity of 40-fold diluted serum to mediate cholesterol efflux from cholesterol-loaded human THP-1 macrophages. Cholesterol efflux capacity was reduced progressively by 4% to 11% (P<0.0001) as a function of the increasing number of coexisting criteria for the metabolic syndrome from 1 to 5. This observation was primarily related to reductions in scavenger receptor class B member 1 and ATP binding cassette subfamily G member 1-dependent efflux. Multivariate analyses indicate that serum efflux capacity was significantly associated with established metabolic syndrome (odds ratio 0.45; 95% CI 0.28-0.72; P=0.009) independent of age, low-density lipoprotein cholesterol, status with regard to lipid-lowering therapy, smoking status, and alcohol consumption. Our study revealed that individual criteria of metabolic syndrome are closely related synergistically to cholesterol efflux capacity. In addition, established metabolic syndrome and cholesterol efflux capacity were independently associated with clinical features of atherosclerosis. © 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

Variability in soil CO2 efflux across distinct urban land cover types

NASA Astrophysics Data System (ADS)

Weissert, Lena F.; Salmond, Jennifer A.; Schwendenmann, Luitgard

2015-04-01

As a main source of greenhouse gases urban areas play an important role in the global carbon cycle. To assess the potential role of urban vegetation in mitigating carbon emissions we need information on the magnitude of biogenic CO2 emissions and its driving factors. We examined how urban land use types (urban forest, parklands, sportsfields) vary in their soil CO2 efflux. We measured soil CO2 efflux and its isotopic signature, soil temperature and soil moisture over a complete growing season in Auckland, New Zealand. Soil physical and chemical properties and vegetation characteristics were also measured. Mean soil CO2 efflux ranged from 4.15 to 12 μmol m-2 s-1. We did not find significant differences in soil CO2 efflux among land cover types due to high spatial variability in soil CO2 efflux among plots. Soil (soil carbon and nitrogen density, texture, soil carbon:nitrogen ratio) and vegetation characteristics (basal area, litter carbon density, grass biomass) were not significantly correlated with soil CO2 efflux. We found a distinct seasonal pattern with significantly higher soil CO2 efflux in autumn (Apr/May) and spring (Oct). In urban forests and sportsfields over 80% of the temporal variation was explained by soil temperature and soil water content. The δ13C signature of CO2 respired from parklands and sportsfields (-20 permil - -25 permil) were more positive compared to forest plots (-29 permil) indicating that parkland and sportsfields had a considerable proportion of C4 grasses. Despite the large intra-urban variability, our results compare to values reported from other, often climatically different cities, supporting the hypothesis of homogenization across urban areas as a result of human management practices.

Vertical gradients and seasonal variation in stem CO2 efflux within a Norway spruce stand.

PubMed

Tarvainen, Lasse; Räntfors, Mats; Wallin, Göran

2014-05-01

Stem CO2 efflux is known to vary seasonally and vertically along tree stems. However, annual tree- and stand-scale efflux estimates are commonly based on measurements made only a few times a year, during daytime and at breast height. In this study, the effect of these simplifying assumptions on annual efflux estimates and their influence on the estimates of the importance of stems in stand-scale carbon cycling are evaluated. In order to assess the strength of seasonal, diurnal and along-stem variability in CO2 efflux, half-hourly measurements were carried out at three heights on three mature Norway spruce (Picea abies (L.) Karst.) trees over a period of 3 years. Making the common assumption of breast height efflux rates being representative of the entire stem was found to result in underestimations of 10-17% in the annual tree-scale CO2 efflux. Upscaling using only daytime measurements from breast height increased the underestimation to 15-20%. Furthermore, the results show that the strength of the vertical gradient varies seasonally, being strongest in the early summer and non-existent during the cool months. The observed seasonality in the vertical CO2 efflux gradient could not be explained by variation in stem temperature, temperature response of the CO2 efflux (Q10), outer-bark permeability, CO2 transport in the xylem or CO2 release from the phloem. However, the estimated CO2 concentration immediately beneath the bark was considerably higher in the upper stem during the main period of diameter growth, coinciding with the strongest vertical efflux gradient. These results suggest that higher growth rates in the upper stem are the main cause for the observed vertical variation in the stem CO2 effluxes. Furthermore, the results indicate that accounting for the vertical efflux variation is essential for assessments of the importance of stems in stand-scale carbon cycling. © The Author 2014. Published by Oxford University Press. All rights reserved.

Temporal and Spatial Variations in Soil CO2 Effluxes of Different Ecosystems

NASA Astrophysics Data System (ADS)

Liang, N.; Kim, S.; Shimoyama, K.; Kim, Y.; Hirano, T.; Takagi, K.; Suto, H.; Fujinuma, Y.; Inoue, G.

2005-12-01

Regional networks for measuring carbon sequestration or loss by terrestrial ecosystems on a year round basis have been in operation since the mid-1990s. However, continuous measurements of soil CO2 efflux, the largest component of ecosystem respiration have only been reported over similar time scales at a few of the sites. Reasons include the lack of automated measurement systems that are commercially available, and the need for frequent servicing to ensure accurate measurements. We have developed a multichannel automated chamber system that can be used for continuous measuring soil CO2 efflux during snow-free seasons. We installed the chamber systems in boreal forest in Alaska, tundra in west Siberia, temperate and cool-temperate forests in Japan and Korea, tropical seasonal forest in Thailand, and tropical rainforest in Malaysia. Annual soil CO2 efflux were measured to be about 5-6 tC ha-1 y-1 in the boreal and cool-temperate forests, 10 tC ha-1 y-1 in the temperate forests, and 26 tC ha-1 y-1 in the tropical rainforests. Efflux showed significant seasonality in the boreal and temperate forest that corresponding with the seasonal soil temperature. However, the wavelike efflux rates in the tropical forests were correlated with the seasonality of soil moisture. Soil CO2 efflux of forest ecosystems showed large spatial variation and was correlated with vegetation type and the chamber size.

Efflux proteins at the blood-brain barrier: review and bioinformatics analysis.

PubMed

Saidijam, Massoud; Karimi Dermani, Fatemeh; Sohrabi, Sareh; Patching, Simon G

2018-05-01

1. Efflux proteins at the blood-brain barrier provide a mechanism for export of waste products of normal metabolism from the brain and help to maintain brain homeostasis. They also prevent entry into the brain of a wide range of potentially harmful compounds such as drugs and xenobiotics. 2. Conversely, efflux proteins also hinder delivery of therapeutic drugs to the brain and central nervous system used to treat brain tumours and neurological disorders. For bypassing efflux proteins, a comprehensive understanding of their structures, functions and molecular mechanisms is necessary, along with new strategies and technologies for delivery of drugs across the blood-brain barrier. 3. We review efflux proteins at the blood-brain barrier, classified as either ATP-binding cassette (ABC) transporters (P-gp, BCRP, MRPs) or solute carrier (SLC) transporters (OATP1A2, OATP1A4, OATP1C1, OATP2B1, OAT3, EAATs, PMAT/hENT4 and MATE1). 4. This includes information about substrate and inhibitor specificity, structural organisation and mechanism, membrane localisation, regulation of expression and activity, effects of diseases and conditions and the principal technique used for in vivo analysis of efflux protein activity: positron emission tomography (PET). 5. We also performed analyses of evolutionary relationships, membrane topologies and amino acid compositions of the proteins, and linked these to structure and function.

Wood CO2 efflux and foliar respiration for Eucalyptus in Hawaii and Brazil

Treesearch

Michael G. Ryan; Molly A. Cavaleri; Auro C. Almeida; Ricardo Penchel; Randy S. Senock; Jose Luiz Stape

2009-01-01

We measured CO2 efflux from wood for Eucalyptus in Hawaii for 7 years and compared these measurements with those on three- and four-and-a-halfyear- old Eucalyptus in Brazil. In Hawaii, CO2 efflux from wood per unit biomass declined ~10x from age two to age five, twice as much as the decline in tree growth. The CO2 efflux from wood in Brazil was 8-10· lower than that...

Oxaloacetate- and acetoacetate-induced calcium efflux from mitochondria occurs by reversal of the uptake pathway.

PubMed Central

Bardsley, M E; Brand, M D

1982-01-01

1. Addition of oxaloacetate or acetoacetate to isolated rat liver mitochondria results in an efflux of Ca2+. Concomitant with this efflux is an immediate oxidation of endogenous nicotinamide nucleotides, a fall in the mitochondrial membrane potential and an increase in the rate of respiration. The primary effect in this sequence may be either (a) physiologically important stimulation of a Ca2+-efflux carrier, followed by Ca2+ re-uptake, a fall in membrane potential and increased respiration, or (b) physiologically unimportant damage to mitochondrial integrity, followed by a fall in membrane potential, increased respiration and Ca2+ efflux. 2. Ruthenium Red and EGTA will restore the increased respiratory rate to one approximating to the control rate of respiration. However, addition of lanthanide, at a concentration which inhibits the uptake but not the normal efflux of Ca2+, inhibits the rate of Ca2+ efflux induced by oxaloacetate or acetoacetate. Therefore the observed efflux is occurring by a reversal of the uptake pathway (uniporter) and thus follows the fall in membrane potential. 3. From these results we conclude that the decrease in membrane potential and increase in the rate of respiration seen during oxaloacetate- or acetoacetate-induced Ca2+ efflux cannot be accounted for by rapid Ca2+ cycling, but are due to damage to mitochondrial integrity. PMID:7082307

Tapping the Sugar Maple--Learning and Appreciating

ERIC Educational Resources Information Center

Malone, Charles

1976-01-01

The article discusses how to tap a maple tree. Tapping a maple tree to produce maple syrup can: (1) lead to better understanding in many subject areas, (2) develop skills through participation in a rewarding activity, and (3) help students appreciate the many important roles that trees play in our environment and daily lives. (NQ)

Paired Synchronous Rhythmic Finger Tapping without an External Timing Cue Shows Greater Speed Increases Relative to Those for Solo Tapping

PubMed Central

Okano, Masahiro; Shinya, Masahiro; Kudo, Kazutoshi

2017-01-01

In solo synchronization-continuation (SC) tasks, intertap intervals (ITI) are known to drift from the initial tempo. It has been demonstrated that people in paired and group contexts modulate their action timing unconsciously in various situations such as choice reaction tasks, rhythmic body sway, and hand clapping in concerts, which suggests the possibility that ITI drift is also affected by paired context. We conducted solo and paired SC tapping experiments with three tempos (75, 120, and 200 bpm) and examined whether tempo-keeping performance changed according to tempo and/or the number of players. Results indicated that those tapping in the paired conditions were faster, relative to those observed in the solo conditions, for all tempos. For the faster participants, the degree of ITI drift in the solo conditions was strongly correlated with that in the paired conditions. Regression analyses suggested that both faster and slower participants adapted their tap timing to that of their partners. A possible explanation for these results is that the participants reset the phase of their internal clocks according to the faster beat between their own tap and the partners’ tap. Our results indicated that paired context could bias the direction of ITI drift toward decreasing. PMID:28276461

Paired Synchronous Rhythmic Finger Tapping without an External Timing Cue Shows Greater Speed Increases Relative to Those for Solo Tapping.

PubMed

Okano, Masahiro; Shinya, Masahiro; Kudo, Kazutoshi

2017-03-09

In solo synchronization-continuation (SC) tasks, intertap intervals (ITI) are known to drift from the initial tempo. It has been demonstrated that people in paired and group contexts modulate their action timing unconsciously in various situations such as choice reaction tasks, rhythmic body sway, and hand clapping in concerts, which suggests the possibility that ITI drift is also affected by paired context. We conducted solo and paired SC tapping experiments with three tempos (75, 120, and 200 bpm) and examined whether tempo-keeping performance changed according to tempo and/or the number of players. Results indicated that those tapping in the paired conditions were faster, relative to those observed in the solo conditions, for all tempos. For the faster participants, the degree of ITI drift in the solo conditions was strongly correlated with that in the paired conditions. Regression analyses suggested that both faster and slower participants adapted their tap timing to that of their partners. A possible explanation for these results is that the participants reset the phase of their internal clocks according to the faster beat between their own tap and the partners' tap. Our results indicated that paired context could bias the direction of ITI drift toward decreasing.

Microenvironment acidity as a major determinant of tumor chemoresistance: Proton pump inhibitors (PPIs) as a novel therapeutic approach.

PubMed

Taylor, Sophie; Spugnini, Enrico Pierluigi; Assaraf, Yehuda G; Azzarito, Tommaso; Rauch, Cyril; Fais, Stefano

2015-11-01

Despite the major progresses in biomedical research and the development of novel therapeutics and treatment strategies, cancer is still among the dominant causes of death worldwide. One of the crucial challenges in the clinical management of cancer is primary (intrinsic) and secondary (acquired) resistance to both conventional and targeted chemotherapeutics. Multiple mechanisms have been identifiedthat underlie intrinsic and acquired chemoresistance: these include impaired drug uptake, increased drug efflux, deletion of receptors, altered drug metabolism, quantitative and qualitative alterations in drug targets, increased DNA damage repair and various mechanisms of anti-apoptosis. The fast efflux of anticancer drugs mediated by multidrug efflux pumps and the partial or complete reversibility of chemoresistance combined with the absence of genetic mutations suggests a multifactorial process. However, a growing body of recent evidence suggests that chemoresistance is often triggered by the highly acidic microenvironment of tumors. The vast majority of drugs, including conventional chemotherapeutics and more recent biological agents, are weak bases that are quickly protonated and neutralized in acidic environments, such as the extracellular microenvironment and the acidic organelles of tumor cells. It is therefore essential to develop new strategies to overcome the entrapment and neutralization of weak base drugs. One such strategy is the use of proton pump inhibitors which can enhance tumor chemosensitivity by increasing the pH of the tumor microenvironment. Recent clinical trials in animals with spontaneous tumors have indicated that patient alkalization is capable of reversing acquired chemoresistance in a large percentage of tumors that are refractory to chemotherapy. Of particular interest was the benefit of alkalization for patients undergoing metronomic regimens which are becoming more widely used in veterinary medicine. Overall, these results provide

A sensitive assay for ABCA1-mediated cholesterol efflux using BODIPY -cholesterol

USDA-ARS?s Scientific Manuscript database

Studies have shown a negative association between cellular cholesterol efflux and coronary artery disease (CAD). Standard protocol for quantifying cholesterol efflux involves labeling cells with [(3)H]cholesterol and measuring release of the labeled sterol. Using [(3)H]cholesterol is not ideal for...

Technical Assistance Plan (TAP)

EPA Pesticide Factsheets

A Technical Assistance Plan (TAP) enables community groups to retain the services of an independent technical advisor and to provide resources for a community group to help inform other community members about site decisions.

Analgesic Effect Of Bilateral Subcostal Tap Block After Laparoscopic Cholecystectomy.

PubMed

Khan, Karima Karam; Khan, Robyna Irshad

2018-01-01

Pain after laparoscopic cholecystectomy is mild to moderate in intensity. Several modalities are employed for achieving safe and effective postoperative analgesia, the benefits of which adds to the early recovery of the patients. As a part of multimodal analgesia, various approaches of Transversus abdominis plane (TAP) block has been used for management of parietal and incisional components of pain after laparoscopic cholecystectomy. This study was designed to compare the analgesic efficacy of two different approaches of ultrasound guided TAP block, i.e., Subcostal-TAP block technique with ultrasound guided Posterior-TAP block for postoperative pain management in patients undergoing laparoscopic cholecystectomy under general anaesthesia. In this double blinded randomized controlled study, consecutive nonprobability sampling was done and a total of 126 patients admitted for elective laparoscopic cholecystectomy fulfilling the inclusion criteria were selected. After induction of general anaesthesia, patients were randomized through draw method and received either ultrasound guided posterior TAP block with 0.375% bupivacaine (20ml volume) on each side of the abdomen or subcostal TAP block bilaterally with the same. Up to 24 hours postoperatively, static and dynamic numeric rating pain scores were assessed. We found statistically significant difference in mean static pain scores over 24 hours postoperatively in subcostal TAP group, suggesting improved analgesia. However, mean dynamic postoperative pain scores were comparable between the two groups. Whereas, patients in both groups were satisfied with pain management. Ultrasound guided subcostal TAP block provides better postoperative analgesia as compared to the Posterior TAP block in laparoscopic cholecystectomy. Otherwise both of the approaches improve patient outcomes towards early recovery and discharge from hospital.

The neural substrates of impaired finger tapping regularity after stroke.

PubMed

Calautti, Cinzia; Jones, P Simon; Guincestre, Jean-Yves; Naccarato, Marcello; Sharma, Nikhil; Day, Diana J; Carpenter, T Adrian; Warburton, Elizabeth A; Baron, Jean-Claude

2010-03-01

Not only finger tapping speed, but also tapping regularity can be impaired after stroke, contributing to reduced dexterity. The neural substrates of impaired tapping regularity after stroke are unknown. Previous work suggests damage to the dorsal premotor cortex (PMd) and prefrontal cortex (PFCx) affects externally-cued hand movement. We tested the hypothesis that these two areas are involved in impaired post-stroke tapping regularity. In 19 right-handed patients (15 men/4 women; age 45-80 years; purely subcortical in 16) partially to fully recovered from hemiparetic stroke, tri-axial accelerometric quantitative assessment of tapping regularity and BOLD fMRI were obtained during fixed-rate auditory-cued index-thumb tapping, in a single session 10-230 days after stroke. A strong random-effect correlation between tapping regularity index and fMRI signal was found in contralesional PMd such that the worse the regularity the stronger the activation. A significant correlation in the opposite direction was also present within contralesional PFCx. Both correlations were maintained if maximal index tapping speed, degree of paresis and time since stroke were added as potential confounds. Thus, the contralesional PMd and PFCx appear to be involved in the impaired ability of stroke patients to fingertap in pace with external cues. The findings for PMd are consistent with repetitive TMS investigations in stroke suggesting a role for this area in affected-hand movement timing. The inverse relationship with tapping regularity observed for the PFCx and the PMd suggests these two anatomically-connected areas negatively co-operate. These findings have implications for understanding the disruption and reorganization of the motor systems after stroke. Copyright (c) 2009 Elsevier Inc. All rights reserved.

Multidrug efflux transporter activity in sea urchin embryos:Does localization provide a diffusive advantage?

NASA Astrophysics Data System (ADS)

Song, Xianfeng; Setayeshgar, Sima; Cole, Bryan; Hamdoun, Amro; Epel, David

2008-03-01

Experiments have shown upregulation of multidrug efflux transporter activity approximately 30 min after fertilization in the sea urchin embryo [1]. These ATP-hydrolyzing transporter proteins pump moderately hydrophobic molecules out of the cell and represent the cell's first line of defense againstexogenous toxins. It has also been shown that transporters are moved in vesicles along microfilaments and localized to tips of microvilli prior to activation. We have constructed a geometrically realistic model of the embryo, including microvilli, to explore the functional role of this localization in the efficient elimination of toxins from the standpoint of diffusion. We compute diffusion of toxins in extracellular, membrane and intracellular spaces coupled with transporter activity, using experimentally derived values for physical parameters. For transporters uniformly distributed along microvilli and tip-localized transporters we compare regions in parameter space where each distribution provides diffusive advantage, and comment on the physically expected conditions. [1] A. M. Hamdoun, G. N. Cherr, T. A. Roepke and D. Epel, Developmental Biology 276 452 (2004).

Hot tap thermowell installation

NASA Technical Reports Server (NTRS)

Romero, C. A.

1971-01-01

System permits valve housings or other fillings to be installed in live steam lines or water pipes without interrupting their operation, thus eliminating current tapping restrictions. Two basic assemblies for installation under pressure are described.

Hematological characteristics in neonates with twin anemia-polycythemia sequence (TAPS).

PubMed

Lopriore, E; Slaghekke, F; Oepkes, D; Middeldorp, J M; Vandenbussche, F P H A; Walther, F J

2010-03-01

To evaluate the neonatal hematological features of monochorionic twins with twin anemia-polycythemia sequence (TAPS) and to determine the additional diagnostic value of reticulocyte count measurement. A cohort of consecutive monochorionic twins with TAPS (n = 19) was included in the study and each twin pair was compared with two monochorionic twin pairs (n = 38) unaffected by TAPS or twin-twin transfusion syndrome (TTTS), matched for gestational age at birth. We measured full blood counts on day 1 and determined the incidence of anemia, polycythemia, reticulocytosis and thrombocytopenia. Median inter-twin hemoglobin (Hb) difference in monochorionic twins with and without TAPS was 13.7 g/dL and 2.4 g/dL, respectively (p < 0.01). Median inter-twin reticulocyte count ratio in twins with and without TAPS was 3.1 and 1.0, respectively (p < 0.01). Thrombocytopenia (platelet count < 150 x 10(9)/L) occurred more often in the TAPS group than in the control group, 45% (17/38) versus 11% (11/38), respectively (p < 0.01). In the TAPS group, mean platelet count was significantly lower in recipients than in donors, 133 x 10(9)/L versus 218 x 10(9)/L, respectively (p < 0.01). TAPS twins have a large inter-twin Hb difference in combination with a large inter-twin reticulocyte count ratio. Recipients are more often thrombocytopenic than donors, probably due to polycythemia. Copyright (c) 2010 John Wiley & Sons, Ltd.

Astronomy on Tap: science engagement in the pub

NASA Astrophysics Data System (ADS)

Livermore, Rachael C.; Silverman, Jeffrey Michael

2015-08-01

Astronomy on Tap is a series of free lectures by astronomers in the pub, aimed at disseminating the latest research to the public in an informal setting. Started in New York City in 2013, Astronomy on Tap has now expanded to seven cities across North and South America. Organized by local astronomers, each event features talks by astronomers from local institutions or visitors, or others whose professions or hobbies intersect with astronomy, along with games and opportunities for the public to interact with professional astronomers. The largest Astronomy on Tap events are in Austin, Texas, attracting over 150 people each month, which consists of populations outside of the self-selected groups that might be reached by more formal EPO activities. The organisers of Astronomy on Tap in Austin (AoTATX) will discuss the impact of and feedback from all of the locations, and present information on setting up new satellite locations.

Altered Expression of TAP-1 and Major Histocompatibility Complex Class I in Laryngeal Papillomatosis: Correlation of TAP-1 with Disease

PubMed Central

Vambutas, Andrea; Bonagura, Vincent R.; Steinberg, Bettie M.

2000-01-01

Recurrent respiratory papillomatosis (RRP) is an insidious disease caused by human papillomavirus (HPV) infection. It is characterized by a variable clinical course that can include frequent disease recurrence, significant morbidity, and occasional mortality. The mechanisms responsible for the variability in the clinical course and the persistence of latent HPV infection remain unknown. Effective T-cell-mediated clearance of HPV-infected cells may be defective in patients with RRP, leading to recurrent disease and failure to suppress latent HPV reactivation. This study describes the down-regulation of the transporter associated with antigen presentation (TAP-1) and the major histocompatibility complex (MHC) class I protein expression in laryngeal papilloma tissue biopsies and cell culture of primary explants. There was a statistically significant correlation between reduction of TAP-1 expression in biopsy tissues and rapid recurrence of disease. Patients with RRP had less frequent recurrence if their papillomas expressed TAP-1 at levels close to that of normal tissue, compared with those with very low expression of TAP-1, who had frequent recurrence (32 versus 5 weeks to the next surgical intervention). These findings suggest that HPV may evade immune recognition by down-regulating class I MHC cell surface expression via decreased TAP-1 levels. Expression of TAP-1 could be used for prognostic evaluation of disease severity. Gamma interferon was able to restore class I MHC expression at the surfaces of laryngeal papilloma cells in culture. This up-regulation of class I MHC antigen at the cell surface potentially allows the infected cell to become a target for the immune system again. This finding provides some promise for nonsurgical treatment of laryngeal papillomas. PMID:10618282

Transversus abdominal plane (TAP) block for postoperative pain management: a review.

PubMed

Jakobsson, Jan; Wickerts, Liselott; Forsberg, Sune; Ledin, Gustaf

2015-01-01

Transversus abdominal plane (TAP) block has a long history and there is currently extensive clinical experience around TAP blocks. The aim of this review is to provide a summary of the present evidence on the effects of TAP block and to provide suggestions for further studies. There are several approaches to performing abdominal wall blocks, with the rapid implementation of ultrasound-guided technique facilitating a major difference in TAP block performance. During surgery, an abdominal wall block may also be applied by the surgeon from inside the abdominal cavity. Today, there are more than 11 meta-analyses providing a compiled evidence base around the effects of TAP block. These analyses include different procedures, different techniques of TAP block administration and, importantly, they compare the TAP block with a variety of alternative analgesic regimes. The effects of TAP block during laparoscopic cholecystectomy seem to be equivalent to local infiltration analgesia and also seem to be beneficial during laparoscopic colon resection. The effects of TAP are more pronounced when it is provided prior to surgery and these effects are local anaesthesia dose-dependent. TAP block seems an interesting alternative in patients with, for example, severe obesity where epidural or spinal anaesthesia/analgesia is technically difficult and/or poses a risk. There is an obvious need for further high-quality studies comparing TAP block prior to surgery with local infiltration analgesia, single-shot spinal analgesia, and epidural analgesia. These studies should be procedure-specific and the effects should be evaluated, both regarding short-term pain and analgesic requirement and also including the effects on postoperative nausea and vomiting, recovery of bowel function, ambulation, discharge, and protracted recovery outcomes (assessed by e.g., postoperative quality of recovery scale).

Antibacterial activity of berberine-NorA pump inhibitor hybrids with a methylene ether linking group.

PubMed

Samosorn, Siritron; Tanwirat, Bongkot; Muhamad, Nussara; Casadei, Gabriele; Tomkiewicz, Danuta; Lewis, Kim; Suksamrarn, Apichart; Prammananan, Therdsak; Gornall, Karina C; Beck, Jennifer L; Bremner, John B

2009-06-01

Conjugation of the NorA substrate berberine and the NorA inhibitor 5-nitro-2-phenyl-1H-indole via a methylene ether linking group gave the 13-substituted berberine-NorA inhibitor hybrid, 3. A series of simpler arylmethyl ether hybrid structures were also synthesized. The hybrid 3 showed excellent antibacterial activity (MIC Staphylococcus aureus, 1.7 microM), which was over 382-fold more active than the parent antibacterial berberine, against this bacterium. This compound was also shown to block the NorA efflux pump in S. aureus.

Computerized measures of finger tapping: effects of hand dominance, age, and sex.

PubMed

Hubel, Kerry A; Reed, Bruce; Yund, E William; Herron, Timothy J; Woods, David L

2013-06-01

Computerized measures of digit tapping rate were obtained over 3 successive, 10-sec. periods in the right and left index fingers, from a community sample of 1,519 participants (ages 18 to 65 years; 607 men, 912 women). Differences between the dominant and non-dominant hands were found for tapping rate, movement initiation, and button down times, and the decline in tapping rate over the successive, 10-sec. periods. Declines were found in tapping rate in older participants in association with increased intertap variability. Men had higher tapping rates than women in all age ranges. The computerized finger tapping test is an efficient and precise measure of tapping speed and kinetics of potential utility in research and clinical studies of motor performance.

Probing and Tapping: Are We Inserting Pedicle Screws Correctly?

PubMed

Prasad, Vishal; Mesfin, Addisu; Lee, Robert; Reigrut, Julie; Schmidt, John

2016-11-01

Although there are a significant number of research publications on the topic of bone morphology and the strength of bone, the clinical significance of a failed pedicle screw is often revision surgery and the potential for further postoperative complications; especially in elderly patients with osteoporotic bone. The purpose of this report is to quantify the mechanical strength of the foam-screw interface by assessing probe/pilot hole diameter and tap sizes using statistically relevant sample sizes under highly controlled test conditions. The study consisted of two experiments and used up to three different densities of reference-grade polyurethane foam (ASTM 1839), including 0.16, 0.24, and 0.32 g/cm 3 . All screws and rods were provided by K2M Inc. and screws were inserted to a depth of 25 mm. A series of pilot holes, 1.5, 2.2, 2.7, 3.2, 3.7, 4.2, 5.0, and 6.0 mm in diameter were drilled through the entire depth of the material. A 6.5 × 45-mm pedicle screw was inserted and axially pulled from the material (n = 720). A 3.0-mm pilot hole was drilled and tapped with: no tap, 3.5-, 4.5-, 5.5-, and 6.5-mm taps. A 6.5 × 45-mm pedicle screw was inserted and axially pulled from the material (n = 300). The size of the probe/pilot hole had a nonlinear, parabolic effect on pullout strength. This shape suggests an optimum-sized probe hole for a given size pedicle screw. Too large or too small of a probe hole causes a rapid falloff in pullout strength. The tap data demonstrated that not tapping and undertapping by two or three sizes did not significantly alter the pullout strength of the screws. The data showed an exponential falloff of pullout strength when as tap size increased to the diameter of the screw. In the current study, the data show that an ideal pilot hole size half the diameter of the screw is a starting point. Also, that if tapping was necessary, to use a tap two sizes smaller than the screw being implanted. A similar optimum pilot hole or tap size may be

Efflux of hydraulically lifted water from mycorrhizal fungal hyphae during imposed drought

PubMed Central

Querejeta, José Ignacio; Allen, Michael F

2008-01-01

Apart from improving plant and soil water status during drought, it has been suggested that hydraulic lift (HL) could enhance plant nutrient capture through the flow of mineral nutrients directly from the soil to plant roots, or by maintaining the functioning of mycorrhizal fungi. We evaluated the extent to which the diel cycle of water availability created by HL covaries with the efflux of HL water from the tips of extramatrical (external) mycorrhizal hyphae, and the possible effects on biogeochemical processes. Phenotypic mycorrhizal fungal variables, such as total and live hyphal lengths, were positively correlated with HL efflux from hyphae, soil water potential (dawn), and plant response variables (foliar 15N). The efflux of HL water from hyphae was also correlated with bacterial abundance and soil enzyme activity (P), and the moistening of soil organic matter. Such findings indicate that the efflux of HL water from the external mycorrhizal mycelia may be a complementary explanation for plant nutrient acquisition and survival during drought. PMID:19704776

Oral and inhaled corticosteroids: Differences in P-glycoprotein (ABCB1) mediated efflux

DOE Office of Scientific and Technical Information (OSTI.GOV)

Crowe, Andrew, E-mail: a.p.crowe@curtin.edu.au; Tan, Ai May

There is concern that P-glycoprotein mediated efflux contributes to steroid resistance. Therefore, this study examined bidirectional corticosteroid transport and induction capabilities for P-glycoprotein (P-gp) to understand which of the systemic and inhaled corticosteroids interacted with P-gp to the greatest extent. Hydrocortisone, prednisolone, prednisone, methylprednisolone, and dexamethasone represented systemically active drugs, while fluticasone propionate, beclomethasone dipropionate, ciclesonide and budesonide represented inhaled corticosteroids. Aldosterone and fludrocortisone represented mineralocorticoids. All drugs were detected using individually optimised HPLC protocols. Transport studies were conducted through Caco-2 monolayers. Hydrocortisone and aldosterone had efflux ratios below 1.5, while prednisone showed a P-gp mediated efflux ratio of onlymore » 1.8 compared to its active drug, prednisolone, with an efflux ratio of 4.5. Dexamethasone and beclomethasone had efflux ratios of 2.1 and 3.3 respectively, while this increased to 5.1 for methylprednisolone. Fluticasone showed an efflux ratio of 2.3. Protein expression studies suggested that all of the inhaled corticosteroids were able to induce P-gp expression, from 1.6 to 2 times control levels. Most of the systemic corticosteroids had higher passive permeability (> 20 × 10{sup −6} cm/s) compared to the inhaled corticosteroids (> 5 × 10{sup −6} cm/s), except for budesonide, with permeability similar to the systemic corticosteroids. Inhaled corticosteroids are not transported by P-gp to the same extent as systemic corticosteroids. However, they are able to induce P-gp production. Thus, inhaled corticosteroids may have greater interactions with other P-gp substrates, but P-gp itself is less likely to influence resistance to the drugs. -- Highlights: ► Inhaled corticosteroids are only weak substrates for P-gp, including budesonide. ► Inhaled corticosteroid potent P-gp inducers especially

Vertical profile of branch CO2 efflux in a Norway spruce tree: a case study

NASA Astrophysics Data System (ADS)

Acosta, M.; Pavelka, M.

2012-04-01

Despite woody-tissue CO2 effluxes having been recognized as an important component of forest carbon budget due to the fraction of assimilates used and the dramatic increase in woody with stand development, there is limited research to determine the CO2 efflux vertical variability of woody-tissue components. For a better understanding and quantification of branch woody-tissue CO2 efflux in forest ecosystems, it is necessary to identify the environmental factors influencing it and the role of the branch distribution within the canopy. The proper assessment of this forest component will improve the knowledge of the ratio between ecosystem respiration and gross primary production at forest ecosystem. In order to achieve this goal, branch CO2 efflux of Norway spruce tree was measured in ten branches at five different whorls during the growing season 2004 (from June till October) in campaigns of 3-4 times per month at the Beskydy Mts., the Czech Republic, using a portable infrared gas analyzer operating as a closed system. Branch woody tissue temperature was measured continuously in ten minutes intervals for each sample position during the whole experiment period. On the basis of relation between CO2 efflux rate and woody tissue temperature a value of Q10 and normalized CO2 efflux rate (E10 - CO2 efflux rate at 10° C) were calculated for each sampled position. Estimated Q10 values ranged from 2.12 to 2.89 and E10 ranged from 0.41 to 1.19 ?molCO2m-2 s-1. Differences in branch CO2 efflux were found between orientations; East side branches presented higher efflux rate than west side branches. The highest branch CO2 efflux rate values were measured in August and the lowest in October, which were connected with woody tissue temperature and ontogenetic processes during these periods. Branch CO2 efflux was significantly and positively correlated with branch position within canopy and woody tissue temperature. Branches from the upper whorls showed higher respiration activity

Synchronized tapping facilitates learning sound sequences as indexed by the P300.

PubMed

Kamiyama, Keiko S; Okanoya, Kazuo

2014-01-01

The purpose of the present study was to determine whether and how single finger tapping in synchrony with sound sequences contributed to the auditory processing of them. The participants learned two unfamiliar sound sequences via different methods. In the tapping condition, they learned an auditory sequence while they tapped in synchrony with each sound onset. In the no tapping condition, they learned another sequence while they kept pressing a key until the sequence ended. After these learning sessions, we presented the two melodies again and recorded event-related potentials (ERPs). During the ERP recordings, 10% of the tones within each melody deviated from the original tones. An analysis of the grand average ERPs showed that deviant stimuli elicited a significant P300 in the tapping but not in the no-tapping condition. In addition, the significance of the P300 effect in the tapping condition increased as the participants showed highly synchronized tapping behavior during the learning sessions. These results indicated that single finger tapping promoted the conscious detection and evaluation of deviants within the learned sequences. The effect was related to individuals' musical ability to coordinate their finger movements along with external auditory events.

Synchronized tapping facilitates learning sound sequences as indexed by the P300

PubMed Central

Kamiyama, Keiko S.; Okanoya, Kazuo

2014-01-01

The purpose of the present study was to determine whether and how single finger tapping in synchrony with sound sequences contributed to the auditory processing of them. The participants learned two unfamiliar sound sequences via different methods. In the tapping condition, they learned an auditory sequence while they tapped in synchrony with each sound onset. In the no tapping condition, they learned another sequence while they kept pressing a key until the sequence ended. After these learning sessions, we presented the two melodies again and recorded event-related potentials (ERPs). During the ERP recordings, 10% of the tones within each melody deviated from the original tones. An analysis of the grand average ERPs showed that deviant stimuli elicited a significant P300 in the tapping but not in the no-tapping condition. In addition, the significance of the P300 effect in the tapping condition increased as the participants showed highly synchronized tapping behavior during the learning sessions. These results indicated that single finger tapping promoted the conscious detection and evaluation of deviants within the learned sequences. The effect was related to individuals’ musical ability to coordinate their finger movements along with external auditory events. PMID:25400564

Military Transition Assistance Program (TAP): An Overview

DTIC Science & Technology

2017-03-15

Title II of P.L. 112-56) which made a pre-separation counseling program mandatory for all servicemembers who have served at least 180 continuous days...anticipated retirement date or 12-month period preceding the anticipated separation date. It also specifies that pre-separation counseling should...in the development, management oversight, and strategic planning of TAP. TAP Counseling Requirements Over time, Congress has increased the

Switch loop flexibility affects substrate transport of the AcrB efflux pump

DOE PAGES

Muller, Reinke T.; Travers, Timothy; Cha, Hi-jea; ...

2017-10-05

The functionally important switch-loop of the trimeric multidrug transporter AcrB separates the access and deep drug binding pockets in every protomer. This loop, comprising 11 amino acid residues, has been shown to be crucial for substrate transport, as drugs have to travel past the loop to reach the deep binding pocket and from there are transported outside the cell via the connected AcrA and TolC channels. It contains four symmetrically arranged glycine residues suggesting that flexibility is a key feature for pump activity. Upon combinatorial substitution of these glycine residues to proline, functional and structural asymmetry was observed. Proline substitutionsmore » on the PC1 proximal side completely abolished transport and reduced backbone flexibility of the switch loop, which adopted a conformation restricting the pathway towards the deep binding pocket. Here, two phenylalanine residues located adjacent to the substitution sensitive glycine residues play a role in blocking the pathway upon rigidification of the loop, since the removal of the phenyl rings from the rigid loop restores drug transport activity.« less

Switch loop flexibility affects substrate transport of the AcrB efflux pump

DOE Office of Scientific and Technical Information (OSTI.GOV)

Muller, Reinke T.; Travers, Timothy; Cha, Hi-jea

The functionally important switch-loop of the trimeric multidrug transporter AcrB separates the access and deep drug binding pockets in every protomer. This loop, comprising 11 amino acid residues, has been shown to be crucial for substrate transport, as drugs have to travel past the loop to reach the deep binding pocket and from there are transported outside the cell via the connected AcrA and TolC channels. It contains four symmetrically arranged glycine residues suggesting that flexibility is a key feature for pump activity. Upon combinatorial substitution of these glycine residues to proline, functional and structural asymmetry was observed. Proline substitutionsmore » on the PC1 proximal side completely abolished transport and reduced backbone flexibility of the switch loop, which adopted a conformation restricting the pathway towards the deep binding pocket. Here, two phenylalanine residues located adjacent to the substitution sensitive glycine residues play a role in blocking the pathway upon rigidification of the loop, since the removal of the phenyl rings from the rigid loop restores drug transport activity.« less

Investigation of Waikele well no 2401-01, Oahu, Hawaii; pumping test, well logs and water quality

USGS Publications Warehouse

Eyre, P.R.

1983-01-01

Field tests indicate that an abandoned well (No. 2401-01) near the confluence of Waikele and Kipapa Streams, Oahu, Hawaii, can be reactivated to produce potable water at a rate of 400-500 gallons per minute. Previous tests in 1946 and 1954 indicated that the well tapped the brackish transition zone which inderlies the Ghyben-Herzberg lens of the Pearl Harbor aquifer. Results of this study, based on geologic and geophysical logs of the wall, as well as on pumping test and water-quality data, indicate that the slightly brackish water produced by the well results from brackish irrigation return water. It does not appear that pumping from this well will cause seawater upconing or intrusion. (USGS)

Eliciting Cervical Vestibular-Evoked Myogenic Potentials by Bone-Conducted Vibration via Various Tapping Sites.

PubMed

Tseng, Chia-Chen; Young, Yi-Ho

2016-01-01

This study compared bone-conducted vibration (BCV) cervical vestibular-evoked myogenic potentials (cVEMPs) via tapping at various skull sites in healthy subjects and patients with vestibular migraine (VM) to optimize stimulation conditions. Twenty healthy subjects underwent a series of cVEMP tests by BCV tapping via a minishaker at the Fz (forehead), Cz (vertex), and inion (occiput) sites in a randomized order of tapping sites. Another 20 VM patients were also enrolled in this study for comparison. All 20 healthy subjects had clear BCV cVEMPs when tapping at the inion (100%) or Cz (100%), but not at the Fz (75%). Mean p13 and n23 latencies from the Cz tapping were significantly longer than those from the Fz tapping, but not longer than those from the inion tapping. Unlike healthy subjects, tapping at the Cz (95%) elicited a significantly higher response rate of present cVEMPs than tapping at the inion (78%) in 20 VM patients (40 ears), because seven of nine VM ears with absent cVEMPs by inion tapping turned out to be present cVEMPs by Cz tapping. While both inion and Cz tapping elicited 100% response rate of cVEMPs for healthy individuals, Cz tapping had a higher response rate of cVEMPs than inion tapping for the VM group. In cases of total loss of saccular function, cVEMPs could not be activated by either inion or Cz tapping. However, if residual saccular function remains, Cz tapping may activate saccular afferents more efficiently than inion tapping.

Physics on Tap

ERIC Educational Resources Information Center

Wheeler, Andrew P. S.

2012-01-01

This article aims to describe how to visualize surface tension effects in liquid jets. A simple experiment is proposed using the liquid jet flow from a mains water tap/faucet. Using a modern digital camera with a high shutter speed, it is possible to visualize the instabilities (capillary waves) that form within the jet due to the action of…

Acanthamoeba keratitis: the role of domestic tap water contamination in the United Kingdom.

PubMed

Kilvington, Simon; Gray, Trevor; Dart, John; Morlet, Nigel; Beeching, John R; Frazer, David G; Matheson, Melville

2004-01-01

The incidence of acanthamoeba keratitis (AK) in the UK is some 15 times that in the United States and seven times that in Holland. To investigate reasons for this higher frequency, a study of the role of domestic tap water as a potential source of AK was undertaken. Tap outlets from the homes of 27 patients with culture-proven AK were sampled and cultured for free-living amoebae (FLA). For all Acanthamoeba isolates, mitochondrial DNA (mtDNA) restriction fragment length polymorphisms (RFLPs) and cytochrome oxidase (cox 1/2) sequence typing was performed to determine the similarity between corneal and tap water isolates. FLA, including Acanthamoeba, were isolated from 24 (89%) of 27 homes, and the presence within the homes varied significantly with tap water temperature and location: 19 (76%) of 25 bathroom sink cold taps sampled compared with 6 (24%) of 25 hot and 9 (47%) of 19 kitchen cold taps compared with 3 (16%) of 19 of hot kitchen taps. Acanthamoeba were isolated from 8 (30%) of 27 homes (five bathroom sink cold taps, one cloakroom cold tap, one bath, and one bedroom sink mixer [hot/cold] taps). In six cases, identical Acanthamoeba mtDNA profiles were found for the clinical and home tap water isolates. In keeping with UK plumbing practice, 24 of 27 homes had internal roof water storage tanks to supply domestic taps, but the mains fed the kitchen cold tap. Water storage tanks promote colonization of domestic water with FLA, including Acanthamoeba, and hence increase the risk of AK. This accounts for the significantly greater incidence of AK in the UK and supports advice to avoid using tap water in contact lens care routines.

Cholesterol efflux is differentially regulated in neurons and astrocytes: implications for brain cholesterol homeostasis

PubMed Central

Chen, Jing; Zhang, Xiaolu; Kusumo, Handojo; Costa, Lucio G.; Guizzetti, Marina

2012-01-01

Disruption of cholesterol homeostasis in the central nervous system (CNS) has been associated with neurological, neurodegenerative, and neurodevelopmental disorders. The CNS is a closed system with regard to cholesterol homeostasis, as cholesterol-delivering lipoproteins from the periphery cannot pass the blood-brain-barrier and enter the brain. Different cell types in the brain have different functions in the regulation of cholesterol homeostasis, with astrocytes producing and releasing apolipoprotein E and lipoproteins, and neurons metabolizing cholesterol to 24(S)-hydroxycholesterol. We present evidence that astrocytes and neurons adopt different mechanisms also in regulating cholesterol efflux. We found that in astrocytes cholesterol efflux is induced by both lipid-free apolipoproteins and lipoproteins, while cholesterol removal from neurons is triggered only by lipoproteins. The main pathway by which apolipoproteins induce cholesterol efflux is through ABCA1. By upregulating ABCA1 levels and by inhibiting its activity and silencing its expression, we show that ABCA1 is involved in cholesterol efflux from astrocytes but not from neurons. Furthermore, our results suggest that ABCG1 is involved in cholesterol efflux to apolipoproteins and lipoproteins from astrocytes but not from neurons, while ABCG4, whose expression is much higher in neurons than astrocytes, is involved in cholesterol efflux from neurons but not astrocytes. These results indicate that different mechanisms regulate cholesterol efflux from neurons and astrocytes, reflecting the different roles that these cell types play in brain cholesterol homeostasis. These results are important in understanding cellular targets of therapeutic drugs under development for the treatments of conditions associated with altered cholesterol homeostasis in the CNS. PMID:23010475

Glucocorticoid receptors in the prefrontal cortex regulate stress-evoked dopamine efflux and aspects of executive function.

PubMed

Butts, Kelly A; Weinberg, Joanne; Young, Allan H; Phillips, Anthony G

2011-11-08

Enhanced dopamine efflux in the prefrontal cortex is a well-documented response to acute stress. However, the underlying mechanism(s) for this response is unknown. Using in vivo microdialysis, we demonstrate that blocking glucocorticoid receptors locally within the rat prefrontal cortex results in a reduction in stress-evoked dopamine efflux. In contrast, blocking glucocorticoid receptors in the ventral tegmental area did not affect stress-evoked dopamine efflux in the prefrontal cortex. Additionally, local administration of corticosterone into the prefrontal cortex increased prefrontal dopamine efflux. The functional impact of enhanced dopamine efflux evoked by acute stress was demonstrated using a cognitive task dependent on the prefrontal cortex and sensitive to impairment in working memory. Notably, stress-induced impairments in cognition were attenuated by blockade of glucocorticoid receptors in the prefrontal cortex. Taken together, these data demonstrate that glucocorticoids act locally within the prefrontal cortex to modulate mesocortical dopamine efflux leading to the cognitive impairments observed during acute stress.

Bimanual tapping of a syncopated rhythm reveals hemispheric preferences for relative movement frequencies.

PubMed

Pflug, Anja; Gompf, Florian; Kell, Christian Alexander

2017-08-01

In bimanual multifrequency tapping, right-handers commonly use the right hand to tap the relatively higher rate and the left hand to tap the relatively lower rate. This could be due to hemispheric specializations for the processing of relative frequencies. An extension of the double-filtering-by-frequency theory to motor control proposes a left hemispheric specialization for the control of relatively high and a right hemispheric specialization for the control of relatively low tapping rates. We investigated timing variability and rhythmic accentuation in right handers tapping mono- and multifrequent bimanual rhythms to test the predictions of the double-filtering-by-frequency theory. Yet, hemispheric specializations for the processing of relative tapping rates could be masked by a left hemispheric dominance for the control of known sequences. Tapping was thus either performed in an overlearned quadruple meter (tap of the slow rhythm on the first auditory beat) or in a syncopated quadruple meter (tap of the slow rhythm on the fourth auditory beat). Independent of syncopation, the right hand outperformed the left hand in timing accuracy for fast tapping. A left hand timing benefit for slow tapping rates as predicted by the double-filtering-by-frequency theory was only found in the syncopated tapping group. This suggests a right hemisphere preference for the control of slow tapping rates when rhythms are not overlearned. Error rates indicate that overlearned rhythms represent hierarchically structured meters that are controlled by a single timer that could potentially reside in the left hemisphere. Copyright © 2017 Elsevier B.V. All rights reserved.

In Vitro Infection Model Characterizing the Effect of Efflux Pump Inhibition on Prevention of Resistance to Levofloxacin and Ciprofloxacin in Streptococcus pneumoniae▿

PubMed Central

Louie, Arnold; Brown, David L.; Liu, Weiguo; Kulawy, Robert W.; Deziel, Mark R.; Drusano, George L.

2007-01-01

The prevalence of fluoroquinolone-resistant Streptococcus pneumoniae is slowly rising as a consequence of the increased use of fluoroquinolone antibiotics to treat community-acquired pneumonia. We tested the hypothesis that increased efflux pump (EP) expression by S. pneumoniae may facilitate the emergence of fluoroquinolone resistance. By using an in vitro pharmacodynamic infection system, a wild-type S. pneumoniae strain (Spn-058) and an isogenic strain with EP overexpression (Spn-RC2) were treated for 10 days with ciprofloxacin or levofloxacin in the presence or absence of the EP inhibitor reserpine to evaluate the effect of EP inhibition on the emergence of resistance. Cultures of Spn-058 and Spn-RC2 were exposed to concentration-time profiles simulating those in humans treated with a regimen of ciprofloxacin at 750 mg orally once every 12 h and with regimens of levofloxacin at 500 and 750 mg orally once daily (QD; with or without continuous infusions of 20 μg of reserpine/ml). The MICs of ciprofloxacin and levofloxacin for Spn-058 were both 1 μg/ml when susceptibility testing was conducted with each antibiotic alone and with each antibiotic in the presence of reserpine. For Spn-RC2, the MIC of levofloxacin alone and with reserpine was also 1 μg/ml; the MICs of ciprofloxacin were 2 and 1 μg/ml, respectively, when determined with ciprofloxacin alone and in combination with reserpine. Reserpine, alone, had no effect on the growth of Spn-058 and Spn-RC2. For Spn-058, simulated regimens of ciprofloxacin at 750 mg every 12 h or levofloxacin at 500 mg QD were associated with the emergence of fluoroquinolone resistance. However, the use of ciprofloxacin at 750 mg every 12 h and levofloxacin at 500 mg QD in combination with reserpine rapidly killed Spn-058 and prevented the emergence of resistance. For Spn-RC2, levofloxacin at 500 mg QD was associated with the emergence of resistance, but again, the resistance was prevented when this levofloxacin regimen was

In vitro infection model characterizing the effect of efflux pump inhibition on prevention of resistance to levofloxacin and ciprofloxacin in Streptococcus pneumoniae.

PubMed

Louie, Arnold; Brown, David L; Liu, Weiguo; Kulawy, Robert W; Deziel, Mark R; Drusano, George L

2007-11-01

The prevalence of fluoroquinolone-resistant Streptococcus pneumoniae is slowly rising as a consequence of the increased use of fluoroquinolone antibiotics to treat community-acquired pneumonia. We tested the hypothesis that increased efflux pump (EP) expression by S. pneumoniae may facilitate the emergence of fluoroquinolone resistance. By using an in vitro pharmacodynamic infection system, a wild-type S. pneumoniae strain (Spn-058) and an isogenic strain with EP overexpression (Spn-RC2) were treated for 10 days with ciprofloxacin or levofloxacin in the presence or absence of the EP inhibitor reserpine to evaluate the effect of EP inhibition on the emergence of resistance. Cultures of Spn-058 and Spn-RC2 were exposed to concentration-time profiles simulating those in humans treated with a regimen of ciprofloxacin at 750 mg orally once every 12 h and with regimens of levofloxacin at 500 and 750 mg orally once daily (QD; with or without continuous infusions of 20 microg of reserpine/ml). The MICs of ciprofloxacin and levofloxacin for Spn-058 were both 1 microg/ml when susceptibility testing was conducted with each antibiotic alone and with each antibiotic in the presence of reserpine. For Spn-RC2, the MIC of levofloxacin alone and with reserpine was also 1 mug/ml; the MICs of ciprofloxacin were 2 and 1 microg/ml, respectively, when determined with ciprofloxacin alone and in combination with reserpine. Reserpine, alone, had no effect on the growth of Spn-058 and Spn-RC2. For Spn-058, simulated regimens of ciprofloxacin at 750 mg every 12 h or levofloxacin at 500 mg QD were associated with the emergence of fluoroquinolone resistance. However, the use of ciprofloxacin at 750 mg every 12 h and levofloxacin at 500 mg QD in combination with reserpine rapidly killed Spn-058 and prevented the emergence of resistance. For Spn-RC2, levofloxacin at 500 mg QD was associated with the emergence of resistance, but again, the resistance was prevented when this levofloxacin regimen

Impact analysis of tap switch out of step for converter transformer

NASA Astrophysics Data System (ADS)

Hong-yue, ZHANG; Zhen-hua, ZHANG; Zhang-xue, XIONG; Gao-wang, YU

2017-06-01

AC transformer load regulation is mainly used to adjust the load side voltage level, improve the quality of power supply, the voltage range is relatively narrow. In DC system, converter transformer is the core equipment of AC and DC power converter and inverter. converter transformer tap adjustment can maintain the normal operation of the converter in small angle range control, the absorption of reactive power, economic operation, valve less stress, valve damping circuit loss, AC / DC harmonic component is also smaller. In this way, the tap switch action is more frequent, and a large range of the tap switch adjustment is required. Converter transformer with a more load voltage regulation switch, the voltage regulation range of the switch is generally 20~30%, the adjustment of each file is 1%~2%. Recently it is often found that the tap switch of Converter Transformers is out of step in Converter station. In this paper, it is analyzed in detail the impact of tap switch out of step for differential protection, overexcitation protection and zero sequence over current protection. Analysis results show that: the tap switch out of step has no effect on the differential protection and the overexcitation protection including the tap switch. But the tap switch out of step has effect on zero sequence overcurrent protection of out of step star-angle converter transformer. The zero sequence overcurrent protection will trip when the tap switch out of step is greater than 3 for out of step star-angle converter transformer.

A statistical characterization of the finger tapping test: modeling, estimation, and applications.

PubMed

Austin, Daniel; McNames, James; Klein, Krystal; Jimison, Holly; Pavel, Misha

2015-03-01

Sensory-motor performance is indicative of both cognitive and physical function. The Halstead-Reitan finger tapping test is a measure of sensory-motor speed commonly used to assess function as part of a neuropsychological evaluation. Despite the widespread use of this test, the underlying motor and cognitive processes driving tapping behavior during the test are not well characterized or understood. This lack of understanding may make clinical inferences from test results about health or disease state less accurate because important aspects of the task such as variability or fatigue are unmeasured. To overcome these limitations, we enhanced the tapper with a sensor that enables us to more fully characterize all the aspects of tapping. This modification enabled us to decompose the tapping performance into six component phases and represent each phase with a set of parameters having clear functional interpretation. This results in a set of 29 total parameters for each trial, including change in tapping over time, and trial-to-trial and tap-to-tap variability. These parameters can be used to more precisely link different aspects of cognition or motor function to tapping behavior. We demonstrate the benefits of this new instrument with a simple hypothesis-driven trial comparing single and dual-task tapping.

Finger tapping and pre-attentive sensorimotor timing in adults with ADHD.

PubMed

Hove, Michael J; Gravel, Nickolas; Spencer, Rebecca M C; Valera, Eve M

2017-12-01

Sensorimotor timing deficits are considered central to attention-deficit/hyperactivity disorder (ADHD). However, the tasks establishing timing impairments often involve interconnected processes, including low-level sensorimotor timing and higher level executive processes such as attention. Thus, the source of timing deficits in ADHD remains unclear. Low-level sensorimotor timing can be isolated from higher level processes in a finger-tapping task that examines the motor response to unexpected shifts of metronome onsets. In this study, adults with ADHD and ADHD-like symptoms (n = 25) and controls (n = 26) performed two finger-tapping tasks. The first assessed tapping variability in a standard tapping task (metronome-paced and unpaced). In the other task, participants tapped along with a metronome that contained unexpected shifts (±15, 50 ms); the timing adjustment on the tap following the shift captures pre-attentive sensorimotor timing (i.e., phase correction) and thus should be free of potential higher order confounds (e.g., attention). In the standard tapping task, as expected, the ADHD group had higher timing variability in both paced and unpaced tappings. However, in the pre-attentive task, performance did not differ between the ADHD and control groups. Together, results suggest that low-level sensorimotor timing and phase correction are largely preserved in ADHD and that some timing impairments observed in ADHD may stem from higher level factors (such as sustained attention).

Hydrodynamic Controls on Carbon Dioxide Efflux from Inland Waters

NASA Astrophysics Data System (ADS)

Long, H. E.; Waldron, S.; Hoey, T.; Newton, J.; Quemin, S.

2013-12-01

Intensive research has been undertaken on carbon dioxide efflux from lakes, estuaries and oceans, but much less attention has been given to rivers and streams, especially lower order streams. River systems are often over-saturated with carbon dioxide and so tend to act as sources of carbon dioxide to the atmosphere. It has been thought that rivers act as pipes carrying this terrestrial carbon to the oceans. However, recent studies have shown that a significant amount of the carbon is reprocessed within the system in a series of transformations and losses. Fluvial evasion of carbon dioxide is now recognised to be a significant component of carbon cycles, however the factors controlling carbon dioxide efflux and its magnitude remain poorly understood and quantified. This research aims to quantify, and better understand the controls on, freshwater carbon dioxide evasion. Data are presented here from field measurements that commenced in Sept 2013 in two contrasting Scottish rivers: the River Kelvin which has a large (335 km.sq) part-urban catchment with predominantly non-peat soils and Drumtee Water, a small (9.6 km.sq) rural catchment of peat soils and agricultural land. Using a floating chamber with the headspace connected to an infrared gas analyser to measure changes in carbon dioxide concentration, efflux rates from 0.22 - 47.4 μmol CO2/m.sq/sec were measured, these close to the middle of the range of previously reported values. At one site on the River Kelvin in May 2013 an influx of -0.61 - -3.53 μmol CO2/m.sq/sec was recorded. Whereas previous research finds carbon dioxide efflux to increase with decreasing river size and a more organic-rich soil catchment, here the controls on carbon dioxide evasion are similar across the contrasting catchments. Carbon dioxide evasion shows seasonality, with maximum fluxes in the summer months being up to twice as high as the winter maxima. Linear regression demonstrates that evasion increases with increased flow velocity

Is the Ca2+-ATPase from sarcoplasmic reticulum also a heat pump?

PubMed

Kjelstrup, Signe; de Meis, Leopoldo; Bedeaux, Dick; Simon, Jean-Marc

2008-11-01

We calculate, using the first law of thermodynamics, the membrane heat fluxes during active transport of Ca(2+) in the Ca(2+)-ATPase in leaky and intact vesicles, during ATP hydrolysis or synthesis conditions. The results show that the vesicle interior may cool down during hydrolysis and Ca(2+)-uptake, and heat up during ATP synthesis and Ca(2+)-efflux. The heat flux varies with the SERCA isoform. Electroneutral processes and rapid equilibration of water were assumed. The results are consistent with the second law of thermodynamics for the overall processes. The expression for the heat flux and experimental data, show that important contributions come from the enthalpy of hydrolysis for the medium in question, and from proton transport between the vesicle interior and exterior. The analysis give quantitative support to earlier proposals that certain, but not all, Ca(2+)-ATPases, not only act as Ca(2+)-pumps, but also as heat pumps. It can thus help explain why SERCA 1 type enzymes dominate in tissues where thermal regulation is important, while SERCA 2 type enzymes, with their lower activity and better ability to use the energy from the reaction to pump ions, dominate in tissues where this is not an issue.

Membrane Fusion Proteins of Type I Secretion System and Tripartite Efflux Pumps Share a Binding Motif for TolC in Gram-Negative Bacteria

PubMed Central

Yoon, Bo-Young; Song, Saemee; Lee, Kangseok; Ha, Nam-Chul

2012-01-01

The Hly translocator complex of Escherichia coli catalyzes type I secretion of the toxin hemolysin A (HlyA). In this complex, HlyB is an inner membrane ABC (ATP Binding Cassette)-type transporter, TolC is an outer membrane channel protein, and HlyD is a periplasmic adaptor anchored in the inner membrane that bridges HlyB to TolC. This tripartite organization is reminiscent of that of drug efflux systems such as AcrA-AcrB-TolC and MacA-MacB-TolC of E. coli. We have previously shown the crucial role of conserved residues located at the hairpin tip region of AcrA and MacA adaptors during assembly of their cognate systems. In this study, we investigated the role of the putative tip region of HlyD using HlyD mutants with single amino acid substitutions at the conserved positions. In vivo and in vitro data show that all mutations abolished HlyD binding to TolC and resulted in the absence of HlyA secretion. Together, our results suggest that, similarly to AcrA and MacA, HlyD interacts with TolC in a tip-to-tip manner. A general model in which these conserved interactions induce opening of TolC during drug efflux and type I secretion is discussed. PMID:22792337

Large-Scale Femtoliter Droplet Array for Single Cell Efflux Assay of Bacteria.

PubMed

Iino, Ryota; Sakakihara, Shouichi; Matsumoto, Yoshimi; Nishino, Kunihiko

2018-01-01

Large-scale femtoliter droplet array as a platform for single cell efflux assay of bacteria is described. Device microfabrication, femtoliter droplet array formation and concomitant enclosure of single bacterial cells, fluorescence-based detection of efflux activity at the single cell level, and collection of single cells from droplet and subsequent gene analysis are described in detail.

On-line tool breakage monitoring of vibration tapping using spindle motor current

NASA Astrophysics Data System (ADS)

Li, Guangjun; Lu, Huimin; Liu, Gang

2008-10-01

Input current of driving motor has been employed successfully as monitoring the cutting state in manufacturing processes for more than a decade. In vibration tapping, however, the method of on-line monitoring motor electric current has not been reported. In this paper, a tap failure prediction method is proposed to monitor the vibration tapping process using the electrical current signal of the spindle motor. The process of vibration tapping is firstly described. Then the relationship between the torque of vibration tapping and the electric current of motor is investigated by theoretic deducing and experimental measurement. According to those results, a monitoring method of tool's breakage is proposed through monitoring the ratio of the current amplitudes during adjacent vibration tapping periods. Finally, a low frequency vibration tapping system with motor current monitoring is built up using a servo motor B-106B and its driver CR06. The proposed method has been demonstrated with experiment data of vibration tapping in titanic alloys. The result of experiments shows that the method, which can avoid the tool breakage and giving a few error alarms when the threshold of amplitude ratio is 1.2 and there is at least 2 times overrun among 50 adjacent periods, is feasible for tool breakage monitoring in the process of vibration tapping small thread holes.

Suppression of asymmetric acid efflux and gravitropism in maize roots treated with auxin transport inhibitors of sodium orthovanadate

NASA Technical Reports Server (NTRS)

Mulkey, T. J.; Evans, M. L.

1982-01-01

In gravitropically stimulated roots of maize (Zea mays L., hybrid WF9 x 38MS), there is more acid efflux on the rapidly growing upper side than on the slowly growing lower side. In light of the Cholodny/Went hypothesis of gravitropism which states that gravitropic curvature results from lateral redistribution of auxin, the effects of auxin transport inhibitors on the development of acid efflux asymmetry and curvature in gravistimulated roots were examined. All the transport inhibitors tested prevented both gravitropism and the development of asymmetric acid efflux in gravistimulated roots. The results indicate that auxin redistribution may cause the asymmetry of acid efflux, a finding consistent with the Cholodny/Went hypothesis of gravitropism. As further evidence that auxin-induced acid efflux asymmetry may mediate gravitropic curvature, sodium orthovanadate, an inhibitor of auxin-induced H+ efflux was found to prevent both gravitropism and the development of asymmetric acid efflux in gravistimulated roots.

Amphipathic Polyproline Peptides Stimulate Cholesterol Efflux by the ABCA1 Transporter

PubMed Central

Sviridov, D.O.; Drake, S.K.; Freeman, L.A.; Remaley, A.T.

2016-01-01

ApoA-I mimetics are short synthetic peptides that contain an amphipathic αα-helix and stimulate cholesterol efflux by the ABCA1 transporter in a detergent-like extraction mechanism. We investigated the use of amphipathic peptides with a polypro helix for stimulating cholesterol efflux by ABCA1. Polypro peptides were synthesized with modified prolines, containing either a hydrophobic phenol group (Prop) or a polar N-acetylgalactosamine (Prog) attached to the pyrrolidine ring and were designated as either PP-2, 3, 4, or 5, depending on the number of 3 amino acid repeat units (Prop - Prog - Prop). Based on molecular modeling, these peptides were predicted to be relatively rigid and to bind to a phospholipid bilayer. By CD spectroscopy, PP peptides formed a Type-II polypro helix in an aqueous solution. PP-2 was inactive in promoting cholesterol efflux, but peptides with more than 2 repeat units were active. PP-4 showed a similar Vmax as a much longer amphipathic α-αhelical peptide, containing 37 amino acids, but had a Km that was approximately 20-fold lower. PP peptides were specific in that they did not stimulate cholesterol efflux from cells not expressing ABCA1 and were also non-cytotoxic. Addition of PP-3, 4 and 5 to serum promoted the formation of smaller size HDL species (7 nM) and increased its capacity for ABCA1-dependent cholesterol efflux by approximately 20-35% (p<0.05). Because of their relatively small size and increased potency, amphipathic peptides with a polypro helix may represent an alternative structural motif for the development of apoA-I mimetic peptides. PMID:26879139

A computer vision framework for finger-tapping evaluation in Parkinson's disease.

PubMed

Khan, Taha; Nyholm, Dag; Westin, Jerker; Dougherty, Mark

2014-01-01

The rapid finger-tapping test (RFT) is an important method for clinical evaluation of movement disorders, including Parkinson's disease (PD). In clinical practice, the naked-eye evaluation of RFT results in a coarse judgment of symptom scores. We introduce a novel computer-vision (CV) method for quantification of tapping symptoms through motion analysis of index-fingers. The method is unique as it utilizes facial features to calibrate tapping amplitude for normalization of distance variation between the camera and subject. The study involved 387 video footages of RFT recorded from 13 patients diagnosed with advanced PD. Tapping performance in these videos was rated by two clinicians between the symptom severity levels ('0: normal' to '3: severe') using the unified Parkinson's disease rating scale motor examination of finger-tapping (UPDRS-FT). Another set of recordings in this study consisted of 84 videos of RFT recorded from 6 healthy controls. These videos were processed by a CV algorithm that tracks the index-finger motion between the video-frames to produce a tapping time-series. Different features were computed from this time series to estimate speed, amplitude, rhythm and fatigue in tapping. The features were trained in a support vector machine (1) to categorize the patient group between UPDRS-FT symptom severity levels, and (2) to discriminate between PD patients and healthy controls. A new representative feature of tapping rhythm, 'cross-correlation between the normalized peaks' showed strong Guttman correlation (μ2=-0.80) with the clinical ratings. The classification of tapping features using the support vector machine classifier and 10-fold cross validation categorized the patient samples between UPDRS-FT levels with an accuracy of 88%. The same classification scheme discriminated between RFT samples of healthy controls and PD patients with an accuracy of 95%. The work supports the feasibility of the approach, which is presumed suitable for PD monitoring

In vitro activity of levofloxacin against planktonic and biofilm Stenotrophomonas maltophilia lifestyles under conditions relevant to pulmonary infection in cystic fibrosis, and relationship with SmeDEF multidrug efflux pump expression.

PubMed

Pompilio, Arianna; Crocetta, Valentina; Verginelli, Fabio; Di Bonaventura, Giovanni

2016-07-01

The activity of levofloxacin against planktonic and biofilm Stenotrophomonas maltophilia cells and the role played by the multidrug efflux pump SmeDEF were evaluated under conditions relevant to the cystic fibrosis (CF) lung. MIC, MBC and MBEC of levofloxacin were assessed, against five CF strains, under 'standard' (CLSI-recommended) and 'CF-like' (pH 6.8, 5% CO2, in a synthetic CF sputum) conditions. Levofloxacin was tested against biofilms at concentrations (10, 50 and 100 μg mL(-1)) corresponding to achievable serum levels and sputum levels by aerosolisation. smeD expression was evaluated, under both conditions, in planktonic and biofilm cells by RT-PCR. The bactericidal effect of levofloxacin was decreased, in three out of five strains tested, under 'CF-like' conditions (MBC: 2-4 vs 8-16 μg mL(-1), under 'standard' and 'CF-like' conditions, respectively). Biofilm was intrinsically resistant to levofloxacin, regardless of conditions tested (MBECs ≥ 100 μg mL(-1) for all strains). Only under 'CF-like' conditions, smeD expression increased during planktonic-to-biofilm transition, and in biofilm cells compared to stationary planktonic cells. Our findings confirmed that S. maltophilia biofilm is intrinsically resistant to therapeutic concentrations of levofloxacin. Under conditions relevant to CF, smeD overexpression could contribute to levofloxacin resistance. Further studies are warranted to define the clinical relevance of our findings. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Crystal structure of an antigenic outer-membrane protein from Salmonella Typhi suggests a potential antigenic loop and an efflux mechanism.

PubMed

Guan, Hong-Hsiang; Yoshimura, Masato; Chuankhayan, Phimonphan; Lin, Chien-Chih; Chen, Nai-Chi; Yang, Ming-Chi; Ismail, Asma; Fun, Hoong-Kun; Chen, Chun-Jung

2015-11-13

ST50, an outer-membrane component of the multi-drug efflux system from Salmonella enterica serovar Typhi, is an obligatory diagnostic antigen for typhoid fever. ST50 is an excellent and unique diagnostic antigen with 95% specificity and 90% sensitivity and is used in the commercial diagnosis test kit (TYPHIDOT(TM)). The crystal structure of ST50 at a resolution of 2.98 Å reveals a trimer that forms an α-helical tunnel and a β-barrel transmembrane channel traversing the periplasmic space and outer membrane. Structural investigations suggest significant conformational variations in the extracellular loop regions, especially extracellular loop 2. This is the location of the most plausible antibody-binding domain that could be used to target the design of new antigenic epitopes for the development of better diagnostics or drugs for the treatment of typhoid fever. A molecule of the detergent n-octyl-β-D-glucoside is observed in the D-cage, which comprises three sets of Asp361 and Asp371 residues at the periplasmic entrance. These structural insights suggest a possible substrate transport mechanism in which the substrate first binds at the periplasmic entrance of ST50 and subsequently, via iris-like structural movements to open the periplasmic end, penetrates the periplasmic domain for efflux pumping of molecules, including poisonous metabolites or xenobiotics, for excretion outside the pathogen.

Novel insight into antimicrobial resistance and sensitivity phenotypes associated to qac and norA genotypes in Staphylococcus aureus.

PubMed

Marchi, Emmanuela; Furi, Leonardo; Arioli, Stefania; Morrissey, Ian; Di Lorenzo, Valeria; Mora, Diego; Giovannetti, Luciana; Oggioni, Marco Rinaldo; Viti, Carlo

2015-01-01

Staphylococcus aureus strains harboring QacA, QacB, QacC, QacG transporters and norA promoter up-regulating mutations were characterized by phenotype microarray (PM), standard methods for susceptibility testing, and ethidium bromide efflux assays, in order to increase knowledge on phenotypes associated to efflux pumps and their substrates. PM data and standard susceptibility testing lead to the identification of new potential efflux targets, such as guanidine hydrochloride or 8-hydroxyquinoline for QacA and QacC pumps, respectively. The identification of compounds to which the presence of efflux pumps induced increased susceptibility opens new perspectives for potential adjunct anti-resistance treatment (i.e. strains bearing QacB transporters showed increased susceptibility to thioridazine, amitriptyline and orphenadrine). Although the tested isolates were characterized by high degree of heterogeneity, a hallmark of clinical isolates, direct ethidium bromide efflux assays were effective in highlighting differences in efflux efficiency among strains. These data add to characterization of substrate specificity in the different classes of staphylococcal multidrug efflux systems conferring specific substrate profiles and efflux features to each of them. Copyright © 2014 Elsevier GmbH. All rights reserved.

Vertical Finger Displacement Is Reduced in Index Finger Tapping During Repeated Bout Rate Enhancement.

PubMed

Mora-Jensen, Mark Holten; Madeleine, Pascal; Hansen, Ernst Albin

2017-10-01

The present study analyzed (a) whether a recently reported phenomenon of repeated bout rate enhancement in finger tapping (i.e., a cumulating increase in freely chosen finger tapping frequency following submaximal muscle activation in the form of externally unloaded voluntary tapping) could be replicated and (b) the hypotheses that the faster tapping was accompanied by changed vertical displacement of the fingertip and changed peak force during tapping. Right-handed, healthy, and recreationally active individuals (n = 24) performed two 3-min index finger tapping bouts at freely chosen tapping frequency, separated by 10-min rest. The recently reported phenomenon of repeated bout rate enhancement was replicated. The faster tapping (8.8 ± 18.7 taps/min, corresponding to 6.0 ± 11.0%, p = .033) was accompanied by reduced vertical displacement (1.6 ± 2.9 mm, corresponding to 6.3 ± 14.9%, p = .012) of the fingertip. Concurrently, peak force was unchanged. The present study points at separate control mechanisms governing kinematics and kinetics during finger tapping.

Bilirubin Decreases Macrophage Cholesterol Efflux and ATP-Binding Cassette Transporter A1 Protein Expression.

PubMed

Wang, Dongdong; Tosevska, Anela; Heiß, Elke H; Ladurner, Angela; Mölzer, Christine; Wallner, Marlies; Bulmer, Andrew; Wagner, Karl-Heinz; Dirsch, Verena M; Atanasov, Atanas G

2017-04-28

Mild but chronically elevated circulating unconjugated bilirubin is associated with reduced total and low-density lipoprotein cholesterol concentration, which is associated with reduced cardiovascular disease risk. We aimed to investigate whether unconjugated bilirubin influences macrophage cholesterol efflux, as a potential mechanism for the altered circulating lipoprotein concentrations observed in hyperbilirubinemic individuals. Cholesterol efflux from THP-1 macrophages was assessed using plasma obtained from normo- and hyperbilirubinemic (Gilbert syndrome) humans (n=60 per group) or (heterozygote/homozygote Gunn) rats (n=20 per group) as an acceptor. Hyperbilirubinemic plasma from patients with Gilbert syndrome and Gunn rats induced significantly reduced cholesterol efflux compared with normobilirubinemic plasma. Unconjugated bilirubin (3-17.1 μmol/L) exogenously added to plasma- or apolipoprotein A1-supplemented media also decreased macrophage cholesterol efflux in a concentration- and time-dependent manner. We also showed reduced protein expression of the ATP-binding cassette transporter A1 (ABCA1), a transmembrane cholesterol transporter involved in apolipoprotein A1-mediated cholesterol efflux, in THP-1 macrophages treated with unconjugated bilirubin and in peripheral blood mononuclear cells obtained from hyperbilirubinemic individuals. Furthermore, we demonstrated that bilirubin accelerates the degradation rate of the ABCA1 protein in THP-1 macrophages. Cholesterol efflux from THP-1 macrophages is decreased in the presence of plasma obtained from humans and rats with mild hyperbilirubinemia. A direct effect of unconjugated bilirubin on cholesterol efflux was demonstrated and is associated with decreased ABCA1 protein expression. These data improve our knowledge concerning bilirubin's impact on cholesterol transport and represent an important advancement in our understanding of bilirubin's role in cardiovascular disease. © 2017 The Authors. Published on

CO2 Efflux from Shrimp Ponds in Indonesia

PubMed Central

Sidik, Frida; Lovelock, Catherine E.

2013-01-01

The conversion of mangrove forest to aquaculture ponds has been increasing in recent decades. One of major concerns of this habitat loss is the release of stored ‘blue’ carbon from mangrove soils to the atmosphere. In this study, we assessed carbon dioxide (CO2) efflux from soil in intensive shrimp ponds in Bali, Indonesia. We measured CO2 efflux from the floors and walls of shrimp ponds. Rates of CO2 efflux within shrimp ponds were 4.37 kg CO2 m−2 y−1 from the walls and 1.60 kg CO2 m−2 y−1 from the floors. Combining our findings with published data of aquaculture land use in Indonesia, we estimated that shrimp ponds in this region result in CO2 emissions to the atmosphere between 5.76 and 13.95 Tg y−1. The results indicate that conversion of mangrove forests to aquaculture ponds contributes to greenhouse gas emissions that are comparable to peat forest conversion to other land uses in Indonesia. Higher magnitudes of CO2 emission may be released to atmosphere where ponds are constructed in newly cleared mangrove forests. This study indicates the need for incentives that can meet the target of aquaculture industry without expanding the converted mangrove areas, which will lead to increased CO2 released to atmosphere. PMID:23755306

TAp63 is a master transcriptional regulator of lipid and glucose metabolism

PubMed Central

Su, Xiaohua; Gi, Young Jin; Chakravarti, Deepavali; Chan, Io Long; Zhang, Aijun; Xia, Xuefeng; Tsai, Kenneth Y.; Flores, Elsa R.

2012-01-01

SUMMARY TAp63 prevents premature aging suggesting a link to genes that regulate longevity. Further characterization of TAp63−/− mice revealed that these mice develop obesity, insulin resistance, and glucose intolerance, similar to those seen in mice lacking two key metabolic regulators, Silent information regulator T1 (Sirt1) and AMPK. While the roles of Sirt1 and AMPK in metabolism have been well studied, their upstream regulators are not well understood. We found that TAp63 is important in regulating energy metabolism by accumulating in response to metabolic stress and transcriptionally activating Sirt1, AMPKα2, and LKB1 resulting in increased fatty acid synthesis and decreased fatty acid oxidation. Moreover, we found that TAp63 lowers blood glucose levels in response to metformin. Restoration of Sirt1, AMPKα2, and LKB1 in TAp63−/− mice rescued some of the metabolic defects of the TAp63−/− mice. Our study defines a role for TAp63 in metabolism and weight control. PMID:23040072

X-ray microtomography study of the compaction process of rods under tapping.

PubMed

Fu, Yang; Xi, Yan; Cao, Yixin; Wang, Yujie

2012-05-01

We present an x-ray microtomography study of the compaction process of cylindrical rods under tapping. The process is monitored by measuring the evolution of the orientational order parameter, local, and overall packing densities as a function of the tapping number for different tapping intensities. The slow relaxation dynamics of the orientational order parameter can be well fitted with a stretched-exponential law with stretching exponents ranging from 0.9 to 1.6. The corresponding relaxation time versus tapping intensity follows an Arrhenius behavior which is reminiscent of the slow dynamics in thermal glassy systems. We also investigated the boundary effect on the ordering process and found that boundary rods order faster than interior ones. In searching for the underlying mechanism of the slow dynamics, we estimated the initial random velocities of the rods under tapping and found that the ordering process is compatible with a diffusion mechanism. The average coordination number as a function of the tapping number at different tapping intensities has also been measured, which spans a range from 6 to 8.

Tap Testing Hammer using Unmanned Aerial Systems (UASs)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mason, JaMein DeShon; Ayorinde, Emmanuel Temiloluwa; Mascarenas, David Dennis

This is the final poster for a Student Symposium at Los Alamos National Laboratory. This research describes the development, validation, and testing of a remote concrete tapping mechanism enabled by UAS. The conclusion is the following: The results quantify for the first time concrete tapping data collected remotely with UAS, enabling cost-effective, safer and sustainable upgrade prioritization of railroad bridges inventories.

Peptide mediators of cholesterol efflux

DOEpatents

Bielicki, John K.; Johansson, Jan

2013-04-09

The present invention provides a family of non-naturally occurring polypeptides having cholesterol efflux activity that parallels that of full-length apolipoproteins (e.g., Apo AI and Apo E), and having high selectivity for ABAC1 that parallels that of full-length apolipoproteins. The invention also provides compositions comprising such polypeptides, methods of identifying, screening and synthesizing such polypeptides, and methods of treating, preventing or diagnosing diseases and disorders associated with dyslipidemia, hypercholesterolemia and inflammation.

An estimation of finger-tapping rates and load capacities and the effects of various factors.

PubMed

Ekşioğlu, Mahmut; İşeri, Ali

2015-06-01

The aim of this study was to estimate the finger-tapping rates and finger load capacities of eight fingers (excluding thumbs) for a healthy adult population and investigate the effects of various factors on tapping rate. Finger-tapping rate, the total number of finger taps per unit of time, can be used as a design parameter of various products and also as a psychomotor test for evaluating patients with neurologic problems. A 1-min tapping task was performed by 148 participants with maximum volitional tempo for each of eight fingers. For each of the tapping tasks, the participant with the corresponding finger tapped the associated key in the standard position on the home row of a conventional keyboard for touch typing. The index and middle fingers were the fastest fingers for both hands, and little fingers the slowest. All dominant-hand fingers, except little finger, had higher tapping rates than the fastest finger of the nondominant hand. Tapping rate decreased with age and smokers tapped faster than nonsmokers. Tapping duration and exercise had also significant effect on tapping rate. Normative data of tapping rates and load capacities of eight fingers were estimated for the adult population. In designs of psychomotor tests that require the use of tapping rate or finger load capacity data, the effects of finger, age, smoking, and tapping duration need to be taken into account. The findings can be used for ergonomic designs requiring finger-tapping capacity and also as a reference in psychomotor tests. © 2015, Human Factors and Ergonomics Society.

Enhanced bacterial efflux system is the first step to the development of metronidazole resistance in Helicobacter pylori.

PubMed

Tsugawa, Hitoshi; Suzuki, Hidekazu; Muraoka, Hiroe; Ikeda, Fumiaki; Hirata, Kenro; Matsuzaki, Juntaro; Saito, Yoshimasa; Hibi, Toshifumi

2011-01-14

Although metronidazole (Mtz) is an important component of Helicobacter pylori eradication regimens, it has been pointed out that the increasing use of Mtz may result in increase in the incidence of Mtz-resistant strains. The present study was designed to examine the initial mechanism of resistance acquisition of H. pylori to Mtz. After 10 Mtz-susceptible strains were cultured on plates containing sub-inhibitory concentrations of Mtz, the MIC of Mtz for 9 of the 10 strains increased to levels of the Mtz-resistant strains. In the Mtz-resistance-induced strains, the expression of the TolC efflux pump (hefA) was significantly increased under Mtz exposure, without the reduction of the Mtz-reductive activity. Our finding suggests that overexpression of hefA may be the initial step in the acquisition of Mtz resistance in H. pylori. Copyright © 2010 Elsevier Inc. All rights reserved.

Comparative effects of auxin and abscisic acid on growth, hydrogen ion efflux and gravitropism in primary roots of maize

NASA Technical Reports Server (NTRS)

Evans, M. L.; Mulkey, T. J.

1984-01-01

In order to test the idea that auxin action on root growth may be mediated by H(+) movement, the correlation of auxin action on growth and H(+) movement in roots was examined along with changes in H(+) efflux patterns associated with the asymmetric growth which occurs during gravitropism. The effects of indoleacetic acid (IAA) and abscisic acid (AbA) on growth, H(+) secretion, and gravitropism in roots were compared. Results show a close correlation existent between H(+) efflux and growth in maize roots. In intact roots there is strong H(+) efflux from the elongation zone. Growth-promoting concentrations of IAA stimulate H(+) efflux. During gravitropism the H(+) efflux from the elongation zone becomes asymmetric; the evidence indicates that auxin redistribution contributes to the development of acid efflux asymmetry. That AbA stimulates root growth is reflected in its ability to stimulate H(+) efflux from apical root segments.

Interactional synchrony in chimpanzees: Examination through a finger-tapping experiment.

PubMed

Yu, Lira; Tomonaga, Masaki

2015-05-11

Humans often unconsciously coordinate behaviour with that of others in daily life. This interpersonal coordination, including mimicry and interactional synchrony, has been suggested to play a fundamental role in social interaction. If this coordinative behavior is socially adaptive, it may be shared with other highly social animal species. The current study targeted chimpanzees, which phylogenetically are the closest living relatives of humans and live in complex social groups, and examined whether interactional synchrony would emerge in pairs of chimpanzees when auditory information about a partner's movement was provided. A finger-tapping task was introduced via touch panels to elicit repetitive and rhythmic movement from each chimpanzee. We found that one of four chimpanzees produced significant changes in both tapping tempo and timing of the tapping relative to its partner's tap when auditory sounds were provided. Although the current results may have limitations in generalizing to chimpanzees as a species, we suggest that a finger-tapping task is one potential method to investigate interactional synchrony in chimpanzees under a laboratory setup.

Effect of vildagliptin and pravastatin combination on cholesterol efflux in adipocytes.

PubMed

Mostafa, Ahmed M; Hamdy, Nadia M; Abdel-Rahman, Sherif Z; El-Mesallamy, Hala O

2016-07-01

Many reports suggested that some statins are almost ineffective in reducing triglycerides or enhancing HDL-C plasma levels, although statin treatment was still efficacious in reducing LDL-C. In diabetic dyslipidemic patients, it may therefore be necessary to use a combination therapy with other drugs to achieve either LDL-C- and triglyceride-lowering or HDL-C-enhancing goals. Such ineffectiveness of statins can be attributed to their effect on the liver X receptor (LXR) which regulates the expression of the ATP-binding cassette (ABC) transporters ABCA1 and ABCG1. A decrease in the expression of these transporters eventually leads to decreased cholesterol efflux from peripheral tissues leading to low levels of HDL-C. Although manipulating the LXR pathway may complement the effects of statins, LXR synthetic ligands as T091317 have shown significant hypertriglyceridemic action which limits their use. We recently found that the antidiabetic drug vildagliptin stimulates LXR expression leading to increased ABCB1/ABCG1 expression which improves cholesterol efflux from adipocytes. Therefore, a combination of vildagliptin and statin may provide a solution without the hypertriglyceridemic action observed with LXR agonist. We hypothesize that a combination of vildagliptin and pravastatin will improve cholesterol efflux in adipocytes. Statin-treated 3T3-L1 adipocytes were treated with vildagliptin, and the expression of LXR-ABCA1/ABCG1 cascade and the cholesterol efflux were then determined. Our data indicate that a combination of vildagliptin and pravastatin significantly induces the expression of LXR-ABCA1/ABCG1 cascade and improves cholesterol efflux (P > 0.05) in adipocytes. Our data may explain, at least in part, the improvement in HDL-C levels observed in patients receiving both medications. © 2016 IUBMB Life, 68(7):535-543, 2016. © 2016 International Union of Biochemistry and Molecular Biology.

HNP-3 enhanced the antimicrobial activity of CIP by promoting ATP efflux from P. aeruginosa cells.

PubMed

Wang, Hao; Dong, Birong; Lou, Li

2011-04-01

To establish a novel strategy of P. aeruginosa control, we acquired recombination HNP-3 by gene recombination. Then we examined HNP-3 bio-activities and the influences of antimicrobial peptide on the efflux of ATP. Consequently, we obtained target protein with a molecular mass of 3,000 D consistent with the Anticipation. FIC index of Ciprofloxacin added HNP-3 was less than 0.5 and HNP3 synergistically cooperated with CIP to suppress P. aeruginosa colony formation revealed that there was significant synergy. ATP efflux was however up-regulated by low concentrations of HNP-3, although CIP did not exert any influence on ATP efflux. Conclusively, recombination protein HNP-3 displayed antimicrobial and synergic effects. HNP-3 enhanced the antimicrobial activity of CIP by promoting ATP efflux from P. aeruginosa cells and decreasing efflux of the drugs, which could have useful clinical applications.

Perioperative transversus abdominis plane (TAP) blocks for analgesia after abdominal surgery.

PubMed

Charlton, Shona; Cyna, Allan M; Middleton, Philippa; Griffiths, James D

2010-12-08

The transversus abdominis plane (TAP) block is a peripheral nerve block which anaesthetises the abdominal wall. The increasing use of TAP block, as a form of pain relief after abdominal surgery warrants evaluation of its effectiveness as an adjunctive technique to routine care and, when compared with other analgesic techniques. To assess effects of TAP blocks (and variants) on postoperative analgesia requirements after abdominal surgery. We searched specialised registers of Cochrane Anaesthesia and Cochrane Pain, Palliative and Supportive Care Review Groups, CENTRAL, MEDLINE, EMBASE and CINAHL to June 2010. We included randomised controlled trials (RCTs) comparing TAP block or rectus sheath block with: no TAP or rectus sheath block; placebo; systemic, epidural or any other analgesia. At least two review authors assessed study eligibility and risk of bias, and extracted data. We included eight studies (358 participants), five assessing TAP blocks, three assessing rectus sheath blocks; with moderate risk of bias overall. All studies had a background of general anaesthesia in both arms in most cases.Compared with no TAP block or saline placebo, TAP block resulted in significantly less postoperative requirement for morphine at 24 hours (mean difference (MD) -21.95 mg, 95% confidence interval (CI) -37.91 to 5.96; five studies, 236 participants) and 48 hours (MD -28.50, 95% CI -38.92 to -18.08; one study of 50 participants) but not at two hours (all random-effects analyses). Pain at rest was significantly reduced in two studies, but not a third.Only one of three included studies of rectus sheath blocks found a reduction in postoperative analgesic requirements in participants receiving blocks. One study, assessing number of participants who were pain-free after their surgery, found more participants who received a rectus sheath block to be pain-free for up to 10 hours postoperatively. As with TAP blocks, rectus sheath blocks made no apparent impact on nausea and vomiting

Molecular mechanism and species specificity of TAP inhibition by herpes simplex virus ICP47.

PubMed Central

Ahn, K; Meyer, T H; Uebel, S; Sempé, P; Djaballah, H; Yang, Y; Peterson, P A; Früh, K; Tampé, R

1996-01-01

The immediate early protein ICP47 of herpes simplex virus (HSV) inhibits the transporter for antigen processing (TAP)-mediated translocation of antigen-derived peptides across the endoplasmic reticulum (ER) membrane. This interference prevents assembly of peptides with class I MHC molecules in the ER and ultimately recognition of HSV-infected cells by cytotoxic T-lymphocytes, potentially leading to immune evasion of the virus. Here, we demonstrate that recombinant, purified ICP47 containing a hexahistidine tag inhibits peptide import into microsomes of insect cells expressing human TAP, whereas inhibition of peptide transport by murine TAP was much less effective. This finding indicates an intrinsic species-specificity of ICP47 and suggests that no additional proteins interacting specifically with either ICP47 or TAP are required for inhibition of peptide transport. Since neither purified nor induced ICP47 inhibited photocrosslinking of 8-azido-ATP to TAP1 and TAP2 it seems that ICP47 does not prevent ATP from binding to TAP. By contrast, peptide binding was completely blocked by ICP47 as shown both by photoaffinity crosslinking of peptides to TAP and peptide binding to microsomes from TAP-transfected insect cells. Competition experiments indicated that ICP47 binds to human TAP with a higher affinity (50 nM) than peptides whereas the affinity to murine TAP was 100-fold lower. Our data suggest that ICP47 prevents peptides from being translocated by blocking their binding to the substrate-binding site of TAP. Images PMID:8670825

Effect of proinflammatory cytokine IL-6 on efflux transport of rebamipide in Caco-2 cells.

PubMed

Miyake, Masateru; Nakai, Daisuke

2017-09-01

1. Effect of IL-6, a pro-inflammatory cytokine, on efflux transport of rebamipide, an antiulcer drug, was investigated in Caco-2 cells. 2. Rebamipide had a greater basal-to-apical than apical-to-basal transport rate. Efflux transport of rebamipide was inhibited by cyclosporine A, a P-gp inhibitor, and probenecid, which is a general MRP inhibitor, but not by Ko143, a BCRP inhibitor. 3. By the addition of IL-6, mannitol transport was slightly increased in a concentration-dependent manner in both directions of absorption and efflux. The addition of IL-6 did not change efflux transport of rebamipide even though efflux transport of digoxin, a typical substrate of P-gp, was significantly decreased by the addition of IL-6, indicating decrease of the function of P-gp. 4. Therefore, it was suggested that increase of MRP(s)-mediated transport compensates for the decrease of P-gp mediated transport of rebamipide. These findings suggested that rebamipide absorption is unlikely to be changed in IBD patients.

Winter CO2 efflux from cold semiarid sagebrush shrublands distributed across the rain-to-snow transition zone

NASA Astrophysics Data System (ADS)

Fellows, A.; Flerchinger, G. N.; Lohse, K. A.; Seyfried, M. S.

2017-12-01

Predicting winter CO2 efflux across the rain-to-snow transition zone is challenging in the cold semiarid northern Great Basin, USA, complicated by steep environmental gradients and marked heterogeneity in ecosystem properties. We therefore examined winter CO2 efflux over 9 site-years using 4 eddy covariance towers located in the Reynolds Creek Critical Zone Observatory. The sites were sagebrush shrublands located at 1425, 1680, 2098, and 2111 m, and spanned a large part of the rain-to-snow transition zone. We focused on two objectives. First, we quantified winter CO2 efflux at the sites, and considered how these varied with elevation. Second, we used a within-site and cross-site analysis to examine the biological and physical factors that impact winter CO2 efflux. Winter conditions were identified using temperature, snow depth, and CO2 exchange measurements and included 12,922 observations. The duration of winter conditions increased from 90 to 180 days with elevation. Peak snow depth increased from < 30 to > 100 cm with elevation. Cumulative winter CO2 efflux accounted for > 10% of the total annual CO2 efflux, increased with elevation, and was a key component of net ecosystem production at some sites in some years. The importance of winter CO2 efflux was accentuated by the region's long winters and also dry summers that decreased water availability and decomposition during non-winter periods. Preliminary regressions examining air temperature, soil temperature, wind speed, snow depth, and gross carbon uptake indicated some of these factors control the rate of winter CO2 efflux and require consideration, but that additional work is needed to disentangle co-linearity and assess the importance of these factors within and between sites. These findings suggest a consideration of winter CO2 efflux is warranted in cold winter-wet semiarid ecosystems, particularly where winters are long and non-winter CO2 efflux is strongly limited by water availability.

The Impact of Diesel Oil Pollution on the Hydrophobicity and CO2 Efflux of Forest Soils.

PubMed

Hewelke, Edyta; Szatyłowicz, Jan; Hewelke, Piotr; Gnatowski, Tomasz; Aghalarov, Rufat

2018-01-01

The contamination of soil with petroleum products is a major environmental problem. Petroleum products are common soil contaminants as a result of human activities, and they are causing substantial changes in the biological (particularly microbiological) processes, chemical composition, structure and physical properties of soil. The main objective of this study was to assess the impact of soil moisture on CO 2 efflux from diesel-contaminated albic podzol soils. Two contamination treatments (3000 and 9000 mg of diesel oil per kg of soil) were prepared for four horizons from two forest study sites with different initial levels of soil water repellency. CO 2 emissions were measured using a portable infrared gas analyser (LCpro+, ADC BioScientific, UK) while the soil samples were drying under laboratory conditions (from saturation to air-dry). The assessment of soil water repellency was performed using the water drop penetration time test. An analysis of variance (ANVOA) was conducted for the CO 2 efflux data. The obtained results show that CO 2 efflux from diesel-contaminated soils is higher than efflux from uncontaminated soils. The initially water-repellent soils were found to have a bigger CO 2 efflux. The non-linear relationship between soil moisture content and CO 2 efflux only existed for the upper soil horizons, while for deeper soil horizons, the efflux is practically independent of soil moisture content. The contamination of soil by diesel leads to increased soil water repellency.

Cerebellar modulation of frontal cortex dopamine efflux in mice: relevance to autism and schizophrenia.

PubMed

Mittleman, Guy; Goldowitz, Daniel; Heck, Detlef H; Blaha, Charles D

2008-07-01

Cerebellar and frontal cortical pathologies have been commonly reported in schizophrenia, autism, and other developmental disorders. Whether there is a relationship between prefrontal and cerebellar pathologies is unknown. Using fixed potential amperometry, dopamine (DA) efflux evoked by cerebellar or, dentate nucleus electrical stimulation (50 Hz, 200 muA) was recorded in prefrontal cortex of urethane anesthetized lurcher (Lc/+) mice with 100% loss of cerebellar Purkinje cells and wildtype (+/+) control mice. Cerebellar stimulation with 25 and 100 pulses evoked prefrontal cortex DA efflux in +/+ mice that persisted for 12 and 25 s poststimulation, respectively. In contrast, 25 pulse cerebellar stimulation failed to evoke prefrontal cortex DA efflux in Lc/+ mice indicating a dependency on cerebellar Purkinje cell outputs. Dentate nucleus stimulation (25 pulses) evoked a comparable but briefer (baseline recovery within 7 s) increase in prefrontal cortex DA efflux compared to similar cerebellar stimulation in +/+ mice. However, in Lc/+ mice 25 pulse dentate nucleus evoked prefrontal cortex DA efflux was attenuated by 60% with baseline recovery within 4 s suggesting that dentate nucleus outputs to prefrontal cortex remain partially functional. DA reuptake blockade enhanced 100 pulse stimulation evoked prefrontal cortex responses, while serotonin or norepinephrine reuptake blockade were without effect indicating the specificity of the amperometric recordings to DA. Results provide neurochemical evidence that the cerebellum can modulate DA efflux in the prefrontal cortex. Together, these findings may explain why cerebellar and frontal cortical pathologies co-occur, and may provide a mechanism that accounts for the diversity of symptoms common to multiple developmental disorders.

Cerebellar Modulation of Frontal Cortex Dopamine Efflux in Mice: Relevance to Autism and Schizophrenia

PubMed Central

MITTLEMAN, GUY; GOLDOWITZ, DANIEL; HECK, DETLEF H.; BLAHA, CHARLES D.

2013-01-01

Cerebellar and frontal cortical pathologies have been commonly reported in schizophrenia, autism, and other developmental disorders. Whether there is a relationship between prefrontal and cerebellar pathologies is unknown. Using fixed potential amperometry, dopamine (DA) efflux evoked by cerebellar or, dentate nucleus electrical stimulation (50 Hz, 200 μA) was recorded in prefrontal cortex of urethane anesthetized lurcher (Lc/+) mice with 100% loss of cerebellar Purkinje cells and wildtype (+/+) control mice. Cerebellar stimulation with 25 and 100 pulses evoked prefrontal cortex DA efflux in +/+ mice that persisted for 12 and 25 s poststimulation, respectively. In contrast, 25 pulse cerebellar stimulation failed to evoke prefrontal cortex DA efflux in Lc/+ mice indicating a dependency on cerebellar Purkinje cell outputs. Dentate nucleus stimulation (25 pulses) evoked a comparable but briefer (baseline recovery within 7 s) increase in prefrontal cortex DA efflux compared to similar cerebellar stimulation in +/+ mice. However, in Lc/+ mice 25 pulse dentate nucleus evoked prefrontal cortex DA efflux was attenuated by 60% with baseline recovery within 4 s suggesting that dentate nucleus outputs to prefrontal cortex remain partially functional. DA reuptake blockade enhanced 100 pulse stimulation evoked pre-frontal cortex responses, while serotonin or norepinephrine reuptake blockade were without effect indicating the specificity of the amperometric recordings to DA. Results provide neurochemical evidence that the cerebellum can modulate DA efflux in the prefrontal cortex. Together, these findings may explain why cerebellar and frontal cortical pathologies co-occur, and may provide a mechanism that accounts for the diversity of symptoms common to multiple developmental disorders. PMID:18435424