Search

Background Common genetic variants of the enzymes and efflux pump involved in tacrolimus disposition have been associated with calcineurin inhibitor nephrotoxicity, but their importance is unclear because of the multifactorial background of renal fibrosis. This study explores the pro-fibrotic response of tacrolimus exposure in relation to the differential capacity for tacrolimus metabolism in proximal tubule cells (PTCs) with a variable (pharmaco)genetic background. Methods PTCs were obtained from protocol allograft biopsies with different combinations of CYP3A5 and ABCB1 variants and were incubated with tacrolimus within the concentration range found in vivo. Gene and protein expression, CYP3A5 and P-glycoprotein function, and tacrolimus metabolites were measured in PTC. Connective tissue growth factor (CTGF) expression was assessed in protocol biopsies of kidney allograft recipients. Results PTCs produce CTGF in response to escalating tacrolimus exposure, which is approximately 2-fold higher in cells with the CYP3A5*1 and ABCB1 TT combination in vitro. Increasing tacrolimus exposure results in relative higher generation of the main tacrolimus metabolite {13-O-desmethyl tacrolimus [M1]} in cells with this same genetic background. Protocol biopsies show a larger increase in in vivo CTGF tissue expression over time in TT vs. CC/CT but was not affected by the CYP3A5 genotype. Conclusions Tacrolimus exposure induces a pro-fibrotic response in a PTC model in function of the donor pharmacogenetic background associated with tacrolimus metabolism. This finding provides a mechanistic insight into the nephrotoxicity associated with tacrolimus treatment and offers opportunities for a tailored immunosuppressive treatment. [ABSTRACT FROM AUTHOR]

Objectives: To determine whether expression of efflux pumps and antibiotic susceptibility are altered in Escherichia coli in response to efflux inhibition.Methods: The promoter regions of nine efflux pump genes (acrAB, acrD, acrEF, emrAB, macAB, cusCFBA, mdtK, mdtABC, mdfA) were fused to gfp in pMW82 and fluorescence from each reporter construct was used as a measure of the transcriptional response to conditions in which AcrB was inhibited, absent or made non-functional. Expression was also determined by RT-qPCR. Drug susceptibility of efflux pump mutants with missense mutations known or predicted to cause loss of function of the encoded efflux pump was investigated.Results: Data from the GFP reporter constructs revealed that no increased expression of the tested efflux pump genes was observed when AcrB was absent, made non-functional, or inhibited by an efflux pump inhibitor/competitive substrate, such as PAβN or chlorpromazine. This was confirmed by RT-qPCR for PAβN and chlorpromazine; however, a small but significant increase in macB gene expression was seen when acrB is deleted. Efflux inhibitors only synergized with antibiotics in the presence of a functional AcrB. When AcrB was absent or non-functional, there was no impact on MICs when other efflux pumps were also made non-functional.Conclusions: Absence, loss-of-function, or inhibition of E. coli AcrB did not significantly increase expression of other efflux pump genes, which suggests there is no compensatory mechanism to overcome efflux inhibition and supports the discovery of inhibitors of AcrB as antibiotic adjuvants. [ABSTRACT FROM AUTHOR]

Background: Tigecycline and carbapenems are regarded as vital antimicrobials to treat serious bacterial infections. Co-occurrence of resistance genes conferring resistance to both tigecycline and carbapenems in Pseudomonas spp. was not investigated.Objectives: To characterize a megaplasmid co-harbouring tmexCD1-toprJ1 and blaVIM-2 in Pseudomonas putida of migratory bird origin.Methods: One tigecycline- and carbapenem-resistant strain was isolated and characterized by antimicrobial susceptibility testing, conjugation assay, WGS and bioinformatics analysis.Results: The strain P. putida ZXPA-20 resistant to meropenem and tigecycline was positive for blaVIM-2 and tmexCD1-toprJ1 genes. The gene blaVIM-2 was inserted into the backbone of the megaplasmid pZXPA-20 within a Tn5090-like structure. The genetic context of tmexCD1-toprJ1 in the megaplasmid was identical to many chromosomal tmexCD1-toprJ1 of Pseudomonas species. Plasmid-mediated tmexCD1-toprJ1 gene cluster in Pseudomonas spp. was more common than that in Klebsiella pneumoniae. To the best of our knowledge, this is the first report of co-occurrence of blaVIM-2 and tmexCD1-toprJ1 in one plasmid.Conclusions: Emergence of plasmid-mediated carbapenem and tigecycline resistance genes in P. putida from migratory birds highlighted the importance of surveillance of novel mobile resistance genes in migratory birds, which may play a vital role in global transmission of novel resistance genes. [ABSTRACT FROM AUTHOR]

Combination therapy is a common antibiotic treatment strategy that aims at minimizing the risk of resistance evolution in several infectious diseases. Nonetheless, evidence supporting its efficacy against the nosocomial opportunistic pathogen Pseudomonas aeruginosa remains elusive. Identification of the possible evolutionary paths to resistance in multidrug environments can help to explain treatment outcome. For this purpose, we here performed whole-genome sequencing of 127 previously evolved populations of P. aeruginosa adapted to sublethal doses of distinct antibiotic combinations and corresponding single-drug treatments, and experimentally characterized several of the identified variants. We found that alterations in the regulation of efflux pumps are the most favored mechanism of resistance, regardless of the environment. Unexpectedly, we repeatedly identified intergenic variants in the adapted populations, often with no additional mutations and usually associated with genes involved in efflux pump expression, possibly indicating a regulatory function of the intergenic regions. The experimental analysis of these variants demonstrated that the intergenic changes caused similar increases in resistance against single and multidrug treatments as those seen for efflux regulatory gene mutants. Surprisingly, we could find no substantial fitness costs for a majority of these variants, most likely enhancing their competitiveness toward sensitive cells, even in antibiotic-free environments. We conclude that the regulation of efflux is a central target of antibiotic-mediated selection in P. aeruginosa and that, importantly, changes in intergenic regions may represent a usually neglected alternative process underlying bacterial resistance evolution, which clearly deserves further attention in the future. [ABSTRACT FROM AUTHOR]

Background: Mutations that inactivate MarR reduce susceptibility to ciprofloxacin and competitive growth fitness in Escherichia coli. Both phenotypes are caused by overexpression of the MarA regulon, which includes the AcrAB-TolC drug efflux pump.Objectives: We asked whether compensatory evolution could reduce the fitness cost of MarR-inactivating mutations without affecting resistance to ciprofloxacin.Methods: The cost of overexpressing the AcrAB-TolC efflux pump was measured independently of MarA overexpression. Experimental evolution of MarR-inactive strains was used to select mutants with increased fitness. The acquired mutations were identified and their effects on drug susceptibility were measured.Results: Overexpression of the AcrAB-TolC efflux pump was found not to contribute to the fitness cost of MarA regulon overexpression. Fitness-compensatory mutations were selected in marA and lon. The mutations reduced the level of MarA protein thus reducing expression of the MarA regulon. They restored growth fitness but also reduced resistance to ciprofloxacin.Conclusions: The fitness cost caused by overexpression of the MarA regulon has multiple contributing factors. Experimental evolution did not identify any single pump-independent cost factor. Instead, efficient fitness compensation occurred only by mechanisms that reduce MarA concentration, which simultaneously reduce the drug resistance phenotype. This resistance/fitness trade-off is a barrier to the successful spread of MarR inactivation mutations in clinical isolates where growth fitness is essential. [ABSTRACT FROM AUTHOR]

Acid stress is an environmental problem for plants and fresh water cyanobacteria like the filamentous, heterocyst forming species Anabaena sp. PCC 7120 (hereafter Anabaena sp.). Heterocyst differentiation, cell–cell communication and nitrogen fixation has been deeply studied in this model organism, but little is known about the cellular response of Anabaena sp. to decreased pH values, causing acid stress. ATP-binding cassette (ABC) transporters are involved in acid stress response in other bacteria, by exporting proteins responsible for survival under acidification. The genome of Anabaena sp. encodes numerous ABC transporter components, whose function is not known yet. Here, we describe the function of the gene all5304 encoding a protein with homology to membrane fusion proteins of tripartite efflux pumps driven by ABC transporters like HlyBD-TolC of Escherichia coli. The all5304 mutant shows less resistance against low pH, even though the expression of the gene is independent from the pH of the medium. We compared the exoproteome of the wild type and mutant cultures and identified three proteins-candidate substrates of the putative transporter. Including the in silico analysis of All5304, our results suggest that All5304 functions as part of an efflux pump, secreting of a protein necessary for acid tolerance in Anabaena sp. [ABSTRACT FROM AUTHOR]

The established role of bitter melon juice (BMJ), a natural product, in activating master metabolic regulator adenosine monophosphate-activated protein kinase in pancreatic cancer (PanC) cells served as a basis for pursuing deeper investigation into the underlying metabolic alterations leading to BMJ efficacy in PanC. We investigated the comparative metabolic profiles of PanC cells with differential KRAS mutational status on BMJ exposure. Specifically, we employed nuclear magnetic resonance (NMR) metabolomics and in vivo imaging platforms to understand the relevance of altered metabolism in PanC management by BMJ. Multinuclear NMR metabolomics was performed, as a function of time, post-BMJ treatment followed by partial least square discriminant analysis assessments on the quantitative metabolic data sets to visualize the treatment group clustering; altered glucose uptake, lactate export and energy state were identified as the key components responsible for cell death induction. We next employed PANC1 xenograft model for assessing in vivo BMJ efficacy against PanC. Positron emission tomography ([18FDG]-PET) and magnetic resonance imaging on PANC1 tumor-bearing animals reiterated the in vitro results, with BMJ-associated significant changes in tumor volumes, tumor cellularity and glucose uptake. Additional studies in BMJ-treated PanC cells and xenografts displayed a strong decrease in the expression of glucose and lactate transporters GLUT1 and MCT4, respectively, supporting their role in metabolic changes by BMJ. Collectively, these results highlight BMJ-induced modification in PanC metabolomics phenotype and establish primarily lactate efflux and glucose metabolism, specifically GLUT1 and MCT4 transporters, as the potential metabolic targets underlying BMJ efficacy in PanC. [ABSTRACT FROM AUTHOR]

Objective: In this study, we develop and apply a high‐throughput screening protocol to investigate the activity of non‐ionic surfactants, with a broad range of hydrophilic–lipophilic balance values, against ABCB1‐mediated efflux transport and ABCC2‐mediated efflux transport. Methods: Caco‐2 cells were grown for 7 days in 96‐well plates, then washed and incubated with the test materials for 2 h in the presence of 2.5 μm of either rhodamine 123 (R‐123) or 5(6)‐Carboxy‐2′,7′ dichlorofluorescein diacetate as probes of ABCB1 and ABCC2, respectively. Key findings: Of the surfactants tested, no activity against ABCC2 was detected and all surfactants showing efficacy against ABCB1 had a HLB value of 22 or below. Inhibition of ABCB1 was seen in the order of efficacy to be poloxamer 335 > poloxamer 40 > Crovol A‐70 > Myrj S‐40 > poloxamer 184 > poloxamer 182 > Etocas 40 > Tween 20 > Etocas 29 > Tween 80 > Acconon C‐44 > Span 20. With regard to this inhibition, the distribution of hydrophilic regions is more important than the HLB value. Conclusion: This work demonstrates a high‐throughput protocol for detecting materials that can modulate ABCB1‐mediated efflux. These surfactants could be exploited to improve oral delivery of drugs prone to efflux. [ABSTRACT FROM AUTHOR]

Objectives: The aim of this research was to assess regional difference in the intestinal absorption of ranitidine HCl as an indicator for the potential effect of P‐glycoprotein (P‐gp) efflux transporters. Methods: In situ rabbit intestinal perfusion was used to investigate absorption of ranitidine HCl, a substrate for P‐gp efflux from duodenum, jejunum, ileum and colon. This was conducted both in the presence and absence of piperine as P‐gp inhibitor. Key findings: Ranitidine HCl was incompletely absorbed from rabbit intestine. The length normalized absorptive clearance (PeA/L) of ranitidine HCl was ranked as colon > duodenum > jejunum > ileum. This is the reverse order of the magnitude of P‐gp expression. Coperfusion of piperine with ranitidine HCl significantly increased the PeA/L of ranitidine HCl from jejunum and ileum with no significant change on the absorption from duodenum and colon. This was confirmed by significant reduction in the length required for complete ranitidine HCl absorption from jejunum and ileum in presence piperine. Conclusions: The results indicate that P‐gp transporters play a major role in determining regional difference in intestinal absorption of ranitidine HCl. Thus, the regional absorption of drugs may be taken as an indirect indication for the role of P‐gp in intestinal absorption. [ABSTRACT FROM AUTHOR]

Background: Reviews assessing the genetic basis of ciprofloxacin resistance in Escherichia coli have mostly been qualitative. However, to predict resistance phenotypes based on genotypic characteristics, it is essential to quantify the contribution of genotypic determinants to resistance.Objectives: We performed a systematic review to assess the relative contribution of known genomic resistance determinants to the MIC of ciprofloxacin in E. coli.Methods: PubMed and Web of Science were searched for English language studies that assessed ciprofloxacin MIC and presence or introduction of genetic determinants of ciprofloxacin resistance in E. coli. We included experimental and observational studies without time restrictions. Medians and ranges of MIC fold changes were calculated for individual resistance determinants and combinations thereof.Results: We included 66 studies, describing 604 E. coli isolates that carried at least one genetic ciprofloxacin resistance determinant. Mutations in gyrA and parC, genes encoding targets of ciprofloxacin, contribute to the largest fold changes in ciprofloxacin resistance in E. coli compared with the WT. Efflux and physical blocking or enzymatic modifications confer smaller increases in ciprofloxacin MIC than mutations in gyrA and parC. However, the presence of these other resistance mechanisms in addition to target alteration mutations further increases ciprofloxacin MIC, thus resulting in ciprofloxacin MIC increases ranging from 250- to 4000-fold.Conclusions: This quantitative review of genomic determinants of ciprofloxacin resistance in E. coli demonstrates the complexity of resistance phenotype prediction from genomic data and serves as a reference point for studies aiming to predict ciprofloxacin MIC from E. coli genomes. [ABSTRACT FROM AUTHOR]

Summary: High expression of the ATP‐binding cassette‐multi‐drug efflux protein 1 (MDR1) is a striking feature of mucosal‐associated invariant T (MAIT) cells, a prominent human innate‐like T cell subset. We demonstrate significantly higher MDR1 expression by CD8 + CD161 ++ Vα7.2 + MAIT cells than the phenotypically and functionally related CD8 + CD161 ++ Vα7.2‐subset and show MDR1 expression to be similarly high throughout MAIT CD4+ , CD8+ , double‐negative (DN) and double‐positive (DP) cell subsets. We demonstrate the MAIT cell‐predominant CD8+CD161++ subset to uniquely and efficiently efflux the cytotoxic anthracycline daunorubicin, retain function on daunorubicin exposure and demonstrate MDR1‐dependent protection from daunorubicin‐induced apoptosis. By contrast, CD8+CD161++Vα7.2+ MAIT cells were not protected from the anti‐proliferative and cytotoxic effects of the immunosuppressive MDR1 substrates tacrolimus and mycophenoic acid, although function following MAIT cell‐specific T cell receptor (TCR)‐dependent and ‐independent stimulation was preserved on in‐vitro exposure to these agents. Overall, our data further define MDR1 expression by CD161++ T and MAIT cells and demonstrate the potential for high MDR1 expression by MAIT cells to confer resistance to cytotoxic MDR1 substrates in vivo. As our understanding of the importance of MAIT cells in human immunity and immunopathology grows, this is an important observation for clinical contexts such as the treatment of malignancy, autoimmunity and post‐transplant immunosuppression. Mucosal Associated Invariant T (MAIT) cells express the multidrug efflux protein MDR1 across all subsets and at a significantly higher level than by other CD8+ T cells, including those that are CD161++ but Vα7.2α. This uniquely enabled MAIT cells to resist the cytotoxic effects of the anthracycline daunorubicin, but not the anti‐proliferative effects of immunosuppressants such as tacrolimus. Nonetheless, MAIT cell function was preserved upon exposure to both drug classes, which has important implications for the treatment of pathologies including malignancy, autoimmunity and transplant‐associated immunosuppression [ABSTRACT FROM AUTHOR]

Motivation Efflux protein plays a key role in pumping xenobiotics out of the cells. The prediction of efflux family proteins involved in transport process of compounds is crucial for understanding family structures, functions and energy dependencies. Many methods have been proposed to classify efflux pump transporters without considerations of any pump specific of efflux protein families. In other words, efflux proteins protect cells from extrusion of foreign chemicals. Moreover, almost all efflux protein families have the same structure based on the analysis of significant motifs. The motif sequences consisting of the same amount of residues will have high degrees of residue similarity and thus will affect the classification process. Consequently, it is challenging but vital to recognize the structures and determine energy dependencies of efflux protein families. In order to efficiently identify efflux protein families with considering about pump specific, we developed a 2 D convolutional neural network (2 D CNN) model called DeepEfflux. DeepEfflux tried to capture the motifs of sequences around hidden target residues to use as hidden features of families. In addition, the 2 D CNN model uses a position-specific scoring matrix (PSSM) as an input. Three different datasets, each for one family of efflux protein, was fed into DeepEfflux, and then a 5-fold cross validation approach was used to evaluate the training performance. Results The model evaluation results show that DeepEfflux outperforms traditional machine learning algorithms. Furthermore, the accuracy of 96.02%, 94.89% and 90.34% for classes A, B and C, respectively, in the independent test results show that our model can perform well and can be used as a reliable tool for identifying families of efflux proteins in transporters. Availability and implementation The online version of deepefflux is available at http://deepefflux.irit.fr. The source code of deepefflux is available both on the deepefflux website and at http://140.138.155.216/deepefflux/. Supplementary information Supplementary data are available at Bioinformatics online. [ABSTRACT FROM AUTHOR]

This study presents results from continuous measurements of stem CO2 efflux carried out for seven experimental seasons (from May to October) in a young Norway spruce forest. The objectives of the study were to determine variability in the response of stem CO2 efflux to stem temperature over the season and to observe differences in the calculated relationship between stem temperature and CO2 efflux based on full growing season data or on data divided into periods according to stem growth rate. Temperature sensitivity of stem CO2 efflux (Q 10) calculated for the established periods ranged between 1.61 and 3.46 and varied over the season, with the lowest values occurring in July and August. Q 10 calculated using data from the full growing seasons ranged between 2.30 and 2.94 and was often significantly higher than Q 10 calculated for the individual periods. Temperature-normalized stem CO2 efflux (R 10) determined using Q 10 from growing season data was overestimated when the temperature was below 10 °C and underestimated when the temperature was above 10 °C, compared with R 10 calculated using Q 10 established for the individual periods. The differences in daily mean R 10 calculated by these two approaches ranged between −0.9 and 0.2 μmol CO2 m−2 s−1. The results of this study confirm that long periods for determining the temperature dependence of stem CO2 efflux encompass different statuses of the wood (especially in relation to stem growth). This may cause bias in models using this relationship for estimating stem CO2 efflux as a function of temperature. [ABSTRACT FROM AUTHOR]

Efflux pumps are widely implicated in antibiotic resistance because they can extrude the majority of clinically relevant antibiotics from within cells to the extracellular environment. However, there is increasing evidence from many studies to suggest that the pumps also play a role in biofilm formation. These studies have involved investigating the effects of efflux pump gene mutagenesis and efflux pump inhibitors on biofilm formation, and measuring the levels of efflux pump gene expression in biofilms. In particular, several key pathogenic species associated with increasing multidrug resistance, such as Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus, have been investigated, whilst other studies have focused on Salmonella enterica serovar Typhimurium as a model organism and problematic pathogen. Studies have shown that efflux pumps, including AcrAB-TolC of E. coli, MexAB-OprM of P. aeruginosa, AdeFGH of A. baumannii and AcrD of S. enterica, play important roles in biofilm formation. The substrates for such pumps, and whether changes in their efflux activity affect biofilm formation directly or indirectly, remain to be determined. By understanding the roles that efflux pumps play in biofilm formation, novel therapeutic strategies can be developed to inhibit their function, to help disrupt biofilms and improve the treatment of infections. This review will discuss and evaluate the evidence for the roles of efflux pumps in biofilm formation and the potential approaches to overcome the increasing problem of biofilm-based infections. [ABSTRACT FROM AUTHOR]

Objectives: Staphylococcus aureus native efflux pump Tet38 confers resistance to tetracycline when overexpressed. tet38 expression is selectively upregulated in infection sites. This study evaluated the effect of Tet38 on tetracycline response in a murine subcutaneous abscess model.Methods: S. aureus MW2 and its tet38 mutant were injected subcutaneously on the opposite flanks of each mouse. The infected mice were treated with tetracycline (10 mg/kg) or PBS (control) intraperitoneally every 12 h. The efficacy of tetracycline against S. aureus was measured by the relative change in viable bacteria in the abscesses 24 h after infection compared with the initial inoculum. Plasmid-based tet38-complemented strains were created and used to infect the mice followed by tetracycline or PBS treatment.Results: The increased bacterial loads of S. aureus MW2 and its tet38 mutant in the abscess after 24 h were similar. Tetracycline produced significant decreases of both MW2 and the tet38 mutant compared with control. Although the tetracycline MICs for MW2 and the tet38 mutant did not differ in vitro, the antibacterial effect of tetracycline was significantly 2-fold greater in the tet38 mutant compared with the MW2 parental strain in vivo with a decrease of 0.67 ± 0.21 and 0.35 ± 0.19 log10 cfu/abscess, respectively (P < 0.05). The increased tetracycline activity in the tet38 mutant was complemented by plasmid-encoded tet38.Conclusions: This study demonstrated that selective increased expression of tet38 in an abscess can affect tetracycline efficacy against S. aureus in vivo, highlighting an effect of native efflux pumps on response to therapy not reflected by testing in vitro. [ABSTRACT FROM AUTHOR]

Simocyclinones are antibiotics produced by Streptomyces and Kitasatospora species that inhibit the validated drug target DNA gyrase in a unique way, and they are thus of therapeutic interest. Structural approaches have revealed their mode of action, the inducible-efflux mechanism in the producing organism, and given insight into one step in their biosynthesis. The crystal structures of simocyclinones bound to their target (gyrase), the transcriptional repressor SimR and the biosynthetic enzyme SimC7 reveal fascinating insight into how molecular recognition is achieved with these three unrelated proteins. [ABSTRACT FROM AUTHOR]

Background. Macrolide efflux encoded by mef(E)/mel and ribosomal methylation encoded by erm(B) confer most macrolide resistance in Streptococcus pneumoniae. Introduction of the heptavalent pneumococcal conjugate vaccine (PCV7) in 2000 reduced macrolide-resistant invasive pneumococcal disease (MR-IPD) due to PCV7 serotypes (6B, 9V, 14, 19F, and 23F). Methods. In this study, the impact of PCV7 and PCV13 on MR-IPD was prospectively assessed. A 20-year study of IPD performed in metropolitan Atlanta, Georgia, using active, population-based surveillance formed the basis for this study. Genetic determinants of macrolide resistance were evaluated using established techniques. Results. During the decade of PCV7 use (2000–2009), MR-IPD decreased rapidly until 2002 and subsequently stabilized until the introduction of PCV13 in 2010 when MR-IPD incidence decreased further from 3.71 to 2.45/100 000 population. In 2003, serotype 19A CC320 isolates containing both mef(E)/mel and erm(B) were observed and rapidly expanded in 2005–2009, peaking in 2010 (incidence 1.38/100 000 population), accounting for 36.1% of MR-IPD and 11.7% of all IPD isolates. Following PCV13 introduction, dual macrolide-resistant IPD decreased 74.1% (incidence 0.32/100 000 in 2013). However, other macrolide-resistant serotypes (eg, 15A and 35B) not currently represented in PCV formulations increased modestly. Conclusions. The selective pressures of widespread macrolide use and PCV7 and PCV13 introductions on S. pneumoniae were associated with changes in macrolide resistance and the molecular basis over time in our population. Durable surveillance and programs that emphasize the judicious use of antibiotics need to continue to be a focus of public health strategies directed at S. pneumoniae. [ABSTRACT FROM AUTHOR]

Background: Recent studies have shown that the cholesterol efflux capacity of HDL is a better predictor of cardiovascular disease (CVD) than HDL cholesterol. However, the standard procedures used for measuring cholesterol efflux capacity involve radioisotope-labeled cholesterol and cultured macrophages. Thus, a simpler method to measure HDL functionality is needed for clinical application. Methods: We established a cell-free assay system to evaluate the capacity of HDL to accept additional cholesterol, which we named cholesterol "uptake capacity," using fluorescently labeled cholesterol and an anti-apolipoprotein A1 antibody. We quantified cholesterol uptake capacity of apolipoprotein B (apoB)-depleted serum samples from patients with coronary artery disease who had previously undergone revascularization. Results: This assay system exhibited high reproducibility (CV <10%) and a short processing time (<6 h). The myeloperoxidase- mediated oxidation of apoB-depleted serum impaired cholesterol uptake capacity. Cholesterol uptake capacity correlated significantly with cholesterol efflux capacity (r² = 0.47, n = 30). Furthermore, cholesterol uptake capacity correlated inversely with the requirement for revascularization because of recurrence of coronary lesions in patients with optimal control of LDL cholesterol (P < 0.01, n = 156). A multivariate analysis adjusted for traditional coronary risk factors showed that only cholesterol uptake capacity remained significant (odds ratio, 0.48; 95% CI, 0.29-0.80; P = 0.0048). Conclusions: Cholesterol uptake capacity assay evaluates the functionality of HDL in a sensitive and high-throughput manner without using radioisotope label and cells. This assay system could be used for the assessment of CVD risk in the clinical settings. [ABSTRACT FROM AUTHOR]

Polar auxin transport together with local synthesis and turnover are crucial for establishing auxin gradients, which determine an array of plant developmental pathways. Transcription factor-mediated control of the genes involved in these processes is steadily receiving increased attention, including efforts to find regulators determining the expression of major auxin efflux carriers of the PIN family. Now, Kong et al. (2017) have provided evidence of a direct link between auxin metabolism and transport mediated by PINs that is controlled by the transcription factor WRINKLED1. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

High body burdens of polybrominated diphenyl ethers (PBDEs) in infants and young children have led to increased concern over their potential impact on human development. PBDE exposure can alter the expression of genes involved in thyroid homeostasis, including those of ATP-binding cassette (ABC) transporters, which mediate cellular xenobiotic efflux. However, little information exists on how PBDEs interact with ABC transporters such as P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). The purpose of this study was to evaluate the interactions of 2,20,4,40-tetrabromodiphenyl ether (BDE-47) and its hydroxylated metabolite 6-OH-BDE-47 with P-gp and BCRP, using human MDR1- and BCRP-expressing membrane vesicles and stably transfected NIH-3T3-MDR1 and MDCK-BCRP cells. In P-gp membranes, BDE-47 did not affect P-gp activity; however, 6-OH-BDE-47 inhibited P-gp activity at low μM concentrations (IC50=11.7 μM). In BCRP membranes, BDE-47 inhibited BCRP activity; however, 6-OH-BDE-47 was a stronger inhibitor [IC50=45.9 μM (BDE-47) vs. IC50=9.4 μM (6-OH-BDE-47)]. Intracellular concentrations of known P-gp and BCRP substrates [(3H)-paclitaxel and (3H)-prazosin, respectively] were significantly higher (indicating less efflux) in NIH-3T3-MDR1 and MDCK-BCRP cells in the presence of 6-OH-BDE-47, but not BDE-47. Collectively, our results indicate that the BDE-47 metabolite 6-OH-BDE-47 is an inhibitor of both P-gp and BCRP efflux activity. These findings suggest that some effects previously attributed to BDE-47 in biological systems may actually be due to 6-OH-BDE-47. Considerations for human exposure are discussed. [ABSTRACT FROM AUTHOR]

Aims 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. Methods and Results Using gene-specific knock-out Camp. jejuni mutants and an efflux pump inhibitor, we showed Cme ABC 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. Conclusions 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. Significance and Impact of the Study 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. [ABSTRACT FROM AUTHOR]

The effect of temperature on the evolution of metabolism has been the subject of debate for a century; however, no consistent patterns have emerged from comparisons of metabolic rate within and among species living at different temperatures. We used experimental evolution to determine how metabolism evolves in populations of Drosophila melanogaster exposed to one of three selective treatments: a constant 16°C, a constant 25°C, or temporal fluctuations between 16 and 25°C. We tested August Krogh's controversial hypothesis that colder environments select for a faster metabolism. Given that colder environments also experience greater seasonality, we also tested the hypothesis that temporal variation in temperature may be the factor that selects for a faster metabolism. We measured the metabolic rate of flies from each selective treatment at 16, 20.5, and 25°C. Although metabolism was faster at higher temperatures, flies from the selective treatments had similar metabolic rates at each measurement temperature. Based on variation among genotypes within populations, heritable variation in metabolism was likely sufficient for adaptation to occur. We conclude that colder or seasonal environments do not necessarily select for a faster metabolism. Rather, other factors besides temperature likely contribute to patterns of metabolic rate over thermal clines in nature. [ABSTRACT FROM AUTHOR]

A properly functioning organism must maintain metabolic homeostasis. Deleterious mutations degrade organismal function, presumably at least in part via effects on metabolic function. Here we present an initial investigation into the mutational structure of the Caenorhabditis elegans metabolome by means of a mutation accumulation experiment. We find that pool sizes of 29 metabolites vary greatly in their vulnerability to mutation, both in terms of the rate of accumulation of genetic variance (the mutational variance, VM) and the rate of change of the trait mean (the mutational bias, Δ M). Strikingly, some metabolites are much more vulnerable to mutation than any other trait previously studied in the same way. Although we cannot statistically assess the strength of mutational correlations between individual metabolites, principal component analysis provides strong evidence that some metabolite pools are genetically correlated, but also that there is substantial scope for independent evolution of different groups of metabolites. Averaged over mutation accumulation lines, PC3 is positively correlated with relative fitness, but a model in which metabolites are uncorrelated with fitness is nearly as good by Akaike's Information Criterion. [ABSTRACT FROM AUTHOR]

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, 100 µg mL-1) corresponding to achievable serum levels and sputum levels by aerosolization. smeD expression was evaluated, under both conditions, in planktonic and biofilm cells by RT-PCR. The bactericidal effect of levofloxacin was decreased, in 3 out of 5 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. [ABSTRACT FROM AUTHOR]

Cell wall impermeability and active efflux of drugs are among the primary reasons for drug resistance in mycobacteria. Efflux pumps are tripartite membrane localized transport proteins that expel drug molecules outside the cells. Several of such efflux pumps are annotated in mycobacteria, but few have been characterized, like MSMEG 2991, a putative efflux pump permease of Mycobacterium smegmatis. To substantiate this, we overexpressed MSMEG 2991 protein in Escherichia coli 2443. Expression of MSMEG 2991 elevated the resistance towards structurally unrelated groups of antibiotics. An active antibiotic efflux pump nature of MSMEG 2991 was revealed by assessing the acquisition of ciprofloxacin in the absence and presence of the efflux pump inhibitor, carbonyl cyanide m-chlorophenyl hydrazone, indicating the involvement of proton-motive force (pmf) during the efflux activity. MSMEG 2991 expression elevated biofilm formation in E. coli by 4-fold, keeping parity to some of the earlier reported efflux pumps. In silico analysis suggested the presence of 12 transmembrane helices in MSMEG 2991 resembling EmrD efflux pump of E. coli. Based on in vivo and in silico analyses, MSMEG 2991 may be designated as a pmf-mediated multidrug efflux pump protein that expels diverse groups of antibiotics and might as well be involved in the biofilm enhancement. [ABSTRACT FROM AUTHOR]

Piscirickettsia salmonis is a fastidious intracellular pathogen responsible for high mortality rates in farmed salmonids, with serious economic consequences for the Chilean aquaculture industry. Oxytetracycline and florfenicol are the most frequently used antibiotics against P. salmonis, but routine use could contribute to drug resistance. This study identified differentiated florfenicol susceptibilities in two P. salmonis strains, LF-89 and AUSTRAL-005. The less susceptible isolate, AUSTRAL-005, also showed a high ethidium bromide efflux rate, indicating a higher activity of general efflux pump genes than LF-89. The P. salmonis genome presented resistance nodulation division (RND) family members, a family containing typical multidrug resistance-related efflux pumps in Gram-negative bacteria. Additionally, efflux pump acrAB genes were overexpressed in AUSTRAL-005 following exposure to the tolerated maximal concentration of florfenicol, in contrast to LF-89. These results indicate that tolerated maximum concentrations of florfenicol can modulate RND gene expression and increase efflux pump activity. We propose that the acrAB efflux pump is essential for P. salmonis survival at critical florfenicol concentrations and for the generation of antibiotic-resistant bacterial strains. [ABSTRACT FROM AUTHOR]

Objectives: The objective of this study was to determine the fitness of Escherichia coli mutants with reduced susceptibility to tigecycline after exposure to adverse conditions in vitro and in vivo.Methods: Survival in response to low pH, bile salts, oxidative stress and human serum was examined for E. coli mutants with reduced susceptibility to tigecycline due to single mutations that caused increased efflux (marR, lon) or impaired LPS (rfaC, rfaE, lpcA). An in vitro competition assay was used to determine growth fitness defects. Competitive fitness was assessed using mouse infection models. MICs, exponential growth rates and expression levels of efflux-related genes were measured for genetically reconstructed double and triple mutants.Results: The LPS mutants were 48-85-fold more susceptible to bile salts compared with the ERN mutants and the WT. As shown by in vitro competitions, the fitness reduction was 0.3%-13% for ERN mutants and ∼24% for LPS mutants. During in vivo survival experiments, LPS mutants were outcompeted by the WT strain in the thigh infection model. Constructed double ERN and LPS mutants showed additive and synergistic increases in tigecycline MICs.Conclusions: Generally, reduced susceptibility to tigecycline caused a decrease in fitness under stressful in vitro and in vivo conditions with ERN mutants being fitter than LPS mutants. When combined, ERN mutations caused a synergistic increase in the MIC of tigecycline. These findings could explain why clinical resistance to tigecycline in E. coli is mainly associated with up-regulation of the AcrAB efflux system. [ABSTRACT FROM AUTHOR]

Objectives: Drug efflux pumps are one of the key machineries in bacterial drug resistance. Although quite a few of these transport systems have been functionally characterized in various organisms, due to large-scale genome sequencing efforts and improved prediction pipelines there are increasing numbers of putative drug efflux genes annotated. For phenotype identification of the proteins encoded by these genes, we developed a novel high-throughput phenotype screening strategy and demonstrated its utility in identifying phenotypes for putative efflux systems encoded by the human pathogen Acinetobacter baumannii.Methods: Seventeen putative drug efflux systems from A. baumannii were heterologously expressed in Escherichia coli. For rapid and economical phenotype screening we employed a combination of multiplexed Biolog Phenotype Microarrays and quantitative PCR. Using this method we screened these putative drug efflux pumps against 240 antimicrobial conditions, equating to 4080 simultaneous phenotypic tests.Results: Of the 17 putative drug efflux systems, phenotypes were confirmed for two pumps and novel drug resistance phenotypes were identified for three new A. baumannii drug transporters, which exemplified the power of this method as a high-throughput screening technique. One of the phenotypically characterized putative drug efflux systems was classified within the ATP-binding cassette superfamily of transport proteins and represents the first drug resistance protein characterized from this superfamily in A. baumannii. The remaining two proteins were members of the major facilitator superfamily of efflux pumps.Conclusions: This method has broad potential for high-throughput phenotype characterization of putative drug efflux systems in a range of organisms. [ABSTRACT FROM AUTHOR]

Background: The transmembrane ATP-binding cassette (ABC) transporters actively efflux an array of clinically relevant compounds across biological barriers, and modulate biodistribution of many physiological and pharmacological factors. To date, over 48 ABC transporters have been identified and shown to be directly and indirectly involved in peri-implantation events and fetal/placental development. They efflux cholesterol, steroid hormones, vitamins, cytokines, chemokines, prostaglandins, diverse xenobiotics and environmental toxins, playing a critical role in regulating drug disposition, immunological responses and lipid trafficking, as well as preventing fetal accumulation of drugs and environmental toxins.Methods: This review examines ABC transporters as important mediators of placental barrier functions and key reproductive processes. Expression, localization and function of all identified ABC transporters were systematically reviewed using PubMed and Google Scholar websites to identify relevant studies examining ABC transporters in reproductive tissues in physiological and pathophysiological states. Only reports written in English were incorporated with no restriction on year of publication. While a major focus has been placed on the human, extensive evidence from animal studies is utilized to describe current understanding of the regulation and function of ABC transporters relevant to human reproduction.Results: ABC transporters are modulators of steroidogenesis, fertilization, implantation, nutrient transport and immunological responses, and function as 'gatekeepers' at various barrier sites (i.e. blood-testes barrier and placenta) against potentially harmful xenobiotic factors, including drugs and environmental toxins. These roles appear to be species dependent and change as a function of gestation and development. The best-described ABC transporters in reproductive tissues (primarily in the placenta) are the multidrug transporters p-glycoprotein and breast cancer-related protein, the multidrug resistance proteins 1 through 5 and the cholesterol transporters ABCA1 and ABCG1.Conclusions: The ABC transporters have various roles across multiple reproductive tissues. Knowledge of efflux direction, tissue distribution, substrate specificity and regulation of the ABC transporters in the placenta and other reproductive tissues is rapidly expanding. This will allow better understanding of the disposition of specific substrates within reproductive tissues, and facilitate development of novel treatments for reproductive disorders as well as improved approaches to protecting the developing fetus. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is a ubiquitous environmental bacterium and an opportunistic pathogen with the ability to rapidly develop multidrug resistance under selective pressure. Previous work demonstrated that upon exposure to the environmental contaminant pentachlorophenol (PCP), P. aeruginosa PAO1 increases expression of multiple multidrug efflux pumps, including the MexAB-OprM pump. The current study describes increases in the antibiotic resistance of PAO1 upon exposure to PCP and other chlorinated organics, including triclosan. Only exposure to chlorinated phenols induced the mexAB-oprM-mediated antibiotic-resistant phenotype. Thus, chlorinated phenols have the potential to contribute to transient phenotypic increases of antibiotic resistance that are relevant when both compounds are present in the environment. [ABSTRACT FROM AUTHOR]

The accumulation of succinate under hypoxic/ischaemic conditions is conserved across diverse tissues and species, 2 and succinate accumulatedduringischaemiaplaysacentralroleinmediatingdamageupontissue reperfusion, i.e., ischaemia-reperfusion (IR) injury. 3 There are two proposed mechanisms of ischaemic succinate accumulation: in one, reversal of mitochondrial succinate dehydrogenase (complex II) reduces fumarate to succinate, while in the other, succinate originates from canonicalKrebs'cycleactivitywithcomplexIIinhibitedduetoreductionof the co-enzyme Q pool. [Extracted from the article]

Background Biocides play an integral role in infection control. Paralleling concern about rising incidence of multidrug-resistant (MDR) organisms is a concern for resistance to biocides. In small studies, several genes involved in the production of efflux pump proteins have been identified as markers of biocide resistance in Klebsiella spp. namely cepA , qacA , qacE , qac∆E , and acrA. This study aimed to analyze the Klebsiella spp. isolates of a previously defined military trauma group with a high incidence of MDR organisms for the presence of these genes and their correlation with other resistance. Methods All infecting K. pneumoniae, K. variicola , and K. quasipneumoniae isolates archived by the Trauma Infectious Disease Outcomes Study (June 2009–December 2014) were selected. Additionally, all colonizing isolates linked with infecting isolates were included; the remainder to total 50 MDR and 46 non-MDR colonizing isolates were chosen randomly. Antimicrobial identification and susceptibilities were determined by CLSI criteria using the BD Phoenix Automated Microbiology System. PCR according to published methods for cepA , qacA , qacE , qac∆E , and acrA was accomplished in duplicate. MDR was defined as either resistance to ≥3 classes of aminoglycosides, β-lactams, carbapenems and/or fluoroquinolones or production of an ESBL or KPC. Results A total of 237 isolates (221 K. pneumoniae , 10 K. variicola , 6 K. quasipneumoniae) met inclusion criteria, of which 149 (63%) were MDR. All isolates had been exposed to antimicrobials prior to isolation. Of all isolates, 234 (98%) carried cepA : 218 (98%) K. pneumoniae carried cepA, 10 (100%) K. variicola carried cepA, and 6 (100%) of K. quasipneumoniae carried cepA. In addition, 148 (62%) isolates with cepA were MDR. One (10%) K. variicola isolate carried qacE along with cepA. This isolate was the only MDR K. variicola. None of the isolates carried qacA, qac∆E, or acrA. Conclusion We confirmed the near universal presence of the cepA biocide resistance gene in Klebsiella spp. isolated from trauma patients in Iraq and Afghanistan. In the largest evaluation of biocide resistance genes in Klebsiella spp. to our knowledge, the presence of qacA , qacE , qac∆E , and acrA was less common than has been reported elsewhere. Disclosures All authors: No reported disclosures. [ABSTRACT FROM AUTHOR]

We previously observed that PRRSV infection increased plasma Met and Thr flux but decreased Lys flux in growing pigs. These changes reflect modification of AA utilization during ISS. This study evaluated the effects of ISS induced by LPS on whole body protein deposition (PD) and plasma free amino acid (AA) flux and pool size. Ten gilts (BW 9.4 ± 1.1 kg) were surgically fitted with venous catheters, individually housed in metabolism crates, and feed restricted (550 g/d) on a corn-SBM based diet (ME 14.3 MJ/kg, SID Lys 11.5 g/kg). ISS was induced by two intramuscular injections of increasing amounts of LPS (30 and 36 ug/kg BW) given 48 h apart. Blood samples were collected at 0 and 72 h after initiation of ISS and assayed for hematology and blood chemistry. Body temperature (BT) was monitored on a daily basis. N-balances were determined during a 3-d pre-ISS and a 3-d ISS period. At the end of each N-balance period, a single dose of [U-13C, U-15N] AA mixture (Lys, Met, Thr, Trp, He, Leu, Val, Phe, Gin) was infused intravenously, and serial blood samples were taken at 0, 2.5, 5, 7.5, 10, 15, 20, 30, and 45 min after tracer administration to determine isotopic enrichment. A double exponential model was fitted to the plasma enrichment for each pig and AA, and equation parameters were used to estimate plasma AA flux and pool size. Blood chemistry, hematology, and BT results indicated that LPS induced effective ISS in pigs (P < 0.05). ISS had no effect on PD (59.4 vs. 55.7 g/d; P = 0.31), but it decreased plasma flux (umol/kg BW/h) for He (112 vs. 76; P < 0.05) and Phe (126 vs. 79; P < 0.05). In agreement with PRRSV challenged pigs, LPS tended to reduce the plasma Lys flux (from 394 to 325, P = 0.08). Plasma flux of other AA was not affected by ISS. ISS increased and tended to increase the pool size (umol/kg BW) for free Leu (22 vs. 34, P < 0.05) and Gin (16 vs. 25, P = 0.11), respectively, but reduced the pool size for free He (13 vs. 9.0, P < 0.05). Collectively, these results suggest that ISS induced by LPS alters AA flux and pool size in growing pigs. The decrease in Lys, Phe, and He flux in LPS induced ISS pigs may be attributed to a reduction in whole body protein synthesis or decreased catabolism of these AA. NPB project 13-082. [ABSTRACT FROM AUTHOR]