Efflux pump

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Xenon for tunnelling analysis of the efflux pump component OprN

Xenon for tunnelling analysis of the efflux pump component OprN

☯ These authors contributed equally to this work. * isabelle.broutin@parisdescartes.fr (IB); thierry.prange@parisdescartes.fr (TP) Abstract 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 mecha- nism 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.
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Mechanistic and Structural Insights Into the Unique TetR-Dependent Regulation of a Drug Efflux Pump in Mycobacterium abscessus

Mechanistic and Structural Insights Into the Unique TetR-Dependent Regulation of a Drug Efflux Pump in Mycobacterium abscessus

MAB_4384 Specifically Regulates Susceptibility to TAC Analogs in M. abscessus We recently showed that mutations in the MAB_4384 regulator were associated with the transcriptional induction of the divergently oriented adjacent genes coding for an MmpS5/MmpL5 efflux pump and accounting for high resistance levels toward various TAC analogs ( Halloum et al., 2017 ). To gain more insight into this drug resistance mechanism in M. abscessus, detailed genetic, functional and structural characterizations of the MAB_4384 regulator were undertaken. First, the expression profile of 19 mmpL genes was analyzed by qRT-PCR using the M. abscessus D15_S4 strain which possesses an early stop codon in MAB_4384 resulting in high resistance levels to the TAC analogs D6, D15 and D17 (MIC > 200 µg/mL), presumably due to derepression of the MmpS5/MmpL5 efflux pump machinery ( Halloum et al., 2017 ). The results clearly showed a pronounced increase in the expression level of MAB_4382c (mmpL5) mRNA in D15_S4 in comparison to the wild-type strain as reported previously, while no marked effect on the remaining mmpL genes was detected (Figure 1A). The expression levels of tgs1, encoding the primary triacylglycerol synthase responsible for the accumulation of triglycerides in M. abscessus ( Viljoen et al., 2016 ) was included as unrelated gene control. As expected, expression of tgs1 stayed unchanged (Figure 1A). To further confirm these results, the MAB_4384 and MAB_4382c genes were inactivated by homologous recombination using the recently developed genetic tool dedicated to facilitate gene disruption in M. abscessus ( Viljoen et al., 2018 ), as illustrated in Supplementary Figures S1A,B. The mutant strain, designated MAB_4384::pUX1, failed to show morphological changes (Supplementary Figure S1C) and grew similarly to its parental strain and the MAB_4382c::pUX1 mutant (Supplementary Figure S1D), suggesting that MAB_4384 does not play a significant role under normal in vitro conditions. However, this mutant exhibited high resistance levels to D6, D15, and D17 (MIC > 200 µg/mL, corresponding to >8-, >32-, and >16- fold-increases in MIC levels, respectively) (Table 2) similarly to our previous results for D15_S4 ( Halloum et al., 2017 ), thus validating the expected phenotype of the strain. Analysis of the transcriptional profile of all 19 mmpL genes in MAB_4384::pUX1 confirmed the results obtained in the D15_S4 strain (Figure 1B). Interestingly, expression of MAB_4384 itself was significantly induced in MAB_4384::pUX1, (Figure 1C), albeit lower than the expression level of mmpL5, thus indicating that MAB_4384 is self-regulated.
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MexEF-OprN efflux pump exports the Pseudomonas quinolone signal (PQS) precursor HHQ (4-hydroxy-2-heptylquinoline)

MexEF-OprN efflux pump exports the Pseudomonas quinolone signal (PQS) precursor HHQ (4-hydroxy-2-heptylquinoline)

Abstract Bacterial cells have evolved the capacity to communicate between each other via small diffusible chemical signals termed autoinducers. Pseudomonas aeruginosa is an opportunistic pathogen involved, among others, in cystic fibrosis complications. Virulence of P. aeruginosa relies on its ability to produce a number of autoinducers, including 4-hydroxy- 2-alkylquinolines (HAQ). In a cell density-dependent manner, accumulated signals induce the expression of multiple targets, especially virulence factors. This phenomenon, called quorum sensing, promotes bacterial capacity to cause disease. Furthermore, P. aeruginosa possesses many multidrug efflux pumps conferring adaptive resistance to antibiotics. Activity of some of these efflux pumps also influences quorum sensing. The present study demonstrates that the MexEF-OprN efflux pump modulates quorum sensing through secretion of a signalling molecule belonging to the HAQ family. Moreover, activation of MexEF-OprN reduces virulence factor expression and swarming motility. Since MexEF-OprN can be activated in infected hosts even in the absence of antibiotic selective pressure, it could promote establishment of chronic infections in the lungs of people suffering from cystic fibrosis, thus diminishing the immune response to virulence factors. Therapeutic drugs that affect multidrug efflux pumps and HAQ-mediated quorum sensing would be valuable tools to shut down bacterial virulence.
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<em>In vitro</em> Investigation of the MexAB Efflux Pump From <em>Pseudomonas aeruginosa</em>

<em>In vitro</em> Investigation of the MexAB Efflux Pump From <em>Pseudomonas aeruginosa</em>

Pseudomonas aeruginosa, which is protected by an outer membrane, efflux transporters are organized as tripartite systems where MexB, the efflux pump located in the inner membrane, works in conjunction with MexA, a periplasmic protein, and OprM, an outer membrane channel. The cytoplasmic inner membrane protein acts as an energy-dependent pump with broad substrate specificity. The outer membrane protein acts as a porin whereas the third one is located in the periplasmic space and is thought to stabilize the whole complex 3 . In the following, we focus on the design of a functional assay for the MexA MexB assembly.
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The overexpression of the efflux pump Tpo1 leads to the bleomycin resistance in Saccharomyces cerevisiae

The overexpression of the efflux pump Tpo1 leads to the bleomycin resistance in Saccharomyces cerevisiae

Based on this finding, we used IMP2 as a tool to make double mutants. Our data revealed that when TPO1, TPO2 and QDR3 are mutated in combination with IMP2, cells become extremely sensitive to bleomycin and polyamines, as well as the imp2 mutant alone. This led us to propose that Imp2 might regulate the expression of an efflux pump whether or not it is the only Tpo1. In contrast with this hypothesis, other reports suggest that the hypersensitivity exhibited by the imp2 mutant towards bleomycin, does not result from the deficiency of efflux pumps expression [94-100, 131] as we thought. Nor is it a consequence of a defect in antioxidant activity [101], and it is not due to unrepaired DNA strand breaks [40], since imp2 mutants are known to be insensitive to ionizing radiations and MMS [92, 93].
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Characterization of a Novel Pyranopyridine Inhibitor of the AcrAB Efflux Pump of Escherichia coli

Characterization of a Novel Pyranopyridine Inhibitor of the AcrAB Efflux Pump of Escherichia coli

3. Nagano K, Nikaido H. 2009. Kinetic behavior of the major multidrug efflux pump AcrB of Escherichia coli. Proc Natl Acad Sci U S A 106:5854-5858. 4. Dewitt SK, Adelberg EA. 1962. The Occurrence of a Genetic Transposition in a Strain of Escherichia Coli. Genetics 47:577-585.

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Quantum dot probes for the quantitative study of drug transport by the MacAB TolC efflux pump in lipid scaffolds

Quantum dot probes for the quantitative study of drug transport by the MacAB TolC efflux pump in lipid scaffolds

Efflux pumps are composed of an inner membrane transporter, a periplasmic protein attached to the inner membrane, and an extrusion channel inserted in the outer membrane. They assemble to allow for an efficient transport of drugs, dyes or detergents outside the cell, bypassing the periplasm. The first line of defense is composed of efflux pumps that belong to the Resistance-Nodulation-Cell-Division (RND) superfamily, with, as most studied members AcrAB-TolC tripartite system from E. coli or MexAB OprM from Pseudomonas aeruginosa. Recently, the MacAB TolC tripartite efflux pump from Escherichia coli raised additional and increasing interest 2 . By contrast to the RND efflux pumps that perform transport by consuming
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Sorafenib Overcomes Irinotecan Resistance in Colorectal Cancer by Inhibiting the ABCG2 Drug-Efflux Pump

Sorafenib Overcomes Irinotecan Resistance in Colorectal Cancer by Inhibiting the ABCG2 Drug-Efflux Pump

Abstract Despite recent advances in the treatment of colorectal cancer (CRC), tumor resistance is a frequent cause of chemotherapy failure. Therefore, new treatment options are needed to improve survival of patients with irinotecan-refractory CRCs, particularly those bearing KRAS mutations that preclude the use of anti-EGFR therapies. In this study, we investigated whether sorafenib could reverse irinotecan resistance, thereby enhancing the therapeutic efficacy of routinely used irinotecan-based chemotherapy. We used both in vitro (the HCT116, SW48, SW620, and HT29 colon adenocarcinoma cell lines and four SN-38–resistant HCT-116 and SW48 clones) and in vivo models (nude mice xenografted with SN-38–resistant HCT116 cells) to test the efficacy of sorafenib alone or in combination with irinotecan or its active metabolite, SN-38. We have shown that sorafenib improved the antitumoral activity of irinotecan in vitro, in both parental and SN-38–resistant colon adenocarcinoma cell lines independently of their KRAS status, as well as in vivo, in xenografted mice. By inhibiting the drug-efflux pump ABCG2, sorafenib favors irinotecan intracellular accumulation and enhances its toxicity. Moreover, we found that sorafenib improved the efficacy of irinotecan by inhibiting the irinotecan- mediated p38 and ERK activation. In conclusion, our results show that sorafenib can suppress resistance to irinotecan and suggest that sorafenib could be used to overcome resistance to irinotecan-based chemotherapies in CRC, particularly in KRAS-mutated tumors. Mol Cancer Ther; 12(10); 2121–34. 2013 AACR.
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Functional investigation of the efflux pump MexA–MexB-OprM of Pseudomonas aeruginosa

Functional investigation of the efflux pump MexA–MexB-OprM of Pseudomonas aeruginosa

4.3 The tripartite assembly of efflux pumps from resistance nodulation division family As developed in the previous paragraph, efflux pumps from the RND family consist of three main partners. Investigating each of them separately was the first logic thing to start with and, thanks to independent investigations of the RND, MFP and OMF, we now have an overall understanding of each of these fascinating proteins. Nevertheless, since efflux pump work as a tripartite machinery, it is indispensable to investigate the three proteins together in order to deeply understand their mutual way of action. Interestingly, more and more articles about interactions between elements of the pump are published and models of the pump assembly have been proposed. To date the stoichiometry of the MFP within the efflux pump is still a matter of debate even though one hypothesis is getting more and more credit. The sequence of events leading to the assembly of the pump remains an open question. The three best studied multidrug efflux pumps are AcrA-AcrB-TolC, CusB-CusA- CusC from E. coli and MexA-MexB-OprM from P. aeruginosa (with respectively: the MFP-the RND transporter-the OMF). The substrate specificities of AcrAB-TolC and MexAB-OprM are very similar. However, when expressed under the same genetic background as in E coli, the resistance pattern of the MexAB-OprM system differs
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In vitro transport activity of the fully assembled MexAB-OprM efflux pump from Pseudomonas aeruginosa

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

is assembled, MexB might adopt a conformation that allows the recruitment of substrate at a lower concentration than in the absence of OprM. The first in vitro reconstitution of a tripartite efflux pump is presented here. This achievement is highly beneficial for the study of multidrug efflux pumps. The assay relies on the use of ethidium bromide as a substrate for MexB but this is not a limitation. Indeed, this assay could very well be adapted to any substrate because the RNA scaffold encapsulated in the OprM proteoliposomes can be modified. The RNA scaffold has a restriction site in which any nucleic acid material can be inserted 25 . Furthermore, DNA/RNA aptamers specific for an increasing number of targets are now available, and it has been shown that it is possible to rationally convert non-fluorescent aptamers into fluorescent reporters 26 . It has also been shown that fluorescent aptamers can be selected directly from random sequence DNA libraries in vitro 27 . Hence, aptamers specific for any substrate could be used as probes to monitor assembly and transport activity.
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Antimicrobial and Efflux Pump Inhibitory Activity of Caffeoylquinic Acids from Artemisia absinthium against Gram-Positive Pathogenic Bacteria

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

Methodology/Principal Findings: In this study we report the identification and characterization of 49,59-O-dicaffeoylquinic acid (49,59-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, 39,59-ODCQA, 49,59-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 49,59-ODCQA with pump inhibitory activity whereas 39,59-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.
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Sorafenib overcomes irinotecan resistance in colorectal cancer by inhibiting the ABCG2 drug-efflux pump.: Sorafenib inhibits ABCG2 and overcome irinotecan resistance.

Sorafenib overcomes irinotecan resistance in colorectal cancer by inhibiting the ABCG2 drug-efflux pump.: Sorafenib inhibits ABCG2 and overcome irinotecan resistance.

These results indicate that, in SN-38 resistant HCT116-SN50 xenografts, the combination of sorafenib plus irinotecan inhibits tumor cell proliferation and angiogenesi[r]

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Leakage flow simulation in a specific pump model

Leakage flow simulation in a specific pump model

[11] Eisele K., Zhang Z., Casey M. V., Gülich J., Schachenmann A. 1997 Flow analysis in a Pump Diffuser Part 1 : LDA and PTV Measurements of the Unsteady FlowTransactions of ASME, Journal of Fluids EngineeringVol. 119 p. 968-977 [12] Culver R., Liu F. 2009 Plane Method for Flutter Computation in Multi-stage Turbomachines47th AIAA Aerospace Sciences Meeting Including The New Horizons Forum and Aerospace Exposition January5-8, (Orlando, Florida)

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Polarization-resolved pump-probe spectroscopy with high harmonics

Polarization-resolved pump-probe spectroscopy with high harmonics

Figure 7. Pump-probe scans for H21 in a 50 /50 mixture of argon and nitrogen, around the half revival of N 2 . The blue line corresponds to the conventional configuration and the red line to the polarization-resolved configuration. We have shown that polarization-resolved spectroscopy could be used to increase the contrast in the detection of alignment revivals in nitrogen. Performing complete polarimetric measurements as a function of the pump–probe delay would allow an additional contrast enhancement. Small variations of the polarization angle can be determined with a very high accuracy through the fits with the Malus’ law (figure 4 ). Knowing the total harmonic signal, it is then possible to calculate the value of the orthogonal component of the harmonic field. In that case there is no background: the orthogonal component is zero if the polarization angle is zero. As soon as a small degree of molecular alignment exists, the polarization angle deviates and creates a non-zero orthogonal background. This method could be used to study subtle effects in the rotational wavepacket dynamics.
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Parametric gain shaping from a structured pump pulse

Parametric gain shaping from a structured pump pulse

D. Bigourd, C. Fourcade-Dutin and H. Maillotte are with the Institut FEMTO- ST, Département d’Optique, UMR 6174 CNRS-Université Bourgogne Copyright (c) 2018 IEEE representative of the spectral gain) is recorded with a spectrograph, the gain temporal structure cannot be directly inferred since the measurement is a time integrated (averaged) spectrum. Therefore, in our case, the structured gain is measured thanks to a chirped signal where the instantaneous frequency evolves quasi-linearly with time. The temporal gain shape influences directly the signal in the temporal and spectral domains. This scheme should be of prime interest to tune a pulse in any experiment requiring a chirped signal as in lidar [13], optical time- stretch imaging [14] or laser amplifier [15]. The letter is organized as follows. Section II explains the principle of the temporal gain shaping from a structured pump pulse through numerical simulations. Section III presents the experimental set-up and some examples of shaped gains. Finally, Section IV provides concluding remarks.
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Innovative linear pulsatile pump for heart assistance circulatory

Innovative linear pulsatile pump for heart assistance circulatory

The present contribution deals with the development of a magneto-active pump devoted to ventricular assist device. The actuator studied is a cylindrical linear switched reluctance motor equipped with a unidirectional mechanical valve inside the tubular mover in order to allow a pulsatile flow. On the bases of theoretical results, a demonstrator has been designed and built in the laboratory. Finite element analysis of this demonstrator has been performed and preliminary tests are detailed showing the flow and pressure characteristics.
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Antibiotic efflux pumps in Gram-negative bacteria: the inhibitor response strategy

Antibiotic efflux pumps in Gram-negative bacteria: the inhibitor response strategy

recently proposed mechanisms including the ‘peristaltic pump mechanism’ and the ‘functionally rotating ordered multidrug binding change mechanism’ 27,31 are very promising but not totally satisfactory for the definition of suitable physico- chemical characteristics for a future efficient inhibitor. To date, the activity of putative EPIs is deduced from various measures using the susceptibilities towards different antibiotics in the absence and presence of the agent. Similarly, the degree of inhi- bition of antibiotic efflux is determined by the variation of intra- cellular concentrations induced by the addition of tested compounds and compared to the level obtained in the presence of a poison that collapses the membrane energy necessary for the drug expulsion. To quantify the effect of EPIs on an efflux pump, more effective assays are needed for determination of kinetic parameters and their relationships to the structure of the component of the efflux pump that is affected. 52 The definition
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NRC wave pump tests for numerical model development

NRC wave pump tests for numerical model development

released in bubbles (flashing) and are reabsorbed as the bubbles collapse (cavitation). These implosions cause severe pressure spikes that pit and damage pump internal parts and cause vibrations. Video of the pump cylinder was recorded for most of the tests. Bubbles are ob- served in the pump cylinder in tests with a period less than 4 seconds. Cavitation is not modelled in the numerical model. While this may not affect overall cylinder pressures, it is possible to fracture a fluid cylinder and/or piping and damage the pump drive end internals with high pressure surges that occur when fluid is cavitating. [4]
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Innovative linear pulsatile pump for heart assistance circulatory

Innovative linear pulsatile pump for heart assistance circulatory

1 I NTRODUCTION Heart failure is one of the most common diseases in developed countries. Because the number of donor heart is limited, patients suffering from end-stage heart failure require mechanical circulatory support as bridge to transplant, bridge to recovery or destination therapy. Most of the time, the mechanical circulatory support is a left ventricular assist device (VAD) or artificial heart pump developed to aid the failed left ventricle by supplying additional flow of blood to the body. In case of severe heart failure, a total artificial heart (TAH) completely replaces the native heart and assumes the function of both ventricles [1].
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Computer simulation of ground coupled heat pump systems

Computer simulation of ground coupled heat pump systems

L’accès à ce site Web et l’utilisation de son contenu sont assujettis aux conditions présentées dans le site LISEZ CES CONDITIONS ATTENTIVEMENT AVANT D’UTILISER CE SITE WEB. NRC Publicat[r]

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